Cystic Fibrosis (Lung Biology in Health and Disease)

Cystic Fibrosis

LUNG BIOLOGY IN HEALTH AND DISEASE

Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

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The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

Cystic Fibrosis Edited by Julian L. Allen University of Pennsylvania School of Medicine The Children’s Hospital of Philadelphia Philadelphia, Pennsylvania, U.S.A. Howard B. Panitch University of Pennsylvania School of Medicine The Children’s Hospital of Philadelphia Philadelphia, Pennsylvania, U.S.A. Ronald C. Rubenstein University of Pennsylvania School of Medicine The Children’s Hospital of Philadelphia Philadelphia, Pennsylvania, U.S.A.

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2010 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business Cover illustration courtesy Marlene Galzin No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-4398-0181-9 (Hardcover) International Standard Book Number-13: 978-1-4398-0181-9 (Hardcover) Thisbookcontainsinformationobtainedfromauthenticandhighlyregardedsources.Reprintedmaterial is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Cystic fibrosis / edited by Julian L. Allen, Howard B. Panitch, and Ronald C. Rubenstein. p. ; cm. — (Lung biology in health and disease ; 242) Includes bibliographical references and index. ISBN-13: 978-1-4398-0181-9 (hb : alk. paper) ISBN-10: 1-4398-0181-9 (hb : alk. paper) 1. Cystic fibrosis. I. Allen, Julian Lewis. II. Panitch, Howard B. III Rubenstein, Ronald. IV. Series: Lung biology in health and disease ; v. 242. [DNLM: 1. Cystic Fibrosis. W1 LU62 v.242 2010 / WI 820 C99685 2010] RC858.C95C9332 2010 616.30 72—dc22 2009051589 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

To Debby, Eli and Jeremy, whose love, understanding and humor have enabled me to complete this book; to my parents, Beatrice and Emmanuel, who taught me to love music and science... JLA To Mary, Oren and Becky, for your encouragement and understanding; to my parents for demonstrating their love of learning... HBP To Andrea, Gavriel, Adina and Watson, for sharing this journey; to my parents, Honora and Murray for helping the journey start... RCR ...and to our patients and their families, who have taught us so much.

Preface

Improvement in the outcome of people with cystic fibrosis (CF) over the past 50 years is truly remarkable. What was once an almost universally fatal childhood genetic disorder has, through advances in research and clinical care, become a chronic illness whereby presently almost half of the people living with CF are adults. The known disease-causing mutations of the cystic fibrosis transmembrane regulator (CFTR) protein gene, which was cloned in 1989, have expanded from just over 150 in 1993 to over 1400 in 2009. Many of the physiologic processes influenced by abnormal CFTR function, from alterations in electrolyte transport to abnormalities of innate airways defense, are now recognized. Nevertheless, CF remains a disease that shortens and alters the quality of the lives of most affected individuals, and much progress remains to be made. In addition to new insights into the basic mechanisms that cause the CF phenotype, our understanding of the pathogenesis of both pulmonary and extrapulmonary manifestations of the disease have advanced. Concurrently, new techniques have been developed to detect CF lung disease at its earliest stages, well before it is clinically apparent. The confluence of these discoveries has led to the development of new therapeutic agents and approaches for the care of people with CF. This book attempts to detail recent insights and knowledge of CF pathogenesis, treatment, and health care systems approaches. We hope to make accessible to caregivers an increased understanding of both the molecular basis of CF and its expanding clinical features. We also hope to provide a common knowledge base that will allow the generation of testable hypotheses for future basic, clinical and translational research. We have sought to highlight new challenges for improving care and outcomes; new approaches for diagnosing, assessing, and treating CF; and the “new” clinical manifestations that have resulted from greater longevity of people with CF. We have also endeavored to relate the underlying cellular and molecular pathophysiologies to their relevant clinical phenotypes, and thereby provide the rationale for novel interventions. In this way, we hope this volume gives the reader an idea of how far we have come in the care of people living with CF, how far we still have to go, and some ideas about how we might get there.

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Preface

A book such as this is not possible without the contributions of many. We would like to thank the editorial staff at Informa Healthcare, especially Joseph Stubenrauch and Aimee Laussen, for their efficiency and especially their willingness to accommodate numerous last-minute changes. And, of course, we would like to thank the authors, whose knowledge and expertise are extraordinary. Julian L. Allen Howard B. Panitch Ronald C. Rubenstein

Contributors

Julian L. Allen University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. David Barker University of Miami, Coral Gables, Florida, U.S.A. Suzanne E. Beck University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Alan S. Brody Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A. A. Whitney Brown University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Andrew Bush Imperial School of Medicine at National Heart and Lung Institute and Royal Brompton Hospital, London, U.K. Andrew C. Calabria University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. James F. Chmiel Case Western Reserve University School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A. John P. Clancy University of Alabama at Birmingham and Children’s Hospital of Alabama, Birmingham, Alabama, U.S.A. Susan Coffin University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Stephanie D. Davis University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Linda A. DiMeglio Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, Indiana, U.S.A.

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Contributors

Scott H. Donaldson University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Kelly Dougherty University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Mitchell Drumm Case Western Reserve University, Cleveland, Ohio, U.S.A. Peter R. Durie University of Toronto and the Hospital for Sick Children, Toronto, Ontario, Canada Andrew P. Feranchak University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. Terence R. Flotte University of Massachusetts Medical Center, Worcester, Massachusetts, U.S.A. J. Kevin Foskett University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A. Ioanna K. Gisone The Children’s Hospital of Philadelphia, Craig-Dalsimer Division of Adolescent Medicine, Philadelphia, Pennsylvania, U.S.A. Samuel Goldfarb University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Nadine G. Haddad Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, Indiana, U.S.A. Wynton Hoover University of Alabama at Birmingham and Children’s Hospital of Alabama, Birmingham, Alabama, U.S.A. Andrea Kelly University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Jeffrey C. Klick University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Michael W. Konstan Case Western Reserve University School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A.

Contributors

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James L. Kreindler University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Craig Lapin Connecticut Children’s Medical Center, University of Connecticut, Hartford, Connecticut, U.S.A. Asim Maqbool University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Maria Mascarenhas University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Oscar H. Mayer University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Susanna A. McColley Northwestern University Feinberg School of Medicine, Chicago, Illinois, U.S.A. Suzanne H. Michel University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Christian Mueller University of Massachusetts Medical Center, Worcester, Massachusetts, U.S.A. Joshua P. Needleman Weill Cornell Medical College, New York, New York, U.S.A. Brian P. O’Sullivan University of Massachusetts Medical School and UMass Memorial Health Care, Worcester, Massachusetts, U.S.A. Chee Y. Ooi (Keith) The Hospital for Sick Children, Toronto, Ontario, Canada Howard B. Panitch University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Jessica E. Pittman University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Paul J. Planet Columbia University, New York, New York, U.S.A. Alexandra L. Quittner University of Miami, Coral Gables, Florida, U.S.A.

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Contributors

Molly Raske Seattle Children’s Hospital, Seattle, Washington, U.S.A. Felix Ratjen Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Clement L. Ren University of Rochester, Rochester, New York, U.S.A. Susan Rettig The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Fariba Rezaee University of Rochester, Rochester, New York, U.S.A. Walter M. Robinson Vanderbilt Children’s Hospital, Center for Biomedical Ethics and Society, Vanderbilt University, Nashville, Tennessee, U.S.A. Margaret Rosenfeld Seattle Children’s Hospital, Seattle, Washington, U.S.A. Alisha J. Rovner Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, U.S.A. Ronald C. Rubenstein University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Lisa Saiman Columbia University, New York, New York, U.S.A. Don B. Sanders University of Wisconsin, Madison, Wisconsin, U.S.A. Meghana N. Sathe University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. Michael S. Schechter Emory University and Children’s Healthcare of Atlanta, Atlanta, Georgia, U.S.A. Virginia A. Stallings University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Laurence Suaud University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A. Sherstin G. Truitt University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

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Lisa K. Tuchman Children’s National Medical Center, Division of Adolescent and Young Adult Medicine, Children’s Research Institute, Center for Clinical and Community Research, Washington, D.C., U.S.A. Elizabeth Tullis University of Toronto and St. Michael’s Hospital, Toronto, Ontario, Canada James R. Yankaskas University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

Contents

Preface . . . . ix Contributors . . . . .

xi

Part I Pathophysiology 1. The Genetics of Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . Laurence Suaud and Ronald C. Rubenstein

1

2. Ion Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . James L. Kreindler and J. Kevin Foskett

11

3. Mucus Abnormalities and Ciliary Dysfunction . . . . . . . . . . A. Whitney Brown and Scott H. Donaldson

24

4. Microbiology in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . Paul J. Planet and Lisa Saiman

36

5. Inflammation in the Cystic Fibrosis Lung . . . . . . . . . . . . . James F. Chmiel and Michael W. Konstan

57

6. Modifier Genes of Cystic Fibrosis . . . . . . . . . . . . . . . . . . . Mitchell Drumm

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Part II Diagnostics 7. Cystic Fibrosis: Diagnosis, Sweat Testing, and Newborn Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brian P. O’Sullivan

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8. Diagnostic Approach to Diseases Associated with Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chee Y. Ooi (Keith), Elizabeth Tullis, and Peter R. Durie 9. Lung Function Testing in Infants . . . . . . . . . . . . . . . . . . . . Jessica E. Pittman and Stephanie D. Davis

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10. Assessment of Lung Function in Young Children with Cystic Fibrosis . . . . . . . . . . . . . . . . . Fariba Rezaee and Clement L. Ren

148

11. Lung Function Testing in School-Age Children with Cystic Fibrosis . . . . . . . . . . . . . . Oscar H. Mayer and Julian L. Allen

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12. Thoracic Imaging in Cystic Fibrosis Pulmonary Disease . . . Molly Raske and Alan S. Brody

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Part III Clinical Manifestations and Treatment 13. Pulmonary Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . Andrew Bush 14. Treatment Strategies for Maintaining Pulmonary Health in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Susanna A. McColley

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15. Mucolytic Therapy and Airway Clearance Techniques . . . . Felix Ratjen and Craig Lapin

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16. Pulmonary Exacerbations . . . . . . . . . . . . . . . . . . . . . . . . . Don B. Sanders and Margaret Rosenfeld

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17. Gastrointestinal Complications of Cystic Fibrosis . . . . . . . . Maria Mascarenhas and Asim Maqbool

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18. Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Meghana N. Sathe and Andrew P. Feranchak

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19. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Asim Maqbool, Kelly Dougherty, Alisha J. Rovner, Suzanne H. Michel, and Virginia A. Stallings

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20. Bone Health and Treatment . . . . . . . . . . . . . . . . . . . . . . . . Nadine G. Haddad and Linda A. DiMeglio

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21. Cystic Fibrosis–Related Diabetes and Management . . . . . . . Andrea Kelly and Andrew C. Calabria

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22. Other Extrapulmonary Complications and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Joshua P. Needleman and Suzanne E. Beck 23. Chronic Respiratory Failure and the Roles of Noninvasive Ventilation and Lung Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Samuel Goldfarb and Howard B. Panitch 24. Gene Repair: Past, Present, and Future . . . . . . . . . . . . . . . Christian Mueller and Terence R. Flotte 25. Restoration of CFTR Function with Small-Molecule Modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wynton Hoover and John P. Clancy

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Part IV Psychosocial Considerations and Care Systems 26. Quality Improvement in Cystic Fibrosis Care . . . . . . . . . . . Michael S. Schechter

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27. Cystic Fibrosis and Infection Control . . . . . . . . . . . . . . . . . Susan Rettig and Susan Coffin

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28. Transition to Adult Care . . . . . . . . . . . . . . . . . . . . . . . . . . Sherstin G. Truitt and James R. Yankaskas

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29. Reproduction, Sexuality, and Fertility . . . . . . . . . . . . . . . . Lisa K. Tuchman and Ioanna K. Gisone

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30. A Biopsychosocial Model of Cystic Fibrosis: Social and Emotional Functioning, Adherence, and Quality of Life . . . David Barker and Alexandra L. Quittner 31. Palliative and End-of-Life Care in Cystic Fibrosis . . . . . . . Walter M. Robinson and Jeffrey C. Klick Index . . . . 497

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1 The Genetics of Cystic Fibrosis LAURENCE SUAUD and RONALD C. RUBENSTEIN University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

The CF Gene

Over recent years there has been a dramatic increase in our understanding of the genetics of cystic fibrosis (CF). In 1949, Lowe et al. postulated that CF must be caused by a defect in a single gene on the basis of the autosomal recessive pattern of inheritance of the disease (1). The identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 1989 by Collins et al. (2–4) created new hope for curative treatment. The CFTR gene is localized on the long arm of chromosome 7 (7q21-34), spanning approximately 190 kb of genomic DNA (5). The gene consists of 27 exons and encodes a mature mRNA transcript of 6.5 kb that is translated into a 1480–amino acid protein. CFTR is found in various epithelial cell types at the apical surface, including respiratory epithelia and submucosal glands, exocrine pancreas, liver, sweat ducts, and the reproductive tract. In these locations, its main function is to act as a cAMP-mediated chloride channel that regulates the ion and water balance across epithelia (6,7). It has also been reported to be present and to have function in the brain (8), neonatal murine cardiac myocytes (9), erythrocytes (10,11), and macrophages (12), among other cell types. CFTR is a member of the ATP-binding cassette (ABC) membrane transporter superfamily that includes proteins such as the multiple drug resistance protein (MDR) and bacterial periplasmic permeases. Like other ABC transporters, the CFTR protein contains two homologous halves comprising two membrane-spanning domains, each with six helices, and two nucleotide-binding domains (NBDs) (3). However, unlike other ABC transporters, CFTR has a unique regulatory (R) domain that separates the homologous halves and contains many charged amino acids and consensus sites for phosphorylation by protein kinases (Fig. 1). The two membrane-spanning domains form a low-conductance chloride channel pore (Fig. 1). CFTR is activated by protein kinase A (PKA), and is probably also regulated by protein kinase C (PKC), with phosphorylation occurring at multiple sites located in the R domain. As phosphorylation by PKA is mandatory for channel activity, CFTR channel is considered a “cAMP-activated channel.”

II.

Incidence

CF remains one of the commonest life-threatening autosomal recessive conditions affecting Caucasians. There are approximately 30,000 affected individuals in the United States, and about 1000 new cases are diagnosed each year. The incidence is 1/2500 to

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Suaud and Rubenstein

Figure 1 Proposed domain structure of the CFTR protein within the cell membrane. Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; MSD, membrane-spanning domain; NBD, nucleotide-binding domain; R, regulatory domain.

Table 1 CF Incidence Across the Globe

Country Finland Mexico Sweden Poland North Ireland Russia Denmark Norway The Netherlands Spain Greece Germany United States Czech republic United Kingdom Australia Italy France Switzerland Ireland Abbreviation: CF, cystic fibrosis. Source: From Ref. 13.

Incidence (1 case per x birth) 25000 8500 7300 6000 5350 4900 4700 4500 3650 3500 3500 3300 2835 2833 2600 2500 2438 2350 2000 1800

The Genetics of Cystic Fibrosis

3

1/90,000, and varies between populations of different ethnicity (Table 1): in Caucasians the incidence is estimated to be 1/3200; in African-Americans 1/15,000; and in AsianAmericans 1/31,000. Even among northern European Caucasian populations, the incidence varies significantly; the incidences in Ireland and Sweden are 1/1800 and 1/7300 live births, respectively (14). The geographical distribution of CFTR mutations also varies worldwide. These variations are likely to be due to founder effects and subsequent patterns of migration and settlement. The high prevalence of the CF gene in certain populations has led to speculations that there may be some selective advantage for heterozygotic carriers (15,16). For example, mutations in the CFTR gene are hypothesized to provide increased resistance to infectious diseases, thereby maintaining the mutant alleles at high frequency in selected populations. Pier and colleagues hypothesized that heterozygote carriers of CFTR mutations had increased resistance to infection with intracellular organisms such as Salmonella (17), while others hypothesized that carriers have a selective advantage because of resistance to diarrhea-causing enterotoxins elaborated by Vibrio cholera and Escherichia coli (18). Cholera toxin (CT) and the heat-labile toxin elaborated by enterotoxic E. coli cause irreversible activation of the stimulatory guanine nucleotide–binding protein, Gs, which in turn activates membrane-bound adenylyl cyclase. This greatly elevates cellular cAMP levels and causes subsequent activation of PKA, phosphorylation of R domain of CFTR, and opening of the CFTR channel, which is the predominant cAMP-regulated Cl
channel in the intestinal and colonic epithelia. The end result of this signaling cascade is massive Cl
secretion into the intestinal lumen and potentially lethal secretory diarrhea characterized by, in the case of cholera, “rice water stools.” The data of Gabriel and colleagues from cftr null and heterozygote carrier mice support this selective advantage hypothesis, as well as a central role of CFTR in the Cl
secretory response of the intestinal epithelia to CT. In response to intraluminal injection of CT, heterozygous CF-carrier mice secreted 50% less volume of fluid into their intestines than wild-type mice under the same conditions. The cftr
/
knockout mice had no fluid-secretory response to intraluminal injection of CT. However, this hypothesis was disputed by Hogenauer et al., who measured the in vivo basal (unstimulated) and prostaglandin-stimulated jejunal chloride secretion in normal human subjects, CF heterozygote carriers, and subjects with CF (19); prostaglandins similarly act to increase cellular cAMP. These data indicated that while subjects with CF had essentially no active chloride secretion in response to the prostaglandin secretagogue, individuals who were heterozygous carriers of a CF mutation secreted chloride at the same rate as people without a CF mutation. These data thus contradicted the earlier mouse model data. Furthermore, it is not clear how such resistance to cholera as the selective advantage for heterozygote carriers would have been significantly beneficial in the populations with the highest CFTR mutation carrier frequencies, as cholera is not endemic in those geographic regions.

III.

Classes of CFTR Mutations

More than 1500 CF mutations have been reported. Many of these mutations are rare in the population, with only a few affected individuals reported. There are also a number of silent and nonsilent changes in the coding sequence that are not clearly demonstrated to

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Suaud and Rubenstein

Table 2 Most Common Mutations Found in the Total U.S. Population

Mutation

Prevalence (%)

DeltaF508 G542X G551D W1282X N1303K

68.6 2.4 2.1 1.4 1.3

Source: From Ref. 13.

induce CFTR dysfunction; such changes are therefore considered CFTR polymorphisms. Thus, the number of true disease-causing mutations is likely fewer [see the CFTR mutations database (20)]. The disease-causing mutations are situated throughout the entire coding region of the gene, and also the promoter and intronic regions, although there are “hot spot” regions where mutations are more common, such as the NBDs, the cytoplasmic loops of the transmembrane domains that are hypothesized to interact with the NBDs (21), and the regulatory (R) domain. The most common disease-causing CFTR mutation worldwide is DF508, which occurs on approximately 70% of mutant CFTR alleles. This absence of phenylalanine at position 508 of the CFTR protein results from an in-frame deletion of three base pairs from exon 10. The DF508 mutation results in an abnormal protein that is defective with regards to intracellular processing. This leads to absence of CFTR channels from the membrane and, as a result, absent CFTR function. This is discussed further below. The majority of the other mutations have significantly lower allele frequencies of less than 2% to 3% (Table 2), although this can clearly vary greatly within populations or ethnic groups suggesting founder effects. For example, the W1282X mutation has an allele frequency of 1% to 2% in the North American Population, but is the predominant allele in Israel and in those of Ashkenazi Jewish descent (22). Many of other mutations are unique to a particular individual or family or have been found in only a handful of cases across the world. Given the large and complicated makeup of the CFTR gene and promoter, as well as the large number of described rare mutations found by sequencing the CFTR coding region and targeted areas of intronic and promoter DNA, it is not surprising that such gene sequencing does not always identify two CFTR mutations in people with CF. Using present commercial techniques, up to 1% of people with a clinical diagnosis of CF may not have two identifiable CFTR mutations. It has become convenient to classify the large number of described CFTR mutations into six different groups according to the mechanism by which they disrupt CFTR Cl
transport function (Fig. 2) (23). However, for the vast majority of CFTR mutations, especially rarer mutations, mechanistic data on which to properly classify such mutations according to this scheme is lacking. Furthermore, there are a number of mutations that either have mechanistic defects that overlap two classes or frankly defy classification according to this scheme. Nevertheless, we briefly discuss this classification scheme here, as it has been clearly useful in assisting clinicians and investigators in thinking about genotype-phenotype correlation in people with CF- or CFTR-related disorders, and in guiding the development of novel, mutation-directed therapies for CF (discussed in Chapter 25 by Hoover and Clancy).

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5

Figure 2 Classes of mutant CFTR. Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator.

Class I mutations abolish protein production and result in complete loss of CFTR protein, and therefore CFTR function. About half of all mutations in CFTR (encompassing gene deletions, exon skipping due to aberrant mRNA splicing, and single nucleotide or smaller deletions leading to reading frame shifts) are thought to fall into this class. Class I also includes mutations that generate premature stop codons, including G542X and W1282X, that lead to early termination of protein translation and rapid mRNA degradation (24). Combined, these “X” mutations have an overall allele frequency of approximately 10%, and, as described in Chapter 25, may be targets for therapies that allow the ribosome to “read through” or suppress these premature termination codons. Class II CFTR mutations are characterized by aberrant intracellular trafficking of the CFTR protein, and therefore the absence of the functional protein from the apical membrane of epithelia. Mutations of this class include the common DF508, and N1303K. In the case of DF508, the mutant CFTR is recognized by the cell quality control mechanism and subsequently degraded. Interestingly, DF508 retains the ability to transport Cl
across intracellular membranes (25), and a number of physical and chemical maneuvers (26), including reduced temperature incubation (27), allow it to reach the apical membrane. However, both DF508 (28) and N1303K (29) appear to have abnormal functions even when at the plasma membrane. This suggests that these mutations make CFTR dysfunctional by multiple mechanisms, and that these mutations may, in fact, have characteristics of more than one CFTR mutation class. Class III mutations disrupt activation and regulation of mutant CFTRs, which are appropriately localized at the plasma membrane. Thus, biosynthesis, trafficking, and

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processing are undisturbed, but the channel may be defective with respect to phosphorylation by PKA or the subsequent regulation of channel opening. This class of dysfunction tends to result from missense mutations in key areas of the protein that are important for regulation of CFTR function (30). The most common mutation of this class is G551D, which has an allele frequency of approximately 2%. G551D is a mutation in CFTR’s first nucleotide-binding fold (NBD-1), which results in a very low channel open probability. This leads to essentially absent CFTR function despite G551D-CFTR’s appropriate localization at the apical membrane of epithelia. Class IV CFTR mutants are defined by aberrant or reduced chloride conduction. These mutants tend to result from more conservative missense mutations of CFTR, and retain normal intracellular location of the mutant protein at the apical epithelial membrane (31). The most common of these class IV mutations is R117H, which is the substitution of a slightly less strongly positively charged histidine for a strongly positively charged arginine residue. Class V mutations reduce the amount of normal CFTR protein in the cell and at the apical membrane by decreasing, but not eliminating, protein production. However, in general, the CFTR protein that is produced by mutations of this class functions normally. Such effects may result from mutations in the promoter or by inefficient mRNA splicing. One example of a class V mutation is the 3849 þ 10 kb C?T mutation found in intron 19, which reduces the splicing efficiency of the CFTR mRNA to approximately 8% of normal (32). Another intronic mutation, 2789 þ 5 kb G?A reduces mRNA splicing efficiency to approximately 4% by altering the splice donor site of exon 14b (33). Class VI mutations, like class V mutations, reduce the amount of functional CFTR protein at the apical membrane. However, in contrast to class V mutations that decrease CFTR production, class VI mutations cause an increased rate of CFTR’s removal from the apical plasma membrane. Mutations leading to this type of CFTR defect are uncommon and include N287Y (34) as well as mutations that delete the carboxyl terminus of the CFTR protein.

IV.

Genotype/Phenotype Correlation

In general, mutations of classes I, II, and III are associated with absent CFTR function. Thus, any person with CF who is homozygous for a mutation of one of these classes, or who is compound heterozygous for any combination of class I, II, or III mutations, is expected to have an overall absence of CFTR function. This typically results in a “severe” or “classic” CF phenotype including exocrine pancreatic insufficiency. This phenotype is present in 85% to 90% of people with CF. Further attempts to delineate genotype/phenotype correlations in this group of people with CF have been less successful, perhaps because almost 50% of people with CF are DF508-homozygotes, and additional approximately one-third are DF508 compound heterozygotes. On the other hand, even among the people with CF who are DF508-homozygous, there is tremendous variability in phenotype and clinical course. This has led to the hypothesis and recent demonstration that polymorphisms in other genes, such as transforming growth factor b (TGF-b), are associated with significantly different clinical CF phenotypes (35). The topic of genes that modify the CF phenotype is discussed in more depth elsewhere in Chapter 6 by Drumm.

The Genetics of Cystic Fibrosis

7

The presence of class IV and V CFTR mutations are associated with significant residual CFTR function, even when present with a second allele where function is absent (i.e., a class I, II, or III mutation). This residual CFTR function of the class IV or V allele usually manifests clinically as phenotypically milder CF, often with exocrine pancreatic sufficiency and less sinopulmonary symptomatology. This milder phenotype occurs in the remaining 10% to 15% of people with CF. Interestingly, the amount of residual function of a class IV or V mutant CFTR, such as R117H, and subsequently clinical phenotype can be significantly modified by polymorphisms that influence how much mRNA encoding R117H, and therefore how much R117H protein is made. One well-studied example of this is exon 8-9 splicing. Failure to correctly splice exon 8 to exon 9 results in a nonfunctional CFTR, and the efficiency and fidelity of this reaction is influenced by a polythymidine sequence within intron 8 preceding the exon 9 splice acceptor site (36). This polythymidine tract is polymorphic with sequences of 5, 7, or 9 thymidines (5T, 7T, or 9T, respectively), and the efficiency of exon 8-9 splicing is directly proportional to the length of the thymidine sequences. The 9T variant allows the greatest proportion of normal exon 8-9 splicing and functional CFTR production, while the 5T variant is associated with the highest level of mRNA missplicing and nonfunctional CFTR protein production (36). The commonest polymorphism is the 7T variant, which has a splicing efficiency intermediate to that of 5T and 9T. The DF508 mutation occurs exclusively associated with the 9T variant, while other class I, II, or III mutations are more often found in cis with 9T than is wild-type CFTR (37). These data suggest that evolution has either sensed that more CFTR function is needed when CFTR is mutant, or that CFTR, when present, should have limited expression. In the case of R117H, this intron 8 polymorphism can clearly modulate phenotype. For a person with one R117H allele and a second nonfunctional allele of class I, II, or III, such as W1282X, DF508, or G551D, those with 5T in cis with R117H typically have more clinical symptoms of CF, including sweat chloride elevation, than do people with 7T in cis with R117H, who may even have normal or borderline abnormal sweat tests. Those with 9T in cis with R117H can, in fact, have minimal, if any, outward symptoms of classical or even pancreatic sufficient CF (36,38,39). Interestingly, the penetrance of the 5T polymorphism in men with congenital bilateral absence of the vas deferens (CBAVD) can be significantly influenced by a number of TG repeats directly adjacent to and upstream of 5T tract. In men with CBAVD who had a severe CFTR mutation (class I, II, or III) on one allele and a normal CFTR in cis with 5T on the other allele, a greater number of TG repeats, 12 or 13, adjacent to 5T were associated with a higher likelihood of CBAVD than if 11 TG repeats were present (40). This is the likely result of a greater number of TG repeats in cis with 5T, causing a further increase in exon 9 skipping during mRNA splicing (41). This would further decrease production of functional CFTR protein. This TG repeat polymorphism may also influence the penetrance of the R117H mutation. These issues are discussed further in Chapter 8 by Ooi, Tullis and Durie. The phenotypic implications of class VI mutations are less well defined. N287Y (34) is associated with a pancreatic sufficient phenotype according to the CFTR mutation database (20). In contrast, mutations that delete the carboxyl terminus of CFTR may be associated with a greater impairment of CFTR function and pancreatic insufficiency.

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V. Heterozygote Carriers of CFTR Mutations It has been generally accepted that heterozygote carriers of CFTR mutations were “healthy” and essentially unaffected by having one dysfunctional CFTR allele. Recent data have challenged this belief and have suggested that there is an increased risk of clinically apparent disease in heterozygous carriers of CFTR mutations in tissues where CFTR has important epithelial function. For example, people with chronic rhinosinusitis have an increased frequency of being heterozygous carriers of CFTR mutations than does the general population (42). Similarly, heterozygote carriers of the DF508 mutation have an increased prevalence of asthma compared with the general population (43), and there is a higher prevalence of CFTR missense mutations in those with asthma than in the general population (44). Nonrespiratory epithelia also appear at risk in heterozygote CFTR mutation carriers. There are increased incidences of CFTR mutations found in people with chronic idiopathic pancreatitis (45), primary sclerosing cholangitis (46), and CAVBD (47). These recently recognized CFTR-related disorders will be explored in more depth in Chapter 8.

VI.

Summary

CF is a monogenic, autosomal recessive condition that results from the absence of the CFTR. Its classic presentation is associated with essentially absent CFTR function. NonCFTR genetic loci can significantly modulate the clinical manifestations of CF. Recent data also suggest that relatively small modulations of CFTR function may significantly alter the CF phenotype. Our understanding of a potential selective advantage for heterozygote carriers that has allowed persistence of CFTR mutations in the population is limited. This is especially apparent since heterozygote carriers of CFTR mutations also appear at an increased risk for clinical manifestations related to dysfunction of their CFTR-expressing epithelia.

Acknowledgment This work is supported by grants from the NIH/NIDDK, the American Heart Association, and the Cystic Fibrosis Foundation.

References 1. Lowe CU, May CD, Reed SC. Fibrosis of the pancreas in infants and children; a statistical study of clinical and hereditary features. Am J Dis Child 1949; 78(3):349–374. 2. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245(4922):1073–1080. 3. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245(4922):1066–1073. 4. Rommens JM, Iannuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989; 245(4922):1059–1065. 5. Ellsworth RE, Jamison DC, Touchman JW, et al. Comparative genomic sequence analysis of the human and mouse cystic fibrosis transmembrane conductance regulator genes. Proc Natl Acad Sci U S A 2000; 97(3):1172–1177.

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6. McIntosh I, Cutting GR. Cystic fibrosis transmembrane conductance regulator and the etiology and pathogenesis of cystic fibrosis. FASEB J 1992; 6(10):2775–2782. 7. Sheppard DN, Welsh MJ. Structure and function of the CFTR chloride channel. Physiol Rev 1999; 79(1 suppl):S23–S45. 8. Mulberg AE, Wiedner EB, Bao X, et al. Cystic fibrosis transmembrane conductance regulator protein expression in brain. Neuroreport 1994; 5(13):1684–1688. 9. Lader AS, Wang Y, Jackson GR Jr., et al. cAMP-activated anion conductance is associated with expression of CFTR in neonatal mouse cardiac myocytes. Am J Physiol Cell Physiol 2000; 278(2):C436–C450. 10. Sprague RS, Ellsworth ML, Stephenson AH, et al. Deformation-induced ATP release from red blood cells requires CFTR activity. Am J Physiol 1998; 275(5 pt 2):H1726–H1732. 11. Lange T, Jungmann P, Haberle J, et al. Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients. Mol Membr Biol 2006; 23(4):317–323. 12. Di A, Brown ME, Deriy LV, et al. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 2006; 8(9):933–944. 13. Bobadilla JL, Macek M, Fine JP, et al. Cystic fibrosis: a worldwide analysis of CFTR mutations—Correlation with incidence data and application to screening. Hum Mutat 2002; 19:575–606. 14. Tsui LC. The spectrum of cystic fibrosis mutations. Trends Genet 1992; 8(11):392–398. 15. Jorde LB, Lathrop GM. A test of the heterozygote-advantage hypothesis in cystic fibrosis carriers. Am J Hum Genet 1988; 42(6):808–815. 16. Pritchard DJ. Cystic fibrosis allele frequency, sex ratio anomalies and fertility: a new theory for the dissemination of mutant alleles. Hum Genet 1991; 87(6):671–676. 17. Pier GB, Grout M, Zaidi T, et al. Salmonella typhi uses CFTR to enter intestinal epithelial cells. Nature 1998; 393(6680):79–82. 18. Gabriel SE, Brigman KN, Koller BH, et al. Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 1994; 266(5182):107–109. 19. Hogenauer C, Santa Ana CA, Porter JL, et al. Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion. Am J Hum Genet 2000; 67(6):1422–1427. 20. Cystic Fibrosis Mutation Database. Available at: http://www.genet.sickkids.on.ca/cftr/app. 21. Riordan JR. CFTR function and prospects for therapy. Annu Rev Biochem 2008; 77:701–726. 22. Kerem E, Kalman YM, Yahav Y, et al. Highly variable incidence of cystic fibrosis and different mutation distribution among different Jewish ethnic groups in Israel. Hum Genet 1995; 96(2):193–197. 23. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 1993; 73:1251–1254. 24. Hamosh A, Rosenstein BJ, Cutting GR. CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Hum Mol Genet 1992; 1:542–544. 25. Pasyk EA, Foskett JK. Mutant (delta F508) cystic fibrosis transmembrane conductance regulator Cl- channel is functional when retained in endoplasmic reticulum of mammalian cells. J Biol Chem 1995; 270:12347–12350. 26. Rubenstein RC. Targeted therapy for cystic fibrosis: cystic fibrosis transmembrane conductance regulator mutation-specific pharmacologic strategies. Mol Diagn Ther 2006; 10(5):293–301. 27. Denning GM, Anderson MP, Amara JF, et al. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 1992; 358(6389):761–764. 28. Dalemans W, Barbry P, Champigny G, et al. Altered chloride channel kinetics associated with the deltaF508 cystic fibrosis mutation. Nature 1991; 354:526–528. 29. Randak C, Welsh MJ. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR. Cell 2003; 115(7):837–850.

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30. Logan J, Hiestand D, Daram P, et al. Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding. J Clin Invest 1994; 94:228–236. 31. Sheppard DN, Rich DR, Ostergaard LS, et al. Mutations in CFTR associated with milddisease-form Cl
channels with altered pore properties. Nature 1993; 362:160–164. 32. Highsmith WE, Burch LH, Zhou Z, et al. A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N Engl J Med 1994; 331(15):974–980. 33. Highsmith WE Jr., Burch LH, Zhou Z, et al. Identification of a splice site mutation (2789 þ5 G > A) associated with small amounts of normal CFTR mRNA and mild cystic fibrosis. Hum Mutat 1997; 9(4):332–338. 34. Silvis MR, Picciano JA, Bertrand C, et al. A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates. J Biol Chem 2003; 278(13):11554–11560. 35. Drumm ML, Konstan MW, Schluchter MD, et al. Genetic modifiers of lung disease in cystic fibrosis. N Engl J Med 2005; 353(14):1443–1453. 36. Massie RJ, Poplawski N, Wilcken B, et al. Intron-8 polythymidine sequence in Australasian individuals with CF mutations R117H and R117C. Eur Respir J 2001; 17(6):1195–1200. 37. Chu CS, Trapnell BC, Curristin S, et al. Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA. Nat Genet 1993; 3(2):151–156. 38. Rave-Harel N, Kerem E, Nissim-Rafinia M, et al. The molecular basis of partial penetrance of splicing mutations in cystic fibrosis. Am J Hum Genet 1997; 60(1):87–94. 39. Kiesewetter S, Macek M Jr., Davis C, et al. A mutation in CFTR produces different phenotypes depending on chromosomal background. Nat Genet 1993; 5(3):274–278. 40. Groman JD, Hefferon TW, Casals T, et al. Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign. Am J Hum Genet 2004; 74(1):176–179. 41. Cuppens H, Lin W, Jaspers M, et al. Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest 1998; 101(2):487–496. 42. Wang X, Moylan B, Leopold DA, et al. Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population. JAMA 2000; 284(14): 1814–1819. 43. Dahl M, Tybjaerg-Hansen A, Lange P, et al. DeltaF508 heterozygosity in cystic fibrosis and susceptibility to asthma. Lancet 1998; 351(9120):1911–1913. 44. Lazaro C, de Cid R, Sunyer J, et al. Missense mutations in the cystic fibrosis gene in adult patients with asthma. Hum Mutat 1999; 14(6):510–519. 45. Cohn JA, Friedman KJ, Noone PG, et al. Relation between mutations of the cystic fibrosis gene and idiopathic pancreatitis. N Engl J Med 1998; 339(10):653–658. 46. Sheth S, Shea JC, Bishop MD, et al. Increased prevalence of CFTR mutations and variants and decreased chloride secretion in primary sclerosing cholangitis. Hum Genet 2003; 113(3): 286–292. 47. Dumur V, Gervais R, Rigot JM, et al. Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype. Hum Genet 1996; 97(1):7–10.

2 Ion Transport JAMES L. KREINDLER University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

J. KEVIN FOSKETT University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Cystic fibrosis (CF) is a disease of abnormal ion transport. Specifically, abnormalities in the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) result in abnormal salt and water transport across epithelial surfaces in the gastrointestinal and hepatobiliary systems, respiratory tract, reproductive system, and sweat glands. With the exception of the sweat glands, abnormal salt and water transport eventuate in end-organ damage causing significant morbidity and severely shortening life span. The connection between abnormally high salt concentration in sweat and fatal disease has been recognized since the Middle Ages. If a child tasted salty when kissed on the forehead, the child was “bewitched or fascinated and was feared to die soon” (1). This saying is assumed to reflect the existence and recognition of CF (2). We now know much more about the child who tastes salty as well as the genetic and molecular mechanisms underlying CF. Through this understanding and with advances in nutritional and pulmonary therapies, mean predicted survival in CF is now more than 37 years. In this chapter, we review the evolution of our knowledge of ion transport defects in CF, describe the ion channel properties of normal CFTR, discuss how changes in the amino acid sequence of CFTR result in channel dysfunction, and review how loss of CFTR function results in end-organ damage, especially in the lungs.

II.

Establishing A Role for Ion Transport in CF

The first description of CF as a pathological, genetic entity in the United States was published in 1938 by Dorothy Andersen, MD, a pathologist at The Babies’ and Children’s Hospital of Columbia University in New York City. This paper entitled “Cystic fibrosis of the pancreas and its relation to celiac disease (3)” firmly established CF of the pancreas as a diagnosis separate and apart from celiac disease. It was not until more than a decade later, however, that the connection was made between salt transport and CF of the pancreas. In 1951, Kessler and Andersen reported on 12 children admitted to Babies’ Hospital with heat prostration who were in relatively good health before a heat wave, and who presented acutely with vomiting and signs of shock without evidence of infection. All of these children, except for one who died, responded quickly to rehydration (4). In patients for whom laboratory data were available, serum electrolyte analyses showed low Cl
and high HCO3
concentrations that were reversed with 11

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therapy. These findings supported an etiological hypothesis that “fibrocystic disease is associated with widespread abnormality of epithelial glands (4).” Following these observations, Paul di Sant‘Agnese, MD, also at Columbia University, prospectively studied sweat electrolyte levels in 43 patients and 50 controls. His results demonstrated that Naþ, Kþ, and Cl
all were elevated in the sweat of CF patients, with sweat Naþ and Cl
levels being markedly elevated (5). The authors also demonstrated that the elevated sweat Naþ and Cl
levels were not the secondary result of pancreatic dysfunction, pulmonary disease, adrenal dysfunction, or renal disease, and concluded that the increased susceptibility to dehydration in CF was due to increased salt loss from sweat glands. These findings led directly to the development of the sweat test as a diagnostic test for CF (6).

III.

Finding The Gene

The paper by di Sant‘Agnese and colleagues included a statement that CF was a disease of altered ion transport, as opposed to one of abnormal mucus composition or secretion, because, although sweat glands had normal morphology, their abnormal function distinguished patients with CF from others (5). Nonetheless, there remained the clinical observations that end-organ damage in the pancreas and lung was characterized by thick, sticky mucus. The question therefore arose of how to rectify these two apparently disparate observations. Over the last five decades, this question has inspired more than 30,000 papers indexed in PubMed. By the early 1980s, observations in the sweat gland, pancreas, and respiratory tract began to suggest that CF was, at its essence, a disease of altered anion transport. Multiple studies confirmed the findings of di Sant‘Agnese that sweat electrolyte concentrations were abnormal in CF. In separate studies using different techniques, Quinton (7) and later Fromter (8) concluded that CF sweat glands had decreased ductal Cl
permeability and reduced secretion in response to adrenergic stimulation. Similarly, studies of pancreatic HCO3
secretion in CF patients concluded that abnormal pancreatic secretion in CF could be attributed at least in part to altered Cl
secretion (9). Knowles and colleagues at the University of North Carolina reported that the electrical potential across the nasal epithelia of CF patients was more electronegative than controls and did not respond appropriately to adrenergic stimulation (10). It seemed, then, that the basic defect in three different organ systems could be attributed to altered Cl
permeability. Armed with this knowledge, CF researchers began to search for the affected gene. In 1985, two laboratories using different markers for linkage analysis localized the gene to the long arm of chromosome 7 (11,12). In 1989, Tsui and colleagues discovered the gene responsible for CF (13) and found that in the majority of patients the gene was missing three nucleotides that resulted in the in-frame deletion of a phenylalanine residue at position 508 of the polypeptide chain (DF508) (14). They designated the protein as the CFTR (14). In doing so, the group recognized that if CFTR was not itself a Cl
channel, then the protein would almost certainly function as a regulator of Cl
channel activity.

IV.

Normal CFTR

Even before the CFTR gene was cloned, it was known that cAMP-stimulated Cl
secretion was defective in CF epithelial cells (15). Shortly after the CFTR gene was identified, data emerged that this defective cAMP-mediated Cl
secretion could be corrected by expression of normal CFTR, but not by expression of DF508 CFTR. These data supported the hypothesis that CFTR was a Cl
channel, but still left open the

Ion Transport

13

possibility that it was functioning as a positive regulator of another Cl
channel (16,17). In 1991, Anderson and colleagues at the University of Iowa expressed recombinant CFTR in three different cell lines, and conferred on those cells a cAMP-activated Cl
conductance that was not found in cells expressing DF508 CFTR (18). These results were independently confirmed in other cell lines (19,20). Demonstration that mutating specific amino acids in CFTR altered the anion selectivity of the ion permeation pathway conferred on cells in which CFTR was heterologously expressed also strongly suggested that CFTR was a Cl
channel (21). Finally, Bear and colleagues purified the CFTR protein, expressed it in isolated planar lipid bilayers, and demonstrated that it had ion permeation and gating (opening and closing activity) properties identical to those of CFTR heterologously expressed in cell culture (22). When studied by standard electrophysiological techniques in either native or heterologous systems, CFTR has a characteristic biophysical profile. It is an anionselective channel with a single channel Cl
conductance of 6 to 10 picosiemens (pS) in approximately 120 mM Cl
and a permeability selectivity sequence Br
 Cl
> I
> F
(23,24). CFTR can also conduct HCO3
(25,26). When studied by patch clamp electrophysiology in symmetrical Cl
-containing solutions, CFTR channels demonstrate a linear current-voltage relationship (Fig. 1) (18). The opening of the anion permeation pathway in CFTR requires phosphorylation of the channel, particularly by cAMPdependent protein kinase A (27), as well as the presence of ATP (28). CFTR is a unique member of the ATP-binding cassette family of transporters (ABC transporters), which ordinarily use energy from ATP hydrolysis to pump substrates actively through the protein and across the membrane (29). CFTR has seven domains: cytoplasmic amino and carboxyl termini, two membrane-spanning domains

Figure 1 Current-voltage (I-V) curve from an excised, inside-out patch containing a single wild-

type CFTR channel bathed in symmetrical Cl
solutions at 358C. Note the linear relationship between current and voltage. The calculated single channel conductance is 6 to 10 pS. Source: From Ref. 24.

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Figure 2 (A) Commonly accepted model of CFTR structure based on its amino acid sequence. (B) Recently published three-dimensional structure of CFTR based on homology modeling. Source: Part A adapted from Ref. 14 and part B from Ref. 30.

that each contain six membrane-spanning segments, two nucleotide binding domains (NBD1 and NBD2) and an R, or regulatory, domain (14). A high-resolution structure of full-length CFTR has not yet been determined, but homology modeling based on crystal structures of bacterial ABC transporters has provided clues about CFTR’s possible three-dimensional architecture in cell membranes (Fig. 2) (30). In addition, functional

Ion Transport

15

studies have revealed how each domain plays a role in the function or regulation of the channel. The putative 12 transmembrane helices provide the anion permeation pathway and contain the gate that controls transmembrane anion flux. The two NBDs of CFTR bind and/or hydrolyze ATP to modulate channel activity in a manner that is not yet completely determined, but likely involves dimerization of the two NBDs (31). Nucleotide binding and/or hydrolysis induces conformational changes of the NBDs that are somehow communicated to the channel gate in the transmembrane domain that result in its opening and closing. This communication may be mediated by extensions of the transmembrane helices that interact with the NBDs. The R-domain of CFTR, unique among ABC transporter family members, is rich in consensus phosphorylation sites, mainly for protein kinases A and C (32). However, other kinases can also phosphorylate CFTR (33). Phosphorylation of CFTR is necessary for CFTR activation (28), and CFTR channels are deactivated upon dephosphorylation carried out by protein phosphatases (34,35). The amino and carboxyl terminal regions have specific amino acid residues that allow CFTR to bind to intracellular proteins (36). For example, the carboxyl terminus interacts with the scaffolding protein NHERF-1 (37), which modulates channel gating (38) and enables CFTR to interact with other proteins (39). In addition to CFTR channel activity regulation by nucleotide binding, phosphorylation, and protein interactions, the amount of CFTR in the plasma membrane is also regulated by its trafficking and recycling in and out of the apical membrane of epithelial cells (40,41).

V. Abnormal CFTR and Tissue-Specific Ion Transport Abnormalities As discussed elsewhere in this volume, CF is an autosomal recessive disorder, meaning that a person must have two abnormal CFTR genes to manifest the abnormal epithelial ion transport characteristic of the disease. A patient’s clinical phenotype will usually reflect full loss of CFTR ion transport function, or if there is residual ion transport function afforded by one of the mutant alleles. For example, patients who have a single R117H CFTR allele have less severe reduction of plasma membrane anion permeability (42,43) and generally have milder disease than patients where CFTR function is absent. The pathophysiology of end-organ damage in CF patients differs from one organ system to the next; however, the basic defect—a lack of apical plasma membrane Cl
and HCO3
permeability in epithelial cells—remains the same. Although CF pancreatic and pulmonary disease are characterized by luminal obstruction and fibrotic parenchyma, the sweat gland is unique in that it does not demonstrate any macroscopic pathological defects such as luminal obstruction or scarring. This may be because the sweat gland does not secrete significant amounts of protein or mucus that need to be flushed from its lumen, as is the case in other tissues affected in CF. That the physiology of the sweat gland is abnormal in CF despite its not being a mucus-secreting epithelium strongly supports the notion that the basic defect in CF is confined to abnormal ion transport rather than extending to basic abnormalities in mucus production or secretion. A. The Sweat Gland

The human sweat gland comprises a coiled secretory acinus connected to an absorptive duct that empties at the surface of the skin. In the secretory acinus, fluid isotonic to plasma is derived primarily by cholinergic-stimulated, CFTR-independent salt secretion.

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Kreindler and Foskett

As isotonic fluid moves up the sweat duct, NaCl is absorbed in a CFTR-dependent manner so that in normal individuals sweat becomes hypotonic to plasma at the skin surface; this allows sweat to easily evaporate from the skin and effect cooling. The sweat gland is unique among epithelia in that CFTR is detected at both apical and basolateral membranes of cells lining the absorptive duct (reviewed in Ref. 2). In CF, lack of Cl
permeability in the sweat duct impairs NaCl reabsorption, causing excretion of sweat with high salt content (5). This salty sweat evaporates less readily and leaves small salt crystal deposits on the skin. In addition to cholinergically mediated secretion, sweat secretion can be stimulated through adrenergic pathways (44). The rate at which sweat is produced after b-adrenergic stimulation is related to the amount of functional CFTR present in the sweat duct. Therefore, in response to b-adrenergic stimulation, patients without CFTR mutations have higher sweat rates, carriers of one CFTR mutation have intermediate rates of sweat production, and CF patients have virtually no sweat production (45,46). This has led some investigators to propose the use of sweat rates as an end point for clinical trials of pharmaceutical agents aimed at correcting the underlying molecular defect in CF, the lack of functional CFTR (47). B. The Pancreas

The pancreas consists of exocrine and endocrine cellular components. Although both can be affected in CF, this section will focus only on the exocrine pancreas because it is primarily affected by the ion transport defect in CF, whereas the endocrine dysfunction is, at least in part, secondary to pancreatic tissue damage. The exocrine pancreas is similar to the sweat duct in that it consists of secretory acini connected to ducts where the ionic composition of acinar secretions is modified. In response to stimuli that elevate intracellular Ca2þ, pancreatic acinar cells release zymogen granules to secrete a digestive enzyme-rich fluid into the lumen of the acinus. This protein-rich fluid is hydrated and alkalinized in the duct lumen by CFTR-dependent Cl
, HCO3
, and fluid secretion that flushes the contents into the intestinal lumen. In CF, the ability of the pancreatic ducts to secrete fluid is severely impaired because of absence of a functional interaction between CFTR and an apical Cl
/HCO3
exchanger (48–50). In this model, CFTR serves at the apical membrane of pancreatic duct cells both as a conductive pathway for Cl
and as a critical regulator of Cl
/HCO3
exchanger activity. As a result of absent ductal HCO3
and fluid secretion, acinar secretions become dehydrated and trapped in pancreatic ducts. This blocks secretion of both fluid and digestive enzymes into the intestine, and allows the digestive enzymes to act on pancreatic parenchyma causing inflammation and scarring that eventuate in destruction and fibrosis of the exocrine pancreas. C. The Intestine

The intestinal epithelium is histologically divided into villi and crypts and glands. CFTR is primarily expressed in intestinal crypts (51) where it promotes Cl
and fluid secretion that hydrates intestinal mucins produced in the glands. The primary mode of fluid secretion in the small intestine is the activation of CFTR at the apical membrane of intestinal epithelial cells and movement of Cl
down its electrochemical gradient into the lumen of the intestine. The opening of CFTR at the apical membrane and subsequent secretion of Cl
into the lumen of the intestine is accompanied by activation of the loop-diuretic (e.g., furosemide)

Ion Transport

17

sensitive Naþ-Kþ-2Cl
cotransporter on the basolateral membrane that provides a mechanism for Cl
entry into the cell (52). Naþ moves across the epithelium through paracellular pathways to maintain electroneutrality, and water follows osmotically. In CF, the absence of functional CFTR results in lack of Cl
secretion and associated fluid secretion so that intestinal mucus and stool are poorly hydrated. Histologically, retained mucins in intestinal crypts seen in pathology specimens are virtually pathognomonic for CF (53). Poorly hydrated intestinal mucus and stool can become trapped in the intestine causing partial or complete luminal obstruction. In the newborn, this process manifests as failure to pass meconium, known as meconium ileus. In the infant and older child, this manifests as inspissations of mucus and stool in the distal small intestine known as distal intestinal obstructive syndrome or DIOS. D. The Liver

Severe liver disease is the most common nonpulmonary cause of mortality in CF. Ion transport abnormalities in the liver result in abnormal salt and water transport resulting in blockage of biliary ducts, and in most patients, cirrhosis of the liver (54). Obstruction of biliary ducts causes periportal inflammation and fibrosis, which occurs in approximately 30% of CF patients (55). In a subset of these patients, liver damage progresses to become multilobular cirrhosis and may result in portal hypertension. This topic is discussed in detail in chapter 18. E.

The Reproductive Tract

More than 90% of males with CF have congenital bilateral absence of the vas deferens (CBAVD). Furthermore, 1% to 2% of non-CF male infertility is due to CBAVD, and these patients have a higher incidence of CFTR mutations than do patients without CBAVD (56). Therefore, the link between CFTR and CBAVD is firmly established. The role of abnormal ion transport in the pathophysiology of CBAVD is less clear. It may be that absence of normal salt and water transport in the vas deferens leads to blockage of the lumen during development of the vas deferens causing subsequent involution. F.

The Lungs

The mean age of predicted survival in CF has increased from less than a year in the 1950s to almost 40 years today. When CF patients died in infancy, it was largely the result of malnutrition or heat prostration, though most showed signs of lung disease at autopsy (3). As patients have survived longer, lung disease has emerged as by far the most common cause of morbidity and mortality in CF. The end result of CFTR dysfunction in the lung is often described as a vicious cycle of infection, inflammation, and tissue damage resulting from obstruction of the airways by thick, sticky mucus. What is the entry point into the vicious cycle? There are a number of hypotheses that try to answer this question. One well-accepted hypothesis states that lack of CFTR function in the apical membrane of airway surface epithelial cells results in hyperabsorption of NaCl from the airway surface liquid (ASL) and subsequent dehydration of airway lining fluid and mucus. This allows airway mucus to appose airway epithelial cells, which in turn flattens cilia and renders them unable to beat. Accordingly, airway mucus becomes trapped and inhaled bacteria are not cleared appropriately.

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This hypothesis encompasses two, not mutually distinct, hypotheses. The first of these hypotheses is that the lack of CFTR results in overactivation of the cellular Naþ absorption pathway via the epithelial sodium channel (ENaC). This overactive Naþ absorption drives Cl
absorption through the paracellular pathway to maintain electroneutrality. The second hypothesis includes a normal role for CFTR as a secretory pathway for Cl
; this Cl
secretion drives fluid secretion as is described earlier for the intestine. Both of these hypotheses have substantial in vitro and in vivo scientific support. In vivo evidence includes the elevated sensitivity to the ENaC-blocker amiloride in nasal potential difference measurements in CF epithelia compared to control epithelia (10,57) and the finding that overexpression of ENaC in mice caused CF-like airway disease (58). To study the effect of CFTR dysfunction on airways ion transport physiology in vitro, researchers have relied on airway epithelial cells grown and differentiated on permeable supports and exposed to air on their mucosal surface. First described using guinea pig cells (59) and later adapted for human cells (60), this model system recapitulates many of the normal functions of the airway epithelium (Fig. 3). In vitro measurements of ASL height in these well-differentiated, polarized cells strongly suggest that ASL height is reduced in cells from CF donors compared with normals (61). This model system has also been used in attempts to dissect the mechanisms by which Naþ absorption is increased in CF epithelial cells. One hypothesis is that absence of CFTR leads to elevated concentrations of proteases in the ASL that activate ENaC (62,63). Another hypothesis suggests there is a reciprocal relationship between CFTR

Figure 3 Simplified model of epithelial sodium and chloride transport. For Naþ absorption, Naþ

enters from the lumen through the epithelial sodium channel (ENaC, #1) and is pumped out of the cell by the Naþ/Kþ-ATPase (#5), which maintains ionic gradients at the expense of ATP hydrolysis. For Cl
secretion, Cl
enters the cell from the blood on the Naþ-Kþ-2Cl
cotransporter (NKCC-1, #6) and can exit the cell to the lumen through two separate channels. cAMPmediated secretion occurs via CFTR (#2), which is the predominant Cl
conductance in most epithelia. Ca2þ-activated Cl
secretion occurs via the Ca2þ-activated Cl
channel (TMEM16A, #3). Both Naþ absorption and Cl
secretion are dependent on apical membrane hyperpolarization by basolateral Kþ channels (#4), which serve as a shunt pathway for Kþ that enters the cell on the Naþ/Kþ-ATPase. Electroneutrality is maintained by paracellular movements of Naþ and Cl
.

Ion Transport

19

and ENaC at the plasma membrane (64). On the other hand, activation of an alternative apical membrane Cl
channel in the absence of CFTR can stabilize ASL volume (65). These data suggest that CFTR may normally play a role in counteracting Naþ-driven fluid absorption by providing a mechanism for Cl
-mediated fluid secretion. Lack of this counterbalance results in excessive salt and water absorption. The salt hyper-absorption hypotheses address the function of surface epithelial cells in removal of salt and water from the airway surface, but they do not address the possible roles of submucosal glands in the pathophysiology of CF. Anatomically, submucosal glands are found in highest density in the trachea and bronchi, but they can be found throughout the conducting airways, including small bronchi. Submucosal glands may secrete the majority of mucus and fluid that make up the ASL. CFTR is highly expressed in the serous cells of submucosal gland acini, even more highly than in the airway epithelia (66), where it plays a role in Cl
, HCO3
, and fluid secretion (67). Some evidence suggests that hyposecretion from submucosal glands in CF is a proximal cause of lung disease, and that this lack of secretion is due to absence of Cl
and HCO3
secretion (68). Hyposecretion from submucosal glands could also contribute to an imbalance of proteases and antiproteases in the CF airway (68), which could impinge on the activity of ENaC, as discussed earlier. A role for altered HCO3
secretion by airways epithelial cells and submucosal glands has been hypothesized in the pathophysiology of CF lung disease. In vitro, lack of CFTR-mediated HCO3
secretion by cultured bronchial epithelial cells results in defective alkalinization after acid challenge (69). In airway submucosal glands ex vivo, inhibition of HCO3
transport results in decreased fluid secretion (67). Furthermore, submucosal glands from CF patients secrete a more acidic fluid (70). Both pH and concentration may affect the manner in which macromolecules such as mucins and proteases behave. Taken together, these data suggest that altered HCO3
secretion may play a significant role in the pathogenesis of airways disease in CF.

VI.

Summary

In summary, our understanding of CF pathophysiology has progressed from folklore to scientifically based understanding of organ and tissue pathophysiology to subcellular and molecular identification of the basic cellular defects. CF is a disease of altered ion transport resulting from abnormal expression and function of CFTR, an anion channel found in apical membrane of many epithelia. In each affected organ, absence of CFTR is the proximate cause of disease. In some cases, such as the sweat gland, we understand completely how absence of CFTR causes altered organ function. In other cases, such as the lungs, there are multiple effects of CFTR absence that may impact on the disease, and questions remain regarding the underlying molecular mechanisms. Despite these questions, our understanding of how basic defects in CFTR lead to end-organ damage is much greater, and patients have benefited directly.

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4. Kessler WR, Andersen DH. Heat prostration in fibrocystic disease of the pancreas and other conditions. Pediatrics 1951; 8:648–656. 5. di Sant’Agnese PA, Darling RC, Perera GA, et al. Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas: clinical significance and relationship to the disease. Pediatrics 1953; 12:549–563. 6. Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959; 23:545–549. 7. Quinton PM, Bijman J. Higher bioelectric potentials due to decreased chloride absorption in the sweat glands of patients with cystic fibrosis. N Engl J Med 1983; 308:1185–1189. 8. Bijman J, Fromter E. Direct demonstration of high transepithelial chloride-conductance in normal human sweat duct which is absent in cystic fibrosis. Pflugers Arch 1986; 407(suppl 2): S123–S127. 9. Kopelman H, Corey M, Gaskin K, et al. Impaired chloride secretion, as well as bicarbonate secretion, underlies the fluid secretory defect in the cystic fibrosis pancreas. Gastroenterology 1988; 95:349–355. 10. Knowles M, Gatzy J, Boucher R. Relative ion permeability of normal and cystic fibrosis nasal epithelium. J Clin Invest 1983; 71:1410–1417. 11. Knowlton RG, Cohen-Haguenauer O, Van Cong N, et al. A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7. Nature 1985; 318:380–382. 12. Wainwright BJ, Scambler PJ, Schmidtke J, et al. Localization of cystic fibrosis locus to human chromosome 7cen-q22. Nature 1985; 318:384–385. 13. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245:1073–1080. 14. Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989; 245:1066–1073. 15. Frizzell RA, Rechkemmer G, Shoemaker RL. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 1986; 233:558–560. 16. Rich DP, Anderson MP, Gregory RJ, et al. Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature 1990; 347:358–363. 17. Drumm ML, Pope HA, Cliff WH, et al. Correction of the cystic fibrosis defect in vitro by retrovirus-mediated gene transfer. Cell 1990; 62:1227–1233. 18. Anderson MP, Rich DP, Gregory RJ, et al. Generation of cAMP-activated chloride currents by expression of CFTR. Science 1991; 251:679–682. 19. Rommens JM, Dho S, Bear CE, et al. cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci 1991; 88:7500–7504. 20. Kartner N, Hanrahan JW, Jensen TJ, et al. Expression of the cystic fibrosis gene in nonepithelial invertebrate cells produces a regulated anion conductance. Cell 1991; 64:681–691. 21. Anderson MP, Gregory RJ, Thompson S, et al. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 1991; 253:202–205. 22. Bear CE, Li CH, Kartner N, et al. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 1992; 68:809–818. 23. Quinton PM. Physiological basis of cystic fibrosis: a historical perspective. Physiol Rev 1999; 79:S3–S22. 24. Sheppard DN, Welsh MJ. Structure and function of the CFTR chloride channel. Physiol Rev 1999; 79:23–45. 25. Poulsen JH, Fischer H, Illek B, et al. Bicarbonate conductance and pH regulatory capability of cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 1994; 91:5340–5344.

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26. Smith JJ, Welsh MJ. cAMP stimulates bicarbonate secretion across normal, but not cystic fibrosis airway epithelia. J Clin Invest 1992; 89:1148–1153. 27. Cheng SH, Rich DP, Marshall J, et al. Phosphorylation of the R domain by cAMPdependent protein kinase regulates the CFTR chloride channel. Cell 1991; 66:1027–1036. 28. Anderson MP, Berger HA, Rich DP, et al. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 1991; 67:775–784. 29. Gadsby DC, Vergani P, Csanady L. The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 2006; 440:477–483. 30. Serohijos AW, Hegedus T, Aleksandrov AA, et al. Phenylalanine-508 mediates a cytoplasmic-membrane domain contact in the CFTR 3D structure crucial to assembly and channel function. Proc Natl Acad Sci U S A 2008; 105:3256–3261. 31. Vergani P, Lockless SW, Nairn AC, et al. CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 2005; 433:876–880. 32. Gadsby DC, Nairn AC. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 1999; 79:S77–S107. 33. Picciotto MR, Cohn JA, Bertuzzi G, et al. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 1992; 267:12742–12752. 34. Berger HA, Travis SM, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. J Biol Chem 1993; 268:2037–2047. 35. Reddy MM, Quinton PM. Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct. Am J Physiol Cell Physiol 1996; 270:C474–C480. 36. Guggino WB, Banks-Schlegel SP. Macromolecular interactions and ion transport in cystic fibrosis. Am J Respir Crit Care Med 2004; 170:815–820. 37. Hall RA, Ostedgaard LS, Premont RT, et al. A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Naþ/Hþ exchanger regulatory factor family of PDZ proteins. Proc Natl Acad Sci U S A 1998; 95:8496–8501. 38. Raghuram V, Mak DO, Foskett JK. Regulation of cystic fibrosis transmembrane conductance regulator single-channel gating by bivalent PDZ-domain-mediated interaction. Proc Natl Acad Sci U S A 2001; 98:1300–1305. 39. Sun F, Hug MJ, Lewarchik CM, et al. E3KARP mediates the association of ezrin and protein kinase A with the cystic fibrosis transmembrane conductance regulator in airway cells. J Biol Chem 2000; 275:29539–29546. 40. Bradbury NA, Cohn JA, Venglarik CJ, et al. Biochemical and biophysical identification of cystic fibrosis transmembrane conductance regulator chloride channels as components of endocytic clathrin-coated vesicles. J Biol Chem 1994; 269:8296–8302. 41. Prince LS, Workman RB Jr., Marchase RB. Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel. Proc Natl Acad Sci U S A 1994; 91:5192–5196. 42. Reddy MM, Quinton PM. Control of dynamic CFTR selectivity by glutamate and ATP in epithelial cells. Nature 2003; 423:756–760. 43. Sheppard DN, Rich DP, Ostedgaard LS, et al. Mutations in CFTR associated with milddisease-form CI- channels with altered pore properties. Nature 1993; 362:160–164. 44. Emrich HM, Stoll E, Friolet B, et al. Sweat composition in relation to rate of sweating in patients with cystic fibrosis of the pancreas. Pediatr Res 1968; 2:464–478. 45. Behm JK, Hagiwara G, Lewiston NJ, et al. Hyposecretion of beta-adrenergically induced sweating in cystic fibrosis heterozygotes. Pediatr Res 1987; 22:271–276. 46. Sato K, Sato F. Defective beta adrenergic response of cystic fibrosis sweat glands in vivo and in vitro. J Clin Invest 1984; 73:1763–1771.

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47. Callen A, Diener-West M, Zeitlin PL, et al. A simplified cyclic adenosine monophosphatemediated sweat rate test for quantitative measure of cystic fibrosis transmembrane regulator (CFTR) function. J Pediatr 2000; 137:849–855. 48. Ko SB, Shcheynikov N, Choi JY, et al. A molecular mechanism for aberrant CFTRdependent HCO(3)(-) transport in cystic fibrosis. EMBO J 2002; 21:5662–5672. 49. Ko SB, Zeng W, Dorwart MR, et al. Gating of CFTR by the STAS domain of SLC26 transporters. Nat Cell Biol 2004; 6:343–350. 50. Wang Y, Soyombo AA, Shcheynikov N, et al. Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis. EMBO J 2006; 25:5049–5057. 51. Strong TV, Boehm K, Collins FS. Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization. J Clin Invest 1994; 93:347–354. 52. Weymer A, Huott P, Liu W, et al. Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. J Clin Invest 1985; 76:1828–1836. 53. Orenstein DM, Rosenstein BJ, Stern RC. Cystic Fibrosis: Medical Care. Philadelphia: Lippincott Williams & Wilkins, 2000. 54. Maurage C, Lenaerts C, Weber A, et al. Meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis. J Pediatr Gastroenterol Nutr 1989; 9:17–20. 55. Colombo C. Liver disease in cystic fibrosis. Curr Opin Pulm Med 2007; 13:529–536. 56. Dork T, Dworniczak B, Aulehla-Scholz C, et al. Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens. Hum Genet 1997; 100:365–377. 57. Knowles MR, Stutts MJ, Spock A, et al. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science 1983; 221:1067–1070. 58. Mall M, Grubb BR, Harkema JR, et al. Increased airway epithelial Naþ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10:487–493. 59. Whitcutt MJ, Adler KB, Wu R. A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells. In Vitro Cell Dev Biol 1988; 24:420–428. 60. Gray TE, Guzman K, Davis CW, et al. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 1996; 14:104–112. 61. Matsui H, Grubb BR, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95:1005–1015. 62. Bridges RJ, Newton BB, Pilewski JM, et al. Naþ transport in normal and CF human bronchial epithelial cells is inhibited by BAY 39-9437. Am J Physiol Lung Cell Mol Physiol 2001; 281:L16–L23. 63. Myerburg MM, Butterworth MB, McKenna EE, et al. Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance: a mechanism for sodium hyperabsorption in cystic fibrosis. J Biol Chem 2006; 281:27942–27949. 64. Yan W, Samaha FF, Ramkumar M, et al. Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in xenopus oocytes. J Biol Chem 2004; 279:23183–23192. 65. Tarran R, Button B, Picher M, et al. Normal and cystic fibrosis airway surface liquid homeostasis. The effects of phasic shear stress and viral infections. J Biol Chem 2005; 280:35751–35759. 66. Engelhardt JF, Zepeda M, Cohn JA, et al. Expression of the cystic fibrosis gene in adult human lung. J Clin Invest 1994; 93:737–749. 67. Ballard ST, Trout L, Bebok Z, et al. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol 1999; 277:L694–L699.

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68. Joo NS, Irokawa T, Robbins RC, et al. Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands. J Biol Chem 2006; 281:7392–7398. 69. Coakley RD, Grubb BR, Paradiso AM, et al. Abnormal surface liquid pH regulation by cultured cystic fibrosis bronchial epithelium. Proc Natl Acad Sci U S A 2003; 100:16083– 16088. 70. Song Y, Salinas D, Nielson DW, et al. Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis. Am J Physiol Cell Physiol 2006; 290:C741–C749.

3 Mucus Abnormalities and Ciliary Dysfunction A. WHITNEY BROWN and SCOTT H. DONALDSON University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

I.

Introduction to Mucus and Mucociliary Clearance

The unavoidable development of chronic bacterial lung infections in cystic fibrosis (CF) results from a significant defect in lung host defenses. Because adaptive defenses appear to be intact in patients with CF, a search for mechanisms to explain impaired innate defense of the lung has been vigorously pursued. The mucus clearance apparatus plays a key role in lung defense, thus it is not surprising that it has become a prime focus of study and therapeutic development for CF. Effective mucus clearance depends on coordinated ciliary motion and the formation of an airway surface liquid (ASL) layer that is capable of supporting mucus transport via cilia-driven and cough clearance. Traditionally, the ASL has been depicted as the aggregate of two distinct phases: a superficial mucus layer and an aqueous periciliary layer (PCL) that approximates the height of extended cilia. In this paradigm, mucus serves to entrap foreign particles and pathogens, and dissolve noxious gases. The PCL, in turn, supports the transport of mucus out of the lung by providing a lowviscosity environment for cilia beating (1) (Fig. 1). In this chapter, we will review our current understanding of the structure and function of the ASL, with particular attention to changes that may result in impaired host defense in CF.

II.

The Mucus Layer

Secreted mucus is a nonhomogeneous, adhesive, viscoelastic gel composed of water, carbohydrates, proteins, and lipids. Respiratory mucus also contains a host of antimicrobial factors (2). The mucus layer is most pronounced in the intermediate to large airways and is approximately 2 to 10 mm thick in the trachea (2). The major constituents of mucus are gigantic peptidoglycan biopolymers known as mucins. These mucins provide the structural framework of the mucus barrier and are largely responsible for the rheologic properties of mucus. They prevent barrier dehydration, sequester pathogens, and act as a reservoir for host-protective proteins and peptides (1). With molecular weights ranging from 3 to 7 million Da, these enormous hydrophilic molecules consist of a core polypeptide chain plus numerous sugar side chains. In fact, 70% of their mass is composed of carbohydrates (1). Mucin macromolecules are well suited for trapping inhaled particles, at least in part, due to diversity of their carbohydrate side chains, which essentially provide a library of ligands for pathogen binding (3). Intramolecular and intermolecular bonds, including disulfide linkages, and ionic and sugar-sugar

24

Mucus Abnormalities and Ciliary Dysfunction

25

Figure 1 Traditional and revised model of the ASL. The ASL is composed of the mucus layer and the PCL. The mucus layer is viscoelastic, heterogeneous gel layer primarily composed of gelforming mucins that serve to trap inhaled particles for clearance. Traditionally, the PCL was thought to be an aqueous solution that facilitated cilia beating (left image). A more contemporary “two-gel” view depicts the PCL more accurately as a grafted-gel structure of cell surface (tethered) mucins and glycolipids that coat the cilia and microvilli (right image). The gel-like PCL not only serves as a lubricating layer for cilia, but also as a barrier between inhaled particles and the cell surface through the protection provided by close approximation of the long-branched mucins. Abbreviations: ASL, airway surface liquid; PCL, periciliary layer.

interactions produce a complex, tangled structure that translates into tangible viscoelastic properties (4). MUC5B and MUC5AC are the major gel-forming, secreted mucins produced in the lung and together comprise approximately 90% of the mucin content of sputum (5), both in pathologic and normal states. Only small amounts of MUC2, another secreted mucin, have been detected in the lung (5). MUC5B is typically thought to originate from submucosal glands, whereas MUC5AC is produced and released by goblet cells of the surface epithelium. These cell types are normally restricted to large, conducting airways. The concentration of mucus-secreting cells in the larger airways brings into question the source and necessity of mucus for the defense of small airways, and the possibility that other cell types (e.g., Clara cells) could be an additional source of mucus in these critical lung regions. Mucin secretion from submucosal glands and superficial epithelia appears to be prompted by distinct stimuli (6,7), suggesting that the mucin composition of mucus may be influenced by the relative contribution from each source (1). It has been speculated that MUC5AC may be an acute-response mucin that is produced in response to insults to the upper and central airways (1). In fact, MUC5AC mRNA and protein expression are upregulated by neutrophil elastase, a protease secreted by neutrophils during inflammation (8). MUC5B, on the other hand, has been postulated to play a larger role in the setting of chronic inflammation and persistent infection (1). Confirming these speculations, both MUC5AC and MUC5B are found in the mucus plugs that obstruct CF airways, and both molecules are variably increased in induced sputum from patients with CF and COPD. Further, a low-charge glycoform of MUC5B is also elevated in these disease states and might impact the physical properties of the mucus gel (9,10). After synthesis, MUC5B and MUC5AC are stored in intracellular membranebound granules and await secretion via either constitutive or stimulated mechanisms. This allows the epithelium to respond rapidly to environmental challenges, with no requirement for de novo mucin production (1). Secretion of glycoproteins from submucosal glands is predominately under cholinergic control, although adrenergic agonists and multiple inflammatory mediators can also contribute to release. Mucin secretion

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Brown and Donaldson

from goblet cells, on the other hand, is triggered by increased intracellular calcium levels and therefore responds to stimulation of luminal P2Y2 nucleotide receptors. Certain physiologic conditions can also increase mucus gland secretion, including (i) hypoxia, (ii) stimulation of mechanoreceptors in the stomach, (iii) stimulation of cough receptors in the trachea and bronchi with chemical agents, and (iv) inhalation of a wide variety of irritants (cigarette smoke, ammonia, etc). Many of the stimuli that increase mucus secretion also elicit cough, suggesting that the two mechanisms are linked. Under normal conditions, mucus efficiently protects the airways. However, there are situations in which mucin secretory cell hyperplasia occurs as an adaptive response to chronic inflammation. In chronic airway diseases such as CF, considerable hyperplasia of mucus secreting elements can lead to a pathologic increase in mucus production. In the extreme state, such as status asthmaticus, massive mucin hypersecretion coupled with airway smooth muscle contraction can lead to complete airway obstruction and death (11,12). Although the importance of mucins should not be underestimated, there is more to the airway mucus than mucins alone. This is evidenced by the fact that concentrated solutions of mucins, in isolation, do not reproduce all the physical properties of the mucus gel (13). Instead, mucus is a complex mixture of ions, mucins, glycoproteins, proteins, and lipids. In disease states, this biologic mixture becomes even more complex. It is estimated that there are more than 100 nonmucin proteins present in the mucus layer, with larger numbers expected in the setting of airways disease (14). Although a full characterization of the nonmucin components in the mucus layer has been slow to emerge, it is likely that the number of globular proteins and their associated functions have been greatly underappreciated and are worthy of further investigation. Another critical constituent of mucus lining airways is water. Normal mucus is approximately 98% water (2% solids), and even relatively small changes in water content (e.g., from 98% to 94%) can dramatically alter the physical properties of a mucus gel. After secretion, tightly packaged mucin molecules rapidly expand and form a hydrated gel. The characteristics of this gel are in part determined by the ionic composition and pH of the milieu it encounters upon secretion. Aberrant mucin hydration is of particular interest in patients with CF. Specifically, the water imbalance in epithelial fluid may negatively impact mucin unpackaging and hydration, which may help explain the abnormal properties of CF mucus (15).

III.

The Periciliary Layer

Traditionally, the PCL has been conceptualized as a simple aqueous solution that provides a low viscosity environment conducive to ciliary beating (16). More recently, the PCL has been envisioned as a highly ordered polyelectrolyte gel. The molecules comprising this gel in the periciliary space include tethered mucins, particularly MUC1, MUC4, and MUC16 (17), which possess transmembrane domains that fix them to epithelial cell membranes that cover microvilli and cilia. These tethered mucins are polyanionic, and repulsive forces cause them to take an extended, brush-like configuration around the cilia (Fig. 2). This PCL model is extremely attractive for several reasons. First, the polyelectrolyte brush configuration is expected to dramatically lower frictional forces between beating cilia (16,18) while also providing a means to mechanically couple their movements. Second, a gel-like PCL provides a mechanism

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27

Figure 2 Mucins on the respiratory epithelium. Tethered mucins (MUC1, MUC4, and MUC16) are found primarily along the cell surface in the PCL, but also comprise about 10% of the overlying mucin raft (layer). MUC5AC (from goblet cells) and MUC5B (from submucosal mucus glands) account for the large majority of the mucus raft. Source: From Ref. 17.

that prevents penetration of the mucus layer into the PCL environment and subsequent adhesion to cell surfaces. Third, the PCL gel serves a barrier function against inhaled pathogens with particles larger in diameter than the space between tethered mucin molecules being excluded (i.e., >20–30 nm). Finally, this configuration better explains the observation that excess fluid on airway surfaces does not cause separation of the PCL and mucus layers (i.e., mucus floating off cilia tips) and the consequent predicted decline in cilia-driven clearance (19). Rather, the mucus and PCL layers remain in proximity to each other and mucus transport is increased. The same phenomenon is observed in vivo in the case of patients with pseudohypoaldosteronism (PHA), in whom inherited defects in the epithelium sodium channel (ENaC) cause increased amounts of airway lining fluid and dramatically accelerated rates of mucociliary clearance (MCC) (20). Therefore, current data suggests that once the PCL is fully hydrated, additional water is absorbed by the sponge-like mucus layer, making it more easily transportable. In contrast, when moderate airway dehydration occurs, the mucus layer is able to donate water to the PCL to support its functions. This two-gel system is likely stable until extreme dehydration occurs and the osmotic pressure of the mucus layer exceeds that of

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Figure 3 (A) ASL response to different states of hydration. When there is excess fluid on the

airway surface, the mucus layer absorbs it which makes it more transportable. With dehydration, the mucus layer is concentrated and encroaches into the PCL. (B) Electron micrograph of normal versus cystic fibrosis respiratory epithelia. The normal PCL is adequately hydrated, allowing full extension of cilia. The CF PCL is dehydrated and collapsed, altering cilia configuration and impairing motility. A concentrated mucus layer is seen above the PCL in the CF micrograph. Abbreviations: ASL, airway surface liquid; MCC, mucociliary clearance; CF, cystic fibrosis; PCL, periciliary layer.

the PCL, causing subsequent collapse of the PCL and encroachment of mucin into this space (Fig. 3).

IV.

Mucociliary Clearance

Inhaled particulates and pathogens routinely reach the lower airways, but are typically trapped within the mucus layer and transported out of the lung via mucus clearance mechanisms without the need to mount a potentially deleterious inflammatory response. Mucus clearance, therefore, is widely considered to be the primary innate airway defense mechanism (3). Perhaps the clearest example of the direct relationship between defective MCC and disease is provided by patients with primary ciliary dyskinesia (PCD), who have defective or absent ciliary activity. Over time, these patients develop chronic lower airway infections and bronchiectasis, as well as significant sinus and middle ear infections. Other examples of diseases in which defective mucus transport is felt to play a role in pathogenesis are chronic bronchitis, ventilator-associated pneumonia, and CF. Clearly, the failure to clear mucus renders the airways vulnerable to

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infection, as the antimicrobial substances present in mucus alone are incapable of chronically suppressing bacterial growth in the lung (3). Mucus is transported over airway surfaces via cilia and airflow-driven mechanisms. Cilia-mediated mucus clearance requires an intact PCL and is related to both ciliary beat frequency and mucus rheologic properties needed for optimal mucus transportability. An abnormality in either ciliary beating or mucus rheology can result in defective mucus transport. Cough clearance is independent of ciliary function, but instead requires the generation of adequate airflow velocity as the propulsive force. Cough, therefore, is most effective in the central airways, where airflow velocity is greatest. Like cilia-mediated clearance, cough clearance also is dependent on ASL properties. Cough clearance increases as the hydration of the ASL increases, but decreases with increased ASL viscosity (3). The preservation of cough clearance in PCD and COPD may explain why these diseases are less severe than CF, where both cilia and cough-mediated clearance are impacted by severe ASL dehydration. Given the importance of mucus clearance, it is not surprising that airway epithelial cells coordinate the relevant effector mechanisms (3). The basal rate of MCC is a function of cilia beat frequency and the properties of the overlying mucus, and there are autocrine and/or paracrine airway epithelial signals that help regulate these properties. For example, adenine nucleotides are released by airway epithelia in response to mechanical shear stresses created by tidal respiration and cough (3,21,22). After release, adenosine 50 -triphosphate (ATP) binds to P2Y2 receptors and, via calcium signaling pathways, increases cilia beat frequency and chloride/water secretion. The end result of these physiologic processes is accelerated mucus clearance. Metabolism of ATP by extracellular nucleotidases produces adenosine, which in turn stimulates chloride secretion via CFTR after binding to the A2B adenosine receptor and increasing intracellular cAMP. Likely, other extracellular signals influence MCC as well, perhaps working through distinct signaling pathways. Mucociliary clearance measurements have been performed for years using various methodologies. The inhalation of aerosolized, radiolabeled particles followed by imaging with g-scintigraphy is the most widely accepted method, although until recently there has been little standardization of the technique. This has limited the comparison of results published by different research groups (23). Even so, multiple studies have clearly demonstrated that patients with CF and COPD have defective mucociliary clearance (24). Interestingly, in COPD, the observed mucus clearance defect is most notable in study parameters that reflect large airway clearance (25). However, these patients appear to have intact small airway clearance and robust cough-induced clearance. In contrast, studies of patients with CF have revealed mucus clearance defects that are more pronounced in the small airways, along with markedly reduced cough clearance (26,27). These data, therefore, provide insights into the distinct pathophysiology of these diseases and suggest targets of tailored therapeutic interventions.

V. The Airway Surface Liquid in Cystic Fibrosis The abnormal properties of CF secretions are striking and give rise to duct obstruction in multiple organ systems. However, identifying the mechanisms that underlie this phenomenon in the lung has been problematic because separation of the primary pathophysiologic processes and secondary effects of infection and inflammation is often

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difficult. In fact, most of the previously cited qualitative mucin abnormalities (elevated fucose content, decreased sialic acid content, increased sulfation) appear to reflect the presence of chronic infection and inflammation, rather than a CF-specific defect (28). The current prevailing hypothesis that links CFTR dysfunction to the development of lung disease proposes that dehydration of the PCL and mucus layer, because of altered ion transport, leads to mucus stasis in the lung and subsequent infection and inflammation. Ion transport abnormalities in CF have been thoroughly documented and include the absence of cAMP-mediated chloride secretion (via CFTR) and excessive sodium absorption through EnaC (22). The predicted net effect of altered CFTR and ENaC activity is reduced ASL volume. Support for this hypothesis comes in part from in vitro studies that demonstrate reduced PCL height in cultured CF airway epithelia using confocal microscopy (29). Additional support comes from the creation of transgenic mice that overexpress an ENaC subunit and has accelerated sodium absorption from across airway surfaces. These animals have a reduced ASL height, dehydration of airway secretions, slowed mucus transport, and mucus adhesion with plugging of airways. Interestingly, these mice also develop neutrophilic airways inflammation without overt bacterial infection, suggesting a potential direct link between mucus retention and the development of airway inflammation (30). Finally, data from patients with CF show that these patients do indeed have dehydrated airway secretions and abnormal mucus transport (27,31,32). The more refined view of the ASL as a “two-gel” system allows us to better understand the effect that ASL hydration has on airways function. Using in vitro airway models, studies have revealed that the volume depletion associated with CF occurs sequentially, first from the mucus layer and then from the PCL (19). With progressive dehydration, the osmotic pressure of the mucus layer eventually exceeds that of the PCL, forcing water movement from the PCL into the mucus layer, causing a reduction in PCL height/volume. Similarly, rehydration of airway surfaces, after ASL dehydration has occurred, first corrects the deficiency in PCL volume. Addition of volume thereafter preferentially swells the overlying mucus layer. The consequences of PCL dehydration in CF are likely twofold. First, and perhaps most importantly, once the PCL becomes volume depleted, the overlying mucus layer encroaches into the near-cell environment, and the secreted mucins, MUC5AC and MUC5B, begin to interact with the cell-attached mucins. Interaction between secreted and tethered mucins, and perhaps other cell surface molecules (e. g., glycocalyx), causes adhesion between the mucus layer and cell surfaces, resulting in the development of mucus plaques and plugs. Second, PCL volume contraction distorts the space in which cilia beat, thereby reducing or eliminating the propulsive force normally provided by beating cilia. Importantly, these phenomena are expected to interfere with both cilia and cough driven clearance. Exacerbating the problem, goblet cells and glands continue to secrete mucus in diseased airways, worsening the buildup of mucus. Static endobronchial mucus provides a fertile nidus for infection and, ultimately, motile Pseudomonas aeruginosa penetrates into mucus plaques and thrives in the privileged environment that they create. Pseudomonas quickly adapts to this unique environment, which includes regions characterized by significant hypoxia, by producing an alginate coating, significantly altering gene expression, and thereafter persisting as a bacterial biofilm. The ultimate result is a chronic, incurable bacterial infection.

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The effect of ASL dehydration on the mucus layer should also be considered. Once again, in vitro studies demonstrate that once airway secretions increase beyond approximately 6% solids (normal secretions are approximately 2% solids, 98% water), they become much less transportable. This degree of dehydration is in fact quite relevant, as airway secretions from stable adults with CF are often in the 5% to 10% solids range, with even higher levels observed in secretions harvested directly from airways at the time of lung transplantation. Dehydration of airway mucus also results in a reduction in the pore size of the mucin mesh network. When comparing normally hydrated and dehydrated airway mucus (i.e., 2.5% vs. 6.5% solids), neutrophil migration essentially ceased at the higher mucus concentrations, and bacterial capture and killing were significantly impaired (33). Concentrated airway mucus (8% solids) has also been shown to promote the development of Pseudomonas biofilms by limiting bacterial motility and the diffusion of small molecules, and to further the impairment of secondary immune defenses (e.g., lactoferrin) in the lung (34). These findings may also help to understand the accentuated inflammatory response that has been observed. If mucus is too concentrated to allow penetration and bacterial killing by neutrophils, the cells can remain in an activated state and continue to release cellular products (e.g., elastase) that ultimately damage airways and stimulate mucus secretion. Finally, as neutrophils degrade, they release DNA into the extracellular environment, which is a further impediment to the clearance of secretions because of its effects on mucus viscoelasticity. Although the number of mucus-secreting cells increases in CF, it is worth noting that there does not appear to be a major change in the cellular distribution of MUC5AC and MUC5B. It should also be mentioned that there are significant technical difficulties associated with mucin immunodetection. Not only are these molecules very large and highly glycosylated, but the generation of robust antibodies also has been difficult, and the CF airways environment itself is replete with proteases that degrade mucus proteins and reduce our ability to detect them using the antibodies that are available. As a result, some investigators have reported that CF is associated with a reduced quantity of mucins, leading them to the hypothesis that this could represent a host defense defect in and of itself (35,36). In contrast, others have shown that an abundance of mucins are present in CF secretions (9,10). Further experiments done in the presence of protease inhibitors and using methodologies that do not rely on antibody detection (i.e., mass spectroscopy) may shed light on this issue.

VI.

Cilia in Cystic Fibrosis

In one of the first examinations of the ultrastructural features of respiratory cilia in CF, cilia from patients with CF were noted to appropriately contain dynein arms and radial spokes (37). Minimal ciliary abnormalities were detected including compound cilia, cilia with excess cytoplasmic matrix, rippled cilia, and cilia with abnormal number or arrangement of microtubular doublets (37). Except for the slightly higher occurrence of rippled cilia in these patients, the abnormalities were similar in morphology and incidence to that of a control group of patients with chronic bronchitis (37). Unmistakably, patients with CF do not have ultrastructural ciliary defects like those seen in PCD. Nasal ciliary function and mucociliary clearance have been studied in patient cohorts with CF, sinusitis, non-CF bronchiectasis, and aged-matched controls. Ciliary beat frequency was slower in the patients with non-CF bronchiectasis (p < 0.05)

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compared with patients with CF, sinusitis, and aged-matched controls (38). Nasal mucociliary clearance in CF and non-CF bronchiectasis was slower than that of controls (p < 0.001) and patients with sinusitis (p < 0.01) (38). The finding of a normal beat frequency in CF cilia studied in vitro in combination with abnormal nasal mucociliary clearance measured in vivo in the same patients suggests an abnormality of mucus, rather than of the cilia themselves. More recently, nasal mucociliary clearance in children with CF was studied and compared with that of children with PCD and with simple cardiac but no respiratory disease (considered negative controls). No difference was seen in nasal MCC times between children with CF and those without respiratory disease (39). However, those with PCD universally had delayed nasal MCC times. An adult CF cohort was also examined after being divided into those with and without chronic sinusitis (39). Those with sinusitis, which included 43% of CF adults, had longer nasal MCC times than those without chronic sinusitis (39). These data suggest that there does not appear to be a primary impairment in cilia-mediated mucus clearance in CF subjects. Instead, there is likely secondary impairment of cilia-mediated mucus clearance as a consequence of longstanding mucosal inflammation as evidenced by longer nasal MCC times in adults compared with children with CF, particularly in adults with chronic sinusitis. Therefore, virtually all studies agree that ciliary morphology and function in CF airway epithelia are intrinsically normal (37). This is in stark contrast to PCD in which congenital defects in ciliary structure and function lead to impaired mucociliary clearance and repeated respiratory tract infections (40). The lack of ciliamediated mucus clearance in PCD leads to heavy dependence on cough clearance alone. Patients with CF, in contrast, have impaired mucociliary clearance due to volume depletion of airway surfaces. With progressive dehydration, the PCL fails to support transport of the airway mucus layer, resulting in mucus adhesion. This is catastrophic, because not only are cilia unable to function in this configuration, but cough clearance fails as well.

VII.

Therapies for Defective Mucociliary Clearance in CF

A number of novel CF therapeutics are aimed at improving ASL hydration by either stimulating increased epithelial liquid secretion (e.g., hypertonic saline, dry powder mannitol, denufosol tetrasodium, and small molecules that restore mutant CFTR function), or slowing ASL absorption (e.g., amiloride, PS-552, and other ENaC inhibitors). Together, these novel therapeutics represent a substantial portion of the CF drug discovery pipeline, and signify that an improved understanding of disease pathogenesis is now being translated into interventions that target the underlying cause of disease. In the case of hyperosmotic hydrators, inhaled hypertonic saline and dry powder mannitol have both been shown to improve mucociliary clearance in proportion to the administered dose (41,42). The basis of this dose-response relationship likely reflects a direct relationship between the number of osmoles deposited on airway surfaces and the resulting magnitude and duration of the ASL volume response. Unfortunately, the tolerability of inhaled hyperosmotic solutions is also related (inversely) to the administered dose, with pharyngeal irritation, cough, and bronchospasm being the usual limiting symptoms. Importantly, hypertonic saline has been shown to improve lung function and significantly reduce disease exacerbations (43). Currently, studies are being performed

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that will test the impact of hypertonic saline in CF infants, where the greatest potential benefit may be realized. Sodium channel blockers (amiloride, PS-552) are antagonists of ENaC and work to slow absorption of sodium from the airway lumen. Although amiloride has not been shown to have clinical efficacy in CF (27,44,45), longer-acting, more potent ENaC blockers are now being tested (46). It is also intriguing to consider whether an ENaC inhibitor could act synergistically with a drug that stimulates ASL secretion by accentuating the size and/or duration of the resulting ASL volume response. Denufosol tetrasodium, a selective P2Y2 receptor agonist, bypasses the defective CFTR chloride channel by activating an alternative chloride channel (calcium-dependent chloride channels or CaCC). This is predicted to result in an increase in airway surface epithelial hydration. It appears to be therapeutically promising (47) and is currently in phase 3 clinical development. How it might interact with hyperosmotic agents is unknown and difficult to predict a priori, but will be an important issue if approved for clinical use. Recombinant human deoxyribonuclease I (rhDNase, or Pulmozyme), a cloned enzyme that cleaves the DNA residue left by degenerating neutrophils thus reducing sputum viscosity, is another means of altering mucus properties in CF (48). The enzyme was approved by the U.S. Food and Drug Administration in 1994 for use in CF patients. It has since accumulated a considerable history of safe administration and robust evidence for efficacy in CF patients across the disease spectrum. Other therapeutics that target mucus adhesion or other adverse rheologic properties in CF constitute another exciting prospect that is being explored. Perhaps most exciting is the possibility of restoring CFTR function, the most basic defect in CF pathophysiology. An enormous amount of work has gone into identifying orally available small molecules that have the capacity to correct mutant CFTR processing (i.e., DF508) and CFTR function (class III and IV mutations), or to promote readthrough of CFTR stop mutations. These new therapeutic classes, designed to address specific CFTR mutations and systemically restore CFTR function rather than ameliorating the sequelae CFTR dysfunction, hold great promise for revolutionizing the care of CF patients in the future. They are another prime example of how improved understanding of disease pathogenesis is driving the development of therapeutics in CF, particularly at the level of early disease pathogenesis.

References 1. Thornton DJ, Rousseau K, McGuckin MA. Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 2008; 70:459–486. 2. Rubin BK. Physiology of airway mucus clearance. Respir Care 2002; 47(7):761–768. 3. Knowles MR, Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002; 109(5):571–577. 4. Fraser RS. Fraser and Pare´’s Diagnosis of Diseases of the Chest. 4th ed. Philadelphia: Saunder’s, 1999. 5. Hovenberg HW, Davies JR, Carlstedt I. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem J 1996; 318(pt 1):319–324. 6. Fung DC, Rogers DF. Airway submucosal glands: physiology and pharmacology. In: Rogers D, Lethem MI, eds. Airway Mucus: Basic Mechanisms and Clinical Perspectives. Basel: Birkhauser, 1997, 179–210.

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7. Davis CW. Goblet cells: physiology and pharmacology. In: Rogers D, Lethem MI, eds. Airway Mucus: Basic Mechanisms and Clinical Perspectives. Basel: Birkhauser, 1997:149–177. 8. Voynow JA, Young LR, Wang Y, et al. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol 1999; 276(5 pt 1):L835–L843. 9. Kirkham S, Sheehan JK, Knight D, et al. Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J 2002; 361(pt 3):537–546. 10. Burgel PR, Montani D, Danel C, et al. A morphometric study of mucins and small airway plugging in cystic fibrosis. Thorax 2007; 62(2):153–161. 11. Sheehan JK, Richardson PS, Fung DC, et al. Analysis of respiratory mucus glycoproteins in asthma: a detailed study from a patient who died in status asthmaticus. Am J Respir Cell Mol Biol 1995; 13(6):748–756. 12. Rogers DF. Airway mucus hypersecretion in asthma: an undervalued pathology? Curr Opin Pharmacol 2004; 4(3):241–250. 13. Raynal BD, Hardingham TE, Thornton DJ, et al. Concentrated solutions of salivary MUC5B mucin do not replicate the gel-forming properties of saliva. Biochem J 2002; 362(pt 2):289– 296. 14. Sheehan JK, Kesimer M, Pickles R. Innate immunity and mucus structure and function. Novartis Found Symp 2006; 279:155–219. 15. Thornton DJ, Sheehan JK. From mucins to mucus: toward a more coherent understanding of this essential barrier. Proc Am Thorac Soc 2004; 1(1):54–61. 16. Randell SH, Boucher RC. Effective mucus clearance is essential for respiratory health. Am J Respir Cell Mol Biol 2006; 35(1):20–28. 17. Hattrup CL, Gendler SJ. Structure and function of the cell surface (tethered) mucins. Annu Rev Physiol 2008; 70:431–457. 18. Raviv U, Giasson S, Kampf N, et al. Lubrication by charged polymers. Nature 2003; 425 (6954):163–165. 19. Tarran R, Grubb BR, Gatzy JT, et al. The relative roles of passive surface forces and active ion transport in the modulation of airway surface liquid volume and composition. J Gen Physiol 2001; 118(2):223–236. 20. Kerem E, Bistritzer T, Hanukoglu A, et al. Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J Med 1999; 341(3):156–162. 21. Lazarowski ER, Tarran R, Grubb BR, et al. Nucleotide release provides a mechanism for airway surface liquid homeostasis. J Biol Chem 2004; 279(35):36855–36864. 22. Tarran R, Button B, Picher M, et al. Normal and cystic fibrosis airway surface liquid homeostasis: the effects of phasic shear stress and viral infections. J Biol Chem 2005; 280 (42):35751–35759. 23. Donaldson SH, Corcoran TE, Laube BL, et al. Mucociliary clearance as an outcome measure for cystic fibrosis clinical research. Proc Am Thorac Soc 2007; 4(4):399–405. 24. Robinson M, Eberl S, Tomlinson C, et al. Regional mucociliary clearance in patients with cystic fibrosis. J Aerosol Med 2000; 13(2):73–86. 25. Smaldone GC, Foster WM, O’Riordan TG, et al. Regional impairment of mucociliary clearance in chronic obstructive pulmonary disease. Chest 1993; 103(5):1390–1396. 26. Bennett WD, Olivier KN, Zeman KL, et al. Effect of uridine 50 -triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis. Am J Respir Crit Care Med 1996; 153(6 pt 1):1796–1801. 27. Donaldson SH, Bennett WD, Zeman KL, et al. Mucus clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354(3):241–250. 28. Davril M, Degroote S, Humbert P, et al. The sialylation of bronchial mucins secreted by patients suffering from cystic fibrosis or from chronic bronchitis is related to the severity of airway infection. Glycobiology 1999; 9(3):311–321.

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29. Matsui H, Grubb B, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 1998; 95(7):1005–1015. 30. Mall M, Grubb BR, Harkema JR, et al. Increased airway epithelial Naþ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004; 10(5):487–493. 31. Redding GJ, Kishioka C, Martinez P, et al. Physical and transport properties of sputum from children with idiopathic bronchiectasis. Chest 2008; 134(6):1129–1134. 32. Donaldson SH. Hydrator therapies for cystic fibrosis lung disease. Pediatr Pulmonol 2008; 43:S18–S23. 33. Matsui H, Verghese MW, Kesimer M, et al. Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces. J Immunol 2005; 175(2):1090–1099. 34. Matsui H, Wagner VE, Hill DB, et al. A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms. Proc Natl Acad Sci U S A 2006; 103(48):18131–18136. 35. Henke MO, Renner A, Huber RM, et al. MUC5AC and MUC5B mucins are decreased in cystic fibrosis airway secretions. Am J Respir Cell Mol Biol 2004; 31(1):86–91. 36. Henke MO, John G, Germann M, et al. MUC5AC and MUC5B mucins increase in cystic fibrosis airway secretions during pulmonary exacerbation. Am J Respir Crit Care Med 2007; 175(8):816–821. 37. Katz SM, Holsclaw DS Jr. Ultrastructural features of respiratory cilia in cystic fibrosis. Am J Clin Pathol 1980; 73(5):682–685. 38. Rutland J, Cole PJ. Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis. Thorax 1981; 36(9):654–658. 39. McShane D, Davies JC, Wodehouse T, et al. Normal nasal mucociliary clearance in CF children: evidence against a CFTR-related defect. Eur Respir J 2004; 24(1):95–100. 40. Noone PG, Leigh MW, Sannuti A, et al. Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med 2004; 169(4):459–467. 41. Robinson M, Hemming AL, Regnis JA, et al. Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with cystic fibrosis. Thorax 1997; 52(10):900–903. 42. Daviskas E, Anderson SD, Eberl S, et al. Effect of increasing doses of mannitol on mucus clearance in patients with bronchiectasis. Eur Respir J 2008; 31(4):765–772. 43. Elkins MR, Robinson M, Rose BR, et al. A controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006; 354(3):229–240. 44. Knowles M, Church N, Waltner W, et al. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl J Med 1990; 322(17):1189–1194. 45. Graham A, Hasani A, Alton E, et al. No added benefit from nebulized amiloride in patients with cystic fibrosis. Eur Respir J 1993; 6(9):1243–1248. 46. Hirsh AJ, Zhang J, Zamurs A, et al. Pharmacological properties of N-(3,5-diamino-6chloropyrazine-2-carbonyl)-N0 -4-[4-(2,3-dihydroxypropoxy) phenyl]butyl-guanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for cystic fibrosis lung disease. J Pharmacol Exp Ther 2008; 325(1):77–88. 47. Deterding RR, Lavange LM, Engels JM, et al. Phase 2 randomized safety and efficacy trial of nebulized denufosol tetrasodium in cystic fibrosis. Am J Respir Crit Care Med 2007; 176 (4):362–369. 48. Fuchs HJ, Borowitz DS, Christiansen DH, et al. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. The Pulmozyme Study Group [see comments]. N Engl J Med 1994; 331(10): 637–642.

4 Microbiology in Cystic Fibrosis PAUL J. PLANET and LISA SAIMAN Columbia University, New York, New York, U.S.A.

I.

Introduction

The unique microbiology of cystic fibrosis (CF) lung disease has been appreciated for decades. Fulminant pneumonia with Staphylococcus aureus was described in infants with CF in the 1940s along with the initial descriptions of CF (1), and S. aureus was the most common pathogen isolated (2). Descriptions of the epidemiology of Pseudomonas aeruginosa were first provided in the 1960s, including the unique mucoid phenotype (3–5). Initial reports of the “cepacia syndrome” appeared in the late 1970s and early 1980s (6,7). Numerous improvements in diagnostic and therapeutic strategies for CF patients have been made over the past 20 years, which have been associated with decreased morbidity and increased life expectancy as well as changes in the microbiology of CF pathogens. These changing strategies have included improved nutrition (8); inhaled DNase (9,10); suppressive therapy for chronic infection with P. aeruginosa, that is, inhaled tobramycin (11) and oral azithromycin (12); implementation of standardized microbiology laboratory protocols to improve detection and identification of microorganisms from respiratory tract specimens (13,14); increased surveillance by quarterly cultures (13); improved infection control practices (13,14); and the increased acceptance early eradication therapy for P. aeruginosa (15–17). Changes in microbiology have included not only changes in the epidemiology of classic CF pathogens, but the emergence of several new pathogens (Table 1) and a recent interest in the potential role of noncultivatable organisms, anaerobes, and intracellular organisms in CF disease. This chapter will describe the critical importance of the clinical microbiology laboratory and the efforts needed to ensure appropriate processing of CF respiratory tract specimens. The epidemiology and clinical impact of both classic CF pathogens and emerging pathogens as well as our current understanding of virulence factors will be reviewed. In addition, the potential contribution of culture-independent to our understanding of CF lung disease will be discussed.

II.

Role of Clinical Microbiology Laboratory

A. Detection and Identification of Pathogens

Accurate detection and identification of the microorganisms from the respiratory tract of CF patients are critical to provide appropriate treatment, further the understanding of the epidemiology of CF pathogens, and promote effective infection control.

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Table 1 Classic and Emerging Pathogens in CF

Microorganism

Classic

Emerging

Bacteria

Staphylococcus aureus–methicillin susceptible Haemophilus influenzae Pseudomonas aeruginosa—nonmucoid and mucoid Burkholderia spp.

Staphylococcus aureus– methicillin resistant Stenotrophomonas maltophilia Achromobacter xylosoxidans Ralstonia spp. Streptococcus milleri Pandoraea apista Iniquilinus spp.

Molds

Aspergillus spp., A. fumigatus >> A. terreus, A. nidulans, A. flavus

Scedosporium apiospermum Scedosporium prolificans Exophiala dermatitidis Mucor spp. Penicillium spp.

Mycobacteria

Mycobacteria spp. Mycobacterium avium complex M. abscessus M. intracellulare M. chelonae M. fortuitum

Abbreviation: CF, cystic fibrosis.

Recommendations for appropriate collection and processing of CF specimens are presented in Table 2. The CF care team should be familiar with these recommendations and ensure they are instituted and monitored at the laboratories that process CF specimens. The use of selective media and more frequent culturing has most likely contributed to the increased prevalence of several CF pathogens due to detection bias. In the early 1980s, the use of selective media for Burkholderia spp. was critical to detect these pathogens and unravel patient-to-patient transmission (7). As will be described below, molecular identification methods have greatly facilitated identification of Burkholderia spp. and furthered our understanding of the complex epidemiology of these microorganisms. In the 1990s, it was shown that CF care sites that instituted complete microbiology protocols had a higher prevalence of S. aureus than sites that used incomplete protocols (18). Increased frequency of culturing has also increased “transient colonization” with Burkholderia, methicillin-resistant S. aureus (MRSA), and Stenotrophomonas maltophilia. Detection of nontuberculous mycobacteria (NTM) can be challenging; as many as three specimens may be needed for acid-fast bacilli (AFB) smear and culture. Proficiency studies for clinical laboratories participating in an NTM epidemiology study further elucidated the importance of a decontamination step to prevent overgrowth of NTM samples with P. aeruginosa (19). Cultures may require six to eight weeks to grow NTM, although the rapid growers, Mycobacteria abscessus, M. chelonae, and M. fortuitum can grow within one to two weeks. Molecular detection techniques can be

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Table 2 Appropriate Collection and Processing of CF Respiratory Tract Specimens

Step

Appropriate procedure

Comment

Collection

Specimens collected by experienced personnel Specimens labeled “CF Specimens” to ensure proper processing Cultures sent quarterly or during exacerbations Specimens brought to lab within 2 hr of collection.

Types of specimens include expectorated sputum, deep throat swabs, induced sputum, or bronchoalveolar lavage. Specimens from CF patients following lung transplantation should be processed as CF Specimens If specimens cannot be delivered within 2 hr, place specimens on ice but do not freeze. Specimens must be processed within 24 hr of collection Prolonged incubation may be required to detect more fastidious microorganisms, e.g., Burkholderia

Transport

Selective media

Biochemical identification

Selective media and/or culture conditions for isolation of S. aureus, H. influenzae, Burkholderia spp., and nontuberculous mycobacteria Commercial assays may misidentify or incompletely identify NLFGNR

Misidentification of l

l

l

Molecular identification

Antimicrobial susceptibility testing

Molecular typing

a

Use of reference laboratory to identify multidrug-resistant “NLFGNR” or Burkholderia cepacia complex Agar-based diffusion methods, e.g., Kirby-Bauer disks or Etest, preferred susceptibility testing assays for Pseudomonas aeruginosa Used for epidemiologic investigations to assess potential routes of transmission or success of early eradication strategies

Burkholderia spp. as other NLFGNRa Other NLFGNR as Burkholderia spp. Nonmucoid, nonpigmented P. aeruginosa as other NLFGNR

Molecular strategies can be used to delineate new taxonomy, e.g., emerging Burkholderia spp. Commercial microtiter assays have unacceptable rate of falsesusceptible results

Can be performed on bacterial and fungal species

Other NLFGNR include B. gladioli, Stenotrophomonas, Pseudomonas, Achromobacter, Ralstonia, Flavobacterium, or Chryseobacterium. Abbreviations: CF, cystic fibrosis; NLFGNR, nonlactose fermenting gram-negative rods

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used for AFB smear-positive specimens (direct amplification tests for ribosomal RNA) and for isolates growing in media (DNA probes for species identification). The interested reader is referred to the consensus document titled, “Infection control recommendations for patients with CF: microbiology, important pathogens, and infection control practices to prevent patient-to-patient transmission.” (13) and “Laboratory aspects of management of chronic pulmonary infections in patients with CF” (20) for detailed recommendations for processing CF respiratory cultures. B. Role of Susceptibility Testing

Current guidelines endorse the need for antimicrobial susceptibility testing to guide treatment, to promote appropriate infection control for multidrug-resistance pathogens, and to track the epidemiology of resistance (21). However, over the past several years, there has been growing concern that conventional susceptibility testing for patients with CF may lack clinical utility. This controversy has roots in clinical observations whereby patients having a pulmonary exacerbation clinically improve while being treated with antibiotics to which their pathogens are resistant in vitro and fail to improve while being treated with antibiotics to which their pathogens are susceptible in vitro. In addition, there has been concern that susceptibility testing assays use laboratory conditions that are irrelevant for the conditions within the CF lung. In the laboratory, bacteria are grown planktonically in aerobic conditions, and in enriched media, while in the CF lung, bacteria are thought to exist in biofilms, in stationary phase, and in a nutrient deficient millieu. Thus, there has been a great deal of interest in the usefulness of susceptibility testing performed on isolates grown in biofilms (22,23). However, the clinical utility of such assays has yet to be proven. Unfortunately, data obtained from clinical trials are lacking that support or refute the usefulness of susceptibility testing. Clinical trials assessing the efficacy of antibiotics during pulmonary exacerbations generally enrolled patients harboring pathogens that were susceptible to the agents tested (24), as such inclusion criteria are standard in antibiotic studies. In an attempt to shed light on this issue, a secondary analysis of the aerosolized tobramycin clinical trial data (25) was performed to assess the relationship between susceptibility to tobramycin and ceftazidime and clinical response (measured as change in lung function) (26). During the trial, 77 patients experienced a pulmonary exacerbation and were treated with these agents, 54 patients improved, 14 were unchanged, and 9 worsened. However, susceptibility to these agents did not correlate with clinical response; patients infected with resistant P. aeruginosa were as likely to respond as those with susceptible strains. Thus, while current in vitro susceptibility testing assays are imperfect, no suitable alternative exists at this time. C. Role of Synergy Testing

To date, the role of synergy testing has not been clearly defined. There is only one randomized, controlled trial assessing the clinical efficacy of synergy studies wherein patients were treated for a pulmonary exacerbation using antibiotic combinations guided by minimal bacteriocidal testing versus conventional susceptibility testing (27). There were no differences in the time until next pulmonary exacerbation or improvement in lung function. The pros and cons of synergy testing in CF were recently explored

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(28,29). These authors concluded that routine synergy testing was not justified based on the current evidence, but that synergy testing should be reserved for patients infected with multidrug-resistant P. aeruginosa who are failing conventional therapy, patients with substantial drug allergies, and/or patients with severely impaired lung function.

III.

Pathogens in CF

In CF patients, respiratory tract infections can begin in early infancy (Fig. 1). Heightened awareness of the diagnosis of CF among pediatric health care professionals and the implementation of newborn screening programs for CF around the world have lead to an earlier diagnosis of CF and a better understanding of the respiratory tract flora of young, often asymptomatic infants (30–32). Methicillin-susceptible S. aureus (MSSA), nontypeable Haemophilus influenzae, and P. aeruginosa are the most common organisms isolated during the first decade of life. Infections with P. aeruginosa, particularly infections with the mucoid phenotype, and Burkholderia cepacia complex are associated with a decline in lung function and predict morbidity and mortality in CF (33). A. Staphylococcus aureus

S. aureus is most often the earliest pathogen to be cultured from respiratory cultures of CF patients and can persist despite antibiotic treatment (median duration ~30 months) (34–36). The mechanism of long-term persistence is multifactorial and may include the ability to form biofilms (37,38), intracellular invasion (39,40), increased antibiotic resistance (41,42), decreased growth rate and metabolism, and/or increased rates of evolution (42). In some cases, persistence may also depend on the presence of other organisms (43). These factors are associated with a phenotypic switch that creates a subpopulation of cells identified by their colony morphology, that is, “small colony variants” (SCV) (44).

Figure 1 The age-specific prevalence per age group for each of the reported pathogens. Source:

Courtesy of Cystic Fibrosis Foundation, National Patient Registry 2007.

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Colonization of the upper respiratory tract seems to be an important risk factor for development of pulmonary infection in non-CF patients, but in CF patients the relevant site of colonization may be the oropharynx as opposed to the anterior nares (45). Documented transmission of S. aureus has been seen between CF patients (35). The introduction of effective antibiotic regimens appears to have greatly reduced mortality due to S. aureus, but the role of S. aureus in the progression of CF has not been fully explored (46,47). Some studies have suggested that early prophylaxis with flucloxacillin reduced colonization with S. aureus and reduced respiratory tract symptoms, although these studies have not shown clear long-term benefit (47). In other studies, prophylaxis with cephalexin has been associated with an increased risk of acquisition of P. aeruginosa (47,48). The differences in findings may be due to differences in the antimicrobial agent used or the age at initiation of prophylaxis. B. Haemophilus influenzae

H. influenzae (Hi) is also an early colonizer of young patients with CF. Most often, it is the nonencapsulated, nontypeable H. influenzae, strains that infect CF patients and, therefore, the Hib vaccine, which targets type B strains, does not protect against infection. Both in vivo and in vitro evidence suggests that H. influezae has the capability to form biofilms on airway epithelial cells (49), which may allow it to persist over longer periods of time. What might seem like chronic infection with H. influenzae may in fact represent reinfection over time with different strains thereby mimicking chronic infections (50). Although generally very susceptible to antimicrobial treatment, H. influenzae can acquire resistance to fluoroquinolones and other antibiotics used to treat other classic CF pathogens (50). This exposure and resultant phenotype may be related to increased mutation rates in certain clones, so-called hypermutability (51). It is unclear what role H. influenzae plays in the progression of lung disease in CF. C. Pseudomonas aeruginosa

P. aeruginosa is the pathogen most often cultured from sputum cultures of CF patients. It is also the pathogen for which the most evidence exists for a direct role in pathogenesis. Children and infants with CF who are colonized/infected with P. aeruginosa have reduced 10-year survival and increased pulmonary disease compared with CF controls who are not colonized/infected (52,53). P. aeruginosa strains are thought to persist for long periods, sometimes life-long, in the lungs of CF patients (54–57). Distinct clones can also coexist within the same patient (58,59). It seems likely that strains are acquired from the environment in many cases; P. aeruginosa can be easily cultured from environmental sources such as soil and tap water (60). However, there are documented cases in which P. aeruginosa seems to have been acquired through contact with other CF patients both in and out of the health care setting (58,59,61–65), emphasizing the potential for patient-to-patient transmission of classic CF pathogens. Much work has been done to understand the molecular basis of chronic lung infections with P. aeruginosa in CF. The long-term persistence of P. aeruginosa within the unique milieu of the CF lung provides this microorganism with the opportunity to evolve and adapt in situ (66–68). This evolution occurs both by mutation in existing genes (66) and

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through large-scale genomic rearrangements and acquisition of new genes (67,69). Observed phenotypic changes in persistent infections include development of antibiotic resistance (70,71) and loss of motility and other factors involved in acute infection (66). Increased mutation rate during long-term infection has been observed as well (72). One of the important phenotypic transitions that occurs in chronic P. aeruginosa infection in CF is the switch to the mucoid phenotype in which organisms overproduce the exopolysaccharide alginate and enter a nonmotile state (73,74). This switch is associated with clinical deterioration and a poor prognosis (75). The switch to mucoidy is thought to be important in formation of adherent biofilms, which can resist clearance, evade the defenses of the immune system, and are more resistant to antibiotic killing (73). Other phenotypes associated with biofilms, such as slow-growing, hyperadherent small colony variant, may also have an important role in colonization and persistence (76–79). Bacterial appendages such as pili and flagella aid in biofilm formation (80–82), while quorum-sensing systems (83) allow chemical communication between bacteria to regulate genes needed for virulence and life within a biofilm. In addition to the virulence factors that facilitate chronic infections, P. aeruginosa strains also express several other virulence factors including exotoxin A, exoenzyme S, leukocidin, alkaline protease, type III secretion systems, and pyocyanins (84,85). The precise role of these virulence factors in CF is not fully understood. D. Burkholderia spp.

Several research laboratories and referral centers around the world have been using molecular identification via 16S rRNA gene sequencing and sequence polymorphisms within the protein-coding gene, recA, as well as multilocus sequence typing (MLST) to provide accurate identification and explore the unique epidemiology of the genus Burkholderia and related genera, for example, Ralstonia and Pandoraea spp. (86–89). B. cepacia complex is now known to consist of phenotypically indistinguishable, genetically distinct species, also termed genomovars. To date more than 15 such species have been identified and isolated from patients with CF, but the two most common species detected in CF are B. cenocepacia and B. multivorans (Table 3). Other species are far less common although B. cepacia, B. stabilis, B. vietnamiensis, and B. dolosa are generally more frequently isolated than B. ambifaria, B. anthina, and B. pyrrocinia. Table 3 Burkholderia spp. Isolated from Patients with CF

Genomovar

Species

I II III IV V VI VII VIII IX X Indeterminate

Burkholderia cepacia B. multivorans B. cenocepacia B. stabilis B. vietnamiensis B. dolosa B. ambifaria B. anthina B. pyrrocinia B. ubonensis –

Estimated prevalence (%) 6 17–38 39–67 1 6 3 1 <1 <1 <1 2

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B. cenocepacia is generally thought to be more virulent than other species, that is, infections with this species are more likely to result in a decline in lung function and associated with higher mortality following lung transplant. In addition, B. cenocepacia is more likely to replace B. multivorans in an individual patient (90) and more likely to be transmitted patient-to-patient. However, these observations may be attributed to a specific strain of B. cenocepacia, such as the ET 12 strain, which has worldwide distribution and expresses the cable pilus and the B. cenocepacia pathogenicity island (86,91). Other epidemic strains have been identified in the United States including the Philadelphia– District Columbia (PHDC) and Mid-West strains (92). In addition, B. multivorans (93) and B. dolosa (94) have been associated with patient-to-patient transmission and worsening of lung function and reduced survival. Most recently, there has been an interesting shift in the epidemiology of Burkholderia infections in the CF population in the United Kingdom. Since 1993, the majority of new infections have been caused by genetically unique strains, most often B. multivorans, suggesting acquisition of environmental strains rather than transmissible strains and providing further evidence of the efficacy of infection control practices (95). E.

Methicillin-Resistant Staphylococcus aureus

The incidence and prevalence of MRSA have significantly increased over the past decade in the United States. As reported to the CF Foundation National Patient Registry, in 1997, the overall prevalence of MRSA in CF patients was 2.6%, which increased to 21.2% in 2007. Risk factors for MRSA in CF have included more days of hospitalization, ciprofloxacin, cephalosporin agents, and chronic infection with Aspergillus fumigatus (96). Recently there has been much interest in determining if MRSA is associated with a decline in lung function. Two different investigations have been performed using registry data and somewhat different case definitions and analytic methods (97,98). Using data in the U.S. CF Foundation Patient Registry, Dasenbrook et al. performed a cohort study to determine the impact of new, persistent MRSA infections (defined as
3 positive cultures) on lung function. The rate of decline of forced expiratory volume in one second (FEV1) among patients 8- to 21 years old with MRSA was more rapid (–2.06% per year) than those without MRSA infection (–1.44% per year). Furthermore, among those with transient detection of MRSA defined as a single culture, there was no significant impact on lung function. In contrast, using 2001 data from the Epidemiology Study of Cystic Fibrosis (97), which includes data from CF patients from North America (some of whom are also in the CF Foundation registry), Ren et al. assessed the FEV1 among patients infected with only MRSA versus those infected with only MSSA. Children <18 years old with MRSA had lower mean FEV1 than patients with MSSA, 80.7% versus 89.4% predicted, respectively. In an era of community-acquired (CA)-MRSA, there has been much recent interest in the epidemiology of the staphylococcal chromosomal cassette (SCC), which contains the methicillin-resistance gene, mecA, among isolates of MRSA recovered from CF patients. This methodology can distinguish hospital-associated (HA) MRSA (SCCmec type II) versus community-associated MRSA (SCCmec type IV) strains. In the New York metropolitan area, a third of MRSA strains (10/31) from children and adolescents with CF and their household contacts were SCCmec type IVa (99). In Dallas

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and Chicago, 33% of 33 typeable MRSA isolates from pediatric CF patients were SCCmec type IV, which was associated with new MRSA acquisition (100). In St. Louis, 25% of MRSA isolates (n ¼ 40) were SCCmec type IV (101) and in Belgium 45% of typeable isolates (n ¼ 29) were SCCmec type IV or IVa (102). Further studies are needed to determine if CA-MRSA are more virulent than HA-MRSA. However, MRSA strains expressing Panton–Valentine leukocidin (PVL), a common virulence factor in CA-MRSA strains, have been associated with a more rapid decline in lung function and focal radiographic findings, including cavitary lung disease (101). F.

Achromobacter xylosoxidans and Stenotrophomonas maltophilia

Pseudomonas appears to be a successful pathogen in CF because it is ubiquitous and adaptable as described above. This model of pathogenesis can extend to other microorganisms with similar properties that have more recently been cultured from the lungs of patients with CF. One such organism is Stenotrophomonas maltophilia, an aerobic gram-negative bacillus that can be readily isolated from aquatic environments. In non-CF patients, Stenotrophomonas maltophilia is an opportunistic, hospital-acquired pathogen and can form biofilms. In CF patients, Stenotrophomonas maltophilia is often isolated from those with worse clinical disease, and single-center studies have shown decreased five-year survival rates in patients who have positive sputum cultures for this organism (103). However, multicenter studies have not found an association between Stenotrophomonas maltophilia detection and clinical deterioration (104). Therefore, the clinical relevance of Stenotrophomonas maltophilia remains unclear. Stenotrophomonas maltophilia can have multiple mechanisms for antibiotic resistance and can acquire others under selection pressure (105–107). High levels of antibiotic resistance likely explain why long-term antibiotic use is a risk factor for colonization (108). Similarly, the gram-negative bacillus Achromobacter (formerly Alcaligenes) xylosoxidans can also colonize and persist in the CF lung. Like Stenotrophomonas maltophilia, the clinical impact of A. xylosoxidans is unclear (109). A. xylosoxidans can be cultured from CF patients who are coinfected with P. aeruginosa, which raises questions about microbial interactions and may confound identification of this pathogen. Other gram-negative organisms such as Pandoraea apista (110–112), Ralstonia spp. (e.g., R. mannitolilytica and R. pickettii) (113), and Iniquilinus spp. (114,115) have also been grown from respiratory tract specimens from patients with CF, but further studies are warranted to determine the clinical impact of these microorganisms. To understand the role that these organisms have in CF, it is critical to utilize reliable (often molecular) detection strategies as many of these organisms can be mistaken for others using traditional techniques (116). G. Nontuberculous Mycobacteria

NTM were first described in CF patients in the 1970s (117). NTM appear to be increasing in both CF and non-CF patient populations (118) presumably due to increased host susceptibility, improved laboratory testing, and potentially due to increased environmental exposure. Olivier et al. studied the epidemiology of NTM in 986 American CF patients (119). Overall, 13% (range per center 7–24%) of patients had at least one positive culture for NTM, of which 72% were Mycobacterium avium complex (MAC)

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and 16% were M. abscessus. However, transient “colonization” was common; 90 (70%) of 128 subjects had one of three cultures positive. Patients infected with M. abscessus were more likely to fulfill the American Thoracic Society criteria for disease used when the study was performed (120,121) and this species is thought to be associated with a more virulent course. Among different CF populations, the rate of NTM infection/colonization has varied greatly ranging from 2% to 23% (121,122). Similarly, the predominant species has varied around the world; Pierre-Audigier et al. reported that M. abscessus (39%) was more common than MAC (21%) in a pediatric CF population in Paris (121) and M. simiae was the most common species of NTM detected in CF patients in Israel (123). Risk factors for NTM are not fully understood. When compared with subjects without NTM-positive cultures in a large study conducted in the United States, subjects with NTM colonization/infection were older (by 4 years), had better lung function (by 6% FEV1 predicted), had a higher prevalence of coinfection with S. aureus (by 12%) and a lower prevalence of coinfection with P. aeruginosa (by 10%) (119). In a single-center study, systemic steroids for treatment of APBA were implicated as a possible risk factor for NTM (124). In an era when more patients are being treated with chronic azithromycin, it is critical to screen CF patients for NTM prior to starting azithromycin, annually while on azithromycin, or whenever the clinical or radiographic findings changes and NTM are included in the differential diagnosis. H. Molds and Yeast

Approximately 40% of CF patients will have Aspergillus spp. isolated from respiratory tract specimens during their lifetime and as many as 60% of CF patients listed for transplant will have a history of colonization with Aspergillus (125). A. fumigatus is the most common species recovered, but A. niger, A. flavus, and A. versicolor, may also be cultured from CF patients. Molecular studies in a small number of patients have shown that patients with chronic colonization can harbor a predominant genotype, but multiple genotypes can be present simultaneously, particularly in patients with recent acquisition (126). The majority of CF patients with positive cultures for Aspergillus spp. are thought to be colonized, although a minority (2–8%) will fulfill the diagnostic criteria for allergic bronchopulomonary aspergillosis (ABPA) and fewer still may have invasive disease. In 2007, 3.4% and 5.7% of CF patients <18 years and
18 years of age were reported to the CF Foundation’s Patient Registry with ABPA, although a consistent diagnostic approach was not employed. A recent consensus document for ABPA suggested that the diagnosis may be more common than appreciated (127). However, given the clinical manifestations of respiratory disease in CF, it can be very difficult to make the diagnosis of ABPA. Detection of molds may be improved by selective media and molecular testing (128–130). As a result of such studies, the range of molds detected in CF respiratory specimens has expanded to include Scedosporium spp., most commonly Scedosporium apiospermum, Exophiala dermatitidis, Mucor spp., and Penicillium spp. (Table 1). Prevalence rates for Sced. apiospermum have been as high as 9% in single-center studies (131) and molecular typing has shown that patients are generally colonized with unique and

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persistent genotypes (132). However, it is very difficult to assign a pathogenic role to these microorganisms given the frequency of coinfection, except for case reports that have demonstrated fatal outcomes from disseminated disease following lung transplantation (133) or manifestations similar to those of ABPA, which are suggestive of the potential for this organism to stimulate an inflammatory response (134). Of interest, S. apiospermum was often recovered from patients who were previously colonized with A. fumigatus further complicating the ability to unravel the pathogenic role of Scedosporim spp. While Candida spp. may also be cultured very frequently from respiratory tract specimens, these yeasts are not considered CF pathogens. I.

Microbial Communities, Culture Independent Techniques, and New Technologies

An important new area of investigation centers on the communities of microorganisms living in the lungs of CF patients. The microbial ecology of the CF airway is complex. Patients are often colonized by more than one major pathogen (70,117,135,136). There is evidence to suggest that pathogens could act synergistically to enhance colonization, or affect the virulence of other potential pathogens (137–141), or in contrast, affect other organisms antagonistically through competition (142–145). Thus, it seems likely that infection of the CF lung is a disease for which ecological concepts such as community structure, diversity, competition/synergism, and succession, (i.e., a progression of ecological patterns over time) could apply (146,147). Microbial communities are often composed of multiple species, some of which can be difficult to isolate using standard culture techniques. For instance, modified culture techniques have recently been shown to detect large numbers of obligate anaerobes (including members of the Actinomyces, Prevotella, Propionibacterium, and Veillonella genera) that would have been missed in CF sputa using traditional culture techniques (148,149). Likewise, a novel culture medium (McKay Agar) has helped to define a possible role for “Streptococcus milleri” group (SMG) colonization/infection in CF pulmonary exacerbations (150,151). More recently, there has been much interest in the use of culture-independent molecular techniques to detect potential pathogens in patients with CF. Most such techniques rely on amplification of the ubiquitous gene for the 16S small subunit of ribosomal RNA. Because this gene has highly conserved regions at the nucleotide level, primers can be designed that amplify the gene “blindly” from all bacterial members of a community. Once amplified and separated (usually by cloning or gel electrophoresis), the species from which the 16S molecules are derived can be identified. One of the first techniques used for 16S identification and microbial community profiling in CF was terminal restriction length polymorphism (T-RFLP) (151–155). T-RFLP typing is advantageous because, at relatively lower cost, 16S diversity can be quickly and reliably profiled in a clinical sample. However, T-RFLP is limited by its discriminating power; bands with the same molecular weight can be derived from vastly different species, and sometimes it is impossible to distinguish closely related species. A more powerful and precise (but costly) way to identify species from 16S amplification is to sequence each amplified 16S gene and find its most similar match in a database of all known 16S sequences (152,156–158). T-RFLP and sequence-based studies together have suggested that there is much greater microbial diversity in the CF lung than previously appreciated. In general,

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Table 4 Concordance of microorganisms in studies assessing culture independent strategies for

CF respiratory specimens Type of microorganism

Species detected

Number (þ) of studies organism detected (n ¼ 7 studies)

Classic CF pathogens Pseudomonas aeruginosa Haemophilus influenzae Burkholderia spp. (cepacia, multivorans, gladioli)a Staphylococcus aureus

þþþþþþþ þþþþþþ þþþþ þþþþ

Emerging pathogens

Stenotrophomonas maltophilia Streptococcus milleri group Achromobacter xylosoxidans

þþþþþ þþþþ þþþ

Anaerobic bacteria

Prevotella spp. (oris, salivae, melaninogenica, denticola) Actinomyces spp. (odontolyticus) Fusobacterium spp. (gonidoformans, nucleatum) Veilonella spp. (atypica, dispar, parvula) Porphyromonas spp. Peptostreptococcus spp.

þþþþþ

Abiotrophia defectiva Neisseria spp. (elongata, subflava) Escherichia coli Methylobacterium spp. Streptococcus sanguinus Bifidobacterium spp. Bacteroides spp. Streptococcus gordonii Strep. cristatus Strep. parasanguis Strep. pneumonia Rickettsia spp. Bergeyella spp. Rothia spp. (dentocariosa, mucilaginosa) Campylobacter consisus Comamonas testosteroni Gemella sanguinis Granulicatellens adiacens

þþþþ þþþþ þþþ þþþ þþþ þþþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ þþ

Otherb

þþþþþ þþþþ þþþþ þþþ þþ

a

Examples of species identified are provided. Detected in
2 studies. Source: From Ref. 151–152 and 154–158. b

studies have estimated the average number of organisms in any given CF patient to be approximately 7 to 15 species, with more than 30 species detected in some patients and only one species noted in others. Perhaps more importantly, however, these studies show significant variation in microbial diversity between individuals and among samples taken from the same individual at different times.

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As seen in Table 4, the culture-independent techniques often detected both classic and emerging pathogens and had similar or slightly better sensitivities than culture, although in a minority of instances, cultures detected organisms not identified by molecular techniques (156,157). Culture-independent techniques readily detect organisms such as anaerobic bacteria that require specialized culture techniques. Therefore, a potential advantage of culture-independent techniques is the ability to uncover novel pathogens as these techniques are not constrained by needing to know the microorganisms being sought. A major technical concern in culture-independent studies is the detection of contaminating bacteria especially from the oropharynx (153), but also from skin and equipment. Many of the organisms detected in these studies are known commensal organisms from the upper airway and mouth. Interestingly, when different cultureindependent studies are compared, the majority of species (75%, 140 of 186 species) have been detected in only one study. Thus, it remains unclear whether or not these organisms are contaminants, commensals, or rare but clinically important pathogens. As sequencing techniques become increasingly economical and more data are gathered to implicate novel bacteria in disease, culture-independent techniques will become increasingly attractive.

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32. Wang SS, FitzSimmons SC, O’Leary LA, et al. Early diagnosis of cystic fibrosis in the newborn period and risk of Pseudomonas aeruginosa acquisition in the first 10 years of life: a registry-based longitudinal study. Pediatrics 2001; 107:274–279. 33. Emerson J, Rosenfeld M, McNamara S, et al. Pseudomonas aeruginosa and other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatr Pulmonol 2002; 34:91–100. 34. Kahl BC, Duebbers A, Lubritz G, et al. Population dynamics of persistent Staphylococcus aureus isolated from the airways of cystic fibrosis patients during a 6-year prospective study. J Clin Microbiol 2003; 41:4424–4427. 35. Schlichting C, Branger C, Fournier JM, et al. Typing of Staphylococcus aureus by pulsedfield gel electrophoresis, zymotyping, capsular typing, and phage typing: resolution of clonal relationships. J Clin Microbiol 1993; 31:227–232. 36. Branger C, Gardye C, Lambert-Zechovsky N. Persistence of Staphylococcus aureus strains among cystic fibrosis patients over extended periods of time. J Med Microbiol 1996; 45:294–301. 37. Molina A, Del Campo R, Maiz L, et al. High prevalence in cystic fibrosis patients of multiresistant hospital-acquired methicillin-resistant Staphylococcus aureus ST228SCCmecI capable of biofilm formation. J Antimicrob Chemother 2008; 62:961–967. 38. Tre-Hardy M, Mace C, El Manssouri N, et al. Effect of antibiotic co-administration on young and mature biofilms of cystic fibrosis clinical isolates: the importance of the biofilm model. Int J Antimicrob Agents 2009; 33:40–45. 39. Kahl BC, Goulian M, van Wamel W, et al. Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line. Infect Immun 2000; 68:5385–5392. 40. Zander J, Besier S, Saum SH, et al. Influence of dTMP on the phenotypic appearance and intracellular persistence of Staphylococcus aureus. Infect Immun 2008; 76:1333–1339. 41. Kahl B, Herrmann M, Everding AS, et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis 1998; 177:1023–1029. 42. Besier S, Zander J, Kahl BC, et al. The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates. Antimicrob Agents Chemother 2008; 52:2183–2189. 43. Hoffman LR, Deziel E, D’Argenio DA, et al. Selection for Staphylococcus aureus smallcolony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2006; 103:19890–19895. 44. Besier S, Smaczny C, von Mallinckrodt C, et al. Prevalence and clinical significance of Staphylococcus aureus small-colony variants in cystic fibrosis lung disease. J Clin Microbiol 2007; 45:168–172. 45. Ridder-Schaphorn S, Ratjen F, Dubbers A, et al. Nasal Staphylococcus aureus carriage is not a risk factor for lower-airway infection in young cystic fibrosis patients. J Clin Microbiol 2007; 45:2979–29784. 46. McCaffery K, Olver RE, Franklin M, et al. Systematic review of antistaphylococcal antibiotic therapy in cystic fibrosis. Thorax 1999; 54:380–383. 47. Smyth A. Prophylactic antibiotics in cystic fibrosis: a conviction without evidence? Pediatr Pulmonol 2005; 40:471–476. 48. Stutman HR, Lieberman JM, Nussbaum E, et al. Antibiotic prophylaxis in infants and young children with cystic fibrosis: a randomized controlled trial. J Pediatr 2002; 140:299–305. 49. Starner TD, Zhang N, Kim G, et al. Haemophilus influenzae forms biofilms on airway epithelia: implications in cystic fibrosis. Am J Respir Crit Care Med 2006; 174:213–220.

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5 Inflammation in the Cystic Fibrosis Lung JAMES F. CHMIEL and MICHAEL W. KONSTAN Case Western Reserve University School of Medicine, Rainbow Babies and Children’s Hospital, Cleveland, Ohio, U.S.A.

I.

Introduction

Although cystic fibrosis (CF) impacts nearly every organ system, lung disease accounts for most of the morbidity and mortality (1). Epithelial cells, particularly those of exocrine glands, are the principal sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and therefore are most affected by its dysfunction. In the airway, abnormal CFTR leads to a vicious cycle of airway obstruction, chronic bacterial infection, and vigorous inflammation (2). The dysregulated inflammatory response eventually becomes more harmful than protective and actually encourages persistence of pathogens, promotes obstruction of the airway lumen, and causes destruction of airway wall architecture resulting in bronchiectasis (Fig. 1). Although the association between the basic defect in CFTR and the development and progression of the lung disease is not fully elucidated, understanding the pathophysiology of the process within the airways is vital to understanding current and emerging therapies.

II.

Characteristics of the Inflammatory Component of CF Lung Disease CF lung disease is characterized by a self-perpetuating cycle of airway obstruction, chronic bacterial infection, and vigorous inflammation that results in bronchiectasis and ultimately the death of the patient (3). The histopathological process in the CF lung is centered in and around the airways and eventually spreads to involve the submucosa, airway wall, and supporting structures. In contrast, the alveoli are relatively spared until later in the disease process. The airways of patients with CF have a predilection for chronic infection with a limited spectrum of distinctive bacteria such as Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, Burkholderia cepacia complex organisms, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans. In addition, other microorganisms including anaerobic bacteria, nontuberculous mycobacteria, and fungi (i.e., Aspergillus fumigatus) may contribute to the disease process to a greater extent than previously believed. Once present in the CF airway, most of these microbes have the ability to form biofilms thus making their eradication with antiinfectives virtually impossible. While the specific species of bacteria infecting the airway may not alter the magnitude of the host inflammatory response (4), the density of infecting organisms may correlate with the intensity of inflammation (5,6). Since

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Figure 1 Pathologic cascade of cystic fibrosis lung disease. The defective CFTR gene and protein lead to the decreased height of the ASL, which initiates the vicious cycle of obstruction, infection and inflammation, and leads to disruption of the airway wall architecture. In the last panel, a bronchiectatic airway with a large mucus plug and dilated submucosal glands is shown. The white ovals with black shading represent bacteria and the black dots represent neutrophils. Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; ASL, airway surface liquid.

bacterial infection is likely the primary stimulus for the inflammatory response, antibiotics play an important role in the therapy of CF lung disease. Although the lungs of CF patients are structurally normal at birth, plugging of the bronchioles with secretions and hypertrophy of submucosal gland ducts are seen by four months of age (7). Airway obstruction by thick secretions and bacterial infection may begin shortly thereafter. A vigorous inflammatory response is frequently initiated before one year of age (8). When local host defense mechanisms are challenged by intercurrent viral or bacterial infections, massive numbers of neutrophils are recruited into the airway. Although inflammation is meant to contain infection, this ultimately fails in the CF airway, as the exaggerated inflammatory response that ensues appears responsible for much of the pathology in the CF lung. Of note, the inflammatory response in CF begins early in life, becomes persistent, and is excessive relative to the burden of infection. Bronchoalveolar lavage (BAL) fluid from CF patients contains large concentrations of inflammatory mediators and massive amounts of neutrophil chemoattractants, neutrophils, and neutrophil products (9–14). This is not surprising in chronically infected adolescents and young adults, even those with mild disease (14). However, even infants with CF have an impressive inflammatory burden in the airways (8,9,11,12,15,16). BAL fluid from infected infants with CF contains greatly elevated concentrations of neutrophil chemoattractants, neutrophils, and neutrophil products compared to BAL fluid from non-CF infants (8,11,15). Moreover, these inflammatory mediators are present, although at somewhat lower concentrations, in BAL fluid from apparently uninfected infants with CF (11). The presence of inflammation in the absence of detectable pathogens might suggest that the inflammatory response in the CF lung operates independently of an infectious stimulus. However, these findings may also be explained by failure to terminate the inflammatory response once the inciting stimulus has been removed, and may actually represent an abnormal persistence of inflammation after clearance of early, transient infection (11,17).

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In addition to abnormal persistence of the inflammatory response, other studies suggest that the inflammatory response in CF is excessive relative to the burden of infection. Human, animal, and in vitro studies suggest that the CF defect may be directly responsible for dysregulated production of interleukin (IL)-8 and other inflammatory mediators. IL-8, a potent neutrophil chemoattractant, is present in high concentrations in CF airways. BAL fluid from infants with CF infected only with H. influenzae contains more neutrophils and IL-8 for any given burden of H. influenzae than does BAL fluid from infants with underlying conditions other than CF who also are infected with H. influenzae (5,6). Studies demonstrating the beneficial effects of anti-inflammatory therapy on pulmonary disease provide supporting evidence that the inflammatory response in CF is excessive. The first clinical trial of oral corticosteroids in children with CF suggested that corticosteroids preserved lung function, improved weight gain, and lessened hospital admissions (18). In a larger four-year clinical trial, beneficial effects of oral corticosteroids on lung function were observed, particularly in patients with mild lung disease infected with P. aeruginosa (19). However the adverse effects of corticosteroids, including glucose intolerance, growth impairment and cataracts were limiting (19). In neither of the trials of systemic corticosteroids was an increase in infectious complications or sepsis reported in corticosteroid-treated patients. Oral administration of highdose ibuprofen (20–30 mg/kg/dose BID) was also studied in a four-year, double-blind, placebo-controlled clinical trial. Patients treated with ibuprofen had significantly less decline in pulmonary function, better preservation of body weight, fewer hospital admissions, and better Brasfield chest radiograph scores (20). There were no significant differences in adverse effects between the treated group and the placebo group. If the inflammatory response were not excessive and necessary to control infection, one would expect that the administration of immunomodulators would be associated with bacterial invasion into the lung parenchyma and secondary bacteremia. Since this was not observed, it supports the hypothesis that the magnitude of the inflammatory response is excessive relative to the burden of bacteria. Many investigators have used the bacteria-imbedded agarose bead model of chronic endobronchial infection in animals (primarily rodents) to study the relationship between infection and inflammation and the impact of potential therapeutics. When delivered to the airways, the agarose beads markedly decrease bacterial clearance, so chronic inflammation ensues (21–25). This model closely mimics the persistent neutrophil-dominated endobronchial infection in CF. CF mice that received endobronchial inoculations of P. aeruginosa–embedded agarose beads had greater mortality, more drastic weight loss, and greater pro-inflammatory cytokine production compared with their normal littermates (23,24), similar to the findings documented in CF patients. Data in this model also suggested the efficacy of ibuprofen, which subsequently was verified in CF patients (20,21). These observations provide further evidence that the underlying inflammatory response in CF is excessive and may be associated with defects in CFTR itself and that attenuating the inflammatory response does not make infection worse.

III.

Role of the Airway Epithelia in Inflammation

The search for the cause of this excessive inflammatory response has most recently focused on the airway epithelial cell. Once thought to be a relatively inert, innocent bystander in the inflammatory process, the airway epithelial cell is now recognized to

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actively participate in regulating the airway inflammatory response. The response of the airway epithelial cell to the absence of CFTR function may include dysregulation of the inflammatory response. Besides the obvious physical barrier that an intact epithelium and its basement membrane provide, the epithelial cell has other important defense functions in the airway. Epithelial cells regulate salt and water composition of the airway surface liquid (ASL) and can respond to challenges by fluid secretion, thereby “washing away” noxious materials by facilitating mucociliary clearance (see chap. 3). Some are adapted for secretion of mucus, which occurs in response to irritants to trap and eliminate them. In addition, the epithelial cells are the source of many molecules of defense. The secretory immune system is coordinated by the specific transcytosis of IgA and IgM through the epithelium via the polymeric immunoglobulin receptor, which provides one-way passage into the ASL for these immunoglobulins. Epithelial cells are also the source of innate defense molecules such as lysozyme and defensins, and can direct the recruitment and activation of other cells of defense when appropriately stimulated. Epithelial cells can release arachidonic acid or leukotriene (LT) A4 that can in turn be taken up and metabolized to the potent chemoattractant LTB4 in neutrophils. Some cytokine products of epithelial cells, such as TNF-a, IL-1b, IL-6, IL-8, granulocyte macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF), are produced in excess in CF, whereas some, such as IL-10 and regulated on activation normal T cell expressed and secreted (RANTES), appear to be reduced. However, caution must be exercised when interpreting the results of studies performed in primary cells retrieved from a highly inflammatory environment compared with cells retrieved from a more normal milieu since metabolic changes may linger even after weeks in culture. In addition, it is becoming clear that there exist numerous genetic polymorphisms, which have little clinical impact on normal individuals, but which cause substantial differences in cytokine responses upon stimulation (see chap. 6 for further discussion). The chronic endobronchial bacterial infection and associated excessive host inflammatory response orchestrated by the epithelial cell promotes the clinical manifestations of the disease, as summarized in Figure 2. In most cases, the clinical course in CF is one of a progressive decline in pulmonary function as more and larger airways become increasingly obstructed. This continuous slow decline is punctuated by periodic exacerbations, which may be associated with increased cough, increased sputum production, weight loss, and malaise. In addition to serving as important triggers for exacerbations of the chronic bacterial infection, intercurrent viral infections of the respiratory tract may also damage the epithelium and increase obstruction of the airways. While the patient typically returns to pre-exacerbation pulmonary function values following an extended course of parenteral antibiotics, this is not always the case. It is likely that the cumulative effects of multiple pulmonary exacerbations contribute to the decline in lung function and that patients with more frequent and/or severe exacerbations may have shorter life spans (26–28).

IV.

Inflammatory Mediators in the CF Lung

Although bacterial products may be responsible for inciting the initial inflammatory response, host mediators are paramount in the persistent stimulation of neutrophil influx. BAL and sputum from individuals with CF are characterized by extremely high

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Figure 2 Schematic demonstrating the inflammatory response in the cystic fibrosis airway.

P. aeruginosa (or other microorganism including viruses and other bacteria) interacts with the epithelial cell and stimulates the production of the cytokines that are responsible for mediating the harmful inflammation. Macrophages also interact with the bacteria and release TNF-a and IL-1b, which upregulate adhesion molecules (ICAM-1) on vascular endothelium and airway epithelium, thus allowing neutrophils to migrate out of the pulmonary circulation and holding them in the airway. Epithelial cells produce large amounts of the chemoattractant IL-8, thereby stimulating the enormous influx of neutrophils, which release more neutrophil chemoattractants and other products that are noxious to host tissues. Lymphocytes are also present in and around the airways and release multiple mediators including IL-17, which also promotes neutrophil influx. Abbreviations: ICAM-I, intercellular adhesion molecule 1; LTB4, leukotriene B4; GM-CSF, granulocyte macrophage colony-stimulating factor.

concentrations of IL-8, LTB4, and complement-derived chemoattractants (C5a/C5a-desArg) (5,6,10,11,13,29,30). In CF, large concentrations of the neutrophil chemoattractant IL-8 can be measured in BAL fluid (10), even during the first year of life (11). The major source of IL-8 is likely the epithelial cell, and much of the neutrophil chemotactic activity of CF sputum in vitro can be inhibited by anti-IL-8 antibody (31). Some studies have demonstrated that the concentration of IL-8 in BAL fluid correlates with the concentration of neutrophils and neutrophil products (DNA and active elastase) (12,15,16,32). Cytokines (TNF-a and IL-1b), neutrophil elastase, viral products (doublestranded RNA), soluble products of P. aeruginosa, lipopolysaccharide (LPS), and adherence of intact P. aeruginosa all induce bronchial epithelial cell lines to produce IL8 in vitro (33–36). A growing body of evidence suggests that IL-8 production is excessive in CF. For example, human fetal CF tracheal grafts implanted in animal models produced eight times more IL-8 than did similar grafts from non-CF fetuses (37).

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IL-8 protein and mRNA expression are also greater in unstimulated CF bronchial submucosal gland cells compared with unstimulated non-CF bronchial submucosal gland cells in culture (38). The overproduction of IL-8 in CF stimulates the massive influx of neutrophils that release products that are responsible for mediating the airway injury. Although IL-8 plays a major role in the pathology of CF lung disease, other host mediators present in the airways also contribute to additional neutrophil chemotactic activity. These include LTB4, which is produced by both macrophages and neutrophils (13,30,39,40) from arachidonic acid (41,42), bacterial products that resemble the prototypic chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), complement fragments such as C5a and C5a-des-Arg, and peptide fragments derived from fibrin and other plasma proteins. High-mobility group box 1 (HMGB1), which is found in CF sputum and serum in large concentrations, is an inflammatory mediator that contributes to both neutrophil chemotaxis and degradation of the extracellular matrix as determined by proline-glycine-proline (PGP) release (43). PGP, which is also elevated in CF sputum, is likely released as a result of matrix metalloproteinase (MMP) 8, MMP9, and endopeptidase action (44). PGP acts on CXC1 and CXC2 receptors on the neutrophil surface to stimulate chemotaxis. PGP levels in sputum appear to be associated with disease severity and changes upon treatment in the lung disease (44). Given the plethora of neutrophil chemoattractants in the CF lung, it is no surprise that millions of neutrophils per milliliter of BAL fluid are found in almost all CF patients, even those with mild lung disease (14). Therefore, for any anti-inflammatory drug to be effective in CF, it will have to address the neutrophil and its products either directly or indirectly, and targeting one chemoattractant may not be sufficient to effectively reduce neutrophil influx (45). Other pro-inflammatory cytokines, including TNF-a and IL-1b, are present in extremely elevated concentrations in CF BAL fluid (10). Although these cytokines may be of benefit in containing bacterial infection by stimulating host defenses, their continued overproduction eventually has detrimental consequences and likely contributes to the pathologic findings in CF. TNF-a enhances neutrophil oxidative and secretory responses (46,47), and may be responsible for the cachexia associated with CF and many other chronic diseases (48). IL-1b induces fever, stimulates acute phase responses, and promotes muscle protein catabolism. TNF-a and IL-1b also likely “prime” neutrophils and increase their response to chemoattractants. They also increase the expression of the adhesion molecule CD11b/CD18 on neutrophils and its ligand intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. The interaction between this pair of molecules increases the neutrophils’ ability to adhere to endothelial cells and migrate out of blood vessels so that they can arrive at the site of infection in the airway. ICAM-1 expression is also increased on airway epithelial cells, which may serve to retain the neutrophils in the airway. Clearly, these pro-inflammatory mediators are integral components of the inflammatory cascade. Recently, the contribution of the adaptive immune response to the airway pathophysiology in CF has just begun to be studied. B cells and T cells are present in the CF lung in large concentrations and also express CFTR. Of particular interest is a newly described subset of T cells known as TH-17 cells (49). IL-23 and IL-17 are proinflammatory cytokines involved in TH-17 cell signaling and are elevated in CF lungs (human and mouse) in response to common stimuli (50,51). Viscous secretions at the

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apical surface of airway epithelia trap bacteria and may magnify the likelihood of antigen presentation, which, in turn, triggers the IL-23/IL-17 signaling pathway. IL-17 appears important in recruiting neutrophils to the airway in response to bacterial products because anti-IL-17 antibodies can effectively reduce airway neutrophilia in mice stimulated with LPS (52) or P. aeruginosa embedded in agarose beads (53). One recently developed monoclonal antibody, anti-IL-17, has generated much excitement and is planned for comprehensive clinical evaluation. Most cytokines are pleiomorphic, and while some demonstrate a preponderance of pro-inflammatory effects, others demonstrate a preponderance of anti-inflammatory effects. IL-10 is an important anti-inflammatory and immunoregulatory cytokine. It is produced by many cell types including macrophages, epithelial cells, T lymphocytes and B lymphocytes. Airway secretions from CF patients appear deficient in IL-10 compared with airway secretions from non-CF individuals (10,17,54). This deficiency, which may be directly associated with defects in CFTR since it has been reported in uninfected CFTR knockout mice (55), is peculiar because LPS and TNF-a, which are abundant in the CF airway, normally stimulate IL-10 production. IL-10 normally serves to terminate acute inflammatory responses by downregulating production of pro-inflammatory cytokines and chemokines, and by other negative feedback mechanisms. IL-10 also induces neutrophil apoptosis. In a state of relative IL-10 deficiency like the CF airway, neutrophil survival would be prolonged. The impact of IL-10 deficiency on the inflammatory response to chronic endobronchial P. aeruginosa infection has been evaluated using the agarose bead model of chronic pseudomonas infection (22). IL-10 knockout mice inoculated with P. aeruginosa embedded in agarose beads had more drastic weight loss, greater neutrophil infiltration, larger amounts of the lung occupied by inflammatory exudate and higher concentrations of pro-inflammatory cytokines in BAL compared with wild-type mice (22). Administration of IL-10 to wild-type mice reversed all of these effects, without increasing the number of bacteria (22). IL-10 also influences the inflammatory response to an acute transient infection. After receiving an intratracheal inoculation of P. aeruginosa in free suspension (rather than in the agarose beads), IL-10 knockout mice had a more exuberant neutrophil response and developed prolonged inflammation that persisted after the bacterial challenge had been cleared (56). Both of these increases were associated with enhanced pro-inflammatory cytokine and neutrophil chemokine production (56). The prolongation of the inflammatory response, which continued after the bacteria had been eradicated, may partly explain the observations of persistent inflammation in CF infants in the absence of cultureable organisms (11,17). These data suggest that restoring IL-10 to the CF airway or administering it in pharmacologic quantities could be beneficial.

V. Dysregulated Arachidonic Acid Metabolism and Deficiency of Essential Fatty Acids Long considered important in CF airway pathophysiology, the eicosanoids are a diverse family of arachidonic acid metabolites that regulate inflammation and other physiologic responses. While leukotrienes and prostaglandins exert predominately pro-inflammatory effects, lipoxins exert primarily anti-inflammatory effects. Pro-inflammatory eicosanoids, like LTB4, are increased in the sputum and BAL from patients with CF (14). Although it is difficult to determine the relative contribution of each chemoattractant

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present in the CF airway to the massive influx of neutrophils, clearly once neutrophils are present, neutrophil-derived LTB4 likely contributes to further neutrophil infiltration. Neutrophil products such as elastase may also contribute to the chemotactic load by stimulating alveolar macrophages to release more LTB4. In contrast to LTB4, lipoxin A4 (LXA4) is an endogenous anti-inflammatory lipid mediator that promotes the resolution of acute neutrophilic inflammatory responses (57). One study suggested that LXA4 concentrations are reduced in BAL fluid from stable CF patients compared with non-CF inflamed controls (57), although this was not confirmed more recently (58). Administration of a metabolically stable lipoxin analog in cell culture and mouse models of chronic airway infection and inflammation decreased IL-8 production and neutrophilic inflammation without increasing infectious burden (57). These data suggest that the role of LXA4 deficiency in CF lung disease remains controversial. It is possible that the imbalance between leukotrienes and lipoxins is due to an alteration in the composition of the membrane lipids that are the precursors of these mediators. CF cell lines, plasma and tissues of patients with CF, and CF mice have decreased docosahexaenoic acid (DHA) and increased arachidonic acid in their membranes (59). Administration of supplemental DHA reversed the excessive inflammatory response to inhaled LPS in cftr-/- mice (60). The agreement between findings in different experimental systems suggests that the alteration in the ratio of arachidonic acid to DHA may be directly related to defects in CFTR rather than being secondary to alterations in intestinal fat absorption related to pancreatic insufficiency. Interestingly, DHA has also been shown to increase chloride conductance in intestinal epithelial cell lines (61). The possibility that dietary supplementation may ameliorate disorders of inflammatory mediator production in CF certainly makes this an important subject of further investigation.

VI.

Disordered Intracellular Signaling as a Cause of Excessive Inflammation The early onset and unusual characteristics of the infectious and inflammatory processes in CF clearly suggest that the microenvironment of the CF lung is unique, and that abnormalities in CFTR must play an important role in the local pathophysiology. Previous studies suggest that inhibition of CFTR production or CFTR function in epithelial cell lines result both in increased activation of the pro-inflammatory transcription factor NF-kB and increased secretion of IL-8 upon stimulation of epithelial cell lines with proinflammatory stimuli (36,62–64). These results provide a strong indication that alterations in the function of CFTR cause dysregulation of the intracellular signaling pathways and pro-inflammatory cytokine production. Several major intracellular pathways that regulate pro-inflammatory gene expression appear disturbed in CF epithelial cells (Fig. 3). In addition to NF-kB, the transcription factors that participate in these signaling pathways include perioxisome proliferator–activated receptor (PPAR), signal transducers and activators of transcription (STATs), and nuclear factor E2–related factor 2 (Nrf2). Although any of these individually might be expected to lead to dysregulation of production of extracellular signals that influence inflammation, exact predictions are difficult because there is a great deal of cross-talk between pathways, and gene transcription is rarely dependent on a single factor.

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Figure 3 Schematic representation of some of the signal transduction pathways involved in the

generation of the excessive and persistent inflammatory response in the CF airway. Pathways have been simplified for illustration purposes and interact more than is indicated here. The dotted arrow from the receptor to NIK represents multiple steps that are not included in the figure. Multiple stimuli are capable of inducing the activation of the transcription factor NF-kB. This is achieved when I-kK phosphorylates I-kB, causing it to dissociate from NF-kB and allowing the transcription factor to translocate into the nucleus to promote the transcription of multiple proinflammatory genes. Nrf2 is a transcription factor also typically sequestered in the cytoplasm by an inhibitory protein (KEAP1). When Nrf2 is activated, it translocates to the nucleus to induce the transcription of multiple antioxidant and cytoprotective genes. This response is believed to be central to mitigating and ultimately halting acute inflammation. In CF, abnormalities exist in these signaling pathways, including increased NF-kB activation through increased I-kK activity. In addition, bacterial adherence, decreased IL-10, decreased PPAR, and decreased nitric oxide promote the activation of NF-kB and transcription of pro-inflammatory cytokines in the airway epithelial cell. Contrary to other conditions with increased oxidative stress, CF epithelial cells have decreased Nrf2 activity. These abnormalities likely help to explain the heightened and prolonged inflammatory state. Abbreviations: GM-CSF, granulocyte macrophage colony-stimulating factor; KEAP1, Kelch-like ECH-associated protein 1; STAT, signal transducers and activators of transcription.

NF-kB is usually maintained as an inactive complex with I-kB (inhibitor of NF-kB) in the cytoplasm. Activation of NF-kB occurs when a stimulus, such as LPS (via Toll-like receptor 4, or TLR4), pseudomonas flagellin (via TLR 5), or TNF-a and/or IL-1b (through MyD88 and upstream linkages to their individual receptors) cause

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activation of I-k kinase (I-kK), which phosphorylates I-kB. Phosphorylation of I-kB by I-kK promotes dissociation of I-kB from NF-kB, and subsequent I-kB degradation. Removal of I-kB exposes a nuclear translocation sequence on NF-kB, which can then move into the nucleus and activate transcription of genes to whose promoters it binds. NF-kB may not be solely responsible for controlling the regulation of these pro-inflammatory cytokine genes, but it may work in concert with other transcription factors including AP-1 and NF-IL6. Increased activation of I-kK and/or failure to regenerate I-kB protein are key factors in the excessive activation of NF-kB in CF cells (65,66). Some investigators have reported greater activation of NF-kB in vitro when CF epithelial cell lines are challenged with either P. aeruginosa or TNF-a compared with similarly challenged cells with normal CFTR activity (67,68). Interestingly, some studies found that CF airways are relatively deficient in IL-10 and nitric oxide (NO) (17,69–71), both of which preserve the function of I-kB. In situations where either or both IL-10 and NO might be deficient, less I-kB would be available to inhibit NF-kB, and pro-inflammatory mediator production would increase. Therefore, an imbalance between I-kB and NF-kB would result in the prolonged and excessive production of the mediators responsible for the damaging inflammation. Further exacerbating this condition is that CF tissues appear to be deficient in PPAR (72,73), which may counteract NF-kB. PPAR is a nuclear receptor that is a member of the steroid hormone receptor family of ligand-activated transcription factors and exists as one of four isoforms (a, b, g, and d) (74). PPAR inhibits NF-kB activity via upregulation of I-kB, or by competition with NF-kB for helicases (75). In CF T cells and airway epithelial cells, decreased PPAR expression leads to an imbalance between I-kB and NF-kB that favors increased inflammation (72,76). Abnormalities in the fatty acid content of CF cells, with deficiencies of both DHA and linoleic acid, may also contribute to the impaired PPARg signaling and the hyperinflammatory state (77). Multiple studies have demonstrated decreased NO in exhaled breath (eNO) from patients with CF (70,71). This was initially surprising since eNO is typically increased in inflammatory lung diseases (78). Decreased NO may contribute to many of the pathologies seen in CF. First, NO may impact on the ASL content by regulation of ion transport across airway epithelium. When NO is decreased, sodium absorption is increased and chloride secretion is decreased, as in CF (79). Second, decreased NO impairs the ability of airway smooth muscle to relax in CFTR knockout mice (80). This may contribute to bronchial obstruction. Third, NO may have a role in host defense. NO combines with reactive oxygen molecules to form a natural antimicrobial agent. The combination of NO with superoxide anion is more effective in killing B. cepacia in vitro than either component alone (81). Although NO probably does not play a large role in controlling chronic bacterial infection, it may be important in the events leading to the establishment of infection when only a few bacteria are present in the airway. Fourth, NO stabilizes IkB in macrophages, thereby dampening the inflammatory response. If this also occurs in epithelial cells, reduced NO could contribute to the aggressive inflammatory response of airway epithelial cells. Finally, decreased NO may directly contribute to the immunopathology of CF by promoting the sequestration of activated neutrophils into the lung (82). Neutrophil accumulation increases the damage to surrounding host tissues from the release of neutrophil products like proteases and oxygen radicals. Although it seems clear that eNO is decreased in CF, the question remains as to why. Studies suggest that CF epithelial cells are unable to adequately produce NO (83–86). In the normal lung and gastrointestinal tract, NO is constitutively produced by epithelial cells by nitric oxide

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synthetase-2 (NOS-2). In CF mouse and human tissues, epithelial production of NO is decreased because of decreased expression of NOS-2, which likely depends on the presence and function of CFTR (83,84). Finally, Nrf2, a transcription factor active in respiratory epithelia and pivotal to mitigating the acute inflammatory response, has deficient activity in CF cells (87). Triterpenoids are synthetic derivatives of naturally occurring compounds used in Asian medicines for centuries. The natural compounds possess relatively modest antiinflammatory and anti-carcinogenic properties and the synthetic derivatives appear to be much more anti-inflammatory through their ability to inhibit both I-kK and Kelch-like ECH-associated protein 1 (KEAP1, an inhibitor of Nrf2) (88–92). By inhibiting I-kK, these drugs prevent the degradation of I-kB, thereby inhibiting NF-kB activation. By inhibiting KEAP1, the triterpenoids also would increase Nrf2 activity, where Nrf2 is a central regulator of many antioxidant and cytoprotective genes that mitigate and ultimately halt acute inflammation, similar to the effects of IL-10. Because both I-kK and Nrf2 appear abnormal in CF models (87,93–96), small molecules like the synthetic triterpenoids that target these pro-inflammatory signaling abnormalities may be excellent anti-inflammatory drug candidates. To this end, early work in CF mice has demonstrated that the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) greatly reduces the inflammatory response after direct airway administration of either LPS or flagellin. In vitro, nanomolar concentrations of CDDO decrease NF-kB activation by approximately 50% in CF-like epithelial cells stimulated with TNF-a, and this mitigating effect is greater in CFTR-deficient cells than that in matched non-CF cells (95). The above mechanisms are not mutually exclusive, and a combination of these processes may coalesce to fuel the damaging inflammatory cascade. Many pathways, including those that activate as well as those that terminate the inflammatory response, seem dysfunctional in CF. It is unlikely that there is an independent fundamental defect in any one specific pathway that accounts for the exaggerated inflammatory response. The end result is that the normal delicate balance between the pro- and anti-inflammatory arms of the host response is disrupted and pathways promoting inflammation are favored. Because cytokine overproduction in the CF airway likely contributes to the excessive inflammation in CF, it seems logical to target these underlying signaling pathways.

VII.

Pathologic Consequences of the Neutrophil as the Major Inflammatory Cell in the CF Airway One of the most striking characteristics of CF lung disease is the continuous predominance of neutrophils as a result of both increased influx and decreased clearance. In that sense, CF seems to represent a prolonged primary inflammatory response as would be seen in acute infection, as opposed to a maturing “round cell,” or granulomatous response as would occur in other chronic infections Neutrophils are short-lived terminally differentiated cells, and once they leave the vascular system, they are unable to return. Thus, neutrophils must be either physically removed (i.e., by coughing) or they must be disposed of by local mechanisms. Normally, the neutrophil influx into the lung that accompanies acute pneumonia, for example, is resolved by cough clearance and by phagocytosis of apoptotic neutrophils by alveolar macrophages. In CF, cough clearance is impaired by physicochemical abnormalities in the mucus and by ciliary dysfunction, and the ability of

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macrophages to scavenge apoptotic neutrophils may be inhibited by large concentrations of proteases that are present in the airway. Furthermore, the accumulation of neutrophils is so great that even early on, neutrophils vastly outnumber macrophages. Exacerbations of CF lung disease are accompanied by increased purulence of sputum primarily because of an increase in neutrophil numbers. Subsequent release of large amounts of intracellular contents from decomposing neutrophils, including actin and long-stranded DNA, then contribute to the high viscosity of CF sputum, which further impairs mucociliary clearance. The highly anionic nature of the extracellular free DNA leads to many ionic interactions with other constituents in CF sputum. Intact DNA also may bind aminoglycoside antibiotics and decrease their efficacy. Neutrophils release other products that have adverse effects including oxidants (H2O2 and O2
) and proteases, which contribute to local tissue damage both individually and in combination. For example, elevated levels of oxidant products have been detected in the airways of CF mice (97) and patients (98). Neutrophils also release the neutrophil chemoattractants LTB4 and IL-8, which attract more neutrophils into the airway and further exacerbate the problem. A. Protease-Anti-protease Imbalance

Of the neutrophil products listed in Table 1, the proteases deserve special mention. Early in life, neutrophils infiltrate the CF airway and release massive amounts of active Table 1 Mediators of Neutrophil Influx and Neutrophil Products that

Contribute to CF Lung Disease I. Mediators Cytokines that prime neutrophils and induce adhesion molecule expression TNF-a IL-1b Chemoattractants IL-8 LTB4 Complement components: C5a, C5a-des-Arg Bacterial products: N-formyl-Met-Leu-Phe II. Neutrophil products (pathologic consequences listed below each) Proteases (elastase) Damage airway wall Impair opsonophagocytosis Increase secretions Impair mucociliary clearance DNA Increase viscosity of secretions Oxidants (H2O2 and O2
) Damage tissue Inactivate a1-antiprotease (a1-antitrypsin) IL-8 Attracts more neutrophils LTB4 Attracts more neutrophils Abbreviation: LTB4, leukotriene B4.

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proteases that overwhelm local anti-protease defenses, particularly alpha-1-protease inhibitor (a1-PI, also known as a-1-antitrypsin) and secretory leukocyte protease inhibitor (SLPI). a1-PI diffuses into the smaller airways and alveoli from the blood and is carried up the airway by the mucociliary escalator. SLPI is produced and secreted by airway mucosal cells and is the predominant protease inhibitor in the conducting airways. Both SLPI and a1-PI also can be inactivated by pseudomonas proteases and oxidants generated during neutrophil activation, which may limit their efficacy in CF, although the extent to which this occurs in vivo is difficult to assess. The concentration of a1-PI in the BAL fluid of CF patients is several-fold higher than in healthy subjects, but even in mild patients there is a several hundred to several thousand fold excess of elastase and other neutrophil proteases, which vastly exceeds the capacity of the inhibitors (2,9,14). The imbalance between the proteases and their inhibitors becomes worse during exacerbations, and, as is described here for elastase, likely contributes to the patients’ deterioration in a number of ways. First, elastase promotes structural damage by promoting detachment of epithelial cells from the basement membrane, creating microulcerations and disrupting tight junctions between epithelial cells. This permits increased access of neutrophil products, inflammatory mediators, and other airway contents to the submucosa. Destruction of elastin in the airway wall decreases the recoil of the airway and leads to dilatation. Together these structural changes begin a process that progresses to irreversible bronchiectasis. Second, elastase contributes to the impairment in mucociliary clearance because it is a potent mucus secretagogue (99,100), and it directly hinders ciliary beating (101,102). Third, elastase fuels the vicious cycle of inflammation by inducing synthesis of neutrophil chemoattractants, such as IL-8 by epithelial cells, and by releasing a C5a-like peptide from C5 (32,34). Fourth, elastase activates the sodium channel (ENaC) located on the apical surface of the epithelial cell thereby increasing sodium hyperabsorption and decreasing ASL height resulting in further impairment of mucociliary clearance (103). Finally, elastase promotes bacterial persistence by crippling normal host opsonophagocytotic mechanisms. Since effective phagocytic defense mechanisms depend on the interactions of opsonic proteins such as immunoglobulins and complement fragments on the surface of the bacteria with specific receptors that are also proteins on the surface of the phagocytes, these mechanisms are quite susceptible to component cleavage by uninhibited extracellular proteases (104–107). The challenge of inhibiting the enormous burden of active proteases in the CF airway is substantial. As neutrophils reach the airway by migrating through the epithelium, much of the release of proteases will occur beneath the thick layer of mucus, which is difficult for exogenous inhibitors to penetrate. However, in model systems, this can be overcome by using the polymeric immunoglobulin receptor to deliver systemically administered a1-antitrypsin to the luminal side of the airway epithelium (108). To date promising data demonstrate that anti-protease therapy has the potential to significantly impact inflammation in CF. To be effective, however, it may be necessary to inhibit all of the active elastase in the airways because even minute amounts interfere with opsonophagocytosis.

VIII.

Implications for Anti-Inflammatory Therapy

In addition to the regular use of antibiotics to treat the chronic bacterial infection and airway clearance techniques to remove harmful secretions from the CF airway,

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addressing the overzealous inflammatory response directly seems warranted because it is the major process leading to the destruction of the lungs. There are several steps in the inflammatory process at which intervention may be targeted, and efficacy at any step may result in benefit to a patient with CF. The most appropriate time to treat the excessive inflammatory response may be relatively early in life, prior to the establishment of the vicious cycle of obstruction, infection, and inflammation, and prior to the onset of structural damage to the airways. However, it is not known whether limiting inflammation in infancy would promote or retard the establishment of chronic infection. While the inflammatory response is likely protective early in the course of disease, published clinical trial data suggest that anti-inflammatory therapy may be most helpful early in the course of disease (18–20). To date, attention has focused primarily on the therapeutic potential of systemic and inhaled corticosteroids (ICSs) and the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen. Corticosteroids possess several potent general anti-inflammatory effects, including reducing the formation of mucus and edema, inhibiting chemotaxis, adhesion, and activation of leukocytes, inhibiting NF-kB activation, and interfering with the synthesis or actions of inflammatory mediators. In fact, corticosteroid use is associated with better lung function, improved weight gain, and fewer hospital admissions, particularly in patients infected with P. aeruginosa (18,19). Unfortunately, adverse effects, including glucose intolerance, growth impairment, and cataracts, limit the long-term use of systemic corticosteroids. However, these studies utilized relatively high doses (1–2 mg/kg every other day of prednisone). Perhaps lower doses (0.1–0.25 mg/kg every other day) might yield significant beneficial effects without the severe side effects documented in earlier studies. Unfortunately, these studies might be difficult to undertake given the experience of the CF community with the previous corticosteroid studies. Adverse effects associated with systemic corticosteroids prompted investigations of ICSs (reviewed in Ref. 109). Because of familiarity with ICS for the treatment of asthma, they have been widely prescribed as anti-inflammatory agents in CF (109). The inhaled route might afford benefit with less risk than systemic administration, but systemic administration may be necessary to affect neutrophil migration. No trials of ICS in CF have demonstrated convincing effects with respect to lung function or inflammatory markers in airway secretions (110), although these studies are hampered by relatively small study populations and short observation periods. Furthermore, no clinical data presently exist that support the efficacy and safety of long-term ICS for slowing the progression of lung disease in CF, nor have the potential long-term complications of ICSbeen adequately evaluated in patients with CF. A Cystic Fibrosis Foundation expert panel recently advised against long-term use of ICS in patients with CF six years of age and older who do not have coexistent asthma or ABPA (111). NSAIDs possess anti-inflammatory properties similar to corticosteroids, but generally have fewer adverse effects making them more suitable for long-term use. Ibuprofen, at high doses (those that achieve peak plasma concentration >50 mg/mL), inhibits neutrophil migration in an animal model of chronic pseudomonas endobronchial infection (21) and in patients with CF (20). High concentrations of ibuprofen also inhibit the activation of NF-kB and AP-1, two important pro-inflammatory transcription factors. In addition, ibuprofen may exert some of its anti-inflammatory effect by activating the transcription factors PPARa and PPARg (72,112). Thus, ibuprofen is a broadspectrum anti-inflammatory agent, much like corticosteroids. In addition to its direct

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anti-inflammatory effects, ibuprofen enhances mucociliary clearance in CF by counteracting the ability of P. aeruginosa to downregulate P2Y receptors, which are found on the apical surface of airway epithelia and believed to be the major coordinator of mucociliary clearance in the lung (113). The demonstration that oral administration of ibuprofen twice daily (in doses sufficient to interfere with neutrophil migration) to rats with endobronchial pseudomonas infection significantly decreased inflammation without increasing the lung burden of organisms (21) led to a four-year, double-blind, placebo-controlled clinical trial of twice daily high-dose ibuprofen in 85 CF patients (20). Compared to placebo, ibuprofen-treated patients had significantly less decline in pulmonary function measures, better preservation of body weight, fewer hospital admissions, and better Brasfield chest radiograph scores (20). These data were confirmed by a subsequent two-year trial in children performed in Canada (114). A Cystic Fibrosis Foundation expert panel recommended considering high-dose ibuprofen for CF patients with mild lung disease (111), and a recent Cochrane review concluded that “high-dose ibuprofen can slow the progression of lung disease in people with CF, especially in children, and this suggests that strategies to modulate lung inflammation can be beneficial for people with CF” (115). The fact that neither corticosteroids nor ibuprofen were associated with infectious complications supports the hypothesis that inflammation is excessive relative to the burden of infection in CF.

IX.

Conclusion

CF is an autosomal recessive disease that is due to defects in CFTR, a regulatory transmembrane protein that controls the transport of ions across the airway epithelium. Airway obstruction, persistent bacterial infection, and vigorous neutrophilic inflammation characterize CF lung disease. Eventually, the inflammatory response escapes homeostatic control, becomes excessive and causes collateral damage to host tissues, and, ultimately, the demise of the patient. Accumulating evidence suggests that CFTR plays a broader role in the downstream effects than originally thought. Links between CFTR and bacterial adherence, as well as immunoregulation are becoming established, and defects in CFTR may cause a chain of events that affect multiple processes and signaling cascades. Because a number of products mediate the excessive and damaging effects of the inflammatory response in CF, inhibition of any single one likely will not greatly impact disease pathology. Since inflammation begins early in life, anti-inflammatory medications that are safe and effective for children also must be developed. Either a combination of anti-inflammatory medications or the development of agents that alter the signaling mechanisms responsible for promoting the transcription of proinflammatory mediators without resulting in significant untoward effects will be necessary to significantly modify the inflammatory response and natural history of CF until a cure is discovered.

Acknowledgments Grant support from the National Institutes of Health Grant P30-DK27651 and the U.S. Cystic Fibrosis Foundation is gratefully acknowledged.

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6 Modifier Genes of Cystic Fibrosis MITCHELL DRUMM Case Western Reserve University, Cleveland, Ohio, U.S.A.

I.

Introduction

The biology of cystic fibrosis (CF) is quite complex, involving multiple organ systems and tissue types. In addition to the primary cellular and physiologic dysfunctions caused by the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) protein, there are clearly temporal components and environmental factors that contribute to the clinical phenotype. Most patients succumb to the complications of pulmonary disease, but even the pulmonary disease is complex in its etiology and consequently it is not at all clear where the most effective therapeutic targets might lie. One approach for finding those targets is to compare patients with milder attributes of the disease to those with more severe manifestations, and to identify the sources of this variation. Once these sources are identified, the mechanisms behind the variation may be studied and clinical applications developed (e.g., pharmaceuticals), which would target and exploit these mechanisms to effect clinical improvement. Here we discuss genetic approaches to identify genes that modify CF disease severity.

II.

Variation of Clinical Characteristics

CF involves many tissues and organ systems, but ultimately all of the manifestations of the disease are the result of mutations in a single gene, CFTR. Despite the single-gene etiology of the disease, there is a large degree of heterogeneity in disease presentation between patients, and includes variation of the organ systems affected, the degree to which they are affected, and even the timing at which they become affected. As examples, only 15% to 20% of CF patients will have experienced meconium ileus (MI), a prenatal intestinal obstruction usually detected at birth, and only about 15% will retain exocrine pancreatic function. Pulmonary manifestations, such as reduced lung function or bacterial infection or colonization, may be apparent in the first few years of life of some patients, yet in others there may be little appreciable lung disease until the second or third decade of life. CF-related diabetes (CFRD; see chapter 21 by Kelly and Calabria) afflicts a large proportion of CF patients, but the prevalence of CFRD increases with age, and by the end of the third decade nearly a third of CF patients will have been diagnosed with CFRD. Similarly, CF liver disease (see chap. 18 by Sathe and Feranchak) affects only a minority of CF patients.

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Understanding the mechanisms giving rise to the variation of these traits among CF patients has profound clinical implications. If it were possible to understand why some patients are resistant to infections, diabetes, liver disease, etc., then methods to prevent or minimize these manifestations could be more effectively pursued.

III.

Sources of Variation

Human diversity, in health or disease, has many causes. Broadly speaking, those causes are classified as either genetic, nongenetic, or interactions of genetic and nongenetic factors. With regards to genetic variation, the human genome is composed of upward of 30,000 different genes, each of which is estimated, on average, to carry several variants, or polymorphisms, and some fraction of these variants have functional consequences for the gene. Currently, there are over 4 million characterized single-nucleotide polymorphisms (SNPs) scattered through the human genome. While this seems an enormous number, a number of technologic advances have made it logistically and economically realistic to assess the majority of these polymorphisms in large populations. More recently, duplications and deletions of sequences in the genome have been identified, and polymorphisms of this type are referred to as copy number variants (CNVs). While there are increasing numbers of examples in which CNVs have functional effects on a gene, their assessment and potential role in modifying the CF phenotype is in its infancy and will not be further discussed in this chapter. Nongenetic sources of variation are often referred to as “environmental factors.” These factors are usually thought of as environmental exposure to infectious agents, pollutants, toxins, etc., but also include diet, climate, and a multitude of other factors. As more nongenetic factors are identified and genotyping technology advances, the ability to assess interactions between gene and environment is also increasing. For example, it has long been recognized that individuals with mutations in the a1 antitrypsin gene are susceptible to emphysema, but exposure to oxidant stress, such as cigarette smoke or other air pollutants, dramatically increases the susceptibility. This same concept is recognized in more genetically complex disorders, such as type 2 diabetes, where multiple genes are risk factors, and the risk is increased by environmental factors such as diet-induced obesity.

IV.

Genetic Variation in the CF Gene, CFTR

In the case of a single-gene disorder, such as CF, it is useful to partition genetic heterogeneity into two types: CFTR and non-CFTR variation. Upon discovery of the CF gene in 1989, sequence analysis of CFTR indicated that about 70% of CF chromosomes carry the same mutation (1,2), a 3-nucleotide deletion referred to as DF508 because it results in the absence of a phenylalanine at the 508th amino acid position. However, over 1500 other mutations in CFTR have been catalogued since 1989, and comparison of patients with different mutations has shown that many clinical characteristics are dependent on the type or class of CFTR mutation carried by the patient (see also chapter 1 by Suaud and Rubenstein and chap. 8 by Ooi et al.). For instance, exocrine pancreatic insufficiency (PI) is typical in patients with loss-of-function CFTR mutations (3), but not in patients carrying mutations with some residual CFTR function (4). In fact, the correlation between CFTR mutation and exocrine pancreatic status is perhaps the strongest

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of CFTR genotype/phenotype associations found to date. In contrast, pulmonary/airway disease is quite variable, even among patients with the same CFTR genotype (2).

V. Genes That Modify CF The term “modifier gene” is a recently applied term to an old concept in genetics, which is that more than one gene can influence a trait, or phenotypic manifestation. However, the application of this concept to human genetics is novel, as one cannot approach human genetics in the same way as for other species. In nonhuman species, the contribution of genetics and estimates of the number of genes involved, as well as identification of those genes, can be determined by controlled or selected breeding. Such an approach is obviously not appropriate for human genetics, and thus these analyses in humans must be performed differently. Before attempting to identify genetic modifiers, it is first necessary to substantiate their existence. That is, there must be evidence to show that the variation associated with a trait has a genetic basis. The classical approach to this question is to use family members for whom the degree of genetic relatedness is known and compare trait variances among these relatives. The concept is employed most often in twin/sibling studies, in which the variation of a trait between monozygous (identical) twins is compared with the variation between dizygous (fraternal) twins or other siblings. As monozygous twins are essentially identical for all of their genes, variation between them would have nongenetic sources. Dizygous twins and other siblings share half of their genes, and thus their trait variation is due to a combination of genetic and nongenetic factors. The differences in variances between these two groups can be used to calculate an estimate of the genetic component of the variance, referred to as the “heritability” of the trait in question. Such twin/sibling studies have been performed for CF, and they suggest that more than half of the variation in pulmonary disease is genetically entrained. Mekus and colleagues, recognizing that morphometric indices correlate with pulmonary function, used a combined measure of lung function and weight for height to show that identical twins were more similar than other sibling pairs (5). Subsequently, Vanscoy and colleagues assessed twins and other siblings for lung function and estimated the genetic contribution to lung function variance at 50% to 80% (6). Thus, these independent studies confirmed that there are genes, other than CFTR, that contribute to CF lung disease. Lung disease is clearly not the only phenotype of interest in CF, nor is it the only trait amenable to modification by genetic heterogeneity. Meconium ileus in CF neonates has been recognized to have high heritability, and linkage analysis suggests that a relatively small number of genes are likely involved (7). Once a genetic contribution is established for the etiology of a trait or the variability of a trait between individuals, there are two approaches utilized to identify the relevant genes. These are outlined in Figure 1. One strategy is based on knowledge of family structure and is referred to as linkage analysis. In this approach, individuals with a particular trait and their family members are ascertained, and a panel of DNA markers that span the chromosomes are used to track the inheritance of chromosomal regions through the family members. In doing so, one can determine whether a specific region of the genome is inherited more often than expected by chance in individuals with the

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Figure 1 Schematic of the two approaches to identify genes or genomic regions that modify a trait. (Left) Families with multiple affected children are ascertained and typed for polymorphisms along the chromosomes and then the pedigree is examined for concordance of particular regions with the presence or absence of the trait. In the example here, one chromosome with seven biallelic SNPs is followed through a family with three affected individuals (black circles and squares). Only the region containing the CAG haplotype (boxed, dotted line), originating from the mother, is concordant with the two CF subjects manifesting the trait and absent from the patient without the trait. (Right) Association studies examine a group with the trait in question (cases) and compare with a group without the trait (controls). In this example five SNPs are typed and the genotype frequencies compared. Only the forth marker shows a difference, with the TT genotype overrepresented in those with the trait compared with the general population or those without the trait. Abbreviations: SNP, single-nucleotide polymorphism; CF, cystic fibrosis.

trait in question. The second approach is to examine unrelated individuals, comparing those with the trait to those without the trait for differences in the frequency of specific genetic variants.

VI.

Identifying Disease-Modifying Genes

Genetic variants have at least two forms or alleles. Currently, the most common variants used in genetic studies are SNPs. There are millions of SNPs in the human genome, most of which have two alleles (biallelic). The density of these variants throughout the genome, combined with the continually decreasing cost to type them has made SNP analysis a powerful tool to carry out genetic studies. As described above, family-based studies (or linkage analyses) are carried out by tracking chromosomal segments through pedigrees and then determining if a trait follows the inheritance of a particular region. SNPs are used as markers for chromosomal regions and a hypothetical example given in Figure 1. In this example, a chromosome with seven SNPs has been genotyped in a nuclear family with four children, three of whom are afflicted with CF (black symbols). Two of these have presented with a

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CF-related trait (pulmonary infection, intestinal obstruction, CFRD, liver disease, etc.) while the third has not. By genotyping the parents, it is possible to determine which regions of the chromosome in the children originated from each parent, and then to determine if there is concordance between the inheritance of a specific region and a CF-related trait. Such a region is indicated by the dotted rectangle, designating a region of the chromosome originating from the mother, individual I-1. Once a sufficiently large number of families are assessed in this way, one can evaluate the extent to which the co-segregation of the chromosomal region and trait associate in the greater population. The other approach is based on case/control study design and outlined in Figure 1 (right). In any population, the proportion of individuals carrying each of the possible allele combinations, or genotypes, has a certain value. The distribution of these genotypes is expected to be in a random distribution known as Hardy–Weinberg proportions. For a two allele system, this distribution is characterized by the expression p2 þ 2pq þ q2, where p is the frequency of the most common allele, q is the frequency of the other rarer allele. The proportion of individuals expected to be homozygous (carry two identical copies) for the common allele is given by p2 and the expected proportion of homozygotes for the rare allele by q2. The expected proportion of heterozygotes who carry one allele of each is given by the expression 2pq. These proportions are predicted from a random distribution of alleles in the population. To test whether a gene modifies a disease trait, one examines how the genotype frequencies relate to the phenotype. If, in case/control studies, Hardy–Weinberg proportions are not observed in a population, there are multiple possible explanations. These include sampling artifacts (stochastic variation), or that the variants, in fact, do affect the trait in question, or are in proximity on the chromosome to a gene that affects the trait. If the trait is qualitative, such as MI, and a patient either had it or did not, one looks to see if genotype frequencies differ between individuals with or without the trait. For qualitative traits with a continuum of values [e.g., lung function or body-mass index (BMI)], one performs comparisons to determine if the magnitude of the trait significantly differs between individuals with different genotypes. The ability of these types of studies to detect genes with modifying effects is not without limitations. As one might expect, like any analysis that relies on statistical testing, study population or sample size is a major determinant of the study’s power. The nuance of this concept in genetic studies is that many genetic variants are relatively rare in the population and thus there may be very few individuals carrying the rarest genotype. Take, for example, a polymorphism with allele frequencies of 0.9 and 0.1. Only 1% (q2) of individuals will carry the rare genotype. Thus, even in a study cohort of 1000 individuals, which is quite large for a CF study cohort, only 10 would carry the rare genotype. A related limitation is the magnitude of the effect caused by the polymorphism. Typically, genetic associations are used to detect disease-causing variants, and thus the effect of the variant on phenotype is inherently large. Modifiers, by definition, are not factors that cause a phenotypic trait, but rather alter some aspect of the trait, such as its magnitude. It is important to keep in mind that studies seeking disease-modifying genes typically focus on common population variation(s), and therefore the effects of the variants on phenotype are likely to be relatively subtle. Taken together, the likelihood of small phenotypic effect and low genotype frequency impose the need for large study cohorts in modifier studies to achieve sufficient power.

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Assessing the Validity of a Modifier

The above strategies invoke statistical tests to determine if polymorphisms in or around a gene associate with variation in the disease presentation. As such, these tests are subject to the same problems or errors as other statistical analyses. With current advances in technology, it is now possible to screen hundreds of thousands of polymorphisms, and thus the incidence of type I errors, or false positives, is potentially quite high. One can reduce this risk of type I error by choosing stringent statistical thresholds. However, with the expectation that modifiers will often have subtle effects, application of more stringent thresholds will likely result in type II errors, and “real” associations will be overlooked. One strategy to verify a biologic basis of an association is by replication in an independent population. If the same or similar association is observed in independent study populations, it is unlikely to have happened by chance alone. We have used this approach to assess the validity of an association between variants in the gene encoding transforming growth factor b1 (TGFb1) and lung function in CF patients (8). Initially, a cohort of 808 patients was genotyped for polymorphisms in a panel of 10 candidate genes and screened for associations with lung function. Alleles of two polymorphisms in the 50 end of the TGFb1 gene, but nowhere else in that gene or the other genes tested, showed an association. A second group of 498 patients was genotyped for the TGFb1 variants and showed a similar association as the first cohort. Another example of the replication approach to validate an association has been accomplished in independent studies. Garred and colleagues (9) showed an association between survival and inactivating variants in the mannose-binding lectin-2 (MBL2) gene. Subsequent studies by other groups support that observation but suggest that the effect may be age dependent (10) and be influenced by variants in other genes (11). While replication is a key piece of evidence, biologic evidence is perhaps even more compelling. That is, for a variant in a gene to affect a phenotype, it must alter the function of the gene or its encoded product. As an example, a gene involved in neutrophil differentiation, IFRD1, has alleles found to associate with severity of CF lung disease (12). Functional studies on neutrophils isolated from individuals with different IFRD1 genotypes indicated that the ability to mount an oxidative burst (necessary for effective bacterial killing) was associated with IFRD1 genotype. These results provide a potential mechanism underlying the genetic association, as one genotype had a different capacity to combat bacterial infection than another genotype. As another example, polymorphisms in the neutrophil chemoattractant gene IL8 also associate with CF lung disease (13). One of these IL8 variants, located in the promoter of the gene, affects transcription of IL8, with one allele having about two to three times higher transcription rate than the other. The variant expressing greater levels of IL8 associates with worse pulmonary outcome, consistent with the hypothesis that increased neutrophil burden in the lung is deleterious in CF.

VIII.

Modifiers of CF Pulmonary Disease

The earliest reports of CF-modifying genes involved small cohorts of patients and examined a small number of candidate genes. As described elsewhere in this volume, the pathophysiologic cascade of airway disease in CF, at least, in part, involves bacterial infection and neutrophilic infiltrates that release elastase and thus create a proteolytic

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burden on the airway. Genes involving inflammation and innate defense against bacteria, as well as antiproteases and wound healing, were therefore logical candidates for potentially modifying the CF pulmonary disease phenotype. The earliest of these studies looked at a1-antiprotease (14) in a cohort of 215 CF subjects and suggested that bacterial colonization was earlier in individuals with genotypes that reduced antiprotease expression. However, no association was found with lung function, nor was the effect on bacterial colonization reproducible in a much larger study (15). Since then, many reports have been published indicating a number of genes have variants that associate with various aspects of CF disease presentation or severity. Rather than recount all of the studies, we will focus on those that have been carried out in large cohorts and/or have been replicated in multiple populations.

IX.

Candidate Genes Vs. Genome-wide Association Studies

As mentioned earlier, there are roughly 30,000 genes spread across the human genome and several million polymorphisms distributed within and between those genes. Such diversity has financial, logistical, and computational costs that must be considered when designing genetic association studies. Early studies focused on a small number of genes and subjects, partly for logistical reasons but largely due to costs. By their nature, these studies needed to be hypothesis-driven; candidate genes were not chosen at random, but rather because of the likely involvement of their products in CF pathophysiology. These initial candidates focused on lung disease physiology and included cytokines, or mediators of inflammation, such as tumor necrosis factor alpha (TNFa) (16), innate defense against bacterial infection (9), oxidative stress (17), wound repair (18), and airway reactivity (19). The validity of these variants as CF modifiers is uncertain, as assessment in larger cohorts has not reproduced the findings in the original reports. Additional candidate genes in these pathways were examined because they were found to associate with clinical conditions outside of CF. The hypothesis here was that the gene variation leading to functional effects in non-CF lung disease might also have an effect in the context of CF. TGFb1, IL8, and the b2-adrenergic receptor (ADRB2) were all tested in this regard. Variants in TGFb1 found to associate with severity of asthma showed a very similar association with severity of CF lung disease (8). Variants in ADRB2 associated with differential responses to bronchodilators in asthma showed a similar association in CF patients (20), but it should be noted that there was no indication that these differences had any effect on long-term pulmonary function of these patients. Alleles of the IL8 gene found to associate with resistance to respiratory syncytial virus (RSV) infection, and subsequent bronchiolitis (21) were found to have the opposite association in CF (13). That is, alleles associated with RSV resistance were associated with worse pulmonary function in CF, a finding replicated in two independent cohorts of patients (13,21). With advances in genotyping technology and development of statistical methods to handle extremely large data sets, the cost to generate these data sets has decreased and the ability to analyze them has increased. Consequently, genome-wide association studies are now commonly performed. These studies take advantage of the history of variants in human genomes, recognizing that alleles of many polymorphisms cosegregate so often that there are inherent redundancies. Consequently, it is necessary to genotype only a fraction, about one-tenth, of the known variants to obtain most of the

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relevant information. Such studies are underway for CF, and the first glimpse was reported by Gu and colleagues (12), in which they found an association between IFRD1 alleles and severity of lung disease from a scan of only a few hundred subjects for approximately 100,000 polymorphisms. Larger studies comprising several thousand CF subjects genotyped for more than 500,000 polymorphisms are currently underway as part of a consortium between groups in the United States and Canada, but the results of that work are not yet available. Such a study should shed light on the reproducibility of other modifier reports, as well as detect new associations with genes not previously tested and likely not even considered, giving us new insight into the disease.

X. Refinement of Airway Modifiers The bulk of investigations into modifiers of pulmonary disease have focused on pulmonary function as the variable phenotype to correlate with genotype. However, pulmonary function is the sum of many factors affecting the airway, such as infection, mucociliary clearance, airway inflammation, and so on. Using a composite phenotype, such as pulmonary function, allows one to survey a number of mechanisms simultaneously, but at the risk of not detecting true modifiers, because their influence is not detectable above the conglomeration of other effects at play. Consequently, modifiers of weak effect may be more readily detected if the phenotype were more specific. This would conceivably include aspects of pulmonary infection, such as age at first infection, age at colonization, species of bacteria present, etc. Microbiology is typically followed and reported in a patient’s clinical record, and thus data are available for these types of investigations. Such studies are underway and will hopefully uncover previously undetected genes involved in protecting the lungs and airways of CF patients from infection.

XI.

Modifiers of Non-airway Phenotypes

Although the complications of airway disease in CF are the primary source of mortality for this disorder, many other organ systems are also affected, and the interaction between these systems is not well understood. For example, morphometric indices such as height, weight, and BMI show a strong correlation with pulmonary function. While it is speculated that this association underlies a cause and effect relationship between nutritional status and lung function, it is equally plausible that pulmonary infection leads to impaired lung function and has cachectic effects, resulting in reduced body weight. It must also be kept in mind that other, as yet unknown or unidentified mechanisms, may explain the association. Nonetheless, investigating genes involved in metabolic pathways and those that predispose to obesity are obvious candidates to explain differences in body size and composition, and if these variants also associate with pulmonary function they may shed light on the common mechanisms between these otherwise apparently unrelated traits. As the goal of identifying modifier genes is to better understand the pathophysiology of the disease and to identify therapeutic targets, it is logical to investigate the genetics of non-airway phenotypes as well. In addition to height, weight, and BMI,

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such phenotypes include intestinal obstruction, CFRD, liver disease, and low bone mineral density, just to name a few. A small proportion (<5%) of CF patients will develop liver cirrhosis with portal hypertension, referred to CF liver disease, but this does not seem to track appreciably with other aspects of CF, suggesting the involvement of other genes or nongenetic factors. To explore the possible involvement of other genes, Bartlett and colleagues collected DNA from North American and European CF patients with liver disease and compared the genotype frequencies of candidate modifier genes with those without liver disease. They found that a mutation in a1-antitrypsin (SERPINA1), the so-called Z allele that causes the protein to be misfolded and retained in the liver, increases the risk of liver disease in CF patients by as much as five times (22). Intestinal obstructions (MI) occur in about 15% to 20% of CF neonates, or later in life, as distal intestinal obstruction syndrome (DIOS), and these anomalies have also been investigated for genetic risk factors. Twin/sibling studies indicate MI is heritable and that a small number of genes are involved, while the genetic contribution to obstruction later in life is quite low. The genes contributing to MI have not been identified, but early reports using a linkage approach suggested that a gene on chromosome 19 plays a role (23). A subsequent linkage study was not able to replicate the chromosome 19 finding, but instead found some evidence for MI genes on chromosomes 4, 8, 11, 20, and 21 (7). Once candidate genes are identified in the areas of linkage, the validity of these modifiers can be more thoroughly assessed. Like intestinal obstruction, CFRD is quite common, and up to 50% of CF patients will develop CFRD in their lifetime. Blackman and colleagues found that CFRD has a familial component, and that a family history of type-2 diabetes increases the risk for a person with CF developing CFRD. Consequently, the hypothesis that CFRD may share genetic risk factors with type-2 diabetes was explored. A gene, TCF7L2, encoding a transcription factor of pancreatic islet cells, has alleles that are risk factors for type-2 diabetes in the non-CF population. These same alleles increase the risk of developing CFRD nearly threefold with onset about seven years earlier in life (24). These results provide a novel way of thinking about CFRD, as historically it has been perceived as an entity distinct from type-1 and type-2 diabetes. These results suggest that the similarities to type-2 diabetes may have been overlooked and that the CF population is substantially more sensitive to diabetes-causing factors than non-CF individuals. In addition to the traits discussed above, there is increasing evidence that loss of CFTR affects many tissues, some directly and some indirectly, and which were not previously thought to be of much consequence to the disease. Reports that CFTR is expressed in immune cells, such as lymphocytes (25–27), macrophages (28), and neutrophils (29), predict that the immune system may have an intrinsic or primary inability to appropriately resolve pulmonary infections in CF. Similarly, CFTR appears to have a functional role in smooth muscle (30–32), and thus loss of CFTR from smooth muscle– rich tissues may contribute to gastrointestinal transit and obstruction. Neuroendocrine processes are also aberrant in CF, most of which are described for growth and sexual development. Recent evidence shows CFTR expression in brain, particularly in the hypothalamus (33–35) and provides potential explanations for suppressed endocrine signaling. Each of these affected tissues and systems provides phenotypes for modification, and as modifier studies begin to delve into additional clinical manifestations, the genes that contribute to variation will likely reveal themselves.

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Summary

An emerging picture is that CF is complicated by disorders found in the general population, including type-2 diabetes, asthma, liver cirrhosis, just to name a few. For some of these conditions, people with CF appear to have elevated susceptibility or sensitivity to the factors causing these disorders, as is apparent for diabetes where a third to a half of CF patients will develop CFRD. In others, there is a striking similarity between the CF and non-CF population. For example, the genotype frequencies of TGFb1 alleles between emphysema patients (COPD) and those without COPD (36) are strikingly similar to those of CF patients with severe and mild lung disease, respectively (8). Some of the overlaps suggested by the genetic data are diagrammed in Figure 2. This model has several important implications. First is that insight is being gained into disease pathophysiology and accordingly new potential therapeutic targets are becoming apparent. These targets not only include the products of the genes identified, but also the pathways and processes that they affect. The second and perhaps more important implication is that these processes are not specific to CF and that therapeutic approaches, which result as a consequence of these studies, will likely have utility for other diseases, many of which are common in the population.

Figure 2 Conceptual diagram of overlap and relationship between CF modifiers and the etiology of other disorders. Many of the clinical manifestations of CF are disorders themselves, outside of CF. Genetics suggest these phenotypes in CF and non-CF individuals are related, at least in some cases. Alleles of the gene TCF7L2 predisposes both CF and non-CF individuals to type-2 diabetes, but with much greater effect in CF. Similar overlaps are emerging with CF and other disorders, such as pulmonary and airway disorders, gastrointestinal problems, and innate defense to infection. As there are metabolic disturbances in CF, it is anticipated that genes will also be identified that modify CF in some way but also influence metabolic disorders outside of CF, such as obesity, malnutrition, and even low bone mineral density.

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References 1. Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science 1989; 245(4922):1073–1080. 2. Kerem E, Corey M, Kerem BS, et al. The relation between genotype and phenotype in cystic fibrosis–analysis of the most common mutation (delta F508). N Engl J Med 1990; 323(22): 1517–1522. 3. Correlation between genotype and phenotype in patients with cystic fibrosis. The Cystic Fibrosis Genotype-Phenotype Consortium [see comments]. N Engl J Med 1993; 329(18): 1308–1313. 4. Kristidis P, Bozon D, Corey M, et al. Genetic determination of exocrine pancreatic function in cystic fibrosis. Am J Hum Genet 1992; 50(6):1178–1184. 5. Mekus N, Ballmann M, Bronsveld I, et al. Categories of deltaF508 homozygous cystic fibrosis twin and sibling pairs with distinct phenotypic characteristics. Twin Res 2000; 3(4): 277–293. 6. Vanscoy LL, Blackman SM, Collaco JM, et al. Heritability of lung disease severity in cystic fibrosis. Am J Respir Crit Care Med 2007; 175(10):1036–1043. 7. Blackman SM, Deering-Brose R, McWilliams R, et al. Relative contribution of genetic and nongenetic modifiers to intestinal obstruction in cystic fibrosis. Gastroenterology 2006; 131(4): 1030–1039. 8. Drumm ML, Konstan MW, Schluchter MD, et al. Genetic modifiers of lung disease in cystic fibrosis. N Engl J Med 2005; 353(14):1443–1453. 9. Garred P, Pressler T, Madsen HO, et al. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis [see comments]. J Clin Invest 1999; 104(4):431–437. 10. Davies JC, Turner MW, Klein N. Impaired pulmonary status in cystic fibrosis adults with two mutated MBL-2 alleles. Eur Respir J 2004; 24(5):798–804. 11. Dorfman R, Sandford A, Taylor C, et al. Complex two-gene modulation of lung disease severity in children with cystic fibrosis. J Clin Invest 2008; 118(3):1040–1049. 12. Gu Y, Harley IT, Henderson LB, et al. Identification of IFRD1 as a modifier gene for cystic fibrosis lung disease. Nature 2009; 458(7241):1039–1042. 13. Hillian AD, Londono D, Dunn JM, et al. Modulation of cystic fibrosis lung disease by variants in interleukin-8. Genes Immun 2008; 9(6):501–508. 14. Doring G, Krogh Johansen H, Weidinger S, et al. Allotypes of alpha 1-antitrypsin in patients with cystic fibrosis, homozygous and heterozygous for deltaF508. Pediatr Pulmonol 1994; 18(1):3–7. 15. Frangolias DD, Ruan J, Wilcox PJ, et al. Alpha 1-antitrypsin deficiency alleles in cystic fibrosis lung disease. Am J Respir Cell Mol Biol 2003; 29(3 pt 1):390–396. 16. Hull J, Thomson AH. Contribution of genetic factors other than CFTR to disease severity in cystic fibrosis. Thorax 1998; 53(12):1018–1021. 17. Baranov VS, Ivaschenko T, Bakay B, et al. Proportion of the GSTM1 0/0 genotype in some Slavic populations and its correlation with cystic fibrosis and some multifactorial diseases. Hum Genet 1996; 97(4):516–520. 18. Arkwright PD, Laurie S, Super M, et al. TGF-beta(1) genotype and accelerated decline in lung function of patients with cystic fibrosis [see comments]. Thorax 2000; 55(6):459–462. 19. Grasemann H, Knauer N, Buscher R, et al. Airway nitric oxide levels in cystic fibrosis patients are related to a polymorphism in the neuronal nitric oxide synthase gene. Am J Respir Crit Care Med 2000; 162(6):2172–2176. 20. Hart MA, Konstan MW, Darrah RJ, et al. Beta 2 adrenergic receptor polymorphisms in cystic fibrosis. Pediatr Pulmonol 2005; 39(6):544–550. 21. Hull J, Ackerman H, Isles K, et al. Unusual haplotypic structure of IL8, a susceptibility locus for a common respiratory virus. Am J Hum Genet 2001; 69(2):413–419.

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22. Bartlett J. Genetic modifiers of liver disease in cystic fibrosis. JAMA 2009; 302(10): 1076–1083. 23. Zielenski J, Corey M, Rozmahel R, et al. Detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13 [letter]. Nat Genet 1999; 22(2):128–129. 24. Blackman SM, Hsu S, Ritter SE, et al. A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis. Diabetologia 2009; 52(9): 1858–1865. 25. Krauss RD, Bubien JK, Drumm ML, et al. Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle. EMBO J 1992; 11(3):875–883. 26. McDonald TV, Nghiem PT, Gardner P, et al. Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current. J Biol Chem 1992; 267(5):3242–3248. 27. Yoshimura K, Nakamura H, Trapnell BC, et al. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acids Res 1991; 19(19):5417–5423. 28. Di A, Brown ME, Deriy LV, et al. CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 2006; 8(9):933–944. 29. Painter RG, Valentine VG, Lanson NA Jr., et al. CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis. Biochemistry 2006; 45(34): 10260–10269. 30. Warth JD, Collier ML, Hart P, et al. CFTR chloride channels in human and simian heart. Cardiovasc Res 1996; 31(4):615–624. 31. Robert R, Norez C, Becq F. Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cl- transport of mouse aortic smooth muscle cells. J Physiol 2005; 568(pt 2):483–495. 32. Robert R, Savineau JP, Norez C, et al. Expression and function of cystic fibrosis transmembrane conductance regulator in rat intrapulmonary arteries. Eur Respir J 2007; 30(5): 857–864. 33. Mulberg AE, Weyler RT, Altschuler SM, et al. Cystic fibrosis transmembrane conductance regulator expression in human hypothalamus. Neuroreport 1998; 9(1):141–144. 34. Mulberg AE, Wiedner EB, Bao X, et al. Cystic fibrosis transmembrane conductance regulator protein expression in brain. Neuroreport 1994; 5(13):1684–1688. 35. Weyler RT, Yurko-Mauro KA, Rubenstein R, et al. CFTR is functionally active in GnRHexpressing GT1-7 hypothalamic neurons. Am J Physiol 1999; 277(3 pt 1):C563–C571. 36. Celedon JC, Lange C, Raby BA, et al. The transforming growth factor-beta1 (TGFB1) gene is associated with chronic obstructive pulmonary disease (COPD). Hum Mol Genet 2004; 13(15):1649–1656.

7 Cystic Fibrosis: Diagnosis, Sweat Testing, and Newborn Screening BRIAN P. O’SULLIVAN University of Massachusetts Medical School and UMass Memorial Health Care, Worcester, Massachusetts, U.S.A.

I.

Diagnosis

Making the diagnosis of cystic fibrosis (CF) has lifelong implications for affected individuals and their families, for both medical and insurance reasons. Therefore, it is imperative that the clinician approach this problem with sensitivity and seriousness of purpose. The diagnosis should be considered in any child or adult who presents with the signs or symptoms listed in Table 1. Classical CF presents in childhood with the combination of poor growth and respiratory symptoms. A variety of other signs and symptoms may develop over time (Fig. 1) (1). Approximately 15% of infants with CF are born with meconium ileus, an obstructive phenomenon secondary to inspissated material in the small and large bowels. Although other causes of intestinal obstruction are sometimes mislabeled as meconium ileus, CF is essentially the only cause of true meconium ileus associated with microcolon. Eighty-five to ninety percent of infants with CF develop pancreatic insufficiency (PI), which may be present at birth or evolve over the first year of life (2). Typical signs of PI are greasy stools, flatulence, abdominal bloating, and poor weight gain. PI leads to steatorrhea, fat-soluble vitamin deficiency, and malnutrition. Thus, any child being evaluated for failure to thrive should have a sweat test to rule out CF. Because of associated vitamin deficiencies, children with CF can also present with retinal problems, anemia, idiopathic increased intracranial pressure, or acrodermatitis. Respiratory symptoms can be present very early in life or not appear until much later. Initial symptoms may be wheezing secondary to bacterial infection of small airways (bacterial bronchiolitis), cough, and sputum production. Digital clubbing is the result of chronic, progressive disease and can be seen in older children and adults in whom the diagnosis has been missed. Recurrent pneumonia with the recovery of typical CF respiratory pathogens from sputum or throat cultures should increase the suspicion of CF. Pseudomonas aeruginosa (PA) is occasionally recovered from infants without CF and is a common cause of ventilator-associated pneumonia in older children and adults (3,4); however, the mucoid strain of PA is almost exclusively found in CF patients. Thus, the recovery of mucoid PA in secretions from a child or adult with respiratory symptoms is virtually pathognomonic for CF. Non-CF infants may have Staphylococcus aureus or gram-negative organisms in their oropharynx (3), but the presence of these

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Table 1 Signs and Symptoms of Cystic Fibrosis

General (any age) Family history of CF Salty-tasting skin Clubbing of the digits Chronic cough with sputum production Chronic/recurrent wheezing Mucoid Pseudomonas aeruginosa isolated from airway secretions Hyponatremic, hypochloremic metabolic alkalosis Neonatal Meconium ileus Prolonged jaundice (direct or conjugated hyperbilirubinemia) Abdominal/scrotal calcifications Intestinal atresia Infancy Persistent or recurrent opacities on chest roentgenogram Failure to thrive Anasarca/hypoproteinemia Chronic diarrhea Abdominal distention Cholestasis Staphylococcus aureus pneumonia Vitamin A deficiency (pseudotumor cerebri, retinal changes) Vitamin E deficiency (anemia) Childhood Chronic pansinusitis/nasal polyposis Steatorrhea Rectal prolapse Distal intestinal obstruction syndrome/intussusception Idiopathic recurrent or chronic pancreatitis Liver disease Adolescence and Adulthood Allergic bronchopulmonary aspergillosis Chronic pansinusitis/nasal polyposis Bronchiectasis Hemoptysis Idiopathic/recurrent pancreatitis Portal hypertension Delayed puberty Azoospermia secondary to congenital bilateral absence of the vas deferens

organisms in a throat culture from a symptomatic infant increases the likelihood that the child has CF. Metabolic disturbances, particularly dehydration with hyponatremic, hypochloremic metabolic alkalosis, can result from excess salt and water losses from sweat glands. This problem is most common in the summer months and was the impetus for development of the sweat test (5,6). Children and adults with milder forms of the disease

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Figure 1 Time frame of appearance of symptoms of CF. Abbreviations: ABPA, allergic bron-

chopulmonary aspergillosis; CBAVD, congenital bilateral absence of the vas deferens; CFRD, cystic fibrosis–related diabetes mellitus; DIOS, distal intestinal obstruction syndrome; HPOA, hypertrophic osteoarthritis.

can present with nasal polyposis, chronic pancreatitis, difficult to control “asthma,” and congenital bilateral absence of the vas deferens (CBAVD) (1). The median age of diagnosis in children with classical CF is approximately six months. Nonclassical forms of the disease can present with milder symptoms and their diagnosis can be delayed. Five to ten percent of individuals who likely have CF will present with atypical symptoms and indeterminate diagnostic test results. These individuals may not come to clinical attention until adulthood (see chap. 8). With the increasingly widespread implementation of newborn screening for CF, it is likely that in the near future nonclassical CF will be the most common form of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction diagnosed outside of the newborn period. However, given that newborn testing is a screening tool only (with an inherent false-negative rate) and is not yet universally available, clinicians must remain aware of the possibility of presentation of classical CF in older children and adults who have respiratory, gastrointestinal, metabolic, and reproductive problems (7). Diagnostic algorithms for classical and nonclassical CF have been published by the European Union CF Diagnostic Working Group and the U.S. Cystic Fibrosis Foundation (Figs. 2 and 3) (7,8). These documents concur that the diagnosis of CF consists of finding specific clinical (phenotypic) characteristics in combination with biochemical or genetic markers of CFTR dysfunction. In general, a diagnosis of CF can be made in a patient with phenotypic features of CF if the sweat chloride is either greater than 60 mmol/L or in the intermediate range and two disease-causing CFTR mutations

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Figure 2 Diagnostic algorithm proposed by the European Union Diagnostic Working Group. Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; FU, follow-up; PD, potential difference; WHO, World Health Organization. Source: From Ref. 8.

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Figure 3 Diagnostic algorithm for CF based on newborn screening. Abbreviations: CF, cystic

fibrosis; CFTR cystic fibrosis transmembrane conductance regulator; IRT, immunoreactive trypsinogen, PCP, primary care provider. Source: From Ref. 7.

are identified (7). Diagnostic dilemmas occur when sweat chloride values are in the intermediate range and only one disease-causing mutation is identified. In such cases, ancillary tests of CFTR function such as nasal potential difference (NPD) measurement may prove helpful.

II.

Sweat Testing

During a heat wave in New York City in 1948, Paul di Sant’Agnese noticed that several infants known to have CF presented with heat prostration. This led him to hypothesize that sweat salt and water loses were abnormal in children with CF. He went on to demonstrate elevated sweat electrolyte concentrations in children with CF (5). It was quickly recognized that measurement of sweat chloride could be used as a diagnostic tool for CF, and a standardized practical process of sweat induction using pilocarpine iontophoresis was developed by Gibson and Cooke (9). Fifty years later, the sweat chloride test remains the most readily available and clinically useful way of making the

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diagnosis of CF, provided it is performed according to strict guidelines (10,11). Specifically, sweat should be collected from the flexor surface of the forearm, although the thighs may be used in very young infants or where the arms are eczematous or otherwise unsuitable. Sweat production should be stimulated using USP grade pilocarpine or a disposable Pilogel1 electrode delivered via iontophoresis for five minutes with a current of 3.0 to 4.0 mA. Skin burns can occur if a current greater than 4 mA is used (12). The iontophoresis current source should be battery operated for safety reasons and the current should never cross the heart. Two-by-two inch filter paper or gauze or a Macroduct1 coil (Wescor, Logan, Utah, U.S.) should be used to collect the sweat. The collection period must not exceed 30 minutes and a minimum sweat collection is 75 mg for filter paper or gauze or 15 mL for the Macroduct coil. Adherence to time and amount of sweat collected is crucial as too prolonged a collection or too low an amount may lead to erroneous results. Most CF centers collect samples from two limbs at the same time to provide internal control and to assure that at least one adequate sample is obtained. Samples from two sites should not be combined to achieve an adequate amount of sweat. Once collected, the sweat should be analyzed using a quantitative test of chloride concentration using coulometric titration with a chloridometer, the mercuric nitrate procedure of Schales and Schales, or an automated chloride analyzer that has been validated against one of the other methods (11). In one study, conductivity correlated well with chloride concentration, but this test has too great a rate of false-positive results to be recommended for ultimate determination of the diagnosis of CF (13). Measures of conductivity or application of a chloride electrode directly to a patient’s skin are not considered accurate enough for use in diagnosis of CF. Similarly, measurement of sweat osmolality, potassium, or sodium without chloride determination are not reliable methods of diagnosing CF (11). Sweat testing is feasible in even very young infants, but there is an increased rate of insufficient samples when done in very small or very young infants. Generally, one can obtain good samples with children greater than two weeks of age. Insufficient sweat quantities are more likely in African-American infants, infants weighing less than 2 kg, and premature infants less than 36 weeks postconceptual age (14). Care must be taken in interpreting the results of the sweat test. A sweat chloride concentration greater than 60 mmol/L (60 mEq/L) is diagnostic of CF in all age groups (7,8,15). Both the U.S. and EU consensus statements make 29 mmol/L the upper limit of normal for a sweat chloride in an infant less than six months of age. In these children, 30 to 59 mmol/L is considered a borderline result and a sweat chloride concentration greater than 60 mmol/L is consistent with the diagnosis of CF. In older children and adults, the upper limit of normal is generally considered to be 39 mmol/L, with 40 to 59 mmol/L considered borderline and greater than 60 mmol/L diagnostic of CF. Recent data, however, have shown that these reference ranges cannot be universally applied to patients of all ages (15). Normal sweat values in people with no known CF mutations increase with age, with the upper limit of normal approaching 59 mmol/L in patients over 20 years of age. Thus, adults with a sweat chloride value between 40 and 60 mmol/L may have normal CFTR function and remain unaffected. The different normative values for very young infants are based on results of sweat chloride tests from several large cohorts of patients with no or only one known CF mutation (14,16–18). Using a value of 29 mmol/L as the upper limit of normal has been challenged by one group, who demonstrated that children with one known CF mutation

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Table 2 Conditions Associated with a False-Positive or False-Negative Sweat Test

False positive

False negative

Atopic dermatitis (eczema) Malnutrition Congenital adrenal hyperplasia Mauriac syndrome Fucosidosis Ectodermal dysplasia Klinefelter syndrome Nephrogenic diabetes insipidus Adrenal insufficiency Hypothyroidism Autonomic dysfunction Environmental deprivation Munchausen by proxy

Dilution of sample Malnutrition Peripheral edema Low sweat rate (quantity not sufficient) Hypoproteinemia Dehydration CFTR mutations with preserved sweat duct function (e.g., 3849 þ 10 kb C>T; R117H-7T)

Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator.

and a shortened thymidine track in intron 8 of the CFTR gene on the other allele could have sweat chloride values in the 20 to 30 mmol/L range (19). These authors suggested lowering the upper limit of normal for sweat test results in newborns to 23 mmol/L. However, one must balance the desire to not miss a truly affected child against the angst and insurance issues raised by telling a family that their child has CF when, in fact, he or she may never develop symptoms. It seems most reasonable to keep the upper limit of normal 29 mmol/L in infants (20). Changes in sweat chloride values over the first year of life have been reported. Therefore, in a child for whom the diagnosis is suspected but the initial sweat chloride is in the borderline range, repeating the sweat test a few months later may clarify the situation. There are a limited number of causes of false-negative or false-positive sweat tests and most of these will be clinically apparent (Table 2). Diagnosis of CF can be aided by performing CFTR mutation analysis. A number of analytical methods for CFTR mutation detection are commercially available. In general, use of a discrete panel of mutations is faster and less costly than full gene sequencing. Incorporation of the 23 mutations demonstrated to cause sufficient loss of CFTR function to confer CF disease will detect 85% of affected individuals in most populations (Table 3) (7,21). A concern with limited analysis is that the diagnosis will be Table 3 The Most Common CFTR Mutations Demonstrated to Cause Sufficient Loss of

Function to Confer CF Disease Missense, deletion, and stop mutations

Splicing and frameshift mutations

G85E R117H R334W R347P A455E

621 þ 1G>T 711 þ 1G>T 1717  1G>A 1898 þ 1G>A 2184delA

I507del F508del G542X G551D R553X

R560T R1162X W1282X N1303K

2789 þ 5G > A 3120 þ 1G > A 3659delC 3849 þ 10kbC > T

Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator.

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missed if the subject is affected by a mutation that is not included on the panel in use. For this reason, regional mutation panels are often modified to include particular mutations unique to the population being tested. Full sequence analysis will detect virtually all CFTR mutations; however, it raises the specter of discovering polymorphisms and novel mutations whose significance is not known. Furthermore, such sequencing only includes the exons and flanking intronic sequences, meaning that deep intronic mutations will be missed. A helpful tool in assessing subjects who may have CF but who do not meet classical diagnostic criteria is measurement of transepithelial NPD (7,8). NPD is performed by determining the electrical potential difference across the nasal epithelium compared with a subcutaneous reference. This potential difference is generated by the transport of ions across the respiratory epithelium and is abnormal in subjects lacking functional CFTR protein. Bathing the nasal mucosa in various solutions while recording potential differences allows for estimation of the change in current attributable to epithelial chloride and sodium channels. At baseline, CF patients have a more negative potential difference than non-CF controls owing to enhanced sodium reabsorption (22). Perfusion of the nose with the epithelial sodium channel blocking agent amiloride causes a greater change in potential difference (less negative) in CF patients than is seen in nonCF subjects. Application of a chloride-free solution causes a negative deflection of the potential difference in a subject without CF, but causes no such change in a CF patient. Finally, a CF patient will have little or no response to perfusion of the nose with isoproterenol, a drug that stimulates CFTR function, whereas a non-CF subject will have a large negative change in potential difference with this agent (22). NPD can be quite useful for confirming or negating the presence of a CFTR abnormality in a patient with uncertain symptoms and a borderline sweat test. Unfortunately, NPD is labor intensive, technically difficult, and is not available at all CF centers.

III.

Newborn Screening

In the early 1990s, only a handful of regions worldwide were performing newborn screening for CF. By the end of the first decade of this century, newborn screening has become commonplace, with almost all states in the United States and many EU countries performing this test. Children diagnosed via newborn screening have improved growth, reduced therapeutic burden, and reduced morbidity when compared with children diagnosed later in life due to clinical symptoms (23–25). Additionally, two U.K. CF Trust registry–based studies have shown both the clinical benefit and cost-effectiveness of newborn screening (23,26). Thus, the benefits of newborn screening for CF are no longer in doubt (27), and the Centers for Disease Control and Prevention has supported adoption of CF newborn screening throughout the United States (28). In regions performing CF newborn screening, it has become highly unusual to see patients present with symptoms of classical CF, like chronic or recurrent respiratory disease and emaciation. Similarly, problems related to electrolyte abnormalities or occult vitamin deficiency due to undiagnosed CF have become almost nonexistent. Since children identified by newborn screening are diagnosed when asymptomatic or with only mild growth retardation, it is possible to help them maintain or recover normal somatic growth. This is crucial because registry studies have shown an association between better preschool nutritional status and higher pulmonary function test results at

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school age (29,30). Indeed, multiple studies have shown that newborn screening for CF leads to improved nutritional outcomes (31,32), and screened infants have better weight gain than do malnourished nonscreened infants with CF (33). Furthermore, recovery of weight z-scores within two years of diagnosis in both screened and nonscreened infants is associated with improved pulmonary status at six years of age (34). Therefore, it is presumed that the improved nutritional status in infancy as a result of newborn screening will lead to improved pulmonary function later in life and, in fact, there are data supporting this hypothesis (35,36). Implementation of CF newborn screening takes considerable foreplanning with cooperation being vital between the newborn screening laboratory, regional CF centers, geneticists, and parents (37,38). Newborn screening is performed by measuring immunoreactive trypsinogen (IRT) in blood spots taken from newborn infants. A very high IRT implies pancreatic injury consistent with (but not specific for) CF. This marker is elevated even in infants with CFTR mutations associated with pancreatic sufficiency. Those infants who have an elevated IRT on initial testing then have further evaluation via a repeat IRT one to three weeks later (IRT/IRT) or by analysis of the initial blood spot for a specified panel of CFTR mutations (IRT/DNA) (39,40). Most screening programs identify infants in the top 5% of values each day for further IRT or DNA analysis. Because of lot-to-lot and seasonal variation in IRT results, it is best to designate a percentage of IRT levels each day as being positive (for example, the top 5%) rather than using a static absolute cutoff value (41). Use of a higher cutoff value (e.g., 2–3% of the highest daily values) will decrease both the number of falsepositive screening tests and the number of unaffected carriers detected. This advantage must be weighed against a loss of sensitivity and an increased chance that an infant with CF will be missed. Of note, the positive predictive value for an elevated IRT is lower in African-American children than in European-American children (42). The reason for higher IRT values in non-CF affected African-American children is unclear, but must be taken into account when initiating newborn screening programs in areas with a high density of families of African origin. Low-birth-weight infants also have higher IRT values and retesting of premature infants who have an initially elevated IRT is appropriate. An advantage of the IRT/IRT method of screening is that it avoids the problems associated with detecting mutations of uncertain clinical significance and of having to counsel families in regard to carrier status or nonpaternity. Unfortunately, the IRT/IRT algorithm requires obtaining a second blood spot one to three weeks after the first one, which may be a major logistical problem in some populations. In contrast, the IRT/DNA method allows complete testing using the initial blood specimen only. Furthermore, IRT/IRT testing has a lower sensitivity (80.2%) than IRT/DNA screening (96.2%) (41) (keeping in mind that the sensitivity of the IRT/ DNA method depend on the number of mutations included in the DNA panel). For regions instituting DNA-based newborn screening, the mutations included on the DNA panel should reflect the frequency of specific CF mutations in the local population (21,40). Again, a balance must be struck between sensitivity and specificity; including too many mutations in the panel can result in identification of more carriers or of children with CFTR dysfunction of questionable clinical significance. Inclusion of only a few of the most common mutations can lead to missing children who truly have CF, especially those in minority populations.

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Not surprisingly, there is much controversy about which mutations should be included on newborn screening panels. Some advocate for only including mutations that cause known, serious dysfunction in the CFTR protein. Others recommend more complete screening with even full CFTR sequence analysis proposed for regions with extremely diverse populations. Perhaps most hotly debated is inclusion of the R117H mutation on screening panels. This mutation has variable expression, in part determined by its association with the polythymidine tract in intron 8. When found in association with a five polythymidine tract (5T), the R117H mutation can cause CF symptoms; when associated with the 7T tract, symptoms may be limited, not appear until adulthood, or be absent. Scotet et al. (43) reported nine infants with the R117H-7T variant in combination with a severe mutation on the other allele. None of these children had developed symptoms of CF by a mean age of seven years. Given the relatively high frequency of the R117H allele, the uncertainty that it is a disease-causing mutation and the difficulty of counseling families about what may occur in the future, the authors recommended against including it in newborn screening panels. In contrast, Lording et al. (44) and O’Sullivan et al. (45) have described compound heterozygote children with a severe CF mutation on one allele and the R117H-7T variant on the other who developed respiratory symptoms and became infected with PA early in life. On the basis of this information, it seems imprudent to ignore the R117H mutation; however, care should be taken when discussing its significance with parents, as the clinical outcome of a child with this mutation is uncertain (46). A positive newborn screening result by either IRT/IRT or IRT/DNA coupled with identification of only one known mutation indicates that a child is at increased risk for CF, not that the child has the disease. Unfortunately, many families and some primary care providers mistakenly believe that a report of a positive newborn screen indicates that the child has CF. A sweat test or expanded mutation analysis must be done to confirm the diagnosis. Identification of two mutations known to be CF disease-causing using the IRT/DNA method is tantamount to a diagnosis of CF; however, there is a minute chance that two different mutations might be inherited in cis from the same parent. Thus, even these children should have a sweat test performed to confirm the diagnosis. Genetic counseling is an integral part of a newborn screening program. Once a child is identified as having CF, the parents are automatically determined to be carriers and siblings and other relatives are at risk for being affected with CF or being carriers of one mutated allele. Although most CF care providers feel comfortable discussing the basics of CF genetics, it is best to involve a trained genetic counselor to advise the family as to their options for further testing (47). It is most appropriate to test young siblings with a sweat test rather than mutation analysis since the decision as to whether or not they want to know their carrier status is best left to them when they reach the age of majority. Siblings who have a borderline sweat test can have the test repeated or, provided the proband’s genetics are known, can have mutation analysis performed to definitively rule in or out the diagnosis of CF. Parents of a child who has a positive IRT and one CF mutation identified and who has a negative sweat test (i.e., a child who is a carrier) must be educated that they may each be carriers since 50% of infants born to two carriers will be unaffected carriers. The genetic counselor can help these parents arrange to get genetic testing if they so desire. Obviously, this is an area where the IRT/IRT newborn screening method provides less information but also creates fewer complications.

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IV.

Conclusion

CF, once considered easy to diagnose—malabsorption, respiratory disease, and a positive sweat test sealed the diagnosis—is now recognized as a multifaceted metabolic problem with many permutations. Diagnosis involves multiple layers of testing with uncertainty possible at all levels including clinical symptoms, sweat chloride values, and genetic testing. Still, CF remains a clinical disease and diagnosis must be based on the presence of clinical symptoms with supporting laboratory values. These supporting tests (newborn screening, sweat testing, mutation analysis, and NPD) must be performed appropriately and interpreted cautiously. The sweat chloride test remains the gold standard for CF diagnosis but does not always give a clear answer. Algorithms are available to the clinician to help determine if a patient has classical CF, atypical (nonclassical) CF, or does not have CF at all.

References 1. O’Sullivan BP, Freedman SD. Cystic fibrosis. Lancet 2009; 373:1891–1904. 2. Borowitz D, Baker SS, Duffy L, et al. Use of fecal elastase-1 to classify pancreatic status in patients with cystic fibrosis. J Pediatr 2004; 145:322–326. 3. Carlson D, McKeen E, Mitchell M, et al. Oropharyngeal flora in healthy infants: observations and implications for cystic fibrosis care. Pediatr Pulmonol 2009; 44:497–502. 4. El Solh AA, Akinnusi ME, Wiener-Kronish JP, et al. Persistent Infection with Pseudomonas aeruginosa in ventilator-associated pneumonia. Am J Respir Crit Care Med 2008; 178:513–519. 5. di Sant’Agnese PA, Darling RC, Perera GA, et al. Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas. Pediatrics 1953; 12:549–563. 6. Bates CM, Baum M, Quigley R. Cystic fibrosis presenting with hypokalemia and metabolic alkalosis in a previously healthy adolescent. J Am Soc Nephrol 1997; 8:352–356. 7. Farrell PM, Rosenstein BJ, White TB, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation Consensus Report. J Pediatr 2008; 153:S4–S14. 8. De Boeck K, Wilschanski M, Castellani C, et al. Cystic fibrosis: terminology and diagnostic algorithms. Thorax 2006; 61:627–635. 9. Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat of cystic fibrosis of the pancreas utilizing pilocarpine iontophoresis. Pediatrics 1959; 23:545–549. 10. Green A, Kirk J. Guidelines for the performance of the sweat test for the diagnosis of cystic fibrosis. Ann Clin Biochem 2007; 44:25–34. 11. LeGrys VA, Yankaskas JR, Quittell LM, et al. Diagnostic sweat testing: the Cystic Fibrosis Foundation guidelines. J Pediatr 2007; 151(1):85–89. 12. LeGrys VA. Sweat testing for the diagnosis of cystic fibrosis: practical considerations. J Pediatr 1996; 129:892–897. 13. Lezana JL, Vargas MH, Karam-Bechara J, et al. Sweat conductivity and chloride titration for cystic fibrosis diagnosis in 3834 subjects. J Cyst Fibros 2003; 2:1–7. 14. Eng W, LeGrys VA, Schechter MS, et al. Sweat-testing in preterm and full-term infants less than 6 weeks of age. Pediatr Pulmonol 2005; 40:64–67. 15. Mishra A, Greaves RF, Carlin J, et al. The diagnosis of cystic fibrosis by sweat test: age-specific reference intervals. J Pediatr 2008; 153:758–763. 16. Farrell PM, Koscik RE. Sweat chloride concentrations in infants homozygous or heterozygous for dF508 cystic fibrosis. Pediatrics 1996; 97:524–528. 17. Massie J, Gaskin K, Van Asperen P, et al. Sweat testing following newborn screening for cystic fibrosis. Pediatr Pulmonol 2000; 29:452–456.

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18. Parad RB, Comeau AM, Dorkin HL, et al. Sweat testing infants detected by cystic fibrosis newborn screening. J Pediatr 2005; 147:S69–S72. 19. Soultan ZN, Foster MM, Newman NB, et al. Sweat chloride testing in infants identified as heterozygote carriers by newborn screening. J Pediatr 2008; 153:857–859. 20. O’Sullivan BP, Zwerdling RG. By the sweat of our brows: how salty should a person be? J Pediatr 2008; 153:735–736. 21. Watson MS, Cutting GR, Desnick RJ, et al. Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med 2004; 6:387–391. 22. Wallis C. Diagnosis of cystic fibrosis. In: Hodson M, Geddes DM, Bush A, eds. Cystic Fibrosis. 3rd ed. London: Edward Arnold, Ltd, 2007:99–108. 23. Sims EJ, Mugford M, Clark A, et al. Economic implications of newborn screening for cystic fibrosis: a cost of illness retrospective cohort study. Lancet 2007; 369:1187–1195. 24. Siret D, Bretaudeau G, Branger B, et al. Comparing the clinical evolution of cystic fibrosis screened neonatally to that of cystic fibrosis diagnosed from clinical symptoms: a 10-year retrospective study in a French region (Brittany). Pediatr Pulmonol 2003; 35:342–349. 25. Accurso FJ, Sontag MK, Wagener JS. Complications associated with symptomatic diagnosis in infants with cystic fibrosis. J Pediatr 2005; 147:S37–S41. 26. Sims EJ, McCormick J, Mehta G, et al. Neonatal screening for cystic fibrosis is beneficial even in the context of modern treatment. J Pediatr 2005; 147(suppl 1):S42–S46. 27. Farrell PM. Cystic fibrosis newborn screening: shifting the key question from “should we screen?” to “how should we screen?” Pediatrics 2004; 113(6):1811–1812. 28. Grosse SD, Boyle CA, Botkin JR, et al. Newborn screening for cystic fibrosis: evaluation of benefits and risks and recommendations for state newborn screening programs. MMWR Recomm Rep 2004; 53:1–36. 29. Konstan MW, Butler SM, Wohl MEB, et al. Growth and nutritional indexes in early life predict pulmonary function in cystic fibrosis. J Pediatr 2003; 142(6):624–630. 30. Padman R, McColley SA, Miller DP, et al. Infant care patterns at epidemiologic study of cystic fibrosis sites that achieve superior childhood lung function. Pediatrics 2007; 119(3): E531–E537. 31. Farrell PM, Kosorok MR, Laxova A, et al. Nutritional benefits of neonatal screening for cystic fibrosis. Wisconsin Cystic Fibrosis Neonatal Screening Study Group. N Engl J Med 1997; 337(14):963–969. 32. Farrell PM, Kosorok MR, Rock MJ, et al. Early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition and improves long-term growth. Pediatrics 2001; 107:1–13. 33. Shoff SM, Ahn H-Y, Davis L, et al. Temporal associations among energy intake, plasma linoleic acid, and growth improvement in response to treatment initiation after diagnosis of cystic fibrosis. Pediatrics 2006; 117:391–400. 34. Lai HJ, Shoff SM, Farrell PM. Recovery of birth weight z score within 2 years of diagnosis is positively associated with pulmonary status at 6 years of age in children with cystic fibrosis. Pediatrics 2009; 123:714–722. 35. McKay KO, Waters DL, Gaskin KJ. The influence of newborn screening for cystic fibrosis on pulmonary outcomes in New South Wales. J Pediatr 2005; 147:S47–S50. 36. Collins MS, Abbott M-A, Wakefield DB, et al. Improved pulmonary and growth outcomes in cystic fibrosis by newborn screening. Pediatr Pulmonol 2008; 43:648–655. 37. Comeau AM, Parad R, Gerstle R, et al. Challenges in implementing a successful newborn cystic fibrosis screening program. J Pediatr 2005; 147:S89–S93. 38. Comeau AM, Parad R, Gerstle R, et al. Communications systems and their models: Massachusetts parent compliance with recommended specialty care after positive cystic fibrosis newborn screening result. J Pediatr 2005; 147:S98–S100.

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39. Therrell BL, Lloyd-Puryear MA, Mann MY. Understanding newborn screening system issues with emphasis on cystic fibrosis screening. J Pediatr 2005; 147(3 suppl 1):S6–S10. 40. Comeau AM, Accurso FJ, White TB, et al. Guidelines for implementation of cystic fibrosis newborn screening programs: Cystic Fibrosis Foundation workshop report. Pediatrics 2007; 119:e495–e518. 41. Kloosterboer M, Hoffman G, Rock M, et al. Clarification of laboratory and clinical variables that influence cystic fibrosis newborn screening with initial analysis of immunoreactive trypsinogen. Pediatrics 2009; 123:e338–e346. 42. Giusti R. Consortium NYSCFNS. Elevated IRT levels in African-American infants: implications for newborn screening in an ethnically diverse population. Pediatr Pulmonol 2008; 43:638–641. 43. Scotet V, Audrezet M-P, Roussey M, et al. Immunoreactive trypsin/DNA newborn screening for cystic fibrosis: should the R117H variant be included in CFTR mutation panels? Pediatrics 2006; 118:e1523–e1529. 44. Lording A, McGaw M, Dalton A, et al. Pulmonary infection in mild variant cystic fibrosis: implications for care. J Cyst Fibros 2006; 5:101–104. 45. O’Sullivan BP, Zwerdling RG, Dorkin H, et al. Early pulmonary manifestation of cystic fibrosis in children with the dF508/R117H-7T genotype. Pediatrics 2006; 118:1260–1265. 46. Curnow L, Savarirayan R, Massie J. Genetic counseling after carrier detection by newborn screening when one parent carries DeltaF508 and the other R117H. Arch Dis Child 2003; 88:886–888. 47. Moskowitz SM, Chmiel JF, Sternen DL, et al. Clinical practice and genetic counseling for cystic fibrosis and CFTR-related disorders. Genet Med 2008; 10:851–868.

8 Diagnostic Approach to Diseases Associated with Cystic Fibrosis Transmembrane Conductance Regulator Gene Mutations CHEE Y. OOI (KEITH) The Hospital for Sick Children, Toronto, Ontario, Canada

ELIZABETH TULLIS University of Toronto and St. Michael’s Hospital, Toronto, Ontario, Canada

PETER R. DURIE University of Toronto and the Hospital for Sick Children, Toronto, Ontario, Canada

I.

Introduction

The discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene led to a significant increase in our knowledge and understanding of the spectrum of disorders associated with functional alterations in the CFTR gene. Prior to this discovery, cystic fibrosis (CF) was thought to be a multisystem disease that manifests clinically either at birth (with intestinal obstruction) or in infancy/early childhood (with failure to thrive and recurrent pulmonary symptoms) (1). Great advances in molecular analysis techniques have led to not only the identification of more than 1550 CFTR mutations (2) but also the recognition of a wide spectrum of diseases associated with mutations in the CFTR gene. What was once considered a disease of infants and young childhood is no longer the case as individuals with symptoms manifesting in adolescence and adulthood are receiving a diagnosis of CF. This has been complicated by the fact that several diseases that resemble CF at an organ-specific level have also been found to be strongly associated with mutations in the CFTR gene. Consequently, the clinical diagnosis of CF disease is not always straightforward because progress in molecular genetics has not been matched by equal progress in our understanding of the clinical consequences of the majority of known CFTR mutations. A. Nomenclature

In effect, as knowledge of the range of phenotypes associated with CFTR gene mutations has expanded the demarcation line between patients with CF disease and those not classified as CF by current diagnostic criteria has blurred. This is further complicated by the introduction of various confusing terms to describe the range of severity of CF and CFTR-related conditions. These include terms such as typical CF, atypical CF, CF variant, pre-CF, and classic and nonclassic CF. Furthermore, these classifications are misleading, and are no longer relevant in the age of newborn screening and to the current state of knowledge of CF disease. CF is no longer considered an unambiguous disease

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entity resulting in death in early childhood, and it is not possible to determine individual prognosis using currently available diagnostic tests. In fact, individuals who display few or no deleterious health effects earlier in life can later develop severe disease in one or more organ systems. Furthermore, there is emerging evidence to suggest that the pulmonary outcomes of individuals classified as having “mild” CF may be as severe as those with “severe” CF. The results of two recent consensus conferences with participants from Europe, North America, and Australasia (3,4) agreed that this classification should be limited to two terms: (i) CF disease, to describe patients who fulfill the current diagnostic criteria, and (ii) “CFTR-related disorder,” to describe individuals with a CF phenotype in at least one affected organ as well as evidence of CFTR dysfunction (CFTR mutations and/or evidence of CFTR ion channel dysfunction) that do not fulfill the diagnostic criteria for CF disease. Nonetheless, as we describe in detail below, individuals who carry mutations in the CFTR gene show an overlapping clinical spectrum, ranging from no clinical disease at one extreme through those with CFTR-related disorders to CF disease with and without sufficient pancreatic function at the other extreme. Similarly ion channel measurements (ICMs) in the sweat gland and nasal epithelium show a continuum of values, with no clear delineating point, ranging from those observed in healthy controls and obligate heterozygotes to values for pancreatic sufficient (PS) and insufficient (PI) CF. Consequently, our ability to establish or exclude CF disease has become increasingly problematic. B. CFTR Function and Disease

The CFTR protein is a cyclic AMP-dependent chloride channel, located in the apical membrane of secretory and absorptive epithelial cells of the airway, pancreas, intestine, liver, vas deferens, and sweat glands. The manifestations of CF predominantly arise from glandular or ductal plugging due to an inability to hydrate macromolecules within the lumen. There is a continuous spectrum of CFTR dysfunction that results in a complex and heterogeneous disease (Fig. 1). In addition, there is a clear relation between the number and functional severity of CFTR gene mutations with the range of CFTR-mediated ion channel abnormalities (5). This observation is providing insight into our understanding of the threshold of CFTR-mediated ion channel function that is required for disease pathogenesis and diagnosis. Different mutations in the CFTR gene have varying effects on CFTR function. A five class system was devised to predict how mutations influence CFTR-mediated ion channel function (6) (Fig. 2). Class I mutations include nonsense or frameshift mutations that cause defective protein biosynthesis while class II mutations (e.g., DF508) result in misfolded functional CFTR protein, which is largely degraded intracellularly. Class III mutations affect channel activation by preventing binding and hydrolysis of ATP at one of the two nucleotide-binding domains. Class IV mutations result in impaired protein function due to abnormal anion conductance. Class V mutations include abnormal splicing, promoter mutations, or inefficient trafficking that lead to a reduced number of normally functioning protein. In general, patients homozygous or compound heterozygous for mutations in classes I, II, and III (which confer loss of CFTR function) are more susceptible to severe clinical consequences.

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Figure 1 Spectrum of disorders associated with level of CFTR function. Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator.

Figure 2 Classification of CFTR mutations according to the functional consequences of the gene product. Severe mutations (classes I–III and VI) confer little or no functional CFTR at the apical membrane, while mild mutations (classes IV and V) confer some partial CFTR function (see text for explanation). Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator. Source: Adapted from Ref. 7.

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The exocrine pancreas is the most reliable phenotypic barometer of CFTR function, and most patients with the PI phenotype carry classes I, II, or III mutations on both alleles. Patients who are homozygous or compound heterozygous for severe mutations are also more likely to develop meconium ileus and severe liver disease (cirrhosis and portal hypertension). Class IV or V mutations confer some residual but highly variable CFTR function. Patients carrying a class IV or V mutation on at least one allele usually have sufficient pancreatic function and commonly do not experience significant intestinal and hepatic complications. These patients usually have the PS form of CF or a CFTR-related disorder. Nonetheless, it is important to note that genotype may not be closely associated with pancreatic phenotype in very young infants, particularly those identified by newborn screening. This is almost certainly due to the fact that some infants carrying severe mutations on both alleles have some residual exocrine pancreatic function. However, almost all of the patients who are homozygous or compound heterozygous for class I, II, or III mutations will develop PI before two years of life (8). While this classification system for CFTR mutations is useful as a conceptual framework, its limitations are acknowledged (e.g., class IV and V mutations can have overlapping consequences, and inferred properties of many mutations remain to be confirmed by functional studies). In addition, the heterogeneous CF disease spectrum in other organs, especially the airways, is poorly explained by CFTR genotype and is also influenced by environmental and other genetic modifying factors (9–11).

II.

Diagnosis of CF and CFTR-Related Disorders

According to the most recent United States Cystic Fibrosis Foundation (USCFF) consensus report (3), a diagnosis of CF can be made in individuals who present with a characteristic clinical phenotype or a history of CF in a sibling plus an abnormal sweat chloride value
60 mmol/L and/or two CF-causing mutations. It is important to note that the diagnosis of CF cannot be made on the basis of identification of CFTR mutations that are not designated as CF-causing. The confirmation or exclusion of CF is usually straightforward, particularly among individuals in the extreme ends of the CFTR function spectrum (i.e., normal healthy individuals and heterozygotes at one extreme and CF patients homozygous or compound heterozygous for severe mutations at the other). However, the diagnosis of CF disease may be less clear-cut among individuals who have residual function of the CFTR protein. A significant number of individuals who have diseases associated with CFTR mutations conferring partial function do not meet the current diagnostic criteria for CF disease. These individuals, who are considered to have a CFTR-related disorder, characteristically present with symptoms of CFTR dysfunction (e.g., chronic sinopulmonary disease; obstructive azoospermia; or acute, recurrent, or chronic pancreatitis) at an older age, and have either sweat chloride values in the intermediate range (40–59 mmol/L for individuals above six months of age) and/or one or two CFTR mutations that are not classified as CF-causing mutations (3). While discussion of excluding or establishing a diagnosis of CF in the era of newborn screening test is covered in a companion chapter by O’Sullivan, it is worthy to note that the diagnosis of CF will be uncertain in a subset of asymptomatic infants that screen positive for CF. Despite the fact that neonatal screening programs for CF have been in existence since the 1980s, the prevalence and natural history of infants with an

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uncertain diagnosis of CF is not well defined. We are unaware of any careful, prospective longitudinal studies of screen-positive infants with an uncertain diagnosis of CF. A single retrospective study has been performed in screen-positive infants who carried one CFTR mutation and a normal or intermediate sweat chloride concentration (12). Extensive CFTR mutation analysis revealed that 20/32 (62.5%) of screen-positive newborns carried a change in the CFTR gene on the second allele (compound heterozygote), but none of these changes were established as CF-causing and may represent functional polymorphisms. Nine of 20 compound heterozygotes evaluated in follow-up at four years of age displayed at least one clinical feature of CF disease, which is not surprising since there are reports of an increased incidence of chronic rhinosinusitis, diffuse bronchiectasis, and idiopathic pancreatitis in individuals who carry at least one CFTR mutation (discussed in more detail below). Three of these patients had a sweat chloride test that was consistent with CF. This study was limited by incomplete ascertainment at follow-up. Only a subset of patients underwent extensive CFTR mutation analysis and an even smaller number of subjects were recalled for follow-up evaluation. Clearly, a carefully performed prospective longitudinal assessment of newborn screen-positive individuals is warranted to determine the true prevalence of CF-disease and CFTR-related disorders in this population.

III.

Diagnostic Challenges

There is considerable debate and difficulty on where to draw the diagnostic line between CF and CFTR-related disorder. Although the following discussion separates them into two entities, they form part of a continuous spectrum of diseases associated with CFTR dysfunction (Fig. 1). A. Cystic Fibrosis Diagnosed in Adolescence and Adulthood

CF may manifest at different ages, in different organs, with variable severity and includes chronic sinopulmonary disease, pancreatic exocrine insufficiency, intestinal diseases, pancreatitis, hepatobiliary disease, and obstructive azoospermia in men (1,5,7,13–22,24). In its traditionally recognized form, CF disease either presents with intestinal obstruction at birth or with failure to thrive and recurrent pulmonary symptoms in infancy/early childhood. However, a subset of patients is diagnosed with CF in adolescence and adulthood, with a wide spectrum of symptoms and severity of disease that does not resemble the typical clinical presentation in infancy or early childhood (1,13–15). In fact, they may first come to the attention of an urologist, otolaryngologist, or gastroenterologist, rather than a pulmonologist. With increasing awareness, the number of CF patients receiving a diagnosis in adolescence and adulthood continues to increase. Gilljam et al. prospectively examined all patients with a CF diagnosis made at the Toronto CF clinics (adult and pediatric) over a 41-year period and compared patients who received a diagnosis of CF in adulthood with those who were diagnosed in infancy or childhood (13). Among all patients who were diagnosed with CF between 1960 and 1989, only 3% were over 16 years of age. In contrast, in the subsequent decade (1990 and 2001), the proportion of subjects diagnosed in adulthood rose to 18% (Table 1) (13). Among patients older than 16 years of age at clinical presentation, the median age of CF diagnosis was 32  11 years (range, 16–58 years), with no age difference between

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Table 1 Proportion of Patients with CF Diagnosed in Adulthood and Childhood at the Toronto

CF Clinics Between 1960 and 2001.

1960–1989: Number of diagnoses (%) 1990–2001: Number of diagnoses (%)

Childhood

Adulthood

767 (97) 211 (82)

27 (3) 46 (18)

Abbreviation: CF, cystic fibrosis. Source: Adapted from Ref. 2.

males (54%) and females (13). Patients who received a diagnosis of CF at an older age tended to have more subtle clinical features, present with single-organ manifestations, have lower sweat chloride levels, and were more likely to be PS (73%) (13). Among patients who were diagnosed in adulthood between 1990 and 2001, the majority (39%) presented with pulmonary symptoms, whereas obstructive azoospermia (men) and pancreatitis accounted for 26% and 4% of the population, respectively (13). Pulmonary symptoms at the time of diagnosis may include chronic productive or nonproductive cough, recurrent airway infections requiring antibiotics, dyspnea, hemoptysis, and wheezing (13). CF-associated pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus may be cultured from sputum in a large percentage of these individuals (13). Approximately 30% have normal chest radiographic findings and 50% had FEV1 >87% predicted (13). However, a number had well-established bronchopulmonary disease that progressed to pulmonary failure and premature death or a life-saving lung transplant (13,25). Gilljam et al. also observed that the smaller number of individuals diagnosed with PI-CF at an older age were likely to have concurrently severe pulmonary disease. Among adolescents and adults who have sufficient pancreatic function at presentation, pancreatitis may be the primary manifestation of CF disease (26). Severe CF-related liver disease, as defined by the development of cirrhosis with or without portal hypertension, is a very uncommon presentation in those diagnosed de novo in adolescence and adulthood (13,27), due largely to the fact that the most of CF patients with severe liver disease have established cirrhosis before the age of 15 (28).

B. CFTR-Related Diseases

Adolescents and adults with a CF phenotype in one or more CF-affected organs carry a much higher frequency of CFTR gene mutations than the general population (5,7,16). These include men with infertility due to obstructive azoospermia due to bilateral absence of the vas deferens (5,7,17), patients with idiopathic recurrent acute and chronic pancreatitis (13,18,19), and a variety of chronic sinopulmonary disorders including chronic rhinosinusitis and bronchiectasis (13,20–22). There is currently insufficient evidence to support a strong association between diseases such as asthma, allergic bronchopulmonary aspergillosis, and primary sclerosing cholangitis with mutation(s) in the CFTR gene, such that, if there is a link, loss of CFTR function is likely to play only a small role in the pathogenesis of these complex genetic diseases (7,22). Of all the organs that are affected by reduced CFTR function, the male reproductive tract appears to be the most sensitive to very minor defects in function (29–34). This is most likely due to several factors, including the narrow lumen, length, and

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Table 2 Diagnosis of CF in Patients with CBAVD or Idiopathic Pancreatitis by Disease-Causing

Mutations, Sweat Chloride, and NPD Group

n

Two CF diseasecausing mutations, n (%)

Sweat chloride > 60 mmol/ L, n (%)

NPD DCl Any test, -free þ Iso n (%) < 7.65 mV, n (%)

All CBAVD CBAVD with no CFTR mutation CBAVD with 1 CFTR mutation CBAVD with 2 CFTR mutations All pancreatitis Pancreatitis with no CFTR mutation Pancreatitis with 1 CFTR mutation Pancreatitis with 2 CFTR mutations

60 6 18 36 56 32 18 6

2 0 0 2 2 0 0 2

17 (28) 0 (0) 6 (33) 11 (31) 5/54 (9) 0/30 (0) 3/18 (17) 2/6 (33)

26 (43) 0 (0) 7 (39) 19 (53) 9/42 (21) 2/23 (9) 4/14 (29) 3/5 (60)

(3) (0) (0) (6) (4) (0) (0) (33)

34 (57) 0 (0) 10 (56) 24 (67) 12 (21) 2/32 (6) 6/18 (33) 4/6 (66)

CF disease-causing mutations according to the 1998 consensus statement on the diagnosis of CF. Chloride-free and isoproterenol-stimulated chloride diffusion potential on the nasal epithelium (DCl -free þ Iso) <99% confidence limits for controls (less than 7.65 mV) was used as an indicator of CF disease. Abbreviations: CBAVD, congenital bilateral absence of the vas deferens; NPD, nasal potential difference; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator. Source: Adapted from Refs. 5 and 16.

tortuosity of the vas deferens; dependence on CFTR for ion transport and fluid secretion; and the high concentration of intraluminal macromolecules that predisposes to luminal obstruction (35). As such, it is not surprising that many studies have demonstrated that infertile men with obstructive azoospermia from CBAVD (congenital bilateral absence of the vas deferens), congenital unilateral absence of vas deferens, and even epididymal obstruction have a higher incidence of mutations in the CFTR gene compared with the general population (29–34,36). Wilschanski et al. prospectively and comprehensively evaluated 60 men with CBAVD by performing ion channel function testing (sweat chloride and nasal transepithelial potential difference measurements) and extensive genotyping, and compared the findings with those of healthy controls, obligate CF heterozygotes, and patients with a known diagnosis of CF (PS and PI phenotypes) (5). Ninety percent of men with CBAVD carried at least one CFTR mutation: 30% carried a mutation on one allele and 60% had mutations on both alleles (5). Among men who carried one and two mutations in the study, 39% and 83% of these men, respectively, carried at least one CF-causing mutation and/or variants, but only two men carried CFcausing mutations in both alleles (5). Among men who present with CBAVD, a significant proportion (57%) would be considered to carry a diagnosis of CF following comprehensive evaluation by sweat chloride testing and/or nasal potential difference (NPD) measurements (Table 2). Similarly, several studies have shown that individuals with idiopathic recurrent acute and chronic pancreatitis have an increased incidence of mutations in the CFTR gene (16,18,37–39). Bishop et al. showed that 43% and 11% of patients with idiopathic recurrent acute or chronic pancreatitis carried at least one and two CFTR mutations

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(and/or variants), respectively; the diagnostic criteria of CF could be fulfilled in 21% of these patients (Table 2) (16). There is also literature on patients with chronic sinopulmonary disease or bronchiectasis in whom CF and other etiologies have been ruled out, but in whom there is evidence of CFTR mutation and/or dysfunction (40–42). In a small prospective study of adult patients with bronchiectasis, approximately 40% of patients had a CFTR-related disorder (43). Not surprisingly, in an organ system that involves complex genetic and environmental interactions in the development of disease, there has been at least one report that found the frequency of CFTR mutations in patients with idiopathic bronchiectasis was not significantly different from the general population (44).

IV.

Diagnostic Tests

In the majority of cases, the main role of diagnostic tests in CF is to confirm the clinical diagnosis of CF. In patients who do not present with the typical presentation of CF in infancy or childhood or who manifest single-organ disease phenotypes, diagnostic testing is necessary to exclude or support the diagnosis of CF and help clinicians consider other differential diagnoses. The sweat chloride test remains the reference standard test for CF and should be used as the first-line test for CF. It is commonly available in most diagnostic laboratories and is internationally standardized. However, the role of CFTR mutation analysis in clarifying the diagnosis of CF has been overestimated by many clinicians. In fact, in patients with a suspected, but unproven diagnosis of CF, mutation testing including complete sequencing of the CFTR gene appears to be the least helpful diagnostic test, as will be described in detail below. The use of the transepithelial NPD and intestinal ICMs in clarifying the diagnosis of CF will also be discussed. Despite the lack of validated reference values and standardization, NPD has been proposed for use by both the USCFF and European CF Society (ECFS) working group in difficult diagnostic cases (3,45). Similarly, intestinal ICM has been introduced in clinical practice, particularly in some European CF centers, although it has neither been standardized nor validated for clinical use. While the use of these alternative measures of CFTR function may provide very helpful information, there remains a lack of the basic and fundamental requirements required for proper use outside a research setting. A. Ion Channel Measurements

The sweat chloride test, which has been in clinical use for more than 50 years, remains the principal test for the confirmation of the diagnosis of CF in the genomic era (46,47). Although referred to as the “gold” standard test for CF, the sweat test is not a perfect test. Current age-related reference range for sweat chloride concentration was defined by studies in CF patients presenting with classical symptoms of CF at an early age (23,48,49). Until recently, there has been no study that had measured the age-related range of sweat chloride concentrations in a normal population (50). Mishra et al. established reference ranges for sweat chloride in healthy children, adolescents, and adults by performing sweat tests on 282 healthy individuals (aged from 5 to >50 years) and compared them with a cohort of established CF patients with PI (aged 15–41 years) (51). In children aged 5 to 9 years, the upper limit (97.5th percentile) of the reference interval for sweat chloride concentration was 39.5 mmol/L, which is remarkably consistent with

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those used to exclude CF (i.e., <40 mmol/L) (51). However, the upper limit of the reference interval for sweat chloride concentration in older individuals (from 10 years old) was found to be within the consensus-defined intermediate range of 40 to 60 mmol/L, making the intermediate range unreliable in the adolescent and adult population (51). This finding is consistent with those reported by Wilschanski et al. (Fig. 3) (5). Unfortunately, the study by Mishra et al. failed to exclude obligate heterozygotes from the group of healthy individuals and did not assess or compare patients with CFTRrelated disease and PS-CF, which constitute, by far the greatest diagnostic challenge. In this regard, it has been well established that PS-CF patients, who are frequently diagnosed at an older age, with more subtle disease manifestations, have mean sweat chloride values that are significantly lower than the current reference criteria (<60 mmol/L) (3,13). Other previous studies have also reported normal or intermediate sweat chloride values to be associated with the diagnosis of CF (PS and PI) (52–55). No study has accurately determined the reference ranges for sweat chloride in infants and young children <5 years. It is important to consider this when assessing infants who test positive on newborn screening for CF. On the basis of limited data and studies not designed to define age-related reference range for sweat chloride concentration, the two consensus statements from North America and Europe recommend the upper limit of normal for sweat chloride in infants <6 months as 29 mmol/L (3,45). However, it is possible that as more information becomes available and our understanding of the long-term outcome of infants with an uncertain diagnosis of CF increases, the normal reference range may require revision. NPD is an electrophysiological test that assesses CFTR activity by measuring transepithelial potential difference of the nasal epithelium based on the characteristic bioelectric properties of amiloride-dependent sodium absorption (via ENaC) and CFTRmediated Cl diffusion. NPD has been utilized in CF research for decades but only recently introduced into clinical practice (56) for assistance in clarifying the diagnosis of CF, including for individuals with intermediate sweat chloride values (57). NPD is measured between a fluid-filled exploring bridge on the nasal mucosa and a reference bridge inserted into subcutaneous space. Both bridges are linked by Ag/AgCl electrodes (or saturated calomel half cells) to a high-impedance voltmeter. Using direct vision with an otoscope, the tip of the exploring catheter is placed onto respiratory mucosa under the concha nasalis inferior. Basal potential difference is recorded once consistent baseline NPD measurements are obtained. Changes in transepithelial potential difference as a response to superfusion of the nasal mucosa with different solutions/drugs via the exploring catheter are measured. The responses to perfusion of (i) amiloride that reflects activity of inwardly directed Naþ transport (DAmil); (ii) a chloride-free solution with amiloride to measure basal chloride diffusion; and (iii) isoproterenol added to the chloridefree solution with amiloride to measure maximal chloride diffusion (DCl-free þ Iso) are recorded (Fig. 4). The findings of a high potential difference during baseline measurements followed by an absent or very low voltage response to zero-chloride plus isoproterenol perfusion (DCl-free þ Iso) are indicative of CF (Fig. 4). NPD is a sensitive determinant of CFTR function, but it has major limitations for clinical utilization. While NPD has been standardized as an outcome measure in research studies, the clinical interpretation of the results should be interpreted with caution, because this technique has neither been standardized nor validated for clinical use. In addition, NPD measurements may be equivocal in patients with equivocal sweat

Figure 3 The range and variability of sweat chloride (A) and chloride-free with isoproterenolstimulated chloride diffusion NPD measurements (DCl-free þ Iso) (B) in men with CBAVD are shown. The relationship of each group with one another is demonstrated by box plots for each group along the x-axis according to the value of the median. Each box plot represents values between the 25th and 75th percentiles. Values outside this range are represented by circles. Values more than 1.5 above and below the 75th and 25th percentiles are shown as solid circles. Horizontal dashed lines depict the 95% confidence intervals for the medians of each group. The healthy controls are 25 men with no identifiable CFTR gene mutations. The reference ranges for sweat chloride and NPD DCl-free þ Iso are represented by the long horizontal and vertical dashed lines: sweat chloride values were interpreted as normal (<40 mmol/L), borderline (40–60 mmol/L) or abnormal (>60 mmol/L); DCl-free þ Iso. Since the diagnostic reference limits for NPD have not been established, values less than 99% confidence limits for controls (less than 7.65 mV) was used as an indicator of CF disease. Abbreviations: NPD, nasal potential difference; CBAVD, congenital bilateral absence of the vas deferens; CF-PI, pancreatic insufficient CF; CFPS, pancreatic sufficient CF; HET, heterozygotes. Source: Adapted from Ref. 5.

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Figure 4 Characteristic nasal potential difference tracings of a healthy individual and a patient

with cystic fibrosis. Changes in transepithelial potential difference as a response to sequential superfusion of the nasal mucosa with amiloride then a chloride-free solution with amiloride, followed by the addition of isoproterenol are measured. A high potential difference during baseline measurements followed by an absent or very low voltage response to zero-chloride plus isoproterenol perfusion (DCl-free þ Iso) is indicative of cystic fibrosis.

chloride and/or CFTR mutation analysis, and consensus of what constitutes a borderline result remains lacking. In fact, despite being advocated by both the USCFF and the ECFS in their diagnostic algorithms (3,45), there is no national or international consensus on the standard operating procedures or the diagnostic reference values for NPD measurements. In addition, it is complex to perform, labor intensive, and operator dependent. False-positive results can occur with incorrect location of the measuring catheter, minor perturbations of the nasal epithelium, allergies, respiratory infections, and smoking (58). In individuals who present with a CF phenotype in at least one affected organ, ICMs such as sweat chloride and NPD tests show a wide spectrum of values, ranging from values for healthy controls and obligate heterozygotes to those for PS and PI patients with CF. In general, the number of CFTR mutations influenced ICMs in patients. For instance, in the study by Wilschanski et al., all sweat chloride and NPD measurements for patients with CBAVD and no identified CFTR mutations were normal in contrast to a large number of CBAVD patients with one or two CFTR mutations who fell within the abnormal range for sweat chloride, NPD, or both (5). Furthermore, the sweat chloride and NPD measurements for CBAVD patients with one or two CFTR mutations overlapped with PS patients with an established diagnosis of CF (Fig. 3) (5). When Wilschanski et al. compared sweat chloride testing in establishing a diagnosis of CF among healthy controls, obligate heterozygotes, individuals with CBAVD, and patients with CF (PS and PI), the sweat test was able to exclude CF in all healthy controls and obligate heterozygotes, and confirm the diagnosis of CF in all CF patients with PI (5). However, only 79% of PS patients with a previously established diagnosis of CF had a sweat chloride concentration >60 mmol/L (5). Gilljam et al. reported a similar

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observation in a cohort of 46 adults with CF, where only 65% had sweat chloride levels >60 mmol/L (13). Similarly, Wilschanski et al. showed that NPD measurements (DCl-free þ isoproterenol) was able to accurately distinguish all healthy controls and obligate heterozygotes from those with PI-CF (5). However, NPD appeared superior to sweat testing as a diagnostic measure, as a diagnosis of CF could be made on NPD alone in at least 90% of adults who presented with symptoms of CF (5,13). To our knowledge, the studies by Wilschanski et al. and Bishop et al. are the only ones that have attempted to establish reference values for NPD based on appropriate cohorts. Nonetheless, the limitations of NPD, as mentioned previously, restrict its application for predominantly research purposes. The role of intestinal ICM in clarifying the diagnosis of CF has been reported in cases of uncertain diagnosis of CF (59–61). ICM is an ex vivo test performed in an Ussing chamber using a freshly obtained rectal biopsy and measuring its electrical response to a series of secretagogues. Intestinal epithelia of CF subjects have been evaluated by two different laboratory methods (circulating vs. continuously perfused) (62,63). The common finding of both approaches demonstrates absent or diminished chloride secretion after stimulation with agonists that act via intracellular cAMP. There is currently no comparison study to determine which protocol is superior. In addition, while it appears that this approach is being used primarily in Europe as a clinical diagnostic aid, the absence of well-established reference values for either test limits its use to predominantly research purposes and warrants careful interpretation of results in the clinical setting. Nonetheless, it may prove to be a useful adjunctive diagnostic tool in infancy and early childhood, because rectal tissue can be obtained safely and painlessly and alternative tests such as NPD measurements require a high degree of patient cooperation. B. CFTR Genotyping

After the CFTR gene was cloned (64,65), there was considerable optimism that genotyping would supersede the traditional sweat test. Not only has this sense of optimism not been realized, there remains considerable misunderstanding of the role of CFTR genotyping as a diagnostic aid in CF. Here, we use the term “mutation” to refer simply to a molecular alteration in the DNA sequence of a gene. For a large number of CFTR mutations, no inference can be made concerning the effect of this alteration on gene expression or the function of the protein product. This is particularly true for missense mutations, which represent approximately 40% of the more than 1550 identified mutations in the CFTR gene. To address this very issue, the ECFS recently published a consensus on the use and interpretation of CFTR mutation analysis in clinical practice (4). On the basis of the ECFS consensus, CFTR mutations may be categorized into four groups according to their predicted clinical consequences: (i) CF-causing mutations, (ii) mutations associated with CFTR-related disease, (iii) mutations with no known clinical consequence, and (iv) mutations with unknown clinical relevance. The current USCFF consensus report lists only 23 mutations in the CFTR gene as proven CF-causing mutations (Table 3) (3). Other mutations can be predicted to be disease-causing if the molecular alteration results in a change in amino acid sequence that severely affects CFTR synthesis and/or function, or introduces a premature termination signal, or alters invariant nucleotides of

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Table 3 List of CF-Causing Mutations Based on Direct or Empiric Evidence

G85E R117H R334W R347P A455E

I507del F508del G542X G551D R553X

R560T R1162X W1282X N1303K

621 þ 1G > T 711 þ 1G > T 1717  1G > A 1898 þ 1G > A 2184delA

2789 þ 5G > A 3120 þ 1G > A 3659delC 3849 þ 10kbC > T

Abbreviation: CF, cystic fibrosis. Source: Adapted from Ref. 3.

intron splice sites (3,4). While a limited number of additional CFTR mutations may fulfill the strict criteria as accepted CF-causing mutations, the majority do not (4). Additional molecular factors contribute to difficulties in interpreting the clinical consequences of CFTR mutations. For example, variable penetrance of the R117H mutation in the CFTR gene is dependent on the length of the polythymidine tract in intron 8 (IVS8-5T, 7T, and 9T) which have a major effect on the splicing efficiency. The R117H-5T, which is associated with poor splicing efficiency, is common in CF patients with pancreatic sufficiency, but can also be seen in subjects with a CFTR-related disorder. The variable penetrance is partially explained by the influence of the number of TG repeat adjacent to the 5T tract (66). R117H in cis with 7T, which has intermediate levels of splicing efficiency, is more frequent among individuals with CFTR-related disorders such as CBAVD and pancreatitis. The 9T allele is most efficient and associated with normal levels of CFTR protein. Also, some mutations can exist as complex alleles. The missense mutation I148T was initially identified as a severe CF-causing allele based on genotype-phenotype associations. In vitro evidence suggests that, despite normal chloride transport function, I148T-CFTR has aberrant function with regards to regulation of bicarbonate transport (67) and regulation of the epithelial sodium channel (68). However, subsequently a disease-causing deletion 3199del6 in exon 20, which cosegregates in 1% of all I148T CFTR genes in the general Caucasian population, is now considered to be the underlying disease-causing mutation in most CF patients carrying the I148T CFTR mutation (69,70). Consequently, the recent ECFS consensus report currently considered I148T alone as a neutral mutation with no clinical consequence (4). Furthermore, the American College of Medical Genetics have recommended removing I148T from the list of mutations recommended for screening in CF carrier testing (71). The difficulties with interpreting and predicting the clinical consequences of IVS8 variants and complex alleles provide further evidence that mere identification of changes in the CFTR gene is insufficient to diagnose CF and that correlations with clinical phenotype and ICMs are necessary. In older patients with a suspected, but unproven diagnosis of CF, complete sequencing of the CFTR gene appears to be least sensitive and specific than carefully performed sweat chloride testing (5,16). CF mutation screening panels usually include only the more common disease-causing mutations associated with childhood onset of CF. Patients presenting in adolescence and adulthood and/or those who present with monosymptomatic diseases in CF-affected organs frequently carry one or more rare CFTR gene mutations, which are often not included in most screening panels. While many of these rare mutations may be detected by complete sequencing of the CFTR

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gene, the diagnosis often still remains unclear because the gene changes found may not be proven CF-causing mutations (3,4). On the basis of the stringent criteria for designating CF-causing mutations, CFTR genotyping alone carried the lowest diagnostic sensitivity. In a cohort of CF patients diagnosed in adulthood, only 33% fulfilled the diagnostic criteria of CF based on genotyping alone (13). When combined with sweat testing, the diagnostic sensitivity improved to 67% (13).

V. Clinical Assessment In cases of diagnostic uncertainty, referral should be made to a CF care center that can provide comprehensive clinical testing to help establish or exclude the diagnosis of CF disease or CFTR-related disorder. The role of additional testing is to accurately define the phenotype and its severity, exclude other diseases, and identify ion channel abnormalities through alternative method(s). Depending on the nature of the clinical presentation, more detailed assessment of affected organ systems is necessary. For example, patients with pulmonary symptoms should be referred for pulmonary function testing, chest imaging (which may include computed tomography of the chest), and testing to exclude conditions such as immunodeficiency states and primary ciliary dyskinesia. It is valuable to clarify the presence of CF-associated pathogens in the respiratory tract from a clinical care perspective, although the presence of these pathogens is neither sensitive nor specific for the diagnosis of CF. It is similarly important to assess the exocrine pancreas through imaging and pancreatic function testing, and to assess males for evidence of obstructive azoospermia. The degree of exocrine pancreatic dysfunction is difficult to assess due to relative inaccessibility of its secretions. The exocrine pancreas also has a large reserve capacity and requires between 90% and 99% of enzyme secretory capacity to be lost before a subject develops clinical manifestations of PI. The direct pancreatic stimulation test is considered the gold standard of pancreatic function tests and allows assessment of both pancreatic acinar (enzyme) and ductular (electrolyte and fluid) status. However, there is no standardized methodology for direct pancreatic stimulation testing, and various techniques are used throughout the world. If not done accurately, measurements and diagnosis of exocrine pancreatic status based on pancreatic stimulation testing warrants careful interpretation and can be misleading. The quantitative pancreatic stimulation test technique that uses perfusion markers with multiple sampling periods is sensitive, highly specific, and capable of evaluating the entire range of pancreatic function (72). With the marker perfusion technique, a double lumen catheter is inserted under fluoroscopic guidance into the duodenum with a proximal lumen positioned at the ampulla of Vater and the other lumen positioned distally in proximity to the ligament of Treitz. Through the proximal opening, a nonabsorbable marker solution is infused into the duodenum throughout the test. Pancreatic juice mixed with infused marker solution is aspirated distally over a timed sampling period while continuously infusing secretin/cholecystokinin. A separate nasogastric tube is placed to aspirate gastric juice and prevent contamination of duodenal contents. The marker perfusion technique offers an ability to quantify recovery of pancreatic secretions and, therefore, allows accurate quantification of pancreatic acinar and ductular secretions. This technique is regarded as the true gold standard in direct pancreatic

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stimulation testing. Nevertheless, this test is time consuming, technically complex to perform and not routinely available. Consequently, alternative and simpler direct pancreatic stimulation techniques (nonquantitative) are used at many clinical centers. These generally utilize a singlelumen duodenal tube or endoscope to aspirate duodenal pancreatic secretions as a spot sample and/or over a timed sampling period (73–76). Despite their popularity, we do not recommend the use of nonquantitative techniques, because they are predicted to have a high false-positive rate for misdiagnosing PI and have not been proven superior over noninvasive indirect pancreatic function tests. In fact, Schibli et al. demonstrated that among PS patients with a secretory capacity between the threshold of PI and the lower limit of normal, these techniques carry the greatest risk of misclassifying a PS patient as PI (77). Currently, noninvasive pancreatic function tests are routinely utilized for defining the CF pancreatic phenotype. The 72-hour fecal fat balance test, expressed as the coefficient of fat absorption (CFA), is considered as the gold standard as far as noninvasive tests are concerned. However, this test is inconvenient and not well accepted by patients or laboratory personnel. Fecal elastase is considered an ideal indirect stool marker of pancreatic function because elastase, an endogenous pancreatic secretion enzyme, is resistant to degradation within the gastrointestinal tract. Previous studies have found it to be a good measure of exocrine pancreatic function. Using a cutoff of 200 mg/g stool, the sensitivity was 63% for mild, 100% for moderate, 100% for severe, and 93% for all patients with exocrine pancreatic insufficiency, and specificity was 93% (78). In another study, which compared fecal elastase and 72-hour fecal fat balance studies in children with CF and non-CF pancreatic disease (PI and PS) as well as disease controls with documented steatorrhea due to nonpancreatic causes, it was found that fecal elastase values below 100 mg/g stool were highly indicative of pancreatic insufficiency, when compared with the 72-hour fecal fat (79). Serum trypsinogen have been used for newborn screening for more than two decades (80) and shown to help define pancreatic functional status (after the age of 7 years) and predict the progression of pancreatic status from PS to PI (81). Serum trypsinogen is also capable of identifying presence of pancreatic disease and pancreatic dysfunction (loss of pancreatic reserve or pancreatitis) in individuals with a CFTRrelated disorder. For example, serum trypsinogen levels may be elevated in patients with CF-like phenotype providing evidence of pancreatic disease, while levels will be low in patients with chronic pancreatitis due to a loss of pancreatic reserve. Despite exocrine pancreatic insufficiency being uncommon in this population of patients, imaging of the pancreas and testing of exocrine pancreatic function may be helpful for diagnostic and therapeutic purposes. However, the finding of PI alone is not diagnostic of CF even in patients who have intermediate sweat chloride values, considering the substantial test and biologic variability in sweat chloride values among normal healthy individuals (51). PI due to chronic pancreatitis unrelated to CF and inherited conditions such as Shwachman–Diamond syndrome, which may also be associated with recurrent respiratory infections, should still be considered (82). Individuals who are PS at presentation need to be assessed periodically or when new symptoms suggestive of PI develop, as some may progress to PI (13). All male patients should be assessed by semen analysis for evidence of obstructive azoospermia. Referral to an experienced urologist for full assessment and careful

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physical examination of the genital tract and transrectal ultrasound may be warranted. Because of the high prevalence of CFTR mutations in men with obstructive azoospermia, affected men and their partners require comprehensive assessment and genetic counseling if assisted reproductive technology is being considered.

VI.

Recommended Approach to Management

There is clear evidence that, following comprehensive assessment, the diagnosis of CF disease will remain uncertain in a significant subset of patients who present with a CF phenotype. We strongly recommend that this uncertainty should be communicated to affected individuals, and the rationale and plan for appropriate follow-up discussed. The inability to confirm a diagnosis of CF in patients with CFTR-related disorders should not exclude them from periodic monitoring as they may be at risk of developing significant disease in CF-affected organs, and early treatment may improve quality of life and longterm outcome (especially in those with pulmonary disease). Over time, further evaluation may confirm a CF diagnosis in some of these individuals, especially infants with an uncertain diagnosis of CF following a positive newborn screening process. Ultimately, the decision to treat an individual should be based on clinical indication(s) rather than the diagnostic label used, assuming alternative diagnoses have been excluded. For instance, an individual with a CFTR-related disorder who has bronchiectasis and is chronically infected with P. aeruginosa would be more appropriately followed in a CF center and offered the same treatments that are available for CF patients with pulmonary disease.

VII.

Conclusion

The discovery of the CFTR gene has led to a significant expansion in knowledge of the wide range of phenotypes associated with CFTR dysfunction. A significant number of individuals who have diseases associated with CFTR mutations that confer partial loss of function typically present in adolescence and adulthood, usually with single-organ manifestations of CF. Many of these subjects do not meet the current diagnostic criteria for CF disease. There are limitations in the traditional diagnostic tests, and the diagnosis of CF cannot simply be made based on the presence of two CFTR mutations. CFTR genotyping alone functions poorly as a diagnostic test especially in the setting of an uncertain diagnosis of CF or CFTR-related disorder. Although sweat chloride testing effectively diagnoses CF patients with the severe PI phenotype, it has limited ability to establish or exclude a diagnosis of CF in PS patients and those with CFTR-related disorders. While NPD and possibly intestinal ICM testing appear to be superior to sweat testing as a diagnostic measure, they still lack the basic and fundamental requirements needed for proper use outside a research setting and remain to be adequately standardized and validated as diagnostic tools. Nevertheless, we expect that there would be ongoing challenges and diagnostic dilemmas related to diseases associated with CFTR mutations, even in the presence of a well-validated, highly sensitive and specific test, due to the continuous spectrum of disorders associated with CFTR mutations and the contributory effects of various modifier genes and environmental factors on the clinical phenotype. Consequently, it is important and reasonable for this uncertainty to be conveyed to affected individuals. Referral to a tertiary CF center for further assessment is

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recommended, and periodic reassessment may be necessary. In selected cases, especially among those with pulmonary disease and pancreatic disease, follow-up and treatment may be best provided in the setting of a CF center, even if the diagnostic criteria for CF cannot be fulfilled.

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9 Lung Function Testing in Infants JESSICA E. PITTMAN and STEPHANIE D. DAVIS University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

I.

Introduction

Recent studies have confirmed that cystic fibrosis (CF) lung disease begins during infancy (1–3). Historically, a paucity of sensitive, repeatable physiologic techniques for evaluating early lung disease led to a lack of detection and recognition of the “silent” airway damage in CF (4,5). Considerable progress in infant pulmonary function testing (PFT) within the past 20 to 30 years has furthered the understanding of pulmonary physiology in infants and toddlers, thereby revealing previously hidden disease burden in the lungs of the youngest CF population (4–7). This chapter discusses the techniques currently available for measurement of lung function in infants, the particular challenges associated with these studies, and pertinent research findings utilizing physiologic measures in young children with CF.

II.

Challenges of Studying Infants

In infants with CF, evidence of airway inflammation, obstruction, and damage as severe as bronchiectasis has been found through bronchoscopic, physiologic, and radiologic studies (1–3), demonstrating that pulmonary disease begins at an early age. The widespread availability of newborn screening in the United States, Europe, and Australia has led to a decline in the age of CF diagnosis and may lead to an increased number of individuals diagnosed with CF prior to the presence of any respiratory symptoms (8). Several studies have shown evidence of airway disease in infants and young children with CF even in the absence of a history of respiratory symptoms (3,9,10). As a result, infancy is often a period of silent lung disease in children with CF, where an absence of clinical symptoms belies the underlying airway damage that is occurring (4). Measuring pulmonary function in infancy thus serves a vital role in the care of the CF patient. The ability to quantify pulmonary disease in infants with CF offers the potential for aggressive intervention before permanent airway damage has occurred, with possible improvement in CF morbidity and mortality. Obstacles faced when assessing lung function in infants are the following: (i) differences in respiratory physiology between infants and older children, (ii) the need for sedation secondary to infants’ inability to cooperate with study maneuvers, (iii) the considerable time and expertise required for study execution and interpretation, and (iv) limited reference data in healthy controls (4,7).

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Respiratory mechanics in infants are affected by preferential nasal breathing and increased chest wall compliance compared with older children and adults (7,11). Nasal resistance, which contributes up to 50% of the total airway resistance in infants, may effectively conceal subtle disease in the lower airways (7). The increased chest wall compliance of infants results in greater net inward-recoil forces of the respiratory system, which in turn leads to exhalation to a lower lung volume relative to total lung capacity (TLC) compared to older children. This subsequently contributes to collapse of peripheral airways and variability of functional residual capacity (FRC). In normal infants, this phenomenon is countered by dynamic elevation of the end-expiratory volume, which is accomplished by initiation of inspiration before reaching equilibration of outward elastic recoil of the chest wall and inward recoil of the lung (7,12). An increased respiratory rate in this age group promotes dynamic elevation of FRC. Another obstacle encountered when evaluating lung function in infants is the obvious lack of cooperation of these patients. Most of the testing performed in infants requires sedation, which adds an increased risk to any procedure, as well as increased personnel and monitoring demands (7). Sedation may also alter airway mechanics by impairing diaphragmatic tone, which is important to the normal, dynamic elevation of end-expiratory volumes in infants (7,13). PFT in infants requires significant expertise on the part of the physician, respiratory therapist, and/or lung function technician. These tests demand technical proficiency, specialized equipment, and a comprehensive understanding of infant pulmonary physiology (14–16). Dedicated personnel are necessary to ensure appropriate testing technique and accurate study interpretation. As such, infant PFT methods are predominantly limited to large tertiary care centers (4,5). Finally, the lack of comprehensive normal reference data for infant PFT, due to, in large part, the ethical issues raised by the prospect of sedating healthy infants (4), is a major challenge. The majority of reference equations available are from small, singlecenter trials, bringing into question the generalizability of reference ranges among different patient populations and between pulmonary function laboratories (4,17–19). Many single-center studies circumvent this issue by including healthy controls as a comparison group (20–22); however, lack of standardized reference equations and normal values is an obvious hindrance to multicenter studies and to the use of infant PFT in clinical practice. The dearth of normal values and of longitudinal data also raises the question of prognostic importance of infant PFT. While airway disease has clearly been demonstrated when comparing infants with CF to healthy controls, to date only one study has investigated how findings on infant PFTs translate into the preschool years (3), and it remains to be seen how these changes will progress and translate to adult-type spirometric measures in later childhood.

III.

Pulmonary Manifestations of Cystic Fibrosis in Infancy

CF begins as an obstructive process in the distal airways caused by mucus plugging due to poor mucociliary clearance, chronic inflammation, and chronic or recurrent infection. These processes eventually lead to bronchial wall thickening and bronchiectasis, as well as obstructive lung disease eventually complicated by restriction. Though many infants with CF diagnosed by newborn screening are asymptomatic at the time of diagnosis, there is evidence that airways disease may be present even in infants with no respiratory

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symptoms (3,23,24). In these asymptomatic infants, early disease is likely heterogeneous and confined to small, peripheral airways, making these abnormalities difficult to detect (25). Inhomogeneity in the distribution of CF lung disease presents a challenge for accurate measurement of pulmonary function. Minor changes in an already small disease burden may be masked by the predominance of relatively normal (particularly more central) airways in young infants (6,7). Mucus plugging resulting in peripheral airway obstruction can actually lead to exclusion of those airways from sampling by some methods of infant lung function testing (7). Certain measurements of lung function in infants (e.g., resistance and compliance) are thought to more closely measure proximal airway caliber than small, distal airways, and this can lead to underestimation of disease in infants with predominantly peripheral airways involvement (11).

IV.

Techniques for Infant Pulmonary Function Testing

A. Preparing the Infant and Devices

Infant lung function testing typically requires oral sedation, usually using chloral hydrate. Because sedation imparts increased risk to the patient, appropriate safety measures must be taken, including pulse oximetry and heart rate monitoring, as well as having oxygen and bag-mask ventilation materials on standby (16,26). A minimum of two dedicated and trained personnel need to be present throughout the procedure to assure adequate data acquisition and close monitoring of the sedated child (26). Institutional sedation policies should direct NPO rules, sedation administration, and monitoring policies. To maximize the likelihood of inducing a sleep state, procedures should be scheduled at the child’s naptime whenever possible, and the infant should be sleepdeprived prior to the study (26,27). All forms of infant PFT require specialized devices, with particular attention paid to minimizing dead space to improve accuracy (14–16,28–32). Face masks are required for infants, as they are obligatory nasal breathers, and putty is typically fitted to the face mask to prevent leaks during measurements. Dead space attributed to the face mask must be accounted for, and should not exceed a maximum of 1 to 1.5 mL/kg (14,33). Resistance of the measuring device must also be minimized (34). These measures require a pneumotachometer calibrated over the appropriate flow range, and computers for data acquisition and analysis (14). In addition, for plethysmographic measures an infant body box is needed to calculate pressure-volume changes (14). Forced expiratory maneuvers require a “squeeze jacket” or vest fitted with an appropriately sized bladder, as well as a pressure reservoir for rapid inflation (16,35). For multiple-breath washout (MBW), a study gas mixture and method of gas analysis (mass spectrometry, ultrasound, or photoacoustic spectroscopy) are necessary (Table 1) (12,36). B. Plethysmography

Plethysmography measures thoracic gas volume (TGV) or FRC (also known as FRCpleth). The infant is placed in an airtight plethysmograph and observed during tidal breathing. Using the principles of Dubois and colleagues (41), TGV is determined on the basis of changes in plethysmograph and mouth (approximating alveolar) pressures during respiratory efforts against an airway typically occluded at end-inspiration (7,32). FRC can be calculated by subtracting the tidal volume at the time of occlusion from the measured

Raw, Crs

Occlusion techniques

V0 maxFRC

FRC

Plethysmography

Partial flow-volume curves

Indices measured

Technique

. Assumes respiratory system functions as a single compartment . May not detect early peripheral airway disease . Assumption that airway opening pressure is equal to alveolar pressure not valid in severe obstruction . Unstable FRC may lead to variability in V0 maxFRC measurements . Inhalation prior to reaching RV . May not achieve flow limitation . Measuring flows over small lung volumes

459 (38)

MOT: 283 SOT: 54 (6)

. Overestimation of FRC in severe obstruction . Expensive equipment

. Commercially available equipment . Measures gas in communicating and noncommunicating airways . Commercially available equipment . Simple, noninvasive

. Commercially available equipment . Uses tidal breathing

311 (19,37)

Disadvantages

No. of healthy infants studied

Advantages

Table 1 Summary of Infant Pulmonary Function Testing Techniques

Yes (38)

No

Yes (19,37)

Reference equations available

126 Pittman and Davis

FVC, FEV0.4, FEV0.5, FEV0.75, FEF25–75, FEF50, FEF75

FRC

LCI

RV, TLC, FRC/ TLC, RV/TLC

RVRTC

Gas dilution

Multiple-breath washout

Fractional lung volumes

Yes

22 (19)

Yes (37,40)

No

. May not measure noncommunicating (completely obstructed) airways . May underestimate gas trapping . No FDA approved device in the United States for use in infants . Requires inert gases . Minimal reference data . Minimal reference data . Requires RVRTC technique and plethysmography . Expensive equipment

Yes (17)

none

469 (21,36,37, 39)

. Requires active inflation . Requires extensive expertise and personnel . Expensive equipment

. Commercially available equipment . Simulates adult-type flowvolume curves . Achieves flow limitation . Measuring flows from a consistent lung volume . Full exhalation to RV is achieved . Requires only tidal breathing . No specialized study gas requirement (N2 washout) . Commercial equipment

. Requires only tidal breathing . LCI may be sensitive marker for early peripheral airways disease . Provides information on air trapping . Detection of restrictive lung disease

433 (16)

Disadvantages

Advantages

Reference equations available

Abbreviations: FRC, functional residual capacity (by plethysmography or gas dilution); Raw, airways resistance; Crs, respiratory system compliance; FVC, forced vital capacity; FEV, forced expiratory volume; TLC, total lung capacity; LCI, lung clearance index; FEF, forced expiratory flow; V0 maxFRC, maximal flow at functional residual capacity.

Indices measured

Technique

No. of healthy infants studied

Lung Function Testing in Infants 127

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TGV (7,32,34). Specific airways resistance (sRaw) can also be calculated via plethysmography as the relationship between change in volume of the plethysmograph (Vpleth) and change in the flow (32,34). C. Occlusion Techniques

During the occlusion techniques, the Herring–Breuer reflex helps elicit relaxation of the respiratory system (11,29). Airflow, pressure, and volume changes are measured at the mouth, and changes noted at the mouth are assumed to be in equilibrium with airways during the induced respiratory pause (11). The single-breath occlusion technique (SOT) is used to evaluate the resistance, compliance, and time constant of the respiratory system (Rrs, Crs, Trs), which are determined by the characteristics of the chest wall, airways, and lung parenchyma (11,29). During this maneuver, the airway is occluded at end-inspiration during tidal breathing. This induces a respiratory pause through the Herring–Breuer reflex, at which point the occlusion is released. Compliance (Crs) is calculated as the change in volume divided by the change in pressure (7,11,29). Resistance (Rrs) is equal to the change in pressure divided by the change in flow. The time constant (Trs) (change in volume divided by change in flow) is defined as the product of Crs and Rrs. Use of the SOT in children with significant obstructive lung disease is challenging both because of their increased respiratory rate and drive, leading to an inability to invoke the Herring–Breuer reflex, and because a respiratory system with significant disease cannot be described by a single time constant, violating one of the assumptions necessary for SOT measurements (7,11,29). Crs is also measured through the multiple occlusion technique (MOT), which allows assessment at several lung volumes. During this technique, occlusions occur at 10 or more different lung volumes above FRC. Airway opening pressure and volume are assessed with each occlusion; the slope of the resultant pressure-volume measurements is the Crs (11,29). D. Forced Expiratory Flows: Partial and Raised Volume Rapid Thoracoabdominal Compression

Forced expiratory flow measures employ the rapid thoracoabdominal compression technique (compression of the thoracic cage by a squeeze jacket containing an inflatable bladder) to mimic the forced exhalations used in adult-type spirometric maneuvers (7,27). The partial flow-volume technique relies on tidal breathing and measures maximal flow at FRC (V0 maxFRC). During tidal breathing, the jacket is inflated, “squeezing” the chest at the end-inspiratory point of a tidal breath to produce a forced exhalation. Jacket pressure is increased until maximal flow no longer increases, and V0 maxFRC is reported from the curves that are presumed to be flow limited (7,27). This technique has several limitations, including (i) variability of FRC during tidal breathing, which may lead to an unstable reference for V0 maxFRC, (ii) the possibility that flow limitation may not be achieved, (iii) measurement of flows over small lung volume ranges (vs. larger ranges with the raised volume technique), and (iv) inhalation before reaching residual volume secondary to lack of adequate control over the respiratory drive (no induction of the Herring–Breuer reflex) (4–6,42). The raised volume rapid thoracoabdominal compression (RVRTC) technique avoids problems of a changing FRC by passively inflating the infant’s lungs to a

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standard pressure (typically 30 cm H2O) and initiating forced exhalation from this elevated volume. First, repeated cycles of inflation and passive exhalation are performed, followed by inflation and forced exhalation elicited through rapid inflation of the jacket bladder (43). The initial cycles of inflation with passive exhalation ensure an adequate respiratory pause (via the Herring–Breuer reflex and/or modest reduction in PaCO2) prior to the forced exhalation maneuver. The absence of an inspiratory effort during the maneuver is critical to achieve full exhalation to residual volume (RV) (16,27). This maneuver mimics adult-type flow-volume curves, is reproducible, and flow limitation has been demonstrated through transesophageal pressure catheter measurements (4,16,44). Moreover, by combining RVRTC with plethysmography, fractional lung volume measurements can be obtained (4). For the RVRTC technique, forced vital capacity (FVC) is defined as the difference between the inflation volume at 30 cmH2O and residual volume. Forced expiratory volume at 0.5 seconds (FEV0.5) (volume exhaled in the first 0.5 seconds) is measured instead of FEV1 secondary to shorter exhalation times in young children; occasionally forced expiratory volume in 0.4 seconds (FEV0.4) or 0.75 seconds (FEV0.75) are reported (16). Forced expiratory flow is measured between 25% and 75% of FVC (FEF25–75), and diminished FEF25–75 may serve as an early marker of airways obstruction. Forced expiratory flows at 25%, 50%, 75%, and 85% of FVC (FEF25, FEF50, FEF75, FEF85) are less frequently reported, but also serve as measures of obstructive airway disease. By combining the RVRTC technique with plethysmography, TLC, RV, and RV/TLC can also be determined (Fig. 1) (4,16,19). E.

Gas Dilution Techniques: FRC Measures and Multiple-Breath Washout

FRC can also be determined in infants by helium dilution or via N2 washout. Helium dilution employs a rebreathing system (with bleeding in of O2 and removal of CO2) in which the patient breathes a known concentration of marker gas (helium) until gas concentrations in the study apparatus and infant lung have equilibrated. FRC is calculated using the change in helium concentration and the known volume of the spirometer or study apparatus (12). Bias flow nitrogen washout is performed by measuring exhaled N2 concentration while having an infant breathe 100% oxygen until exhaled N2 reaches a specified concentration. FRC is calculated from the amount of N2 expired (12). Multiple-breath washout (MBW) measures ventilation inhomogeneity utilizing diffusion of inert (marker) gases [N2, He, or sulfur hexafluoride (SF6)]. The measures most often reported from MBW studies are FRC and lung clearance index (LCI); LCI is a measure of the number of lung volume turnovers necessary to clear the marker gas to a specified concentration (12,46). The degree of inhomogeneity determined by the LCI is considered to be a sensitive indicator of obstructive lung disease. To measure ventilation inhomogeneity using a bias flow system, the subject inhales a study gas mixture with known concentration of inert gas (often 4% SF6) until inhaled and exhaled marker gas levels are equal (the washin phase). At this point, study gas flow is terminated and the patient breathes room air until the exhaled marker gas concentration reaches 1/40 of the level at the end of the washin phase (washout phase) (Fig. 2). The cumulative expired volume divided by the FRC defines the LCI; a higher LCI equates to a larger number of lung volume turnovers necessary to clear the lungs of marker gas, implying ventilation inhomogeneity (12,46,47).

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Figure 1 The RVRTC technique: (A) Sedated infant undergoing pulmonary function testing via

the RVRTC technique. (B) Schematic diagram of equipment for the RVRTC technique. The lungs of the infant are inflated to 30 cm H2O using a pop-off valve that is part of the circuit. After several cycles of inflation and passive exhalation through the open expiratory valve, the jacket bladder wrapped around the infant’s thoracic cavity is rapidly inflated via the pressure reservoir at end-inspiration, leading to a forceful exhalation. Abbreviation: RVRTC, raised volume rapid thoracoabdominal compression. Source: Part A from Ref. 4 and part B from Ref. 4.

Other, less frequently reported measures include moment ratios and concentration-normalized phase III slope analysis, which are less intuitive for interpretation, and, in the case of moment ratios, appear to be no more sensitive than LCI for detection of early obstructive disease (12); discussion of these indices is beyond the scope of this chapter.

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131

Figure 2 The multiple-breath washout technique: (A) During the washin phase, the subject inhales a gas mixture with a known concentration of inert marker gas via a bias flow setup until the exhaled and inhaled concentrations (as measured by the gas analyzer) are in equilibrium. (B) During the washout phase, study gas flow is terminated, and the subject breathes room air until the marker gas concentration has decreased to a predetermined cutoff point

V. Research Studies Employing Infant Pulmonary Function Testing A. Observational Studies

Abnormal lung function in infants with CF has been demonstrated using a variety of lung function testing techniques. Kraemer and colleagues showed diminished specific airway conductance (sGaw) in 26% of their infant cohort with CF evaluated using

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plethysmography (48). Beardsmore et al. showed a strong correlation between sGaw and physicians’ clinical score in CF infants, with those with more prominent symptoms having decreased airway conductance (49). Assessing respiratory system compliance (Crs) using deflation pressure-volume curves from an inflation pressure of 30 cmH2O, Tepper and colleagues found no difference between CF infants and healthy controls (50). In an earlier study, the same group found that CF infants with pulmonary symptoms and failure to thrive at diagnosis had significantly lower Crs (measured via a weighted spirometer technique) than normal controls or CF infants presenting with meconium ileus or isolated failure to thrive (20). The conflicting results between these two studies may be due to differences in Crs measurement techniques, since the weighted spirometer measurement is performed over a smaller range of lung volumes. As this data demonstrates, the effect of CF on airway conductance and elastic properties of the respiratory system in early childhood has yet to be fully elucidated. FRC measured via plethysmography has been shown to be significantly elevated in infants with CF compared with healthy controls (51,52), and an elevated TGV has been correlated with worsening clinical scores (49). Hiatt et al. demonstrated a significant correlation between lower respiratory tract infection and elevated FRC measured via helium dilution in CF infants (21). In addition, a different set of investigators found a significant elevation in FRC values when comparing CF infants to healthy controls (20). Together, these findings suggest that hyperinflation plays an important role in the early pathogenesis of CF lung disease. Many studies in the late 1980s and early 1990s focused on measurements of V0 maxFRC using forced expiratory flow measured from the tidal breath. In a crosssectional study of CF infants designed to investigate associations between symptoms at clinical presentation and lung function, V0 maxFRC was noted to be diminished in CF infants with respiratory symptoms and failure to thrive at presentation as compared with both healthy controls and CF infants presenting with either meconium ileus or failure to thrive (20). Baseline V0 maxFRC has also been demonstrated to be significantly diminished in CF infants compared with healthy controls at a mean age of 10 months, and this difference persisted after the completion of the respiratory viral season despite an equal number of upper respiratory tract illnesses reported in the two groups (CF infants were also more likely to have lower respiratory tract illnesses) (21). Two longitudinal studies of V0 maxFRC in the first year of life showed that diminished V0 maxFRC correlated both with respiratory symptoms at initial CF clinical presentation (53) and with respiratory severity score assigned by a clinician (49). Thus, decreased V0 maxFRC has been shown to be present in the youngest population with CF. The RVRTC technique has superseded the partial flow-volume technique for obtaining forced expiratory flows in infants because of high intra- and intersubject variability of V0 maxFRC (42) compared with flows obtained via the RVRTC technique, as well as the potential increased sensitivity of raised volume measures over those obtained over only the tidal range of breathing. As an illustration, Turner demonstrated that FEV0.5 and FEV0.75 as measured by the RVRTC technique were both significantly diminished in CF infants compared with healthy controls, while there was no significant difference in V0 maxFRC between the two groups (54). More recently, FEV0.5 was noted to be more sensitive at detecting airway disease in CF infants when compared with V0 maxFRC measures, with 13 of 47 CF infants having significantly diminished FEV0.5 values, while only one CF infant had a decreased V0 maxFRC value (55).

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133

Over the past 10 years, the RVRTC technique has been employed to better characterize the pulmonary disease burden in infants with CF. In 2001, a significant reduction in forced expiratory volume in 0.4 seconds (FEV0.4) and forced expiratory flow at 75% of forced vital capacity (FEF75) was demonstrated in infants with CF compared with healthy controls (10). Interestingly, this difference persisted when comparing only those CF infants with no recognized history of a lower respiratory illness to healthy controls (10), suggesting that airway damage from CF lung disease occurs “silently” in early childhood. In a 2004 study employing RVRTC soon after CF diagnosis with measures repeated six months later, the same group found significant reduction in mean FVC, FEV0.5, and FEF75 z-scores in CF subjects at the time of the first study (mean age 28 weeks), which persisted at the return visit (56). Strikingly, 41% of CF subjects had an FEV0.5 below the fifth percentile (z score below 1.64) at both study visits (56). Linnane and colleagues recently showed diminished FEV0.5 and FEF75 in infants with CF diagnosed by newborn screen compared with healthy controls, with differences becoming significant at six months of age (23). These results stress the significant disease burden present in the youngest CF population and also highlight a potential therapeutic window where preventative efforts may have maximal effect. Kozlowska and colleagues recently completed the first longitudinal study of lung function in children with CF in which repeated RVRTC measures (for infants) and incentive spirometry measures (for preschoolers) were performed. Forty-eight children with CF and 33 healthy controls had at least two lung function studies performed before age six. Subjects with CF showed a significant decline in FEV 0.75 and FEF25–75 over time compared with controls. Growth of Pseudomonas aeruginosa on respiratory culture was associated with a further significant decline in FEV0.75 and FEV1, as well as in FEV0.5 and FVC. Wheeze and cough were also associated with a greater decline in FEV0.5 and FEF25–75 (3). This landmark study not only confirms previously found differences in forced expiratory values between CF subjects and healthy controls, but also highlights the variability in the rate of decline of these measurements in CF subjects that may be due, in part, to infectious status, though contributions from genetic modifiers, environmental exposures, and nutrition may also play a role. The MBW technique has recently been evaluated in infants with CF. A 2007 study demonstrated similar sensitivities in identifying CF infants versus healthy controls when comparing the LCI measured by MBW with FEV0.5 and FEF25–75 measured by RVRTC. Over 70% of infants with CF had an abnormal LCI and/or abnormal forced expiratory values (41% had abnormal LCI, 41% had abnormal forced expiratory values, with some overlap). Fifteen percent of infants with CF had an abnormal LCI with normal forced expiratory values, and 15% had abnormal forced expiratory values with normal LCI (24). On the basis of this one published study, the two techniques appear to have similar sensitivity in infants; however, LCI has been shown to be more sensitive than FEV0.5 or FEV1 for identification of small airway disease in CF preschoolers and older children, respectively (57,58). Furthermore, the lack of overlap between the two measures in 30% of infant subjects suggests they may, in part, be assessing different aspects of CF lung disease. These studies show the presence of early physiologic abnormalities in infants with CF, even those who are asymptomatic or have been diagnosed by newborn screening (Table 2). These results emphasize the importance of further study in our youngest

Year

1988

1988

1993

Author

Observational Beardsmore (49)

Tepper (20)

Tepper (53)

AIM: Longitudinal evaluation of partial flow-volume curves, plethysmographic measures, and clinical score in first year of life. FINDINGS: Correlation was seen between all parameters and clinical score at time of initial testing and between sGaw and V0 maxFRC. AIM: Evaluation of CF infants presenting with meconium ileus (mec), failure to thrive (ftt), and pulmonary and FTT (comb) symptoms compared with healthy controls, by partial flow-volume curves, compliance, and He dilution. FINDINGS: CF subjects presenting with pulmonary and FTT symptoms had lower Crs and V0 maxFRC than other CF subjects and controls. AIM: Longitudinal evaluation of CF infants at presentation and 1 yr later, comparing those with and without respiratory symptoms (RESP) at presentation by partial flow-volume curves and He dilution. FINDINGS: At presentation, subjects with respiratory symptoms had lower V0 maxFRC than those without. V0 maxFRC had improved at 1 yr follow-up, but remained lower in the RESP group.

AIM and findings

Table 2 Summary of Selected Studies Involving Infant Pulmonary Function Testing in CF

Healthy

32 (14 RESP) (5.4 mo)a

25 [1 mo (mec), 4 mo (ftt), 5 mo (comb)]a

28 (5–57 wk) repeated in 17

33 (6 mo)a

(Age at initial evaluation)a,b

CF

Number of subjects

. V0 maxFRC . FRC

. V0 maxFRC . FRC . Crs

. sGaw . V0 maxFRC . TGV

Parameters

134 Pittman and Davis

Year

1994

1999

2000

2001

2001

Author

Turner (54)

Hiatt (21)

Kraemer (48)

Davis (22)

Ranganathan (10)

AIM: Comparison of infants with CF with and without previous respiratory illness vs. healthy controls by RVRTC. FINDINGS: FEV0.4 and FEF75 were diminished in infants with CF, with similar reductions in those with and without previous respiratory symptoms.

AIM: Comparison of asymptomatic infants with CF vs. historic healthy controls by RVRTC vs. partial flowvolume curves. FINDINGS: FEV0.5 and FEV0.75 were diminished in the CF group compared with controls. V0 maxFRC did not distinguish between the two groups. AIM: Comparison of CF infants vs. healthy controls preand postrespiratory viral season by partial flow-volume curves and He dilution. FINDINGS: V0 maxFRC was diminished in CF infants vs. healthy controls at baseline; the difference persisted after viral illness. Lower respiratory tract infection resulted in further diminished V0 maxFRC in CF infants. AIM: Longitudinal evaluation by plethysmography in first 2 years of life; correlation of lung function with genotype. FINDINGS: Only 19% of CF infants had normal lung function in the first year of life. Decline in SGaw from 6 to 12 mo of age. AIM: Comparison of CF infants vs. healthy controls by RVRTC breathing room air and heliox. FINDINGS: CF infants had lower FEF50 than controls. Density dependence comparisons and flows measured with heliox did not distinguish between CF and controls.

AIM and findings

Healthy

33 (24.5 wk)a

10 (4–21 mo)

80 (4.6 mo)a repeated in 50

22 (10 mo)a

12 (10 mo)b

V0 maxFRC FEV0.5 FEV0.75 FEV1

. TGV . sGaw

. V0 maxFRC . FRC

. . . .

Parameters

(continued)

21 . FVC (2–24 mo) . FEV0.5 . FEF50 . FEF75 . FEF25–75 . DD50 . DD75 87 . FEV0.4 (8 wk)b . FEF75

27 (10 mo)a

26 (14 mo)b

(Age at initial evaluation)a,b

CF

Number of subjects

Lung Function Testing in Infants 135

Year

2002

2004

2004

2004

Author

Ranganathan (55)

Castile (51)

Ranganathan (56)

Tepper (50)

AIM: Comparison of CF infants vs. healthy controls by RVRTC and partial flow-volume curves. FINDINGS: FEV0.5 was more sensitive for CF than V0 maxFRC (diminished in 13 of 47 and 1 of 47, respectively). RVRTC measures were diminished in CF infants with and without prior lower respiratory tract illnesses. AIM: Comparison of CF infants and healthy controls by RVRTC, plethysmographic FRC, nitrogen washout FRC, and fractional lung volumes FINDINGS: FEF50, FEF75, and FEF25–75 were significantly lower in CF infants vs. controls. RV, FRCpleth, and FRCnitrogen were higher in CF infants. No difference in trapped gas measurements between CF and control subjects. AIM: Longitudinal comparison of CF infants vs. healthy controls by RVRTC. FINDINGS: FVC, FEV0.5, and FEF75 were all diminished both soon after birth and 6 mo later in CF infants compared with controls. AIM: Comparison of respiratory system compliance measured using passive deflation pressure-volume curves and RVRTC in infants with CF vs. controls. FINDINGS: Subjects with CF had lower FEF50, FEF75, FEF25–75. No difference in Crs measured using static deflation pressure-volume curves from an elevated lung volume between CF and controls.

AIM and findings

Healthy

Crs FVC FEF50 FEF75 FEF25–75

. . . . . 10 (68 wk)a

34 (28 wk)a

RV TLC FRCpleth V0 maxFRC FRCnitrogen FEF50 FEF75 FEF25–75 FVC FEV0.5 FEF75

. . . . . . . . . . . 29 (79 wk)a

34 (51 wk)a

32 (7 wk)a

30 (66 wk)a

187 . FVC (1–100 wk) . FEV0.5 . FEF75 . FEF25–75 . V0 maxFRC

Parameters

47 (6–93 wk)

(Age at initial evaluation)a,b

CF

Number of subjects

Table 2 Summary of Selected Studies Involving Infant Pulmonary Function Testing in CF (Continued )

136 Pittman and Davis

2007

2008

Lum (24)

Kozlowska (3)

AIM: Comparison of MBW and RVRTC for evaluation of lung disease in CF vs. healthy controls. FINDINGS: CF subjects had elevated LCI and FRC and lower FVC, FEV0.5, FEF75, and FEF25–75 compared with controls. 72% of CF subjects had abnormal lung function (41% by both techniques, 15% by MBW or RVRTC alone). AIM: Longitudinal evaluation of CF infants vs. healthy controls in the first 6 yrs of life by RVRTC and preschool spirometry. FINDINGS: Subjects with CF had greater decline in FEV0.75 and FEF25–75 than controls. Those who grew Pseudomonas aeruginosa had reduced FVC, FEV0.5, and FEV0.75. Wheeze was associated with diminished FEV0.5, FEV0.75, and FEF25–75. Cough was associated with diminished FEV0.5 and FEF25–75.

AIM and findings

Correlation with other measures of pulmonary disease Rosenfeld (59) 2001 AIM: Correlation of partial flow-volume curves and FRC with BAL findings. FINDINGS: FRC lower at age 2 in subjects with no pathogen growth vs. those with growth. FRC higher and V0 maxFRC lower in those with Pseudomonas aeruginosa on BAL culture. Dakin (60) 2002 AIM: Correlation of single-breath occlusion and nitrogen washout with BAL findings. FINDINGS: Subjects with higher (>105 cfu/mL) pathogen burden on BAL had lower sCrs and higher FRC/TLC. Martinez (61) 2005 AIM: Correlation of HRCT findings and RVRTC in infants with CF. FINDINGS: Higher airway wall area:lumen area ratio (increased wall thickness) was associated with lower FEV0.5, FEF50, and FEF75.

Year

Author

Healthy

33 (0.2 yr)a

48 (0.6 yr)a

11 (17 mo)a

22 (23.2 mo)a

40 (3.9 mo)a (at diagnosis)

21 (37 wk)a

39 (41 wk)a

(Age at initial evaluation)a,b

CF

Number of subjects

FVC FEV0.5 FEV0.75 FEV1 FEF25–75

. . . . .

. . . . . . .

(continued)

sCrs sRrs FRC TLC FEV0.5 FEF50 FEF75

. V0 maxFRC . FRC

LCI FRC FVC FEV0.5 FEF75 FEF25–75

. . . . . .

Parameters

Lung Function Testing in Infants 137

2005

2008

2009

Nixon (62)

Linnane (23)

PetersonCarmichael (63)

AIM: Correlation of RVRTC and BAL findings. FINDINGS: Respiratory pathogen growth in BAL and daily cough were independently, additively associated with reduction in FEV0.5. AIM: Correlation of RVRTC and BAL in CF infants diagnosed by newborn screening. FINDINGS: Subjects with CF demonstrated a decline in FVC, FEV0.5, and FEF75 beginning at 6 mo of age. No association of RVRTC measures with BAL markers or culture results. AIM: Determine correlation of RVRTC and plethysmography with BAL findings in infants with CF undergoing an exacerbation. FINDINGS: Direct correlation between neutrophil percentage on BAL and FRC, RV/TLC, and FRC/TLC. Indirect correlation between neutrophil percentage and FEF75 and FEF25–75. Correlation between increased pathogen density and FRC (increased) and FEF75 and FEF25–75 (decreased).

AIM and findings

Therapeutic/interventional Hiatt (64) 1988 AIM: Determine responsiveness to inhaled bronchodilator in CF infants vs. healthy controls by partial flowvolume curves. FINDINGS: Infants with CF had lower V0 maxFRC at baseline than healthy controls. After inhaled metaproterenol, V0 maxFRC in CF infants was equivalent to healthy controls.

Year

Author

. FEV0.5 . FEV0.75 . FEV1

36 (17.8 mo)a

. V0 maxFRC

FRC FVC FEV0.5 FEF75 FEF25–75 RV TLC

. . . . . . . 16 (86 wk)b

28 (16 mo)a

FVC FEV0.5 FEF75 FEF25–75

. . . .

68 (59 wk)a repeated in 16

22 (13 mo)a

Parameters

Healthy

(Age at initial evaluation)a,b

CF

Number of subjects

Table 2 Summary of Selected Studies Involving Infant Pulmonary Function Testing in CF (Continued )

138 Pittman and Davis

Year

1989

1994

1997

1998

2003

Author

Kraemer (52)

Beardsmore (65)

Tepper (66)

Clayton (67)

Berge (45)

AIM: Investigation of plethysmography and response to oral bronchodilator. FINDINGS: 15 subjects with TGV > 130% predicted, 13 subjects with Raw >130% predicted. Raw, sGaw, and TGV improved with oral salbutamol. AIM: Comparison of plethysmographic measures and partial flow-volume curves in CF infants receiving antistaphylococcal antibiotic prophylaxis vs. those receiving episodic antibiotics with exacerbations. FINDINGS: No difference in measurements between group receiving prophylactic treatment and the group receiving antibiotics only with exacerbations. AIM: Randomized control trial of effect of IV hydrocortisone during pulmonary exacerbation on partial flow-volume curves and FRC measured via nitrogen washout. FINDINGS: V0 maxFRC significantly increased 1–2 mo after hospitalization in treatment (Rx) group. FRC slightly decreased in the treatment group and slightly increased in placebo group. AIM: Characterization of airway disease before and after hospitalization and treatment for pulmonary CF exacerbation. FINDINGS: V0 maxFRC, GL, and CL significantly increased after treatment for CF exacerbation. AIM: Pilot study investigating effect of 2 wk of inhaled DNase vs. normal saline on partial flow-volume curves in CF infants. FINDINGS: Significant increase in V0 maxFRC after 2 wk of inhaled DNase.

AIM and findings

. TGV . sGaw . V0 maxFRC

. FRC . V0 maxFRC

42 (16.4 wk)a

20 [8.8 mo (Rx), 9.5 mo (placebo)]a

9 (1.4 yr)a

(continued)

. V0 maxFRC

. V0 maxFRC . GL . CL

. TGV . Raw . sGaw

24 (2.8 mo)a

17 (15.1 mo)a

Parameters

Healthy

(Age at initial evaluation)a,b

CF

Number of subjects

Lung Function Testing in Infants 139

2007

2008

Subbarao (68)

Dellon (69)

AIM: Investigate safety and tolerability of inhaled 7% hypertonic saline in infants using RVRTC. FINDINGS: No significant change in FVC, FEV0.5, or FEF25–75 after bronchodilator or inhaled hypertonic saline. AIM: Investigate safety and tolerability of 3% and 7% inhaled hypertonic saline using RVRTC. FINDINGS: No change in FVC, FEV0.5, or FEF25–75 following administration of 3% or 7% inhaled hypertonic saline.

AIM and findings

6 (3%), 8 (7%) (1.6 yr)a

13 (81)a

(Age at initial evaluation)a,b

Healthy

. FVC . FEV0.5 . FEF25—75

. FVC . FEV0.5 . FEF25–75

Parameters

b

Mean age reported. If repeated studies, age is at time of first lung function testing. Median age reported. If repeated studies, age is at time of first lung function testing. Abbreviations: FRC, functional residual capacity (by plethysmography or gas dilution); FVC, forced vital capacity; FEV, forced expiratory volume; MOT, multiple occlusion technique; SOT, single occlusion technique; RV, residual volume; FDA, Food and Drug Administration; TLC, total lung capacity; LCI, lung clearance index; CF, cystic fibrosis; FEF, forced expiratory flow; RVRTC, raised volume rapid thoracoabdominal compression technique; BAL, bronchoalveolar lavage; CL, lung compliance, Crs, respiratory system compliance; V0 maxFRC, maximal flow at functional residual capacity.

a

Year

Author

CF

Number of subjects

Table 2 Summary of Selected Studies Involving Infant Pulmonary Function Testing in CF (Continued )

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patients with CF to better delineate pulmonary disease burden and progression, as well as the need for investigations that determine risk factors for CF airway damage. B. Correlation with Other Measures of Pulmonary Disease

Measures of infant lung function in CF subjects have been shown to correlate with both structural airway disease seen on high-resolution chest computed tomography (CT) and markers of airway inflammation and infection on bronchoalveolar lavage (BAL). Rosenfeld and colleagues demonstrated that lower FRC was associated with a lack of respiratory pathogen growth as assessed through BAL in two-year-old subjects with CF; FRC was significantly elevated and V0 maxFRC significantly diminished in subjects with a history of P. aeruginosa on BAL fluid culture (59). Diminished specific compliance of the respiratory system (sCrs, or Crs normalized for FRC) has been associated with greater infectious burden (>105 colony-forming units/mL) on BAL (60). Recently, Linnane et al. found no significant association between forced expiratory measures (RVRTC) and either inflammatory markers or culture results on BAL (23). This is in contrast to the findings of Peterson-Carmichael and colleagues, who demonstrated a direct correlation between neutrophil percentage on BAL and FRC, RV/TLC, and FRC/TLC, as well as an inverse correlation with FEF75 and FEF25–75 in subjects experiencing an acute pulmonary exacerbation (63). Though this group did not find a difference in lung function parameters between those with and without infection as determined by BAL, they did note an association between increased pathogen density on BAL and higher FRC, lower FEF75, and lower FEF25–75 (63). The variability of these results may be due to differences in disease burden between the two study populations (baseline vs. pulmonary exacerbation), but also implies that we have yet to definitively answer the question of association between lung function measures and infectious or inflammatory burden as determined by BAL (Table 2). High-resolution chest CT has been shown to correlate both with lung function testing (MBW and spirometry) in older children with CF (57) and with disease burden measured by BAL in CF infants (2); however, limited studies have been done comparing CT and lung function measures in CF infants. Martinez and colleagues compared forced expiratory flows (by RVRTC) and CT findings in 11 infants with CF aged 8 to 33 months. They found a significant negative correlation between the ratio of wall area to lumen area and FEV0.5, FEF50, and FEF75 (61). Thus, airway wall thickening appears to be associated with increased airway obstruction in infants with CF. C. Interventional Studies

There are relatively few interventional studies in infants employing lung function testing as an outcome measure. The lack of studies is likely due to the inherent institutional difficulties involved in studying infants, the technical expertise required for proper use of these techniques, the relatively new development of reliable commercial equipment to perform the techniques (such as RVRTC), and the large sample size requirement for such studies necessitating a multicenter collaboration. Tepper et al. performed a randomized control trial investigating the effect of 10 days of hydrocortisone versus placebo in 20 infants hospitalized with CF exacerbations. Results demonstrated that, while there was no difference between the two groups 10 days into treatment based on lung function measures, the steroid group had significantly increased V0 maxFRC at the one- to two-month follow-up compared with the

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placebo group (66). FRC was noted to have decreased slightly in the treatment group and increased slightly in the placebo group at follow-up, though this finding did not reach statistical significance. The authors suggested that intravenous steroid therapy during an acute exacerbation may diminish airway obstruction and air trapping. Clayton and colleagues assessed change in lung function following hospitalization and antibiotic therapy for CF exacerbation in a cohort of 17 infants and toddlers with CF. They demonstrated a significant improvement in V0 maxFRC following treatment, with those subjects with the most diminished lung function values at study initiation showing the greatest improvement in airway obstruction (67). Lung compliance (CL) and lung conductance (GL), as measured by esophageal balloon catheters, also improved following therapy (67). In a similar study employing RVRTC, investigators found statistically significant improvements in FEF25, FEF50, FEF75, FEF85, FEF25–75, V0 maxFRC, FVC, FRC, and RV/TLC in 44 CF infants compared before and after a mean of 13 days of antibiotic therapy for pulmonary exacerbation (70). Two groups have recently published tolerability studies of inhaled 3% and 7% hypertonic saline in a total of 25 children under three years of age with CF (68,69). Both centers performed RVRTC maneuvers postbronchodilator and repeated the measures post hypertonic saline. One of the groups also performed the RVRTC maneuvers prior to any inhaled therapies (68,69). Results from both studies showed no significant change in FEV0.5 or FEF25–75 post 3% or 7% hypertonic saline. The authors of both studies concluded that inhaled hypertonic saline was acutely well tolerated in infants with CF. These studies show that the RVRTC technique may be used to generate outcome measures both for short-term and long-term interventional trials in infants with CF (Table 2).

VI.

Clinical Uses

The role of PFT in the clinical management of infants with CF has yet to be determined. Many CF centers perform physiologic measures as a clinical tool in infants and toddlers with CF on an annual or semiannual basis, though this has not yet been adopted as standard of care. On the basis of our experience and recently published data (71), it appears that results of lung function testing may influence clinical decision making in the care of infants and toddlers with CF. There is no commercially available, FDA-approved device for performing multiple-breath washout maneuvers in infants in the United States. Though available in Europe, MBW is, much like RVRTC, limited to large tertiary care centers with active research in pediatric pulmonary disease. While a few experienced European physicians have begun using MBW in clinical practice, further study is necessary to determine its role in guiding clinical management.

VII.

Future Directions

Infant PFT shows significant promise in detecting burden of lung disease in infants with CF. However, further study is needed to determine the role these tests will assume in regular clinical practice. In particular, further correlation of RVRTC measures with preschool and adult-type spirometry findings later in life and longitudinal studies of the MBW technique in infants and preschoolers are necessary to determine the long-term

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clinical significance of early abnormalities in lung function measures. Correlation with inflammatory mediators, infectious status, pulmonary exacerbations, and findings on chest imaging will also help better define the importance of abnormal lung function measures in infants with CF. As newborn screening is now widely available in the United States, Europe, and Australia, this is an ideal time to undertake such studies. Questions that might be addressed include the impact of infant PFT on management of the CF infant (including antibiotic use and the use of chronic therapies), the effect of PFTs on nutritional status in infancy, and the impact of the clinical use of infant PFTs on pulmonary function in later childhood. There is a surprising paucity of therapeutic trials in CF utilizing infant PFT as an outcome measure. Because it is now recognized that CF pulmonary disease begins in infancy, it will become increasingly important to determine the efficacy and long-term benefits of therapeutic interventions in the infant population. This will require larger, multicenter trials to obtain adequate subject numbers, which raises questions of feasibility and quality assurance. Davis and colleagues have recently shown the feasibility of performing infant PFTs using the RVRTC technique coupled with plethysmography in a 10 site multicenter trial, with 72% to 88% of studies performed deemed to be of research quality (72). This was accomplished through rigorous attention to detailed standardized operating protocols, training, quality assurance, and overreading of studies by a central, lead site. A multicenter study of inhaled hypertonic saline in infants with CF using FEV0.5 as an exploratory outcome measure is currently enrolling. Infant PFT has tremendous research and clinical potential in the CF population. With proper training and personnel, these techniques will reveal unseen aspects of CF lung disease in our youngest, most vulnerable patients, where clinically silent damage to airways may be occurring during rapid lung growth (6). As a tool in longitudinal studies, therapeutic trials, and eventually clinical management, infant PFT may aid in further improvements in the morbidity associated with CF lung disease.

Abbreviations BAL CL Crs CT DD FEF25-75 FEF25 FEF50 FEF75 FEF85 FEV0.5 FEV0.75 FEV1 FRC FRCpleth FVC GL

bronchoalveolar lavage lung compliance respiratory system compliance computed tomography density dependence forced expiratory flow between 25 and 75% of FVC forced expiratory flow at 25% of FVC forced expiratory flow at 50% of FVC forced expiratory flow at 75% of FVC forced expiratory flow at 85% of FVC forced expiratory volume in 0.5 seconds forced expiratory volume in 0.75 seconds forced expiratory volume in 1 second functional residual capacity (by plethysmography or gas dilution) functional residual capacity measured by plethysmography forced vital capacity lung conductance

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LCI MBW MOT PFT Raw Rrs RVRTC sCrs sGaw sRrs SOT TGV TLC Trs V0 maxFRC

lung clearance index Multiple-breath washout multiple occlusion technique pulmonary function testing airways resistance resistance of the respiratory system raised volume rapid thoracoabdominal compression specific compliance of the respiratory system specific airways conductance specific resistance of the respiratory system single-breath occlusion technique thoracic gas volume total lung capacity time constant of the respiratory system maximal flow at functional residual capacity

References 1. Armstrong DS, Hook SM, Jamsen KM, et al. Lower airway inflammation in infants with cystic fibrosis detected by newborn screening. Pediatr Pulmonol 2005; 40(6):500–510. 2. Davis SD, Fordham LA, Brody AS, et al. Computed tomography reflects lower airway inflammation and tracks changes in early cystic fibrosis. Am J Respir Crit Care Med 2007; 175(9):943–950. 3. Kozlowska WJ, Bush A, Wade A, et al. Lung function from infancy to the preschool years following clinical diagnosis of cystic fibrosis. Am J Respir Crit Care Med 2008; 178(1):42–49. 4. Davis SD, Brody AS, Emond MJ, et al. Endpoints for clinical trials in young children with cystic fibrosis. Proc Am Thorac Soc 2007; 4(4):418–430. 5. Ranganathan S, Linnane B, Nolan G, et al. Early detection of lung disease in children with cystic fibrosis using lung function. Paediatr Respir Rev 2008; 9(3):160–167. 6. Gappa M, Ranganathan SC, Stocks J. Lung function testing in infants with cystic fibrosis: lessons from the past and future directions. Pediatr Pulmonol 2001; 32(3):228–245. 7. Stocks J. Pulmonary function tests in infants and preschool children. In: Chernick VBT, Wilmott RW, Bush A, eds. Kendig’s Disorders of the Respiratory Tract in Children. 7th ed. Philadelphia: Saunders Elsevier, 2006:129–167. 8. Stick SM. Clinical trials in infants with cystic fibrosis detected following newborn screening. Paediatr Respir Rev 2008; 9(3):176–180. 9. Linnane B, Robinson P, Ranganathan S, et al. Role of high-resolution computed tomography in the detection of early cystic fibrosis lung disease. Paediatr Respir Rev 2008; 9(3):168–174; quiz 174–175. 10. Ranganathan SC, Dezateux C, Bush A, et al. Airway function in infants newly diagnosed with cystic fibrosis. Lancet 2001; 358(9297):1964–1965. 11. Davis S, Gappa M, Rosenfeld M. Respiratory mechanics. In: Hammer J, Eber E, eds. Pediatric Pulmonary Function Testing. New York: Karger, 2005:20–33. 12. Gustafsson P, Ljungberg H. Measurement of functional residual capacity and ventilation inhomogeneity by gas dilution techniques. In: Hammer J, Eber E, eds. Pediatric Pulmonary Function Testing. New York: Karger, 2005:54–65. 13. Hammer J, Eber E. The peculiarities of infant respiratory physiology. In: Hammer J, Eber E, eds. Pediatric Pulmonary Function Testing. Basel: Karger, 2005:2–7.

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14. Frey U, Stocks J, Coates A, et al. Specifications for equipment used for infant pulmonary function testing. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J 2000; 16(4):731–740. 15. Frey U, Stocks J, Sly P, et al. Specification for signal processing and data handling used for infant pulmonary function testing. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J 2000; 16(5):1016–1022. 16. ATS/ERS statement: raised volume forced expirations in infants: guidelines for current practice. Am J Respir Crit Care Med 2005; 172(11):1463–71. 17. Jones M, Castile R, Davis S, et al. Forced expiratory flows and volumes in infants. Normative data and lung growth. Am J Respir Crit Care Med 2000; 161(2 pt 1):353–359. 18. Ranganathan SC, Hoo AF, Lum SY, et al. Exploring the relationship between forced maximal flow at functional residual capacity and parameters of forced expiration from raised lung volume in healthy infants. Pediatr Pulmonol 2002; 33(6):419–428. 19. Castile R, Filbrun D, Flucke R, et al. Adult-type pulmonary function tests in infants without respiratory disease. Pediatr Pulmonol 2000; 30(3):215–227. 20. Tepper RS, Hiatt P, Eigen H, et al. Infants with cystic fibrosis: pulmonary function at diagnosis. Pediatr Pulmonol 1988; 5(1):15–8. 21. Hiatt PW, Grace SC, Kozinetz CA, et al. Effects of viral lower respiratory tract infection on lung function in infants with cystic fibrosis. Pediatrics 1999; 103(3):619–626. 22. Davis S, Jones M, Kisling J, et al. Comparison of normal infants and infants with cystic fibrosis using forced expiratory flows breathing air and heliox. Pediatr Pulmonol 2001; 31(1):17–23. 23. Linnane BM, Hall GL, Nolan G, et al. Lung function in infants with cystic fibrosis diagnosed by newborn screening. Am J Respir Crit Care Med 2008; 178(12):1238–1244. 24. Lum S, Gustafsson P, Ljungberg H, et al. Early detection of cystic fibrosis lung disease: multiple-breath washout versus raised volume tests. Thorax 2007; 62(4):341–347. 25. Davis P. Pulmonary disease in cystic fibrosis. In: Chernick VBT, Wilmott RW, Bush A, eds. Kendig’s Diseases of the Respiratory Tract in Children. New York: Saunders Elsevier, 2006:873–886. 26. Gaultier CFM, Beardsmore C, Motoyama E, et al. Measurement conditions. In: Stocks JSP, Tepper RS, Morgan WJ, eds. Infant Respiratory Function Testing. New York: Wiley-Liss, 1996:29–44. 27. Modl M, Eber E. Forced expiratory flow-volume measurements. In: Hammer J, Eber E, eds. Pediatric Pulmonary Function Testing. New York: Karger, 2005:34–43. 28. Bates JH, Schmalisch G, Filbrun D, et al. Tidal breath analysis for infant pulmonary function testing. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J 2000; 16(6):1180–1192. 29. Gappa M, Colin AA, Goetz I, et al. Passive respiratory mechanics: the occlusion techniques. Eur Respir J 2001; 17(1):141–148. 30. Morris MG, Gustafsson P, Tepper R, et al. The bias flow nitrogen washout technique for measuring the functional residual capacity in infants. ERS/ATS task force on standards for infant respiratory function testing. Eur Respir J 2001; 17(3):529–536. 31. Sly PD, Tepper R, Henschen M, et al. Tidal forced expirations. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J 2000; 16(4):741–748. 32. Stocks J, Godfrey S, Beardsmore C, et al. Plethysmographic measurements of lung volume and airway resistance. ERS/ATS task force on standards for infant respiratory function testing. European respiratory society/American thoracic society. Eur Respir J 2001; 17(2):302–312. 33. Sly PD, Davis G. Equipment requirements for infant respiratory function testing. In: Stocks JSP, Tepper RS, Morgan WJ, eds. Infant Respiratory Function Testing. New York: Wiley-Liss, 1996:45–80.

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34. Gappa M, Hulskamp, G. Infant whole-body plethysmography. In: Hammer J, Eber E, eds. Pediatric Pulmonary Function Testing. New York: Karger, 2005:44–53. 35. LeSouef PN CR, Turner DJ, Motoyama E, et al. Forced expiratory maneuvers. In: Stocks J SP, Tepper RS, Morgan WJ, eds. Infant Respiratory Function Testing. New York: WileyLiss, 1996:379–409. 36. Pillow JJ, Ljungberg H, Hulskamp G, et al. Functional residual capacity measurements in healthy infants: ultrasonic flow meter versus a mass spectrometer. Eur Respir J 2004; 23(5):763–768. 37. Stocks J, Quanjer PH. Reference values for residual volume, functional residual capacity and total lung capacity. ATS workshop on lung volume measurements. Official statement of the European respiratory society. Eur Respir J 1995; 8(3):492–506. 38. Hoo AF, Dezateux C, Hanrahan JP, et al. Sex-specific prediction equations for Vmax(FRC) in infancy: a multicenter collaborative study. Am J Respir Crit Care Med 2002; 165(8): 1084–1092. 39. Latzin P, Sauteur L, Thamrin C, et al. Optimized temperature and deadspace correction improve analysis of multiple-breath washout measurements by ultrasonic flowmeter in infants. Pediatr Pulmonol 2007; 42(10):888–897. 40. Tepper RS, Reister T. Forced expiratory flows and lung volumes in normal infants. Pediatr Pulmonol 1993; 15(6):357–361. 41. Dubois AB, Botelho SY, Bedell GN, et al. A rapid plethysmographic method for measuring thoracic gas volume: a comparison with a nitrogen washout method for measuring functional residual capacity in normal subjects. J Clin Invest 1956; 35(3):322–326. 42. Henschen M, Stocks J. Assessment of airway function using partial expiratory flow-volume curves: how reliable are measurements of maximal expiratory flow at FRC during early infancy? Am J Respir Crit Care Med 1999; 159(2):480–486. 43. Davis SD JR, Flucke RL, Kisling JA, et al. AARC clinical practice guideline: infant/toddler pulmonary function tests—2008 Revision & Update. Respir Care 2008; 53(7):929–945. 44. Feher A, Castile R, Kisling J, et al. Flow limitation in normal infants: a new method for forced expiratory maneuvers from raised lung volumes. J Appl Physiol 1996; 80(6):2019–2025. 45. Berge MT, Wiel E, Tiddens HA, et al. DNase in stable cystic fibrosis infants: a pilot study. J Cyst Fibros 2003; 2(4):183–188. 46. Gustafsson PM, Kallman S, Ljungberg H, et al. Method for assessment of volume of trapped gas in infants during multiple-breath inert gas washout. Pediatr Pulmonol 2003; 35(1):42–49. 47. Aurora P, Gustafsson P, Bush A, et al. Multiple-breath inert gas washout as a measure of ventilation distribution in children with cystic fibrosis. Thorax 2004; 59(12):1068–1073. 48. Kraemer R, Aebi C, Casaulta Aebischer C, et al. Early detection of lung disease and its association with the nutritional status, genetic background and life events in patients with cystic fibrosis. Respiration 2000; 67(5):477–490. 49. Beardsmore CS, Bar-Yishay E, Maayan C, et al. Lung function in infants with cystic fibrosis. Thorax 1988; 43(7):545–551. 50. Tepper RS, Weist A, Williams-Nkomo T, et al. Elastic properties of the respiratory system in infants with cystic fibrosis. Am J Respir Crit Care Med 2004; 170(5):505–507. 51. Castile RG, Iram D, McCoy KS. Gas trapping in normal infants and in infants with cystic fibrosis. Pediatr Pulmonol 2004; 37(5):461–469. 52. Kraemer R. Early detection of lung function abnormalities in infants with cystic fibrosis. J R Soc Med 1989; 82(suppl 16):21–25. 53. Tepper RS, Montgomery GL, Ackerman V, et al. Longitudinal evaluation of pulmonary function in infants and very young children with cystic fibrosis. Pediatr Pulmonol 1993; 16(2): 96–100.

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54. Turner DJ, Lanteri CJ, LeSouef PN, et al. Improved detection of abnormal respiratory function using forced expiration from raised lung volume in infants with cystic fibrosis. Eur Respir J 1994; 7(11):1995–1999. 55. Ranganathan SC, Bush A, Dezateux C, et al. Relative ability of full and partial forced expiratory maneuvers to identify diminished airway function in infants with cystic fibrosis. Am J Respir Crit Care Med 2002; 166(10):1350–1357. 56. Ranganathan SC, Stocks J, Dezateux C, et al. The evolution of airway function in early childhood following clinical diagnosis of cystic fibrosis. Am J Respir Crit Care Med 2004; 169(8):928–933. 57. Gustafsson PM, De Jong PA, Tiddens HA, et al. Multiple-breath inert gas washout and spirometry versus structural lung disease in cystic fibrosis. Thorax 2008; 63(2):129–134. 58. Aurora P, Bush A, Gustafsson P, et al. Multiple-breath washout as a marker of lung disease in preschool children with cystic fibrosis. Am J Respir Crit Care Med 2005; 171(3):249–256. 59. Rosenfeld M, Gibson RL, McNamara S, et al. Early pulmonary infection, inflammation, and clinical outcomes in infants with cystic fibrosis. Pediatr Pulmonol 2001; 32(5):356–366. 60. Dakin CJ, Numa AH, Wang H, et al. Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 2002; 165(7):904–910. 61. Martinez TM, Llapur CJ, Williams TH, et al. High-resolution computed tomography imaging of airway disease in infants with cystic fibrosis. Am J Respir Crit Care Med 2005; 172(9):1133–1138. 62. Nixon GM, Armstrong DS, Carzino R, et al. Early airway infection, inflammation, and lung function in cystic fibrosis. Arch Dis Child 2002; 87(4):306–311. 63. Peterson-Carmichael SL, Harris WT, Goel R, et al. The association of lower airway inflammation with physiologic findings in young children with cystic fibrosis. Pediatr Pulmonol 2009; 44(5):503–511. 64. Hiatt P, Eigen H, Yu P, et al. Bronchodilator responsiveness in infants and young children with cystic fibrosis. Am Rev Respir Dis 1988; 137(1):119–122. 65. Beardsmore CS, Thompson JR, Williams A, et al. Pulmonary function in infants with cystic fibrosis: the effect of antibiotic treatment. Arch Dis Child 1994; 71(2):133–137. 66. Tepper RS, Eigen H, Stevens J, et al. Lower respiratory illness in infants and young children with cystic fibrosis: evaluation of treatment with intravenous hydrocortisone. Pediatr Pulmonol 1997; 24(1):48–51. 67. Clayton RG Sr., Diaz CE, Bashir NS, et al. Pulmonary function in hospitalized infants and toddlers with cystic fibrosis. J Pediatr 1998; 132(3 pt 1):405–408. 68. Subbarao P, Balkovec S, Solomon M, et al. Pilot study of safety and tolerability of inhaled hypertonic saline in infants with cystic fibrosis. Pediatr Pulmonol 2007; 42(5):471–476. 69. Dellon EP, Donaldson SH, Johnson R, et al. Safety and tolerability of inhaled hypertonic saline in young children with cystic fibrosis. Pediatr Pulmonol 2008; 43(11):1100–1106. 70. Sheikh S, Flucke R, McCoy K, et al. Changes in lung function during treatment for pulmonary exacerbations in infants with cystic fibrosis. Pediatr Pulmonol 2000; S20(suppl):296 (abstr 429). 71. Binder A, Bordeaux KA, Ren CL. Do infant pulmonary function test (PFT) results affect treatment in cystic fibrosis (CF)? Am J Respir Crit Care Med 2009; 179(abstracts issue): A1780. 72. Davis S, Kerby G, Acton J, et al. Feasibility, sensitivity, and variability of adult-type pulmonary function tests in infants with CF in a multicenter, longitudinal trial. Pediatric Pulmonol 2006; (suppl 29):A360.

10 Assessment of Lung Function in Young Children with Cystic Fibrosis FARIBA REZAEE and CLEMENT L. REN University of Rochester, Rochester, New York, U.S.A.

I.

Introduction

Advances in our understanding of the pathophysiology of cystic fibrosis (CF) lung disease have resulted in the development of therapies to restore airway surface liquid (1), improve mucociliary clearance (2–4), reduce inflammation (5,6), and control infection (7,8). At the same time, CF newborn screening has been recommended in the United States and other countries (9) and is anticipated to be performed in almost every state in the United States by 2010 (P. M. Farrell, personal communication). These developments will result in a growing population of infants and young children with CF for whom methods to assess disease progression and to serve as outcome measures for clinical trials of new therapeutics will be critical. For older CF patients, measurement of the forced expiratory volume in one second (FEV1) by spirometry is widely accepted as a pulmonary function test (PFT) for both clinical practice and research (10). Techniques for PFTs in infants and toddlers less than three years old have been recently developed as well (see chap. 9). Young children between the ages of three to six years (preschool aged) pose special challenges to pulmonary function testing. They are too old for infant PFT techniques, but they are also not as capable of performing the maneuvers required for standard adult PFTs. However, in recent years, several techniques that require less active cooperation have been developed to assess lung function in this age group. A joint working group of the American Thoracic Society and European Respiratory Society (ATS/ERS) recently published recommendations for performing preschool PFTs and their application to clinical care and research (11). In this chapter, we will review the ATS/ERS recommendations with specific application to preschool CF patients.

II.

Modified Spirometry

Spirometry is the most frequently used method for measuring lung function. It is commonly performed in adults and children greater than six years old, and there are well-established criteria for its performance in this age group (10). Preschool children cannot consistently perform the maximal forced expiratory maneuvers required to meet the criteria for acceptable adult spirometry. However, more recent studies have shown

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that acceptable flow-volume curves can be generated by preschool children using modified criteria. Since spirometry equipment is present in most PFT laboratories, modified spirometry is an attractive option for preschool pulmonary function testing. Standard spirometry incorporates three key elements to obtain flow limitation and consistent flow-volume curves: inspiration to total lung capacity (TLC), expiration down to residual volume (RV), and continuous maximal forced expiration. A minimum of six seconds of expiratory effort is required to meet these criteria in adults. Reproducibility is established by generating a minimum of three flow-volume curves whose values are within 10% of each other. Modifications to these criteria in preschool children are based both on their inability to comply with all of these criteria and considerations of developmental pulmonary physiology. Several groups have conducted studies in preschool children using modified spirometry criteria (12–14). The criteria vary somewhat from site to site, but Table 1 summarizes some of the commonly used criteria for modified spirometry. In adults, a forced expiratory time of at least six seconds is usually required to ensure complete emptying of the lungs (10). Compared with adults, children have a higher specific conductance and empty their lungs much faster (15). Because of this physiologic difference and the difficulty that preschool children have in maintaining forced expiration, investigators have accepted a shorter forced expiratory time than six seconds. Some studies have utilized an expiratory time of 1 second (12), while others will accept times as short as 0.5 seconds as long as a plateau in the volume-time curve can be seen (16). Only two reproducible curves are required, and a single maneuver can be accepted provided it meets the other criteria for acceptable testing. A cessation of flow at >10% of peak flow is a sign of early termination, but a convex shape of the descending limb of the flow-volume curve should not be interpreted as early termination, since this is a common finding in many healthy preschool children (13,14,16). An objective measure of the start of test quality is the back-extrapolated volume (VBE) (Fig. 1). High values reflect a slow start of test, indicating suboptimal effort. The VBE is normally a larger proportion of the total forced vital capacity (FVC) in preschool children compared with adults, and this criterion should be adjusted accordingly. Reference values in normal, healthy preschool children have been generated using criteria outlined in Table 1 (12). Several investigators have applied modified spirometry to the study of young children with CF (17–20). The results of these studies are summarized in Table 2. A consistent finding in all of these studies is clear evidence that by preschool age, Table 1 Quality Control Criteria That Have Been Used for Modified Spirometry l l l l l l l

Volume of back extrapolation
80 mL or 12.5% of FVC Expiratory time 1 sec At least 2 reproducible maneuvers with values within 10% of each other Clearly defined PEF Cessation of flow occurs at
10% of PEF Flow-volume curve demonstrates a rapid rise to PEF and a smooth descending limb No evidence of cough or glottic closure

Abbreviations: FVC, forced vital capacity; PEF, peak expiratory flow. Source: Adapted from Refs. 12 and 16.

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Table 2 Spirometric Findings in Young Children with CF

Study

Mayer et al. (20)

Marostica et al. (17)

Nielsen et al. (19)

Vilozni et al. (18)

Subjects Age(years) Success rate FVC FEV1 FEF25–75

49 3.1–6.9 79.5% 1.07  1.33 1.28  1.36 0.49  1.31

33 3.7–6.9 87.8% 0.75  1.63 1.23  1.97 0.74  1.36

30 4.9–7.5 100%a NRb 1.2  1.2 NRb

79 2.5–6.9 81.7% 0.36  0.58 0.36  0.72 NRb

Values for FVC, FEV1, and FEF25–75 are reported as mean Z score  SD for the study population compared with normal controls. The p value for all Z scores was <0.001 compared with normal controls. a Spirometry was not performed on patients under 4.9 years old. b NR, not reported. Abbreviations: FVC, forced vital capacity; FEV1, forced expiratory volume in one second; FEF, forced expiratory flow.

children with CF already have detectable airflow obstruction. Not only was the mean FEV1 lower than normal controls, the proportion of children whose FEV1 was <2 SD below the normal mean ranged from 18% to 42%. Many of these children with abnormal expiratory flows did not have respiratory symptoms. These findings are consistent with studies in CF infants where detectable lung disease has already begun (21–23) and strongly suggest that future clinical trials should be focused on this age group. The overall success rate in obtaining acceptable spirometry varied from study to study (Table 2) and was also highly dependent on age. For example, Mayer et al. found

Figure 1 Calculation of VBE. Source: Adapted from Ref. 17.

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that almost 100% of six-year-olds could perform acceptable modified spirometry, but only 50% of three-year-olds could do so (20). In the study by Nielsen et al., spirometry was not even attempted in the younger children (19). Other investigators have reported low success rates in preschool children with other respiratory diseases (24), and it remains to be seen how successful modified spirometry can be in the clinical setting. These studies have also identified several key elements necessary for obtaining successful spirometric data from preschoolers. Young children have short attention spans and are easily distracted. They need to be engaged and encouraged by the operator to participate in the test. Interactive computerized incentives may be used to encourage the maneuver (25), but the use of such incentives is not essential. In fact, some centers have reported that incentives may even result in a lower success rate (26). More important than incentives are the testing environment and personnel. The environment should be child friendly, relaxing, and nonthreatening, and ample time should be afforded to complete the test. The operator must establish a trusting relationship with the child and exhibit considerable patience and understanding. As discussed above, young children have a higher specific conductance than adults (15). This leads to rapid emptying times and a higher FEV1/FVC ratio, so FEV1 may not be the best outcome measure. FEV0.5 or FEV0.75 may be better outcome measures, but the best spirometric measure for preschoolers has not been established. In summary, modified spirometry is a technique with potential clinical and research applications in preschool children with CF. However, enthusiasm for this technique should be tempered by the variable success rates reported by investigators, especially in the younger age range. Centers that plan to use modified spirometry for clinical care must be certain to adhere to appropriate quality control criteria and devote the necessary time and effort needed to generate acceptable spirometric data. Other issues that need future resolution include a consensus on a consistent set of quality control criteria and the choice of appropriate outcome measures.

A. Forced Oscillometry

Forced oscillometry (FO) measures the total respiratory system impedance (Z) by measuring changes in pressure and flow in response to an oscillatory pressure signal applied at the airway opening (27,28). Z is a generalized concept of resistance and exists as a complex number made up of a real component that is in phase with flow and an imaginary component that is out of phase with flow. The real component consists of resistance (R), while the imaginary component is represented by reactance (X) (Fig. 2). R represents the forces of resistance associated with frictional losses in the airways and the lung parenchyma. X is the out-of-phase component or imaginary part, and is composed of an inertive element (Xi), which represents the inertive forces of the respiratory system, and a capacitant element (Xc), which reflects the viscoelastic properties of the lung (27,28). Input signals can be either monofrequency or multifrequency. Monofrequency oscillations provide a measure of Z at a single frequency, whereas multifrequency signals allow operators to detect frequency dependence of Z, a feature of airway obstruction (29). Spectral analysis utilizing fast Fourier transforms converts multifrequency signals from the time domain to the frequency domain (30). Multifrequency signals can be generated as pseudorandom noise composed of multiple harmonics of the

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Figure 2 The relationship between Z, R, and X. The real component of Z is graphed on the horizontal axis and consists of R. The imaginary component is graphed on the vertical axis and consists of X, which in turn is made up of Xi and Xc.

fundamental frequency or impulse oscillations. Earlier studies with FO utilized devices with a single frequency. More recently, commercial devices have been developed that utilize multifrequency signals, typically from 4 to 48 Hz. Normal reference data in the preschool age group are available for both pseudorandom noise and impulse oscillation devices (31,32). Figure 3 shows a representative FO tracing from a healthy four-year-old child. R increases as frequency falls, a phenomenon known as frequency dependence of resistance. Frequency dependence of resistance in adults is a sign of peripheral airway obstruction (29). Because the absolute airway caliber of young children is smaller than adults, frequency dependence of resistance is a normal finding in preschool children, and changes in R with frequency must be compared with normal reference data. At low frequencies, Xc is predominant and total X has a negative value. With increasing

Figure 3 Respiratory system resistance (gray circles) and reactance (black squares) as a function

of frequency in a healthy four-year-old boy. Units are hPa/L/sec.

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frequency, Xi predominates and X takes on a positive value. The frequency at which total X ¼ 0 is the resonant frequency and represents the point at which Xi ¼ Xc. The coherence function (g2) provides an index of the causality between the input and output signals (11). Its value ranges from 0 to 1.0. Lower values of g2 reflect increased noise in the system and indicate increased variability of the measurements. There have been no studies of what g2 threshold will provide the optimal signal-to-noise ratio, especially in young children. Some investigators have used a cutoff of 0.95 (33), while others have used 0.80 (24,34). The selection of g2 cutoff may also depend on the choice of equipment. In some published studies, no coherence threshold is reported, making it difficult to compare results from other studies. FO is a noninvasive technique that can be carried out during tidal breathing. Compared to spirometry, it needs much less patient cooperation. The ATS/ERS preschool PFT recommendations provide guidelines for FO equipment, procedures, and quality control (11). There is no universal consensus on FO quality control criteria, and studies using FO in the preschool population have used a variety of criteria. Table 3 summarizes some of the quality control criteria that have been applied to the preschool population by the U.S. CF Foundation’s Therapeutics Development Network. Using similar criteria, the success rate in obtaining acceptable FO data in preschool children has ranged from 80% to 90% (24,32,33,35). FO has been applied to the study of preschool children with and without CF. In preschool children without CF, FO has been used to demonstrate improvement in lung function in wheezing children treated with inhaled corticosteroids, bronchodilator response, and diminished lung function in preterm children with bronchopulmonary dysplasia (24,36,37). Studies in preschool CF children have yielded conflicting results. Nielson et al. found FO to be relatively insensitive to detecting lung function abnormalities in 30 preschool CF children studied during a time of clinically stable lung disease (19). In contrast, Gangell et al. studied 56 preschool CF children and found abnormalities in both R and X; symptomatic children were more likely to have lower lung function (33). Differences in the equipment used and study protocols may account for the different results from these two studies. Nielson et al. used an impulse oscillation system with frequency analysis at 5 Hz intervals from 5 to 35 Hz, whereas Gangell et al. used a pseudorandom noise signal at 2 Hz intervals from 4 to 48 Hz. As discussed above, low frequency signals reflect peripheral airway obstruction and viscoelastic changes, but at frequencies <10 Hz, g2 falls significantly (11,27,28). Frequencies between 6 to 8 Hz may provide the optimal balance between obtaining a high signal-to-noise ratio and sampling at the most informative frequencies. In older patients (6–18 years) hospitalized Table 3 Quality Control Criteria for FO l l l l l

l l

Steady tidal breathing Good seal around mouthpiece and no occlusion with tongue Minimum of 3 reproducible maneuvers (5 are preferred) Coherence (Co)  0.95 at all frequencies > 6 Hz for all acceptable maneuvers The graph of X vs. frequency crosses the zero axis (i.e., a resonant frequency can be determined) R10 values are within 20% of each other for all acceptable maneuvers Minimum of 8 sec of tidal breathing collection

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for treatment of pulmonary exacerbation, improvements in FO correlated with changes in FEV1 (38). The improvements were most notable in low-frequency reactance. At low frequencies, X predominately reflects the viscoelastic properties of the lung, suggesting that the changes observed reflected improved aeration of the lung periphery and an improvement in lung elastance, which arose from reduction in mucus plugging and airway inflammation. The results of this study suggest that FO may be able to serve as an outcome measure for clinical trials of CF therapeutics. The above studies suggest that FO may be able to serve as a PFT for preschool CF children. However, some outstanding issues remain to be determined. The short-term and long-term variability has not been defined, and the clinical relevance of abnormal X and R values has also not been determined.

III.

The Interrupter Technique

Pulmonary resistance measurement by the airway interruption method was first described in the 1920s (11), and commercially produced devices are now available. The interrupter technique is based on the concept that if expiratory airflow during tidal breathing is interrupted, mouth pressure (Pmo) and alveolar pressure (Palv) will equilibrate. Upon airway occlusion at the mouth there is a rapid rise in airway opening pressure (Pinit) followed by a slower pressure change (Pdif) up to a plateau (Pel) (Fig. 4). Pdif represents the viscoelastic properties of the respiratory system, which generate pressure oscillations until equilibrium is achieved (Fig. 4) (39). By measuring flow just before the interruption (V0 int), the interrupter resistance (Rint) can be calculated by dividing Pinit by V0 int. The duration of the interruption is typically 100 milliseconds, which is usually long enough for Pmo and Palv to equilibrate, but short enough to avoid initiation of the next tidal breath. The

Figure 4 Pressure-time tracing during Rint. Pinit is calculated by back extrapolation of Pmo to the beginning of the interruption (straight line). Source: Courtesy of Dr N. Beydon.

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oscillations in Pmo after occlusion make it difficult to determine Pinit, and different methods have been suggested to back-extrapolate to the beginning of the interruption (11). The ATS/ERS working group has developed guidelines for Rint equipment and procedures, and normal reference values are available (11). However, there is no consensus on the best algorithm to calculate Pmo during the occlusion. One central assumption of Rint is that Pmo and Palv equilibrate during the occlusion. However, in the setting of obstructive lung disease, equilibration may take a longer period of time and Palv may be underestimated. Therefore, Rint may not reflect actual airway resistance, and the degree of underestimation may affect accuracy of the test. Similar to FO, Rint requires minimal cooperation on the part of the patient, and it has been used in many studies of preschool children (11). Nielson et al. measured Rint longitudinally during times of clinical stability in a single-center cohort of 30 preschool children with CF and did not observe consistently abnormal values compared with normal controls (19). In a multicenter cross-sectional study, Beydon et al. compared Rint in 39 preschool CF children with 79 normal controls and found a higher mean Rint in the CF subjects (40). Symptomatic CF children were more likely to have elevated Rint. These studies suggest Rint may be a useful technique to assess lung function in preschool CF children, but further data are needed to determine the sensitivity and clinical utility of Rint, and a consensus for determination of Pinit must be developed. A. Gas Mixing Techniques

Ventilation abnormalities arising from mucus plugging of the small peripheral airways and airway wall thickening occur early in CF lung disease and can be measured by gas mixing techniques. The multiple-breath washout (MBW) technique involves equilibration inside the lungs of an inert tracer gas followed by washout by room air tidal breathing (41). Inert gases that have been used include nitrogen (N2), argon (Ar), helium (He), and sulfur hexafluoride (SF6) (41). Commercial MBW equipment is available, but most published studies have used devices built by the investigators’ site. An increase in the time taken to completely wash out the tracer gas reflects impaired ventilation and can be expressed as an elevated lung clearance index (LCI) (11). The functional residual capacity can also be calculated by dividing the net amount of gas exhaled during washout by the difference in marker gas concentration at start and end of washout. Studies in preschool CF children have demonstrated elevated LCI compared with normal controls (42,43), and one study found that MBW was more sensitive in detecting lung function abnormalities than either specific airway resistance or spirometry (44). The success rate in obtaining acceptable MBW data has ranged from 50% to 75%. Higher success rates can be achieved using masks sealed with therapeutic putty and performing testing in a quiet, dim room with appropriate distractions (e.g., children’s entertainment video). MBW has great promise as a noninvasive method to assess airway function in preschool CF children. However, further work is needed to allow widespread adoption and multicenter use.

IV.

Specific Airway Resistance

A standard whole-body plethysmograph (WBP) can be used to measure airway resistance (Raw) (45). Measurement of Raw requires panting against a closed shutter to

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obtain the alveolar pressure. Young children usually cannot perform this maneuver. However, specific airway resistance (sRaw), which is corrected for total lung volume, can be determined by measuring flow and changes in box volume with the shutter open, avoiding the need for the panting maneuver. When measuring sRaw, minimal cooperation is needed so that the acceptability is high, and PFT laboratories can use standard equipment with minimal adaptations. Children who are anxious can perform the maneuver while seated in their parents’ lap. Nielson et al. found that sRaw was a sensitive test to detect early airflow obstruction in preschool CF children (19). Body heat results in increased pressure in the WBP due to gas expansion, and this is accounted for by compensation algorithms in the computerized software used to calculate sRaw (46). Because these algorithms vary with different WBPs, a uniform set of reference data is not available, and results from one laboratory cannot always be directly compared with another. These features limit the use of sRaw into general clinical practice and multicenter clinical trials. This technique may be useful at single centers that can obtain their own normal reference values and track their patients’ results over time.

V. Tidal Breathing Techniques Tidal breathing patterns can be measured either by having subjects breath through a pneumotachometer or by respiratory inductance plethysmography (RIP) (11,47). RIP uses impedance bands around the rib cage and abdomen to measure their excursion. Analysis of both expiratory flow and thoracoabdominal motion can be performed. The ratio of time to peak expiratory flow over total expiratory time (Tpef/Te) can be measured from analysis of tidal expiratory flow data and is lower in patients with airflow obstruction (47). The mechanism for this observation is unclear, and it may result from both alterations in respiratory mechanics and neural control of breathing. The primary measure used in analysis of thoracoabdominal motion is the phase angle (j), a quantitative measure of thoracoabdominal asynchrony (TAA). In the absence of TAA, j ¼ 0, whereas a j of 1808 represents paradoxical breathing. Other measures of thoracoabdominal motion include the percentage contribution to tidal volume of the rib cage, labored breathing index, and maximal compartmental amplitude. Guidelines for tidal breathing measurements and normal reference values in preschool children have been published (11,48). Tidal breathing analysis has principally been applied to the study of older adults with chronic obstructive pulmonary disease and infants (49–52). However, there have been some studies in CF. In a study using RIP in 15 adolescents and young adults with CF, there was no significant difference in Tpef/Te compared with normal controls, but j was significantly high in the CF patients (53). Mayer et al. conducted a longitudinal study of RIP and spirometry in preschool CF children (20). They were able to obtain acceptable RIP data in 77% of the study subjects. Consistent with other studies, they found that spirometry in the CF children was abnormal, but there were no significant differences in j or Tpef/Te. However, j did increase during pulmonary exacerbations. These results suggest that RIP may not be a sensitive measure of early lung disease in CF preschoolers, but it may be useful in detecting changes in lung function either due to exacerbation or in response to treatment. It also remains to be determined whether j, or other measures of tidal breathing, is the most useful outcome measure.

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Table 4 Comparison of Different Preschool PFT Techniques

Technique

Sensitivity of Suitable for detecting lung multicenter Feasibility disease trials

Normal Commercially reference data available available

Modified spirometry FO RIP Rint sRaw MBW

Moderate High High High High High

Yes Yes Yesa Yesb Yesc Yes

High Mod Low Mod–high High High

Moderate High Moderate High Low Lowd

Yes Yes Yes Limited No Limited

a

Not FDA cleared for preschool-aged children. Not FDA cleared in the United States. c Commercial whole-body plethysmographs are available, but because different WBPs use different compensation algorithms, the results cannot be compared. d A standardized protocol for multicenter MBW has not been developed yet. Abbreviations: PFT, pulmonary function test; FO, forced oscillometry; RIP, respiratory inductance plethysmography; Rint, interrupter resistance; sRaw, specific airway resistance; MBW, multiple-breath washout. b

VI.

Summary of Preschool PFTs and Future Directions

Several preschool PFT techniques have been developed in recent years and applied to the study of young children with CF. The features of different preschool PFT techniques are summarized in Table 4. Studies, to date, so far have clearly shown that preschool children with CF have detectable alterations in lung function, suggesting that therapeutic intervention for this age group should be considered. Studies with different techniques have yielded variable results, perhaps because they have been mainly single-center studies with relatively small sample sizes. The U.S. CF Foundation is currently conducting a longitudinal multicenter study of 100 preschool CF children comparing FO, modified spirometry, and RIP. A randomized controlled trial of short-term dornase alfa therapy in CF preschoolers has recently been initiated (Clinicaltrials.gov ID# NCT00680316). The primary outcome measure for this study will be X at 8 Hz measured by FO, and modified spirometry will be a secondary outcome measure. Data from these studies will provide further information on the utility of preschool PFTs in multicenter research trials.

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5. Chmiel JF, Berger M, Konstan MW. The role of inflammation in the pathophysiology of CF lung disease. Clin Rev Allergy Immunol 2002; 23:5–27. 6. Boucher RC. Airway surface dehydration in cystic fibrosis: pathogenesis and therapy. Annu Rev Med 2007; 58:157–170. 7. Retsch-Bogart GZ, Burns JL, Otto KL, et al. A phase 2 study of aztreonam lysine for inhalation to treat patients with cystic fibrosis and Pseudomonas aeruginosa infection. Pediatr Pulmonol 2007; 43:47–58. 8. Ramsey BW, Pepe MS, Quan JM, et al. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin Study Group. N Engl J Med 1999; 340:23–30. 9. Grosse SD, Boyle CA, Botkin JR, et al. Newborn screening for cystic fibrosis: evaluation of benefits and risks and recommendations for state newborn screening programs. MMWR Recomm Rep 2004; 53:1–36. 10. Miller MR, Hankinson J, Brusasco V, et al. Standardisation of spirometry. Eur Respir J 2005; 26:319–338. 11. Beydon N, Davis SD, Lombardi E, et al. An official American Thoracic Society/European Respiratory Society statement: pulmonary function testing in preschool children. Am J Respir Crit Care Med 2007; 175:1304–1345. 12. Eigen H, Bieler H, Grant D, et al. Spirometric pulmonary function in healthy preschool children. Am J Respir Crit Care Med 2001; 163:619–623. 13. Vilozni D, Barker M, Jellouschek H, et al. An interactive computer-animated system (SpiroGame) facilitates spirometry in preschool children. Am J Respir Crit Care Med 2001; 164:2200–2205. 14. Zapletal A, Chalupova J. Forced expiratory parameters in healthy preschool children (3-6 years of age). Pediatr Pulmonol 2003; 35:200–207. 15. Mead J. Dysanapsis in normal lungs assessed by the relationship between maximal flow, static recoil, and vital capacity. Am Rev Respir Dis 1980; 121:339–342. 16. Aurora P, Stocks J, Oliver C, et al. Quality control for spirometry in preschool children with and without lung disease. Am J Respir Crit Care Med 2004; 169:1152–1159. 17. Marostica PJ, Weist AD, Eigen H, et al. Spirometry in 3- to 6-year-old children with cystic fibrosis. Am J Respir Crit Care Med 2002; 166:67–71. 18. Vilozni D, Bentur L, Efrati O, et al. Spirometry in early childhood in cystic fibrosis patients. Chest 2007; 131:356–361. 19. Nielsen KG, Pressler T, Klug B, et al. Serial lung function and responsiveness in cystic fibrosis during early childhood. Am J Respir Crit Care Med 2004; 169:1209–1216. 20. Mayer OH, Jawad AF, McDonough J, et al. Lung function in 3-5-year-old children with cystic fibrosis. Pediatr Pulmonol 2008; 43:1214–1223. 21. Castile RG, Iram D, McCoy KS. Gas trapping in normal infants and in infants with cystic fibrosis. Pediatr Pulmonol 2004; 37:461–469. 22. Linnane BM, Hall GL, Nolan G, et al. Lung function in infants with cystic fibrosis diagnosed by newborn screening. Am J Respir Crit Care Med 2008; 178:1238–1244. 23. Sharp j, Sheehan D, Ren CL. Lung function in infants with CF diagnosed by newborn screening. Pediatr Pulmonol 2007; S30:334. 24. Guilbert TW, Morgan WJ, Zeiger RS, et al. Long-term inhaled corticosteroids in preschool children at high risk for asthma. N Engl J Med 2006; 354:1985–1997. 25. Vilozni D, Barak A, Efrati O, et al. The role of computer games in measuring spirometry in healthy and “asthmatic” preschool children. Chest 2005; 128:1146–1155. 26. Gracchi V, Boel M, van der LJ, et al. Spirometry in young children: should computeranimation programs be used during testing? Eur Respir J 2003; 21:872–875. 27. Goldman MD. Clinical application of forced oscillation. Pulm Pharmacol Ther 2001; 14:341–350.

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28. Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodology, recommendations and future developments. Eur Respir J 2003; 22:1026–1041. 29. Grimby G, Takishima T, Graham W, et al. Frequency dependence of flow resistance in patients with obstructive lung disease. J Clin Invest 1968; 47:1455–1465. 30. Michaelson ED, Grassman ED, Peters WR. Pulmonary mechanics by spectral analysis of forced random noise. J Clin Invest 1975; 56:1210–1230. 31. Frei J, Jutla J, Kramer G, et al. Impulse oscillometry: reference values in children 100 to 150 cm in height and 3 to 10 years of age. Chest 2005; 128:1266–1273. 32. Hall GL, Sly PD, Fukushima T, et al. Respiratory function in healthy young children using forced oscillations. Thorax 2007; 62:521–526. 33. Gangell CL, Horak F Jr., Patterson HJ, et al. Respiratory impedance in children with cystic fibrosis using forced oscillations in clinic. Eur Respir J 2007; 30:892–897. 34. Marotta A, Klinnert MD, Price MR, et al. Impulse oscillometry provides an effective measure of lung dysfunction in 4-year-old children at risk for persistent asthma. J Allergy Clin Immunol 2003; 112:317–322. 35. Klug B, Bisgaard H. Specific airway resistance, interrupter resistance, and respiratory impedance in healthy children aged 2-7 years. Pediatr Pulmonol 1998; 25:322–331. 36. Thamrin C, Gangell CL, Udomittipong K, et al. Assessment of bronchodilator responsiveness in preschool children using forced oscillations. Thorax 2007; 62:814–819. 37. Vrijlandt EJ, Boezen HM, Gerritsen J, et al. Respiratory health in prematurely born preschool children with and without bronchopulmonary dysplasia. J Pediatr 2007; 150:256–261. 38. Ren CL, Brucker JL, Rovitelli AK, et al. Changes in lung function measured by spirometry and the forced oscillation technique in cystic fibrosis patients undergoing treatment for respiratory tract exacerbation. Pediatr Pulmonol 2006; 41:345–349. 39. Sly PD, Bates JH. Computer analysis of physical factors affecting the use of the interrupter technique in infants. Pediatr Pulmonol 1988; 4:219–224. 40. Beydon N, Amsallem F, Bellet M, et al. Pulmonary function tests in preschool children with cystic fibrosis. Am J Respir Crit Care Med 2002; 166:1099–1104. 41. Gustafsson PM. Inert gas washout in preschool children. Paediatr Respir Rev 2005; 6:239–245. 42. Aurora P, Gustafsson P, Bush A, et al. Multiple breath inert gas washout as a measure of ventilation distribution in children with cystic fibrosis. Thorax 2004; 59:1068–1073. 43. Gustafsson PM, Aurora P, Lindblad A. Evaluation of ventilation maldistribution as an early indicator of lung disease in children with cystic fibrosis. Eur Respir J 2003; 22:972–979. 44. Aurora P, Bush A, Gustafsson P, et al. Multiple-breath washout as a marker of lung disease in preschool children with cystic fibrosis. Am J Respir Crit Care Med 2005; 171:249–256. 45. Bisgaard H, Nielsen KG. Plethysmographic measurements of specific airway resistance in young children. Chest 2005; 128:355–362. 46. Klug B, Bisgaard H. Measurement of the specific airway resistance by plethysmography in young children accompanied by an adult. Eur Respir J 1997; 10:1599–1605. 47. J Palmer, J Allen, O Mayer. Tidal breathing analysis. NeoReviews 2004; 5:E186–E193. 48. Mayer OH, Clayton RG Sr., Jawad AF, et al. Respiratory inductance plethysmography in healthy 3- to 5-year-old children. Chest 2003; 124:1812–1819. 49. Gonzalez H, Haller B, Watson HL, et al. Accuracy of respiratory inductive plethysmograph over wide range of rib cage and abdominal compartmental contributions to tidal volume in normal subjects and in patients with chronic obstructive pulmonary disease. Am Rev Respir Dis 1984; 130:171–174. 50. Allen JL, Wolfson MR, McDowell K, et al. Thoracoabdominal asynchrony in infants with airflow obstruction. Am Rev Respir Dis 1990; 141:337–342.

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11 Lung Function Testing in School-Age Children with Cystic Fibrosis OSCAR H. MAYER and JULIAN L. ALLEN University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Lung function testing is a central part of the pulmonary evaluation of patients with cystic fibrosis (CF) and an important outcome measurement for many intervention trials. Accurate lung function testing from infancy is important since lung disease often develops before testing is performed at school age (1). Now there are a variety of comparable pulmonary function techniques that allow longitudinal measurement from infancy through the preschool years and later childhood (2–5). With these developments, we have effective ways to measure lung function longitudinally in CF patients to diagnose and manage lung disease in its earliest stages. The discussion that follows will focus on the techniques available to the cooperative older patient.

II.

Measurements of Airway Obstruction

Spirometry has been one of the most widely used techniques for assessing obstructive lung disease. More recently, other measurements of airway obstruction have been used, such as single- and multiple-breath inert gas washouts for the measurement of the lung clearance index (LCI), airways resistance measurements by the interrupter technique or plethysmography, and forced oscillometry for respiratory system reactance and resistance. A. Spirometry

Accurate and reliable spirometry requires a maximal expiratory maneuver involving first an inspiration to total lung capacity (TLC) and then forceful exhalation to residual volume (RV). The “maximal” exhalation must be performed to the point of flow limitation, where airflow is limited by the mechanics of the airways and not by the driving force of the patient’s exhalation (Fig. 1). The recommended technique, for both patient and technician, has been well established by the American Thoracic Society (ATS) and European Respiratory Society (ERS), and revised criteria have been proposed for use in children younger than 10 years old (6). A maximal forced expiratory maneuver is composed of three discrete stages, each of which has quality control criteria: inhalation to TLC, rapid initiation of exhalation to the point of flow limitation, and complete exhalation to RV. After a period of calm tidal breathing, the inhalation should be performed from functional residual capacity (FRC) up to TLC. Then, with little pause, the patient is coaxed to “blast” the air from the lungs

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Figure 1 Progressive increase in expiratory flow to the point of flow limitation. The highest two

curves represent flow limitation, in which greater effort no longer increases flow rates.

and is coached and encouraged to exhale smoothly and with maximal effort. Exhalation continues until there is a plateau on the volume-time curve indicating no further air can be exhaled. This typically takes a minimum of six seconds in patients 10 years and over, but may occur in as little as three seconds in children between 6 and 10 years. After the trial is complete, there are expiratory timing and flow-volume curve morphology criteria that need to be satisfied for a trial to be acceptable. First, the onset of exhalation must begin without significant delay, as determined by visual evidence on the flow-volume curve and a volume of back-extrapolation of <150 mL or <5% of forced vital capacity (FVC) (6). Different criteria, however, exist for younger subjects (7). The expiratory flow pattern should be smooth and without variability, and there should be no abrupt cessation of expiratory flow. After a trial is accepted, the maneuver should be repeated at least two more times. Reproducibility of the measurements exists when the two highest FVC and forced expiratory volume in one second (FEV1) values are within 150 mL of each other (6). These last two criteria suggest, but do not confirm, the presence of expiratory flow limitation. The FVC, FEV1, FEV1/FVC, and forced expiratory flow (FEF) between 25% and 75% of vital capacity (FEF25–75%) are typically reported, and interpreted by standard criteria (6). While FEV1 and FEF25–75% are both indexes of obstructive lung disease, FEF25–75% is felt to reflect small airway function (8). Because the usual course of CF lung disease begins with small airway obstruction, the FEF25–75% often decreases into the abnormal range before the FEV1 does. It is also important to review the shape of the flow-volume curve since concavity toward the volume axis, indicating early obstructive lung disease, can occur before either the FEV1 or FEF25–75% is abnormal.

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FEV1 has been widely used as an index of obstructive lung disease in longitudinal analyses (9) and prognostic assessments (10–12). It is the primary lung function assessment tool used among CF centers by the Cystic Fibrosis Foundation in the United States, the Cystic Fibrosis Trust in the United Kingdom, and within the Epidemiologic Study of Cystic Fibrosis (13,14). It has also been used as the primary outcome measurement in some of the seminal drug intervention studies in CF (15–18). B. Airway Resistance

In healthy older children and adults, the majority of airway resistance resides in the central airways, where the total cross-sectional area at a given airway generational level is smaller than in the peripheral airways. Thus, the utility of resistance measurements in detecting early peripheral airflow obstruction is limited. Such measures may, however, be quite useful in more advanced disease, or in subjects unable to perform maximal forced expiratory maneuvers. Airway resistance can be determined in a body plethysmograph (RAW) or by the interrupter technique (RINT). Calculating RAW is a two-step process that utilizes Boyle’s law, which states that the product of pressure and volume of a gas in a closed system remains constant. The first step involves panting through a pneumotachometer inside a closed plethysmograph, to assess change in pressure in the plethysmograph (DPPLETH) relative to change in flow at the airway opening (DV0 ), DPPLETH/DV0 . The second step, breathing against a closed shutter, measures the relationship between DPPLETH and the change in pressure at the airway opening (Pao). When the airway is occluded with the glottis open and there is no flow, Pao will be equal to alveolar pressure (PALV). Therefore, multiplying the value from the first maneuver (DPPLETH/DV0 ) and the second maneuver (PALV/PPLETH) relates flow to PALV and gives a measurement of resistance: DPPLETH DPALV  ¼ RAW DV 0 DPPLETH

ð1Þ

Solving for equation (1) yields: DPALV ¼ RAW DV 0 Airway resistance can also be measured using the interrupter technique (RINT), in which alveolar pressure is measured by obstructing the airway at the mouth during a tidal breathing expiration. Pressure at the mouth reaches a plateau after interruption; the back extrapolation of this plateau to time zero of the interruption is assumed to equal alveolar pressure, which when divided by the flow immediately preceding the interruption yields airway resistance. RAW by plethysmography correlates with other measures of tidal breathing airflow obstruction (19,20). It underestimates resistance measured by RINT in children without airflow obstruction, but the measurements are closer in subjects with more severe airflow obstruction (21). Airway conductance (GAW) is the reciprocal of resistance. Because airways resistance is dependent on the lung volume at which it is measured and because both conductance and lung volume increase with age, airway resistive properties are sometimes expressed as specific conductance, sGAW (GAW/FRC). In normal subjects sGAW is relatively constant with increasing age beyond infancy. sGAW is reduced in patients with

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reduced elastic recoil, supporting the concepts that lung recoil is important in both airway driving pressure and tethering, and that abnormalities in these contribute to the decreased flow rates seen in patients with CF (22,23). C. Forced Oscillometry

The theory and technique of forced oscillometry (FO) are reviewed in chapter 10. Older patients with CF have elevated overall impedance (24) (the pressure cost of minute ventilation), consistent with studies in younger children with CF (25,26). Respiratory system resistance (RRS) measured by the impedance technique may be less sensitive in subjects with predominantly peripheral airway obstruction than in those with central obstruction (27). After bronchodilator administration, the decreases in RRS measured by FO or in RAW by plethysmography in CF subjects are similar (28). Baseline lung function measurements by FO also correlate with spirometry in subjects with CF (29,30). D. Gas Washout Lung Clearance Index

The LCI represents the number of lung volumes (FRC) the patient needs to exhale to effectively empty the lungs of the gas being washed out. As such it reflects the degree of ventilation inhomogeneity and has been used as an index of small airway obstruction (31–33). The LCI is calculated using the cumulative expired volume (CEV) required to clear an inert gas from the lungs divided by the FRC. LCI ¼

CEV FRC

Initially the LCI was described by the CEV from a nitrogen washout maneuver using 100% oxygen. Because of the potential decrease in respiratory rate from breathing 100% oxygen during the washout phase, other nonbiological gasses such as sulfur hexafluoride (SF6) and helium (He) have been used as tracer gasses. In this technique, the patient breathes on a circuit that contains a pneumotachometer to measure flow, a port for administering tracer gas, and a separate port for sampling gas concentration. The patient inspires the tracer gas during quiet breathing until a target concentration is reached, and then breathes ambient air free of tracer gas until the exhaled concentration of the tracer gas is below 0.1%. The LCI has demonstrated lung function abnormalities in a wide age range of patients with CF before they can be detected by more standard measurements such as FEV1, FEF25–75%, and sRAW (31–35). Gustafsson et al. demonstrated a sensitivity for detecting lung disease by LCI relative to high-resolution computed tomography (HRCT) scan scoring of 93%, while the sensitivity of FEV1 was 26% and of FEF25–75% was 63% (35). On the basis of this, it has been suggested that LCI could be used as a screening test of early lung disease with the same power as HRCT (35). LCI has been used to discriminate patients with CF and healthy children (35). It also has potential to be a very useful longitudinal measurement since the range of normal does not change with age (36).

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Slope of Phase 3

During a single-breath nitrogen washout maneuver, the patient inhales 100% oxygen from RV up to TLC and then exhales slowly and fully to RV again. There are four separate phases of exhalation that represent gasses from different portions of the respiratory tract (Fig. 2A). During phase 1, nitrogen concentration at the mouth is zero as the anatomic dead space empties 100% oxygen. In phase 2, the nitrogen concentration starts to rise as physiologic dead space and late filling alveoli empty. The concentration approaches a plateau during phase 3 as mixed alveolar gas is exhaled. In the absence of obstruction, the slope of phase 3 is nearly flat; however, with peripheral obstruction and heterogeneous filling and emptying of acini, the slope of phase 3 is more positive. During phase 4, the

Figure 2 Single-breath nitrogen washout with phases 1, 2, 3, and 4. (A) Lower slope of phase 3 indicating more homogenous ventilation and (B) a higher slope of phase 3 and earlier onset of phase 4 indicating more heterogeneous ventilation. Abbreviations: RV, residual volume; TLC, total lung capacity.

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concentration of nitrogen rises sharply as alveoli in the apical portion of the lung empty. This is because at the beginning of the prior inspiration of 100% oxygen, high nitrogen dead space gas is preferentially directed toward the apical alveoli because the airways in the bases are closed. Phase 4 starts earlier in expiration (i.e., at a higher lung volume) in subjects with small airway disease (Fig. 2B), in whom airway closure in the basal segments of the lung occurs at a higher lung volume than normal. Slope of phase 3 analysis has been applied to each breath during a multiple-breath exhalation of nitrogen, and also helium, and SF6 after wash in. After normalizing the slope of phase 3 to the mean gas concentration through phase 3, the normalized slope (SnIII) can be compared from breath to breath to get a measure of homogeneity of ventilation (34,37,38). The breath-to-breath change in SnIII in healthy lungs increases slightly throughout the washout; however, in CF with increasing peripheral obstruction, SnIII can increase markedly (34,37,38). In addition, the difference between the SnIII from He and SF6 multiple-breath exhalation trials has been used as a further index of peripheral airway obstruction (39).

III.

Measurements of Static Lung Volumes

Lung volumes can be measured by gas dilution or by body plethysmography. The helium dilution and nitrogen washout techniques differ from plethysmography in that they only measure the lung volume that communicates with the airway opening and do not measure

Figure 3 Fractional lung volumes. Abbreviations: TLC, total lung capacity; VC, vital capacity;

FRC, functional residual capacity; IC, inspiratory capacity; ERV, expiratory reserve volume; RV, residual volume.

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lung volume in regions of air trapping. Therefore, with obstructive lung disease, TLC determined by helium dilution or nitrogen washout may be underestimated. Outside of moderate to severe obstructive lung disease, the difference should be minimal. Each technique produces a measurement of FRC, from which TLC is calculated by one of two methods. The first subtracts expiratory reserve volume (ERV) from FRC to calculate RV. The vital capacity is then added to get TLC (Fig. 3). The second method adds inspiratory capacity (IC) to the FRC to obtain TLC (Fig. 3). A. Plethysmography

FRC is calculated using plethysmography by applying Boyle’s law, which states that the product of volume and pressure of a gas in a closed system will remain constant such that V1 P1 ¼ V2 P2 Here, V1 and V2 are lung volume before and after an expiratory maneuver against a closed shutter, and P1 and P2 are alveolar pressure before and after the maneuver. Thus, V1 P1 ¼ ðV1  DVÞðP1 þ DPÞ DP is the pressure change at the mouth. Since the airway opening is occluded, there is no flow and so no resistive pressure drop between the mouth and alveoli; thus, the change in mouth pressure equals alveolar pressure. DV is the volume change of the lungs inferred from the pressure change in the closed plethysmograph. Hence, V1 P1 ¼ V1 P1 þ V1 DP  DVP1  DVDP and V1 DP ¼ DVP1 þ DVDP If one assumes DVDP to be negligibly small compared to V1 and P1, the equation simplifies to V1 ¼

DVP1 DP

where V1 is lung volume at the point of airway occlusion (usually performed at FRC), P1 is atmospheric pressure, DV is the change in lung volume (as compressed gas volume), and DP is the pressure change at the mouth during the closed shutter panting maneuver starting from FRC. Some of the assumptions of plethysmography can be violated in patients with moderate or severe airway obstruction. First, the assumption that DVDP is negligibly small may not be true in subjects with airway obstruction (40). Second, high airway compliance or very inhomogeneous regional time constants result in airflow during the panting maneuver, violating the assumption that pressure swings at the mouth equal those in the alveoli (41). However, body plethysmography remains a very useful tool in the assessment of lung volumes and air trapping in patients with CF. An elevated RV and RV/TLC ratio are felt to be sensitive measurements of abnormal small airway function and increase before TLC does, because small airway obstruction is more apparent at low lung volumes (23). Increased TLC is present in about 40% of 6- to 8-year-olds, and in nearly 70% of 18-year-olds with CF (42). The

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evolution of lung hyperinflation is influenced by factors such as initial pulmonary function, colonizing organisms, and genotype (42). Hyperinflation in CF patients can persist after lung transplantation, and may reflect a chest wall adaptation to chronic hyperinflation prior to transplantation (43). B. Gas Dilution Helium

Helium dilution is performed by having a patient breathe from FRC into a closed system of known volume (V1) and with a known concentration of helium (C1) until the helium concentration stabilizes (C2). The final volume (V2) is the sum of V1 and the starting lung volume of the patient (FRC). Since the relationship between concentration and volume is constant (i.e., the amount of helium in the closed system does not change during the maneuver), FRC can then be solved mathematically as C1 V1 ¼ C2 V2 or; C1 V1 ¼ C2 ðV1 þ FRCÞ C1 V1  C2 V1 ¼ C2 FRC ðC1 V1  C2 V1 Þ FRC ¼ C2 Nitrogen Washout

Nitrogen washout is performed by having a patient breathe 100% oxygen over the tidal range beginning at FRC, through an open circuit from which both nitrogen concentration and exhaled volume can be measured. The patient then continues to breathe until the exhaled nitrogen concentration decreases below 1.5% (from the 79% of ambient air). Since the amount of nitrogen in the lung is conserved in the total exhaled volume, FRC can be calculated as FRC ¼

Exhaled volume of N2 ½N2initial   ½N2final 

As mentioned above in the section on the LCI, the technique of nitrogen washout produces data that can be further analyzed in sum or as individual breaths to assess for heterogeneous ventilation (34,35,37,38).

IV.

Measurement of Diffusion

A. Diffusion Limitation to Carbon Monoxide

The diffusion capacity of the lung for carbon monoxide (DLCO), or the transfer factor, is the standard test to measure the diffusion capacity of the lung. Carbon monoxide (CO) is used because it strongly and irreversibly binds to hemoglobin and its concentration in the serum remains extremely low. This means that its movement into the blood stream is not limited by perfusion, but instead the diffusion across the alveolarcapillary barrier. The DLCO is performed by having a subject inhale a known volume of CO and an inert nondiffusible tracer gas, such as helium (He), from FRC. The standard measurement

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is made after a single inhalation to TLC followed by a 10 second breath hold. The exhaled gas is then collected and the amount of CO and He measured, from which the amount of diffused CO and lung volume can be assessed. The DLCO can then be corrected for the surface area for diffusion (in a relative sense by lung volume) and for the CO reservoir or hemoglobin, and there are normative data for each correction. The measurement assumes that all the inhaled gas that is not diffused is exhaled and can be compared to the inhaled gas concentration to determine the amount of CO that has diffused. In patients with obstructive lung disease and significant air trapping, this may not be the case, and less CO will be exhaled making the reported DLCO falsely high. In the presence of significant pulmonary hemorrhage, CO will bind to the hemoglobin in the airspaces, less will be exhaled, and the DLCO can be falsely high as well. While DLCO can yield useful information in both obstructive and interstitial lung diseases, its utility in CF for both acute (44) and longitudinal evaluations is questionable (9,45).

V. Challenge Testing A. Airway Responsiveness/Reactivity Bronchodilator Responsiveness

A significant difference in pre- and postbronchodilator lung function is an important component of a diagnosis of asthma, but is a variable part of the pulmonary pathology in CF. Testing for bronchodilator responsiveness can be performed when the patient has not taken short-acting bronchodilators (adrenergic agonist or cholinergic antagonist) for at least 4 hours or long-acting bronchodilators for at least 12 hours before the trial (46). In school-age and older children, the standard testing technique uses spirometry followed by administration of an appropriate dose of bronchodilator, with repeat spirometry testing performed 15 minutes later (46). A significant effect is defined as a 12% improvement in FVC or FEV1 (46). Bronchodilator responsiveness can be present in patients with CF (47), but it is not universal, and instead clinical response is felt to be a more useful determinant of bronchodilator response and effectiveness (48–50). Although albuterol is most commonly used therapeutically in CF and is felt to be more effective than ipratropium, ipratropium may be more useful in the subset of CF patients with bronchial hyperreactivity (BHR) (49,50). Both FO and RINT have been used to test bronchodilator sensitivity in healthy children (51) and those with asthma (52,53) and CF (28); however, there is no consensus on what constitutes a significant response (54). While both FO and RINT can demonstrate a bronchodilator response, they do not always correlate with a similar change in FEV1 (53,55). Bronchial Hyperreactivity

Although BHR testing has been performed using numerous agents such as mannitol and histamine, methacholine challenge is the standard recommended by the ATS due to its better side effect profile (56). Methacholine acts directly on the smooth muscle as a cholinergic agonist, and the challenge consists of grading the decrease in FEV1 in response to increasing concentrations of methacholine. Hypertonic saline (4.5–10%) challenge has also been used to evaluate BHR in CF. It is felt to test for BHR indirectly

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through an atopic mechanism by stimulating release of bronchoconstrictive mediators from mast cells and eosinophils (57). BHR is a central component of a diagnosis of asthma, but its presence in the CF lung and its significance remain controversial (50,58). The BHR in CF may be due to true atopy from a coincident diagnosis of asthma, but may also be due to an inflammatory response and epithelial damage from chronic bronchopulmonary bacterial colonization (50,58). Unlike asthma patients, patients with CF typically have a greater bronchoconstrictive response to methacholine compared with histamine (50). In addition, the effect can be attenuated by pretreatment with ipratropium bromide nebulization (49,50), which suggests a cholinergic mechanism for BHR in patients with CF (50). In a further deviation from the normal asthmatic response, the bronchoconstrictive effect of hypertonic saline nebulization in CF patients can be short lived and correct to baseline before bronchodilator administration (57). While BHR testing may be helpful in further evaluating patients with CF that have a significant bronchodilator response, the etiology of BHR (atopy vs. inflammation from chronic bronchopulmonary colonization) will still need to be determined. B. Exercise

Exercise is an important part of maintaining respiratory function and optimizing cardiopulmonary fitness in CF (59,60). An inability to follow an exercise routine due to respiratory limitation can be an early indication of worsening lung function. Exercise testing can be a useful tool to refine an exercise protocol or simply to test capacity, which can be helpful in establishing overall respiratory health and prognosis (59,60). Three ways of measuring exercise capacity are the six-minute walk test, cardiopulmonary exercise testing (CPET) to measure peak oxygen uptake (VO2max) and maximal level of exertion, and a test to detect exercise-induced bronchospasm (EIB). Although there is good correlation between the six-minute walk test and VO2max, they measure exertion at very different levels (61). While CPET measures “peak” exercise capacity that is driven externally by a technician, the six-minute walk test is a test of submaximal exertion that is driven by the patient with no external encouragement. As such, the six-minute walk test is felt to be a measurement of “everyday” cardiopulmonary capacity, and is a major component of the evaluation for lung transplantation. It is a self-paced walk without external coaching for six minutes with continuous pulse oximetry monitoring on a flat nonmoving surface that is 100 ft in length (61). The total distance walked is the primary functional outcome (62). It is used prognostically in assessing the rehabilitation potential of patients being considered for lung transplantation and has been used to assess respiratory compromise in patients with CF (60). Although the six-minute walk test is a sensitive measure of respiratory limitation in patients with lung disease, the CPET may be more sensitive in assessing early stages of exertional limitation in that the patient effort is driven externally by a technician (60,62). Although CPET has been shown to be reproducible in adults (63), its utility as an outcome measure in CF, compared with more standard testing like spirometry and lung volume measurements, has not been demonstrated (64). EIB can also be evaluated independently. The ATS has specific guidelines for the challenge itself and the duration of postchallenge observation and lung function testing

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(10 minutes) to maximize the sensitivity and specificity of the challenge (62). Unlike patients with asthma, patients with CF typically have a bronchodilatory response to exercise (48,50).

VI.

Measurement of Respiratory Muscle Strength/Endurance

A. Maximal Inspiratory Pressure/Maximal Expiratory Pressure

Respiratory muscle strength can be easily assessed by measuring the maximum sustained pressures, over one second, at the airway opening by either maximal inspiratory or maximal expiratory efforts against a closed shutter. These pressures are lung volume dependent: maximal inspiratory pressure (MIP) is highest when generated from RV as opposed to from FRC, because the length-tension characteristics of the diaphragm are most favorable and the outward chest wall recoil is greatest at RV. Conversely, the greatest expiratory pressures are measured at TLC. Therefore, it is critical to compare MIP or MEP (maximal expiratory pressure) measurements made from the same starting relative lung volumes. It is important to keep these considerations in mind when measuring MIP and MEP in subjects with CF, who may have hyperinflation and gas trapping due to peripheral airway obstruction (65). While some studies in CF patients show preserved respiratory muscle strength despite malnutrition and decreased nonrespiratory skeletal muscle strength (65–69), other studies have shown a correlation between diaphragm strength and nutritional status (70,71), and subjects with reduced fat-free body mass also have evidence of reduced diaphragm thickness (72). In any event, respiratory muscle strength tends to be preserved better than peripheral muscle strength in subjects with CF, perhaps because the resistive load of chronic airflow obstruction has a training effect on the respiratory muscles (68). This is also true in CF patients who have undergone lung transplantation (73). Physical therapy and respiratory muscle training can improve respiratory muscle strength in CF patients (70,74,75). B. Tension Time Index

The tension time index (TTI) of the diaphragm and of the respiratory muscles (TTmus) are measures of respiratory muscle endurance and the susceptibility to muscle fatigue or failure. The TTI and TTmus differ from each other because the TTI assesses only diaphragm function and requires placement of esophageal and gastric balloons, whereas the TTmus evaluates all of the inspiratory muscles and is measured noninvasively. Both are high in patients with chronic obstructive respiratory disease, indicating a susceptibility to respiratory muscle fatigue. The susceptibility to fatigue can also be thought of as a measure of respiratory muscle endurance, i.e., the ability to sustain a given level of muscular contraction. TTmus is expressed as PiMEAN Ti  PiMAX Ttot PiMEAN can be measured noninvasively, by extrapolating the P100 (respiratory occlusion pressure at the mouth measured at 100 milliseconds of a tidal inspiration) to what the mean value would be over the entire breath (Fig. 4). PiMAX is the MIP sustained

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Figure 4 The relationship between the variables involved in calculating TTMUS. PiMEAN represents the mean inspiratory pressure during a normal tidal breath, extrapolated from the occlusion pressure after the first 100 milliseconds (P100).

over one second. Ti/Ttot is the ratio of inspiratory time to entire respiratory cycle time and represents the percent of the respiratory cycle that the inspiratory muscles are active (the “duty cycle”). Respiratory endurance can also be expressed as the sustained maximal inspiratory pressure (SMIP). Here, a sustained pressure is measured during a slow inspiration against a high resistance between RV and TLC; the SMIP is the area under the pressuretime curve thus generated. In CF subjects, TTmus is high (76,77). During exercise, CF patients adopt breathing strategies (e.g., a reduced duty cycle), which help limit fatigue (78). CF subjects with low BMI have reduced SMIP despite preserved MIP (79). Inspiratory muscle training can improve endurance in subjects with CF (80).

VII.

Application of Pulmonary Function Testing

The progressive pathophysiology of CF lung disease begins with small airway obstruction and progresses to significant air trapping with bronchiectasis and cystic lung disease. Therefore, acute and longitudinal measurements of lung function need to focus on both direct and indirect measurements of airway obstruction. However, for broad longitudinal evaluation, the ideal technique to use is one that can be properly performed from infancy through childhood and is robust enough to show small early changes in lung function that precede clinical deterioration. The ATS and ERS have published guidelines on a variety of different lung function testing techniques that cover infancy (81) through the preschool years (82) into later childhood and to adulthood (6,46,83–85). Pulmonary function testing is a critical component of guidelines to describe pulmonary exacerbations (86) and for prognostic modeling (10–12). The primary measurements that are used to follow CF lung disease longitudinally are FEV1 and FEV1/FVC, with values 2 standard deviations below the mean

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indicating obstructive lung disease (46). FEV1 remains the standard measurement that is tracked both longitudinally and acutely. FEV1/FVC is not valid in infants and preschool children, since the time to exhale vital capacity decreases to the point that FEV1/FVC equals 1.00. As a surrogate measurement to FEV1, FEV0.75 and FEV0.5 have been used in younger children. Additionally, FEF25–75% can be used as an index of airway obstruction, and is felt to represent small airway function. It is perhaps not as robust as the FEV1 for a longitudinal or acute measurement since it is more variable and may not be as sensitive to small changes in lung function as is the FEV1. While FEV1 is the “standard” PFT measurement for acute and longitudinal evaluations (9,14), other measurements have been proposed in the hope of identifying earlier and perhaps preclinical airway and parenchymal disease and to allow reliable testing in patients for whom spirometry may be difficult to perform. The LCI has been shown to be more sensitive than spirometry in detecting pulmonary function abnormalities (33,34,87); however, there is not enough experience with it yet, to demonstrate its utility for longitudinal evaluation. It does offer the potential advantage of testing within the preschool population. Additionally, FO (26) and resistance measurements by plethysmography (88) and the interrupter technique (88) have also been used both for longitudinal and acute measurements of lung function.

VIII.

Standards for Testing

Whichever test of lung function is used, data quality is of paramount importance. This begins with regular daily calibration of equipment and includes the use of pulmonary function technicians trained in performing pulmonary function testing on children. The laboratory must follow established guidelines for testing and data quality. Standards for performing pulmonary function testing have been well described in the adult population in a series of statements published by the ATS and ERS (6,46,83–85). However, many of these recommendations have been modified to describe proper testing technique in younger school-age children and preschool children (6). Strict criteria are important for data integrity and “research quality” testing in multicenter and intervention studies to allow comparisons with confidence and minimized variability. If all criteria are not fulfilled, however, results of testing are not rendered useless. Within the guidelines for acceptability are modifications that suggest what data might be useful from a trial that does not fulfill strict ATS/ERS criteria. For instance, a maximal expiratory maneuver in which there is a normal volume of back extrapolation, peak flow, and full exhalation, but a cough artifact causing a spuriously high or low FEF25–75%, can produce an acceptable FEV1, FVC, and FEV1/FVC (6). Data management utilizing a centralized database is critical in a modern pulmonary function testing laboratory. From a data quality standpoint, it allows for easy overreading of data, tracking of technical deficiencies, and prompt remediation, and is a model that has been used successfully in multicenter intervention studies (89). More importantly, electronic linkage of testing stations and workstations to a centralized source allows prompt interpretation and reporting of results.

IX.

Normative Data and Multicenter Studies

Criteria for the selection of reference equations for lung function have been recently discussed (46). In addition to the quality control criteria mentioned above, reference

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equations of the laboratory should be appropriate for the age, ethnicity, and socioeconomic status of the populations being tested. Age extrapolation of reference equations beyond the limits of the patient populations on which they are based can lead to serious predicted equation errors and should not be done. Height extrapolation has similar consequences. This can be a particular problem in a patient population in which height for age values are significantly lower than the population in which normal values were derived, as is the case in CF. This has led to the proposed use of CF-specific lung function predicted values, in which children’s lung function is compared to age- and height-matched children with CF (90). One must remain cognizant of the normal change in physiological predicted values with age. For example, the FEV1/FVC ratio is normally higher in younger children and adolescents than that in adults. Choosing a fixed lower limit of normal value, of say 75%, may be incorrect for young children but correct for older adults. Because of the differences in truncal height to total height ratios among Caucasians, African-Americans, and Asians, the lung volume normalized for height can be different, and different normative equations can be used. For multicenter studies, similar equipment, testing methods, and reference equations must be used. There are many different reference equations used by pulmonary function laboratories (91–93). The ATS/ERS statement on interpretation of lung function testing reviews different sets of reference data commonly used in the United States and Europe (46). The Cystic Fibrosis Foundation has elected to use two sets of reference equations in reporting center-specific data. One set of data are used for 6- to 17-year-old males and 6- to 15-year-old females (94). The NHANES spirometric data are used for 18 years and older males and 16 years and older females (95). The selection of appropriate reference equations is particularly important in multicenter clinical trials. There are many outcome variables that are useful in clinical trials, but FEV1 remains a crucial one and is a component of nearly every seminal trial in CF therapeutics, including studies showing the efficacy of inhaled tobramycin (96), recombinant human DNAse (16), hypertonic saline (97), and the non-CFTR chloride channel activator denufosol (98). FEV1 has been shown to correlate with sputum biomarkers of inflammation (99). FEV1 has also shown no significant response in one phase 2B trial of the gene therapy vector, adeno-associated virus AAV-CFTR (100). The use of the Cystic Fibrosis Foundation’s national patient registry has allowed examination of the relationship between bacterial colonization and lung function. For example, one such study has shown that Stenotrophomonas maltophilia acquisition is associated with lower lung function, but not a more rapid decline in lung function over time (101). Several problems with FEV1 as an outcome measure are worth mentioning. First, whether to use absolute FEV1 in liters or as a percent predicted value is controversial. Absolute FEV1 can only be used in closely matched placebo and intervention groups in whom age, height, sex, race, and baseline lung function are similar. The use of percent predicted values are often used instead, but are subject to errors as the coefficient of variation of FEV1 can be different at different ages. The latter issue can be addressed by the use of standard deviation or z-scores (102). Second, the phenomenon of regression toward the mean must be taken into account in any clinical trial that has FEV1 as an outcome variable. This is the tendency

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for FEV1 to fall after enrollment in any clinical trial that has minimum FEV1 entry criteria, since subjects are likely to be at their highest FEV1 at enrollment; comparisons with baseline values are therefore subject to underestimation (103). This error should be minimized by an inclusion of placebo groups in all trials of therapeutic agents employing FEV1 as an outcome variable.

X. Longitudinal Change In time, the rate of lung growth of most patients with CF slows relative to normal. Furthermore, with more severe pulmonary disease lung growth can easily be overcome by the irreparable lung damage that occurs because of the repeated cycles of inflammation and infection. Therefore, understanding what is “normal” lung growth in CF is important when comparing the impact of an intervention longitudinally. In mixed pediatric and adult CF populations, the annual rate of decrease in FEV1 as percent predicted ranges from 1.25% to 3.6% per year (104). Additionally, there are significant sex effects with females having a more rapid loss of lung function than males (105). Additionally, patients with pancreatic insufficiency also have a more rapid loss of growth (105) (Fig. 5A, B). The trend of progressive loss of lung function is exceptionally important to recognize early as there is an inverse correlation between the rate of loss of lung function and age of death in patients with CF (105). Related to this, Rosenbluth et al. demonstrated that rate of decline of the best FEV1 over the previous four years is a more practical and better predictive model for referral for lung transplantation than the point at which the FEV1 dropped below 30% of predicted (107). Xu et al. and Que et al. examined five-year birth cohorts of patients between 18 and 22 years of age with CF and showed similar progressive decreases in the annual rate of FEV1 decline in the 1960 to 1964 cohort through the 1985 to 1989 cohort (104,108). Interestingly, VanDevanter et al. analyzed data from the Epidemiological Study of Cystic Fibrosis and showed that while there has been an increase in the mean lung function in all age groups from 1995 to 2005, the rate of loss of lung function has remained relatively constant (14). Data from the Cystic Fibrosis Foundation Patient Registry, 2007 Annual Data Report (Fig. 5C), show a similar trend when comparing longitudinal data from 1990 to 2007 (106). In this report, the FEV1 at six years of age was higher in the 2007 cohort, but the progression of lung function loss continued at about the same rate as in the 1990 cohort (106). Any reduction in the rate of lung function loss could affect the interpretation of intervention studies in which decreasing the trend of lung function loss is the primary outcome measure, as more subjects will be needed to demonstrate a statistically significant response (109).

XI.

Conclusion

Lung function testing is a critical component of a comprehensive acute and longitudinal evaluation of patients with CF. The standard for many years had been to study patients when they were able to perform a maximal expiratory maneuver. However, now there have been a number of modifications to the techniques described for adults, and development of new techniques that make lung function testing available for children from infancy through adulthood.

Figure 5 The longitudinal change in FEV1 with age relative to (A) sex, (B) pancreatic sufficiency (105), and (C) 1990 versus 2007 epoch (106). Abbreviations: PS, pancreatic sufficient; PI, pancreatic insufficient.

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44. Espiritu JD, Ruppel G, Shrestha Y, et al. The diffusing capacity in adult cystic fibrosis. Respir Med 2003; 97(6):606–611. 45. Merkus PJ, Govaere ES, Hop WH, et al. Preserved diffusion capacity in children with cystic fibrosis. Pediatr Pulmonol 2004; 37(1):56–60. 46. Pellegrino R, Viegi G, Brusasco V, et al. Interpretative strategies for lung function tests. Eur Respir J 2005; 26(5):948–968. 47. Hordvik NL, Konig P, Morris D, et al. A longitudinal study of bronchodilator responsiveness in cystic fibrosis. Am Rev Respir Dis 1985; 131(6):889–893. 48. Brand PL. Bronchodilators in cystic fibrosis. J R Soc Med 2000; 93(suppl 38):37–39. 49. Halfhide C, Evans HJ, Couriel J. Inhaled bronchodilators for cystic fibrosis. Cochrane Database Syst Rev 2005; (4):CD003428. 50. Weinberger M. Airways reactivity in patients with CF. Clin Rev Allergy Immunol 2002; 23(1):77–85. 51. Beydon N, Amsallem F, Bellet M, et al. Pre/postbronchodilator interrupter resistance values in healthy young children. Am J Respir Crit Care Med 2002; 165(10):1388–1394. 52. Black J, Baxter-Jones AD, Gordon J, et al. Assessment of airway function in young children with asthma: comparison of spirometry, interrupter technique, and tidal flow by inductance plethysmography. Pediatr Pulmonol 2004; 37(6):548–553. 53. Delacourt C, Lorino H, Fuhrman C, et al. Comparison of the forced oscillation technique and the interrupter technique for assessing airway obstruction and its reversibility in children. Am J Respir Crit Care Med 2001; 164(6):965–972. 54. Oostveen E, MacLeod D, Lorino H, et al. The forced oscillation technique in clinical practice: methodology, recommendations and future developments. Eur Respir J 2003; 22(6):1026–1041. 55. Davies PL, Doull IJ, Child F. The interrupter technique to assess airway responsiveness in children with cystic fibrosis. Pediatr Pulmonol 2007; 42(1):23–28. 56. Crapo RO, Casaburi R, Coates AL, et al. Guidelines for methacholine and exercise challenge testing-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. Am J Respir Crit Care Med 2000; 161(1):309–329. 57. Rodwell LT, Anderson SD. Airway responsiveness to hyperosmolar saline challenge in cystic fibrosis: a pilot study. Pediatr Pulmonol 1996; 21(5):282–289. 58. Balfour-Lynn IM, Elborn JS. “CF asthma”: what is it and what do we do about it? Thorax 2002; 57(8):742–748. 59. Orenstein DM, Higgins LW. Update on the role of exercise in cystic fibrosis. Curr Opin Pulm Med 2005; 11(6):519–523. 60. Rogers D, Prasad SA, Doull I. Exercise testing in children with cystic fibrosis. J R Soc Med 2003; 96(suppl 43):23–29. 61. American Thoracic Society. ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 2002; 166(1):111–117. 62. Ross RM. ATS/ACCP statement on cardiopulmonary exercise testing. Am J Respir Crit Care Med 2003; 167(10):1451; author reply 1451. 63. Ferrazza AM, Martolini D, Valli G, et al. Cardiopulmonary exercise testing in the functional and prognostic evaluation of patients with pulmonary diseases. Respiration 2009; 77(1):3–17. 64. Barker M, Franke E, Bohle M, et al. Effect of DNase on exercise capacity in cystic fibrosis. Pediatr Pulmonol 2004; 38(1):70–74. 65. Lands L, Desmond KJ, Demizio D, et al. The effects of nutritional status and hyperinflation on respiratory muscle strength in children and young adults. Am Rev Respir Dis 1990; 141(6):1506–1509. 66. Hanning RM, Blimkie CJ, Bar-Or O, et al. Relationships among nutritional status and skeletal and respiratory muscle function in cystic fibrosis: does early dietary supplementation make a difference? Am J Clin Nutr 1993; 57(4):580–587.

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12 Thoracic Imaging in Cystic Fibrosis Pulmonary Disease MOLLY RASKE Seattle Children’s Hospital, Seattle, Washington, U.S.A.

ALAN S. BRODY Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, U.S.A.

I.

Thoracic Imaging in CF Pulmonary Disease

A. Chest Radiography

For decades, the mainstay for imaging cystic fibrosis (CF) pulmonary disease has been chest radiography. Even after the advent of cross-sectional imaging, the use of chest radiography continues to be promoted, its appeal based on its accessibility and relative low doses of radiation. In fact, the 2004 consensus conference report for adult CF care recommends posterior-anterior (PA) and lateral chest radiographs every two to four years in stable CF patients. This report also advises chest radiographs in patients experiencing complications such as pulmonary exacerbation, hemoptysis, pneumothorax, and lobar collapse (1). Greene and colleagues demonstrated that chest radiography was insensitive for detecting acute exacerbations in adults, and suggested that its worth lay in excluding critical complications like pneumothorax (2). Chest radiographs may be most valuable in long-term evaluation. A number of chest radiography scoring systems have been proposed including the Shwachman-Kulczycki (3), the Chrispin-Norman (4), modified Chrispin-Norman (5), the Brasfield (6), the Northern (7), and the Wisconsin (8) scoring systems. These systems score abnormalities including hyperinflation, bronchiectasis, peribronchial thickening, atelectasis, nodules, large parenchymal opacities, and cysts/ bullae. Publications have correlated scores obtained with different systems with different clinical and laboratory parameters (9–11). However, studies by Terheggen-Lagro et al. and others have shown no statistical difference between systems (9–11). TerheggenLagro et al. reported low interobserver variability between the systems with all of them correlating strongly with pulmonary function tests (PFTs) and exacerbation rate (11). The Wisconsin system is the most recently developed. Its creators strove to address a deficiency in the other systems, “the inability to discriminate normality and mild disease on chest radiographs” (8). They proposed this ability would make chest radiographs invaluable in long-term clinical studies. The authors noted that chest radiographs could evaluate the young child who cannot reliably perform PFTs. In an ongoing study of CF neonatal screening, they found that it was more sensitive than spirometry in showing mild disease progression (12–14). They hypothesized that the infection initially affected small airways and that associated morphologic abnormalities

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seen on chest X ray preceded decline by spirometry (12). The Wisconsin system was developed as a research tool and its use on patients in the clinical setting is untested (15). Slattery et al. showed that radiographs obtained over a multiple-year period showed a decrease in lung decline compared with both untreated patients and with the subject’s prior rate of decline (16) (Fig. 1). B. Computed Tomography

Interest in the use of computed tomography (CT) in evaluating CF pulmonary disease began in the late 1980s as articles reported that CT was superior to chest radiography in detecting mild disease and the pattern and extent of bronchiectasis (17–19). Also noted was its utility in distinguishing hilar adenopathy from pulmonary artery enlargement (18). Subsequent CT research improved our understanding of the development of CF lung disease by demonstrating disease in very young children (20) and by showing bronchiectasis, sometimes extensive, in children with normal PFTs (21). A series of publications have commented on the potential for this modality to serve as a research tool and possibly to guide clinical care. Morphologic abnormalities relating to the spectrum of CF pulmonary disease are extensively described in the literature. The most often reported abnormality is bronchiectasis, seen clearly on CT and to a lesser degree on chest radiography. A bronchus should be similar in size or smaller than its accompanying artery and enlargement of this ratio is well demonstrated on CT scan. Other findings identified on CT include bronchial wall thickening, mucus plugging, parenchymal abnormalities (centilobular nodules, atelectasis, consolidation), and air trapping on expiratory images or mosaic attenuation on inspiratory images (19–29) (Fig. 2). Clinical Utility

The early publications of CT imaging in CF patients largely focused on validating the use of CT by reporting its positive correlation with spirometry and defining pulmonary abnormalities in CF patients in their usual state of health. Later studies shaped our notions of how CT can be used to complement spirometry and clinical evaluation (23,30) in the work-up of pulmonary exacerbations and overall disease progression. Several researchers showed that CT positively correlated with clinical measures such as serum immunoglobulin levels (25), bronchoalveolar lavage (BAL) inflammatory markers (31), and clinical scores (25,27,32,33). CT was also shown to demonstrate treatment effects (23,28). Shah and colleagues studied adult CF patients and showed that the finding of air fluid levels within dilated bronchi was limited to patients imaged during an acute exacerbation. They also described that centrilobular nodules, mucus plugging, and peribronchial thickening resolved in a subset of their study population scanned after treatment (28). When studying pediatric CF patients during pulmonary exacerbation, the findings of peribronchial thickening and mucus plugging as well as overall score significantly improved after treatment (23). Essential to interpretation of CT for acute disease is establishing which findings constitute a reversible versus an irreversible change. Some findings, however, are seen to represent both. For example, bronchiectasis is most often classified as an irreversible finding (28,29,34). Yet examination of CT scans obtained during an acute exacerbation in children under four years of age showed a statistically significant improvement in

Figure 1 PA (A) and lateral (B) chest X ray in a patient with CF demonstrates mild hyperinflation, bronchial wall thickening, and bronchiectasis present in all lobes but most prominent in the bilateral upper lobes. Nodular densities are present throughout the lungs, likely reflecting mucus plugging. This radiograph would receive a 12 in the Brasfield scoring system as follows: Subtracting from a total possible score of 25 points:
3 (hyperinflation),
3 (interstitial markings),
2 (cysts/nodules),
2 (focal infiltrate),
3 (overall impression) ¼ 12.

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Figure 2 (A–C) Three representative axial slices from a thoracic HRCT shows characteristic

findings in a patient with CF. All lobes contain dilated, thickened bronchi, mucus plugging, and parenchymal nodules. Several bullae are present in the left lower lobe.

bronchiectasis after treatment (31). Indeed, reversible bronchiectasis has been described in patients with acute pneumonia, resolving four to six months after infection. Similarly, the finding of bronchial wall thickening has been categorized as irreversible by some investigators (29,35) and reversible by others (23). Robinson and colleagues studied a population with relatively severe disease and demonstrated no significant reversibility of this finding after treatment (35). Likewise, Shah and colleagues studied adults and only saw reversibility of this finding in 11% of patients (28). These studies suggest that in more advanced patients bronchial wall thickening likely represents inflammatory infiltration coupled with fibrosis (36). In a population with milder disease, bronchial wall thickening could reflect transient inflammation as 73% of young patients showed resolution of this finding after treatment (23). Mosaic attenuation and air trapping

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Figure 3 Two axial CT acquired at the same level during inhalation (A) and exhalation (B) in a patient with CF. Differences in attenuation throughout the lung, termed “mosaic attenuation,” are subtly seen on the inspiratory image and accentuated on the exhalation image. During exhalation, some regions of the lung appropriately collapse and become higher in attenuation than they were during inhalation whereas the anterior and posterior upper lobes and the apical segment of the right lower lobe remain lucent, reflecting the inability of the acini subserved by the small airways in that area to empty, a process known as “air trapping.”

describe the finding of hyperlucent regions in the lung parenchyma seen on inspiratory and expiratory CT, respectively (Fig. 3). This finding is felt to reflect obstruction of airways too small to be resolved by CT (less than 0.5–1.0 mm) (21). In infants and young children with mild disease, this finding is thought to represent potentially reversible airway obstruction or reactivity (37,38). In more severely affected patients, air trapping did not show significant improvement after treatment for exacerbation (35). In these patients, hyperlucency more likely reflects constrictive or obliterative bronchiolitis, an irreversible end-stage process. Authors disagree on the use of CT scanning in clinical care. In point/counterpoint articles, Tiddens suggested that CT was the method of choice to stage the severity of CF lung disease and should be used routinely for assessment and evaluation of treatment (39). Cooper and MacLean questioned whether the benefit of CT outweighed the radiation risk, and noted that there is “insufficient evidence for the benefit of CT” (40). The Cystic Fibrosis Foundation has yet to list guidelines to define the role of CT in the surveillance of CF pulmonary disease.

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Research Potential

The recognition that CT’s value as a research tool was predicated on converting descriptive observations of structural changes into quantitative data prompted the development of several CT scoring systems over the past decade. These scoring systems share in common the assessment of bronchiectasis, peribronchial thickening, mucus plugging, and parenchymal opacities. Some of these systems also include air trapping, centrilobular nodules, and sacculations (32,41). All scoring systems recognize that CF affects different lung regions with varying degrees of severity and therefore divide the lung into regions, with the sum of the regions’ scores providing a total score. Despite differences in scoring, de Jong and colleagues reported that the scoring systems of Bhalla et al. (22), Castile et al. (42), Helbich et al. (33), Santamaria et al. (32), and Brody et al. (23) were “comparable and robust.” Each system demonstrated good reliability, correlation with the other systems, and correlation with PFTs. Weaknesses included increased variability after a longer interval of time between readings, lower interobserver reproducibility among subscores, and greater disagreement between observers when rating mild disease. It was suggested that repeat training and reference images may enhance recall and help remedy at least some of these issues (24). In an effort to improve objectivity and accuracy, researchers have proposed computerized analysis of several scoring components, specifically air trapping, bronchial wall thickness, and bronchiectasis (38,43,44). Computerized analysis of air trapping has shown the effect of Pulmozyme over a three-month period (38). Computerized analysis shows promise but needs refining and further validation before it can be embraced (45). Outcome Surrogate

Spirometry, a functional study, has long been an accepted standard for detecting disease progression and regression in CF patients. Its use is valued both in clinical work and research. In fact, early studies on chest radiography and CT imaging of pulmonary disease in CF proved positive correlation with PFTs as a way of validating their scoring systems (6,11,24,26,27,29,33,46–48). Toward the late 1990s several studies began to question whether PFT measurements had adequate sensitivity to detect mild disease or slight progression. Helbich and colleagues demonstrated morphologic changes on CT that significantly differed between two age groups, although no significant differences in PFT measurements between the two groups existed (25). Discrepancies between the CT and PFT results have since been proven several times over with studies showing weak correlations between spirometry and CT (21), stable spirometry with deteriorating CT findings (33,34,49,50), and subpopulations that show improvement in PFTs with decline in CT scores (34). A sophisticated chest X-ray scoring system even proved more sensitive to early disease progression than PFT measurements (12). With the discovery that PFT measurements are relatively insensitive to mild change, support for the use of CT as an outcome surrogate emerged. A surrogate endpoint is a surrogate for a true endpoint such as exacerbation or death. Surrogate endpoints can decrease the number of subjects needed and/or shorten the duration of a trial, but must be carefully validated to avoid incorrect results (51,52). The argument in support of CT as a surrogate endpoint encompasses several qualities. CT is biologically plausible as it has been shown to correlate with clinical and laboratory parameters (22,23,26–29,32–34,46,47,53) and with pathologic studies (36). CT is feasible for study

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populations and accuracy can be achieved with established standards in acquisition and interpretation. Brody and colleagues have addressed the correlation of CT scanning and a true endpoint in their study that measured the baseline CT scores in a cohort of CF patients and the change in the CT scores after a two-year period. These data were correlated with the number of pulmonary exacerbations. The study found that baseline CT had a positive predictive value in the number of exacerbations and that the changes in CT scores also positively correlated with number of exacerbations. In contrast, the baseline PFT measurements correlated, but the change in PFT measurements did not correlate with number of exacerbations (54). CT has been used as an outcome surrogate in treatment studies with mixed results. A study using CT findings and PFT measurements to observe treatment efficacy of recombinant human deoxyribonuclease (rhDNase) showed that the observation of air trapping was more discriminatory in detecting change than the total CT or the PFT scores (38). Nasr and colleagues demonstrated a significant difference between CT scores between placebo and treatment group in a study of inhaled rhDNase. In a study designed to assess the effect of inhaled Tobramycin on decreasing disease progression in patients with mild to moderate disease at baseline, a trend was seen but this did not reach statistical significance (p ¼ 0.07) (55). No significant change in CT score was seen in a trial designed to assess drug safety. In this trial other measures such as PFTs and interleukin-8 levels were also not significantly changed, providing a test/retest evaluation of CT (56).

CT Acquisition Technique

Multidetector CT now allows for two options: (i) to volumetrically acquire a dataset through the lungs with the ability to reconstruct the images in multiple planes using thin slices or (ii) perform a high-resolution CT (HRCT), acquiring 0.5 to 1.5 mm slices every 0.5, 1.0, or 2.0 cm with gaps between the slices. Techniques can be combined according to individual center’s protocols. For example, some centers perform the volumetric acquisition during a breath hold after inspiration and the HRCT slices after expiration. A variable number of image slices are used for expiratory acquisition, ranging from 3 to 25 images covering different levels in the lung (57). Expiratory scans are used to assess air trapping while the remainder of the interpretation is obtained from the inspiratory images. In children old enough to cooperate, voluntary breath hold after inspiration and expiration is often used. Several centers opt to use a spirometry controlled technique to standardize lung volumes in an attempt to provide a more reproducible interpretation of air trapping. In infants, controlled ventilation scanning is also employed (58). The advantage of HRCT is a lower dose of radiation delivered to the patient compared with standard CT. This must be balanced against the loss of information obtaining a 1 mm slice every 10 mm, a typical protocol. Because of the gaps between images, the accurate detection of airway abnormalities can be more challenging (20). Similarly, it is reasonable to assume that parenchymal changes could likewise be underestimated on these exams. A study by Robinson and colleagues suggests that increasing the interval between slices to 2.0 cm instead of 1.0 cm causes a decrease in scoring sensitivity and should not be used to reduce radiation if optimal scoring is required (59).

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Radiation Risk

The risk of radiation-induced cancer is always a concern when performing imaging studies that deliver ionizing radiation, such as CT. This risk is greater in pediatric patients than in adult patients. This is due to increased sensitivity of growing organs to radiation, the latency of radiation effects with a longer life span allowing more time for the development of a malignancy, and the higher absorbed dose for the smaller body mass of a child for the same technical factors (60,61). The combination of the increased life expectancy of CF patients as well as the increased use of CT makes concern for radiation-induced cancer in this population a valid one. Estimates for dose acquired during a PA and lateral chest X ray is 0.1 mSV and for a V/Q scan is 2.5 mSV (62). Dose estimates for CT scans in children range from 0.5 to 3.0 mSV depending on the technique used (41,60). To put this in perspective, the background radiation dose per year to most Americans living at sea level is 3 mSV, acquired from cosmic rays and naturally occurring radionuclides. No direct link between CT scanning and increased cancer has been demonstrated. Estimates of cancer risks due to low doses of ionizing radiation are created indirectly by extrapolating from other data, most commonly the Japanese atomic bomb survivors. Estimates for the excess lifetime risk of cancer induced by cumulative radiation exposure from annual CT scans were recently reported as 0.02% for males and 0.07% for females on the basis of a mean survival age of 36 (current CF patient’s life expectancy) and increased to 0.08% and 0.46%, respectively, assuming a median survival of 50 years. The increased risk for females was based on the increased risk of breast cancer (50% of the total cancer risk) and the higher risk of thyroid cancer (63). This past decade has seen the use of CT increase substantially in the pediatric population (64). The use of CT scanning should be considered a risk benefit decision. It is important to emphasize that the risk benefit ratio is favorable for any indicated CT scan. The radiology community strives to reduce risk by lowering radiation dose. Instructions to successfully do so abound in the literature (58,61,65–68).

C. Functional Imaging–Magnetic Resonance Imaging and Nuclear Medicine

CF is known to damage the lungs in an inhomogeneous pattern. It is speculated that the relative insensitivity of a functional study such as spirometry is due to the fact that this test measures overall lung function and is unable to accurately reflect an affliction to one area. However, functional analysis does have its place in the assessment of CF pulmonary disease and functional imaging studies may be helpful in assessing regional changes. The suitability of nuclear medicine studies such as single-photon emission computed tomography (SPECT), fluorodeoxyglucose positron-emission tomography (FDG-PET), and of magnetic resonance imaging (MRI) in detecting functional changes in the lungs of CF patients is promising. MRI has the advantage of also being able to demonstrate structural changes. Findings in a study using SPECT and HRCT suggest that SPECT can reveal regions of decreased lung perfusion (and therefore ventilation) before CT exhibits morphologic abnormalities (69). Early research into using FDG-PET shows that its contribution at present is a somewhat broad measure of pulmonary inflammation in severe disease (70). Both of these functional studies expose patients to radiation, which is even more of a consideration if coupled with CT.

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MRI has the advantage of imaging without radiation and serving the dual role of a functional and morphologic exam. Several of the reasons why MRI has historically been ill suited for pulmonary imaging have been addressed with the advent of rapidly acquired sequences and with the use of contrast agents that improve image quality. Signal generation in MRI relies on large concentrations of polarizable protons, of which the lungs are fairly deficient. However, inhaled hyperpolarized 3He can result in signal-to-noise ratios 100,000 times higher than conventional proton MRI values (71). Using this technique, investigators have observed abnormalities of morphology and ventilation (71,72). Hyperpolarized 3He MRI has been used to calculate regional alveolar partial pressure of oxygen and ventilation/ perfusion ratios in an experimental animal (73). While the ability of MRI and HRCT were shown to be equal in detecting morphologic change in patients with more severe disease (74), MRI is inferior to CT in detecting morphologic changes in milder disease because of its comparatively limited spatial resolution (74,75). Further work comparing the two modalities would be helpful in defining the roles of these modalities.

II.

Sinus Imaging in CF

It is well known that the exocrine dysfunction that leads to thickened mucus in the lungs also thickens the mucus in the sinuses. These thickened secretions predispose CF patients to chronic sinonasal infection and inflammation. The majority of CF patients will suffer chronic rhinosinusitis with a smaller number also afflicted with sinonasal polyposis. Some patients remain asymptomatic of both, and the need for imaging in this population is unclear. When comparing the sensitivity of questionnaire to nasal endoscopy and CT imaging, it was shown that CT was the best of the three options in detecting sinus disease whereas patient’s subjective interpretation of their symptoms was the least correlated to the presence of sinus disease (76). In the group that is symptomatic, CT can be helpful in preoperative planning and to detect the rare complications of polyposis and chronic rhinosinusitis such as periorbital abscess and mucocele (77). CT imaging of the sinuses should also be used in asymptomatic patients in centers that use preoperative sinus surgery as a means to lessen the risk of colonizing the transplanted lung with infection from the upper respiratory tract. Routine sinus imaging in the postoperative period may not be necessary as CT was not shown to be beneficial as an outcome measure in these patients (78). The possibility of sinonasal lymphoproliferative disease in the posttransplant population demands close surveillance. However, CT would likely not discriminate between baseline inflammation and post-transplantation lymphoproliferative disorder, and until there is further data on the role of MRI, clinical rather than imaging surveillance is advised (79).

III.

Gastrointestinal Imaging in CF

Imaging in CF may now begin before birth with fetal ultrasound and MRI. Imaging is an important component of neonatal care for the diagnosis and treatment of meconium ileus (MI). In later life, imaging can be useful in conditions such as distal intestinal obstructive syndrome (DIOS), biliary disease, and suspected masses. A. Prenatal Imaging

The presence of echogenic bowel on second trimester ultrasound is associated with a 2% to 5% incidence of CF (80). This finding is identified on approximately 1% of prenatal

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ultrasound examinations and can be seen as a normal finding. Echogenic bowel can be associated with chromosomal abnormalities and infection as well as CF. Little has been written on fetal MR imaging in CF. MRI has been reported to be superior to ultrasound in the evaluation of small bowel obstruction (81). To our knowledge, no reports have described distinguishing features of MI on fetal MRI (82). B. Neonate and Young Child

The most common indication for imaging the child with CF in the neonatal period is suspected MI. The classic radiographic appearance in MI is a right lower quadrant mass with a “soap bubbly” appearance and a lack of air/fluid levels in the distended bowel. This reflects the dilated, tenacious meconium mixed with gas in loops of distal small bowel (83). However, the presence of air/fluid levels or the absence of the bubbly mass does not exclude MI (84). MI is evaluated and treated with a contrast enema. A diagnostic enema using isotonic water-soluble contrast material demonstrates a small caliber colon (microcolon); a finding that is also encountered in distal small bowel obstruction from other etiologies (85). Reflux of contrast into loops of dilated meconium containing small bowel confirms the diagnosis (86). Small pellets of meconium are frequently seen in the microcolon (Fig. 4). The use of ultrasound has been suggested to differentiate MI from ileal atresia if the diagnosis is not clear following contrast enema (87). Approximately 50% of children with MI have no additional abnormalities of the bowel (simple or uncomplicated MI). The remaining 50% have complications including perforation, volvulus, and intestinal atresia (complicated MI) (88). Complicated MI requires surgical treatment. Uncomplicated MI can be treated with a therapeutic contrast enema following the diagnostic enema, using a hypertonic water soluble contrast material to draw water into the bowel and decrease the meconium’s viscosity. In a survey of pediatric radiologists, the use of hypertonic Gastrografin was associated with increased success (89). The addition of N-acetylcysteine or Tween 80 further increased the success rate (89). A potential complication of hypertonic contrast enema is dehydration due to fluid shifts into the colon. Small children in particular should be well hydrated and monitored closely (90). The incidence of gastroesophageal reflux (GER) is increased in children with CF (91), but also seen in approximately 50% of non-CF infants. The recommendation for children without CF is not to image otherwise well-appearing children with GER (92). Since GER has the potential to damage the lungs and to decrease nutrition, imaging evaluation may be considered more often in CF patients. A barium upper gastrointestinal series or a technetium 99M nuclear medicine reflux study can be used. Esophageal pH monitoring has been reported to be more sensitive (93). C. Older Children and Adults

The most common GI complaint requiring imaging in this group is the DIOS. DIOS is primarily a clinical diagnosis, but plain abdominal radiographs showing small bowel dilation and often a right lower quadrant mass can confirm the diagnosis and distinguish DIOS from severe constipation. If the source of abdominal pain is not clear, CT scanning may be performed, revealing distended stool-filled distal ileum without air fluid levels with a “bubbly” content. Multiple methods, including a water soluble contrast enema, can be used to treat DIOS (94).

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Figure 4 Abdominal radiographs in a neonate with meconium ileus frontal (A), decubitus (B), and cross-table lateral (C) demonstrate multiple dilated bowel loops without air-fluid levels throughout the abdomen and bubbly lucencies present in the right lower quadrant. A contrast enema on a different patient with meconium ileus (D) shows a microcolon with refluxed contrast in the meconium-filled terminal ileum.

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Like other children, the CF patient can be afflicted with appendicitis. Ultrasound and CT are used to evaluate these patients. The uninflamed appendix may be enlarged in children with CF and this must be taken into account when appendicitis is suspected (95). The hepatobiliary system can also be involved in patients with CF. One common finding is hepatic steatosis (fatty infiltration), which can mimic a mass. On crosssectional imaging, the presence of normal vessels passing through the “mass” can be a useful clue to this diagnosis. Pancreatitis is increased in pancreatic sufficient CF patients (96). There are no specific imaging findings of pancreatitis. Ultrasound can detect gallstones, which may precipitate pancreatitis and evaluate for complications of pancreatitis such as pseudocyst formation. MR cholangiopancreatography can also be used to evaluate the biliary tract. The future role of radiographic exams for assessing severity and prognosis in patients with CF is promising. Much progress has already been made in defining the role of CT in research settings. Guidelines for the clinical use of CT scanning need to continue to be sought. Improvements in CT scanning techniques with reduced doses have been published and are being implemented by the radiology community. CT interpretation assistance using computer analysis is also being investigated for more widespread use. Furthermore, the benefits of using MRI techniques in following established disease are becoming increasingly recognized. Yet even as more sophisticated imaging methods are implemented, the important role of chest radiographs need to continue to be analyzed to maximize the value of this low dose and inexpensive study.

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13 Pulmonary Manifestations ANDREW BUSH Imperial School of Medicine at National Heart and Lung Institute and Royal Brompton Hospital, London, U.K.

I.

Introduction

Lung disease is the commonest cause of death in cystic fibrosis (CF) and accounts for much of the morbidity of the condition. This chapter reviews the pathophysiology of CF lung disease, and its pulmonary presentations and complications. Details of the microbiology of the CF airway are discussed in chapter 4.

II.

Pathophysiology of Lung Disease

Although subtle cellular and molecular differences may be detectable (1), for practical purposes the lungs of the CF baby are normal at birth (2–4). However, infection and inflammation develop very shortly after birth in the majority of patients (5). A. Early Airway Disease

Bronchoscopic studies in particular have dispelled the myth of the “normal” lung in the “well” CF baby. Even in screened babies, early bronchoscopy reveals evidence of inflammation and infection (6–12), and increased levels of bronchoalveolar lavage fluid (BALF) DNA (13) and protease activity (14). The Perth group perform early pulmonary function, bronchoscopy, and limited high-resolution computed tomography (HRCT) scans on their new born screening (NBS) babies (15). They have shown that lung functions as shown by raised volume rapid thoracoabdominal compression (RVRTC) technique were normal in an admittedly small number of babies in the first six months of life, but thereafter deteriorated rapidly, falling at a rate faster than one Z (standard deviation) score a year. Furthermore, there were very early HRCT abnormalities detected in these infants. Thus, abnormal structure and function is common even in NBS babies, and there is no excuse for complacency about “well” CF babies. B. Inflammation Vs. Infection

There is lack of clarity about the relationship between infection and inflammation and the role of the host inflammatory response at varying stages of the disease. What is clear is that (i) in moderately advanced CF airway disease there is both infection and an exuberant inflammatory response; (ii) inhibition of that inflammatory response using oral prednisone was shown to be beneficial in proof of concept studies, albeit at the cost of unacceptable side effects (16,17); and (iii) not all anti-inflammatory interventions are beneficial; a large, well-designed, randomized, placebo controlled trial of a leukotriene (LT) B4 receptor 198

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antagonist had to be terminated prematurely because of increased numbers of infective exacerbations in the treatment arm (18). The rest of this section is speculative. The first major question is, what is the relationship between infection and inflammation; does infection come as a response to infection, or is the CF airway intrinsically proinflammatory? The most compelling evidence that infection is primary comes from the Australian bronchoscopic studies, which showed increased inflammatory markers in BALF only in the presence of infection. Indeed, in an “ultra-pure” group who were never infected there was no evidence of inflammation (11). Others believe that the CF airway is itself proinflammatory in the absence of infection; this is based in part on studies of explanted human CF fetal tracheas in the flanks of immunosuppressed (severe combined immunodeficiency) mice (19) and also human explanted airways (20). Before there is any evidence of infection, the CF airway is in a pro-inflammatory state, with increased IL-8 and subepithelial leukocyte accumulation, and the extra stimulus of infection leads to an early and intense inflammatory response. Whether the CF airway is intrinsically pro-inflammatory, it is clear that there is an exuberant inflammatory response to infection. There are at least four hypotheses for the relationship between inflammation and infection in CF, not all mutually exclusive: (i) the CF airway is proinflammatory; (ii) there is a failure to clear infection, leading to an appropriately increased inflammatory response; (iii) there is an increased inflammatory response relative to the burden of infection in CF; and (iv) there is a deficit of one or more molecular pathways that normally lead to the resolution of infection. These hypotheses are dealt with in more detail in chapter 5. An additional possibility is that CF neutrophils may be abnormal. The role of neutrophils in CF lung disease has recently been reviewed (21). Gene expression in blood and sputum neutrophils is altered in CF (22). Many studies are in relatively late stage CF lung disease, making it impossible to determine whether the abnormalities are primarily due to CF, or secondary to the burden of infection and inflammation in the airway. A recent paper using a genome-wide SNP analysis identified interferon-related developmental regulator 1 (IFRD1) as an important modifier gene for CF lung disease severity (23). IFRD1 is a histone deacetylase–dependent transcriptional coregulator expressed during terminal neutrophil differentiation (24). IFRD1 polymorphisms were significantly associated with variation in human neutrophil effector function. This manuscript has firmly shifted the inflammatory focus onto the neutrophil. The second, more practical question is, at what time in the evolution of the disease should there be attempts to modulate inflammation? It seems likely but unproven that at all stages of the disease, the inflammatory process is to some extent beneficial in preventing systemic spread of infection. It also seems likely that the host inflammatory response initially clears infection, and, even when chronic infection is established, contains the local consequences. This greatly complicates the designing of trials of antiinflammatory strategies in CF (25); recruiting all comers will undoubtedly dilute any signal. The role of inflammation in CF lung disease is discussed in more detail in the chapter 5. C. Inflammation/Infection/Obstruction Cycle

CF airway disease progresses to bronchial obstruction and airway wall destruction. The current paradigm is that intraluminal infection and inflammation lead to secondary

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airway wall changes, and undoubtedly this is part of the picture. However, the asthma paradigm of inflammation leads to airway wall remodeling has been challenged (26–29), and new data in CF suggests that there may also be an intrinsic airway wall defect. Children diagnosed with CF because of symptoms have airflow obstruction at presentation, irrespective of whether they have any positive cultures or respiratory symptoms (30–32); this persists through the preschool years, despite treatment in specialist CF centers (33,34). Even in CF children without any respiratory complications, there appears to be a decrement in spirometry (30). Bronchoscopic studies have suggested that there might be at least two forms of structural airway wall abnormalities: matrix destruction, as shown for example by increased BALF glycosaminoglycans, which correlates with neutrophilic airway inflammation, and thickening of the reticular basement membrane, which appears to be independent of infection and inflammation (35). Furthermore, airway smooth muscle is increased in CF, and this is not related to airway inflammation (36). Studies utilizing the CF mouse nose have also demonstrated epithelial changes and increased submucosal lymphocytic infiltration in the absence of an increase in infection or inflammation (37). In summary, although there is clearly a large element of airway wall destruction in CF secondary to infection and inflammation, there may also be components of airway obstruction that are related to an intrinsic CFTR defect in the airway wall. If this is true, it implies that therapeutic strategies additional to antibiotics and anti-inflammatories may be needed to prevent progressive airflow obstruction.

III.

Pulmonary Clinical Presentations

A. Introduction

With the advent of NBS, symptomatic presentation is becoming increasingly rare. However, even in areas where NBS is performed, late diagnosis of CF must be considered. Mild atypical cases (usually pancreatic sufficient) may be missed by screening; the screening test may not have been performed, or there may have been some sort of laboratory error; or the family may have moved from a non-NBS location after the birth of the CF child. As a safe general rule, if anyone (physician or family) thinks that CF is a remotely possible diagnosis, safe practice is to perform at least a sweat test, reserving more sophisticated testing for cases with a higher index of suspicion. At all ages, for purposes of considering most of the pulmonary presentations of CF, the disease may be considered as essentially an immunodeficiency localized to the lungs, and thus the acronym SPUR (Severe, Persistent, Unusual, Recurrent infections) may be useful. Presentation may also be with complications of these infections. Presentation with severe bacterial pneumonia at any age, especially if slow to resolve, should prompt consideration of the diagnosis of CF. If typical CF organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, and Burkholderia cepacia are isolated in the context of either acute respiratory symptoms or chronic sputum production, with or without bronchiectasis, then diagnostic testing for CF is mandatory. The significance of any lower respiratory symptom is heightened if there are also any extrapulmonary problems suggestive of CF, especially chronic diarrhea and failure to thrive; electrolyte disturbance such as hypokalemia, hyponatremia, and chloride depletion; rectal prolapse; and (in children especially) nasal polyps, which are almost diagnostic of CF in this age group. A particularly important physical sign, neglected in

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pediatric but not in adult practice, is digital clubbing. Anecdotally, digital clubbing in small children in particular is often severe before it is noticed. B. Antenatal

There are no pulmonary presentations; gastrointestinal presentations may be diagnosed by antenatal ultrasound examination (38–40). C. At or Soon After Birth

Respiratory disease is rare at birth, and given the pathophysiology of CF is characterized by epithelial sodium channel (ENaC) overactivity (41,42), and that ENaC is pivotal in switching the lung from an intrauterine secretory organ to an extrauterine organ of respiration (43), this is unsurprising. Respiratory distress from birth in a term baby is more suggestive of primary ciliary dyskinesia (PCD) (44,45), a surfactant protein (SP) deficiency (SP-B, SP-C, ABCA3) (46–48), congenitally small lungs from any cause, or other congenital abnormality of the airway (e.g., large airway obstruction), parenchyma (e.g., large congenital thoracic malformation), or chest wall (e.g., undiagnosed diaphragmatic hernia) (49). Congenital heart disease also enters the differential diagnosis. Presentation with bowel obstruction is commoner than respiratory issues in the newborn period (see chap. 17). D. Infancy

Recurrent respiratory infections are normal in early childhood. Indeed, the hardest diagnosis of all may be that of “normal child.” For example, normal infants may have more than 10 viral colds/year and symptoms may last for more than two weeks (50,51). Viral colds are particularly likely if the infant is in a child care facility (52), and firsttime parents in particular may have misconceptions of the pattern of respiratory illness in infancy (53). A really important distinction is between episodic and continuous symptoms; the latter, particularly if relentlessly worsening, should prompt investigation. Productivesounding cough is universal with colds, but the child who at any age has a productive sounding cough or (in older children) daily sputum production lasting for more than eight weeks should be investigated (54). A “wet” cough is strongly predictive of the presence of lower airway secretions (55). In infancy, typically severe and persistent bronchiolitis, often but not invariably due to respiratory syncytial virus (RSV), may be the presenting feature. CF should be excluded in any infant with prolonged oxygen dependency after bronchiolitis. Presentation with severe bacterial pneumonia at this or any age should prompt consideration of the diagnosis of CF. There are old reports of presentation with staphylococcal empyema (56), and although this seems much less common in modern times (57,58), the possibility of CF presenting this way should not be forgotten. This is particularly true in the developed world setting where necrotizing and complicated pneumonia seems to be becoming more common again (59,60). About one-third of infants will wheeze in the first three years of life (61), and most will not require investigation. A number of important points should be considered in the initial evaluation. Firstly, wheeze is used by parents to describe a variety of respiratory noises (62–65), including rattling and other upper airway noises, and efforts

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must be made to determine what exactly the noise is, perhaps with a video questionnaire (64,65). Wheeze may be episodic (viral) or multitrigger (66), and the pattern determined, the latter being more likely a manifestation of CF. Any treatments used, and the response, should be noted. Finally, any associated features suggestive of CF should be sought (above). However, it should be noted that even typical episodic viral symptoms with nothing else to find on history and examination may be the first presentation of CF. Gastroesophageal reflux (GER) is almost universal in infancy, but in particular if it is unusually severe, may be the presenting feature of CF. In addition to easy and recurrent vomiting, symptoms such as poor feeding, arching away from the breast, and crying may be due to GER and esophagitis. Finally, in the days before NBS in the United Kingdom, an all too common presentation of CF was as “neurotic mother”; the mother who frequently sought medical and nursing opinions and was convinced there was something wrong with the baby, and whose persistence lead to this unflattering label. All too frequently, these babies turned out to have CF, and the mother therefore wise and not neurotic. The just anger of these mothers understandably persists for years. E.

Childhood and Adolescence

Presentation may be with “atypical asthma,” established bronchiectasis, or pneumonia with unusual features. Asthma may be the initial consideration because parents have misinterpreted other sounds as wheeze, or “cough variant asthma” (which is grossly overdiagnosed) thought to be the problem. Unfortunately, if the child does not respond to basic asthma therapy, instead of doubting the diagnosis, treatment is often escalated uncritically and the diagnosis delayed. Asthma with any extrapulmonary feature (above) should in particular prompt a diagnostic review. Less usually, airflow obstruction with acute bronchodilator-responsiveness is documented, and this misleads the physician. CF and asthma can coexist (67), although the diagnosis of asthma in this context is very difficult. Bronchiectasis is a worrisome clinical presentation of CF. The airway is essentially normal at birth, but very rapidly, combinations of infection and inflammation begin the process of airway wall destruction (see the preceding text). As the disease progresses, there is hypertrophy of the bronchial circulation, and airway wall destruction. The lumen becomes filled with thick, viscid, adherent secretions. Distal lung destruction becomes prominent, and the combination of airway obstruction, infection, and inflammation leads to parenchymal destruction and cyst formation. Three types of cysts are described (68): the commonest is bronchiectatic, in direct communication with the airways; the second type is interstitial, lined by fibrous tissue; and the third is emphysematous, as a result of parenchymal destruction. Cyst rupture may lead to pneumothorax (see later). It is to be hoped that in a developed world setting, no child would elude diagnosis until established bronchiectasis is present, but a sweat test is part of the workup of bronchiectasis of unknown cause at any age. As in infancy, pneumonia with atypical features, or extrapulmonary findings, should prompt exclusion of CF. Upper airway disease may be the presenting feature of CF in children. In particular, nasal polyps are almost pathognomonic of CF in children; aspirin-sensitive asthma, a cause of polyps, is very rare, and although in some series of PCD patients they are reported as common (69,70), this has not been my personal experience (71).

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Adulthood

A review (72) of the major series (73–80) of more than 1000 late-diagnosed CF patients showed that age at diagnosis ranged from 13 to 73 years, and the majority of studies showed a female predominance. The biggest series (n ¼ 786 patients), comprising data from the USCFF (United States Cystic Fibrosis Foundation) registry showed that the incidence of adult diagnosis (age  18 years) of CF was 7.8% in 1996, with a total prevalence of 10.9% (77). Late diagnosis is becoming more common (81,82). Women were more likely to be diagnosed late. Acute or persistent “respiratory symptoms” and nasal polyps were commoner in adult diagnosed patients, and gastrointestinal features, including failure to thrive, were less common, because they had milder, pancreatic-sufficient mutations. Typical respiratory manifestations of CF presenting in adult life include bronchiectasis; atypical “asthma”; and the isolation of classical CF microorganisms such as S. aureus, P. aeruginosa, and even Burkholderia cepacia (unusual infections). Pulmonary infection with nontuberculous mycobacteria may be the first presentation (83), and isolation of this microorganism should prompt diagnostic testing for CF, including a genotype, given that sweat electrolytes may be nondiagnostic in adults. Upper airway problems, including severe chronic sinusitis and nasal polyps, should prompt consideration of the diagnosis.

IV.

Differential Diagnosis

In this section, the differential diagnosis of the pulmonary presentations is discussed by age group. A. Infancy

Most children with intermittent cough are normal. Congenital or acquired immunodeficiency including PCD enters the differential diagnosis. PCD typically presents with respiratory distress in the newborn period (71,84) and in around 50%, with heterotaxy (85). There are prominent upper airway symptoms. Timing of onset, nature of the infecting organism, and extrapulmonary manifestations may give clues to the nature and type of a systemic immunodeficiency. Shwachman’s syndrome should be considered in the context of neutropenia and pancreatic insufficiency. HIV may present with respiratory infection [often pneumocystis pneumonia (PCP) by Pneumocystis jirovecii] and failure to thrive in the infant of an apparently healthy woman. PCP is suggested by marked tachypnea and hypoxemia in a child who is not systemically unwell. Aspiration syndromes enter the diagnosis, including incoordinate swallow because of bulbar or pseudobulbar palsy, laryngeal cleft, GER, and late-presenting H-type fistula (86,87). Recurrent, usually localized infection may be as a result of airway obstruction or a localized anatomical parenchymal abnormality such as a congenital thoracic malformation. Airway obstruction may be intraluminal (e.g., foreign body, tumor), intramural (e.g., airway malacia or stenosis), or due to extrinsic compression (e.g., by a vascular ring or sling, a congenital cyst, or lymph node enlargement). In a developing world context, tuberculosis will be the major diagnostic consideration. Extrapulmonary manifestations suggestive of CF, for example rectal prolapse (88) and severe dehydration (89), combined with even atypical respiratory manifestations, should prompt diagnostic testing for CF. Many of these considerations will also enter the differential diagnosis in older children.

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CF should be considered in any child with undiagnosed bronchiectasis, atypical asthma (in particular if nonatopic), and prominent upper airway disease. There are numerous potential causes of bronchiectasis that enter the differential diagnosis of the respiratory manifestations of CF (90,91). A chronic productive cough with neutrophilic airway cytology and chronic infection typically with Haemophilus influenzae may be due to persistent bacterial bronchitis (92–94). CF should always be excluded as part of the workup of this condition. C. Adulthood

In adults, unlike in children, nasal polyps are more likely part of the aspirin-sensitive asthma diathesis. The differential diagnostic considerations are same as for children, with the additional clue that male infertility may be part of the picture.

V. Diagnostic Approach If there is any likelihood that a patient has CF, then further diagnostic testing is mandatory. In addition to the diagnostic considerations above, the presence of airway obstruction for no apparent reason on spirometry should prompt consideration of CF. Chest X ray (CXR) abnormalities may be suggestive, but are not pathognomonic. If there is marked airway wall thickening, severe hyperinflation, or upper lobe disease in an “asthmatic,” then CF must be excluded.

VI.

Pulmonary Complications of CF

A. Pneumothorax

This is a potentially major complication of CF, usually seen in those with severe chest disease, and should always be taken seriously. Prevalence in Children

The largest pediatric series from Melbourne (95) reported that in a clinic of around 500 children, there were 17 pneumothoraces experienced by 11 children over a 15-year period. All had evidence of moderate or severe CF lung disease, with chronic infection with mucoid or nonmucoid P. aeruginosa, or B. cepacia. Spirometry was mainly less than 60% predicted and declined further after the pneumothorax. Four of 11 were dead within a year. Treatments included needle aspiration, chest tube insertion, and pleurodesis. No new cases were reported after September 2000, presumably reflecting improved lung function with modern treatment. Prevalence in Adults

Data from the USCFF Registry (n ¼ 28,858 patients, adults, and children) reported an annual incidence of 0.64%, and that overall 3.4% of patients (n ¼ 965) suffered one or more pneumothoraces (96). Pneumothorax was commoner in older patients (73% were over age 21), especially if forced expiratory volume in one second (FEV1) <40% predicted (a feature of > 75% patients, one of the principle risk factors). Unlike in children (mentioned earlier) there were no apparent secular prevalence changes. Risk

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factors included infection with P. aeruginosa, B. cepacia, or Aspergillus fumigatus. Other risk factors, cosegregating with bad lung disease, include low socioeconomic status, need for enteral feeding, pancreatic insufficiency, allergic bronchopulmonary aspergillosis (ABPA), and massive hemoptysis. Pathophysiology

The pathophysiology of this condition in CF is unclear, but it is thought to be related to severe airflow obstruction leading to air trapping and in some cases, the rupture of subpleural blebs and cysts. In general, pneumothorax is a feature of severe CF lung disease. It may coincidentally occur spontaneously in well CF children. Presentation

The patient may complain of sudden onset chest pain and dyspnea, although asymptomatic pneumothorax has been described. The diagnosis is often easily made on CXR, but sometimes HRCT may be required to demonstrate the air leak. Management

CF patients with all but the most trivial pneumothorax should be admitted and given intravenous antibiotics, with extra attention to sputum clearance and hydration. Prolonged immobilization, with poor respiratory excursions because of pain and the discomfort of the chest drain, must be avoided. A variety of specific treatments have been proposed, including simple observation if the pneumothorax is small (<20%), needle aspiration, thoracostomy tube placement, chemical or abrasion pleurodesis, and pleurectomy. A consensus document has been published (97), advocating early surgery if there is not rapid resolution of the pneumothorax. Surgical pleurodesis is preferred, unless the patient is too sick to tolerate operation. There are no randomized controlled trial (RCT) data on any aspect of management. A special case, with particular management difficulties, is pneumothorax in CF patients being treated with noninvasive ventilation [nasal intermittent positive pressure ventilation (NIPPV)] (98). The positive airway pressures may worsen the condition, and NIPPV may need to be withheld temporarily. The management dilemmas have recently been summarized (98). Prognosis

Pneumothorax adversely affects prognosis, with a drop in lung function, and between 6.3% and 14.3% attributable risk of death within two years. Four-year mortality after a pneumothorax was 40%. Treatment decisions have been influenced by the likelihood that the patient will need a lung transplant in the near future, and there have been worries that over-vigorous surgical pleural procedures may lead to major bleeding during transplantation. However, an observational study showed that CF patients withpneumothorax, irrespective of whether they had undergone a pleural procedure, had dense adhesions compared with CF patients without a pneumothorax or other diagnostic groups such as pulmonary fibrosis who underwent transplantation. However, surgical outcomes including use of blood products were not any worse in this group (99). Thus previous pneumothorax and pleural procedures do not contraindicate lung transplantation.

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The usual source of bleeding in CF is the hypertrophied bronchial circulation. Bronchial blood flow is more than 10-fold increased in CF (100). Minor blood streaking of the sputum is common in CF and requires no additional management. Massive hemoptysis, defined arbitrarily at all ages as bleeding > 240 mL in a single day, or recurrent bleeding over several days or weeks of > 100 mL/day (101), is frightening for the patient and may require active management. Really massive hemoptysis may cause life-threatening hypotension or airway obstruction. Massive hemoptysis is usually but not invariably a complication of severe lung disease. Less severe chronic hemoptysis interfering with the patient’s lifestyle and activities may also merit active treatment. Prevalence in Children

A large retrospective series from Australia reported on approximately 750 CF children studied over 20 years (102). There were 102 bleeding episodes in 51 children. Only four were younger than 10 years, age at first bleed was at a mean of 15 years, range 7 to 19. There was a mean of two episodes per child, range 1 to 7. Fifty-eight percent had severe lung disease, but massive hemoptysis was also seen in milder patients. Most patients had chronic infection with P. aeruginosa. Prevalence in Adults

Data from the USCFF Registry (n ¼ 28,858 patients, adults, and children) reported an annual incidence of 0.87%, and that overall 4.1% of patients (n ¼ 1153) suffered one or more episodes of massive hemoptysis (103). As with pneumothorax, there was no change in prevalence over the time period of the study. More than 60% of patients had an FEV1 <40% predicted. However, 11% had normal lung function. Although there was a minor increased odds ratio of bleeding in patients with diabetes or infection with S. aureus, there were no particularly obvious predictive or protective factors. The apparent protective effect of infection with P. aeruginosa and B. cepacia may simply be a survivor effect. Pathophysiology

The source of bleeding is from the bronchial arteries or nonbronchial collateral arteries. Bronchial arterial hypertrophy is a physiological response to airway inflammation (104). The combination of areas of infection and inflammation, and hypertrophied arteries at systemic pressure, is thought to lead to bleeding. However, this hypothesis, although plausible, does not account for the admittedly rarer cases of massive hemoptysis in patients with relatively mild lung disease. It is possible that there is a modifier gene effect, with candidates being angiogenic growth factors (105,106), but this is to date speculative. Presentation

Hemoptysis is usually obvious but needs to be distinguished from hematemesis (usually from varices) and upper airway bleeding. The patient often can feel which lung is the source of bleeding, and this impression is usually accurate.

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Management

For all but trivial hemoptysis, admission to hospital is advisable. Resuscitation of the patient as appropriate is the first priority. Blood should be taken for a platelet count and coagulation studies, and cross-matched in case transfusion is necessary. Any coagulopathy should be treated. CXR may localize the site of bleeding. HRCT is unlikely to contribute much to the acute management of the patient. Bronchoscopy is generally not a useful investigation, and should only be performed if there is real doubt as to whether bleeding is from the airway. Any medications that may exacerbate bleeding should be discontinued, but only after careful consideration of risk and benefit, given the total lack of any evidence for this recommendation. Indeed, there was no association between use of inhaled medications and massive hemoptysis in the U.S. Registry study (103). Intravenous antibiotics are commenced; these should probably include cover for S. aureus (103), and cautious airway clearance must be continued under the guidance of an experienced respiratory physiotherapist. It is better to clear secretions in a controlled manner rather than cough uncontrollably as sputum builds up. Many patients will settle with conservative management. If active bleeding continues, then bronchial artery embolization should be considered (107–109). All large vessels should be occluded, irrespective of whether they are thought to be bleeding. The major risk is spillover of embolic material causing neurological side effects (109); spillover into the spinal arteries may cause paraplegia. Initial success rate is good. Ninety-eight percent children stopped bleeding with the first procedure but nearly half will require a further procedure (108); recurrence rate was the same irrespective of whether embolization was performed. In adults, multiple sessions were commonly needed and recurrence was also common (107). Lobectomy is a last resort (110), and with the availability of modern interventional radiology it should rarely, if ever, be needed. There are anecdotal reports of treatment with tranexamic acid (111–114). As with so many aspects of CF there are no RCT data to guide management. Prognosis

Four of 51 children died in the acute phase of bleeding (109). Median survival was 103 months after bronchial artery embolization, compared with 52 months after conservative treatment, but this cannot be taken to prove the superiority of embolization. Also, with modern treatments, a better prognosis is now likely. However, patients with hemoptysis requiring admission to ICU have a poor prognosis (114). In the USCFF dataset (103), massive hemoptysis impacted adversely on lung function and survival. The two-year attributable mortality was between 5.8% and 16.1%, and there is a more than seven-fold risk of death in patients with massive hemoptysis and an FEV1 > 50% compared with those with similar lung function and no massive hemoptysis. One group suggested that the increase in respiratory failure in the time period after embolization may relate to loss of the carbon dioxide–exchanging function of the very hypertrophied bronchial circulation (115). Spirometry also declined after hemoptysis, and there was a greater subsequent use of medical resources. The U.S. study (108) suggested there might be fewer exacerbations after embolization, but this observation requires confirmation. At the present time, the need for embolization should be judged on the current clinical state and the need to control ongoing hemorrhage; any additional long-term benefit should be seen as a bonus.

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A. fumigatus is ubiquitous in the environment. The manifestations of A. fumigatus pulmonary disease in CF are protean and include isolated positive sputum cultures [up to 60% in some series (116)], which are by no means as benign as was once thought (117,118); an allergen provoking wheeze [skin test sensitivity in up to 60%, positive radioallergosorbent test (RAST) in nearly 50%]; large airway plugging; ABPA; and rarely, mycetoma and invasive aspergillosis. IgG-positive precipitins may be found in up to 70%. Only ABPA is covered in this chapter; other manifestations are discussed in chapter 4. An ABPA-like picture has rarely been reported as being caused by other fungi, for example, other strains of Aspergillus and non-Aspergillus species such as Scedosporium apiospermum (119). Definition

Diagnostic criteria are debated because many of the symptoms and signs of ABPA may be part of the underlying CF (117,120). Furthermore, markers of sensitization to A. fumigatus and positive sputum cultures are common in CF. Major and minor criteria for ABPA have been proposed, but it is clear that atypical cases may not meet classical criteria but still require treatment. Certainly, treatment should not be delayed if the index of suspicion is high but “classical” criteria are not met. Prevalence of ABPA

This is difficult to determine because of the different diagnostic criteria used and the different indices of suspicion prevailing in the various clinics. A large database study (117) gave a prevalence of 2% across the whole age range, almost certainly, as the authors concluded, an underestimate. Smaller series have reported prevalences of up to nearly 20% (121,122). Nonetheless, despite the possible diagnostic inaccuracies, this database study gives useful information. There was an increasing prevalence with age up to 20, thereafter there was a slight fall off in prevalence. Ten percent patients had a normal FEV1 and more than 80% did not wheeze. There was a high prevalence of infection with P. aeruginosa (around 70%). The relationship between the two organisms is complex—P. aeruginosa exotoxins may inhibit the hyphal growth of A. fumigatus, which could either lead to the release of A. fumigatus antigens and worse ABPA, or lead to a lesser likelihood of A. fumigatus airway infection and thus less likelihood of ABPA (123). Only one-third had a positive sputum culture for A. fumigatus [just less than half in the European study (124)]. Males were marginally more likely to have ABPA than females (not the case in the European study). The European registry study (967 ABPA patients in a total of 12,447 CF patients in the registry) also had a peak in the 13- to 18year age range, with a low prevalence below the age of six years. There were wide variations in prevalence across Europe (overall 7.8%, range 2.1–13.6%). An ABPA diagnosis was associated with a poorer general clinical condition (10% lower FEV1, lower weight Z-score, more commonly infected with P. aeruginosa, B. cepacia, Stenotrophomonas maltophilia, and, surprisingly, H. influenza; there was no association with S. aureus infection) and there was an association with pneumothorax and massive hemoptysis, presumably related to the underlying severe lung disease. There were no associations with particular genotypes, although unfortunately no analysis by class of genotype was performed, which might have been informative (125). Finally, ABPA is a

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risk factor for infection with atypical Mycobacteria, although whether this is just because of steroid therapy rather than the underlying disease cannot currently be determined (126). Pathophysiology

A. fumigatus spores (diameter 3 mm) are aerodynamically capable of impacting in the distal airways, and unusually, the organism grows at body temperature. A. fumigatus may provoke an intense allergic response leading to secondary airway damage, but proteolytic and other enzymes may be released, leading to nonallergic, direct pulmonary toxicity (120). ABPA is the result of a skewed CD4þ, TH2 T-cell response to A. fumigatus leading to IL-4 and IL-5 production and hence elevation of serum IgE, and airway eosinophilia, contrasting with the CD4þ TH1, IgG response in the nonatopic patient (127). Familial occurrence has been reported, suggesting a genetic component (128). Class 2 HLA antigens have a crucial role interacting with antigen specific T cells. One study showed that HLA-DR2 and 5, and possibly 4 and 7, confer susceptibility to ABPA in the setting of CF, whereas HLA-DQ2 confers resistance (129). IL-10 is an anti-inflammatory cytokine, which is important in controlling T-cell responses. The –1082GG polymorphism in the promoter region of the IL-10 gene leads to increased production of IL-10 and is associated with A. fumigatus infection and ABPA (130). The hypothesis would be that increased IL-10 levels lead to downregulation of the T-cell response to A. fumigatus and hence a greater risk of infection and complications. Diagnostic Criteria

It should be accepted that, although tables of major and minor criteria are useful guides to the diagnosis of ABPA, they are no more than guides, and atypical cases will continue to be diagnosed on an individual clinical basis. The USCFF Consensus Conference (120), which is a useful although now outdated reference guide, proposed criteria for classic ABPA, minimal diagnostic criteria, and recommendations for screening (see Text Box). These are a useful guide to the clinician and very helpful for ensuring uniformity of diagnosis in registries, but cannot be considered definitive under all circumstances.

Classical ABPA Acute or subacute clinical deterioration not attributable to another cause Serum total IgE > 1000 IU/mL in a patient not receiving oral corticosteroids Positive skin prick test to A. fumigatus Positive IgG precipitins to A. fumigatus New or recent CXR abnormalities not clearing with conventional therapy such as physiotherapy and antibiotics Minimal diagnostic criteria for ABPA Acute or subacute clinical deterioration not attributable to another cause Serum total IgE >500 (retest in 1–3 months if 200–500) Positive skin prick test to A. fumigatus One of positive IgG precipitins to A. fumigatus or new or recent CXR abnormalities not clearing with conventional therapy such as physiotherapy and antibiotics

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ABPA should be suspected in patients with an increase in respiratory symptoms, particularly if there is wheeze, chest tightness, or pleuritic chest pain. If this last symptom is present, a pleural rub may be audible. Exceptionally, pleural effusion has been described in ABPA (131). There may be a sharp decline in spirometry. The CXR typically shows one or more new soft fluffy shadows, with a “gloved finger” appearance of mucus impaction in the airways; these would be very unusual in a standard CF chest exacerbation. In our hands, the single most useful test has been an abrupt, more than fourfold rise in total IgE to more than 500 (132); we also showed that a fall in IgE may occur with treatment, but this is not inevitable and serial IgE measurements should be used with caution to determine steroid tapering (133). A high level of IgG-precipitating antibodies to A. fumigatus may be helpful (>90 mg/mL, Immunocap) (134), but in this study, as with so many in this field, there was no second population to validate the findings in the discovery population. Others have used the pragmatic but minimalist criteria of new radiological shadowing that does not respond to intravenous antibiotics. A. fumigatus antigens may be secreted or cytoplasmic, and it is the latter that may be particularly elevated in ABPA patients. There is evidence that using targeted allergy testing to specific purified A. fumigatus antigens obtained from cDNA may be helpful, but the literature is unclear as to which antigens should be used. One study using intracutaneous testing suggested that Asp f4 and Asp f6 gave the optimal diagnostic results (135). Receiver operating curves in one study suggested that sensitivity to Asp f3 and Asp f4 gave the best sensitivity and specificity for ABPA in CF (136); unfortunately, this study lacked a second population in which to test this hypothesis. A further drawback is that the levels did not fall with remission of ABPA, making them not useful in following the effects of treatment (133). Another proposed marker, again needing validation in a second population, is serum thymus and activated regulated chemokine (TARC) levels, proposed to be better than IgE, Asp f4, or f6 levels (137). TARC is involved in the modulation of the T-cell response to fungal infection and is known to be elevated in ABPA. TARC levels may be useful in monitoring the response to treatment of ABPA; other cytokines were not useful. Screening

An annual measurement of total IgE is recommended, with further investigation if IgE >500, or 200 to 500 and the index of suspicion is high. Management

Avoidance of risk where practical is advisable, specifically playing or working in damp places such as stables where A. fumigatus spores are in high concentrations (138). There is no satisfactory evidence base on which to recommend the nature and duration of treatment of ABPA. If there is any doubt about the diagnosis, then intravenous antibiotics should be given first. The mainstay of treatment is oral prednisolone, which may need to be given in high dose for a prolonged period of time. A typical regime would be 2 mg/kg for two weeks (maximum 60 mg), then 1 mg/kg for two weeks, then 1 mg/kg on alternate days for two weeks, followed by a slow taper. My practice is to add an oral antifungal agent, usually itraconazole, for two reasons. Although there is no RCT in ABPA complicating CF, in ABPA complicating asthma there is clear evidence that this

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is beneficial (139,140). There is also less strong, retrospective evidence of benefit in CF (141). However, it should be noted that oral absorption is poor and that serum levels should be measured if practical; there is also a drug interaction with inhaled budesonide, namely itraconazole inhibition of the cytochrome p450 enzyme CYP3A, which can lead to Cushing’s syndrome and iatrogenic adrenal suppression (142). Secondly, rare cases of invasive aspergillosis complicating CF have been described (143,144), and in the context of COPD, airway obstruction and prednisolone therapy are sufficient to trigger this complication (145), and at least in theory the addition of itraconazole may prevent this. If steroid side effects become intolerable, which is usually in the context of recurrent episodes of ABPA, anecdotally, other treatments used have included pulsed methyl prednisolone (146,147), nebulized amphotericin (which may be combined with nebulized budesonide) (148), oral voriconazole (149), a prolonged course of intravenous liposomal amphotericin, and the anti-IgE monoclonal antibody omalizumab (xolairTM) (150,151). Inhaled corticosteroids are commonly employed (124), but there is only the most limited evidence that they are beneficial. Prognosis

Some cases of ABPA may resolve spontaneously, but the majority have relapses after treatment. ABPA can be divided into five stages with different prognoses (Text Box) (120); it is arguable whether these are clinically useful. They are not a chronological progression in clinical practice. If not aggressively treated, ABPA may lead to severe proximal bronchiectasis. Rate of change of lung function during follow-up of ABPA was not affected by the diagnosis in the European Registry study (124); however, this was not the case in another study (152) so prognosis must be guarded.

Stages of ABPA Stage 1: Acute phase. There are acute infiltrates, which clear completely with prednisolone Stage 2: Remission. No prednisone therapy or infiltrates for six months Stage 3: Recurrent exacerbation similar in type to stage 1 Stage 4: Phase of steroid-dependent asthma Stage 5: Fibrotic disease, no longer completely responds to prednisolone therapy

D. Lung or Lobar Collapse

This should be relatively uncommon complication of CF if modern treatments are applied. The diagnosis should always lead to consideration of whether ABPA is the underlying cause. Lobar collapse may be due to proximal mucus plugging, or distal collapse and fibrosis, with the proximal airways patent; a CT scan can distinguish the two. Standard therapy includes the use of intensive physiotherapy, possibly augmented by positive airway pressure; either or both of rhDNase and hypertonic saline; and appropriate intravenous and nebulized antibiotics. If this fails, bronchoscopy should be considered. However, if the CT scan shows that the collapsed lobe contains a prominent

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air bronchogram, it is unlikely that bronchoscopy will be effective. In the case of extensive proximal mucus plugging, there is evidence from case reports that directly instilling rhDNase endobronchially may be beneficial (153,154). If there are really tenacious plugs of sputum that cannot be removed using the fiberoptic bronchoscope, rigid bronchoscopy may be considered. Surgical removal of a chronically and irreversibly collapsed lobe is occasionally contemplated in the patient with otherwise mild disease elsewhere. However, in general a CT scan will reveal that the disease is not truly isolated, and there is widespread bronchiectasis. The occasional patient may get shortterm benefit from lobectomy (155), but only in the rare case of a destroyed nonfunctional lobe with otherwise well-preserved lung architecture. E.

Antibiotic Allergy and Desensitization

Aggressive use of antibiotics has brought many benefits to the CF patient, but inevitable problems including the selection of resistant organisms (156), subtle long-term toxic effects such as chronic renal failure (157), and antibiotic allergy (158–160). Antibiotic allergy is a frequent problem due to multiple exposures; there is no evidence that the CF patient is more likely to become sensitized than a normal person. Typically, it becomes a problem in teenagers and young adults, and multiple allergies are common. Most reactions occur within 24 hours of commencing treatment, pruritis and rash being the most common. Frequency increases with age, reaching up to 36% in adults, exposure and decrement in lung function. Piperacillin is particularly likely to lead to allergic reactions, including cross-allergy to other antibiotics (161). Allergic reactions are commonest for penicillins and cephalosporins, intermediate for carbapenems, and lowest for aztreonam. Aminoglycosides rarely cause allergic sensitization. Treatment is with desensitization, which is time-consuming and requires detailed monitoring, because there may be severe reactions in up to 20% of cases; detailed protocols have been published (160,162). However, in most, desensitization is successful, but in up to 25%, usually because allergy is non-IgE mediated, it fails (163). The use of corticosteroids and anti-histamines may increase the likelihood of successful desensitization.

VII.

The Lung Under Stress

A. Air Travel and Altitude

Issues for the CF patient contemplating a journey include (i) tolerance of the conditions of the journey itself, including hypoxemia in commercial jets, immobility, and potential lack of access to treatment for a prolonged period and (ii) the risks of the actual destination. The risks include whether there will be an access to CF care if needed, the risks of high altitude, and the risks of acquiring a new infection. It should be remembered that it is always easy to trigger an acute deterioration, but it may be much harder to regain lost ground; “holiday disasters” are well described (164) and should be preventable by careful planning. In particular during long flights, dehydration must be avoided by drinking liberal amounts of nonalcoholic fluids. Dehydration may predispose to bowel obstruction, and, combined with immobility, to pulmonary embolism. Salt supplements are recommended in hot climates. Commercial jet flights mean breathing the equivalent of 15% oxygen, so assessment of the need for supplemental oxygen is important (note that in nonpressurized cabins, the potential for hypoxemia is much greater. Unsurprisingly (165), the patients with the worst lung disease are more likely to need oxygen, but there is no one good predictive

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clinical parameter. It is not known how best to assess this (164,166–168); predictive equations may be substantially overestimate the need for oxygen (169). A sea-level hypoxic challenge with 15% oxygen has been advocated (164,166), but this is not as sensitive or specific as one would wish; however, for all its imperfections, this remain our preferred test, and the level of oxygen supplementation can be titrated on the same visit. Furthermore, hypoxemia may be exacerbated if the patient falls asleep in the aeroplane (166). However, no one knows what level and duration of hypoxemia in an aeroplane is safe. Most commercial airlines will arrange in-flight oxygen if it is booked in advance, but the price charged varies considerably. It should be noted that the fiscal cost of diverting a commercial flight to make an emergency landing may exceed $50,000, quite apart from the inconvenience to other passengers, so the decision to sign a “fit to fly” letter is not a light one! If the CF patient is holidaying at altitude, hypoxemia becomes even more important. The ideal would be to do a hypoxic challenge during overnight sleep (169), but this is rarely feasible. If there is any suggestion of sleep desaturation on room air at sea level, prudence suggests that overnight oxygen might be wise at altitude; however, this is not evidence based advice. Pneumothorax is said to be a risk if the patient has cystic changes in the lung, because of fears of cyst rupture secondary to expansion of the cyst as cabin pressure drops. This fear is largely based on loose physiological thinking. By definition, the cystic areas must be in communication with the outside air, albeit with a long time constant, or else the air would be absorbed and the cyst would collapse. So during take-off and landing, when cabin pressures gradually change, there should be enough time for intracyst pressure to track. This is borne out by the extreme rarity of in-flight pneumothorax under any circumstances. Another important risk is acquisition of infection. Usual infections may be acquired in the cramped conditions of an aeroplane, but on arrival, new risks may become significant. There have been a number of reports of the acquisition of B. pseudomallei infection in, for example, Southeast Asia, northern Australia, and Brazil (170,171). Patients need to take an informed decision about these potential risks. Finally, an adequate supply of all medications needs to be taken on holiday. Patients should be warned about potential side effects, especially photosensitivity with ciprofloxacin and minocycline (172). It is important that they be equipped with a nebulizer that is compatible with local electricity supplies. Many units have devised a “holiday pack” of protocols, travel tips, and contact details, for the increasing number of CF patients who go abroad (173). B. During General Anesthesia

Surgery may need to be performed as a result of CF-related complications or for a coincidental, unrelated condition. In all cases, the anesthetic, surgical and CF teams need to work closely together; firstly to see if the surgery is truly necessary and secondly to ensure CF-related complications of anesthesia and surgery are detected early, and appropriate treatment is continued. It is easy for important CF-related issues to be forgotten in a nonspecialist CF surgical unit (174). Preoperative Planning and Assessment of the Patient

It is worth briefly considering whether other procedures should be performed opportunistically while the primary surgery is performed, particularly obtaining lower airway

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secretions either by blind suction below the vocal cords performed by the anesthetist or by fiberoptic bronchoscopy. CF issues should not distract from the routine preoperative checks normally carried out by the anesthetist. Ideally, the anesthetist and the CF physician should jointly see the patient. If this is not possible, at least a discussion of the current CF issues should take place. In addition, there are a number of special CF-related issues that should be considered. Oxygen saturation and spirometry should be measured in all those over age five, unless pain or another severe illness precludes spirometry, and the results should be compared with the patient’s usual values. For elective procedures, the patient’s respiratory status should first be optimized with intensive physiotherapy and appropriate antibiotics. A sputum or cough swab culture should be obtained prior to surgery, if time permits. For all but minor procedures in well patients, a course of intravenous antibiotics should be considered, starting at least 48 hours prior to the procedure and continuing until the patient is pain free and has made a complete recovery. The need for per- and postoperative steroid treatment should be judged on standard criteria. A preoperative CXR should be obtained. Nutritional status should be assessed and optimized as the urgency of the surgical situation permits, and any electrolyte imbalance corrected. Any constipation should be treated vigorously to avoid postoperative distal intestinal obstruction syndrome (DIOS). The presence of GER should be noted. CFRD should be managed using standard protocols; the stress of surgery may precipitate hyperglycemia in the CF patient with borderline endocrine pancreatic function. Finally, the potential for drug interaction should be noted. Intraoperative Management

Suxamethonium should probably be avoided, because this agent may cause postoperative pain that will impede the performance of efficient physiotherapy. Standard intraoperative monitoring is performed as usual. The patient should be ventilated in such a way so as to prevent postoperative atelectasis as far as possible. The anesthetist should be aware of the possibility of the build up of secretions causing V:Q mismatch or even blocking the endotracheal tube. In general, preoperative chest physiotherapy while the patient is anesthetized has not been shown to be useful (175). Intra- and postoperatively, oxygen and other inhaled gases should be humidified. Drugs that might cause postoperative suppression of cough, or constipation, should be avoided. Regional blockade may reduce the need for opiate analgesia. Postoperative Care

Adequate pain relief without cough suppression is essential. Chest physiotherapy is crucial. Opiates and dehydration may predispose to constipation, which should be treated. If the postoperative phase is prolonged and nutrition is difficult, a nasogastric tube can be used for feeding using a predigested diet. If a prolonged period of ileus or other cause of failure of enteral nutrition is anticipated, then total parenteral nutrition should be instituted early. C. In the Intensive Care Unit

This section discusses the indications for intensive care in the CF patient, the details of the intensive care unit (ICU) course are discussed elsewhere. Admission to ICU is unequivocally indicated in the sick child in whom the diagnosis is in doubt and for

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ventilation after surgical procedures. Infants and children aged four years and under with CF who need ventilation generally have a good outcome (176). ICU admission for invasive ventilation (intubation, tracheostomy) is in my view firmly contraindicated in CF patients who have deteriorated progressively and remorselessly despite optimal medical care, and are terminally ill. The position may be difficult if the patient is waitlisted for lung transplantation and is desperately hanging on and hoping for a donor organ, but the results of transplanting an invasively ventilated CF patient are significantly worse (177), and it is difficult to argue ethically that, in a situation of donor organ shortage, it is right to transplant a poor prognosis group. Having said that, prolonged survival after transplantation during the course of positive pressure ventilation has been described. The CF patient who has an acute severe deterioration on the background of relatively good health should be managed maximally without ventilation, but if ventilation is necessary, the results are reasonable. If deterioration is due to hematemesis, pneumothorax, or surgery, the results are good (178). Hemoptysis may carry a worse prognosis (114,176). Overall mortality has decreased in recent years, but the need for invasive ventilation is still a poor prognostic feature (a more than 16-fold increased risk of dying in this series, with a mortality of nearly 60%, which has not changed over time). Interestingly, ICU outcome is not predictable from standard prognostic markers of CF lung disease severity in at least some (179) but not all (114) series. Some have reported an adverse effect of malnutrition (174) and diabetes (180,181) Overall, ICU survival is similar in CF to a general ICU population (around 80%), although clearly the CF ICU population is highly selected. Prognosis for a pulmonary exacerbation severe enough to warrant ICU treatment is poor both during the ICU course and in the succeeding year in those who survive (182). Many ICU survivors (including those having been intubated) will go on to be transplanted in the following 12 months (183), with no apparent effect of previous ICU admission on at least short-term outcome. Use of non-invasive ventilation (NIV) after ICU care may anecdotally be beneficial in improving survival (114).

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126. Mussaffi H, Rivlin J, Shalit I, et al. Nontuberculous mycobacteria in cystic fibrosis associated with allergic bronchopulmonary aspergillosis and steroid therapy. Eur Respir J 2005; 25:324–328. 127. Chauhan B, Knutsen AP, Hutcheson PS, et al. T cell subsets, epitope mapping, and HLArestriction in patients with allergic bronchopulmonary aspergillosis. J Clin Invest 1996; 97:2324–2331. 128. Chauhan B, Santiago L, Kirschmann DA, et al. The association of HLA-DR alleles and T cell activation with allergic bronchopulmonary aspergillosis. J Immunol 1997; 159: 4072–4076. 129. Chauhan B, Santiago L, Hutcheson PS, et al. Evidence for the involvement of two different MHC class II regions in susceptibility or protection in allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol 2000; 106:723–729. 130. Brouard J, Knauer N, Boelle P-Y, et al. Influence of interleukin-10 on Aspergillus fumigatus infection in patients with cystic fibrosis. J Infect Dis 2005; 199:1989–1991. 131. O’Connor TM, O’Donnell A, Hurley M, et al. Allergic bronchopulmonary aspergillosis: a rare cause of pleural effusion. Respirology 2001; 6:361–363. 132. Marchant JL, Warner JO, Bush A. Rise in total IgE as an indicator of allergic bronchopulmonary aspergillosis in cystic fibrosis. Thorax 1994; 49:1002–1005. 133. Casaulta C, Fluckiger S, Crameri R, et al. Time course of antibody response to recombinant Aspergillus fumigatus antigens in cystic fibrosis with and without ABPA. Pediatr Allergy Immunol 2005; 16:217–225. 134. Barton RC, Hobson RP, Denton M, et al. Serologic diagnosis of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis through the detection of immunoglobulin G to Aspergillus fumigatus. Diagn Microbiol Infect Dis 2008; 62:287–291. 135. Nikolaizik WH, Welchel M, Blaser K, et al. Intracutaneous tests with recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis and Aspergillus allergy. Am J Respir Crit Care Med 2002; 165:916–921. 136. Knutsen AP, Hutcheson PS, Slavin RG, et al. IgE antibody to Aspergillus fumigatus recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis. Allergy 2004; 59:198–203. 137. Hartl D, Latzin P, Zissel G, et al. Chemokines indicate allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. Am J Respir Crit Care Med 2006; 173:1370–1376. 138. Simmonds EJ, Littlewood JM, Hopwood V, et al. Aspergillus fumigatus colonisation and population density of place of residence in cystic fibrosis. Arch Dis Child 1994; 70:139– 140. 139. Denning DW, Van Wye JE, Lewiston NJ, et al. Adjunctive therapy of allergic bronchopulmonary aspergillosis with itraconazole. Chest 1991; 100:813–819. 140. Wark PA, Hensley MJ, Saltos N, et al. Anti-inflammatory effect of itraconazole in stable allergic bronchopulmonary aspergillosis: a randomized controlled trial. J Allergy Clin Immunol 2003; 111:952–957. 141. Skov M, Hoiby N, Koch C. Itraconaziole treatment of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. Allergy 2002; 57:723–728. 142. De Wachter E, Malfroot A, De Schutter I, et al. Inhaled budesonide induced Cushing’s syndrome in cystic fibrosis patients, due to drug inhibition of cytochrome P450. J Cyst Fibros 2003; 2:72–75. 143. Chung Y, Kraut JR, Stone AM, et al. Disseminated aspergillus in a patient with cystic fibrosis and allergic bronchopulmonary aspergillosis. Pediatr Pulmonol 1994; 17:131–134. 144. Brown K, Rosenthal M, Bush A. Fatal invasive aspergillosis in an adolescent with cystic fibrosis. Pediatr Pulmonol 1999; 27:130–133. 145. Palmer L, Greenberg H, Schiff MM. Corticosteroid treatment is a risk for invasive aspergillosis. Thorax 1991; 46:15–20.

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14 Treatment Strategies for Maintaining Pulmonary Health in Cystic Fibrosis SUSANNA A. MCCOLLEY Northwestern University Feinberg School of Medicine, Chicago, Illinois, U.S.A.

I.

Introduction

Progressive lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). To address this, significant effort has been put forth in understanding the pathophysiology of the disease and in developing therapies. In this chapter, a conceptual framework for maintenance treatment of CF pulmonary disease is presented. This framework includes both models of the progression of CF lung disease and strategies to maintain and improve lung health. Specific therapies are discussed, but the reader is encouraged to recognize that therapies for CF lung disease are constantly evolving.

II.

Principals of Maintenance Therapy for CF Lung Disease

A. Goals of Treatment to Maintain Pulmonary Health in CF

The specific goals of pulmonary therapies for CF will vary according to the stage of disease of the affected individual. All patients will benefit from therapies that maintain or improve pulmonary function, reduce the risk of pulmonary exacerbation, and reduce the rate of pulmonary function decline. It is important to recognize that many therapies for CF lung disease are expensive, time consuming, or otherwise burdensome. It is essential that clinicians, patients, and families communicate well about the risks and benefits of specific therapies and make decisions on treatment together. Suggested general goals are presented in Table 1. B. Approaches to Maintenance of Pulmonary Health in CF

Conceptually, it is useful to consider two different models of the progression of lung disease. Neither of these models alone leads to a single therapeutic approach to maintain pulmonary health in CF. Taken together, however, they allow the clinician or researcher to rationally consider interventions that may result in better clinical outcomes. The first, a pathophysiologic model, was first proposed by Dr Jeffrey Wine (1) and is presented in Figure 1. In this construct, the pathophysiologic cascade begins with the mutant cystic fibrosis transmembrane regulator (CFTR) gene, resulting in absent or abnormally functioning CFTR protein. The lack of a normally functioning protein results in abnormal mucociliary clearance, chronic bacterial infection, and intense airway inflammation. Destruction of the lung parenchyma and bronchiectasis result and worsen over time. Eventually, progression of lung disease leads to respiratory

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Table 1 Goals for Pulmonary Maintenance Therapies in Cystic Fibrosis

Lung disease severity Asymptomatic

Mild

Moderate

Severe

Short-term goal

Long-term goal

Remain asymptomatic Avoid chronic infection Reduce the risk of exacerbation Reduce symptoms Avoid or reduce the burden of chronic infection Reduce the risk of exacerbation Reduce symptoms Maintain or improve quality of life Reduce the risk of complications, e.g., hemoptysis Reduce the risk of exacerbation Reduce the risk of respiratory failure Reduce symptoms Maintain or improve quality of life Reduce the risk of complications, e.g., hemoptysis Reduce the risk of exacerbation Reduce the risk of respiratory failure

Maintain normal lung function Reduce the rate of lung function decline Maintain or improve lung function Reduce the rate of lung function decline

Maintain or improve lung function Reduce the rate of lung function decline Reduce the risk of mortality

Reduce the risk of mortality Improve the outcome of lung transplantation, when applicable

insufficiency and failure, leading to death unless successful lung transplantation occurs. In this model, therapies are targeted toward specific abnormalities along the cascade. This model is extremely useful for understanding the development of therapeutic targets, including replacement of the mutant gene with a normal copy of CFTR, replacement or improvement in protein function, and treatments targeted toward improving mucociliary transport and reducing inflammation and infection, and, ultimately, replacement of lung through transplantation. A second model of CF lung disease is proposed in Table 2. This developmental model of onset and progression serves to summarize the stages of CF lung disease. The CF lung is essentially normal at birth; other than some hypertrophy of submucosal glands, there are no significant structural or functional abnormalities. Young infants with CF diagnosed by newborn screening show normal airways function, measured by forced expiratory volume in first 0.5 seconds (FEV0.5) until about six months of age, at which time lung function drops below age-matched infants without CF (2); this decline continues over subsequent years. During infancy and early childhood, there is significant airway inflammation, as measured by increased neutrophils and proinflammatory cytokines, notably IL-8, in bronchoalveolar lavage (BAL) fluid (3,4). Importantly, although infection clearly intensifies the inflammatory response, inflammation is seen even in the absence of infection detected by conventional techniques (5). Structural and

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Table 2 Stages of CF Lung Disease

Approximate age or stage

Pulmonary pathophysiology

Newborn through early infancy (birth to 6 mo) Later infancy, preschool (6 mo–5 yr)

Nearly normal structure, normal function, intermittent infection Mildly abnormal pulmonary function, air trapping, early bronchiectasis, intermittent to chronic infection Variable pulmonary function, air trapping, early to moderate bronchiectasis, intermittent to chronic infection Variable, but often declining lung function, symptomatic bronchiectasis, often chronic infection Variable, but often abnormal lung function, symptomatic bronchiectasis, chronic infection Severe obstructive lung disease, bronchiectasis with chronic abscesses, abnormal gas exchange

Childhood (6–12 yr)

Adolescence (13–17 yr)

Adulthood (18 yr and older) End stage (childhood to elderly)

Figure 1 Pathophysiologic cascade of CF lung disease.

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functional abnormalities are first seen in the smallest airways, with bronchiolar inflammation causing air trapping. The mucociliary clearance defect promotes airways infection, which ultimately becomes chronic. Bronchiectasis is evident early in life (6,7) and progresses with time. In this conceptual model, therapies are targeted toward reducing the progression of lung disease from an earlier to a later stage or toward maintaining health when disease is advanced.

III.

General Principles of Therapy

A. Frequent Health Care Supervision by a Multidisciplinary Care Team

CF is a complex, chronic, lifelong disorder. Positive family adjustment, support of family, and development of self-management strategies are essential to promote optimal health. The multidisciplinary care team, which includes physicians, nurses and/or nurse practitioners, physical and respiratory therapists, dieticians, social workers, and, often, clinical psychologists, is an essential resource to meet these goals. Family and patient education, emphasizing both knowledge of disease management and practical skills, and psychosocial support are ongoing activities for the CF team. Specific methods of disease education and self-management support in CF are varied, but must be sensitive and responsive to the family and patient’s belief systems, educational level, and primary language and culture. Team meetings to review patient status and plan outpatient sessions are helpful in assuring that patients and their families receive optimal care in a cohesive manner. Frequent monitoring allows for anticipatory guidance and also provides the clinician an opportunity to detect subtle changes in health status before overt symptoms occur. Frequent monitoring of respiratory tract cultures and pulmonary function tests are important strategies that are associated with better pulmonary outcomes in CF populations (8). Obviously, performing these measurements alone does not improve outcomes, but acting on these data allows the opportunity for additional diagnostic testing and earlier treatment of abnormalities. B. Rationale for “Aggressive” Care

There is significant variability in important health outcomes for CF patients treated at different CF clinical programs. Most of this variability is not readily explained by nonmodifiable differences in patient populations, such as genotype and socioeconomic status. The relationship between median pulmonary function (FEV1) in CF programs and care patterns, including frequency of outpatient visits and testing, prescription of various therapies, and utilization of intravenous antibiotics, was studied at 132 sites enrolled in the North American Epidemiologic Study of Cystic Fibrosis (8), a large, longitudinal observational study. Sites reporting median FEV1 in the highest quartile were compared with sites reporting median FEV1 in the lowest quartile. Those in the highest quartile saw patients more frequently, performed more spirometry and respiratory tract cultures, and had more frequent hospitalizations and courses of intravenous (IV) antibiotics than sites in the lowest quartile. Not surprisingly, sites with the highest FEV1 also reported more resistant bacteria on cultures. Utilization of other specific therapies did not vary significantly between sites. These data suggest that the general strategy of frequent monitoring and frequent use of IV antibiotics, which is probably a marker for diagnosis and treatment of pulmonary exacerbation, is more important than utilization of a given therapy.

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McColley C. The Benefit and Challenge of “Additive” Therapies

The first therapy specifically developed for maintenance therapy in CF lung disease was dornase alfa (Pulmozyme1), which was approved for use by the U.S. Food and Drug Administration in 1993. Since that time, an increasing number of maintenance therapies have been developed or adopted for CF. In most cases, each therapy has been studied as additive to “usual care,” for example, patients enrolled in clinical trials have continued prior therapies such as dornase alfa. This leads to challenges both in the design and interpretation of clinical trials. Furthermore, in situations where a patient will only take a limited number of therapies due to financial or time constraints, it can be difficult to decide what an “optimal” therapeutic regimen contains. Nevertheless, the development of new therapies for CF has had a strong temporal association with increasing life expectancy for those affected. D. The Benefits and Limitations of Practice Guidelines

Consensus conferences and practice guidelines have been developed for a plethora of acute and chronic diseases, including CF. As of early 2009, 21 practice guidelines related to CF had been published; among these, 13 specifically address patient care. These publications are intended to guide clinicians, health systems, and third party payers. In some cases, lay summaries are created, intended to benefit patients and families. Consensus conferences are intended to provide guidance for care in circumstances when adequate literature does not yet provide a strong evidence basis. The process of developing evidence-based practice guidelines has evolved over the years and now generally includes a systematic review of the literature using specific and transparent strategies, and grading of evidence. While practice guidelines are useful to provide guidance on an approach to care, the utility of guidelines is limited in that recommendations can only be made when adequate research is available to review. Medications for maintenance of lung health were addressed in a consensus conference guideline of the Cystic Fibrosis Foundation in 2007 (9). These guidelines are limited to patients aged six and older because most randomized, controlled trials of CF pulmonary therapies have been designed to detect improvement in FEV1. Therefore, they include only patients who are able to perform this maneuver (generally, those aged 6 or older) and patients who have at least mildly impaired pulmonary function. As a result, there is little literature to suggest what therapies may benefit patients less than six years of age or patients who have normal FEV1. Furthermore, therapies only received a strong recommendation if a large cohort of patients in one or more clinical trials demonstrated a significant benefit. This means that a substantial research effort must have occurred before a strong recommendation can be made. To date, research has primarily focused on patients with measurably abnormal FEV1 so that improvement can be detected. Thus, a clinician must weigh guidelines in the context of current advances and use clinical judgment when decided whether, and when, to prescribe therapies that have less robust recommendations.

IV.

Preventive and Maintenance Treatment of Infection in CF Lung Disease The role of infection in CF is unique in human disease. The microenvironment of the CF respiratory tract allows chronic, high-level bacterial infection without a high rate of systemic dissemination. Pathogens are isolated from upper and lower airways early in

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life. Staphylococcus aureus is the most common organism isolated from the CF respiratory tract in infancy and childhood. Pseudomonas aeruginosa can be isolated as early as the first year of life and becomes more prevalent over time; by early adulthood, it is the most commonly isolated pathogen. Further information about the microbiology of CF lung disease is found in chapter 4 by Planet and Saiman. The presence of chronic infection increases inflammation and is therefore an important target for intervention. One result of chronic infection is pulmonary exacerbation of CF; this is reviewed in chapter 16 by Sanders and Rosenfeld. Primary prevention of, or prophylaxis against, infections with specific pathogens is hypothesized to have the most potential impact. Treatment of initial infection, with the goal of eradication and prevention or delay of chronic infection, is another promising strategy. Chronic suppressive therapy has been widely studied and practiced, especially for patients with chronic P. aeruginosa infection. Preventive strategies have most often been employed for prevention of S. aureus infections in infants and young children with CF. A number of publications, using a variety of study designs and drugs, have shown various potential benefits of prophylactic anti-staphylococcal antibiotics. Weaver et al. (10) conducted a randomized study of daily flucloxacillin versus clinically indicated antibiotics alone in 38 CF infants diagnosed by newborn screening. Infants in the flucloxacillin group had a lower prevalence of positive cultures for S. aureus and lower hospital admission rates and lengths of stay than patients in the clinically indicated antibiotics group. A subsequent study of the same cohort revealed no difference in pulmonary function at three to four months and one year of age (11). Studies (12) that used microbiology as an outcome measure have consistently shown that anti-staphylococcal prophylaxis reduces the rate of isolation of S. aureus from respiratory tract cultures. One small study showed an improvement in weight gain, but no studies have shown an impact on pulmonary function. The largest study (13) of anti-staphylococcal prophylaxis was a double blind, placebo-controlled study of cephalexin. Among 209 children enrolled during the second year of life, 119 completed the study at six years of age. There were no differences in the clinical outcomes of the two groups, which included hospital days, other antibiotics, symptoms, signs, nutritional status, or pulmonary function. The treatment group, however, had an increased rate of respiratory tract cultures for P. aeruginosa. A retrospective study of pediatric CF patients without P. aeruginosa at baseline also showed an increased rate of P. aeruginosa infection in patients receiving various drugs for anti-staphylococcal prophylaxis (14). Other studies of prophylaxis have not shown this association. Some studies, such as Weaver et al. (10), have shown a reduction in symptoms and hospital days, though others have not. Interpretation of published literature is vexing due to methodological issues. Overall, there are insufficient data to formulate a strong conclusion regarding the effectiveness of anti-staphylococcal prophylaxis (12). Prevention of P. aeruginosa has also been explored using various techniques. Among the most promising strategies is vaccination. To date, while some vaccines have shown encouraging results (15), none has been adequately effective to develop for widespread clinical use. Primary prevention via routine inhalation of anti-pseudomonal antibiotics has also been suggested on the basis of clinical observations (16), but has not been adequately studied in randomized, prospective trials. Eradication strategies for CF infection have focused on treatment of patients with P. aeruginosa. This strategy was pioneered in Denmark, where treatment of initial

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positive cultures for P. aeruginosa with a three-month course of ciprofloxacin and colistin was implemented on a widespread basis. Compared with historical controls, treated patients had a marked reduction in chronic infection with P. aeruginosa, improved pulmonary function, and a possible survival benefit (17); as controls were historical, the effects of eradication of P. aeruginosa could not be fully separated from other advances in care. Although this specific treatment strategy was not uniformly adopted across different countries, there is now broad consensus that it may be helpful to treat patients with new infections with P. aeruginosa with antibiotics, with a goal of eradication. Various antibiotics have been studied in this context, either as mono- or combination therapy. Gibson et al. (18) performed BAL in infants and young children with positive oropharyngeal cultures for P. aeruginosa. Those with P. aeruginosa isolated from BAL cultures were randomized to receive tobramycin inhalation solution (TIS) or placebo for 28 days. When BAL was repeated at the end of therapy, 8 of 8 subjects receiving TIS had cultures that were negative for P. aeruginosa, whereas only 1 of 13 subjects receiving placebo had a negative culture. In a subsequent study (19), subjects with positive BAL cultures for P. aeruginosa received either 28 or 56 days of TIS; BAL was repeated 4, 8 or 12 weeks following cessation of therapy. In this study, the majority of subjects had BAL cultures that were negative for P. aeruginosa up to 12 weeks after completing either 28 or 56 days of treatment. Thus, TIS appears to be an effective therapy for treatment of P. aeruginosa after initial infection or reinfection. Whether or not scheduled anti-pseudomonal antibiotic therapy after negative cultures are achieved has clinical benefit is under investigation. Chronic suppressive antibiotic therapy has been employed for many years in the treatment of CF lung disease. Studies on this strategy for S. aureus are limited, because most include both uninfected patients and infected patients; thus the prophylactic versus suppressive effects cannot be easily separated. Colistin via inhalation was the first antipseudomonal antibiotic employed for chronic suppression of P. aeruginosa, but no welldesigned double-blind, placebo-controlled clinical trials have been performed. Preclinical studies of inhaled tobramycin showed good potential for chronic suppression but predicted that very high doses would be required for adequate bacterial killing, because of biofilm formation and the properties of CF sputum. Ramsey et al. (20) reported improved pulmonary function after 28 days of high-dose tobramycin, 600 mg thrice daily via ultrasonic nebulizer, in a crossover study of patients with chronic P. aeruginosa infection. On the basis of these findings, TIS was developed. Two randomized, placebocontrolled clinical trials in patients with FEV1 25% to 75% predicted and chronic P. aeruginosa showed improvement in pulmonary function and reduction in exacerbation frequency in patients receiving TIS at a dose of 300 mg twice daily during alternating four-week periods compared with those receiving placebo (21). A subsequent randomized, open label study of subjects with mild lung disease was terminated early, after patients in the active treatment group showed a greater than twofold decrease in number of pulmonary exacerbations compared with a group receiving no treatment. Chronic therapy with TIS has become a common treatment strategy for patients with chronic P. aeruginosa infection; however, studies spanning several years are lacking. A number of other inhaled antibiotics are currently under development for chronic suppressive therapy of P. aeruginosa in CF. These include inhaled preparations of

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aztreonam and fluoroquinolones as well as a liposomal preparation of amikacin. Some may have activity against other pathogens as well.

V. Other Specific Therapies for Maintenance of Pulmonary Health A. Airway Clearance and Mucoactive Therapies

Techniques to clear mucus from the airways via mechanical methods are among the oldest CF therapies in use. Therapies that improve mucociliary clearance, including dornase alfa and hypertonic saline, are widely used in CF care. Dornase alfa decreases sputum viscosity by degrading DNA that is released from dying neutrophils in the airway that were recruited to combat the airways infection. Hypertonic saline, in contrast, improves the defective mucociliary clearance of CF airways. These are discussed in more detail in chapter 15 by Ratjen and Lapin. B. Exercise

Exercise is an important adjunct to maintenance of pulmonary health in CF. Increased peak oxygen consumption (VO2 peak), a measure of aerobic fitness, is a significant and independent predictor of survival in CF (22). Long-term aerobic and anaerobic training are with improved measures of fitness, but variable results are seen in other measures such as weight, body composition, and pulmonary function, and it is unclear whether physical training is an adequate substitute for conventional airway clearance techniques (23). While benefits of exercise are clear, the best way to promote activity and adherence is not; individualized plans based on patient preferences and resources may be the optimal method, but have not been adequately studied (24). C. b-Agonists

Short-acting b-agonists, such as albuterol, have been used for many years for maintenance therapy of CF lung disease; long-acting b-agonists have been more recently introduced. The rationale for b-agonist therapy includes bronchodilation and enhanced mucociliary clearance through increasing ciliary beat frequency. Numerous studies have shown that administration of b-agonists improves FEV1; these have been reviewed in a Cochrane Collaborative report (25). While most of the randomized, clinical trials have been of short duration and are mixed in terms of inclusion of subjects with or without demonstrable bronchodilator responsiveness, both short- and longer-term studies have suggested better lung function in patients receiving short- or long-acting chronic b-agonist therapy. D. Anticholinergic Agents

Fewer studies of anticholinergics have been performed, all using short-acting anticholinergic drugs. These were included in the Cochrane Collaborative report mentioned above (25). Improvement in FEV1 has been seen in these studies; however, most are single-dose or very short-term studies, and other clinical outcomes have not been reported. There is ongoing interest in the use of anti-cholinergic agents, driven in part by their benefit in chronic obstructive pulmonary disease, and further clinical trials are anticipated.

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Corticosteroid Therapy

Early studies of oral corticosteroid therapy of CF lung disease appeared promising, with improvement in both pulmonary function and nutritional indices (26). A subsequent large, multiyear clinical trial of alternate day prednisone (27) confirmed a small but significant benefit in pulmonary function; however, this was accompanied by a high rate of serious side effects, including abnormal glucose metabolism, linear growth retardation, cataracts, and an increased rate of cultures positive for P. aeruginosa. As a result, chronic therapy with oral corticosteroids is usually reserved for treatment of complicating conditions of CF, such as allergic bronchopulmonary aspergillosis. Inhaled corticosteroids (ICS), a mainstay of therapy for persistent asthma, are frequently prescribed to patients with CF (28). A number of studies have failed to show benefit on pulmonary function or on pulmonary exacerbation rate in CF. A withdrawal study that assessed the effect of discontinuing budesonide versus continuing therapy in 117 children with CF showed no difference in time to exacerbation, the primary endpoint, between the two groups. However, in the Epidemiologic Study of Cystic Fibrosis, addition of inhaled steroid therapy in children with CF was associated with a decreased rate of FEV1 decline compared with prior to initiation of ICS therapy (29) after controlling for use of other therapies, including intravenous antibiotics. Importantly, children starting ICS therapy had decreased linear growth and an increase in use of insulin and oral hypoglycemic agents. This suggests the possibility that some patients may benefit, but there are insufficient data to specifically identify which patients may be candidates for this therapy, and corticosteroid side effects are a risk of therapy. F.

Nonsteroidal Anti-inflammatory Therapy

Ibuprofen therapy for CF was first described in a four-year, double-blind, placebocontrolled study at a single CF center in the United States (30). The main outcome was a decrease in the rate of FEV1 decline in the patients randomized to ibuprofen. A subsequent, larger, multicenter trial in Canada showed similar positive benefits of ibuprofen (31). An analysis of U.S. Cystic Fibrosis Foundation Registry Data confirmed a reduction in the rate of pulmonary function decline in a cohort of patients taking high-dose ibuprofen therapy compared with patients in the same age range not taking ibuprofen (32). G. Macrolides

Several clinical trials have shown that chronic administration of low-dose azithromycin, a macrolide antibiotic, improves pulmonary function and reduces pulmonary exacerbation frequency compared with placebo (33–35), particularly in patients with chronic infection with P. aeruginosa. Some studies have included a substantial proportion of patients without P. aeruginosa (36), and a large study of chronic azithromycin therapy for patients with CF who are not colonized with P. aeruginosa is nearing completion. Chronic administration of azithromycin is associated with resistance to azithromycin in S. aureus strains (37). H. Ion Channel Therapies

Therapies that increase chloride transport or reduce sodium absorption across the airway epithelial surface are of great interest for pulmonary maintenance therapy. Clinical trials of several agents have been undertaken. One of these agents, denufosol tetrasodium, is a

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P2Y2 receptor agonist that activates chloride transport via an alternate chloride channel. Phase 2 and 3 clinical trials showed a benefit in pulmonary function in patients with mild pulmonary disease by spirometric criteria (38,39). A phase 3 registration trial is in progress. I.

CFTR Mutation Directed Therapy and Gene Therapy

Improvement of function of mutant CFTR protein or incorporation of a normal copy of the CFTR gene into airway epithelial cells have been targets of drug development since the CFTR gene was first discovered. These are discussed in chapters 24 and 25 by Mueller and Flotte, and Hoover and Clancy, respectively.

VI.

Summary

CF lung disease has complex pathophysiology that remains incompletely understood 20 years after the discovery of the CFTR gene. Maintenance treatment is continually evolving. Prevention of pulmonary disease in CF remains an elusive goal; however, with an increasing number of therapies available, the opportunity to reduce the impact of lung disease is tremendous.

References 1. Wine JJ. Basic aspects of cystic fibrosis. Clin Rev Allergy 1991; 9:1–28. 2. Linnane BM, Hall GL, Nolan G, et al. Lung function in infants with cystic fibrosis diagnosed by newborn screening. Am J Respir Crit Care Med 2008; 178:1238–1244. 3. Khan TZ, Wagener J, Bost T, et al. Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med 1995; 151:1075–1082. 4. Kirchner KK, Wagener JS, Khan TZ, et al. Increased DNA levels in bronchoalveolar lavage fluid obtained from infants with cystic fibrosis. Am J Respir Crit Care Med 1996; 154: 1426–1429. 5. Armstrong DS, Grimwood K, Carlin JB, et al. Lower airway inflammation in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 1997; 156:1197–1204. 6. Long FR, Williams RS, Castile RG. Structural abnormalities in infants and young children with cystic fibrosis. J Pediatr 2004; 144:154–161. 7. Tiddens HAWM. Detecting early structural lung damage in cystic fibrosis. Pediatr Pulmonol 2002; 34:228–213. 8. Johnson C, Butler SM, Konstan MW, et al. Factors influencing outcomes in cystic fibrosis: a center based analysis. Chest 2003; 123:20–27. 9. Flume PA, O’Sullivan BP, Robinson KA, et al. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lug health. Am J Respir Crit Care Med 2007; 176:957–969. 10. Weaver LT, Green MR, Nicholson K, et al. Prognosis in cystic fibrosis treated with continuous flucloxacillin from the neonatal period. Arch Dis Child 1994; 70:84–89. 11. Beardsmore CS, Thompson JR, Williams A, et al. Pulmonary function in infants with cystic fibrosis: the effect of antibiotic treatment. Arch Dis Child 1994; 71:133–137. 12. McCaffery K, Olver RE, Franklin M, et al. Systematic review of antistaphylococcal antibiotic therapy in cystic fibrosis. Thorax 1999; 54:380–383. 13. Stutman HR, Lieberman JM, Nussbaum E, et al. Antibiotic prophylaxis in infants and young children with cystic fibrosis: a randomized controlled trial. J Pediatr 2002; 140:299–305. 14. Ratjen F, Comes G, Paul K, et al. Effect of continuous anti-staphylococcal therapy on the rate of P. aeruginosa acquisition in patients with cystic fibrosis: a randomized controlled trial. Pediatr Pulmonol 2001; 31:13–16.

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15. Zuercher AW, Horn MP, Que JU, et al. Antibody responses induced by long-term vaccination with an octovalent conjugate Pseudomonas aeruginosa vaccine in children with cystic fibrosis. FEMS Immunol Med Microbiol 2006; 47:302–308. 16. Lebecque P, Leal T, Zylberberg K, et al. Towards zero prevalence of chronic Pseudomonas aeruginosa infection in children with cystic fibrosis. J Cyst Fibros 2006; 5:237–244. 17. Frederiksen B, Koch C, Hoiby N. Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 1997; 23:330–335. 18. Gibson RL, Emerson J, McNamara S, et al. Significant microbiologic effect of inhaled tobramycin in young children with cystic fibrosis. Am J Respir Crit Care Med 2003; 167:841–879. 19. Gibson RL, Emerson J, Mayer-Hamblett N, et al. Duration of treatment effect after tobramycin solution for inhalation in young children with cystic fibrosis. Pediatr Pulmonol 2007; 42:610–623. 20. Ramsey BW, Dorkin HL, Eisenberg JD, et al. Efficacy of aerosolized tobramycin in patients with cystic fibrosis. N Engl J Med 1993; 328:1740–1746. 21. Ramsey BW, Pepe MS, Quan JM, et al. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic fibrosis inhaled tobramycin study group. N Engl J Med 1999; 340:23–30. 22. Nixon PA, Orenstein DM, Kelsey SF, et al. The prognostic value of exercise testing in patients with cystic fibrosis. N Engl J Med 1992; 327:1785–1788. 23. Bradley JM, Moran FM, Elborn JS. Evidence for physical therapies (airway clearance and physical training) in cystic fibrosis: an overview of five Cochrane systematic reviews. Respir Med 2006; 100:191–201. 24. Orenstein DM, Higgins LW. Update on the role of exercise in cystic fibrosis. Curr Opin Pulm Med 2005; 11:519–523. 25. Halfhide C, Evans HJ, Couriel J. Inhaled bronchodilators for cystic fibrosis. Cochrane Database Syst Rev 2005; 4:CD00348. Available at: http://www.cochrane.org/reviews/clibintro.htm. 26. Auerbach HS, Williams M, Kirkpatrick JA, et al. Alternate-day prednisone reduces morbidity and improves pulmonary function in cystic fibrosis. Lancet 1985; 2:686–688. 27. Eigen H, Rosenstein BJ, FitzSimmons S, et al. Cystic Fibrosis Foundation Prednisone Trial Group. A multicenter study of alternate-day prednisone therapy in patients with cystic fibrosis. J Pediatr 1995; 126:515–523. 28. Oermann CM, Sockrider MM, Konstan MW. The use of anti-inflammatory medications in cystic fibrosis: trends and physician attitudes. Chest 1999; 115:1053–1058. 29. Ren CL, Pasta DJ, Rasouliyan L, et al. Relationship between inhaled corticosteroid therapy and rate of lung function decline in children with cystic fibrosis. J Pediatr 2008; 153:746–751. 30. Konstan MW, Byard PJ, Hoppel CL, et al. Effect of high-dose ibuprofen in patients with cystic fibrosis. N Engl J Med 1995; 332:848–854. 31. Lands LC, Milner R, Cantin A, et al. High dose ibuprofen in cystic fibrosis: Canadian safety and effectiveness trial. J Pediatr 2007; 149:249–254. 32. Konstan MW, Schulcter MD, Xue W, et al. Clinical use of ibuprofen is associated with slower FEV1 decline in children with cystic fibrosis. Am J Respir Crit Care Med 2007; 176:1084–1089. 33. Saiman L, Marshall BC, Mayer-Hamblett N, et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. J Am Med Assoc 2003; 290:1749–1756. 34. Wolter J, Seeney S, Bell S, et al. Effect of long term treatment with azithromycin on disease parameters in cystic fibrosis: a randomized trial. Thorax 2002; 57:212–216. 35. Equi A, Balfour-Lynn JM, Bush A, et al. Long term azithromycin in children with cystic fibrosis: a randomized, placebo-controlled crossover trial. Lancet 2002; 360:978–984.

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36. Clement A, Tamalet A, Leroux E, et al. Long term effects of azithromycin in patients with cystic fibrosis: a double blind, placebo controlled trial. Thorax 2006; 61:895–902. 37. Tramper-Stranders GA, Wolfs TFW, Fleer A, et al. Maintenance azithromycin treatment in pediatric patients with cystic fibrosis. Long term outcomes related to macrolide resistance and pulmonary function. Pediatr Infect Dis J 2007; 26:8–12. 38. Deterding R, Retsch-Bogart G, Milgram L, et al. Cystic Fibrosis Foundation Therapeutics Development Network. Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y2 receptor agonist: results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis. Pediatr Pulmonol 2005; 39:339–348. 39. Deterding RR, Lavange LM, Engels JM, et al. ; for the Cystic Fibrosis Therapeutics Development Network and the Inspire 08-103 Working Group. Phase 2 randomized safety and efficacy trial of denufosol tetrasodium in cystic fibrosis. Am J Respir Crit Care Med 2007; 176:362–369.

15 Mucolytic Therapy and Airway Clearance Techniques FELIX RATJEN Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

CRAIG LAPIN Connecticut Children’s Medical Center, University of Connecticut, Hartford, Connecticut, U.S.A.

I.

Mucolytic Therapy

Airway mucus is a two compartment liquid consisting of both an aqueous layer that supports the epithelial cilia and a gel layer that mainly contains glycoproteins (mucins) (1). The term “mucus” is also used for secretions produced in respiratory diseases such as cystic fibrosis (CF), although this term may actually be inappropriate as the composition of airway secretions in CF differs greatly from normal physiological content. The goal of mucolytic therapy is to facilitate physiological clearance by optimizing the viscoelasticity of mucus. Optimizing viscoelasticity does not equal maximal reduction of both viscosity and elasticity: therapy that lowers mucus viscosity beyond the physiological state has the potential to negatively affect its transportability, as secretions with viscosity close to that of water cannot be cleared effectively by either mucociliary or cough clearance (2). Overall, mucociliary transportability of sputum can be improved by reducing sputum viscosity, given that elasticity is preserved, while cough clearability of secretions requires higher viscosity. Several agents, described below, have been used in an effort to enhance the removal of secretions from the CF airway. A. N-Acetylcysteine

Accumulation of mucopurulent secretions in the lower airways is a key feature of CF lung disease. Classic mucolytic drugs reduce the elasticity and viscosity of mucus by breaking down the gel structure of mucus. However, the major component of airway secretions in CF is not mucin derived from mucus-producing cells but rather it is pus derived from degraded neutrophils (3). While there is ongoing controversy as to whether mucins are reduced in CF airway secretions or not (4,5), there is little evidence that classic mucolytics have any efficacy in CF. The best studied compound is N-acetylcysteine (NAC), which lowers both the viscosity and elasticity of the mucus by substituting free sulfhydryl groups for the disulfide bonds connecting mucin proteins (6). Despite in vitro mucolytic activity in airways diseases including CF, there are no convincing data to support the use of inhaled NAC in CF lung disease (7). On the other hand, oral acetylcysteine has been shown to replete glutathione stores in CF patients and may potentially play a role as an antioxidant (8).

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B. Dornase alfa

CF sputum as well as bronchoalveolar lavage fluid have been shown to contain significant amounts of DNA (9,10). Extracellular DNA is mainly derived from neutrophils recruited into the airway lumen as part of the inflammatory response. Extracellular DNA polymerizes and contributes to the viscid airway secretions in CF patients (11). Dornase alfa is a recombinant form of the human DNase 1 enzyme and digests extracellular DNA. When mixed with purulent sputum from subjects with CF, dornase alfa has been shown to lower the viscosity and adhesivity of sputum in vitro (9). This effect has also been found in studies on sputum samples from patients inhaling dornase alfa, although variability in response exists. Reduction in viscosity is associated with fragmentation of DNA in the sputum that is seen in the majority, but not all cases (12). The change in sputum viscosity with the addition of dornase alfa is dose dependent, with greater reduction occurring at higher drug concentrations. Interestingly, while dornase alfa increased transportability of mucus in an in vitro model (the excised frog palate), in vivo studies in CF patients did not demonstrate any effect on mucociliary clearance (13,14). Initial studies with dornase alfa have demonstrated improvements in pulmonary function in patients during treatment, an effect that is lost once therapy is stopped (15). This resulted in the concept of designing dornase alfa as a long-term therapy for CF. The clinical response to dornase alfa treatment is well documented. In a large phase III trial, inhalation of dornase alfa was found to reduce the frequency of respiratory infections requiring parenteral antibiotics and to improve pulmonary function in CF patients with moderate disease severity (16). A subsequent study in patients with milder disease demonstrated similar effects on both pulmonary exacerbations and lung function (17). The safety and benefit of dornase alfa has also been demonstrated in patients with severe lung disease (18). On the basis of these positive study results dornase alfa is one of the compounds that has been recommended by the Cystic Fibrosis Foundation therapy guidelines committee (19). Dornase alfa treatment appears to have indirect effects on downstream aspects of CF lung disease such as infection and inflammation. A study using bronchoalveolar lavage to assess the evolution of airway inflammation in CF patients with normal lung function demonstrated a positive effect on inflammation in the treated patients (20). DNA burden was also reduced as were matrix proteinases (10,21). A recent study also suggested that treatment may lower the rate of Staphylococcus aureus infection in CF patients (22). Treatment with dornase alfa is well tolerated, and its side effects are rare. Adverse events occurring in higher frequency in treated patients include voice alterations, pharyngitis, or laryngitis; these are usually transient despite continuation of treatment (16). Because of its effect on mobilizing secretions, dornase alfa often induces cough during the initial phase of therapy and should not be given right before bedtime.

II.

Airway Surface Liquid Hydration Therapy

Effective mucociliary clearance depends on an adequate volume of airway surface liquid (ASL) as outlined in chapter 3. As CF is characterized by depletion of the ASL layer, osmotic agents that improve surface hydration by increasing the water content of the epithelial surface and therefore of the liquid phase of mucus can improve clearance of airway secretions (23). The osmotic effect is not limited to the ASL layer as treatment

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will also improve hydration of airway mucus thereby reducing its viscosity. However, the main function of these agents is thought to be the result of increasing ASL water content. Because of their mechanism of action, these agents are therefore considered to be hydrators rather than mucolytic agents.

A. Hypertonic Saline

Hypertonic saline has been shown to improve mucociliary clearance in CF patients when administered as a single dose (24). These positive effects were concentration dependent, and mucociliary clearance continued to increase up to a concentration of 7%. Higher concentrations did not further improve mucociliary clearance, but were associated with a higher rate of side effects; therefore, a 7% solution has been used in most clinical studies (25). A series of smaller studies using hypertonic saline showed promising short-term benefits on lung function in CF patients (26,27). Initially, the effect of hypertonic saline was thought to be rather short lived, since sodium applied to the epithelial surface was expected to be rapidly taken up through the epithelial sodium channel, whose activity is upregulated in CF. Surprisingly, a recent study demonstrated that hypertonic saline not only had a prolonged effect on ASL height of CF epithelium in vitro, but also resulted in a sustained improvement of mucociliary clearance in CF patients: improved mucociliary clearance was observed up to eight hours after the last dose (28). A large Australian trial with 7% hypertonic saline addressed the question of longterm benefit of this treatment (29). After 48 weeks of inhaled hypertonic saline, patients had a modest improvement in FEV1 compared with the isotonic saline control group. A more impressive effect was seen on pulmonary exacerbations, which were significantly reduced in patients receiving hypertonic saline. Notably, the beneficial effects seen in the study were independent of concurrent dornase alfa use. These studies did not clarify whether the improvement in mucociliary clearance arises from an increase in ASL, from improvements in airway clearance through induction of cough, or a combination of the two mechanisms. If hydration of the airways is the major effect, hypertonic saline could be an attractive early intervention strategy to be initiated before major airway disease is established. Two single-center studies recently showed that inhaled hypertonic saline can be used safely in infants and young children (30,31). A multicenter trial to assess the effect of inhaled hypertonic saline in infants is currently under way. Hypertonic saline can lead to bronchospasm and patients should be pretreated with bronchodilators. Testing of acute treatment response is recommended to assess tolerability in individual patients before initiating long-term treatment. Although salty taste, nausea, dyspnea, and chest pain have been reported, hypertonic saline has been found to be well tolerated in clinical studies. Similar to other medications administered via inhalation, deposition of an osmotic agent such as hypertonic saline will preferentially occur in areas not obstructed by mucous plugs. The most affected areas may therefore not benefit from this treatment approach. Moreover, hypertonic droplets exhibit hydroscopic growth in a water vapor saturated environment such as the lower airways, which will increase their particle size during inhalation and favor a more central deposition unless the nebulizer system is adjusted accordingly to produce a smaller particle size (32). This makes it less likely that adequate amounts of the osmotic agent enter the smaller airways, the area where CF lung disease is

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thought to originate. In addition, the small airways have a large overall surface area, by far exceeding the surface area of the central airways. It is currently not clear what quantity of an osmotic active agent is needed to restore ASL in CF patients. There is ongoing debate whether hypertonic saline should be used as an alternative or adjunct to other therapies addressing airway clearance. As outlined earlier, the only other medication with proven efficacy on airway clearance in CF patients is recombinant DNase (dornase alfa). Data for a head-to-head comparison of these two agents are rather limited. A crossover trial has found hypertonic saline to be inferior to dornase alfa with respect to lung function improvements. However, this study did not assess pulmonary exacerbations as an outcome parameter (33). In vitro studies have shown beneficial effects of combining hypertonic saline and dornase alfa, and the large Australian study found similar treatment effects of hypertonic saline in patients on dornase alfa therapy compared with those treated with hypertonic saline alone (14). B. Mannitol

Mannitol is a monosaccharide that is available as a dry powder for inhalation. Mannitol has primarily been developed as a simple alternative to histamine or methacholine to test for bronchial hyperresponsiveness, therefore its tolerability may be less favorable in patients with airway hyperreactivity (34). Similar to hypertonic saline, dry powder mannitol is hyperosmolar and can cause an influx of water into the CF airway thereby increasing ASL volume (35). Similar to hypertonic saline, mannitol has been shown to improve mucociliary clearance in CF patients (36). A recent phase II trial demonstrated improvements in lung function in treated patients, but the long-term benefit of inhaled mannitol are currently unknown (37). While tolerability was reported to be adequate in this trial, another study reported that one quarter of CF children showed a significant fall in FEV1 in response to a test dose of mannitol, which could potentially limit the broad applicability of this treatment (38). There is potential concern that chronic mannitol inhalation can contribute to the proliferation of bacteria in the lower airways, as mannitol can act as a carbon source for bacteria such as Pseudomonas. No evidence for this was seen in the recent phase II study, but further evidence is needed to clarify this in a trial of longer duration.

III.

Airway Clearance

Airway clearance therapy (ACT) is considered to be part of standard of care in CF, but this is not based on strong scientific data proving its efficacy. Many studies show no significant change between treated groups or the control, and it is unlikely that ACT will ever be completely evidence based in CF. Two excellent reviews have recently questioned the evidence supporting the use of any type of airway clearance (39,40). Most ACT studies were of short duration and/or included a small number of patients, thus decreasing their ability (power) to demonstrate a difference, but short-term benefits have been reported (41). The widespread acceptance of ACT in the CF community has reduced the possibility of performing trials that include an untreated control group, and most studies have therefore compared different modalities to each other. There is little consensus regarding which outcome measures should be used in trials evaluating ACTs. Pulmonary function tests generally are not sensitive enough for short-term studies to indicate superiority of one treatment over another, and few long-term studies have ever been attempted. Mucociliary clearance studies using

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nuclear medicine have shown differences between ACT modalities; unfortunately they are expensive, require expertise, and are not standardized. Secretion production, measured by wet or dry sputum weight, has been used in many studies; theoretically less mucus should mean less obstruction, less inflammation, and better preservation of health. The mixture of mucus with saliva during expectoration complicates the assessment of true clearance, and there is no clear correlation between sputum quantity and other clinically significant outcomes, making mucus production a difficult outcome to validate. Other outcome measures such as pulmonary exacerbation rates and quality of life have rarely been assessed in studies to date. Debate is ongoing as to whether airway clearance should be initiated in the asymptomatic infant, a discussion that has intensified with the wider utilization of newborn screening (42). There are several studies that document early signs of disease even in infants (43–45), but there are few studies that have considered airway clearance at this age (10,46,47).

A. Airway Clearance Physiology

Airway clearance is affected by a number of factors including the quantity and viscosity of mucus, size of bronchi and their apertures, ciliary beat frequency, as well as shear forces generated by airflow from ventilation. The greater the shear forces, the more the mucus movement. Airway clearance techniques cannot enhance secretion clearance without airflow (48). Factors that can affect airflow include airway resistance, expiratory pressures, bronchial wall stability, and elastic recoil. Airway clearance techniques employ maneuvers that can affect these factors. Many techniques utilize the concepts of asynchronous ventilation, collateral ventilation channels, and choke points (CP). CPs explain flow limitation in the wavespeed theory of pulmonary physiology (49). For this chapter, CP terminology will be used synonymously with the “equal pressure point” used in earlier studies. The CPs are the regions where the pressure within the airway has decreased to equal the pleural (extramural) pressure and hence the pressure difference across the airway wall is zero. With breathing at tidal volume to functional residual capacity (FRC), the CP lies in the trachea and main bronchi; as exhalation moves into lower lung volumes (expiratory reserve volume), the point at which dynamic compression takes place moves more peripherally (50,51). Coughing is the body’s natural mechanism for airway clearance. CPs play an extremely important part in the effectiveness of cough because a significant jump in airflow velocity occurs at these points of narrowing. High linear airflow velocity is associated with turbulent flow, high shearing forces at the airway walls, and high kinetic energies. Another means to produce supramaximal airflow and high linear velocities is a “huff.” This is a forced expiratory maneuver, usually initiated from mid-to-low lung volumes and is performed with an open glottis. The huff maneuver does not generate as high a transmural pressure as does cough, and so it may be more effective in helping to clear secretions when airway walls are unstable because of cartilage destruction, by causing less airway collapse. A huff can also be instituted at different lung volumes, thus allowing the shift of CPs to different areas. Mucus can be mobilized with expiratory airflow velocities of 1.0 to 2.5 m/sec for annular flow, or >2.5 m/sec for mist flow moving sputum droplets suspended as an

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Figure 1 Flow volume loops and flow transients with cough and huff.

aerosol (52). Airflow of sufficient magnitude to mobilize secretions and mucus transport itself can occur during forced exhalations (huffs) or even with tidal breathing in some circumstances (53). Bennett and Zeman determined that airway clearance with huff was faster than control and similar to that generated by voluntary cough (54) (Fig. 1). Asynchronous ventilation is related to different filling times for different regions of the lung—faster for healthier and unobstructed areas, slower for more diseased and obstructed regions. Alternate or collateral ventilation channels exist between bronchi and/or alveoli in older children and adults (55), but are not present in infants and toddlers. In healthy lungs, little gas movement occurs through these channels during regular breathing. Breath-holding maneuvers can improve aeration in obstructed, less healthy areas by maximizing filling time for slow-filling regions, promoting ventilation through collateral channels and interdependence (Pendelluft) (56). B. Airway Clearance Techniques Postural Drainage and Percussion

Postural drainage and percussion (PD&P) has been synonymous with chest physical therapy in CF for decades. There are 12 different positions in which a patient can be placed to optimize central movement of mucus from bronchopulmonary segments. Percussion, vibration, and shaking may be used as adjuncts. In each position, the chest is percussed between 2 to 10 minutes, usually followed by deep breathing exercises and huffing on exhalation in older patients. Studies have shown PD&P to be an effective technique for clearing excessive bronchial secretions in patients with CF (57–59). Potential problems associated with PD&P include time and energy used, discomfort and pain, hypoxemia (60), arrhythmias (61), and bronchospasm (62). The head-down position has become controversial because of concerns over gastroesophageal reflux and increased pulmonary disease (63,64). Modified PD&P excludes this position unless a specific focal lesion warrants it. It is now suggested that modified PD&P (omitting the

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head-down position) be performed in all patients, especially those with any symptoms of gastroesophageal reflux. Active cycle of breathing techniques

The active cycle of breathing technique (ACBT) is a cycle of breathing control (BC), thoracic expansion exercises (TEE), and the forced expiration technique (FET = huffing combined with BC) (65) (Fig. 2). The greatest strength of ACBT is its adaptability for different lung pathophysiology. TEE (with breath-hold) results in asynchronous ventilation; this component should be augmented in the cycle when atelectasis or emphysema is present. FET moves the secretions from different parts of the airways, and is used more often in patients with excess secretions. ACBT was initially known as the FET (65); however, this predisposed to a misinterpretation, with the BC and TEE components of the cycle sometimes being omitted entirely (66). ACBT trials have shown equivalence compared with directed coughing, PD&P, autogenic drainage (AD), positive expiratory pressure (PEP), or oscillating PEP/Flutter (oscPEP) with respect to pulmonary function, mucociliary clearance, and/or wet sputum weight (67–70).

Figure 2 Active cycle of breathing techniques—three cycles for differing lung pathology:

(A) mild disease; (B) increasing secretion; (C) complex disease with significant atelectasis, airway hyperreactivity, and secretions.

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Autogenic Drainage

The underlying concept of AD is to achieve high expiratory flow rates in different generations of bronchi by controlled breathing, but to avoid significant airway closure that may occur with coughing or forced exhalations (71–73). Performing regular tidal volume breaths at low to high lung volumes generates higher flow rates that start in the small peripheral airways and gradually move into larger central ones. These greater flow rates produce higher shear forces than might occur with cough or forced expiratory maneuvers. The incorporation of a breath-hold throughout the technique improves asynchronous ventilation by lengthening the time for alveolar filling and increasing collateral ventilation. The technique of AD requires feedback to the patient to facilitate mucociliary clearance. Preliminary evidence from a study comparing AD and PD&P demonstrated no difference in clinical status or pulmonary function (74); the study ended early after the first year because almost half the AD group refused to change over to PD&P as they felt AD was more effective. Studies comparing AD to PD&P (75), high pressure positive expiratory pressure (HiPEP) (76), or ACBT evaluating oxygenation, pulmonary functions, and mucociliary clearance (69) did not demonstrate significant differences in efficacy between techniques. Positive Expiratory Pressure

PEP is proposed to increase airflow via the collateral ventilatory channels (55). In the presence of lung disease, with breath-holds and when breathing out against a PEP, it is proposed that these channels open more. As a result, the resistance to airflow falls and air can flow along collateral channels, allowing air to build up behind secretions to assist in loosening and mobilizing them. More slowly ventilating lung units can also receive part of their inspired volume from the more rapidly ventilating units via the collateral channels. PEP also should reduce dynamic compression of unstable airways during expiration (77). PEP involves tidal breathing with a slightly active exhalation partially into the expiratory reserve volume through a mask or mouthpiece and one-way valve system. A resistor is attached to the expiratory limb, and this is adjusted to give pressures between 10 and 20 cmH2O in mid-exhalation. After several breaths to build up air behind mucus, the patient huffs or coughs to mobilize secretions. Effectiveness of PEP (78) as an airway clearance regimen was first demonstrated in a study from Denmark. A subsequent one year study (79) showed improved lung function with PEP, compared with a decline in lung function in the group using PD&P. High-pressure PEP is a modification (80) in which 8 to 10 breaths against a fixed resistor with pressures 10 to 20 cm, similar to regular PEP, is followed by 1 to 2 huffs or forced expirations through the resistor. Pressures generated usually are between 50 and 120 cmH2O, and the resistor selected is the one that maximizes forced vital capacity. The regimen of high-pressure PEP requires close and ongoing monitoring, using flow volume loops to select the resistor which optimizes the flow volume loop for highest vital capacity. This modality has been shown to be effective in airway clearance in CF, but superiority over conventional PEP has not been demonstrated (81). In spite of theoretical concern because of raised pressures, there has been no demonstration of increased complications of pneumothorax or hemoptysis using high-pressure PEP (82). Oscillating Positive Expiratory Pressure

This technique combines PEP with high-frequency oscillations (HFOs) generating intermittent acceleration of expiratory airflow. Oscillatory endobronchial pressure

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pulses cause flow transients, microsecond periods when flow exceeds the maximal expiratory flow able to be generated by a forced expiratory maneuver. Coughing and huffing also generate flow transients (Fig. 1). Endobronchial oscillating pressures are proposed to act as “microcoughs,” transiently increasing the airflow and shear forces over the mucus layer, thus promoting mucus clearance (83). The PEP generated may decrease dynamic collapse of unstable airways, and endobronchial vibrations help to dislodge secretions from the airway lumen (84). HFOs are capable of breaking mucus bonds, decreasing viscosity (61), and improving clearance. Finally, it is hypothesized that the oscillations enhance ciliary beat frequency as well as disrupt mucus adhesion to airway walls. The three main devices used in oscillating positive expiratory pressure (OscPEP) are the Flutter, RC Cornet1, and Acapella1; the latter also allows delivery of a fixed exogenous PEP beneath the endobronchial pressure pulses. In vitro comparative studies of the Flutter and Cornet show the Flutter generates higher peak pressures at higher frequencies, but lowers amplitudes of the endobronchial pulse (85). There is no convincing evidence that any of the devices is superior to the others (86), but the Acapella might be easier to use for some patients because its effectiveness is less dependent on technique. The Flutter has been used in several studies that have not demonstrated superiority to PD&P (87,88), ACBT (68), or PEP (89,90). High Frequency Chest Compression

High-frequency chest compression (HFCC) or high-frequency chest wall oscillation (HFCWO) has been shown to increase mucociliary clearance (91,92) theoretically by increasing an expiratory flow bias. This cephalad flow bias is purported to be the main mechanism of mucus clearance in smaller airways with bulk airflow (as opposed to convection) and is associated with a tendency toward shearing mucus and movement centrally (93,94). A comparison of a percussor (producing an unbiased sine wave) to an oscillator showed that the oscillator produced higher flow and a significantly increased mucus velocity (95). HFCC also enhances secretion clearance by decreasing the viscoelasticity of mucus (96). Multiple versions of HFCC vests exist; the waveform of the pressure pulse delivered varies by device (square wave for ThAIRpy, ABI, and Hill-Rom 101 and 102; sine wave for 103, 104, MedPulse, and SmartPulse; and triangle waveform for inCourage System). Because of this, review and evaluation of HFCC literature requires careful attention to the device used (97–99). Lower frequencies oscillate more volume for a given pressure setting, while higher frequencies cause higher oscillating flow rates and therefore more shear force. Increasing pressure settings increases both flows and volumes, but is usually associated with greater discomfort. Vests that generate a sine wave are used at the highest pressure setting that can be tolerated; lower frequencies are used to displace volume, while higher frequencies are used to increase flow rates. For vests generating triangular waveform pressure pulses both the largest airflows and air volumes reportedly occur over the same frequency range of 5 to 11 Hz (98). It is important for the patient to cough or huff during and at the end of treatment to perform the technique properly. Evidence for efficacy is limited and no studies have demonstrated clinically significant differences between the different devices. An observational retrospective analysis suggested that HFCC can increase sputum production (100). Decreased end-expiratory

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lung volumes have been documented with use of the vest (101), but atelectasis is not a reported complication of vest use in CF patients. Using positive end-expiratory pressure improves oscillatory flows with HFCC in CF (102,103). A recent small, short-term study showed better pulmonary function response in patients treated with ACBT compared with HFCC during an acute pulmonary exacerbation (104), but sufficiently powered studies have not been performed to compare HFCC with other treatment modalities. Intrapulmonary Percussive Ventilation

In theory, intrapulmonary percussive ventilation (IPV) improves airway clearance by several means. Mucus viscosity is decreased with exposure to high-HFO. During inspiration, high-frequency air pulses expand the lungs, enlarge the airways, vibrate, provide an increased mean airway pressure, and deliver gas into distal lung segments beyond accumulated mucus (105). It is further hypothesized that due to the percussive amplitude of shaking airways, mucus is dislodged. Similar to HFCWO, IPV should decrease asynchronous ventilation and improve gas exchange by oscillatory movement and migration of oxygen (room air). Use of IPV theoretically would be effective for patients who would benefit from airway distension (e.g., those with widespread bronchiolectasis) since it distends airways while oscillating them. Finally, IPV delivers continuous oral oscillating pressures during both inhalation and exhalation. Therefore, patients should experience flow transients (microcoughs during exhalation) similar to those found with OscPEP use. Studies on IPV in CF are not extensive and do not demonstrate significant differences to other modalities (106,107). No reports of its use in infants or younger children with CF exist. Exercise

Exercise has long been identified as an alternative or adjunct to airway clearance. A meta-analysis of PEP, FET, AD, exercise, and “standard physical therapy” (PD&P) examined outcomes of FEV1, sputum weight, or sputum clearance. PD&P significantly increased sputum expectoration above no treatment, and only exercise coupled with PD&P increased FEV1 above PD&P alone (59). There is little data to suggest precisely how it improves clearance. Recently, exercise was shown to inhibit epithelial sodium channels in patients with CF, causing a partial normalization of potential difference, and therefore improved ion and water movement into airway secretions (108). C. Selecting Airway Clearance Techniques

As outlined above, each technique is based on physiological principles by which it enhances mucociliary clearance; thus, one modality could potentially be more effective than another. There is, however, no specific research regarding which technique is best for a given patient. Given the lack of strong evidence for superiority of one technique versus others, techniques are often chosen to fit the individual patient’s age, life style, family situation, and culture. This increases the likelihood of adherence, which will then impact upon efficacy. Adherence with ACT in CF is notoriously poor, cited to be between 40% and 60% of patients performing ACT as described (109,110). Age plays a major role in the choice of ACT. Passive techniques are generally used in infants and young children. Techniques vary in complexity, with PEP, oscillating PEP, and HFCC being relatively easy to learn, and AD being the most difficult. For a

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working adult, the ability to perform ACT at any time without the need for adjunct devices may be important. Cost must be taken into consideration including financial and time investment. Ultimately, the ACT to which the patient adheres and performs will be more important than the best physiologically sound technique that the patient does not do (107).

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89. van Winden CM, Visser A, Hop W, et al. Effects of flutter and PEP mask physiotherapy on symptoms and lung function in children with cystic fibrosis. Eur Respir J 1998; 12:143–147. 90. McIlwaine PM, Wong LT, Peacock D, et al. Long-term comparative trial of positive expiratory pressure versus oscillating positive expiratory pressure (flutter) physiotherapy in the treatment of cystic fibrosis. J Pediatr 2001; 138:845–850. 91. King M, Phillips DM, Gross D, et al. Enhanced tracheal mucus clearance with high frequency chest wall compression. Am Rev Respir Dis 1983; 128:511–515. 92. King M, Phillips DM, Zidulka A, et al. Tracheal mucus clearance in high frequency oscillation II: chest wall versus mouth oscillation. Am Rev Respir Dis 1984; 130:703–706. 93. Chang HK, Weber ME, King M. Mucus transport by high-frequency nonsymmetrical oscillatory airflow. J Appl Physiol 1988; 65:1203–1209. 94. Gross D, Zidulka A, O’Brien C, et al. Peripheral mucociliary clearance with high frequency chest wall compression. J Appl Physiol 1985; 58:1157–1163. 95. Rubin E, Scantlen GE, Chapman GA, et al. Effect of chest wall oscillation on mucus clearance: comparison of two vibrators. Pediatr Pulmonol 1989; 6:122–126. 96. Dasgupta B, Tomkiewicz RP, Boyd WA, et al. Effects of combined treatment with rhDNase and airflow oscillations on spinnability of cystic fibrosis sputum in vitro. Pediatr Pulmonol 1995; 20:78–82. 97. Milla CM, Hansen LG, Weber A, et al. High frequency chest compression: effect of the third generation compression waveform. Biomed Instrum Technol 2004; 38:322–328. 98. Warwick WJ. High frequency chest compression. In: Button B, McIlwaine M, ed. Airway Clearance Techniques Training Class. North American Cystic Fibrosis Conference, 2005. 99. Kempainen RR, Williams CB, Hazelwood A, et al. Comparison of high frequency chest wall oscillation with differing waveforms for airway clearance in cystic fibrosis. Chest 2007; 132:1227–1232. 100. Warwick WJ, Hansen LG. The long-term effect of high-frequency chest compression therapy on pulmonary complications of cystic fibrosis. Pediatr Pulmonol 1991; 11:265–271. 101. Jones RL, Lester RT, Brown NE. Effects of high frequency chest compression on respiratory system mechanics in normal and cystic fibrosis patients. Can Respir J 1995; 2:40–46. 102. Perry RJ, Man GCW, Jones RL. Effects of positive end-expiratory pressure on oscillated flow rate during high frequency chest compression. Chest 1998; 113:1028–1033. 103. Dosman CF, Zuberbuhler PC, Tabak JI, et al. Effects of positive end-expiratory pressure on oscillated flow rate during high frequency chest compression in children with cystic fibrosis. Can Respir J 2003; 10:94–98. 104. Phillips GE, Pike SE, Jaffe A, et al. Comparison of active cycle of breathing and highfrequency oscillation jacket in children with cystic fibrosis. Pediatr Pulmonol 2004; 37:71–75. 105. Langenderfer B. Alternatives to percussion and postural drainage, autogenic drainage, positive expiratory pressure, flutter valve, intrapulmonary percussive ventilation, and high frequency chest compression with the Thairpy vest. J Cardiopulm Rehabil 1998; 18: 283–289. 106. Newhouse PA, White F, Marks JH, et al. The intrapulmonary percussive ventilator and flutter device compared to standard chest physiotherapy in patients with cystic fibrosis. Clin Pediatr 1998; 37:427–432. 107. Homnick DN, White F, deCastro C. Comparison of the effects of an intrapulmonary percussive ventilator to standard aerosol and chest physiotherapy in cystic fibrosis. Pediatr Pulmonol 1995; 20:50–55. 108. Hebestreit A, Kerstin U, Basler B, et al. Exercise inhibits epithelial sodium channels in patients with cystic fibrosis. Am J Respir Crit Care Med 2001; 164:443–446. 109. Passero MA, Remor B, Salomon J. Patient-reported compliance with cystic fibrosis therapy. Clin Pediatr 1981; 20:264–268. 110. Modi AC, Lim CS, Yu N, et al. A multi-method assessment of treatment adherence for children with cystic fibrosis. J Cyst Fibros 2006; 5:177–185.

16 Pulmonary Exacerbations DON B. SANDERS University of Wisconsin, Madison, Wisconsin, U.S.A.

MARGARET ROSENFELD Seattle Children’s Hospital, Seattle, Washington, U.S.A.

I.

Introduction

Cystic fibrosis (CF) lung disease is characterized by episodic increases in respiratory symptoms such as cough and sputum production, often accompanied by systemic symptoms such as anorexia and fatigue. These episodic changes in signs and symptoms from the patient’s baseline are termed pulmonary exacerbations (1,2). Pulmonary exacerbations have a significant impact on quality of life (3–6), mortality (7–10), and CF health care expenditures (11). Furthermore, they are amenable to therapies aimed both at prevention and treatment. The rate of pulmonary exacerbations has served as a key outcome measure in many clinical trials in CF over the past 15 years (12–18). This chapter reviews the pathophysiology, clinical characteristics, evaluation, treatment, and prevention of pulmonary exacerbations.

II.

Pathophysiology of Pulmonary Exacerbations

Given the importance of pulmonary exacerbations in CF, remarkably little is known about their underlying pathophysiology. A quasi-stable homeostasis exists in the CF lung between endobronchial pathogens and the associated host neutrophilic inflammatory response. Triggers such as viral respiratory infections, other infectious agents, pollutants (19), allergens, and respiratory tract irritants can disrupt this fragile homeostasis, resulting in an increase in endobronchial bacterial burden and airway inflammation, manifesting clinically as a pulmonary exacerbation (Fig. 1). Concentrations of bacteria and inflammatory markers are increased at the onset of an exacerbation and are reduced in respiratory secretions following aggressive antibiotic therapy (20–24). Recent studies suggest that Pseudomonas aeruginosa and other bacterial organisms exist in biofilms in chronic CF endobronchial infection (25). Biofilms are communities of microorganisms encased within an extracellular polysaccharide matrix that adhere to a surface. Bacteria within biofilms are slow growing and communicate by the process of quorum sensing. They demonstrate increased antibiotic resistance relative to planktonic bacteria, likely due to decreased penetration of antibiotics through the extracellular matrix and expression of biofilm-specific resistance mechanisms. In addition, the antibiotic susceptibility profiles of bacteria in biofilms differ markedly from that of the same bacteria growing planktonically.

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Figure 1 Pathophysiology of a pulmonary exacerbation.

Growth of bacteria in biofilms likely explains why chronic infections are rarely eradicated by current antibiotic therapy. However, the proven clinical efficacy of b-lactam antibiotics in treating exacerbations suggests that planktonic bacteria may play a role in pulmonary exacerbations. It has been proposed that bacteria growing in biofilms and in planktonic phase may play a role in chronic infection and exacerbations, respectively (26). Recent evidence that exacerbations in patients chronically infected with P. aeruginosa are generally due to the clonal expansion of strains of microorganisms present at the patient’s stable baseline rather than to the acquisition of new strains also supports this hypothesis (27).

III.

Definition of Pulmonary Exacerbations

Despite the central role that pulmonary exacerbations play in CF patient care and research, no standardized definition exists. Three groups have systematically evaluated which signs and symptoms best characterize a pulmonary exacerbation (28–30). The results of these three studies in different patient populations demonstrate fairly good concordance (Table 1). Some of the characteristics most strongly associated with a pulmonary exacerbation included increased cough, increased sputum production, decreased exercise tolerance, decline in weight-for-age percentile, reduced appetite, hemoptysis, and new adventitial sounds on examination of the chest. In all three studies, symptoms and signs were more predictive of an exacerbation than laboratory data. Pulmonary exacerbations are key clinical efficacy endpoints in many CF therapeutic trials (13–15,17,18,31,32). Participants in a 1992 Cystic Fibrosis Foundation, U.S. Food and Drug Administration (FDA) Consensus Conference on Outcome Measures in CF, recommended that a standardized definition of pulmonary exacerbations be established to strengthen individual trials and facilitate comparison between trials (33). The recommendation was for this definition to include clinical symptoms and not therapeutic interventions, in view of the rapid change in treatment modalities occurring in the field and the marked variability in treatment strategies between clinicians and CF centers (29,34). Yet, 15 years after the initial recommendations, no such universal definition has been established (35). Exacerbation definitions for clinical trials typically require antibiotic administration (most commonly intravenously) in association with a constellation of patient signs and symptoms and laboratory data. Empiric pulmonary exacerbation definitions have been developed for specific clinical trials (13,18,31), but their accuracy and precision have never been measured and there is no universally accepted definition. One limitation of all published definitions is that they are physician-derived interpretations of health outcomes, rather than patient-reported outcomes (PROs). In

– – 74 74 40 – 37 58

4.3 14.1 5.9 2.7i – –

70 62 35 95 30 58

– –

98 91 91 86 79 93

Well child

56 65 49 93 53 49

– –

93 98 88 98 91 91

Advanced diseased

– – 2.8h – 4.1 – –

7.8h – – – –

4.4 – 1.5 – 2.0 1.4g

6–12 yr

– –

3.9 – 2.2 – 3.2 2.2g

<6 yr

3.7 – –

2.7h –

– –

2.3 – 3.0 – 2.3 2.0g

13–17 yr

Rabin et al. (OR)c

b

Odds ratios for association of characteristics with the presence of a pulmonary exacerbation from univariate logistic models. Percentage of questionnaire respondents managing children and adolescents rating item helpful or very helpful (among 43 respondents). c Odds ratios for the presence (vs. absence) of a pulmonary exacerbation with mutual adjustment for all variables. d Scenario involved a patient with established bronchiectasis and chronic sputum production. e Increased fatigue. f Weight loss 1 kg over past month. g Relative decline in weight-for-age percentile. h New crackles. i Decline in FEV1 10% over past month. Source: From Ref. 2.

a

School or work absenteeism Retractions or use of accessory muscles Change in chest sounds Change in respiratory rate Fever Change in spirometry Oxygen saturation CXR changes

100 NA – 91 – 91

24.5 22.4 11.4 15.2e – 15.2 (appetite) 2.1 (weight)f 5.6 12.9

Increased cough Exercise intolerance Change in sputum production Change in activity Hemoptysis Change in appetite or weight

Age <5 yr

Rosenfeld et al. (OR)a

Clinical or laboratory characteristic

Dakin et al. (%)b

Table 1 Signs and Symptoms Associated with a Pulmonary Exacerbation

3.8 – –

2.5h –

– –

2.8 – 1.8 – 2.4 1.7g

18 yr

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response to the increased emphasis by the FDA on PROs, recent efforts have focused on incorporated patient or parent report on symptoms or signs into a pulmonary exacerbation definition (36).

IV.

Epidemiology of Pulmonary Exacerbations

Annual rates of pulmonary exacerbations requiring intravenous antibiotics increase with age: ~18% in infants less than 1 year of age to 44% over the age of 30 (37) (Fig. 2). The percent of patients requiring treatment with any antibiotics (oral, inhaled, and/or intravenous) for an exacerbation also increases with age: during a 6-month observation period, rates were 23% for children under 6 years, 32% for ages 6 to 12 years, 48% for ages 13 to 17 years, and 63% for patients older than 18 years (29). Recurrent pulmonary exacerbations are clearly associated with greater mortality and morbidity. Logistic regression models used to predict short-term survival among CF patients have shown that pulmonary exacerbations requiring hospitalization are an important risk factor for mortality (7,8). Admission for pulmonary exacerbations between one and five years of age is also negatively associated with lung function and risk of death at eight-year follow-up evaluation (9). A recent review of the experience at a single adult center in France with adult CF patients having moderate to severe lung disease revealed a high risk of one-year mortality for those hospitalized in the intensive care unit for a pulmonary exacerbation (10). In contrast, in a study comparing the frequency of patient monitoring and the use of therapeutic interventions between U.S. CF centers in the upper and lower quartiles of age-specific lung function, patients at the upper quartile sites received more frequent intravenous antibiotic therapy than those in the lowest quartile (34). The authors hypothesized that patients at the upper quartile sites may have been treated more

Figure 2 Pulmonary exacerbations in 2007 by age group. Source: From Ref. 36

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Figure 3 Pulmonary exacerbations by age in children (A) and adults (B). Source: From Ref. 38

frequently because they were evaluated more often and because the overall therapeutic strategy was more aggressive at the these sites. They postulated that intensive use of antibiotics might have contributed to the improved outcomes at these sites. On a population level, risk factors for the occurrence of pulmonary exacerbations include viral respiratory infections, reactive airway disease, lower FEV1, advancing age, and infection with P. aeruginosa (9,38,39). The association between advancing age and more frequent pulmonary exacerbations is roughly exponential in children and linear in adults (Fig. 3) (38). Others have found that low socioeconomic status was a risk factor for exacerbations (40). In addition, markers of air pollution such as particulate matter and ozone concentrations are significantly associated with exacerbation rates (19). In addition, factors that may lead to a pulmonary exacerbation in an individual include nonadherence to maintenance treatment, the development of allergic bronchopulmonary aspergillosis (ABPA), and the acquisition of nontuberculous mycobacteria (41).

V. Evaluation of Pulmonary Exacerbations When a clinician makes the diagnosis of a pulmonary exacerbation, he or she must determine the severity of the exacerbation and choose appropriate therapy, including

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type of antibiotics and route of administration, adjunctive therapies, and location of treatment (home vs. hospital). Evaluations summarized below will assist in this decision-making process. A. Pulmonary Function Studies

Spirometry is typically measured at the onset of the exacerbation to aid in determination of severity, during the course of therapy to determine response to treatment, and at the end of therapy to determine whether return to baseline lung function has been achieved. A decline in spirometric measures when compared with recent (6–12 month) periods of clinical stability is frequently used as an indicator of a pulmonary exacerbation. In most clinical trials using pulmonary exacerbations as an endpoint, a relative decrease in forced vital capacity (FVC) or forced expiratory volume in one second (FEV1) > 10% is a criterion in the definition of an exacerbation (33,42). In addition, the degree of improvement in FEV1 seen during the first week of therapy is predictive of the length of treatment and discriminates between patients who will recover within two weeks and those who will require more prolonged antibiotic courses (43). The intrapatient measurement variability in FEV1 during an exacerbation is similar to that in clinically stable outpatients with CF (44). B. Microbiologic Studies

Since antibiotics are a major component of pulmonary exacerbation treatment, a respiratory culture, including antimicrobial susceptibility testing, is used to guide therapeutic decisions. The culture must be performed utilizing the appropriate selective media in a clinical microbiology laboratory with expertise in the identification of CF pathogens. The Cystic Fibrosis Foundation has established reference laboratories to evaluate the antibiotic susceptibility of multiresistant organisms by synergy testing and for the confirmatory speciation of difficult organisms, such as those belonging to the Burkholderia cepacia complex. Previous cultures may be used to guide initial antibiotic selection, but a new culture at the time of hospitalization may be more reflective of the current endobronchial bacterial burden, especially if time has elapsed since the previous culture. In expectorating patients, sputum is an adequate sample for lower respiratory tract flora. In patients too young to produce sputum, oropharyngeal (OP) swabs are widely used, although the limitations in their diagnostic accuracy relative to lower airway cultures must be understood (45). Among 141 CF patients less than five years old, OP cultures had a sensitivity of 44% (95% CI, 14%, 79%) and a specificity of 95% (90%, 99%) for detecting P. aeruginosa compared with bronchoalveolar lavage. A negative OP culture in young patients is reasonably helpful in “ruling out” lower airway Pseudomonas, while a positive OP culture does not necessarily “rule in” lower airway infection. Given its invasive nature and expense, bronchoscopy with bronchoalveolar lavage is generally reserved for patients who have inadequate respiratory cultures and are significantly ill, or those who have failed to respond to appropriate therapy based on upper airway cultures. In patients with refractory signs and symptoms, even young children, respiratory samples should also be cultured for nontuberculous mycobacteria and fungi (46). As our understanding of the ecosystem of chronic bacterial infection in the CF airway grows, the limitations of current clinical respiratory cultures become clearer.

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First, the planktonic conditions in which clinical cultures are performed do not mimic the biofilm conditions in which bacteria in the CF airway generally live (47). Secondly, there is growing evidence of the importance of anaerobic bacteria in the CF airway, which are not detected by current culture techniques (48). Lastly, antibiotic susceptibilities in vitro do not necessarily correlate with clinical outcomes (49). C. Imaging Studies

Chest radiographs are not routinely indicated in the diagnosis of an exacerbation, but may detect new or evolving infiltrates, pneumothoraces, atelectasis, or progressive bronchiectasis. While high-resolution chest tomography (CT) is more sensitive than spirometry for monitoring progression of CF lung disease (50,51), its role in the diagnosis and management of pulmonary exacerbations has not been systematically explored. Chest CTs performed at the end of a pulmonary exacerbation may be more informative about baseline degree of structural damage than CTs obtained at the onset of treatment for an exacerbation. D. Biomarkers

There are multiple candidate biomarkers of CF airway inflammation. Some groups have detected elevated concentrations of sputum neutrophils (52) and inflammatory mediators (53,54) in patients experiencing a pulmonary exacerbation, although results have not been consistent across studies (52,55). Longitudinal data are lacking. Thus, to date, there are no sensitive or specific biomarkers available to assist in the diagnosis of a pulmonary exacerbation (56).

VI.

Evidence-Based Management of Pulmonary Exacerbations Though few well-designed and sufficiently powered studies have been conducted to evaluate the efficacy of different treatment regimens for pulmonary exacerbations, evidence-based guidelines have recently been published (57). In general, trials of pulmonary exacerbation treatment have evaluated patients chronically infected with P. aeruginosa. In these patients, antibiotic therapy for a pulmonary exacerbation decreases the endobronchial burden of P. aeruginosa but cannot completely eradicate the organism (58). On the basis of the results of a few controlled trials, the combination of an aminoglycoside and a b-lactam antibiotic is used to achieve synergistic antibacterial activity (21). Once-daily aminoglycoside dosing appears to provide equivalent efficacy and safety in CF patients compared with thrice-daily dosing. In a study of 219 patients randomized to ceftazidime and either once-daily or thrice-daily tobramycin for 14 days, both regimens were equally effective at improving lung function, and, in children, once-daily aminoglycoside administration was associated with less nephrotoxicity (59). Ototoxicity was similar in both groups. Once-daily dosing takes advantage of the property of aminoglycosides that bacterial killing is dependent on the highest concentration of tobramycin achieved, while nephrotoxicity is related to the serum concentration over time. Once-daily dosing also simplifies the treatment regimen for families administering intravenous antibiotics at home. However, the cumulative toxicity of exposure to recurrent courses of oncedaily aminoglycosides has not been evaluated, nor has the effect of repeated courses on the

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development of antibiotic resistance. Careful monitoring of drug levels, nephrotoxicity, and ototoxicity is important, taking into account that patients with CF are known to have more rapid clearance of antibiotics, particularly aminoglycosides (60), and are likely to have cumulative exposure. Although lung function and other clinical outcomes improve with the use of intravenous anti-Pseudomonas antibiotics (alone or in combination) (58,61,62), the best way to choose these antibiotics remains controversial. A small retrospective study failed to demonstrate an association between antibiotic susceptibility-based treatment and improved outcomes (49,63). Antibiotic choices based on multiple combination bactericidal antibiotic testing, in which double and triple antibiotic combinations are tested for bactericidal activity against multiresistant bacterial isolates, also may not improve clinical outcomes. In a randomized, controlled trial in 251 CF patients chronically infected with multiresistant gram-negative bacteria, antibiotic therapy directed by combination antibiotic susceptibility testing did not result in better clinical or bacteriologic outcomes compared with therapy directed by standard cultures and sensitivity techniques (63). The apparent disconnect between in vitro sensitivity testing and clinical response is likely multifactorial in nature. Bacteria in the CF airway generally grow in biofilms, whereas current in vitro susceptibility testing uses isolates growing planktonically. The nonbactericidal effects of antibiotics (such as anti-inflammatory properties) may also play a role in improving clinical outcomes. Recent investigations examining susceptibility patterns of bacterial biofilms in the airway milieu suggest that traditional antibiotic susceptibility testing, in which individual antibiotics are tested against planktonically growing bacterial isolates from CF sputum, may not be relevant to endobronchial infection or predictive of treatment response (64). Biofilm susceptibility testing methods have been developed (47), and biofilm-based antibiotic choices may differ substantially from traditional culture mediabased antibiotic choices (65). One single-center retrospective study suggested that patient treated with biofilm-effective therapy seemed to have improved clinical outcomes (66); a prospective, randomized, controlled trial exploring whether biofilm susceptibility-based antibiotic selection results in improved clinical outcomes is ongoing (66). Typically, patients are treated with intravenous antibiotics for two to three weeks, the duration required for a plateau in improvement in lung function (67). In most cases, clinical and spirometric improvement is noted within the first week of treatment, although in patients with severe underlying lung disease, spirometry may actually decline before it improves (67). Most patients experience improvements in spirometry with treatment, but not necessarily to previously achieved levels (49). There have been no controlled studies defining the optimal length of therapy. Studies have shown that the gains in pulmonary function can be lost over a period of days to weeks after intravenous antibiotics are discontinued (55). While inhaled antibiotics, such as tobramycin solution for inhalation, are widely used to treat exacerbations, their efficacy in this setting has never to our knowledge been evaluated in a clinical trial. Consideration also must be given to treating non-Pseudomonas pathogens, including Staphylococcus aureus, Haemophilus influenzae, B. cepacia, Stenotrophomonas maltophilia, and nontuberculous mycobacteria, particularly Mycobacterium abscessus. Methicillin-resistant Staphylococcus aureus (MRSA) has become more

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prevalent in recent years and may be associated with more rapid lung function decline and higher rates of treatment with antibiotics (68,69). Recent reports highlight the possibility that CF lung microbiology is even more complex, including potentially pathogenic anaerobic bacteria that may also require treatment (48). The results of small studies that compared the efficacy of treating exacerbations at home versus in the hospital are conflicting. In 2007, one-quarter to one-third of the total number of days of intravenous antibiotic therapy among U.S. CF patients were administered at home (37). Some reports have shown comparable results for home and hospital treatment in clinical outcomes, whereas others have found poorer responses in pulmonary function and more prolonged use of intravenous antibiotics among the group treated at home (70–73). However, most studies have found significant cost savings and important psychosocial benefits for the patients and the family when antibiotics were administered at home (74). Careful patient selection for home therapy of a pulmonary exacerbation is of prime importance (57). Treatment with antibiotics is not the only therapeutic intervention for pulmonary exacerbations. Patients in the placebo arms of controlled antibiotic trials who received three to four times daily airway clearance also experienced significant improvements in clinical parameters from baseline at the time of hospitalization (58,75). Patient preference and availability may determine the choice of chest physiotherapy modality, as studies have failed to demonstrate the superiority of one technique over another (76,77). Exercise may also improve outcomes during an exacerbation. Two small studies reported beneficial effects of a physical training regimen during inpatient management of an exacerbation, although it is unclear how much of the improvement reflected resolution of the underlying infection and inflammation with treatment (78,79). Patients with poor lung function may not tolerate airway clearance and are prone to significant hypoxemia from mucus plugging and ventilation-perfusion inequalities. In these patients, noninvasive positive pressure ventilation during airway clearance may improve respiratory muscle performance and enhance aerosol deposition (80). The increased work of breathing, infectious burden, and inflammatory response that occurs during exacerbations can result in significant increases in energy expenditure, and anorexia and weight loss are common. Thus, aggressive nutritional support is an important component of care. Patients can experience insulin resistance as part of an exacerbation, so it is also advisable to monitor blood glucose concentrations and initiation of insulin therapy or an increase above baseline dose may be required (81). While there are not yet data in CF to suggest that aggressive glycemic control during acute exacerbations improves outcomes, data in other clinical situations suggest that this may be the case (82). Given the heightened inflammatory response that occurs during a pulmonary exacerbation, anti-inflammatory agents could potentially be of therapeutic benefit during exacerbations. However, treatment with anti-inflammatory agents has not shown clear benefit in small studies. A small, controlled study of intravenous hydrocortisone in infants and young children failed to demonstrate significant changes in pulmonary function at the end of in-hospital treatment, although significant differences between the hydrocortisone and placebo groups were found months later (83). In a recent randomized, double-blind, placebo-controlled pilot trial of prednisone therapy among 24 CF patients 10 years of age, hospitalized for a pulmonary exacerbation, no difference was detected between the groups in spirometry or inflammatory markers in induced sputum (84). The authors concluded that a larger clinical trial was warranted.

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Debate remains over the benefits of scheduled intravenous antibiotic therapy versus such episodic intravenous antibiotic treatment for acute exacerbations. Standard of care in the United States is generally to administer intravenous antibiotics for symptomatic acute exacerbations. Scheduled treatment with intravenous antibiotics did not perform better than symptom-based treatment in a randomized control trial (85), although the practice continues in Denmark because of historic improvements in survival after implementation of this practice (86).

VII.

Infection Control

Because of the well-described risk of nosocomial interpatient transmission of CF pathogens (87–92), strict infection control policies must be followed in both the inpatient and ambulatory care settings. Such guidelines have been published by the Cystic Fibrosis Foundation (93) and are discussed in the chapter by Rettig and Coffin.

VIII.

Prevention of Pulmonary Exacerbations

Phase 3 randomized controlled clinical trials of inhaled recombinant human DNase, inhaled antibiotics (tobramycin and aztreonam), oral azithromycin, and inhaled hypertonic saline have showed significant and clinically important reductions in exacerbation rates among subjects in the active arm (13,16,18,31). Thus, all these chronic maintenance therapies may reduce exacerbation rates. Inhaled corticosteroids are widely employed as chronic maintenance therapy, although a well-designed randomized trial of withdrawing inhaled steroids did not show an increase in exacerbation rates (94). These and other approaches to the maintenance of pulmonary health in CF are discussed in chapter 14 by McColley.

IX.

Conclusions

Pulmonary exacerbations, a cardinal feature of CF lung disease, are easily recognized yet difficult to define at a molecular, cellular, or even a clinical level. Considerable variability exists in the definition and treatment of pulmonary exacerbations. Pulmonary exacerbations are used as a key efficacy endpoint in numerous clinical trials, but a universally standardized definition is lacking. A standardized definition has the potential to improve both clinical care and research.

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26. VanDevanter DR, Van Dalfsen JM. How much do Pseudomonas biofilms contribute to symptoms of pulmonary exacerbation in cystic fibrosis? Pediatr Pulmonol 2005; 39:504–506. 27. Aaron SD, Ramotar K, Ferris W, et al. Adult cystic fibrosis exacerbations and new strains of Pseudomonas aeruginosa. Am J Respir Crit Care Med 2004; 169:811–815. 28. Rosenfeld M, Emerson J, Williams-Warren J, et al. Defining a pulmonary exacerbation in cystic fibrosis. J Pediatr 2001; 139:359–365. 29. Rabin HR, Butler SM, Wohl ME, et al. Pulmonary exacerbations in cystic fibrosis. Pediatr Pulmonol 2004; 37:400–406. 30. Dakin C, Henry RL, Field P, et al. Defining an exacerbation of pulmonary disease in cystic fibrosis. Pediatr Pulmonol 2001; 31:436–442. 31. Ramsey BW, Pepe MS, Quan JM, et al. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin Study Group. N Engl J Med 1999; 340:23–30. 32. Wilmott RW, Amin RS, Colin AA, et al. Aerosolized recombinant human DNase in hospitalized cystic fibrosis patients with acute pulmonary exacerbations. Am J Respir Crit Care Med 1996; 153:1914–1917. 33. Ramsey BW, Boat TF. Outcome measures for clinical trials in cystic fibrosis. Summary of a Cystic Fibrosis Foundation Consensus Conference. J Pediatr 1994; 124:177–192. 34. Johnson C, Butler SM, Konstan MW, et al. Factors influencing outcomes in cystic fibrosis: a center-based analysis. Chest 2003; 123:20–27. 35. Mayer-Hamblett N, Ramsey BW, Kronmal RA. Advancing outcome measures for the new era of drug development in cystic fibrosis. Proc Am Thorac Soc 2007; 4:370–377. 36. Goss CH, Quittner AL. Patient-reported outcomes in cystic fibrosis. Proc Am Thorac Soc 2007; 4:378–386. 37. Cystic Fibrosis Foundation. 2007 Annual Data Report to the Center Directors. Bethesda, MD: Cystic Fibrosis Foundation, 2008. 38. Goss CH, Burns JL. Exacerbations in cystic fibrosis. 1: Epidemiology and pathogenesis. Thorax 2007; 62:360–367. 39. Block JK, Vandemheen KL, Tullis E, et al. Predictors of pulmonary exacerbations in patients with cystic fibrosis infected with multi-resistant bacteria. Thorax 2006; 61:969–974. 40. Schechter MS, Shelton BJ, Margolis PA, et al. The association of socioeconomic status with outcomes in cystic fibrosis patients in the United States. Am J Respir Crit Care Med 2001; 163:1331–1337. 41. Smyth A, Elborn JS. Exacerbations in cystic fibrosis: 3–management. Thorax 2008; 63: 180–184. 42. Gozal D, Bailey SL, Keens TG. Evolution of pulmonary function during an acute exacerbation in hospitalized patients with cystic fibrosis. Pediatr Pulmonol 1993; 16:347–353. 43. Rosenberg SM, Schramm CM. Predictive value of pulmonary function testing during pulmonary exacerbations in cystic fibrosis. Pediatr Pulmonol 1993; 16:227–235. 44. Sanders DB, Rosenfeld M, Mayer-Hamblett N, et al. Reproducibility of spirometry during cystic fibrosis pulmonary exacerbations. Pediatr Pulmonol 2008; 43:1142–1146. 45. Rosenfeld M, Emerson J, Accurso F, et al. Diagnostic accuracy of oropharyngeal cultures in infants and young children with cystic fibrosis. Pediatr Pulmonol 1999; 28:321–328. 46. Esther CR Jr., Henry MM, Molina PL, et al. Nontuberculous mycobacterial infection in young children with cystic fibrosis. Pediatr Pulmonol 2005; 40:39–44. 47. Moskowitz SM, Foster JM, Emerson J, et al. Clinically feasible biofilm susceptibility assay for isolates of Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol 2004; 42:1915–1922. 48. Tunney MM, Field TR, Moriarty TF, et al. Detection of anaerobic bacteria in high numbers in sputum from patients with cystic fibrosis. Am J Respir Crit Care Med 2008; 177:995–1001.

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49. Smith AL, Fiel SB, Mayer-Hamblett N, et al. Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis. Chest 2003; 123:1495–1502. 50. Brody AS, Tiddens HA, Castile RG, et al. Computed tomography in the evaluation of cystic fibrosis lung disease. Am J Respir Crit Care Med 2005; 172:1246–1252. 51. de Jong PA, Lindblad A, Rubin L, et al. Progression of lung disease on computed tomography and pulmonary function tests in children and adults with cystic fibrosis. Thorax 2006; 61:80–85. 52. Salva PS, Doyle NA, Graham L, et al. TNF-alpha, IL-8, soluble ICAM-1, and neutrophils in sputum of cystic fibrosis patients. Pediatr Pulmonol 1996; 21:11–19. 53. Schuster A, Haarmann A, Wahn V. Cytokines in neutrophil-dominated airway inflammation in patients with cystic fibrosis. Eur Arch Otorhinolaryngol 1995; 252(suppl 1):S59–S60. 54. Francoeur C, Denis M. Nitric oxide and interleukin-8 as inflammatory components of cystic fibrosis. Inflammation 1995; 19:587–598. 55. Cunningham S, McColm JR, Mallinson A, et al. Duration of effect of intravenous antibiotics on spirometry and sputum cytokines in children with cystic fibrosis. Pediatr Pulmonol 2003; 36:43–48. 56. Sagel SD, Chmiel JF, Konstan MW. Sputum biomarkers of inflammation in cystic fibrosis lung disease. Proc Am Thorac Soc 2007; 4:406–417. 57. Flume PA, Mogayzel PJ, Robinson KA, et al. Cystic fibrosis pulmonary guidelines: treatment of pulmonary exacerbations. Am J Respir Crit Care Med 2009; 180:802–808. 58. Regelmann WE, Elliott GR, Warwick WJ, et al. Reduction of sputum Pseudomonas aeruginosa density by antibiotics improves lung function in cystic fibrosis more than do bronchodilators and chest physiotherapy alone. Am Rev Respir Dis 1990; 141:914–921. 59. Smyth A, Tan KH, Hyman-Taylor P, et al. Once versus three-times daily regimens of tobramycin treatment for pulmonary exacerbations of cystic fibrosis—the TOPIC study: a randomised controlled trial. Lancet 2005; 365:573–578. 60. Horrevorts AM, de Witte J, Degener JE, et al. Tobramycin in patients with cystic fibrosis. Adjustment in dosing interval for effective treatment. Chest 1987; 92:844–848. 61. Padoan R, Brienza A, Crossignani RM, et al. Ceftazidime in treatment of acute pulmonary exacerbations in patients with cystic fibrosis. J Pediatr 1983; 103:320–324. 62. Richard DA, Nousia-Arvanitakis S, Sollich V, et al. Oral ciprofloxacin vs. intravenous ceftazidime plus tobramycin in pediatric cystic fibrosis patients: comparison of antipseudomonas efficacy and assessment of safety with ultrasonography and magnetic resonance imaging. Cystic Fibrosis Study Group. Pediatr Infect Dis J 1997; 16:572–578. 63. Aaron SD, Vandemheen KL, Ferris W, et al. Combination antibiotic susceptibility testing to treat exacerbations of cystic fibrosis associated with multiresistant bacteria: a randomised, double-blind, controlled clinical trial. Lancet 2005; 366:463–471. 64. Aaron SD, Ferris W, Ramotar K, et al. Single and combination antibiotic susceptibilities of planktonic, adherent, and biofilm-grown Pseudomonas aeruginosa isolates cultured from sputa of adults with cystic fibrosis. J Clin Microbiol 2002; 40:4172–4179. 65. Moskowitz SM, Foster JM, Emerson JC, et al. Use of Pseudomonas biofilm susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection. J Antimicrob Chemother 2005; 56:879–886. 66. Keays T, Ferris W, Vandemheen KL, et al. A retrospective analysis of biofilm antibiotic susceptibility testing: a better predictor of clinical response in cystic fibrosis exacerbations. J Cyst Fibros 2009; 8:122–127. 67. Redding GJ, Restuccia R, Cotton EK, et al. Serial changes in pulmonary functions in children hospitalized with cystic fibrosis. Am Rev Respir Dis 1982; 126:31–36. 68. Dasenbrook EC, Merlo CA, Diener-West M, et al. Persistent methicillin-resistant Staphylococcus aureus and rate of FEV1 decline in cystic fibrosis. Am J Respir Crit Care Med 2008; 178:814–821.

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69. Sawicki GS, Rasouliyan L, Pasta DJ, et al. The impact of incident methicillin resistant Staphylococcus aureus detection on pulmonary function in cystic fibrosis. Pediatr Pulmonol 2008; 43:1117–1123. 70. Donati MA, Guenette G, Auerbach H. Prospective controlled study of home and hospital therapy of cystic fibrosis pulmonary disease. J Pediatr 1987; 111:28–33. 71. Pond MN, Newport M, Joanes D, et al. Home versus hospital intravenous antibiotic therapy in the treatment of young adults with cystic fibrosis. Eur Respir J 1994; 7: 1640–1644. 72. Bosworth DG, Nielson DW. Effectiveness of home versus hospital care in the routine treatment of cystic fibrosis. Pediatr Pulmonol 1997; 24:42–47. 73. Thornton J, Elliott R, Tully MP, et al. Long term clinical outcome of home and hospital intravenous antibiotic treatment in adults with cystic fibrosis. Thorax 2004; 59:242–246. 74. Wolter JM, Bowler SD, Nolan PJ, et al. Home intravenous therapy in cystic fibrosis: a prospective randomized trial examining clinical, quality of life and cost aspects. Eur Respir J 1997; 10:896–900. 75. Gold R, Carpenter S, Heurter H, et al. Randomized trial of ceftazidime versus placebo in the management of acute respiratory exacerbations in patients with cystic fibrosis. J Pediatr 1987; 111:907–913. 76. Gondor M, Nixon PA, Mutich R, et al. Comparison of Flutter device and chest physical therapy in the treatment of cystic fibrosis pulmonary exacerbation. Pediatr Pulmonol 1999; 28:255–260. 77. Braggion C, Cappelletti LM, Cornacchia M, et al. Short-term effects of three chest physiotherapy regimens in patients hospitalized for pulmonary exacerbations of cystic fibrosis: a cross-over randomized study. Pediatr Pulmonol 1995; 19:16–22. 78. Cerny FJ. Relative effects of bronchial drainage and exercise for in-hospital care of patients with cystic fibrosis. Phys Ther 1989; 69:633–639. 79. Selvadurai HC, Blimkie CJ, Meyers N, et al. Randomized controlled study of in-hospital exercise training programs in children with cystic fibrosis. Pediatr Pulmonol 2002; 33: 194–200. 80. Fauroux B, Itti E, Pigeot J, et al. Optimization of aerosol deposition by pressure support in children with cystic fibrosis: an experimental and clinical study. Am J Respir Crit Care Med 2000; 162:2265–2271. 81. Cystic Fibrosis Foundation. Clinical Practice Guidelines for Cystic Fibrosis. Bethesda, MD: Cystic Fibrosis Foundation, 1997. 82. van den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in the critically ill patients. N Engl J Med 2001; 345:1359–1367. 83. Tepper R, Eigen H, Stevens J, et al. Lower respiratory illness in infants and young children with cystic fibrosis: evaluation of treatment with intravenous hydrocortisone. Pediatr Pulmonol 1997; 24:48–51. 84. Dovey M, Aitken ML, Emerson J, et al. Oral corticosteroid therapy in cystic fibrosis patients hospitalized for pulmonary exacerbation: a pilot study. Chest 2007; 132:1212–1218. 85. Elborn JS, Prescott RJ, Stack BH, et al. Elective versus symptomatic antibiotic treatment in cystic fibrosis patients with chronic Pseudomonas infection of the lungs. Thorax 2000; 55:355–358. 86. Johansen HK, Norregaard L, Gotzsche PC, et al. Antibody response to Pseudomonas aeruginosa in cystic fibrosis patients: a marker of therapeutic success?—A 30-year cohort study of survival in Danish CF patients after onset of chronic P. aeruginosa lung infection. Pediatr Pulmonol 2004; 37:427–432. 87. Bosshammer J, Fiedler B, Gudowius P, et al. Comparative hygienic surveillance of contamination with pseudomonads in a cystic fibrosis ward over a 4-year period. J Hosp Infect 1995; 31:261–274.

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88. Burdge DR, Nakielna EM, Noble MA. Case-control and vector studies of nosocomial acquisition of Pseudomonas cepacia in adult patients with cystic fibrosis. Infect Control Hosp Epidemiol 1993; 14:127–130. 89. Cheng K, Smyth RL, Govan JR, et al. Spread of beta-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic. Lancet 1996; 348:639–642. 90. Doring G, Jansen S, Noll H, et al. Distribution and transmission of Pseudomonas aeruginosa and Burkholderia cepacia in a hospital ward. Pediatr Pulmonol 1996; 21:90–100. 91. Jones AM, Govan JR, Doherty CJ, et al. Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic. Lancet 2001; 358:557–558. 92. Holmes A, Nolan R, Taylor R, et al. An epidemic of burkholderia cepacia transmitted between patients with and without cystic fibrosis. J Infect Dis 1999; 179:1197–1205. 93. Saiman L, Siegel J. Infection control recommendations for patients with cystic fibrosis: microbiology, important pathogens, and infection control practices to prevent patient-topatient transmission. Infect Control Hosp Epidemiol 2003; 24:S6–S52. 94. Balfour-Lynn IM, Lees B, Hall P, et al. Multicenter randomized controlled trial of withdrawal of inhaled corticosteroids in cystic fibrosis. Am J Respir Crit Care Med 2006; 173:1356–1362.

17 Gastrointestinal Complications of Cystic Fibrosis MARIA MASCARENHAS and ASIM MAQBOOL University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

The gastrointestinal (GI) tract is affected in all patients with cystic fibrosis (CF), whether or not they are pancreatic sufficient (PS) or insufficient (PI). The incidence of the GI manifestations varies depending on the age of the patient, genotype, and presence of pancreatic involvement. Meconium ileus (MI) is the first manifestation of CF and can be detected in utero. The pancreas is affected in most patients. Gastroesophageal reflux (GER), distal intestinal obstruction syndrome (DIOS) or MI equivalent, constipation, small bowel bacterial overgrowth (SBBO), and pancreatic insufficiency are the most common manifestations seen. Animal models [mouse and piglet (1,2)] provide excellent modalities for looking at GI abnormalities, including DIOS, bacterial overgrowth and intestinal inflammation, MI, focal biliary cirrhosis, and pancreatic destruction with resulting PI. While all models do not necessarily translate well to CF-related disease in humans, they nonetheless pave the way for future exciting research to help elucidate the mechanisms for the GI manifestations of CF.

II.

Malabsorption

There are several reported perturbations in GI function that result in maldigestion and malabsorption in CF (Fig. 1) (1,3). Abnormal gastric acid secretion and dysmotility have been reported in subjects with CF (4). Gastric acid coupled with impaired pancreatic bicarbonate secretion lowers duodenal pH, resulting in suboptimal pancreatic enzyme function (5,6). Pancreatic enzyme deficiency is perhaps the most important of these factors (3). Abnormalities in phospholipase A2 have also been reported in CF, which impact fatty acid metabolism (phospholipid status), and may impair fatty acid absorption (7,8). Cystic fibrosis transmembrane regulator (CFTR) protein is also highly expressed in the GI tract and abnormal CFTR is implicated in hepatobiliary dysfunction, further impacting digestion and absorption (9). Impaired CFTR function results in thickened luminal secretions, plugging ducts and crypts, and results in a thickened, poorly hydrated unstirred layer through which diffusion of micelles may be impaired. Motility may also be altered in CF (10), which, coupled with incomplete digestion and absorption of nutrients, impairs innate host antibacterial Paneth cell activity and host-bacterial interactions. This further contributes to bacterial overgrowth and inflammation that also impairs mucosal functioning (11–14). MI and ileal resections also contribute to the above risk factors. These changes in the intestinal milieu have further impact on absorptive function for fats and may increase risk for vitamin B12 deficiency (15,16). It 266

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Figure 1 Normal digestion and absorption and perturbations in cystic fibrosis. Abbreviation:

CFTR, cystic fibrosis transmembrane regulator.

is unclear if there are deficits at the membrane level that influence fat absorption; there is mixed evidence of long-chain fatty acid malabsorption in subjects with CF (17,18). Fat and fat-soluble vitamin deficiency, in turn, may further impair fat digestion and absorption in CF (19). Some strategies are already well established for improving maldigestion and malabsorption in CF. Pancreatic enzyme replacement therapy (PERT) is discussed in the next section. PERT efficacy may be enhanced by pharmacological acid blockade to reduce gastric and, thereby, duodenal acidity; however, this has not been conclusively established (20,21). Increased hydration of the lumen, as explored in the animal model, may also have beneficial effects (22). It is important to recognize that many factors influence macronutrient and micronutrient absorption, and many perturbations in CF may contribute to malabsorption. In the case of fat malabsorption, it is key to recognize that while PI is a major contributory factor to malabsorption, it is not the sole factor; motility, inflammation, and loss of bile salt are also contributory. These observations help explain why PERT therapy alone will not completely resolve fat malabsorption, and increasing the dose of PERT beyond recommended doses does not facilitate fat absorption and may also increase the risk of fibrosing colonopathy (FC).

III.

Pancreas: PI, PS, and Pancreatitis

The pancreas is one of the major organs impaired by defective CFTR structure and function, resulting in perturbations in chloride transport, bicarbonate exchange, and fluid

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balance in the ductal lumen. The dehydrated intraluminal contents precipitate, resulting in obstruction, inflammation, and fibrosis with destruction of the exocrine pancreas (23–25). Pancreatic function is determined primarily by fat balance studies: normal coefficient of fat absorption is >95%; <93% represents PI. Approximately 85% of subjects with CF are PI, and 15% are PS. In fact, at the time of diagnosis of CF in infants identified by neonatal screening, at least 50% were PI, suggestive of a process that begins in utero (26). With further identification of gene mutations and classification of mutation classes reflecting CFTR function, more insights have been gained into genotype-phenotype relationships with pancreatic disease in CF. The onset and severity of pancreatic disease is associated with CFTR mutation class, with classes I, II, and III associated with nonfunctional CFTR, and classes IV and V with some residual function. Patients with class I and II mutations have early progression of pancreatic damage within the first two years of life (25,27,28). A retrospective study spanning 30 years of data identified 1075 subjects with CF; 87% were PI at diagnosis, 3% were PS and progressed to PI, and 10% of the subjects remained PS (29). All of the subjects with PI had class I, II, or III mutations. All of the subjects with PS had at least one allele associated with a class IV or V mutation, presumably with this allele expressed in a dominant fashion conferring some functionality to CFTR. Pancreatic function testing by means of cholecystokinin (CCK)/secretin infusion and quantification of fluid, trypsin, lipase/colipase, and bicarbonate/chloride secretion has shown better acinar and ductular function in those patients with class IV and V mutations (30). While pancreatic functionality is strongly associated with clinical outcomes in CF, the precise mechanisms and relationships of this are not completely understood. Surveillance of pancreatic status in subjects with PS is prudent. The natural history of progression to PI is variable and unpredictable, and the optimal screening frequency for change in functional status has not been precisely established (29,30). Patients with CF and PI are diagnosed at a younger age, with greater elevations in sweat chloride, lower FEV1, and poorer growth than patients with CF and PS, who have greater median age of survival than their PI counterparts (31). Assessment of exocrine pancreatic function can include objective testing, both direct and indirect. Direct measurement of pancreatic enzyme and bicarbonate stimulation testing following secretin and cholecystokinin stimulation, with recovery via properly placed nasojejunal tube, is the most precise measurement. However, it is invasive, cumbersome, and not commonly practiced. Indirect testing assesses maldigestion of fat as a consequence of impaired pancreatic function. While these tests are more clinically relevant, they are less specific for pancreatic function. The gold standard testing is the coefficient of fat absorption calculated from a three-day stool collection and dietary intake record. Additional testing modalities include radiotracer recovery from labeled fats in breath, blood, and stool. Serum immunoreactive trypsinogen levels are used in the newborn screen. Other methods of indirect testing are reviewed by Walkowiak et al. (27). Pancreatic elastase is not degraded during intestinal transport, and is recoverable and quantifiable in the stool by means of monoclonal or polyclonal antibody testing. Other enzymes such as lipase and chymotrypsin can also be recovered and quantified in the stool, but may be less reliable than elastase (32,33). The monoclonal test is specific for human pancreatic elastase and does not cross-react with porcine-based PERT or other antigens, rendering it as the preferred assay (34–36). The polyclonal assays crossreacts with porcine-based PERT. While there is lack of agreement as to what cut-off

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level should be used to denote PI, the literature supports agreement that fecal elastase <15 mg/g stool most likely denotes PI, whereas >200 mg/g stool more likely supports PS (37–40). There is good agreement between direct exocrine pancreatic assessments, fecal fat quantification, and fecal elastase for subjects with moderate to severe fat malabsorption, but less strong agreement for milder forms of PI with less fat malabsorption (41). While many of these indirect tests of exocrine pancreatic function are promising, the gold standard remains the 72-hour fecal fat quantification expressed as a percentage of dietary fat intake (42). What is less clear is the use of fecal elastase, as proposed by some investigators, to monitor for conversion from PS to PI (43). The use of objective testing to document pancreatic status is key to appropriate diagnosis and management. The sensitivity, specificity, reproducibility, and both intratest and intertest reliability have implications for the correct identification of pancreatic status, surveillance of status changes for PS patients, and for appropriate medical and nutritional management that are strongly associated with clinical outcomes. There are limitations of many of these indirect tests, and they must be interpreted with caution. Recognition that pancreatic function is a central factor, but not the only factor in the pathophysiology of malabsorption in CF, is an important concept (44). PERT provides for some correction of PI-related malabsorption in CF. Various products and formulations are available. These pancreatic enzyme replacement products differ by their whether or not they are enterically coated, and, if so, by their content (such as by the presence of bicarbonate), microencapsulated coating, microcapsular size, and dissolution and dispersion properties. The enteric coated varieties are designed to be acid resistant, thereby, reducing pH and gastric degradation and allowing for more products to be delivered to the duodenum. Acid reduction therapy enhances their efficacy, to approximate endogenous pancreatic enzymatic activity expected in the duodenal luminal milieu. Dosing of PERT is influenced significantly by their potential of adverse effects, specifically, with respect to the risk for FC, with highest risk associated with 50,000 lipase units/kg/day, and probable increased risk with doses >20,000 lipase units/kg/day (or 6000 units/meal). The recommended dose ranges are currently 5 to 10,000 lipase units/kg/day. Common practice is to provide 1000 to 3000 lipase units/kg/ meal (*1800 lipase units/g fat) and not to exceed 10,000 lipase units/kg/day (45,46). Other complications include oral ulcers and perianal rash in infants. Pancreatic transplantation is a newer modality that has recently been pioneered in combination with lung transplantation recently, and may be a modality to increase survival and outcomes in select eligible candidates (47–49).

IV.

Pancreatitis

Pancreatitis is the presenting symptom for an estimated one-third of subjects with CF and PS, with the actual percentage potentially being higher, when unexplained recurrent abdominal pain is the presenting symptom. No specific genotype predicts risk for pancreatitis (29). In fact, idiopathic pancreatitis may be the initial presentation of CF in, otherwise, asymptomatic adults. In one study of patients with idiopathic pancreatitis, CFTR gene mutational analysis revealed at least one CFTR allele mutation in all patients and both allele mutations in three subjects (50). CFTR mutations are one of three identified genetic defects associated with recurrent or chronic pancreatitis; the

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other two are of the serine protease inhibitor Kazal 1 (SPINK1) and of cationic trypsinogen (PRSS1). In a study of 381 subjects with a primary diagnosis of recurrent or chronic pancreatitis, 49% of subjects had one or more mutations associated with hereditary pancreatitis; 32% of the subjects with pancreatitis had CFTR mutation– associated alleles, 6% had SPINK1 mutations, and 8.9% had PRSS1 mutations (51). Interestingly, 5.5% of subjects had mutations in both CFTR and SPINK1, and 1.8% with CFTR and PRSS1 mutations. In this study, 12 novel genes associated with CFTR mutations were identified. While these patients may not have the classical findings and clinical course of CF and may have a longer life expectancy than their PI counterparts, pancreatitis may have long-term consequences not traditionally ascribed to CF.

V. Meconium Ileus MI is neonatal intestinal obstruction due to meconium and is most often due to CF. Meconium from patients with CF has increased viscosity, reduced water content, and increased protein concentration. MI can be detected in utero by ultrasound and is the earliest manifestation of CF. It occurs in 15% of patients with CF. Gene modifiers probably influence the incidence of MI, independent of CFTR. MI also correlates with DIOS, but this is probably caused by nongenetic factors. Regions of suggestive linkage identified by genome-wide association analysis for modifier genes that cause MI are on chromosomes 4q35.1, 8p23.1, and 11q25, and regions that protect from MI are on chromosomes 20p11.22 and 21q22.3. DIOS in older patients is caused by nongenetic factors, but MI is associated with CFTR and two or more modifier genes (52). MI is caused by thick, inspissated, intestinal secretions, and can be associated with intestinal atresias, volvulus, perforation, peritonitis, calcification, and pseudocyst formation. Neonates typically present with symptoms and signs of intestinal obstruction, and the diagnosis of MI can be made by X rays (abdominal films and gastrograffin enema) and laparotomy. Surgery is indicated for neonates who do not clear the meconium obstruction with a gastrograffin enema, and for those with perforation and with persistent obstructive symptoms. In the early 1960s, the Bishop–Koop ileostomy was created because mortality was very high in infants with CF who needed intestinal surgery. Improved survival (37–90%) was noted in infants who had this type of surgery (53). Currently most neonates, who undergo surgery and need a resection, have a primary anastamosis; this has been found to be a safe option (54) and is associated with a reduced length of initial hospital stay (55). The long-term outcome of infants with MI is different (56) from infants without MI. Infants with MI who undergo surgery tend to have feeding difficulties and are at risk for parenteral nutrition–associated cholestasis when enteral feeds cannot be started soon after surgery. Aggressive postoperative nutritional care with use of parenteral nutrition is often required in these patients, and PERT is started when the rate of feeds is significant. Munck et al. (57) have not seen a difference between MI and non-MI patients in terms of genotype, nutritional status, and acquisition of pseudomonas. However, Li et al. (58) have shown more lung disease and more obstructive lung disease at age 8 to 10 years in a group of children with MI, who were diagnosed by newborn screening. This was seen in all infants with MI, including those who did and did not need surgery. In the past, patients with MI had high perinatal mortality, poor growth, and poor long-term survival; now with improvements in care, including surgical interventions, prognosis is improved.

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Distal Intestinal Obstruction Syndrome

DIOS, also called MI equivalent, is a recurrent variable intestinal obstruction starting in the ileocecal region and extending throughout the colon (59). DIOS can be seen in PI or PS patients (60). Abnormal water and electrolyte transport and intestinal mucus, dysmotility, inspissated secretions, increased protein content of mucus, and modifier genes are felt to be responsible for DIOS (61). Prolonged orocecal transit results in stasis in the colon, and stool builds up in the cecum and terminal ileum in a layered fashion. In CFTR knockout mice, MI and DIOS occur due to defective electroneutral sodium absorption, bicarbonate secretion, and defective chloride secretion. Precipitating factors for an episode of DIOS include dehydration, changes in diet, immobilization, respiratory infection, bacterial overgrowth, use of constipating medications, and fat malasorption. Patients with a history of previous GI surgery may be at increased risk during summer months because of intestinal dysmotility, dehydration, and bacterial overgrowth. Patients with lung transplantation are at increased risk for DIOS. Recognition of this complication and institution of preventative measures are important (62). Morton showed an incidence of DIOS of 10% in 121 patients with CF who underwent lung transplantation. Risk factors included a past history of MI or previous laparotomy. Initial treatment consisted of laxatives, stool softners, and bowel preparation formula that was successful in 14 out of 17 patients when the regimen was given for up to 14 days. However, 3 out of 17 patients needed enterotomy and fecal disimpaction. One patient died because of late diagnosis (63). Clinical symptoms of DIOS include chronic, recurrent, crampy abdominal pain frequently in the right lower quadrant, associated with a right lower quadrant palpable mass. No change in bowel movements is often noted. Management of DIOS consists of adequate hydration of fluid and electrolytes, medications (polyethylene glycol), optimization of PERT, and dietary measures. Adequate fluid and electrolyte intake is very important to hydrate intestinal secretions. Polyethylene glycol increases the volume of intestinal secretions and stimulates peristalsis. Prevention and treatment of fat malabsorption and bacterial overgrowth are important as well. For severe episodes of DIOS, including obstruction, patients need to be well hydrated and the obstruction is treated by using a gastrograffin enema. It is important to reflux the gastrograffin into the terminal ileum and to document clearance of stool by obtaining an abdominal X ray. Polyethylene glycol lavage solutions (orally or via nasogastric tube) can also be used to clear the obstruction from above, but several doses may be needed to clear the stool buildup. Passage of clear liquid stool is not an evidence of a good clean out, and an abdominal X ray must be done to document the absence of residual stool (64). Patients who have intestinal obstruction due to DIOS after abdominal surgery may benefit by having N-acetylcysteine instilled via J tube, as close to the obstruction as possible. It is also important in the postoperative period to avoid drugs that slow motility (such as narcotics) and keep patients well hydrated. In severe cases, recurrent DIOS may be treated with a modified antegrade continence enema procedure (65).

VII.

Gastroesophageal Reflux

GER is a normal physiological process. It is considered pathological, or as gastroesophageal reflux disease (GERD), when it results in esophagitis or is associated with dysphagia, odynophagia, failure to thrive, and extraesophageal manifestations, which

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Figure 2 The normal anti-reflux barrier with factors contributing to reflux. Abbreviations: GER, gastroesophageal reflux; LES, lower esophageal sphincter.

may include the lungs (Fig. 2). GER is common in patients with CF, occurring in 25% to 55% of pediatric cases (66,67), in 19% of infants with CF (68), and 94% of adult patients (69). Duodenogastric bile reflux has also been recently observed in CF (70). GER occurs in response to transient relaxation of the lower esophageal sphincter (LES) mediated by vagal innervation, nitric oxide, and cholecystokinin A. Several factors may contribute to GER, including (i) posture and dietary composition; (ii) anatomical abnormalities (e.g., hiatal hernias); and (iii) gastric distention, volume, pressure, and motility (71). The gastric accommodation reflex and intrathoracic pressures influence GER (72). In adults, abnormal esophageal coordination and peristalsis, and decreased basal LES resting pressures on manometry have been seen in 60% of patients (69). Hiatal hernias may also increase the risk of GER, and have been documented in up to 30% of children and adolescents (73,74). Postural drainage techniques may also increase the frequency of GER episodes in infants (75), and similar effects due to supine positioning are noted in adults (69). Additionally, decreased intrathoracic pressure associated with hyperinflation (when present) and increased transdiaphragmatic pressure associated with coughing and wheezing can contribute to reflux. The role of altered gastric pressures, transit, and motility in subjects with CF has been summarized elsewhere (76). Esophagitis has been reported in at least 50% of pediatric cases (67,77). Barrett’s esophagitis, a precancerous condition, has also been reported in adolescent patients with

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CF (73). The relationship of GER with respect to lung disease in CF is complex, and most likely bidirectional. The traditional assessment tools for GER (contrast studies, scintigraphy, endoscopy, pH monitoring, and manometry) are limited and less informative in studying associations of GER with the airway. Recent research and clinical approaches include tracheal pH monitoring. Acidification of the airway and microaspiration as measured by tracheal breath condensate pH monitoring (to pH <5.5) has been documented in adults (69), and may not be specific to GER. This test may be problematic, as the pH threshold proposed is at a higher pH than that considered for esophageal pH measurement (78). Lipid-laden macrophages obtained by bronchoalveolar lavage are neither specific nor sensitive for GER (79). Gastric pepsin may be a more specific marker of aspiration, and is being studied in relationship to airway disease (80–82). The correlation of severity of GER and pulmonary disease in the literature are inconsistent. In infants, pH-documented GER was not associated with radiological pulmonary changes (68). In adolescents, there was a covariation between GER, pulmonary disease, and malnutrition (74). GER has been studied in symptomatic and asymptomatic patients either before or after lung transplantation, using pH measurements (83). A reduction in basal LES resting tone was noted in symptomatic adults with end-stage lung disease, awaiting lung transplantation (84). The prevalence of GER after lung transplantation increased (85,86). In the latter study, the presence of GERD pretransplant predicted GERD 100 days posttransplant. Additionally, some subjects with normal pH studies pretransplant developed GERD following transplantation. An animal model of GER in lung transplantation suggests aspiration of gastric fluid may adversely affect the transplanted tissue and may be associated with allograft dysfunction (87). GER has been described as a risk factor for bronchiolitis obliterans (85). The treatment approach to GER, in general, includes postural therapy, dietary modification, pharmacological therapy (acid reduction and promotility agents), and, in refractory cases, fundoplication. Data suggests that supine positioning may exacerbate symptoms, and reducing the time spent in this position may be helpful (88). While caloric needs are increased in CF, smaller, more frequent meals may be helpful in improving gastric emptying. Symptomatic improvement was noted with the use of either cisapride alone or in combination with ranitidine therapy (67). Higher doses of ranitidine are needed in children and adolescents with CF than those used in healthy subjects for comparable acid suppression (89). Proton pump inhibitors are also frequently used. In one study, cisapride improved esophageal acid clearance in only three out of seven subjects (71), and has been taken off the market because of cardiac arrythmias. Recently, intravenous erythromycin has been shown to improve gastric emptying on antroduodenal manometry testing (90). Fundoplication has traditionally been reserved for subjects who fail to adequately respond to aggressive lifestyle changes and pharmacological therapy, and is frequently recommended. Boesch and Action (91) showed that 27% of patients were able to be weaned off from acid reduction therapy postfundoplication, but 48% developed symptoms of recurrent GERD after surgery. For lung transplant recipients, pulmonary outcomes improved for those who had an FEV1 <60% prior to transplant. Case reports suggest that fundoplication can reverse early allograft reduction and improve pulmonary function (92). Other studies have suggested improved allograft survival with fundoplication done within 90 days of transplant (93,94). While early aggressive management of

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GER in the CF population undergoing lung transplantation appears to reduce the risk of allograft rejection and improve lung function, the precise timing of fundoplication needs to be prospectively studied and better defined.

VIII.

Small Bowel Bacterial Overgrowth

SBBO is frequently seen in patients with CF (1). The exact cause of SBBO in patients with CF is not known, but it is hypothesized that bacteria get trapped in the intestinal mucus layer, preventing the actions of local antibacterial factors. Dysmotility also results in impaired housekeeping mechanisms, resulting in further accumulation of pathogenic bacteria and mucus. While the exact incidence in patients with CF is unknown, Fridge et al. (95) showed that the incidence was as high as 56% in a group of patients with CF when compared with 20% in control subjects when glucose breath tests were used for diagnosis. Baseline breath hydrogen values were 22 and 5 ppm in the CF and control group, respectively. The use of azithromycin was associated with a positive breath test. Decreased risk of SBBO was seen with laxative (polyethylene glycol) use. Lewindon et al. (96) looked at prevalence of carbohydrate malabsorption and bacterial overgrowth in patients with CF using lactose, sucrose, and lactulose breath tests, and compared them to control subjects. Patients with CF had longer orocecal transit times (99 vs. 68 minutes), higher incidence of SBBO (32% vs. 7%) and sucrose malabsorption (47% vs. 14.5%) than controls. Those with SBBO had longer transit times: 123 minutes in patients with CF versus 69 minutes in control subjects (97). Schneider et al. also showed a high incidence of positive glucose breath tests (68%) in patients with CF, but felt that the test was more an indication of malabsorption and intestinal dysmotility than SBBO. Delisle et al. showed altered bowel transit as well as altered Paneth cell function in a CF mouse model. SBBO occurred by day 8 in these mice. Gastric emptying was slightly decreased in mice with CF when compared with wild-type mice, and small bowel transit was dramatically less in mice with CF (13). Clinical symptoms for SBBO include abdominal pain, increased gas and abdominal distension, diarrhea and increased burping resulting in malabsorption, maldigestion, fat-soluble vitamin deficiency, elevated folate levels, and intestinal inflammation. Predisposing factors for SBBO include antibiotic use, previous GI surgery resulting in stasis, dysmotility, intestinal strictures, and adhesions. Diagnosis is made by a combination of clinical symptoms and/or tests (glucose and lactulose breath tests). In a research setting, a duodenal aspirate sent for aerobic and anerobic culture can be obtained, but this is hard to obtain in a clinical setting. Treatment consists of the use of appropriate antibiotics either as a single course or as intermittent therapy.

IX.

GI Infections and Inflammatory Conditions

Some GI infections may be seen more often in patient with CF (1). Possible explanations include altered local immunity and impaired defense mechanisms at the intestinal mucosal level. True Clostridium difficile infection is rare, despite a large carrier rate in patients with CF (98). Infection rates with C. difficile may be higher and clinical symptoms atypical and more severe in patients with CF who have had lung transplantation. Diarrhea was not a presenting symptom in 20% of these patients. Infections were complicated and, in some patients, resulted in death (99). In another series, C. difficile infection was fulminant with a mortality of 50%. Once again presentation

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was atypical with one patient having only abdominal distension as the presenting symptom, making it difficult to diagnose the infection (100). There does not appear to be an increased risk of acquiring Helicobacter pylori (HP) infection in patients with CF. In a study involving 30 patients with CF, the prevalence of HP antigen in stool was 50% less than age-matched control subjects. Patients with less severe mutations had a higher risk of infection when compared with those with severe mutations. This study also showed that most patients (50%) had stool positive for C. difficile toxin A and B, but were asymptomatic and felt to be carriers (101). HP serology is not a reliable method to diagnose HP infection, since there is cross-reactivity with other bacterial antibodies, especially pseudomonas. Prevalence of HP antibodies was between 24% and 47%, depending on the assay used. The incidence of HP antibodies decreased to 0% to 15% once the pseudomonas antibodies were removed, resulting in a true incidence of infection of 8% when antibodies were detected by ELISA. Those with HP antibodies detected by ELISA were proven on upper endoscopy to be true infections (102). Giardia can be seen in patients with CF. Rates of infection with giardia in patients with CF were higher than in normal members of their families (28% vs. 6.3%, respectively), and this disparity increased with age (103). Inflammatory conditions include esophagitis (reflux and eosinophilic), gastritis, peptic ulcer disease including HP infection, celiac disease, inflammatory bowel disease (IBD), and appendicitis. GER, reflux esophagitis, and HP infection were previously described. To date there has been no report of an increased incidence of eosinophilic esophagitis in patients with CF, and there is no evidence to suggest that the incidence will be dissimilar to that of the general population. Reports suggest that peptic ulcer disease is uncommon in CF (104). Predisposing factors include increased acid secretion, altered mucins, impaired alkalinization due to altered hormones, reduced duodenal bicarbonate, duodenal hyperacidity, and possibly the role of CFTR in duodenal bicarbonate secretion. Other factors include use of certain drugs such as asprin, NSAIDS, and alcohol. Peptic ulcer disease appears to be more common in older patients with CF: 1.2% in the 35- to 44-year age range versus 0.1% in the 11- to 17-year age group. HP infection is infrequently involved. Current treatment includes the use of gastric acid blocking medications like histamine-2 receptor antagonists and proton pump inhibitors. Celiac disease is reported in patients with CF (1,105). Recent reports from Norway and Sweden indicate that when systematic screening was performed in patients with CF, a prevalence rate of 1.2% or 1:83 was noted, three times higher than the prevalence of celiac disease in the general population (106). IBD is noted to be 17 times more common in patients with CF (1,107). However, this report was written before FC was described. It has been suggested that DF508 heterozygosity may exert a protective effect in Crohn’s disease (108). Right-sided microscopic colitis has been described in adults with CF (109). Antisaccharomyces cerevisiae antibody (ASCA) seropositivity was significantly higher in CF compared to general population (20.7% vs. 4%); the reasons for this are unknown (110). Wilchanski, in a preliminary report, described diffuse areas of small bowel inflammation that included edematous folds, erythema, mucosal breaks, and frank ulcerations in four out of five adult patients with CF and PI. The significance of these lesions is unknown (111). Appendicitis can be seen in patients with CF, with delayed diagnosis, chronic appendicitis, and abscess formation (112,113). It has been noted that the appendiceal diameter should not be the only factor in diagnosing appendicitis in patients with CF, since the appendiceal diameter was enlarged in the majority of patients with CF (114).

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X. Rectal Prolapse Rectal prolapse is usually seen in patients with CF around the time of diagnosis and when there is malabsorption. Malnutrition, constipation with straining, or diarrhea may be related as well. Passage of bulky large stools with or without straining is important in the pathogenesis. Treatment consists of manual reduction of the prolapse, correction of malabsorption, avoidance of straining by using stool softeners, and improvement of nutritional status. In general, the prolapse occurs less frequently over time. If persistent, then injection of a sclerosant solution in the perirectal tissue or surgical resection may be required (115).

XI.

Intussusception

Patients with CF are at increased risk for intussusception (116) that occurs in 1% to 2% of adult patients with CF, a rate of 10 to 20 times that of the general population. The clinical presentation can vary from severe abdominal pain to an incidental finding in an asymptomatic patient. Intussusceptions can be chronic and recurrent, and are sometimes found as an incidental finding on an upper GI barium study. It is felt that sticky mucus acts as the lead point for the intussusception. In patients with CF, intussusception presents at an older age compared to the general population. Intussuception usually occurs in the ileocolic region, is seen in patients with PI, and may be the presenting complaint of undiagnosed CF (117). Treatment is conservative. It has been shown that air or gastrograffin reduction is safe and well tolerated, and surgery can be prevented in the majority of cases (118).

XII.

Dysmotility

There are varying reports of esophageal and gastric motility abnormalities in patients with CF (1,76). There is little evidence for small bowel and colonic abnormalities to date. Esophageal abnormalities are described in the section on GER. Reports on abnormalities in gastric emptying vary depending on the test and the type of meal used, and both delayed and rapid gastric emptying have been described. Collins et al. (119) reported fast gastric emptying in patients with CF. Cucchiara et al. (120) showed delayed gastric emptying (DGE) in patients with CF, using ultrasound with improvement in DGE after the use of ranitidine. Bodet-Milin et al. (121) showed a high incidence (67%) of DGE for solids in patients with CF, prior to lung transplantation. After surgery, 97% had DGE for liquids. Bentur et al. (122) reported that DGE was seen in all patients with end-stage lung disease, and the DGE worsened after transplantation. He also reported electrogastrography (EGG) and gastric emptying scintigraphy abnormalities. Abnormal EGG patterns were seen in 78% of patients with CF compared to 31% control subjects during fasting and 56% vs. 15%, respectively, after eating. Bradygastria was the main defect seen in patients with CF. Gastric emptying results correlated with EGG results in 9 out of 11 patients. Cisapride improved EGG abnormalities in 83% of patients, suggesting that clinically EGG may be a useful tool. Scha¨ppi et al. (4) noted that tachygastria seen in patients with CF was not corrected with cisapride. Tonelli et al. (90) recently showed that gastroparesis can occur in patients without end-stage lung disease and without CFRD, and that erythromycin was effective in improving gastroparesis.

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Constipation occurs frequently in patients with CF and is three times more common than DIOS. It is most frequently seen in children less than five years of age and in adults (123). Decreased colonic motility and decreased fluid intake are felt to be precipitating factors. Treatment consists of adequate hydration and fiber intake, use of laxatives such as stool softners, lubricants, osmotic agents, and stimulants: polyethylene glycol, lactulose, and milk of magnesia and senna. Mineral oil should never be used due to possible interference with fat-soluble vitamin absorption. Treatment of constipation is similar to that used for patients without CF. Behavior modification and regular toilet sitting are important components of the treatment plan. Anticipatory guidance around toilet training in toddlers may be helpful in preventing stool withholding behavior and constipation. Enemas may need to be used for disimpaction. Oral or nasogastric golytely maybe used for a clean out, and use of this agent has been noted to be safe in patients with CF (123). Slow intestinal transit and mucus accumulation have been noted in the CF mouse model. When treated with an osmotic laxative or N-acetylcysteine, a reduction in the mucus by 43% and 50%, respectively, was noted as well as a decrease in SBBO by 92% and 63%, respectively. Transit was normalized by the osmotic laxative but not by Nacetylcysteine. Additionally, the expression of innate immune response–related genes was normalized suggesting that slow small bowel transit and mucus accumulation are predisposing factors for SBBO (12). DeLisle also showed that while mast cells are not involved with dysmotility, their activation can stimulate small intestinal transit in the CF mouse (124).

XIII.

Fibrosing Colonopathy

FC was first described in the United Kingdom in 1993 and subsequently in 1994 in the United States. In 1992, high-potency pancreatic enzymes were first available clinically and the use of these preparations allowed patients to receive very high dose of PERT. Since 2002, more than 100 cases have been described in the United States. Possible mechanisms for the development of FC include failure of microcapsules to dissolve in the small intestine, high concentration of PERT in the cecum due to stasis, overfilling of PERT capsules, mucosal damage from PERT, and altered GI motility. FC is not associated with any one product but is associated with high dose of PERT, length of therapy, previous GI surgery, use of gastric acid–blocking medications, and pulmozyme therapy (125). FC has been seen in neonates who have not received PERT suggesting that mechanisms other than those listed above may be responsible (126). Treatment consists of lowering PERT dose, a GI evaluation to look for other GI disease, nutrition support, close monitoring, and surgery for severe cases (127).

XIV.

Malignancy

Recurrent and chronic pancreatitis is an identified risk factor for pancreatic cancer. There are now several case series describing primarily GI cancers in CF, and pancreatic cancer in subjects with pancreatitis and CFTR mutations who were previously asymptomatic or had undiagnosed CF. The incidence of these cancers or their reporting may be increasing, in part related to increased survival of patients with CF and increased testing for CFTRrelated gene mutations in patients with idiopathic and recurrent pancreatitis. The age at

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the time of diagnosis with pancreatic cancer in the case reports vary, with reports between 19 and 46 years of age (128–130). Genotype information is not consistently available, which renders interpretation of genotype-phenotype associations with pancreatic cancer unclear (131,132). Pancreatic adenocarcinoma has been reported in a patient with a DF508 mutation (133). Preexisting cystic lesions may pose increased risk for pancreatic cancer. A mucinous cystadenocarcinoma was reported in a 19-year-old DF508 homozygote with CF and PI who had the pancreatic cyst identified 13 years earlier (128). It is not clear if the cancer was due to a specific gene association involved in carcinogenesis or to the cumulative effects of inflammation. The other clinical scenario in which pancreatic cancer is reported in subjects with CF has been following lung transplantation, which may relate to the immunosuppression, or to reoccurrence of a preexisting malignant process (134). Other GI malignancies have been seen in adults with CF: esophageal, stomach, small bowel (fivefold increase) and colon cancers, especially adenocarcinomas (135,136). The age-adjusted risk for patients with CF was 9:1 for malignancies of the digestive tract and 31:1 for pancreatic malignancies. In addition to the above-mentioned malignancies, liver and biliary tract cancers are also seen (131).

XV.

Summary

The GI tract is affected in all patients with CF (PS and PI). Management of the GI complications of CF is an integral part of care. As newer animal models are developed we are learning increasingly more about the pathophysiology of the GI tract and are hopeful that newer therapies will be developed to improve care and quality of life for the patient with CF. As patients with CF are living longer, GI malignancy is an emerging problem.

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74. Gustafsson PM, Fransson SG, Kjellman NI, et al. Gastro-oesophageal reflux and severity of pulmonary disease in cystic fibrosis. Scand J Gastroenterol 1991; 26(5):449–456. 75. Button BM, Heine RG, Catto-Smith AG, et al. Postural drainage in cystic fibrosis: is there a link with gastro-oesophageal reflux? J Paediatr Child Health 1998; 34(4):330–334. 76. Gregory PC. Gastrointestinal pH, motility/transit and permeability in cystic fibrosis. J Pediatr Gastroenterol Nutr 1996; 23(5):513–523. 77. Feigelson J, Girault F, Pecau Y. Gastro-oesophageal reflux and esophagitis in cystic fibrosis. Acta Paediatr Scand 1987; 76(6):989–990. 78. Hunt JF, Gaston B. Airway acidification and gastroesophageal reflux. Curr Allergy Asthma Rep 2008; 8(1):79–84. 79. Rosen R, Fritz J, Nurko A, et al. Lipid-laden macrophage index is not an indicator of gastroesophageal reflux-related respiratory disease in children. Pediatrics 2008; 121(4):e879–e884. 80. Starosta V, Kitz R, Hartl D, et al. Bronchoalveolar pepsin, bile acids, oxidation, and inflammation in children with gastroesophageal reflux disease. Chest 2007; 132(5):1557–1564. 81. Sweet MP, Patti MG, Hoopes C, et al. Gastro-oesophageal reflux and aspiration in patients with advanced lung disease. Thorax 2009; 64(2):167–173. 82. Farrell S, McMaster C, Gibson D, et al. Pepsin in bronchoalveolar lavage fluid: a specific and sensitive method of diagnosing gastro-oesophageal reflux-related pulmonary aspiration. J Pediatr Surg 2006; 41(2):289–293. 83. Button BM, Roberts S, Kotsimbos TC, et al. Gastroesophageal reflux (symptomatic and silent): a potentially significant problem in patients with cystic fibrosis before and after lung transplantation. J Heart Lung Transplant 2005; 24(10):1522–1529. 84. D’Ovidio F, Singer LG, Hadjiliadis D, et al. Prevalence of gastroesophageal reflux in endstage lung disease candidates for lung transplant. Ann Thorac Surg 2005; 80(4):1254–1260. 85. Benden C, Aurora P, Curry J, et al. High prevalence of gastroesophageal reflux in children after lung transplantation. Pediatr Pulmonol 2005; 40(1):68–71. 86. Young LR, Hadjiliadis D, Davis RD, et al. Lung transplantation exacerbates gastroesophageal reflux disease. Chest 2003; 124(5):1689–1693. 87. Hartwig MG, Appel JZ, Li B, et al. Chronic aspiration of gastric fluid accelerates pulmonary allograft dysfunction in a rat model of lung transplantation. J Thorac Cardiovasc Surg 2006; 131(1):209–217. 88. Ledson MJ, Tran J, Walshaw MJ. Prevalence and mechanisms of gastro-oesophageal reflux in adult cystic fibrosis patients. J R Soc Med 1998; 91(1):7–9. 89. James LP, Stowe CD, Farrar HC, et al. The pharmacokinetics of oral ranitidine in children and adolescents with cystic fibrosis. J Clin Pharmacol 1999; 39(12):1242–1247. 90. Tonelli AR, Drane WE, Collins DP, et al. Erythromycin improves gastric emptying halftime in adult cystic fibrosis patients with gastroparesis. J Cyst Fibros 2009; 8(3):193–197. 91. Boesch RP, Action JD. Outcomes of fundoplication in children with cystic fibrosis. J Pediatr Surg 2007; 42:1341–1344. 92. Palmer SM, Miralles AP, Howell DN, et al. Gastroesophageal reflux as a reversible cause of allograft dysfunction after lung transplantation. Chest 2000; 118(4):1214–1217. 93. Cantu E III, Appel JZ III, Hartwig MG, et al. Maxwell chamberlain memorial paper. Early fundoplication prevents chronic allograft dysfunction in patients with gastroesophageal reflux disease. Ann Thorac Surg 2004; 78(4):1142–1151. 94. Davis RD Jr., Lau CL, Eubanks S, et al. Improved lung allograft function after fundoplication in patients with gastroesophageal reflux disease undergoing lung transplantation. J Thorac Cardiovasc Surg 2003; 125(3):533–542. 95. Fridge JL, Conrad C, Gerson L, et al. Risk factors for small bowel bacterial overgrowth in cystic fibrosis. J Pediatr Gastroenterol Nutr 2007; 44(2):212–218. 96. Lewindon PJ, Robb TA, Moore DJ, et al. Bowel dysfunction in CF – Importance of breath testing. J Paediatr Child Health 1998; 34:79–82.

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97. Schneider AR, Kluevber S, Posselt HG, et al. Application of the glucose hydrogen breath test for the detection of bacterial overgrowth in patients with cystic fibrosis—a reliable method? Dig Dis Sci 2008; [Epub ahead of print]. 98. Wu TC, McCarthy VP, Gill VJ. Isolation rate and toxigenic potential of clostridium difficile isolates for patients with Cystic Fibrosis. J Infect Dis 1983; 148:176. 99. Theunissen C, Knoop C, Nonhoff C, et al. Clostridium difficile colitis in cystic fibrosis patients with and without lung transplantation. Transpl Infect Dis 2008; 10(4):240–244. 100. Yates B, Murphy DM, Fisher AJ, et al. Pseudomembranous colitis in four patients with cystic fibrosis following lung transplantation. Thorax 2007; 62(6):554–556. 101. Yahav J, Samra Z, Blau H, et al. Helicobacter pylori and Clostridium difficile in cystic fibrosis patients. Dig Dis Sci 2006; 51(12):2274–2279. 102. Israel NR, Khanna B, Cutler A, et al. Seroprevalence of Helicobacter pylori infection in cystic fibrosis and its cross-reactivity with anti-pseudomonas antibodies. J Pediatr Gastroenterol Nutr 2000; 30(4):426–431. 103. Roberts DM, Craft JC, Mather FJ, et al. Prevalence of giardiasis in patients with cystic fibrosis. J Pediatr 1988; 112(4):555–559. 104. Rosenstein BJ, Perman JA, Kramer SS. Peptic ulcer disease in cystic fibrosis: an unusual occurrence in black adolescents. Am J Dis Child 1986; 140(10):966–969. 105. Kamath BM, Bhargave S, Markowitz JE, et al. Grand rounds: a girl with cystic fibrosis and failure to thrive. J Pediatr 2003; 143:115–19. 106. Fluge G, Olesen HV, Gilljam M, et al. Co-morbidity of cystic fibrosis and celiac disease in Scandinavian cystic fibrosis patients. J Cystic Fibros 2009; 8(3):198–202. 107. Lloyd-Still JD. Crohn‘s disease and cystic fibrosis. Dig Dis Sci 1994; 39(4)880–885. 108. Bresso F, Askling J, Astegiano M, et al. Potential role for the common cystic fibrosis Delta F508 mutation in Crohn’s disease. Inflamm Bowel Dis 2006; 13(5):531–536. 109. Chaun H. Colonic disorders in adult cystic fibrosis. Can J Gastroenterol 2001; 15:586–590. 110. Condino AA, Hoffenberg EJ, Accurso F, et al. Frequency of ASCA seropositivity in children with cystic fibrosis. J Pediatr Gasteroenterol Nutr 2005; 41(1):23–26. 111. Wilchanski M, Kerem E, Adler S, et al. Capsule endoscopy in CF: the first look. Pediatr Pulmonol 2005; (suppl. 28):342. 112. Coughlin JP, Gauderer MW, Stern RC, et al. The spectrum of appendiceal disease in cystic fibrosis. J Pediatr Surg 1990; 25:835–839. 113. Sheilds MD, Levison H, Reisman JJ, et al. Appendicitis in cystic fibrosis. Arch Dis Child 1991; 66:307–310. 114. Lardenoye SW, Puylaert JB, Smit MJ, et al. Appendix in children with cystic fibrosis: US features. Radiology 2004; 232:187–189. 115. Friedlander JA, Mascarenhas MR, Garner LM, et al. Rectal Prolapse. Available at: http:// emedicine.medscape.com/article/931455-overview. 116. Wilchanski M, Durie PR. Patterns of gastrointestinal disease in adulthood associated with mutations in CFTR gene. Gut 2007; 56:1153–1163. 117. Holsclaw DS, Rocmans C, Shwachman H. Intussuception in patients with cystic fibrosis. Pediatrics 1971; 48:51. 118. Pomerantz B, Anupindi S, Wales PW, et al. Radiographic reduction of intussuception in patients with cystic fibrosis. Pediatr Surg Int 2007; 23(8):763–765. 119. Collins CE, Francis JL, Thomas P, et al. Gastric emptying is faster in cystic fibrosis. J Pediatr Gastroenterol Nutr 1997; 25(5):492–498. 120. Cucchiara S, Raia V, Minella R. Ultrasound measurements of gastric emptying time in patients with cystic fibrosis and effect of ranitidine on delayed gastric emptying. J Pediatr 1996; 128(4):485–488.

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121. Bodet-Milin C, Querellou S, Oudoux A, et al. Delayed gastric emptying scintigraphy in cystic fibrosis patients before and after lung transplantation. J Heart Lung Transplant 2006; 24(9):1077–1083. 122. Bentur L, Hino B, Shamir R, et al. Impaired gastric myoelectrical activity in patients with cystic fibrosis. J Cyst Fibros 2006; 5(3):187–191. 123. Rubinstein S, Moss R, Lewiston N. Constipation and meconium ileus equivalent in patients with CF. Pediatrics 1986; 78:473–479. 124. Delisle RC, Meldi L, Roach E, et al. Mast cells and gastrointestinal dysmotility in the cystic fibrosis mouse. PLos One 2009; 4(1):e4283; [doi:10.1371/journal.pone0004283]. 125. Fitzsimmons SC, Burkhart GA, Borowitz D, et al. High dose pancreatic enzyme supplements and fibrosing colonopathy in children with cystic fibrosis. N Engl J Med 1997; 336:1283–1289. 126. Taylor CJ. Fibrosing colonopathy unrelated to pancreatic enzyme supplementation. J Pediatr Gastroenterol Nutr 2002; 35:3. 127. Lloyd-Still JD, Beno DW, Kimura RM. Cystic fibrosis colonopathy. Curr Gastrointest Rep 1999; 1:231–237. 128. Oermann CM, Al-Salmi Q, Seilheimer DK, et al. Mucinous cystadenocarcinoma of the pancreas in an adolescent with cystic fibrosis. Pediatr Dev Pathol 2005; 8(3):391–396. 129. McIntosh JC, Schoumacher RA, Tiller RE. Pancreatic adenocarcinoma in a patient with cystic fibrosis. Am J Med 1988; 85(4):592. 130. Tedesco FJ, Brown R, Schuman BM. Pancreatic carcinoma in a patient with cystic fibrosis. Gastrointest Endosc 1986; 32(1):25–26. 131. Neglia JP, FitzSimmons SC, Maisonneuve P, et al. The risk of cancer among patients with cystic fibrosis. Cystic Fibrosis and Cancer Study Group. N Engl J Med 1995; 332(8):494–499. 132. Sheldon CD, Hodson ME, Carpenter LM, et al. A cohort study of cystic fibrosis and malignancy. Br J Cancer 1993; 68(5):1025–1028. 133. Tsongalis GJ, Faber G, Dalldorf FG, et al. Association of pancreatic adenocarcinoma, mild lung disease, and delta F508 mutation in a cystic fibrosis patient. Clin Chem 1994; 40(10):1972–1974. 134. Petrowsky H, Schuster H, Irani S, et al Pancreatic cancer in cystic fibrosis after bilateral lung transplantation. Pancreas 2006; 33(4):430–432. 135. Maisonneuve P, Fitzsimmons SC, Neglia JP, et al. Cancer risk in non-transplanted and transplanted cystic fibrosis patients. J Natl Cancer Inst 2003; 95(5):381–387. 136. Davis TM, Sawicka EH. Adenocarcinoma in cystic fibrosis. Thorax 1985; 40(3):199–200.

18 Liver Disease MEGHANA N. SATHE and ANDREW P. FERANCHAK University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A.

I.

Introduction

Cystic fibrosis–associated liver disease (CFALD) is a serious complication with significant morbidity and mortality for patients with cystic fibrosis (CF), as well as a vexing entity that continues to challenge the best of scientists and clinicians. Although involvement of the liver and biliary tract has been recognized as a complication of CF since Andersen’s initial description in 1938 (1), the clinical significance of hepatobiliary disease in CF is not well characterized. This is due to the fact that liver involvement in CF is often asymptomatic, though silent in nature, surfacing only once end-stage complications of liver disease. Historically, the pulmonary complications of CF were associated with early mortality and therefore often eclipsed the liver involvement. Although pulmonary disease still accounts for the greatest mortality from CF, CFALD is now considered the third leading cause of death from CF (after pulmonary and transplant complications) (2). Fundamental questions remain however regarding the pathogenesis, detection, natural history, and treatment of CFALD as highlighted in this chapter.

II.

Role of CFTR in Normal Liver Function

Bile formation. One of the major functions of the liver is the production of bile, which is essential for normal digestion of fats and fat-soluble vitamins. The formation of bile depends on complementary interactions between hepatocytes, the main parenchymal cell of the liver, and cholangiocytes, the small intrahepatic biliary epithelial cells that line the bile ducts. Together, these cells constitute the bile secretory unit (3). Bile formation is initiated by transport of bile acids from the hepatocyte canalicular membrane. Subsequently, bile flows into the lumen of intrahepatic ducts where it undergoes alkalinization and dilution as a resultant of cholangiocyte ion and fluid secretion. Although cholangiocytes constitute less than 5% of the nuclear mass of the liver, they form an extensive branching network and are thought to account for up to 40% of bile volume in humans. Increases in the cholangiocyte apical membrane Cl
permeability is felt to provide the driving force for Cl
/HCO3
exchange and water efflux into the duct lumen (3). Consequently, opening or closing of Cl
channels in the apical membrane of cholangiocytes represents an important site for modulation of transepithelial movement of water, alkalinization, and ductular bile formation.

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Utilizing the available cell and epithelial models, the best evidence suggests that the primary role of cystic fibrosis transmembrane conductance regulator (CFTR) in the liver is to contribute to the formation of bile. First, CFTR is located on the apical membrane of cholangiocytes (it is not found on hepatocytes or other cells of the liver) and functions as a cAMP-activated Cl
channel (4,5). Second, binding of the hormone secretin to receptors on the basolateral cholangiocyte membrane causes an increase in intracellular cAMP and activation of CFTR through a PKA-dependent mechanism (6,7). Third, secretin-stimulated increases in cAMP result in a parallel increase in HCO3
transport at the apical membrane of cholangiocytes (8,9). Fourth, increases in cAMP are a stimulus for regulated exocytosis including movement and insertion of aquaporin-1 channels into the apical membrane (10,11). Together these findings support a working model whereby secretin increases intracellular cholangiocyte cAMP levels and stimulates apical membrane Cl
, HCO3
, and H2O transport, thus modulating alkalinization and dilution of bile (Fig. 1). B. Alternate Secretory Pathways

Although the above working model provides a useful mechanism for secretin-mediated bile formation, it fails to explain why only a subset of patients with CF (even patients with the same CFTR mutation) develop clinically significant liver disease. If these patients all have abnormal or absent CFTR on the apical membrane of cholangiocytes, why do they not all exhibit a decrease in biliary secretion and clinical manifestations of the disease? This suggests that alternate Cl
secretory pathways may exist in cholangiocytes and contribute importantly to biliary secretion and bile formation. Indeed, several other Cl
channels (Fig. 1) are present in cholangiocytes and appear to contribute to ductular secretion independent of CFTR, including those activated by Ca2þ/ calmodulin kinase (5,12), increases in cell volume (13), and extracellular nucleotides (13–15). The overall contribution of these alternate secretory pathways to biliary secretion is, at present, unknown. It is attractive to speculate that these alternate Cl
channels may compensate for the Cl
secretory defect in the CF liver. In fact, in the cftr
/
mouse model, increased expression of alternate Cl
channels modulates organ level disease (16). Thus, Cl
channels other than those encoded by CFTR may sustain biliary secretion. Recent data suggest that secretion mediated by extracellular nucleotides (e.g., ATP) acting on purinergic (P2) receptors on the luminal membrane of biliary epithelial cells is functionally important. ATP is present in bile, and binding of ATP to P2 receptors increases Cl
efflux from isolated cholangiocytes (13–15,17) and dramatically increases transepithelial secretion in biliary epithelial monolayers (13,15). Indeed, the magnitude of the secretory response to ATP is two- to threefold greater than that observed with cAMP (15). With recent studies demonstrating that the mechanical effects of fluid-flow and/or shear stress at the apical membrane of biliary epithelial cells is a robust stimulus for ATP release (18), a model emerges in which mechanosensitive ATP release and Cl
secretion is a dominant pathway regulating biliary secretion. If these observations apply to in vivo conditions, a decrease in bile flow associated with CF may be accompanied by alterations in these mechanosensitive pathways, which may further exacerbate abnormalities in Cl
secretion and bile formation.

Figure 1 Model of cholangiocyte secretion. Stimulation of basolateral receptors by secretin results in increases in cAMP and PKA-dependent stimulation of Cl
efflux through CFTR. The transmembrane Cl
gradient drives Cl
/HCO3
exchange. Alternatively, HCO3
may enter bile through a conductive manner or through CFTR. Water is transported via aquaporin (AQP) proteins. The increase in HCO3
and water secretion leads to alkalinization and dilution of bile. On the basolateral membrane, Naþ/Hþ exchange, Naþ-dependent Cl
/HCO3
exchange, and Naþ/HCO3
symport help to maintain intracellular pH and HCO3
concentrations. Cl
uptake is mediated by a Naþ/Kþ/2Cl
cotransporter. Small (SK2) and intermediate (iK) conductance Ca2þ-activated Kþ channels have been identified in the basolateral membrane and may work in parallel with apical Cl
channels to hyperpolarize the membrane and provide the driving force for continued secretion. Alternate Cl
channels have been identified in cholangiocytes including: a G protein–regulated channel, a Ca2þ-activated channel, an osmosensitive channel, and a channel activated by extracellular nucleotides (e.g., ATP) through purinergic receptor (P2) binding. The molecular identity and overall contribution to bile formation is unknown for most of these channels. An apical transporter for bile acids has also been identified (ASBT). Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; AQP, aquaporin; ASBT, apical sodium-dependent bile acid transporter.

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Figure 2 Proposed model for pathogenesis of CF-associated liver disease. Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; GSH, glutathione.

III.

Pathogenesis

The pathophysiology underlying the development of CFALD is still only speculative, and while several hypotheses have been proposed (Fig. 2), studies directly assessing the effects of abnormal CFTR in hepatobiliary function are limited. This reality is disappointing since the genetic underpinnings responsible for CF have been defined for more than 15 years. A study of regulated transport in isolated human cholangiocytes with a compound heterozygous mutation (DF508/G542X) demonstrated abnormal cAMP-stimulated Cl
, Kþ, and HCO3
secretion, but normal Ca2þ-activated Cl
secretion (19). Studies in human CF gallbladder revealed similar abnormalities in cAMP-stimulated Cl
transport, but an intact ATP-stimulated Cl
conductance presumably through a Ca2þ-dependent pathway (20). Although these studies are in agreement with the working model, demonstrating an important role of CFTR in ductular Cl
and HCO3
secretion, they do not provide any further insight into the pathogenesis of the liver disease. A. Abnormal Secretion

Despite many uncertainties, one conventional model proposes that a loss of cAMPstimulated Cl
and HCO3
secretion resulting from an abnormal or absent CFTR protein on the cholangiocyte membrane initiates a series of events, including a decrease in fluid secretion and hence bile flow, subsequent bile duct plugging by thickened secretions, cholangiocyte and hepatocyte injury, activation of hepatic stellate cells, collagen deposition, and ultimately the development of focal biliary cirrhosis leading to multilobular cirrhosis (21) (Fig. 2). The histologic finding of eosinophilic material plugging the intrahepatic bile ducts of patients with CFALD provides some evidence for

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this “bile plugging” model, but fails to explain why only a minority of patients with CF develop clinically significant liver disease. Other factors such as abnormal mucin secretion, stellate cell activation, an altered bile acid pool, retained abnormal protein, or inflammatory mediators have all been proposed as contributors to disease pathogenesis, but a uniform model is still unproven. B. Mucins

Abnormalities in mucin secretion may alter bile viscosity in CF patients potentially contributing to the bile duct plugging or liver injury (22). As mentioned, the accumulation of eosinophilic material in bile ductules is one of the earliest histologic features found in infants and children with CF (23), though a thorough analysis of this material has not been performed. C. Stellate Cell Activation

As progressive fibrosis is a hallmark of CFALD, it is reasonable to implicate stellate cell activation as a contributor to the liver lesion in CF. Stellate cells are responsible for the production of extracellular matrix proteins that result in the formation of scar tissue or fibrosis in most, if not all, chronic fibrosing conditions of the liver (24,25). In CF, stellate cell activation appears to begin focally in the liver near the area of the portal triad, and therefore is termed focal biliary cirrhosis, ultimately progressing to bridging fibrosis, and multilobular cirrhosis. The factors that initiate or propagate stellate cell activation in CF are largely unknown; however, both TGF-b1 and monocyte chemotaxis protein-1 have been shown to recruit and activate stellate cells during cholestasis (26,27). D. Toxic Bile Acids

It has been suggested that retention of hydrophobic bile acids, such as monohydroxy and dihydroxy bile acids, may be responsible for cell membrane injury and contribute to the pathogenesis of liver injury in CF. In fact, a recent study in the DF508 mouse model demonstrated abnormal enterohepatic recirculation of bile acids with a corresponding increase in the synthesis of more hydrophobic, toxic bile acids (28). Retention of hydrophobic bile acids and the presence of increased hepatocellular lipid (steatosis) have been associated with liver cell injury (29). One human study showed that children with CF but no liver disease have higher levels of endogenous ursodeoxycholic acid (UDCA) in bile compared with children with CFALD, suggesting a possible protective role of hydrophilic bile acids (30). E.

Retained Abnormal Protein

Alternatively, the initiating step in CFALD may be direct cholangiocyte injury due to a retained abnormal CFTR protein. The most common mutation DF508 results in protein misfolding and subsequent degradation by the ubiquitin-proteosome pathway. Abnormalities in the cellular “quality control mechanisms” whereby misfolded proteins are targeted and degraded may therefore be playing a role in the pathogenesis of CFALD in the case of disease associated with class II mutations. A specific relationship between class II mutations and an increased incidence of CFALD has not been proven; however, there is significant data to support a relationship between pancreatic insufficiency and CFLAD (31–34), and pancreatic insufficiency is associated with severe mutations such as class II mutations (31–34).

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Recurrent Infection

It should be noted that unlike the progression of lung disease in CF, recurrent or chronic infections do not appear to play a significant role in the progression of liver disease. Cholangitis, or infection of the intrahepatic biliary tree, has not been consistently documented in CF, and inflammatory infiltrates are not a major histologic finding in the liver. However, a careful evaluation of intrahepatic infections as potential contributors to CFALD has not been performed.

IV.

Genetic Modifiers

Although specific gene mutations (genotype) have been associated with the severity of pancreatic involvement, there is no correlation between specific genotype and clinically detectable liver disease in patients with CF. However, there appears to be an overall lower frequency of liver disease in pancreatic sufficient patients with CF (35). Because patients with CF and identical CFTR mutations exhibit variable onset and severity of liver disease, it is postulated that there are other modifying genetic or environmental factors that determine whether clinically significant hepatobiliary involvement will occur. A. Male Gender

A preponderance of male patients has been described in all age groups of patients with CF and liver disease (2,36,37). However, it is unclear if this is a true risk factor for the development of liver disease or represents an over-representation of males given the reported survival advantage (38). A recent study suggests that decreased survival rates in women with CF may be associated with the effect of estrogen on reducing the ability of airway epithelia to respond appropriately to nucleotides and inhibiting Ca2þ signaling and airway surface liquid volume homeostasis airway epithelia by attenuating Ca2þ influx in both non-CF and CF airway epithelia (39). In the liver, estrogens may regulate expression of hepatocyte transporters (40) and scaffolding proteins (41) and thus modulate liver function. The relation of sex hormone–dependent regulation of hepatocyte transport and the development and/or progression of CFALD is unknown. B. Meconium Ileus

Several studies have suggested that a history of meconium ileus as an infant is a risk factor for the subsequent development of liver disease. It is unclear if meconium ileus itself represents an independent risk factor or if the concurring complications, such as abdominal surgery with possible bowel resection, and/or total parental nutrition, contribute to the observed increased risk. Nonetheless, a report suggested an approximate sixfold increase in the development of liver disease in the infants with a history of meconium ileus (34,42,43), though other studies have failed to find such an association (44) and the majority of patients with CF and significant liver disease do not have a history of meconium ileus. Interestingly, several loci modifying the development of meconium ileus have been identified, though specific genes, protein products, or functional consequences of these regions are unknown (45,46). C. Immune System

A possible immune contribution to the pathogenesis of hepatobiliary injury in CF is suggested by the findings of certain human leukocyte antigen (HLA)-type associations

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with the development of liver disease (47). In one multicenter study, DQ6 was found in 66% of patients with CFALD, but in only 33% of CF patients without liver disease, while DR15 and B7 were significant markers for liver disease with portal hypertension in males only (47). Additionally, other studies have shown an association between polymorphisms in genes for TGF-b (48) and angiotensin converting enzyme (ACE), a protein involved in TGF-b activation (49) and deteriorating lung function and the development of portal hypertension. The role of modifiers of the immune response in relationship to the development of liver disease requires further exploration. D. Other Liver Disorders

Another hypothesis suggests that patients with CF who develop significant liver disease are heterozygote carriers of another chromosomal mutation for a common genetic liver disease that renders the liver more susceptible to injury in the presence of abnormal CFTR function. The gene for hemochromatosis, one of the most common genetic liver diseases in the Caucasian population, has been proposed as a candidate modifier locus for CF (50,51), however no confirmatory studies have demonstrated that abnormalities in the hereditary hemochromatosis gene or protein (HFE) lead to CFALD. Wilson disease, a disorder of copper metabolism, has also been proposed as a potential contributor to the pathogenesis of CFALD. Although a case report describes a patient with CF and Wilson disease (52), no larger confirmatory cohorts exist establishing this relationship. Interestingly, the Wilson protein is expressed in human sweat glands and may modulate sweat production, which has been shown to be lower in patients with Wilson disease (53), suggesting a possible interaction between this protein and CFTR and/or other proteins involved in sweat production. a-1 Antitrypsin deficiency is another commonly inherited genetic liver disease and preliminary data suggests an increase in frequency of heterozygote carriers for the a-1-antitrypsin mutation allele among CF patients with liver disease compared with those without (54,55). Additionally, a recent study in transgenic mice with a mutation in a-1-antitrypsin demonstrated an increased susceptibility to fibrosis in a model of induced cholestasis (56). The relationship between abnormal CFTR, a-1-antitrypsin, and the development of CFALD deserves further study. Primary sclerosing cholangitis (PSC) is another cholestatic liver disease with a histologic appearance demonstrating inflammation predominantly in the portal areas. Interestingly, in two studies patients with PSC had an increased likelihood of being heterozygote carriers for CFTR mutations (57,58) that were associated with abnormalities in CFTR-mediated ion transport in one (57). However, other studies have not found any increase in the incidence of common CFTR mutations in patients with PSC (59–61), and one study has suggested that several CFTR-variants may offer protection from the development of PSC (62). In summary, while intriguing, no definitive data exists at present establishing a link between other liver diseases and the development of CFALD. However, further studies are warranted and other genes involved in hepatocyte or cholangiocyte transport (e.g., BSEP, MDR3, FIC-1, cMOAT) should be investigated.

V. Prevalence and Clinical Manifestations A. Prevalence

A confounding feature of the literature upon review is that a universally accepted definition of what constitutes CFALD does not exist. Hence, prevalence estimates are

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Table 1 The Prevalence of Hepatobiliary Manifestations of Cystic Fibrosis

Condition Hepatic Benign elevation of liver enzymes Hepatomegaly Hepatic steatosis Neonatal cholestasis Focal biliary cirrhosis Multilobular cirrhosis Biliary Microgallbladder Cholelithiasis

% Affected

References

10–46 30 20–60 2–38 10–72 7–20

37,63 23,43,64 65 66–68 69,70 37,42,64,70

20–30 1–10

67,71 37,55

difficult to interpret between studies. However, based on several prospective studies, the best current estimate for clinically significant liver disease (based on a constellation of abnormal physical exam, biochemical parameters, and/or imaging) in patients with CF is approximately 13% to 27% (23,34,63,64). In almost all studies, those patients that developed clinically significant liver disease did so by adolescence. The manifestations of CFALD can be divided into liver and biliary manifestations (Table 1). The most common liver manifestations include hepatic steatosis, focal biliary cirrhosis, multilobular cirrhosis, and very rarely hepatocellular carcinoma, while the biliary manifestations include microgallbladder, cholelithiasis, and very rarely cholangiocarcinoma. B. Steatosis

Hepatic steatosis is defined by fatty infiltration of hepatocytes (Fig. 3) (65). The pathogenesis of this condition remains unknown, although there is speculation that it may be related to malnutrition, essential fatty acid (EFA) deficiency, and/or oxidative stress (72–74). Physical exam reveals a large but soft liver in the absence of systemic signs of liver disease (e.g., portal hypertension) (65). Aminotransferase levels range from normal to only mildly elevated. Ultrasonography can aid in diagnosis; however, the sensitivity and specificity in detecting this lesion has not been defined. Previously, this was thought to be a benign, nonprogressive condition, however, with the increasing recognition of the progressive liver injury secondary to other fatty conditions of the liver (such as nonalcoholic steatohepatitis), this requires a more thorough review. C. Focal Biliary Cirrhosis and Multilobular Cirrhosis

Focal biliary cirrhosis is characterized histologically by focal areas of portal inflammation and fibrosis, bile duct obstruction and proliferation, and inclusions of eosinophilic material within the bile ductules (Fig. 3). This plugging of bile ducts by “eosinophilic material” is considered the pathognomonic lesion in CFALD. As described earlier in this chapter, the pathogenesis is unknown. Because of the silent, but progressive, nature of focal biliary cirrhosis, diagnosis requires a high index of suspicion and serial evaluations of physical exam, biochemical markers, and ultrasonographic

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Figure 3 Histopathologic manifestations of CF-associated liver disease. (A) Steatosis in CF. Histologic section demonstrating extensive numbers of hepatic parenchymal cells filled with micro- and macrovesicular fat. (B) Focal biliary cirrhosis. Histology demonstrates significant cholestasis and expansion of the portal triads with bile duct proliferation. Bile duct plugging with “eosinophilic material” is shown at the arrowhead. (C) Focal biliary cirrhosis (gross specimen). Focal areas of scarring and furrowing are shown with interspersed normal liver parenchyma. (D) Multilobular cirrhosis (gross specimen). Pathology reveals lobulated liver parenchyma secondary to extensive broad bands of fibrosis extending between portal areas with adjacent areas of normal liver parenchyma. Source: All images courtesy of Dr Arthur Weinberg, University of Texas Southwestern Medical Center, Dallas, Texas.

imaging. Focal biliary cirrhosis may progress until most of the liver is replaced with fibrotic tissue, ultimately impacting normal function and associated with the signs of portal hypertension (Fig. 3). At this point, the lesion in CF has been termed “multilobular cirrhosis,” though there is nothing that distinguishes this form of cirrhosis from cirrhosis due to other (non-CF) liver disorders. Approximately 7% to 20% of patients with CF and focal biliary cirrhosis will progress to frank cirrhosis (37,43,64,70) and in the majority of patients this develops prior to adolescence (37,43,64). Clinically, patients with cirrhosis present with signs and symptoms of portal hypertension (due to reversal of flow in the portal vein) including splenomegaly and ascites, as well as other signs of chronic liver disease such as clubbing, spider angiomas, and palmar erythema (37,43,64,70). Laboratory evaluation may reveal coagulopathy and evidence of hypersplenism with pancytopenia. It is not known if focal biliary cirrhosis is reversible or if the progression to cirrhosis can be prevented. However, the identification that fibrosis can regress with treatment of the underlying disorder in other liver diseases (73,74) provides hope that successful therapies for CFALD may similarly improve, or reverse, fibrosis.

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Biliary tract abnormalities are common in CF, although the pathogenesis is not well understood. A small or microgallbladder is seen in 20% to 30% of patients (67). Although there seems to be no clinical sequela associated with this finding, it is important to exclude biliary atresia, which is associated with an absent gallbladder in the majority of cases. While cholelithiasis is found in 1% to 10% of CF patients, clinically significant symptoms are seen in less than 4% of cases (75). The main component of CF-associated gallstones is calcium bilirubinate, which is resistant to UDCA therapy, making cholecystectomy the treatment of choice (76). The role of elective cholecystectomy in asymptomatic CF patients is controversial. Some providers advocate elective cholecytectomy in asymptomatic patients with stable pulmonary function to optimize surgical outcomes when there is no respiratory compromise. This approach may decrease the risk of emergent surgery because of potential complications of cholelithiasis (e.g., bile duct obstruction and cholangitis) at a future time when more severe pulmonary disease or respiratory compromise may be present.

VI.

Diagnosis

As mentioned earlier, there are no specific diagnostic criteria that are universally accepted to define CFALD. Therefore, at present the diagnosis is established through the combination of a careful physical examination, biochemical evaluation, radiographic imaging modalities, and occasionally histology (if available). A. Physical Examination

A complete physical examination, including evaluation of liver size and texture (soft or hard), should be conducted at every clinical visit. As hyperinflation of lungs may push the liver edge below the coastal rib margin, the entire liver span should be measured and compared to age appropriate normal values (Table 2). After the top edge is located by percussion and the bottom edge by palpation, the span should be measured at the right midclavicular line (77). Liver involvement is usually associated with hepatomegaly but can also be associated with a small and hard liver in the case of end-stage disease (cirrhosis). It is also imperative to evaluate for other signs of chronic liver disease as highlighted earlier. Table 2 Normal Liver Span at Midclavicular Line

Age

Liver span

Birth 1 year 3 years 12 years Adult

3.0–5.5 cm 4.0–6.0 cm 5.0–7.0 cm 7.0–9.0 cm >12.0 cm = hepatomegaly <6.0 cm = atrophy

Upper limit of normal is 1.5 to 2 cm higher than the mean values noted above Source: From Refs. 77–79.

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B. Biochemical Evaluation

As recommended by the CF Foundation Hepatobiliary Disease Consensus Guidelines, serum liver enzymes (aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, and g-glutamyltransferase) should be performed on a yearly basis (77). While abnormalities in these values should raise the possibility of liver involvement, it is important to note that CF patients with cirrhosis can have normal liver enzymes. Conversely, in 10% to 46% of CF patients, elevations in serum liver enzymes are not correlated with clinical significant liver disease (63,80). Because of this discrepancy between serum liver enzymes and clinically significant liver disease, the CF Foundation Hepatobiliary Disease Consensus Guidelines were proposed as a screening strategy (Fig. 4). When CFALD is suspected, consultation or referral to a pediatric gastroenterologist/hepatologist is warranted for further evaluation to exclude other causes of liver disease (drugs, infection, metabolic, autoimmune, or congenital liver disease) and to direct possible therapy. In addition, although the risk of hepatocellular carcinoma is rare, annual evaluation with a-fetoprotein levels has been suggested (81).

Figure 4 Serum liver enzyme screening protocol based on the CF Foundation Hepatobiliary

Disease Consensus Guidelines. Liver enzymes should be obtained on an annual basis assuming physical exam is normal and patient has no evidence of liver disease. Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, g-glutamyltransferase; PTT, activated partial thromboplastin time; INR, international normalized ratio. Source: From Ref. 77.

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Sathe and Feranchak C. Ultrasonography

The CF Hepatobiliary Consensus Group has recommended liver ultrasonography in all patients with CF in whom CFALD is suspected (78). Ultrasonography is the modality of choice to screen for biliary tract abnormalities including biliary obstruction or stricture, gallstones, and gallbladder abnormalities. When combined with Doppler, ultrasonography has the added benefit of identifying reversal of flow in the portal vein, indicating the presence of portal hypertension. It has been proposed that ultrasonography may be a useful noninvasive technique that can be utilized for both screening and following the progression of CFALD (82–84), even though the sensitivity and specificity to detect and discriminate the liver lesions in CFALD have not been defined in large cohorts. Additionally, there appears to be significant intra- and interobserver variability in defining the lesions (82,84). D. Liver Biopsy

The role of liver biopsy in CFALD is controversial and no clear guidelines exist. One significant issue is that focal biliary cirrhosis is “patchy,” increasing the likelihood of sampling error and thereby underestimating the liver involvement. Potential complications of liver biopsy include pneumothorax (theoretically increased in CF because of lung hyperexpansion) and bleeding, while potential benefits include the possibility of rapidly excluding other causes and defining the predominant lesion (steatosis vs. focal biliary cirrhosis). E.

Biomarkers

To date no reliable and commercially available biomarkers have been established to either diagnose or assess the progression of CFALD. However, reports have shown serum elevations in extracellular matrix proteins including collagen type VI (CL-VI), procollagen III polypeptide (PIIIP), prolyl hydroxylase (PH), and hyaluronic acid (HA) in patients with CFALD (85–88). A recent study showed increases in serum TIMP-1, PH, and CL-VI in patients with CFALD compared with CF patients without liver disease and healthy controls (85). Given the difficulty in detecting CFALD on the basis of clinical parameters, the development of biomarkers to serve as surrogate markers of disease presence and progression would be of immense importance. The use of these serum markers of liver injury, inflammation, and fibrosis require further study to determine the usefulness in screening programs.

VII.

Treatment

The management of CFALD requires a multidisciplinary approach with significant involvement from a pediatric gastroenterologist/hepatologist, experienced CF dietician, and the primary CF care team. Patients with CFALD should be counseled on the general principles of chronic liver disease management including avoiding alcohol, liver-toxic medications, and herbal therapies. All patients should receive hepatitis A and B vaccines. In addition, those with signs of portal hypertension should avoid nonsteroidal antiinflammatory medications (NSAIDs) that may increase risk of gastrointestinal bleeding. A. Nutrition

A recent population study revealed that patients with advanced CFALD were shorter, weighed less, and had reduced fat mass compared with CF patients without liver disease

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(89). Patients with CFALD may have higher resting energy expenditure, micronutrient deficiencies, and increased incidence of glucose intolerance, osteoporosis, and fat malabsorption (due to both pancreatic insufficiency and cholestasis with reduced intestinal micelle formation) (90–92). Additionally, the presence of CFALD has been associated with the development of diabetes in one case controlled study (93) with further potential impacts on nutritional status. Overall malnutrition results in increased morbidity and mortality for all patients with CF, this is especially true of patients with CFALD that require liver transplantation (94). Thus, optimal nutritional support must be at the forefront of CFALD medical management. B. Energy

In general, CF patients require anywhere from 10% to 100% greater calories than their age-matched controls (95,96). However, the exact number of calories should be adjusted to attain and maintain normal body weight (95,96). Supplemental nocturnal enteral feeds via nasogastric or gastrostomy tube should be considered if weight falls below the 85% for ideal body weight for age (97). Careful consideration should be made prior to placement of gastrostomy tubes in patients with CFALD and portal hypertension due to the risk of forming varices around the gastrostomy site with subsequent gastrointestinal hemorrhage. C. Protein

In patients with CFALD, protein intake should be in accordance with the recommended dietary allowance (RDA) for age. Short periods of protein restriction may be necessary during decompensated hepatic failure with hyperammonemia to prevent encephalopathy; however, this must be balanced by the need to maintain adequate protein stores (98). D. Fat

A greater fat intake (35–40% of total calories) along with optimizing pancreatic enzyme replacement therapy (PERT) should be recommended in patients with CFALD. The Cystic Fibrosis Foundation (CFF) consensus committee on PERT has recommended a dosage range of 1000 to 2500 U of lipase/kg/meal for ages four years and younger and 500 to 2500 U of lipase/kg/meal for ages four years and older in patients with CF and pancreatic insufficiency (97). Acid suppression therapy with proton pump inhibitors can enhance the efficacy of PERT (99). E.

Vitamins

Chronic liver disease with impaired bile flow results in altered intestinal micelle formation and therefore increases the risk of EFA deficiency (linoleic and linolenic acid) and deficiency of the fat-soluble vitamins (A, D, E, and K) already common in CF (100). Once fat-soluble vitamin deficiencies have been identified, appropriate supplementation should be provided and levels rechecked within one to two months. Assessment and correction of fat-soluble vitamins is discussed in greater detail in chapter. F.

Ursodeoxycholic Acid

UDCA, a hydrophilic bile acid normally produced in small quantities by the human liver, has been shown to increase bile flow, displace toxic bile acids, and possess immunogenic properties (101–106). Studies in human biliary cells have shown that UDCA stimulates Cl
efflux through opening of Ca2þ-activated Cl
channels (102).

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Another recent study demonstrated that UDCA stimulates ATP release from the apical membrane of biliary epithelium, thereby increasing Cl
efflux via purinergic activation (104). Prospective clinical trials of UDCA in humans demonstrated that doses of 10 to 20 mg/kg/day for 6 to 12 months led to significant improvements in liver enzymes (107– 109). One of these studies also showed alteration of biliary bile acid composition to a more hydrophilic bile acid pool (108). Another two-year uncontrolled study showed histologic improvement, decreased inflammation, and bile duct proliferation in 7 of 10 patients (108). A 10-year prospective study of 70 patients with CFALD suggested the UDCA (20 mg/kg/day) may improve the ultrasongraphic appearance of the liver (110). In addition, positive effects on nutritional status have been reported with UDCA treatment, including improvements in serum EFA and fat-soluble vitamin levels (111). Despite these theoretical benefits and positive results in animal models, UDCA has not yet been shown to alter the progression of liver disease or the development of cirrhosis in CF (112,113). In PSC, UDCA treatment has shown significant improvements in serum transaminases, pruritis, and fatigue (114,115). One recent study of patients with PSC also showed histologic improvement with the use of high dose UDCA (116). However, overall outcomes in PSC, similar to those seen in CFALD, in regard to the development of end-stage liver disease and the need for liver transplantation remains unchanged between groups of patients treated with UDCA and those without (117,118). Despite the lack of objective data in long-term clinical outcomes with the use of UDCA, results of both in vitro and in vivo studies in both CFALD and PSC have shown some therapeutic, as well as substantial theoretical, benefits as described earlier. Thus, the Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group continues to recommend the treatment of patients with CFALD with UDCA (20 mg/kg/day in two divided doses) (77). Long-term trials, which are clearly indicated, have been difficult to conduct in a prospective manner, given patient, practitioner, and ethical concerns regarding placement of patients in a placebo group. This highlights the importance of strategies to identify those patients at risk for the development of CFALD. Biomarkers that identified patients with latent disease or those with potential for more aggressive disease progression would be helpful in selecting populations that warranted early or prophylactic treatment. G. Portal Hypertension

Patients with CFALD rarely develop acute fulminant liver failure; rather the course is often complicated by the insidious onset of portal hypertension with associated splenomegaly, thrombocytopenia, gastrointestinal hemorrhage, chronic and/or acute hemoptysis, and ascites. A long-term study of patients with CFALD reported the development of portal hypertension in 25%, associated with clinically significant variceal hemorrhage (119). Management of portal hypertension is best performed by an experienced pediatric gastroenterologist/hepatologist and includes endoscopic variceal ligation, careful diuresis of ascites, and strategies to prevent gastrointestinal and/or pulmonary hemorrhage. The use of b-blockers (to reduce portal venous pressures) in the patient with CFALD may be contraindicated because of the adverse effect on pulmonary status and need for b-receptor agonist therapy. Surgical portosystemic shunts and transjugular portosystemic shunts (TIPS) have been successfully utilized to decompress the portal venous system and thereby reduce the risk of gastrointestinal bleeding and other complications associated with portal hypertension in patients with CFALD (120).

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H. Liver Transplantation

Liver transplantation should be considered in patients with life-threatening complications of portal hypertension or severe impairment with adequate pulmonary function or those who have developed hepatopulmonary syndrome as a consequence of chronic liver disease. Survival rates have been reported of 80% to 90% at 1 year and 70% to 75% at 5?years (90,94,121), which is similar to the survival rates for pediatric transplants performed for other indications. Many patients with CF have experienced improved pulmonary function post transplantation, possibly because of immunosuppressive therapy contributing to decreased airway inflammation and/or improved lung capacity due to decreased abdominal pressure (from improvement in ascites or hepatomegaly) (90,94,121). Hepatopulmonary syndrome, characterized by chronic liver disease, intrapulmonary vascular dilatation, and arterial hypoxemia, has been shown to be reversible with good outcomes with liver transplantation alone (122,123). Successful combined lung-liver and liver-small bowel transplants have been performed in CF patients; however, the overall experience is still limited (124–128).

VIII.

Future Therapies

Continued research efforts have increased our fundamental understanding of CFTR function within the hepatobiliary system and raise the possibility of new therapeutic strategies in the treatment of CFALD. A. Bile Acids

New synthetic bile acids have been proposed as potential treatment options for CFALD including two conjugates of UDCA: tauroursodeoxycholic acid (TUDCA) and 24-norursodeoxycholic acid (129–133). These bile acids preferentially increase the hydrophilic bile acid pool, increase bile acid–dependent bile formation, and increase bicarbonaterich choleresis. Additionally, they appear to have enhanced anti-inflammatory and antifibrotic properties, though long-term trials are needed. Focusing on the nuclear receptors responsible for regulating the expression of hepatic bile acid transporters is another potentially exciting area for therapeutic intervention (134), though the role in CFALD is unknown. B. Alternate Secretory Pathways

Another area of therapeutic potential includes targeting alternative secretory pathways important in bile formation and secretion. Studies of Ca2þ-activated Cl
channels in cftr
/
mice demonstrated a relationship between the level of expression of Ca2þactivated Cl
channels and protection from organ level injury (16). Studies in isolated cholangiocytes demonstrate a two to fourfold increase in the magnitude of Cl
currents mediated by Ca2þ mobilizing agonists compared with cAMP-activated currents (5,135). Recently, Kþ channels on the cholangiocyte membrane have been shown to play an important role in biliary secretion (135). These channels work in parallel with apical Cl
channels to provide a continued driving force for Cl
efflux (135), and therefore may be a target to modulate secretion in CF. Lastly, purinergic signaling has emerged as a predominant pathway mediating cholangiocyte Cl
secretion (13–15). Importantly, while CF biliary epithelium exhibits abnormal secretin/cAMP-stimulated Cl
secretion, it maintains a robust Cl
secretory response to exogenous nucleotides (136), suggesting

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that targeting the purinergic signaling pathway may represent another strategy to improve biliary secretion in CFALD. C. Preventing Fibrosis

The role of hepatic stellate cells in progressive fibrosis has emerged as another exciting area of possible therapeutic intervention. Several recent studies targeting the stellate cell have focused on the antifibrotic properties of curcumin and orthovandate and the potential of these therapeutic agents in reversing fibrosis associated with other conditions in both animal and cellular models (137–140).

IX.

Summary

Substantial and fundamental questions remain regarding the pathogenesis, natural history, identification/diagnosis, and treatment of the liver disease in CF. The development of new mouse (28,141) and pig (142) CF models (which appear to develop liver disease similar to the human disease), as well as new experimental techniques, promises to be an exciting time for basic scientists and clinicians to work toward the goal of understanding, treating, and ultimately preventing the liver disease associated with CF.

X. Acknowledgments Studies in our laboratory are supported by the Cystic Fibrosis Foundation and the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) of the National Institute of Health.

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19 Nutrition ASIM MAQBOOL and KELLY DOUGHERTY University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

ALISHA J. ROVNER Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, U.S.A.

SUZANNE H. MICHEL and VIRGINIA A. STALLINGS University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Patients with cystic fibrosis (CF) experience a broad range of pathophysiological and medical conditions, especially those who have CF with pancreatic insufficiency (PI). Most therapies focus on the unrelenting lung disease and loss of pulmonary function. In addition, the importance of nutrition in CF care has been established. Nutritional care provided by the CF team is a major factor in selecting the amount and type of food and specific nutrients to support optimal growth (weight, stature), development (lean body mass, fat stores, peak bone mass, puberty, reproduction in females), micronutrient status [fat-soluble vitamins, other micronutrients, essential fatty acids (EFAs)], and modulate immune and inflammatory status. Research, clinical experience, pancreatic enzyme replacement therapy (PERT) and vitamin, mineral and enteral nutritional supplements have lead to major improvements in the nutritional status of children and adults with CF. However, the result is far from optimal—children still do not grow to their genetic potential, suboptimal nutritional status is known to contribute to the pace of lung function loss, and adults with CF are underweight and have complications such as osteoporosis and reduced work and fitness capacity in excess of the degree of lung disease. For this chapter, we focus on selected issues in the nutritional care of people with CF; these were chosen because of advances in science and clinical care over the last decade or so. The aim is to provide the reader an update to encourage improved nutritional care and to remind us of questions that must be answered so that evidence-based care will continue to improve. Recent consensus and evidence-based recommendations from around the world are compared (Table 1) in an effort to stimulate discussions and harmonization. The role of fat-soluble vitamins and specialty CF products are presented as a review of science and a comparison of products on the market (Table 2). Other areas pose clinical questions such as in the current clinical environment, how much dietary fat do patients with CF maldigest? How well are the recommendations for “high calories and high fat” dietary intake followed? How well are children with CF and PI growing?

308

400–800

Vitamin D (IU)

PI and northern countries 400–800, based on serum value

0–1 yr: 10–50 200–400 mgc 1–10 yr: 50–100 mg >10 yr: 100–200 mg Infants: 400 400–800 Children: and adequate 400–800 sunlight Adults: 400–2000

0–12 mo: 40–50 1–3 yr: 80–150 4–8 yr: 100–200 >8 yr: 200–400

Vitamin E (IU)c

2.5–5 mg/wk

100–400

300–500

Vitamin K (mg/day)

35–40% of calories

10,000

NIa

NIa

Consensus

USA CFF 2004 (4) Adult

PI or cholestasis 1 mg/day to 10 mg/wk

NIa

NIa

NIa Base calories on pattern of weight gain and fat stores NIa

Energy

Fat

Evidence and consensus NIa

Consensus

UK CF Trust) 2002 (3) All ages

Consensus

European CFS 2002 (2) All ages

Criteria

USA CFF 2002 (1) Pediatric

Table 1 International Consensus and Evidence-Based Recommendations for CF Care

400–1000

Unrestricted; 100 g/d if >5 yr 0–36 mo: 150–500 >4 yr: 300–500 0–12 mo: 40–80 1–3 yr: 50–150 4–7 yr: 150–300 >8 yr 150–500

120–150% Recommended dietary intake for non-CF

Evidence

Australasian 2006 (5) All ages

NIa b

b

b

NIa

NIa

NIa

(Continued )

Evidence and consensus Calorie dense feedings if wt loss or inadequate wt gain

USA CFF 2009 (7) Birth to 2 yr

NIa

Evidence and consensus 110–200% Recommended intake for non-CF

USA CFF 2008 (6) All ages

Nutrition 309

Supplement if dietary intake is inadequate

NIa

Infants: 1/8 tsp (12.6 mEq) Others: high salt diet

Watersoluble vitamins

Sodium, mEqd

NIa

10,000

<1 yr: 4000 >1 yr: 4000– 10,000

NIa

USA CFF 2004 (4) Adult

UK CF Trust) 2002 (3) All ages

NIa Infants: 1–2 Infants: PRI: 23– mmol/kg/day 46 mg/kg (1–2 if urine Na < mEq/kg) daily 10 Breast-fed: supplement if fever or >2 yr: additional requirement in summer months warmer climates

PI 4000–10,000

0–12 mo 1500 1–3 yr 5000 4–8 yr 5000– 10,000 >8 yr 10,000

European CFS 2002 (2) All ages

Vitamin A (IU)

USA CFF 2002 (1) Pediatric

USA CFF 2008 (6) All ages

0–12 mo: 1500– NIa 2000 1–3 yr: 1500– 2500 >4 yr: 2500– 5000 NIa Supplement if intake is inadequate or evidence of B12 def or with terminal ileum resection Infants: 500– NIa 1000 mg Children: 4000 mg >12 yr: 6000 mg

Australasian 2006 (5) All ages

Table 1 International Consensus and Evidence-Based Recommendations for CF Care (Continued )

0–6mo: 1/8 tsp/day (12.6 mEq) > 6 mo: ¼ tsp/day (25.2 mEq) Up to 4 mEq/kg/ day

NIa

b

USA CFF 2009 (7) Birth to 2 yr

310 Maqbool et al.

Supplement if deficient

NIa

Consider for poor growth or refractory vitamin A deficiency. 6 mo zinc trial

Minimum:1997 IOM recommendations

NIa

Calcium

Pregnancy

Provides information

NIa

Provides information

NIa

As indicated

USA CFF 2004 (4) Adult NIa

USA CFF 2008 (6) All ages

Recommended NIa daily intake or 1500 mg/day with low bone density Provides NIa information

NIa

Australasian 2006 (5) All ages

NIa

Consider for inadequate growth despite PERT and sufficient calories 1 mg/kg/day, 6 month zinc supplementation trial. NIa

USA CFF 2009 (7) Birth to 2 yr

b

Not included in recommendations. Start CF-specific multivitamins shortly after diagnosis. c 1 IU of vitamin E ¼1 mg all-rac-a-tocopherol acetate ¼ 0.67 mg RRR-a tocopherol ¼ 0.74 mg RRR-a tocopherol acetate. The International Unit (IU) system is now outdated. Vitamin E intake as currently defined by the RDA is for a-tocopherol specifically. This includes RRR-a-tocopherol (the only natural form in foods and supplements) and the 2R-stereoisomeric forms of a-tocopherol (that are present in fortified foods and supplements). As per the Dietary Reference Intakes, 1 IU of all-rac-a-tocopherol or its esters = 0.45 mg 2R-a-tocopherol; 1 IU RRR-a-tocopherol or its esters = 0.67 mg 2R-a-tocopherol (8). Vitamin E in esterified form is protected from oxidization and normally has prolonged shelf life compared with unesterified forms. Esters are normally hydrolyzed in the intestine and then absorbed in the unesterified form; this process may be impaired in malabsorptive states, including CF. The DRI recommendations do not include g-tocopherol and other vitamin E compounds, secondary to a lack of evidence of health benefits in humans. d 1 mEq sodium ¼ 23 mg of sodium. Abbreviations: PI, pancreatic insufficient; PRI, Population Reference Intake; tsp, teaspoon; IOM, Institute of Medicine; DRI, Dietary Reference Intake.

a

UK CF Trust) 2002 (3) All ages

Consider with poor NIa growth or severe steatorrhea

European CFS 2002 (2) All ages

Zinc

USA CFF 2002 (1) Pediatric

Nutrition 311

SourceCF1a Drops, Chewables, Softgels

50 (1 mL) 100 (2 mL) 200/chewable

400/2 softgels

500 (1 mL) 1000 (2 mL) 1000/ chewable

2000/2 softgels

>9 years

Vitamin D (IU) 0–12 months 1–3 years 4–8 years

>9 years

32,000/2 softgels 88% BC

Vitamin E (IU)f 0–12 months 1–3 years 4–8 years

>9 years

Vitamin A (IU) retinol and b-carotene (BC) 0–12 months 4627 (1 mL) 75% BC 1–3 years 9254 (2 mL) 75% BC 4–8 years 16,000/chewable 88% BC

Age

800/2 chewables

– – 400/chewable

300/2 chewables

– – 150/chewable

18,000/2 chewables 60% BC

– – 9000/chewable 60% BC

ADEK Chewables1b

400 (1 mL) 800 (2 mL) Ages 4–10 yr: 800 / 1 softgel Ages 10 and up: 1600 / 2 softgels

50 (1 mL)g 100 (2 mL)g Ages 4–10 yr: 150/1 softgelg Ages 10 and up: 300/2 softgelsg

5751 (1 mL) 87% BC 11,502 ( 2 mL) 87% BC Ages 4–10 yr 18,167/1 softgel, 92% BC Ages 10 and up 36,334/ softgels, 92% BC

AquADEKs1c Drops, Softgels

Table 2 Comparison of CF-Specific Vitamin and Mineral Supplements in the United States

800/2 chewables

400 (1 mL) 800 (2 mL) 400/chewable

400/2 chewables

50 (1 mL) 100 (2 mL) 200/chewable

10,000/2 chewables 50% BC

3170 (1 mL) 0%BC 6,340 (2 mL) 0% BC 5000/chewable 50% BC

Vitamax1d Drops, Chewables

800/2 tablets

400 (1 mL) 800 (2 mL) 400/chewable

60/2 tablets

5 (1 mL) 10 (2 mL) 30/chewable

7000/2 tablets 29% BC

1500 (1 mL) 0% BC 3000 (2 mL) 0% BC 3500/chewable 29% BC

Poly-Vi-Sol Drops1e Centrum1 Chewable, Tablet

312 Maqbool et al.

1600/2 softgels

5 (1 mL) 10 (2 mL) 15/chewable 30/2 softgels

>9 years

Zinc (mg) 0–12 months 1–3 years 4–8 years >9 years – – 7.5/chewable 15/2 chewables

300/2 chewables

– – 150/chewable

ADEK Chewables1b

5 (1 mL) 10 (2 mL) For 4–10 yr: 10 / softgel For ages 10+: 20/2 softgels

400 (1 mL) 800 (2 mL) Ages 4–10 yr: 700/1 softgel Ages 10 and up: 1400 / 2 softgels

AquADEKs1c Drops, Softgels

7.5 (1 mL) 15 (2 mL) 7.5/chewable 15/2 chewables

400/2 chewables

300 (1 mL) 600 (2 mL) 200/chewable

Vitamax1d Drops, Chewables

0 0 15/chewable 22/2 tablets

50/2 tablets

0 0 10/chewable

Poly-Vi-Sol Drops1e Centrum1 Chewable, Tablet

The content of this table was confirmed December 2008. Products also contain a full range of water-soluble vitamins. a SourceCF1 Liquid, Chewables, and Softgels are registered trademarks of SourCF1 Inc., a subsidiary of Eurand Pharmaceuticals, Inc. b ADEK1 Chewables is a registered trademark of Axcan Pharma, Inc. c AquADEKs1 Liquid and Softgels are registered trademarks of Yasoo Health Inc. d Vitamax1 Drops and Chewables are registered trademarks of Shear/Kershman Labs. Inc. e Poly-vi-sol Drops1 is a registered trademark of Mead Johnson and Company. Centrum1 Chewables and Tablets are registered trademarks of Wyeth Consumer Care. f a-Tocopherol. g Contains mixed tocopherols. Source: Courtesy of SourceCF1 Inc., a subsidiary of Eurand Pharmaceuticals, Inc.

400 (1 mL) 800 (2 mL) 800/chewable

SourceCF1a Drops, Chewables, Softgels

Vitamin K (mg) 0–12 months 1–3 years 4 – 8 years

Age

Table 2 Comparison of CF-Specific Vitamin and Mineral Supplements in the United States (Continued )

Nutrition 313

314

Maqbool et al.

Table 3 Reported (Mean  SD) Energy and Fat Intake in Subjects with Cystic Fibrosis

Gordon et al., 2007 (10) Colombo et al., 2006 (12) Schall et al., 2006 (13) Olveira et al., 2006 (14) Powers et al., 2002 (15) Fung et al., 1999 (16) Collins et al., 1998 (17)

Sample

Age (yr)

% Energy intakea

% Intake as fat

USA

CF: 64

32  10

120–150

33  7

Italy

CF: 37 C: 68 CF: 75

CF: 8  3 C: 8  1 10  1

CF: 37 C: 37 CF: 35 C: 34 CF: 21

CF: 25  9 C: 26  6 CF: 19  8 C: 18  8 12  3

CF: 126  34 C: 101  21 CF: 115 range: 75–193 CF: 132  27 C: 108  24 CF: 103  26 C: 88  23 CF: 122  27

CF: 33  5 C: 30  4 CF: 37 range: 21–49 CF: 39  5 C: 40  5 CF: 34  6 C: 33  6 CF: 33  5

CF M: 12 CF F:17

M: 13  6 F: 11 5

CF M: 131  32 CF F: 107  16

CF M: 39  6 CF F: 37  6

USA Spain USA USA Australia

a

Energy intake as % country specific recommendations for healthy people. Abbrevaitions: CF, cystic fibrosis; C, control; M, males; F, females.

II.

Caloric and Fat Intake of People with CF

Long-term survival for people with CF is associated with numerous factors including nutritional status (25). To meet the goals of normal growth patterns for children and healthy weight for adults, nutrition recommendations for people with CF and PI include an energy intake of 20% to 50% above that for a healthy person of similar age and gender, and a fat intake of 35% to 40% of total calories (1). A review of studies prior to 1996 (9) indicated that these recommendations were seldom met. More recent studies are summarized in Table 3 and suggest higher energy but not higher fat intake in CF compared with healthy people. This summary demonstrates the variability in energy and fat intake and various international standards used to interpret the data. However, a recent report (18) suggested dietary intake reports from children, and their parents should be interpreted with caution and are often overestimated. Therefore, the accuracy of reported dietary intake of calories and fat may be unreliable and should be used to understand dietary patterns only.

III.

Nutrients Lost in Stool

Virtually all food presented to a healthy gastrointestinal tract is digested via pancreatic enzymes (lipase to digest fat, amylase for starch, and proteases for protein), absorbed by intestinal cells and enters the body for metabolic activities. Stool from healthy children and adults contain small amounts of carbohydrate and protein and < 7% kcal of ingested fat. In CF with PI pancreatic enzymes are not secreted into the duodenum and the loss of ~90% enzyme activity results in PI (19). Intestinal cells have some capacity to digest carbohydrates and proteins, but lack lipase. Lingual and gastric lipase is insufficient to support fat digestion, and CF and PI results in excess stool fat (steatorrhea). Since dietary fat provides approximately twice the energy as carbohydrate or protein per gram, fat malabsorption and steatorrhea lead to important clinical consequences including energy, fat-soluble vitamin and EFA malabsorption, and often to malnutrition and poor growth.

Nutrition

315

Table 4 Coefficient of Fat Absorption (Mean  SD) in Subjects with Cystic Fibrosis and

Pancreatic Insufficiency with and Without Pancreatic Enzyme Replacement Therapy

Existing product studies Patchell et al., 2002 (20)a Chen et al., 2005 (21) Schall et al., 2006 (13)c Cohen et al., 2005 (22)c New product studies Stern et al., 2000 (23)d Borowitz et al., 2006 (24)e

N

Age (yr)

59 53 75 75

10  4 9  1b 91 71

37 117

12–25  1–2 21–22  8–9

CFA (%) 91–95  5–8 86  1 85  13 81  14 PERT No PERT 84–87  2 51–52  6–7 67–70  18 52–56  18–20

a

Study investigating the subject preference between pancreatic enzyme formulations containing different sized spheres. b Standard error of the mean. c Same subjects at different ages: 6 to 9 years (13) and 8 to 11 years (22). d Novel PERT: minimocrospheres with enteric-coating, delayed release action. e Novel PERT: containing lipase, protease, and amylase. Abbreviations: CFA, coefficient of fat absorption; PERT, pancreatic enzyme replacement therapy.

Treatment for PI in patients with CF includes PERT, with the goal of sufficient active lipase for food in the duodenum. A 72-hour fecal fat balance study from which the coefficient of fat absorption (CFA) is derived is currently the most used objective measure of degree of PI and enzyme therapy effectiveness (1). Reported CFA range from 50% to 55% without PERT to 55% to 95% with PERT (Table 4) in people with CF and PI. Although studies have reported CFA for groups of patients in the desirable 85% range, it is evident that for many individual patients, PI is not corrected to this level.

IV.

Growth in Children with CF

The goal for children with CF is to achieve normal growth and optimal nutritional status, since better growth is associated with better health outcomes including reduced infections, improved lung function, and survival (Fig. 1) (6,11,25–27). The CF Foundation (CFF) recognizes that nutritional management is an integral part of CF care and have developed consensus reports that provide recommendations on how to monitor growth and nutritional status, strategies to prevent undernutrition, and interventions for those with nutritional failure. A pediatric nutrition care consensus report was published in 2002 (1) and was an update of the one published 10 years ago (28). More recently, the CFF Subcommittee on Growth and Nutrition conducted an evidenced-based review and performed new analyses using the CFF Patient Registry to update the recommendations for growth and weight-status monitoring for children and adults (6). For adults, broader nutrition screening and treatment recommendations are outlined in the 2004 CF Adult Care Consensus Report (4). The 2002 CFF nutrition guidelines recommend that patients be seen every three months with stature and weight assessed at every visit (1). Since these guidelines were published, the limitations of using percent ideal body weight (%IBW) in CF was

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Figure 1 (A) FEV1 percent predicted versus BMI percentile in children 6 to 20 years old. (B)

FEV1 percent predicted versus BMI percentile in adults 20 to 40 years old. Source: From Ref. 32. Reproduced with permission of the Cystic Fibrosis Foundation.

recognized (29) and %IBW should no longer be used (6). Instead the BMI (percentile or Z-score) method (30) should be used to assess stature and weight (31). In 2008, the CFF Subcommittee on Growth and Nutrition conducted new analyses from the CFF Patient Registry to examine the association of percent predicted FEV1 with weight-forstature status in individuals with CF and PI (6). BMI percentiles were used for children aged 6 to 20 years and BMI for those more than 20 years. The BMI percentile was shown to be sensitive to changes in percent predicted FEV1 and have a stronger association to percent predicted FEV1 than %IBW (29). On the basis of these, findings and the recommendations for growth monitoring in children and weight monitoring in adults were revised. The new recommendations are that children and adolescent 2 to 20 years of age maintain a BMI at or above the 50th percentile (6). For children

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diagnosed before 2 years, a weight-for-length stature of 50th percentile by age 2 years is recommended. For adults, women should maintain a BMI at or above 22 and men should maintain a BMI at or above 23. Despite newborn screening and medical advances, undernutrition is common in people with CF. The most recent CFF Patient Registry reported the median BMI percentile for patients 2 to 20 years of age was 47 and BMI for patients 21 years of age the BMI was 21 (32). When poor growth is identified in children with CF, they should be evaluated more frequently. Children less than two years should be seen every two to four weeks, and children older than two years should be seen every four to six weeks (1). These visits include medical, behavioral, and nutrition assessment, with time to support family education and needed interventions. There are various CF-related medical conditions including active pulmonary or sinus disease, gastroesophageal reflux disease, CF-related diabetes, and liver disease that should be considered for patients who fail to gain weight at the expected velocity or those who have lost weight. PERT should be evaluated since ineffective doses or poor compliance are an easily treated cause of growth in CF. For patients with persistent poor pattern of growth, referral to a gastroenterologist should be considered. The nutrition evaluation in patients with poor growth or weight loss should include evaluation of feeding and eating behaviors and assessing dietary intake patterns. The Behavioral Pediatrics Feeding Assessment Scale is a caregiver self-reported measure of mealtime problems (33). Although estimating dietary intake is challenging, a 24hour diet recall provides a qualitative assessment of dietary patterns and is easily obtained in a clinic setting. To quantitatively assess energy and nutrient intake a three- to five-prospective-day diet record is required, although accuracy may be limited (18) as previously mentioned. Interventions for nutritional failure include behavioral and educational interventions to increase caloric intake, oral supplements, and enteral feedings. The first approach should be to encourage increased food and fat intake. Behavioral strategies that are proven to be effective in CF are increasing calories one meal at a time, teaching parents alternative ways to respond to children who eat slowly or negotiate what he/she eats, and identifying appropriate rewards for improved eating behavior (34). A metaanalysis of nutrition interventions demonstrated that behavioral interventions were as effective in improving weight gain in CF as other medical approaches (35). For those children who are unable to increase calories with food alone, enternal (oral, feeding tube) supplements are used. The goal is to increase intake with oral supplements and avoid the substitution of supplements for established food intake. Limited data exits to determine the superiority of one type of enteral access over another. The nutrition goal is to optimize weight-for-height proportions and to achieve genetic height potential. CFF nutrition guidelines on growth and nutritional monitoring, strategies to prevent undernutrition, and interventions for those with nutritional failure are designed to support optimal care.

V. EFAs and Choline Individuals with CF are prone to polyunsaturated fatty acid (PUFA) abnormalities, which include EFAs, linoleic acid (LA), and a-linolenic acid (ALA), as well as the longchain PUFAs. Humans cannot synthesize EFA, which must be supplied by food.

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Through a series of enzyme desaturation and elongation transformations, EFAs are rendered into longer chain PUFAs, which in turn have structural roles and confer fluidity to cellular membranes, as well as multiple cellular functional roles including cell signaling, gene expression via peroxisome proliferator–activated receptors (PPARs), and inflammatory activity modulation via the eicosanoid pathways. EFA deficiency (EFAD) was first described in CF in 1962 (36). The typical presentation of EFAD was in infancy or childhood at the time of diagnosis, with the classic clinical manifestations of alopecia, easy bruisability, desquamating skin rash, and suboptimal growth. While these classical manifestations still occur, biochemical evidence of EFAD is common in otherwise apparently adequately nourished individuals with CF (37). The etiology of PUFA abnormalities and EFAD in CF is multifactorial and associations with genotype (38) and pancreatic status in different tissue compartments have been described (39). Data suggest unique metabolic consequences of EFAD and PUFA abnormalities in CF; whereas EFAD was associated with a blunting of the inflammatory response in otherwise healthy individuals, a paradoxical, more pronounced inflammatory effect was observed in subjects with CF (40). Compared to healthy individuals, the most consistently described PUFA abnormalities in patients with CF are LA deficiency and decreased docasahexaenoic acid (DHA) (41,42). Serum LA status has been associated with clinically important CF outcomes of growth and pulmonary status (43–45) and such associations were not observed with the triene:tetraene ratio (45). This may indicate that LA status is a better clinical indicator of EFA status than the triene:tetraene ratio. Supplementation studies have demonstrated that improvement of LA status to concentrations observed in healthy individuals (serum level 26 mol% as the most common reference value) is possible (46). LA status and the triene:tetraene ratio are biochemically related, suggesting that in addition to or in lieu of the ratio, LA status may be a more informative biomarker of EFA status in CF (45). DHA is a long-chain omega 3 PUFA associated with retinal and brain development and with decreasing the inflammatory cascade via eicosanoids. DHA status is commonly suboptimal in CF patients compared with healthy subjects (41,42). Arachidonic acid (AA, a PUFA derived from LA) is considered pro-inflammatory; the AA: DHA ratio conveys information regarding inflammatory potential/status via eicosanoidmediated pathways. DHA supplementation studies resulted in improved DHA levels and decreased the AA:DHA ratio. A number of short-term supplementation trials have been performed, and were well tolerated without adverse health effects and associated with improvement of serum and tissue DHA status (47–49). Investigators have demonstrated improvement in anti-inflammatory eicosanoid and cytokine status with DHA supplementation with respect to leukotriene B4 (50,51). Studies have not, however, demonstrated an association of DHA status with clinically significant outcomes of pulmonary and growth status. Attainment of adequate status is the goal in the nutritional management of EFA and PUFA status in CF. An alternative goal is to promote serum PUFA profiles associated with optimal CF clinical outcomes (52), and associated with risk reduction of the general population chronic noncommunicable diseases (53). Choline is an essential nutrient and has a myriad of structural and functional roles including cell membrane functions, methyl donor in folate, homocysteine-methionine metabolism, and as such intersects with DNA repair; choline is additionally relevant to

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hepatocyte and renal tubule function, a precursor to neurogenic compounds and has a role in apoptosis (54,55). Subjects with CF are prone to choline deficiency and to altered membrane phospholipid status (56,57). This may be related to increase in membrane turnover (56) and to dietary phospholipid malabsorption (21). Supplementation with choline or metabolites has increased serum choline approaching that of healthy subjects (58). A prospective supplementation trial employing a novel lipid matrix composed of lysophosphatidylcholine, monoglycerides, and fatty acids in subjects with CF showed improved weight, PUFA, and fat-soluble vitamin status in children with CF (59). Choline and thiol metabolism are associated with PUFA status and oxidative stress in CF (60), and may suggest a unifying hypothesis for the PUFA and membrane phospholipid abnormalities and increased oxidative stress observed in people with CF (61).

VI.

Vitamin D

Vitamin D is a fat-soluble vitamin that naturally occurs in very few commonly consumed foods, is added to others (fortification), and is available as a dietary supplement. Vitamin D is also produced in the skin from exposure to ultraviolet (UV) B radiation from sunlight (62). Various factors influence one’s ability to cutaneously synthesize vitamin D, including latitude, time of day, season, amount of skin exposed, age, skin pigmentation, and the use of a sunscreen (SPF > 8) (62). Supplemental vitamin D is available in two forms, ergocalciferol (D2) and cholecalciferol (D3). Ergocalciferol (D2) is manufactured by irradiation of ergosterol in yeast, and cholecalciferol (D3) by irradiation of 7-dehydrocholesterol from lanolin. Cholecalciferol (D3) is considered more clinically effective, although some uncertainty remains (63,64). Vitamin D from sun, food, and supplement undergoes CF in two hydroxylation steps for activation. The first occurs in the liver and converts vitamin D to 25-hydroxyvitamin D [25(OH)D], also known as calcidiol. The second occurs primarily in the kidney and converts 25(OH)D to 1,25-dihydroxyvitamin D [1,25(OH)2D], also known as calcitriol (65). Classic functions of vitamin D are to enhance calcium absorption and maintain blood levels of calcium and phosphorus. Rickets in children and osteomalacia in adults are the consequences of calcitrophic vitamin D deficiency, and are discussed in more detail in the chapter on bone health (chap. 20); however, vitamin D has additional important health roles. Secondary hyperparathyroidism from suboptimal vitamin D status results in the release of bone calcium to maintain serum concentrations (65), thus reducing bone mass. Health consequences related to vitamin D extend well beyond the skeletal system (66), and recent studies suggest that vitamin D is important for lung function and low concentrations may contribute to wheezing, asthma, and respiratory infections (67). In the third National Health and Nutrition Examination Survey, a doseresponse relationship was found between serum 25(OH)D and FEV1 (68). In addition, adequate concentration of 25(OH)D may reduce the risk of illness such as risk of type 1 diabetes mellitus (69), hypertension (70), and cancer (71). Serum 25(OH)D concentration is the best indicator of vitamin D status since it reflects both dietary intake (D2 and D3) and cutaneous synthesis (D3) (65). No consensus on optimal concentrations of serum 25(OH)D exists; however, a cutoff of 10 to 15 ng/ mL (25–37.5 nmol/L) is typically used to define deficiency when related only to skeletal health. In the 1997, Dietary Reference Intake (DRI), vitamin D deficiency was defined as a 25(OH)D concentration less than 11 ng/mL (27 nmol/L) (65), while others consider

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this too low. Studies in adults have demonstrated that serum parathyroid (PTH) concentrations are at ideal (suppressed) levels when 25(OH)D is more than 32 ng/mL (80 nmol/L)(72). Measuring 25(OH)D concentration is a challenge since variability exists among assays and standardization is needed (73). In a person without kidney disease, 1,25(OH)2D provides little additional insight because of its tight physiological control. Serum 1,25(OH)2D is usually normal in a vitamin D–deficient state since a physiological secondary hyperparathyroidism response increases renal production of 1,25(OH)2D. CFF guidelines recommend supplementation of at least 800 IU vitamin D per day for children older than one year of age (1,74), which is four times the intake recommended for healthy people, and guidelines remain silent on the vitamin D2 versus vitamin D3 issue. Higher supplements may be needed to achieve optimal 25(OH)D concentrations, which the CF defines as between 30 and 60 ng/mL (75–150 nmol/L). Standard of care includes checking 25(OH)D annually in the late autumn or winter when cutaneous synthesis is low. Studies demonstrated that many children with CF have suboptimal vitamin D status (75,76) and that high doses of ergocalciferol (D2) are ineffective for treating deficiency in adults (77). A recent study demonstrated the better effectiveness in CF with D3 supplementation (78). Vitamin D2 absorption was evaluated in 10 adults with CF and PI taking enzymes, and subjects absorbed less than half the D2 than the control subjects (79). Vitamin D3 increases serum 25(OH)D concentrations more efficiently than D2 in healthy people (64,80). To date, no study has examined the outcomes of vitamin D absorption in children with CF. A pilot study was conducted with five people with CF (81) and demonstrated that brief exposure to UV sunlamp light for eight weeks raised 25(OH)D concentrations. In summary, vitamin D is important both for skeletal and for extraskeletal functions. Evidence suggests that people with CF are not achieving 25(OH)D concentrations needed for optimizing bone, and lung and immune status.

VII.

Vitamin A

Vitamin A is a family of compounds important for cellular integrity, growth, immune function, and vision that is composed of preformed retinoid and pro-vitamin A carotenoids. Retinoids are fat-soluble, animal-derived compounds that include retinol, retinal, and retinoic acid. Ingested retinoids are solubilized and esterified into retinyl esters (REs) in the intestine, circulated as both bound RE and unbound retinol, and stored in the liver as retinol. While the serum retinol concentration is homeostatically maintained, hepatic stores accumulate indefinitely with increased ingestion. Conversely, pro-vitamin A carotenoids are water-soluble, plant-derived compounds that include carotene, lutein, and xanthophyll. They are less bioavailable than retinoids and excess amounts are excreted. The relative potency of the vitamin A compounds is expressed as retinol activity equivalents (RAEs); preformed retinol is 12 times as potent as b-carotene. Vitamin A deficiency is well described in CF. Preformed, water miscible vitamin A supplements have been available for about 25 years, and include preformed retinol preparations. Use of water miscible retinol supplements in the healthy population has raised concerns of potential liver toxicity as well as adverse impact to bone (82). Vitamin A status is assessed by serum retinol status, as well as serum retinol

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binding protein, and by functional testing, such as modified relative dose response and dark adaptation tests. Serum retinol is depressed with acute inflammation, such as during pulmonary exacerbations and may be due to decreased release or increased turnover (83,84). There are no prospective randomized controlled trials of vitamin A supplementation on clinically relevant outcomes in (85). Investigators have demonstrated unexpectedly elevated serum retinol in children, adolescents, and young adults with CF (86,87) under current practices of supplementation with preformed, water-miscible vitamin A. Both of these studies documented excess vitamin A intake when compared to the Recommended Daily Allowance and Tolerable Upper Intake Level (82) and in excess of CFF Nutrition Consensus Committee recommendation (1,4). Hepatic vitamin A status of subjects with CF has been considered episodically, since the liver is the main storage. A 1972 study reported significantly higher hepatic and lower serum retinol concentrations in 12 subjects with CF (8–23 years old) compared with healthy controls (88). A 1997 study found normal hepatic retinol concentrations in 15 vitamin A–supplemented Swedish subjects with CF (8–34 years old) compared with healthy controls (89). While serum retinol and retinol binding protein are informative with respect to vitamin A deficiency, they do not reflect vitamin A excess. Measurement of serum REs as a function of serum retinol (normal is < 10%) provides information for risk of toxicity (90). Prospective studies of serum and tissue concentration employing newer, noninvasive techniques (including stable isotopes) are required to determine vitamin A status across compartments and across the clinical status ranges of deficiency to adequacy to toxicity (91). Newer formulations of CF supplements contain b-carotene as well as preformed retinol, and the benefits and risks of this supplement are not well studied in CF.

VIII.

Vitamin E

The vitamin E family of fat-soluble compounds is composed of tocopherols and tocotrienols, which are necessary for normal development, cell membrane stability, prevention of hemolysis, and in antioxidant status. Vitamin E plays a critical role in myelination and CNS development and is postulated to impact cognitive function (92). This increases the importance of preventing deficiency where possible and aggressive early correction when present in infants and children. The CF population is prone to vitamin E deficiency secondary to fat malabsorption and oxidative stress, and deficiency renders the cell membranes susceptible to damage (61). PUFA status has been linked to vitamin E status as well (48). Oxygen radical–scavenging capacity of a-tocopherol is well described, and recent data points to g-tocopherol functioning as a nitrogen radical– quenching entity (93). Defining clinical vitamin E status is not well established and complicates comparisons among studies. Vitamin E is reported as serum/plasma concentration, as a ratio to total cholesterol (decreased in CF), or as a ratio to total lipids (not commonly clinically available) (94). Serum or plasma reference values are available for a-tocopherol; reference values for g-tocopherol and other potentially important vitamin E compounds have not been established. The relationship of vitamin E status to functional and clinical outcomes in CF needs to be explored.

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IX.

Zinc

Zinc plays a critical role in over 300 cellular and metabolic functions including many related to pulmonary health and general immunity (95). Symptoms of zinc deficiency are nonspecific and include lack of appetite, alterations in taste, growth failure, and disturbed immune function (96–98). Zinc homeostasis is affected by nutrient content of the diet and fat malabsorption, therefore placing patients with CF at risk (99,100). The lack of a clinically informative laboratory method and reference values to identify zinc deficiency makes diagnosing deficiency challenging. Most zinc stores are intracellular (101), and investigators (101) suggest red blood cell zinc as a better indicator of zinc status. Reference values for zinc are dependent on the population and methods used (95,102). Prior to the initiation of PERT, infants are at risk of developing zinc deficiency because of their reduced ability to adequately absorb food zinc and endogenous recirculated zinc (103,104). Cases of acrodermatitis enteropathica-like rash due to zinc deficiency in infants and children prior to diagnosis with CF continue to be reported (105,106). Estimates of the prevalence of zinc deficiency in the CF population vary widely (95,102,107). In zinc supplementation trials, patients with lower zinc concentrations prior to intervention had greater benefit from supplementation (95,98,108). Empiric zinc supplementation for six months has been recommended for children with CF who exhibit growth failure, short stature, or have vitamin A deficiency, including night blindness, refractory to vitamin A therapy (1,109).

X. Summary Adequate nutrition is a key component of the standard of care for all patients with CF. Although advances in care have resulted in less undernutriton than in the past, the median BMI in children and adults with CF remains below recommendations. As the full impact of newborn screening (in USA) is realized, and infants and their families are quickly integrated into comprehensive CF care settings, the opportunity for nutritional care to contribute to better growth, lung health, immune and cognitive status, and quality of life will be even greater. As children and adolescents survive and thrive, research is key to discovering the evolving nutritional needs of patients with CF and PI to support optimal health into middle and older age. As further metabolic, cellular, and genomic roles of nutrients are discovered, there will be important roles for studies to determine the doses, nutritional or pharmacological, of vitamins, minerals, and fatty acids to support the well being of people with CF.

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65. Institute of Medicine. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D and Fluoride. Washington, DC: National Academy Press, 1997. 66. Holick MF. The vitamin D deficiency pandemic and consequences for nonskeletal health: mechanisms of action. Mol Aspects Med 2008; 29(6):361–368. 67. Ginde AA, Mansbach JM, Camargo CA Jr. Vitamin D, respiratory infections, and asthma. Curr Allergy Asthma Rep 2009; 9(1):81–87. 68. Black PN, Scragg R. Relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey. Chest 2005; 128(6):3792–3798. 69. Hypponen E, Laara E, Reunanen A, et al. Intake of vitamin D and risk of type 1 diabetes: a birth-cohort study. Lancet 2001; 358(9292):1500–1503. 70. Krause R, Buhring M, Hopfenmuller W, et al. Ultraviolet B and blood pressure. Lancet 1998; 352(9129):709–710. 71. Lappe JM, Travers-Gustafson D, Davies KM, et al. Vitamin D and calcium supplementation reduces cancer risk: results of a randomized trial. Am J Clin Nutr 2007; 85(6):1586–1591. 72. Heaney RP. Functional indices of vitamin D status and ramifications of vitamin D deficiency. Am J Clin Nutr 2004; 80(6 suppl):1706S–1709S. 73. Hollis BW. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr 2008; 88(2):507S–510S. 74. Aris RM, Merkel PA, Bachrach LK, et al. Guide to bone health and disease in cystic fibrosis. J Clin Endocrinol Metab 2005; 90(3):1888–1896. 75. Rovner AJ, Stallings VA, Schall JI, et al. Vitamin D insufficiency in children, adolescents, and young adults with cystic fibrosis despite routine oral supplementation. Am J Clin Nutr 2007; 86(6):1694–1699. 76. Green D, Carson K, Leonard A, et al. Current treatment recommendations for correcting vitamin D deficiency in pediatric patients with cystic fibrosis are inadequate. J Pediatr 2008; 153(4):554–559. 77. Boyle MP, Noschese ML, Watts SL, et al. Failure of high-dose ergocalciferol to correct vitamin D deficiency in adults with cystic fibrosis. Am J Respir Crit Care Med 2005; 172(2):212–217. 78. Stephenson A, Brotherwood M, Robert R, et al. Cholecalciferol significantly increases 25hydroxyvitamin D concentrations in adults with cystic fibrosis. Am J Clin Nutr 2007; 85(5):1307–1311. 79. Aris RM, Lester GE, Dingman S, et al. Altered calcium homeostasis in adults with cystic fibrosis. Osteoporos Int 1999; 10(2):102–108. 80. Houghton LA, Vieth R. The case against ergocalciferol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 2006; 84(4):694–697. 81. Chandra P, Wolfenden LL, Ziegler TR, et al. Treatment of vitamin D deficiency with UV light in patients with malabsorption syndromes: a case series. Photodermatol Photoimmunol Photomed 2007; 23(5):179–185. 82. Institute of Medicine. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. Washington, DC: National Academy Press, 2001. 83. Hakim F, Kerem E, Rivlin J, et al. Vitamins A and E and pulmonary exacerbations in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2007; 45(3):347–353. 84. Duggan C, Colin AA, Agil A, et al. Vitamin A status in acute exacerbations of cystic fibrosis. Am J Clin Nutr 1996; 64(4):635–639. 85. O’Neil C, Shevill E, Chang AB. Vitamin A supplementation for cystic fibrosis. Cochrane Database Syst Rev 2008; (1):CD006751. 86. Graham-Maar RC, Schall JI, Stettler N, et al. Elevated vitamin A intake and serum retinol in preadolescent children with cystic fibrosis. Am J Clin Nutr 2006; 84(1):174–182. 87. Maqbool A, Graham-Maar RC, Schall JI, et al. Vitamin A intake and elevated serum retinol levels in children and young adults with cystic fibrosis. J Cyst Fibros 2008; 7(2):137–141.

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88. Underwood BA, Denning CR. Blood and liver concentrations of vitamins A and E in children with cystic fibrosis of the pancreas. Pediatr Res 1972; 6(1):26–31. 89. Lindblad A, Diczfalusy U, Hultcrantz R, et al. Vitamin A concentration in the liver decreases with age in patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 1997; 24(3):264–270. 90. James DR, Owen G, Campbell IA, et al. Vitamin A absorption in cystic fibrosis: risk of hypervitaminosis A. Gut 1992; 33(5):707–710. 91. Tanumihardjo SA. Assessing vitamin A status: past, present and future. J Nutr 2004; 134(1):290S–293S. 92. Koscik RL, Lai HJ, Laxova A, et al. Preventing early, prolonged vitamin E deficiency: an opportunity for better cognitive outcomes via early diagnosis through neonatal screening. J Pediatr 2005; 147(3 suppl):S51–S56. 93. Christen S, Woodall AA, Shigenaga MK, et al. Gamma-tocopherol traps mutagenic electrophiles such as NO(X) and complements alpha-tocopherol: physiological implications. Proc Natl Acad Sci U S A 1997; 94(7):3217–3222. 94. Huang SH, Schall JI, Zemel BS, et al. Vitamin E status in children with cystic fibrosis and pancreatic insufficiency. J Pediatr 2006; 148(4):556–559. 95. Abdulhamid I, Beck FW, Millard S, et al. Effect of zinc supplementation on respiratory tract infections in children with cystic fibrosis. Pediatr Pulmonol 2008; 43(3):281–287. 96. Mocchegiani E, Provinciali M, Di Stefano G, et al. Role of the low zinc bioavailability on cellular immune effectiveness in cystic fibrosis. Clin Immunol Immunophathol 1995; 75(3):214–224. 97. Cantin AM. Potential for antioxidant therapy of cystic fibrosis. Curr Opin Pulm Med 2004; 10(6):531–536. 98. Van Biervliet S, Van Biervliet JP, Vande VS, et al. Serum zinc concentrations in cystic fibrosis patients aged above 4 years: a cross-sectional evaluation. Biol Trace Elem Res 2007; 119(1):19–26. 99. Krebs NF, Westcott JE, Arnold TD, et al. Abnormalities in zinc homeostasis in young infants with cystic fibrosis. Pediatr Res 2000; 48(2):256–261. 100. Easley D, Krebs N, Jefferson M, et al. Effect of pancreatic enzymes on zinc absorption in cystic fibrosis. J Pediatr Gastroenterol Nutr 1998; 26(2):136–139. 101. Akanli L, Lowenthal DB, Gjonaj S, et al. Plasma and red blood cell zinc in cystic fibrosis. Pediatr Pulmonol 2003; 35(1):2–7. 102. Maqbool A, Schall JI, Zemel BS, et al. Plasma zinc and growth status in preadolescent children with cystic fibrosis. J Pediatr Gastroenterol Nutr 2006; 43(1):95–101. 103. Krebs NF, Sontag M, Accurso FJ, et al. Low plasma zinc concentrations in young infants with cystic fibrosis. J Pediatr 1998; 761–764. 104. Krebs NF. Overview of zinc absorption and excretion in the human gastrointestinal tract. J Nutr 2000; 130(5S suppl):1374S–1377S. 105. Bernstein ML, McCusker MM, Grant-Kels JM. Cutaneous manifestations of cystic fibrosis. Pediatr Dermatol 2008; 25(2):150–157. 106. Crone J, Huber WD, Eichler I, et al. Acrodermatitis enteropathica-like eruption as the presenting sign of cystic fibrosis—case report and review of the literature. Eur J Pediatr 2002; 161(9):475–478. 107. Van Biervliet S, Vanbillemont G, Van Biervliet JP, et al. Relation between fatty acid composition and clinical status or genotype in cystic fibrosis patients. Ann Nutr Metab 2007; 51(6):541–549. 108. Van Biervliet S, Vande VS, Van Biervliet JP, et al. The effect of zinc supplements in cystic fibrosis patients. Ann Nutr Metab 2008; 52(2):152–156. 109. Tinley CG, Withers NJ, Sheldon CD, et al. Zinc therapy for night blindness in cystic fibrosis. J Cyst Fibros 2008; 7(4):333–335.

20 Bone Health and Treatment NADINE G. HADDAD and LINDA A. DIMEGLIO Indiana University School of Medicine and Riley Hospital for Children, Indianapolis, Indiana, U.S.A.

I.

Introduction

As survival improves for persons with cystic fibrosis (CF), bone disease is increasingly recognized as a significant comorbidity. Manifestations of bone disease include low bone mineral density (BMD), bone pain, kyphosis, and fractures. The etiology is multifactorial, including overall malnutrition; deficiencies of vitamin D, vitamin K, and calcium; chronic infection and inflammation; steroid treatment; hypogonadism; low physical activity and reduced lean body mass (LBM); CF-related diabetes; and direct bone effects of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. Prophylactic and therapeutic regimens should address each comorbidity if present, and may involve therapy with bisphosphonates in selected cases. Bone is a highly specialized tissue composed of bone cells (osteoblasts and osteoclasts) and a matrix (osteoid). Osteoblasts are responsible for bone matrix formation and its mineralization whereas osteoclasts resorb mineralized bone. Osteoid is comprised of type I collagen (~94%) and noncollagenous proteins. The hardness of bone results from mineral salts in the osteoid matrix, which is a crystalline complex of calcium and phosphate (hydroxyapatite). Poor bone mineralization in CF may be the result of decreased bone formation, increased resorption, or decreased mineralization.

II.

Clinical Manifestations

A. Bone Mineral Density

BMD is defined as the amount of mineral contained in a certain area or volume of bone. Bone mineralization status is most commonly assessed using dual energy X-ray absorptiometry (DXA). DXA measures bone mineral content (BMC, grams) and bone area (centimeters) at skeletal sites. It then provides a calculated value for areal BMD (g/cm2). In adults, measurements of the spine and proximal femur are the most common sites of assessment. In children, spine and total-body less head measurements are the most accurate and reproducible sites of assessment (1). A diagnosis of osteoporosis in adults is made when a t score (a standard deviation score comparing the BMD of the individual to the measurement in young adults of the same race and gender) is 2.5 or lower (2). Osteopenia is diagnosed when t scores are from 1 to 2.5. In children, the diagnosis of osteoporosis is not made solely by densitometry, but rather requires the presence of both a clinically significant fracture history and low BMC or BMD for age (3).

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Bone densitometry is used to identify individuals at risk for fracture and to monitor and guide therapy for those with compromised bone mass. Epidemiologic studies in healthy adults have demonstrated that risk of fracture increases proportionately to a decrease in BMD t score (4). There are few pediatric data on the relationship between fracture risk and BMD, particularly in children with chronic disease. However, there is increasing evidence for a relationship between DXA measurements and fracture risk in children and adolescents (1,5). BMD measurements are affected by several parameters including height, weight, bone size, LBM, skeletal age, and pubertal stage. For instance, an apparently low BMD in a short person may be due to smaller bones rather than aberrant mineral content for body size. These factors may be more pronounced in children with chronic illness, rendering interpretation of studies in such patients more difficult. Incorporating bone age assessment has been suggested as an approach to adjust for factors related to puberty and growth since bone age corresponds to pubertal maturation. However, bone age readings are subjective, and BMDs corrected for bone age can result in overestimations of z scores in children with delayed bone ages (1). Many adults with CF have either osteopenia or osteoporosis—estimates range from 40% to 70% (6). These rates increase with more severe pulmonary disease and poorer nutritional status and are particularly high in patients with end-stage lung disease and persons undergoing lung transplantation. Osteoporosis can also worsen after lung transplantation because of steroid and calcineurin inhibitor use (7). Studies of children with CF provide mixed results regarding the prevalence of low BMD for age. Some studies have shown normal BMD (8–11). In a study of children with CF and mild pulmonary disease, total-body BMC was similar between patients and a control group of healthy children matched for age, sex, LBM, and height (8). In a cross-sectional study of 20 prepubertal young children with CF, BMC was normal when compared with age- and sex-matched healthy sibling controls. However, patients had less LBM than controls at taller heights, suggesting that as children with CF grow, they fail to accrue bone mass normally (11). Other studies report BMD reductions (12–14). In a study of 136 young patients with CF aged 3 to 24 years, spine and total-body BMD z scores were low (z score < 1) in 66%, independent of sex and age (12). In this study, impaired pulmonary function and total steroid dose (both inhaled and enteral) had the greatest negative influences on bone mass. Similarly, 52% of 25 patients with CF less than six years of age who were well nourished and had mild pulmonary disease were found to have height-adjusted lumbar spine BMDs of < 1, suggesting that a primary, intrinsic bone factor may be partly responsible for low BMD in CF (14). Some of the variability in these results reflect factors involved in DXA interpretation such as height and bone volume (12). Since adolescents with CF commonly have delayed puberty and small stature, unadjusted DXA measurements underestimate BMD. Thus, adjustments for height, body mass index (BMI), and/or bone age are generally necessary to appropriately interpret studies (15). Some of the differences in reports also reflect difference in the ages, severity of lung disease, and nutrition of studied populations. Bone mineral accrual is a continuous process that starts prior to birth and continues through childhood and adolescence. A rapid gain in bone mass occurs during adolescence with up to 25% of peak bone mass being acquired around the period of peak

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height velocity (16). Longitudinal studies suggest that bone disease results from decreased bone formation during childhood and adolescence, and increased bone turnover in adulthood (17). It has been postulated that failure to accrue bone mass during puberty may be the primary factor contributing to low BMD in persons with CF. In a study of 85 patients with CF, aged 5 to 18 years, followed for 2 years, gains in total-body and lumbar spine BMD were significantly lower than controls after adjusting for age, sex, and height z scores (18). In addition, studies in young adults with CF show progressive bone loss. A yearlong study of 55 patients aged 15 to 24 years showed an annualized bone loss rate of 2.5% in the femoral neck and 2.2% in the total hip (17). Thus, these young persons with CF not only fail to accrue bone mass, as would be expected at this young age, but also lose bone at a rate similar to postmenopausal women. Similarly, 59 people with CF aged 25 to 49 years experienced decreases of 1.9% per year at the femoral neck and 1.5% per year at the total hip (17). B. Fractures

A high fracture incidence has been reported in adults with CF (19). One study demonstrated a twofold higher fracture rate in persons aged 16 to 45 years compared with the general population (20). The most commonly reported fractures are vertebral and rib fractures, with rates 10 and 100 times higher, respectively, than in the general population. These fractures are clinically significant since they can alter chest wall structure and impair coughing and airway clearance, which in turn can cause a decline in lung function. In a longitudinal study of 49 adults with CF (mean age 25.2 years), the prevalence of at least one vertebral fracture was 16.3% at baseline and increased to 21.3% at 3-year follow-up (21). Studies of fractures in children are few and show variable results. A high prevalence of fractures and kyphosis were described in earlier studies of children and adolescents with CF (20,22). More recently, fracture risk adjusted for age and sex in a group of young patients with CF and mild-to-moderate pulmonary disease was not greater than that of controls (23). This latter study may reflect effects of improved management of lung disease and nutrition.

III.

Pathogenesis

The pathophysiology of bone disease in CF results from an interplay of factors, detailed below. A. Malnutrition

Adequate nutrient intake is important for achieving and maintaining both bone mass and LBM. A number of studies have shown a correlation between low BMD and low BMI in persons with CF (19,24,25), indicating that adequate nutritional support to maintain an adequate BMI is key for bone health, particularly during puberty when significant bone accrual occurs. B. Vitamin D Deficiency

Vitamin D is essential for calcium absorption and retention and normal bone mineralization. It can either be synthesized in the skin with ultraviolet B (UVB) light exposure or absorbed from dietary sources. The etiology of vitamin D deficiency in CF is

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postulated to result from inadequate absorption (26), decreased conversion of vitamin D to its active forms (27), and limited sun exposure. Current recommendations are for serum 25-hydroxy vitamin D (25-OHD) to be maintained above 75 nmol/L (30 ng/mL) (28). Levels less than 40 nmol/L (16 ng/mL) are considered “deficient” and 41 to 75 nmol/L (17–29 ng/mL) considered “insufficient.” However, despite wide acceptance of supplementation, vitamin D insufficiency and deficiency remain common regardless of latitude or season (28). In an observational study of 81 pediatric patients with CF, 95% had plasma 25-OHD levels below 75 nmol/L and 50% had values less than 40 nmol/L, despite these children being relatively well nourished and having only mildto-moderate lung disease (29). The majority of adults with CF also have vitamin D levels in the insufficient range (28,30). C. Vitamin K Deficiency

Vitamin K also plays a key role in maintaining bone formation through direct stimulatory effects on osteoblasts. It impacts osteocalcin activity by acting as a cofactor for posttranslational carboxylation of osteocalcin glutamic acid residues. Uncarboxylated osteocalcin has a reduced affinity for hydroxyapatite compared with carboxylated forms. Vitamin K insufficiency is common in CF. In a multicenter study of children and adolescents with CF, serum vitamin K levels were found to be low in 65% of patients, with 30% having undetectable levels (29). Similar results were found in a study of 93 children with CF, where 70% had suboptimal vitamin K levels (31). High uncarboxylated osteocalcin levels reflecting vitamin K deficiency have been reported in individuals with CF (32,33). High levels of uncarboxylated osteocalcin also predicted lower lumbar spine BMD in a study of 14, 8- to 12-year-old children with CF (32). D. Calcium Deficiency

Calcium is a critical component of bone mineral, yet it is often deficient in individuals with CF. Deficiency is the result of inadequate amounts of vitamin D or poor absorption of dietary calcium. E.

Chronic Infection and Inflammation

It is difficult to assess the role of chronic infection in isolation on bone health in people with CF, as those subjects with recurrent episodes of infection are also more likely to be more malnourished and have lower pulmonary function. Lung infection in CF increases cytokine release, particularly tumor necrosis factor (TNF)-a and interleukins (IL) 1 and 6. Cytokines affect bone metabolism by increasing bone resorption and decreasing bone formation (34,35). Several studies have shown an independent association between increased numbers of intravenous antibiotic courses and lower BMD in CF patients (19,36), reflecting the influence of chronic infection on BMD. F.

Glucocorticoids

Glucocorticoids worsen bone disease via a variety of complex and incompletely understood mechanisms. Primarily, they reduce bone formation by decreasing osteoblast number and activity and suppressing insulin-like growth factor 1 (IGF-1) secretion and action. They also decrease sex hormone production, inhibit calcium absorption, and decrease calcium renal tubular reabsorption (37). They tend to have the greatest effect

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on sites with high amounts of trabecular bone (spine and ribs) rather than on those with more cortical bone (long bones). Steroid therapy, whether oral or inhaled, has been associated with low BMD in children and adults with CF (12,17,19). However, this association is confounded by disease severity, since sicker individuals are more likely to receive more courses of steroid therapy. G. Delayed Puberty and Hypogonadism

Delayed sexual maturation and early hypogonadism have been described in young adults with CF and are related to disease severity (19). Delayed puberty can result in a delay in attainment of peak bone mass. As management of CF has improved, delayed puberty has become less prevalent and severe (38), and by the time of young adulthood, most persons with CF have normal sex hormone levels (28). H. Physical Activity, Exercise, and LBM

In healthy individuals, weight-bearing exercise is important to increase BMD and maintain bone mass (39). LBM is one of the main determinants of bone development and BMD (40). Individuals with CF are often limited in exercise capacity by respiratory function. In children with CF, LBM is low and strongly correlates with bone mineral accrual and BMD (11,14,18). I.

CF-Related Diabetes

Type 1 diabetes is accompanied by low bone mass, primarily due to a failure of bone formation rather than bone loss (41). No studies have specifically addressed the role of CF-related diabetes (CFRD) in bone accrual or BMD. However, adverse effects on bone accrual from hyperglycemia, inflammation, and amylin (another b-cell hormone) deficiency may magnify other risk factors for osteoporosis. J.

CFTR in Bone

Accumulating in vitro and in vivo evidence suggests a direct role of the CFTR protein in CF-related bone disease. Other chloride channel mutations are implicated in disorders of bone metabolism, such as mutations in the chloride channel 7, which produce osteopetrosis through adverse effects on osteoclast function. The mechanism of action of CFTR in bone homeostasis remains largely unknown. Functional studies suggest that mutations in the CFTR protein can impair normal bone remodeling by adversely affecting parathyroid hormone (PTH) stimulation of osteoblasts (42). In mice, CFTR is expressed in osteoblasts but not osteoclasts (43). CFTR-null mice, despite having minimal pulmonary disease and malabsorption, have severe osteopenia with reduced cortical width and trabecular thinning (44,45). CFTR was recently found to be expressed in human bone osteoblasts, osteocytes, and osteoclasts (46). DF508 mutations, which are associated with worse pulmonary diseases, have been shown to be an independent risk factor for low BMD in persons with CF. In a study of 88 adults with CF, those who had at least one copy of DF508 exhibited significantly lower BMD than those with other mutations (47). Similarly, in a cross-sectional study of 20 prepubertal children with CF, patients homozygous for class I or II mutations, including DF508, had lower BMC and decreased markers of bone formation compared with other combinations (11).

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Biochemical Markers

Studies of bone markers in CFTR-null mice indicate a reduction of bone formation and concomitant increase in bone resorption (44,45). Studies of bone turnover markers in persons with CF also demonstrate increased turnover with high bone resorption (27,48). Markers of bone formation such as osteocalcin and bone alkaline phosphatase have been reported to be low in children and adults with CF (48,49). The rate of calcium deposition in bone in children with CF also appears to be low, contributing to low bone accretion. In a calcium kinetics analysis, calcium deposition in bone was found to be lower among a group of girls with CF compared with healthy children consuming comparable calcium intakes (50).

V. Bone Histopathology Iliac crest bone biopsies in adults with CF demonstrate lower cancellous bone area than controls, with a trend toward a decrease in the connections between bone trabeculae (higher length of trabeculae with greater marrow volume) (51). At the tissue level, bone formation indices are low with decreased mineralizing perimeter and mineral apposition rates and narrower wall widths. Autopsy bone samples demonstrate a decrease in osteoblast number and a decrease in biosynthetic potential of osteoblasts (52). These findings are consistent with the reductions in cortical bone width and trabecular thickness seen in CFTR-null mice (44,45).

VI.

Management

Prophylactic treatment of CF-related bone disease aims to prevent bone fracture, deformity, and bone pain. Early diagnosis is important for effective management. Diagnosis begins with DXA screening. Current guidelines recommend DXA for all adults with CF and for children over eight years of age if they have risk factors for low BMD, including low body weight, low FEV1 (forced expiratory volume in one second), high glucocorticoid use, delayed puberty, or fracture (Fig. 1). Prevention should begin in childhood and includes optimizing nutrition and management of lung disease, encouraging weight-bearing exercise, and adequate sun exposure. Approaches to management of various pathologies are outlined below. A. Vitamin D Replacement

Recommendations are based on serum concentrations of 25-OHD, whereas measurement of 1,25-OHD is not valuable. Vitamin D supplementation should be provided with the goal of achieving a target serum 25-OHD of 75 to 150 nmol/L (30–60 ng/mL) (28). Generally, current recommendations for vitamin D supplementation are achieved using either ergocalciferol (vitamin D2) or cholecalciferol (vitamin D3), and include daily intake of 400 IU of ergocalciferol by infants and 800 IU by individuals older than one year. However, larger vitamin D doses may be needed to optimize serum 25-OHD levels (53). The best vitamin D preparation and dose required to achieve these levels needs further research. Studies on the effectiveness of vitamin D therapy in persons with CF are limited but promising. In one such study, 30 adults with CF who were already receiving a standard vitamin D supplement of 900 IU per day were randomized to receive an

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Figure 1 Recommendation for screening of bone disease in CF. Abbreviations: CF, cystic fib-

rosis; DXA, dual energy X-ray absorptiometry; FEV1, forced expiratory volume in one second; BMI, body mass index; BMD, bone mineral density. Source: From Ref. 28.

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additional 800 IU of cholecalciferol plus 1 g of calcium daily or placebo for 12 months. The treatment group had decreased rates of both bone loss by DXA and bone turnover, although no values reached statistical significance (54). Interestingly, serum 25-OHD levels were not significantly changed by therapy, indicating that possibly larger vitamin D doses may be required to achieve clinically significant effects. Given concerns about decreased vitamin D absorption, small trials have assessed the efficacy of supplements with the hydroxylated forms calcifediol (25-OHD) and calcitriol (1–25 OHD). In a controlled study, 73 CF children and adults with BMD z scores of < 1.5 or < 2.0, respectively, were treated with calcifediol (0.7 mg/kg/ day) and 1 g of calcium. Sixty-seven percent of the calcifediol-treated patients experienced an increase in BMD compared to 32% of controls (55). The efficacy of calcitriol was also assessed in a short-term study of 10 adults with CF who were treated with oral calcitriol (0.5 mg twice daily) for 14 days (56). Calcitriol improved markers of calcium balance by significantly increasing fractional absorption of calcium, lowering PTH levels and suppressing bone resorption. UVB radiation, which converts 7-deoxycholesterol to previtamin D3 in the skin, may also be effective adjuvant therapy for persons with CF (57).

B. Vitamin K Supplementation

The current recommendation for individuals with CF is for a vitamin K supplement of 0.3 to 0.5 mg/day. Although literature on the appropriate dose to prevent vitamin K deficiency is limited, a study of 14 children with CF and pancreatic insufficiency supplemented with 1 mg or 5 mg of vitamin K daily showed that both doses restored normal serum vitamin K levels (58). The efficacy of vitamin K supplementation for improving bone formation rates in children and adolescents with CF has been studied (49). Twenty individuals aged 6 to 17 years were treated with a weekly 10-mg oral dose of vitamin K. Bone turnover markers were examined before and after therapy and compared with controls. Although BMD z scores did not improve with therapy, treated patients experienced an increase in serum levels of bone formation markers and a decrease in serum PTH levels, indicating a potential beneficial role.

C. Bisphosphonates

Bisphosphonates are chemical analogs of pyrophosphate in which the central oxygen has been replaced by a carbon (59). Bisphosphonates reduce bone resorption primarily through inhibitory effects on osteoclastic bone resorption. Side-chain substitutions determine their antiresorptive potency, with introduction of nitrogen atoms (pamidronate, alendronate), particularly within a ring structure (zoledronic acid, risedronate), further accentuating potency. Choice of bisphosphonate for a given clinical indication depends on these potency differences, the availability and expense of a particular compound, and prior experiences/labeling for that drug in a given patient population. Studies using bisphosphonates to improve BMD in adults with CF have yielded encouraging results. Intravenous pamidronate (30 mg every three months) was the first bisphosphonate used in adults with CF, since intravenous administration avoided both the potential for decreased intestinal bioavailability due to malabsorption as well as the risk of esophagitis from gastroesophageal reflux of oral preparations. In a trial of

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30 persons with CF, six months of pamidronate therapy resulted in significant gains in lumbar spine and total hip BMD (mean difference between pamidronate-treated and untreated groups of 5.8% and 3.0% respectively, p < 0.05) (60). Unfortunately, 73% of pamidronate-treated individuals experienced significant bone pain, requiring shortening of the trial. Since patients on glucocorticoids did not experience bone pain, pretreatment with prednisone may be useful. Pamidronate was also shown to improve BMD in patients with CF after lung transplantation (61). Oral alendronate therapy has shown promising results in a few studies (62,63). In a randomized placebo-controlled study, 56 adults with CF (27 in treatment group and 29 controls) received 70 mg of oral alendronate weekly for 12 months in addition to 800 IU of vitamin D and 1 g of calcium daily. Treated patients had significant increases in lumbar spine and total hip BMDs (5.20% and 2.14%, respectively) compared with controls ( 0.08% and 0.3%). Alendronate therapy was well tolerated with no increase in the incidence of adverse events, including gastrointestinal-related events, in the treatment group (62). A retrospective study of eight children and adults with CF with lumbar spine z scores < 2 SD treated with cyclical etidronate for one year demonstrated significant increases in totalbody and lumbar spine BMD z scores without adverse effects (64). D. Growth Hormone Therapy

Growth hormone (GH) therapy has shown promising results on bone accrual in children with CF. In a multicenter, randomized, controlled crossover trial of 61 prepubertal children with CF, GH-treated patients demonstrated significant increases in height, weight, LBM, and BMC compared with the nontreated group (65). Although BMC, not BMD, was measured, improvements remained significant even with correction for height increases, suggesting that GH improves bone mineral accrual. E. Other Potential Therapies Calcium Supplementation

Although calcium intake is important for bone mineral accrual, there have been no randomized controlled studies of calcium supplements in CF to date. The current recommendation is for intake of 1300 to 1500 mg/day (28). Hormone Replacement Therapy

Therapy with testosterone and/or estrogen in adolescents with pubertal delay would be expected to improve bone mass, in addition to promoting growth and pubertal development. However, the effects of androgen and estrogen supplementation on bone mass in persons with CF with delayed puberty or early andro/menopause have not yet been studied. Therefore, such therapies cannot be recommended routinely for the primary purpose of amelioration of bone health in CF. Exercise

The efficacy of exercise interventions for bone health in CF has not been directly studied; however, they likely could play a significant role (66). Parathyroid Hormone

Recombinant intermittently administered PTH is a promising anabolic agent used for osteoporosis in adults. It is not used in children because of concerns about the development

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of osteosarcoma in the growing skeleton. Although no studies of PTH in adults with CF have been conducted to date, it may have promise in this population because of the pronounced anabolic effects of PTH on bone.

VII.

Conclusion

CF-related bone disease is multifactorial. Early screening and management of low bone density is important to prevent sequelae of osteopenia and osteoporosis. Adequate nutrition and supplementation with calcium and vitamin D and K are essential to maintain bone health. Many questions remain regarding optimal regimens for CF bone disease. More research is needed. For now, treatment of additional risk factors and use of bisphosphonates for severely affected individuals should be individualized.

References 1. Gordon CM, Bachrach LK, Carpenter TO, et al. Dual energy X-ray absorptiometry interpretation and reporting in children and adolescents: the 2007 ISCD Pediatric Official Positions. J Clin Densitom 2008; 11:43–58. 2. Genant HK, Cooper C, Poor G, et al. Interim report and recommendations of the world health organization task-force for osteoporosis. Osteoporos Int 1999; 10:259–264. 3. Baim S, Leonard MB, Bianchi ML, et al. Official positions of the international society for clinical densitometry and executive summary of the 2007 ISCD pediatric position development conference. J Clin Densitom 2008; 11:6–21. 4. Raisz LG. Clinical practice. Screening for osteoporosis. N Engl J Med 2005; 353:164–171. 5. Rauch F, Plotkin H, DiMeglio L, et al. Fracture prediction and the definition of osteoporosis in children and adolescents: the ISCD 2007 pediatric official positions. J Clin Densitom 2008; 11:22–28. 6. Doring G, Conway SP. Osteoporosis in cystic fibrosis. J Pediatr 2008; 84:1–3. 7. Hadjiliadis D. Special considerations for patients with cystic fibrosis undergoing lung transplantation. Chest 2007; 131:1224–1231. 8. Hardin DS, Arumugam R, Seilheimer DK, et al. Normal bone mineral density in cystic fibrosis. Arch Dis Child 2001; 84:363–368. 9. Humphries IR, Allen JR, Waters DL, et al. Volumetric bone mineral density in children with cystic fibrosis. Appl Radiat Isot 1998; 49:593–595. 10. Laursen EM, Molgaard C, Michaelsen KF, et al. Bone mineral status in 134 patients with cystic fibrosis [see comment]. Arch Dis Child 1999; 81:235–240. 11. Haddad N, Howenstine M, Peacock M, et al. Bone Status Evaluation in Pre-Pubertal Children with Cystic Fibrosis (abstr). Montreal, Canada: American Society for Bone and Mineral Research, September 2008. 12. Bianchi ML, Romano G, Saraifoger S, et al. BMD and body composition in children and young patients affected by cystic fibrosis. J Bone Miner Res 2006; 21:388–396. 13. Gronowitz E, Garemo M, Lindblad A, et al. Decreased bone mineral density in normalgrowing patients with cystic fibrosis. Acta Paediatr 2003; 92:688–693. 14. Sermet-Gaudelus I, Souberbielle JC, Ruiz JC, et al. Low bone mineral density in young children with cystic fibrosis. Am J Respir Crit Care Med 2007; 175:951–957. 15. Kelly A, Schall JI, Stallings VA, et al. Deficits in bone mineral content in children and adolescents with cystic fibrosis are related to height deficits. J Clin Densitom 2008; 11:581–589. 16. Sabatier JP, Guaydier-Souquieres G, Laroche D, et al. Bone mineral acquisition during adolescence and early adulthood: a study in 574 healthy females 10–24 years of age. Osteoporos Int 1996; 6:141–148.

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17. Haworth CS, Selby PL, Horrocks AW, et al. A prospective study of change in bone mineral density over one year in adults with cystic fibrosis. Thorax 2002; 57:719–723. 18. Buntain HM, Schluter PJ, Bell SC, et al. Controlled longitudinal study of bone mass accrual in children and adolescents with cystic fibrosis. Thorax 2006; 61:146–154. 19. Elkin SL, Fairney A, Burnett S, et al. Vertebral deformities and low bone mineral density in adults with cystic fibrosis: a cross-sectional study. Osteoporos Int 2001; 12: 366–372. 20. Aris RM, Renner JB, Winders AD, et al. Increased rate of fractures and severe kyphosis: sequelae of living into adulthood with cystic fibrosis. Ann Intern Med 1998; 128: 186–193. 21. Papaioannou A, Kennedy CC, Freitag A, et al. Longitudinal analysis of vertebral fracture and BMD in a Canadian cohort of adult cystic fibrosis patients. BMC Musculoskelet Disord 2008; 9:125. 22. Henderson RC, Specter BB. Kyphosis and fractures in children and young adults with cystic fibrosis. J Pediatr 1994; 125:208–212. 23. Rovner AJ, Zemel BS, Leonard MB, et al. Mild to moderate cystic fibrosis is not associated with increased fracture risk in children and adolescents. J Pediatr 2005; 147:327–331. 24. Haworth CS, Selby PL, Webb AK, et al. Low bone mineral density in adults with cystic fibrosis. Thorax 1999; 54:961–967. 25. Gibbens DT, Gilsanz V, Boechat MI, et al. Osteoporosis in cystic fibrosis. J Pediatr 1988; 113:295–300. 26. Lark RK, Lester GE, Ontjes DA, et al. Diminished and erratic absorption of ergocalciferol in adult cystic fibrosis patients. Am J Clin Nutr 2001; 73:602–606. 27. Aris RM, Ontjes DA, Buell HE, et al. Abnormal bone turnover in cystic fibrosis adults. Osteoporos Int 2002; 13:151–157. 28. Aris RM, Merkel PA, Bachrach LK, et al. Guide to bone health and disease in cystic fibrosis. J Clin Endocrinol Metab 2005; 90:1888–1896. 29. Grey V, Atkinson S, Drury D, et al. Prevalence of low bone mass and deficiencies of vitamins D and K in pediatric patients with cystic fibrosis from 3 Canadian centers. Pediatrics 2008; 122:1014–1020. 30. Donovan DS Jr., Papadopoulos A, Staron RB, et al. Bone mass and vitamin D deficiency in adults with advanced cystic fibrosis lung disease. Am J Respir Crit Care Med 1998; 157:1892–1899. 31. Conway SP, Wolfe SP, Brownlee KG, et al. Vitamin K status among children with cystic fibrosis and its relationship to bone mineral density and bone turnover. Pediatrics 2005; 115:1325–1331. 32. Fewtrell MS, Benden C, Williams JE, et al. Undercarboxylated osteocalcin and bone mass in 8–12 year old children with cystic fibrosis. J Cyst Fibros 2008; 7:307–312. 33. Aris RM, Ontjes DA, Brown SA, et al. Carboxylated osteocalcin levels in cystic fibrosis. Am J Respir Crit Care Med 2003; 168:1129. 34. Norman D, Elborn J, Cordon S, et al. Plasma tumor necrosis factor alpha in cystic fibrosis. Thorax 1991; 46:91–95. 35. Manolagas SC. The role of IL-6 type cytokines and their receptors in bone. Ann N Y Acad Sci 1998; 840:194–204. 36. Aris RM, Stephens AR, Ontjes DA, et al. Adverse alterations in bone metabolism are associated with lung infection in adults with cystic fibrosis. Am J Respir Crit Care Med 2000; 162:1674–1678. 37. Ali NJ, Capewell S, Ward MJ. Bone turnover during high dose inhaled corticosteroid treatment. Thorax 1991; 46:160–164.

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38. Buntain HM, Greer RM, Wong JC, et al. Pubertal development and its influences on bone mineral density in Australian children and adolescents with cystic fibrosis. J Paediatr Child Health 2005; 41:317–322. 39. Hind K, Burrows M. Weight-bearing exercise and bone mineral accrual in children and adolescents: a review of controlled trials. Bone 2007; 40:14–27. 40. Young D, Hopper JL, Macinnis RJ, et al. Changes in body composition as determinants of longitudinal changes in bone mineral measures in 8 to 26-year-old female twins. Osteoporos Int 2001; 12:506–515. 41. McCabe LR. Understanding the pathology and mechanisms of type I diabetic bone loss. J Cell Biochem 2007; 102:1343–1357. 42. Kingsley L, McKibbin C, Clines K, et al. CFTR Regulates Osteoblast Bone Formation Independent of Chloride Conductance (abstr). Montreal, Canada: American Society for Bone and Mineral Research, September 2008. 43. Aris RM, Guise TA, Clines G. Murine calvarial osteoblasts express CFTR mRNA in an amount similar to the pancreas. Ped Pulmonol 2006; 29:393. 44. Haston CK, Li W, Li A, et al. Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice. Am J Respir Crit Care Med 2008; 177:309–315. 45. Dif F, Marty C, Baudoin C, et al. Severe osteopenia in CFTR-null mice. Bone 2004; 35: 595–603. 46. Shead EF, Haworth CS, Condliffe AM, et al. Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human bone. Thorax 2007; 62:650–651. 47. King SJ, Topliss DJ, Kotsimbos T, et al. Reduced bone density in cystic fibrosis: deltaF508 mutation is an independent risk factor. Eur Respir J 2005; 25:54–61. 48. Greer RM, Buntain HM, Potter JM, et al. Abnormalities of the PTH-vitamin D axis and bone turnover markers in children, adolescents, and adults with cystic fibrosis: comparison with healthy controls. Osteoporos Int 2003; 14:404–411. 49. Nicolaidou P, Stavrinadis I, Loukou I, et al. The effect of vitamin K supplementation on biochemical markers of bone formation in children and adolescents with cystic fibrosis. Eur J Pediatr 2006; 165:540–545. 50. Schulze KJ, O’Brien KO, Germain-Lee EL, et al. Calcium kinetics are altered in clinically stable girls with cystic fibrosis. J Clin Endocrinol Metab 2004; 89:3385–3391. 51. Elkin SL, Vedi S, Bord S, et al. Histomorphometric analysis of bone biopsies from the iliac crest of adults with cystic fibrosis. Am J Respir Crit Care Med 2002; 166:1470–1474. 52. Haworth CS, Webb AK, Egan JJ, et al. Bone histomorphometry in adult patients with cystic fibrosis. Chest 2000; 118:434–439. 53. Boyle MP, Noschese M, Watts S, et al. Prevalence of 25-hydroxyvitamin D deficiency in adults with CF and effect of high dose ergocalciferol supplementation. Pediatr Pulmonol 2003; 25(suppl):350 (abstr). 54. Haworth CS, Jones AM, Adams JE, et al. Randomised double blind placebo controlled trial investigating the effect of calcium and vitamin D supplementation on bone mineral density and bone metabolism in adult patients with cystic fibrosis. J Cyst Fibros 2004; 3:233–236. 55. Enfissi L, Bianchi ML, Galbiati E, et al. Osteoporosis in CF: calcifediol therapy increases bone mineral density (BMD). Pediatr Pulmonol Suppl 2001; 22:334 (abstr 475). 56. Brown SA, Ontjes DA, Lester GE, et al. Short-term calcitriol administration improves calcium homeostasis in adults with cystic fibrosis. Osteoporos Int 2003; 14:442–449. 57. Gronowitz E, Larko O, Gilljam M, et al. Ultraviolet B radiation improves serum levels of vitamin D in patients with cystic fibrosis. Acta Paediatr 2005; 94:547–552. 58. Drury D, Grey VL, Ferland G, et al. Efficacy of high dose phylloquinone in correcting vitamin K deficiency in cystic fibrosis. J Cyst Fibros 2008; 7:457–459. 59. Russell RG. Bisphophonates: mode of action and pharmacology. Pediatrics 2007; 119(suppl 2): S150–S162.

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60. Haworth CS, Selby PL, Adams JE, et al. Effect of intravenous pamidronate on bone mineral density in adults with cystic fibrosis. Thorax 2001; 56:314–316. 61. Aris RM, Lester GE, Renner JB, et al. Efficacy of pamidronate for osteoporosis in patients with cystic fibrosis following lung transplantation. Am J Respir Crit Care Med 2000; 162:941–946. 62. Papaioannou A, Kennedy CC, Freitag A, et al. Alendronate once weekly for the prevention and treatment of bone loss in Canadian adult cystic fibrosis patients (CFOS trial). Chest 2008; 134:794–800. 63. Aris RM, Lester GE, Caminiti M, et al. Efficacy of alendronate in adults with cystic fibrosis with low bone density. Am J Respir Crit Care Med 2004; 169:77–82. 64. Ringuier B, Leboucher B, Leblanc M, et al. Efficacite des biphosphonates oraux sur la demineralisation osseuse des patients atteints de mucoviscidose. Arch Pediatr 2004; 11: 1445–1449. 65. Hardin DS, Adams-Huet B, Brown D, et al. Growth hormone treatment improves growth and clinical status in prepubertal children with cystic fibrosis: results of a multicenter randomized controlled trial. J Clin Endocrinol Metab 2006; 91:4925–4929. 66. Hind K, Truscott JG, Conway SP. Exercise during childhood and adolescence: a prophylaxis against cystic fibrosis-related low bone mineral density? Exercise for bone health in children with cystic fibrosis. J Cyst Fibros 2008; 7:270–276.

21 Cystic Fibrosis–Related Diabetes and Management ANDREA KELLY and ANDREW C. CALABRIA University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

With improved survival among individuals with cystic fibrosis (CF), diabetes mellitus has emerged as a significant comorbidity. While much of its pathophysiology overlaps that of both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), cystic fibrosis–related diabetes (CFRD) is considered a distinct form of diabetes. An important distinction clinically is the management approach, which is tailored to meet the unique medical needs of the individual with CF.

II.

Insulin Physiology

III.

Diabetes Classification/Clinical Features

Insulin is a peptide hormone secreted by b-cells. These glucose-responsive cells are housed with other hormone-secreting cells in pancreatic islets which are distributed throughout the exocrine pancreas. Integrated with the feeding and fasting cycle, insulin secretion is positioned to govern glucose, amino acid, and fat disposition. In an insulinreplete state, these fuels are targeted for storage (1): insulin stimulates glycogen synthesis and glucose uptake by fat and muscle while suppressing hepatic glycogenolysis and gluconeogenesis, lipolysis, and ketogenesis. In this way, insulin tightly regulates blood glucose concentrations and promotes anabolism. The ability of insulin to induce glucose uptake and suppress hepatic glucose production provides a measure of insulin sensitivity. Conversely, insulin resistance can be defined by a decrease in the ability of insulin to evoke such effects. Insulin sensitivity may be objectively quantified by hyperinsulinemic euglycemic clamp studies as well as other surrogate measures.

Diabetes arises from insulin deficiency, insulin resistance, or a combination of both. The American Diabetes Association (ADA) classifies diabetes mellitus on the basis of their etiology. T1DM arises from b-cell destruction. T2DM arises from insulin resistance coupled with compromised b-cell insulin secretion. Genetic defects of b-cell function, drug-induced diabetes, and exocrine pancreatic disorders, such as CFRD are grouped as “other” (2). A. Type 1 Diabetes Mellitus

T1DM, previously referred to as juvenile or insulin-dependent diabetes mellitus, generally presents in childhood and adolescence. It primarily arises from autoimmune-mediated

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b-cell destruction. Insulin deficiency is severe. Affected individuals depend on exogenous insulin delivery for glucose homeostasis and prevention of ketoacidosis. Thus, insulin replacement is targeted to address both carbohydrate intake and basal requirements. B. Type 2 Diabetes Mellitus

T2DM, formerly referred to as non-insulin-dependent diabetes mellitus, is characterized by insulin resistance superimposed on an insulin secretory defect. Initially, increased insulin secretion compensates for insulin resistance, and euglycemia is maintained. As b-cells are “overtaxed,” insulin secretion defects progress, and b-cells can no longer meet the increased insulin requirements established by insulin resistance. Varying degrees of hyperglycemia ensue. T2DM is treated with weight loss, caloric restriction, and oral medications or insulin, depending on the severity of b-cell compromise. Ketoacidosis occurs only occasionally. C. Cystic Fibrosis–Related Diabetes

CFRD shares features with both T1DM and T2DM. Like T1DM, CFRD is characterized by insulin deficiency arising from b-cell destruction. Unlike T1DM, CFRD is not a b-cell-specific autoimmune process. A possible link has been found, though, between CFRD and T1DM genes associated with inflammation, such as tumor necrosis factor (3). Mouse models have suggested a role for CFTR (cystic fibrosis transmembrane regulator) in the susceptibility toward islet dysfunction in CFRD (4), but twin studies suggest that the risk of CFRD may closely correlate with certain genetic modifiers that are independent of CFTR as well as genes that may be associated with T2DM, such as TCF7L2 (5). Despite the insulin deficiency and frequent need for exogenous insulin in CFRD, ketoacidosis is uncommon. Patients with CF often develop insulin resistance, as occurs with T2DM. However, this insulin resistance rarely arises because of obesity but because of intercurrent illness or systemic glucocorticoid use. As with T2DM, insulin resistance can complicate reduced b-cell mass as well as the defective insulin secretion suspected to occur in CF.

IV.

Diagnosis

Diagnostic criteria for CFRD are similar to those for other forms of diabetes (Table 1). Relatively few patients with CFRD are identified by symptoms. Rather, oral glucose tolerance tests (OGTTs), although only occasionally used as a diagnostic tool for the Table 1 2009 CFRD Diagnostic Criteria

NGT IGT CFRDa Indeterminate a

Fasting plasma glucose (mg/dL)

Two-hour OGTT glucose (mg/dL)


126

<140 140–199
200 <200

Other

HbA1c
6.5%b 2-hr postprandial glucose >200 mg/dLc 1-hr glucose
200

Repeat testing on separate day required for diagnosis. HbA1c <6.5% does not exclude diagnosis of CFRD. c In the hospitalized patient, hyperglycemia must persist for >48 hours for the diagnosis of CFRD. Abbreviations: NGT, normal glucose tolerance; IGT, impaired glucose tolerance; CFRD, cystic fibrosis– related diabetes; OGTT, oral glucose tolerance test. Source: From Ref. 6. b

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diagnosis of T1DM and T2DM, may provide the only means by which to diagnose CFRD. As such, a 2009 North American CFRD Consensus Committee recognized four glucose tolerance categories in CF (Table 1), on the basis of the fasting and two-hour blood glucose during an OGTT (1.75 g/kg glucose as advocated by the World Health Organization; maximum dose 75 g): normal glucose tolerance (NGT), impaired glucose tolerance (IGT), CFRD, and indeterminate. Few patients have completely normal glucose tolerance. The earliest changes involve variable postprandial hyperglycemia, occurring earlier than two hours, thus the indeterminate category which represents an elevated plasma glucose at one-hour but normal two-hour plasma glucose during the OGTT. Whichever screening measure is used, a repeat study should be performed on a separate day to confirm the glucose abnormality. Recognizing that the major outcomes of concern in an individual with CF are nutritional status and pulmonary function and that insulin can improve at least body mass index in individuals with CFRD w/o FH, the 2009 CFF Consensus Statement (6), unlike the 1998 Consensus, no longer distinguishes CFRD forms by presence or absence of fasting hyperglycemia. This distinction arose because it was thought the prognosis might differ between these two groups (7), and at the time of the 1998 consensus, limited data were available on the relationship between mild-to-moderate hyperglycemia and risk of complications in CFRD. Moreover, studies had questioned the appropriateness of diagnosing CFRD on the basis of conventional thresholds derived from epidemiologic studies in nonCF patients (7). In other forms of diabetes, glycemic thresholds defining diabetes were based on glucose concentrations associated with diabetes-specific complications, such as diabetic retinopathy. In T2DM, patients without FH have the same risk of developing microvascular complications as those with FH. Individuals with T2DM also likely suffer from additional comorbidities such as obesity, hypertension, and hyperlipidemia, potential contributors to diabetes complications. Because CF patients are metabolically very different from T2DM patients, they are unlikely to have these other risk factors.

V. Pathology CFRD occurs most commonly in the setting of severe CF mutations associated with exocrine pancreatic insufficiency. Abnormal chloride channel function results in thick viscous secretions that give rise to obstructive damage to the exocrine pancreas. Progressive fibrosis and fatty infiltration ensue, disrupting and destroying islet architecture. Overall, pancreatic islet tissue is reduced leading to decreased insulin secretory capacity. An inherent defect in insulin secretion, perhaps arising from b-cell stress, may also contribute to insulin deficiency. Immunohistochemical studies of islets from patients with CFRD initially revealed a significantly reduced percentage of insulin-producing cells within islets when compared with islets of non-CFRD patients and controls (8–10)—this b-cell-specific destruction characterizes T1DM. A later study by Abdul-Karim et al., however, found an overall decrease in islet number without specific reduction of b-cell content. These retrospective studies have also suggested that the reduction in b-cell mass is variable and does not correlate with the diagnosis of CFRD. Unfortunately, glucose abnormalities were not well characterized in these cohorts, and subjects defined as “normal” may have had CFRD. Immunohistochemistry suggests differences in other islet cells exist in CFRD, but their significance is unclear. While some studies suggest increased amounts of glucagonproducing a-cells are present in islets (8,10), another study found no difference in islet

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content of a-cells (9). In contrast, an increased proportion of somatostatin-producing D-cells has been reported (8–10). Given the variability of b-cell content and lack of direct correlation with the development of CFRD, researchers have speculated that CFRD is not solely related to severity of fibrosis-induced islet damage, and may be influenced by amyloid deposition. Autopsy studies have identified the presence of amyloid in 11 of 16 adult subjects with CFRD and 2 of 12 with IGT, but not in any CF patient with NGT or controls (11). Islet amyloid deposition has also been noted in T2DM, but in neither T1DM nor in the setting of pancreatitis (12). Whether amyloid accumulation is a simple by-product of the disease process, reflects stress on the endoplasmic reticulum (ER) or directly contributes to b-cell dysfunction is not clear (13). Murine studies of Perk, a protein kinase that resides in ER, may provide insight into the role of amyloid in CFRD. Perk is highly expressed in pancreas and is an essential component of ER-associated protein degradation, a process implicated in the mechanism of numerous genetic disorders, including CF (14). Despite normal pancreatic development, Perk / mice develop pancreatic exocrine insufficiency and diabetes. ER stress associated with increased b-cell apoptosis and progressive glucose intolerance has been observed (14). Amyloid may serve as a marker of this ER stress; a direct role of amyloid in ER stress has also been suggested (15,16).

VI.

Pancreatic/Pancreatic-like Hormone Secretion in CF

A. b -Cell Function

Significant alterations in insulin secretion patterns have been appreciated in CF. Delayed and blunted insulin secretion are found during OGTT even in CF patients without CFRD (17). Similarly, delayed and reduced C-peptide responses have been observed during OGTT, even in CF patients with NGT (18,19). The abnormalities are more pronounced with worsening glycemic status (17,19,20). Likewise, intravenous studies have revealed impaired first-phase insulin and C-peptide secretion in response to glucose and other stimulatory agents (19). Of note, levels of the insulin precursor, proinsulin, are elevated in CF patients with abnormal glucose tolerance and may signal progression of b-cell dysfunction (21,22). Basal insulin secretion is generally preserved, except in CFRD with FH. B. Non-b b-Cell Islet Function

As with basal insulin secretion, basal glucagon and somatostatin levels are usually normal in CF patients (17,19). With worsening glucose tolerance, diminished glucagon secretion has been observed in response to OGTT and various other stimuli (17,19), suggesting islet cell destruction is not cell selective and is linked to exocrine pancreas fibrosis. In contrast, stimulated levels of somatostatin may be elevated, consistent with immunohistochemical studies showing increased islet D-cell content. Somatostatin exerts a paracrine inhibitory effect on both insulin and glucagon secretion, but its role in CFRD is not understood (23). C. Incretins

Incretin hormones, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are secreted by neuroendocrine cells in the gastrointestinal tract. These hormones enhance the insulin response to enteral glucose; this “incretin” effect may account for

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50% to 70% of the total insulin response to an oral glucose load. GLP-1 and GIP account for approximately 90% of this incretin response (24). GLP-1 may also enhance b-cell mass, delay gastric emptying, and inhibit glucagon secretion (25,26). T2DM patients have reduced or absent incretin responses (24), and incretin mimetic agents, such as GLP-1 receptor agonists, have generated recent interest as a therapeutic modality. To date, the role of incretins in CF has received limited attention. Normal basal levels of GLP-1 and GIP were detected at all classes of glucose tolerance in CF (17), but hypersecretion of GIP in response to OGTT has been found in adults without CFRD (27). Further studies are needed to clarify any potential role for incretins in CFRD. D. Insulin Sensitivity Vs. Insulin Resistance

While insulin secretion is generally impaired in CF, the role of insulin resistance is unclear. Insulin resistance may represent both decreased peripheral glucose uptake and poor insulin suppression of hepatic glucose production (28,29). Studies using hyperinsulinemic euglycemic clamps, considered the gold standard for assessing insulin sensitivity, have yielded conflicting results. Lanng et al. described normal peripheral sensitivity in adult CF patients with NGT and CFRD (30). Moran et al. found that pancreatic insufficient subjects displayed increased peripheral but reduced hepatic insulin sensitivity (31). Similarly, studies by Hardin et al., using indirect calorimetry, suggest that CF patients have high rates of hepatic glucose production, consistent with hepatic insulin resistance (32). In contrast, decreased peripheral insulin sensitivity has been observed with IGT versus NGT; however, both study groups had decreased insulin-stimulated glucose disposal—suggestive of insulin resistance (29). Lack of consensus likely reflects differences in methodology, glucose categorization, and study populations. While insulin sensitivity may be normal at baseline, with worsening glucose tolerance, patients may initially compensate with enhanced peripheral insulin sensitivity, as illustrated above by Moran et al. (31). Insulin resistance may then be precipitated by situations such as acute pulmonary exacerbation, chronic severe lung disease, and glucocorticoid therapy (33).

VII.

Screening

Because CFRD onset is usually insidious, early screening represents an opportunity to identify asymptomatic patients. Potential symptoms of CFRD include those classically associated with diabetes, such as polyuria and polydipsia, but may also include failure to gain or maintain weight despite nutritional interventions, poor growth velocity, and unexpected pulmonary function decline (7). Because many symptoms are nonspecific, diabetes should be considered in the evaluation of patients with such issues. Whatever method is used in screening for the presence of CFRD, repeat testing on a separate day is required for the diagnosis of CFRD. The CFF recommends fasting glucose and OGTTs be measured annually starting at age 10 years. European centers generally start screening by age 10 years, and some large U.S. centers may screen earlier, starting between six and eight years, on the basis of the prevalence of CFRD in their patient populations. Fasting and postprandial glucose should be measured during hospitalization for acute illness. Hyperglycemia may be intermittent, occurring with physical stressors, infection, intensive nutritional intervention such as high calorie overnight G-tube feeds, or glucocorticoid treatment (33).

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Table 2 Screening Criteria for Cystic Fibrosis-Related Diabetes

Outpatient OGTT: test of choice for routine screening Annually, starting at age 10, for all pancreatic insufficient patients not known to have CFRD All patients with symptoms and normal FPG FPG
126—diagnostic if accompanied by second reading
126 or 2-hr OGTT >200a Hemoglobin A1c
6.5%b Preliminary screening with home glucose monitoring acceptable in high risk situationscc: Continuous gastrostomy feedings Home intravenous antibiotics High-dose glucocorticoids Inpatient All pancreatic insufficient patients to have FPG and 2-hr values measured on multiple days FPG < 126 and 2-hr values < 200: no further action required, routine screening when well If FPG
126 and/or 2-hr values > 200—diagnostic if persists for > 48 hr a

Repeat testing on multiple days required for diagnosis. Hemoglobin A1c < 6.5% does not exclude diagnosis. c Hyperglycemia must be confirmed by laboratory glucose levels. Abbreviations: OGTT, oral glucose tolerance test; CFRD, cystic fibrosis–related diabetes; FPG, fasting plasma glucose. b

Resolution may occur between episodes, but the individual remains at risk for recurrent hyperglycemia. As such, once the diagnosis of CFRD is made this diagnosis persists even if the hyperglycemia resolves. For acutely hospitalized patients, CFRD is diagnosed when hyperglycemia persists for more than 48 hours. Screening strategies for inpatient and outpatient care are outlined in Table 2. A. Hemoglobin A1c

Hemoglobin A1c (HbA1c) has generally not been considered an appropriate screening test for CFRD. The relationship between HbA1c and mean plasma glucose levels is not established as in other forms of diabetes (34). The majority of studies find HbA1c underestimates overall glycemic control in nondiabetic CF patients compared with the non-CF population. In fact, HbA1c has poor sensitivity, being normal in about 70% of CFRD cases diagnosed by OGTT (28). Still, Yung et al. found that only 16% of patients with CFRD diagnosed by OGTT had normal HbA1c values (35). Some authors have speculated that HbA1c may be unreliable because of altered red blood cell turnover in CF patients, but this has never been proven. Nevertheless, the 2009 CFF Consensus adopted as part of its CFRD diagnostic criteria the ADA diagnostic criteria of HbA1C
6.5%. Because of its poor sensitivity in individuals with CF, however, a normal HbA1C cannot exclude the diagnosis of CFRD. Alternative measures of glycemic control, such as fructosamine and GlycoMark, have not been formally tested. B. Oral Glucose Tolerance Tests

The majority of patients with CFRD does not have FH and can only be detected by OGTT. A comprehensive evaluation of 285 patients with CFRD over a 15-year period showed that most patients with CFRD w/o FH progressed to CFRD with FH over time (36). Comparison of OGTT blood glucose in CF subjects and controls reveals similar fasting

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and two-hour values, but marked differences at intermediate values. In CF subjects, 30-, 60-, and 90-minutes values are elevated and may provide a more sensitive method for detecting glucose intolerance than two-hour values (37). Currently, the CFF does not diagnose CFRD based upon these intermediate time points. These elevations fall under the category of indeterminate. C. Casual Blood Glucose

Casual blood glucose testing alone is generally not sufficient to make a diagnosis of diabetes, although hyperglycemia is generally diagnostic of diabetes. Many CF patients will have elevated casual blood sugars in the diabetic range, even with normal OGTTdefined glucose tolerance (38). While alarming to clinicians, this finding should prompt further investigations before a diagnosis of CFRD is made. However, in certain high risk situations, such as continuous gastrostomy feedings, home intravenous antibiotics, and high dose glucocorticoids, preliminary screening with home glucose monitoring can be used if the patient is not hospitalized with later laboratory confirmation of hyperglycemia. D. Continuous Glucose Monitoring

Evidence suggests that the OGTT criteria in CF patients may be missing episodes of hyperglycemia. Some authors report glucose values exceeding 200 mg/dL in CF patients with declining weight and lung function despite normal OGTTs (39). The continuous glucose monitoring system (CGMS) has been validated in non-CF patients and provides a clinically relevant means by which to investigate glucose trends that may previously have been undetected. Its extension to CF patients has identified mean glucose levels higher than age-matched controls (40). In a study of 49 CF patients, CGMS revealed postprandial peaks above 200 mg/dL in 36% of subjects with NGT, in 52% of IGT subjects, and in all patients with CFRD (41). Since CGMS may reveal elevated glucose values that were not previously documented with OGTT (Fig. 1), it may offer a potential tool for the earlier detection of hyperglycemia in CF. Because CGMS does not actually

Figure 1 CGMS in a seven-year-old with CF and normal OGTT (fasting plasma glucose ¼ 83 mg/dL, one hour glucose ¼ 134 mg/dL, and two hour glucose ¼ 83 mg/dL). Despite the normal OGTT, CGMS identified elevated glucose (>200 mg/dL) on two consecutive days. Abbreviations: CF, cystic fibrosis; OGTT, oral glucose tolerance test; CGMS, continuous glucose monitoring system.

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measure plasma glucose and the agreement between interstitial glucose and plasma glucose is not perfect, the CFF does not recommend the use of CGMS for diagnostic purposes. For the patient with established CFRD, however, it may provide adjunct information for identifying blood glucose trends and tailoring diabetes management.

VIII.

Prevalence/Incidence

The prevalence of CFRD may be underestimated because of lack of universal screening at many CF centers. Still, as the median survival has doubled over the past 25 to 37 years as of 2007, CFRD has emerged as the most common comorbidity in CF (42). The 2006 Cystic Fibrosis Foundation Patient Registry Annual Report, which reflects data from 24,487 patients at U.S. accredited CF care centers, reported that 19.5% of CF patients in the United States
14 years of age had CFRD/glucose intolerance (42). In Denmark, where OGTTs are performed annually, 50% of CF patients aged 30 years or older are reported to have CFRD (3). Risk factors include female gender, pancreatic insufficiency, and the DF508 homozygous genotype (43–45). While CFRD is generally uncommon before the age of 10 years, the incidence and prevalence increase markedly with age. Data from the University of Minnesota (Fig. 2) where annual OGTT screening is recommended for all patients
6 years, reveal CFRD prevalences of 9% of 5- to 9-year-olds, 26% of 10- to 20-year-olds, and 43% in individuals 30 years of age and older (7). In a comprehensive 15-year-study of 775 patients
6 years, CFRD was diagnosed by OGTT in 285 patients, 64% of whom had FH (36). Sixty percent of patients with CFRD w/o FH progressed to CFRD with FH within a 10year period (36). Lanng et al. reported that the annual incidence of CFRD increased with age at 5.0% per year in patients >10 years and 9.3% per year in patients >20 years (28).

Figure 2 Glucose tolerance categories in CF, expressed as percent prevalence within age groups

(n ¼ total number of patients studied within that age group). Patients with CFRD with FH include those who chronically required insulin to prevent FH and those who intermittently required insulin during periods of stress. Abbreviations: CF, cystic fibrosis; CFRD, cystic fibrosis–related diabetes; FH, fasting hyperglycemia. Source: From Refs. 5 and 33.

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Figure 3 Survival curves for male subjects with CF but without diabetes (A, median survival

49.5 years), male subjects with CF and diabetes (B, median 47.4 years), female subjects with CF but without diabetes (C, median 47.0 years), and female subjects with CF and diabetes (D, median survival 30.7 years). Abbreviation: CF, cystic fibrosis. Source: From Ref. 41.

The number of patients diagnosed with CFRD is rapidly increasing, possibly reflecting better surveillance and improved survival. Prior studies have suggested greater implications of CFRD for females compared to males, but this has become less apparent over time. A U.K. study, beginning 12 months after diagnosis, showed that female CFRD subjects had a 20% lower FEV1% compared with CF females with NGT (46). Likewise, a U.S. study found median survival of 49.5 years for males without CFRD, 47.4 years for males with CFRD, 47 years for females without CFRD, and 30.7 years for females with CFRD (43) (Fig. 3). However, after 15 years of follow up, this apparent gender difference has disappeared, possibly due to earlier surveillance and more intensive regimens (47). Other studies have similarly not identified a gender disparity (48). Still, the potential for a gender difference in some populations may represent an opportunity for more intensive interventions in high-risk populations.

IX.

Complications

A. Morbidity/Mortality

CFRD has been associated with increased morbidity and up to a sixfold greater mortality rate (49). In the 1980s, Finkelstein et al. found that <25% of CFRD patients survived to age 30 years, compared with 60% of the nondiabetic group (50). CFRD has also been associated with worse clinical status, as indicated by poorer pulmonary function, increased frequency of acute pulmonary exacerbations, increased prevalence of important sputum pathogens, poorer nutritional status, and a higher prevalence of liver disease (44). The presence of CFRD has also been linked to worsening nutritional status and, even more recently than the 1980s, to decreased survival (44,51–54). B. Body Mass Index

Gradual decreases in body mass index (BMI) and in lung function have been found four years prior to the diagnosis of CFRD (52). Slightly lower BMI has been observed in patients older than 15 years with the greatest difference in patients aged 15 to 19 years

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(53). Mean weight for age was also consistently lower in CFRD patients and reached its nadir in the 15- to 19-year-old group (53). Highlighting the potential impact of this CFRD-nutrition effect, studies in the general CF population are demonstrating increased survival at five years in patients with body weight >85% versus those with body weight <85% (55). C. Pulmonary Function

The presence of CFRD is linked to poor pulmonary function regardless of age. An insidious decline in clinical status can occur two to six years before the diagnosis of CFRD (50–52). Using data from the European Epidemiologic Registry of CF, researchers have also shown that mean FEV1 is markedly lower in CFRD patients relative to nondiabetic CF patients (53). The rate of pulmonary deterioration over a fouryear period has been correlated with the degree of insulin deficiency at baseline (51). This finding reinforces the link between the loss of the potent anabolic effects of insulin, subsequent protein catabolism, and clinical deterioration (56). This metabolic impact may be greater to the long-term survival of CF patients than that of hyperglycemia. Marked differences in lung function between CFRD and nondiabetic patients are suggestive of a direct correlation between CFRD and lung disease, but a selection bias may be present if CFRD is investigated and treated more readily in patients with advanced lung disease (53). D. Microvascular Complications

Microvascular complications occur in patients with CFRD, although the prevalence may be lower than in other forms of diabetes. Of 775 patients studied over 15 years at the University of Minnesota, no CFRD patient without FH developed retinopathy or microalbuminuria (36). Conversely, of CFRD patients with FH and diabetes
10 years, 16% had retinopathy and 14% had microalbuminuria (36). The overall prevalence is less than with other forms of diabetes but may be reflective of duration of diabetes and level of glycemic control. This apparent protection from retinopathy and nephropathy may also be due to the persistence of variable degrees of endogenous insulin secretion or a relative absence of metabolic risk factors such as hyperlipidemia and hypertension that are more common in other diabetic forms (36). A smaller subset of patients from the study illustrated that autonomic neuropathy and gastropathy occur as commonly in CFRD as in other forms of diabetes (36). While some microvascular complications may be less common in CFRD, screening is recommended in patients who have had CFRD for five years or if onset is unknown, when fasting hyperglycemia is first diagnosed. Screening should include a spot urine microalbumin to creatinine, dilated retinal exam (done every 2–3 years if initial evaluation normal), and neurologic and foot exam (done every 2–3 years if initial evaluation normal), with blood pressure measured at each visit.

X. Treatment Insulin is the only currently recommended therapy for CFRD. Small studies have shown that insulin therapy may help to improve or stabilize pulmonary function and augment nutritional status in CFRD patients (57,58) and in CF patients with deteriorating clinical status but elevated casual blood glucose, despite normal OGTT (39). Analysis of patients

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in this prediabetic state has revealed relative insulin deficiency correlating with deteriorating clinical status (57). However, the CFF does not currently recommend insulin for IGT and indeterminate glycemia. The suggestion that loss of insulin anabolic effects may concomitantly lead to clinical decline has engendered much interest in treating both CFRD w/o FH and IGT. Preliminary results of a multicenter randomized control trial aimed at testing the hypothesis that insulin therapy improves nutritional status in adults with CFRD w/o FH were presented at the 2008 North American Cystic Fibrosis Conference (59); 75 subjects with CFRD w/o FH and 25 subjects with IGT were randomized to aspart insulin with meals, repaglinide with meals, or placebo. Insulin was associated with a sustained increase in BMI in the CFRD w/o FH group. This finding provides hope that earlier CFRD intervention will have long-term benefit that is pertinent to individuals with CF. Given the already intense medical regimens required of the individual with CF, however, the addition of multiple daily insulin injections may not readily be accepted by all individuals with CF—five of the study subjects in the insulin group dropped out. No insulin regimen is standardly prescribed in CFRD. Much as in T1DM, the choice of insulin regimen is tailored to fit the individual needs and compliance history of the patient as well as the overall treatment program. Basal-bolus regimens are frequently used. Basal insulin (glargine, detemir, NPH) provides background insulin and a continuous anabolic effect. Short-acting coverage provides flexibility for variable eating patterns (60) and helps control postprandial hyperglycemia—often the most significant abnormality in CFRD. For carbohydrate coverage, a common starting dose is 0.5 to 1 units of short-acting insulin for every 15 g of carbohydrates consumed, with adjustments as needed to achieve postprandial blood glucose goals (33). Frequent meals, however (typical of the individual with CF), can translate into frequent injections especially with a glargine or detemir regimen. CFRD has also effectively been treated with an insulin pump (61,62). For patients on nighttime enteral feeds, insulin requirements are increased at night. A combination of isophane and regular insulin can be used to cover the overnight feed (33). Self monitoring of blood glucose levels should occur at least three times daily. Blood glucose goals are similar to other forms of diabetes, and goal hemoglobin A1c should be <7.0%. Oral hypoglycemic agents are currently not recommended in CFRD, and randomized clinical trials evaluating their efficacy are lacking (63). Despite this recommendation, physicians report using oral hypoglycemics for CFRD (64,65). Additionally, in a case series of 20 subjects, outcomes were not different among adults with CFRD treated with insulin versus oral agents, but the study design and sample size preclude the drawing of firm conclusions (66). The side-effect profiles of many of these medications limit their use in CF: metformin (diarrhea, appetite changes), acarbose (diarrhea, anorexia, malabsorption), and thiazolidinediones (osteoporosis). In a study of seven adults with CFRD w/o FH, the glycemic and insulin secretory effects of shortacting insulin, the insulin secretagogue repaglinide, and placebo were compared (67). Repaglinide increased endogenous insulin concentrations but was less effective than short-acting insulin in regulating postprandial hyperglycemia (67). Neither drug completely normalized blood glucose at the doses used (67). These findings helped lead to the multicenter study discussed above, comparing the effects of short-acting insulin, repaglinide, and placebo on CF-specific outcomes.

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Nutritional therapy is an integral part of both CF and CFRD management. Patients often require 120% to 150% or more of normal caloric intake for age and gender to maintain body weight. Since poor nutritional status has been linked to CFRD and malnutrition has been associated with worse outcomes in CF, caloric restriction is not an appropriate option for the treatment of CFRD. Avoidance of simple sugars (soda and sugar-based candies—i.e., those lacking protein and complex carbohydrates) or their consumption with complex carbohydrates, protein, and fats to slow sugar absorption may be indicated, though to minimize hyperglycemia. To optimize caloric intake, carbohydrate counting may be performed and short-acting insulin administered to cover carbohydrate intake.

XI.

Conclusions

As patients with CF survive longer, CFRD will become more prevalent. With time, greater surveillance of CFRD has occurred throughout the United States and Europe, but greater consensus is needed to optimize screening strategies. More research is also needed to clarify the impact of hyperglycemia and its treatment on long-term outcomes germane to the patient with CF.

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36. Schwarzenberg SJ, Thomas W, Olsen TW, et al. Microvascular complications in cystic fibrosis-related diabetes. Diabetes Care 2007; 30(5):1056–1061. 37. Lombardo F, De Luca F, Rosano M, et al. Natural history of glucose tolerance, beta-cell function and peripheral insulin sensitivity in cystic fibrosis patients with fasting euglycemia. Eur J Endocrinol 2003; 149(1):53–59. 38. Yung B, Kemp M, Hooper J, et al. Random blood glucose alone in the diagnosis of cystic fibrosis related diabetes. Lancet 1997; 349(9052):619. 39. Dobson L, Hattersley AT, Tiley S, et al. Clinical improvement in cystic fibrosis with early insulin treatment. Arch Dis Child 2002; 87(5):430–431. 40. Dobson L, Sheldon CD, Hattersley AT. Conventional measures underestimate glycaemia in cystic fibrosis patients. Diabet Med 2004; 21(7):691–696. 41. Moreau F, Weiller MA, Rosner V, et al. Continuous glucose monitoring in cystic fibrosis patients according to the glucose tolerance. Horm Metab Res 2008; 40(7):502–506. 42. Cystic Fibrosis Foundation Patient Registry Annual Data Report. 2006. Accessible though http://cff.org. Cystic Fibrosis Foundation. Bethesda, MD. 43. Milla CE, Billings J, Moran A. Diabetes is associated with dramatically decreased survival in female but not male subjects with cystic fibrosis. Diabetes Care 2005; 28(9):2141–2144. 44. Marshall BC, Butler SM, Stoddard M, et al. Epidemiology of cystic fibrosis-related diabetes. J Pediatr 2005; 146(5):681–687. 45. Adler AI, Shine BS, Chamnan P, et al. Genetic determinants and epidemiology of cystic fibrosis-related diabetes: results from a British cohort of children and adults. Diabetes Care 2008; 31(9):1789–1794. 46. Sims EJ, Green MW, Mehta A. Decreased lung function in female but not male subjects with established cystic fibrosis-related diabetes. Diabetes Care 2005; 28(7):1581–1587. 47. Moran A, Dunitz J, Nathan B, et al. Cystic fibrosis-related diabetes: current trends in prevalence, incidence, and mortality. Diabetes Care 2009; 32(9):1626–1631. 48. Bismuth E, Laborde K, Taupin P, et al. Glucose tolerance and insulin secretion, morbidity, and death in patients with cystic fibrosis. J Pediatr 2008; 152(4):540–545, 5 e1. 49. Moran A, Hardin D, Rodman D, et al. Diagnosis, screening and management of cystic fibrosis related diabetes mellitus: a consensus conference report. Diabetes Res Clin Pract 1999; 45(1):61–73. 50. Finkelstein SM, Wielinski CL, Elliott GR, et al. Diabetes mellitus associated with cystic fibrosis. J Pediatr 1988; 112(3):373–377. 51. Milla CE, Warwick WJ, Moran A. Trends in pulmonary function in patients with cystic fibrosis correlate with the degree of glucose intolerance at baseline. Am J Respir Crit Care Med 2000; 162(3 pt 1):891–895. 52. Lanng S, Thorsteinsson B, Nerup J, et al. Influence of the development of diabetes mellitus on clinical status in patients with cystic fibrosis. Eur J Pediatr 1992; 151(9):684–687. 53. Koch C, Rainisio M, Madessani U, et al. Presence of cystic fibrosis-related diabetes mellitus is tightly linked to poor lung function in patients with cystic fibrosis: data from the European Epidemiologic Registry of Cystic Fibrosis. Pediatr Pulmonol 2001; 32(5):343–350. 54. Rosenecker J, Hofler R, Steinkamp G, et al. Diabetes mellitus in patients with cystic fibrosis: the impact of diabetes mellitus on pulmonary function and clinical outcome. Eur J Med Res 2001; 6(8):345–350. 55. Kerem E, Reisman J, Corey M, et al. Prediction of mortality in patients with cystic fibrosis. N Engl J Med 1992; 326(18):1187–1191. 56. Moran A, Milla C, Ducret R, et al. Protein metabolism in clinically stable adult cystic fibrosis patients with abnormal glucose tolerance. Diabetes 2001; 50(6):1336–1343. 57. Lanng S, Thorsteinsson B, Nerup J, et al. Diabetes mellitus in cystic fibrosis: effect of insulin therapy on lung function and infections. Acta Paediatr 1994; 83(8):849–853.

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58. Nousia-Arvanitakis S, Galli-Tsinopoulou A, Karamouzis M. Insulin improves clinical status of patients with cystic-fibrosis-related diabetes mellitus. Acta Paediatr 2001; 90(5):515–519. 59. Moran A, Pekow P, Grover P, et al. Cystic Fibrosis Related Diabetes Therapy Study Group. Insulin therapy to improve BMI in cystic fibrosis-related diabetes without fasting hyperglycemia: results of the cystic fibrosis related diabetes therapy trial. Diabetes Care 2009; 32(10): 1783–1788. 60. Grover P, Thomas W, Moran A. Glargine versus NPH insulin in cystic fibrosis related diabetes. J Cyst Fibros 2008; 7(2):134–136. 61. Sulli N, Shashaj B. Continuous subcutaneous insulin infusion in children and adolescents with diabetes mellitus: decreased HbA1c with low risk of hypoglycemia. J Pediatr Endocrinol Metab 2003; 16(3):393–399. 62. Sulli N, Bertasi S, Zullo S, et al. Use of continuous subcutaneous insulin infusion in patients with cystic fibrosis related diabetes: three case reports. J Cyst Fibros 2007; 6(3):237–240. 63. Onady GM, Stolfi A. Insulin and oral agents for managing cystic fibrosis-related diabetes. Cochrane Database Syst Rev 2005; (3):CD004730. 64. Allen HF, Gay EC, Klingensmith GJ, et al. Identification and treatment of cystic fibrosisrelated diabetes. A survey of current medical practice in the U.S Diabetes Care 1998; 21(6): 943–948. 65. Mohan K, Miller H, Burhan H, et al. Management of cystic fibrosis related diabetes: a survey of UK cystic fibrosis centers. Pediatr Pulmonol 2008; 43(7):642–647. 66. Onady GM, Langdon LJ. Insulin versus oral agents in the management of Cystic Fibrosis Related Diabetes: a case based study. BMC Endocr Disord 2006; 6:4. 67. Moran A, Phillips J, Milla C. Insulin and glucose excursion following premeal insulin lispro or repaglinide in cystic fibrosis-related diabetes. Diabetes Care 2001; 24(10):1706–1710.

22 Other Extrapulmonary Complications and Treatment JOSHUA P. NEEDLEMAN Weill Cornell Medical College, New York, New York, U.S.A.

SUZANNE E. BECK University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Cystic fibrosis (CF) is a truly multisystem condition requiring care and attention to all aspects of the patient. This chapter will focus on several clinical complications that are not commonly considered a major part of CF, but which can have a marked impact on the health and quality of life of the affected patient.

II.

Cutaneous Complications

Cutaneous manifestations of CF are often underappreciated, but the dermatologic complications of CF can be life-threatening at times and can present as great a challenge as many of the other clinical problems (1). The diverse CF-related skin diseases can be grouped into four main categories (i) nutritional deficiency; (ii) primary skin disorder because of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction; (iii) allergic response; (iv) vasculitic rash. The cystic fibrosis nutrient deficiency dermatitis (CFNDD) occurs between two weeks and six months of age, usually as a presenting feature of CF (2–6). CFNDD is present in patients who have not been diagnosed with CF and are, therefore, not receiving nutritional support. Initially, erythematous papules form in the diaper area and perioral and periorbital regions, often in an annular configuration (2,4). The dermatitis spreads to the extremities and develops into extensive desquamating plaques (5,7). Associated laboratory findings include anemia, hypoproteinemia, elevated transaminases, and low zinc and vitamin E levels. A skin biopsy reveals epidermal hyperplasia, parakeratosis, spongiosis, diminished granular cell layer, and a perivascular infiltrate of mononuclear cells (1,8). The initial appearance of CFNDD is similar to that of acrodermatitis enteropathica, and is often referred to as an acrodermatitis enteropathica-like eruption (7,9). The periorificial distribution and the low zinc levels have distinct similarities, but nails and mucous membranes have been spared in all reported cases of CFNDD. Similarly, the rash of essential fatty acid deficiency has been confused with CFNDD and many CF patients have essential fatty acid deficiency (10). Some patients with CF have presented with a severe case of CFNDD, marked malnutrition, and a diagnosis of kwashiorkor

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(8,11). With clinical features of several nutrient-deficient dermatoses and laboratory findings consistent with protein, zinc, and fatty acid deficiencies, the etiology of CFNDD is likely multifactorial (1). The infants reported to have CFNDD had malabsorption with significant malnutrition. The diagnosis of CF was often delayed because of low clinical suspicion in the absence of any pulmonary symptoms, and difficulties obtaining sweat chloride analysis in a subject with a severe, diffuse rash (6,7,11). In addition, the presence of hypoproteinemia and edema can result in a falsely negative sweat test (12). The dermatitis responds to aggressive nutritional support, including elemental formula, pancreatic enzyme replacement, parenteral nutrition, and vitamin, trace metal, and essential fatty acid supplementation. Nutritional therapy will often need to be initiated prior to confirmation of the diagnosis of CF by sweat testing or genetic analysis to prevent further morbidity or mortality from malnutrition or skin infection (2). Aquagenic wrinkling of the palms (AWP) has been reported in CF patients in an apparent increased frequency compared with the general population (13). The condition is characterized by swelling, thickening, and hyperwrinkling of the palms accompanied by edematous whitish plaques or papules. It has also been reported on the soles of some patients as well. Associated with the skin wrinkling are the symptoms of burning, pruritus, tingling, and, in some cases, hyperhidrosis of the palms (14). Several mechanisms have been hypothesized for the etiology of AWP in CF patients. Elevated sweat chloride content can increase keratin binding to water and lead to swelling of the epidermal layer, or CFTR dysfunction may lead to abnormal regulation of epidermal water channels (13). Notably, one of the patients without CF, reported to have AWP, had borderline elevated sweat chloride concentrations of 35 and 39 mmol/L on two separate tests with no identified CFTR mutations (15). Treatments reported to have some success include topical aluminum chloride, antihistamines, and iontophoresis (1). Patients with CF experience frequent allergic phenomena that can have cutaneous manifestations, which complicate their care. Frequently, they have reactions to the wide variety and large number of agents to which they are exposed for treatment of their CF. The incidence of atopy has been shown to be higher in CF patients compared to their unaffected siblings (16). Despite this, CF patients do not appear to have a higher incidence of atopic dermatitis or urticaria (1). The incidence of drug reactions and antibiotic hypersensitivity is high, however, with as many as 33% of patients experiencing a reaction to antibiotics (17). b-Lactam antibiotics are most likely to cause reactions of rash, pruritus, and urticaria, often with fever. Piperacillin has been shown in some reports to be more allergenic than other antipseudomonal antibiotics, with a higher incidence of acute reactions and serum sickness as well (1,17,18). Patients with CF are also susceptible to the photosensitivity caused by quinolone antibiotics (19). Cutaneous vasculitis in CF patients has been described as maculopapular eruptions, often localized to the lower extremities, macular purpura, and palpable purpura, often associated with arthritis or arthralgias (20–22). Immune complexes have been demonstrated in the serum of affected patients, and skin biopsies reveal a perivascular inflammation. The patients usually respond to nonsteroidal anti-inflammatory drugs, or, when these fail, to systemic corticosteroids (23,24). In summary, the dermatologic complications of CF are varied and only partially understood. The morbidity can be high, especially if the diagnosis is elusive. The

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diagnosis of CFNDD, in particular, can be challenging as it is usually present before the diagnosis of CF is made, but can be fatal if left undiagnosed and not treated aggressively. All other CF-related dermatoses can have significant impact on the quality of life for the CF patient as well.

III.

Metabolic Complications

Metabolic alkalosis has long been recognized as a presenting feature of CF in infancy (25–28). Hypochloremic alkalosis, both with and without dehydration, has been reported (29). More recently, there have been descriptions of adults presenting with new diagnoses of CF and metabolic alkalosis (30). Alkalosis can occur in patients under treatment for CF as well (31,32); one study of infants under treatment for CF found an incidence of hypoelectrolytemia and metabolic alkalosis to be 16% (33). Chloride loss through sweat is the primary cause of metabolic alkalosis in CF. Sodium is then resorbed in the distal renal tubules in association with carbonic anhydrase–mediated bicarbonate production, resulting in increased serum bicarbonate (30,34). Hypoalbuminemia can also be a cause of metabolic alkalosis; thus, those patients with poor nutrition are also at risk for metabolic alkalosis (35). The risk of developing alkalosis is augmented with increased sweating, febrile illnesses, or chloride loss through vomiting (26,30,32). While concern for hypochloremia in babies with CF is often increased during the summer months and in hot climates, Fustik et al. report a high incidence of metabolic alkalosis in all months of the year, emphasizing the need for continued surveillance and a high index of suspicion (33). The clinical impact of a chronic metabolic alkalosis can be significant. Infants and young children can have failure to thrive and developmental delay despite high-calorie diets with appropriate pancreatic enzyme replacement. Anorexia and recurrent vomiting are also common (33,36,37). Holland and colleagues report a series of adults with advanced lung disease in whom metabolic alkalosis appeared to complicate acute hypercapneic respiratory failure with respiratory acidosis (38). In this study, 13 out of 14 patients with hypercapneic respiratory failure and metabolic alkalosis had elevated arterial pH, suggesting a primary metabolic alkalosis and a respiratory acidosis that was, at least in part, compensatory. The treatment of metabolic alkalosis is replacement of sodium chloride and potassium, and correction of dehydration, if present. In severe cases with low serum albumin, the hypoproteinemia may need to be corrected as well before the alkalosis is completely reversed (25,32). Prevention of alkalosis is achieved with salt supplementation and anticipatory guidance regarding change in climate, activity level, hydration, and feeding practices (30,33,37). As with all other aspects of CF care, a high level of suspicion and anticipation of complications or deterioration is important in the prevention and management of metabolic alkalosis.

IV.

Neurologic Complications

Many diverse neurologic illnesses and complaints have been reported in patients with CF. The conditions can be grouped, roughly, into conditions caused by nutrient deficiency, complaints secondary to untoward effects of CF therapy, and neurologic symptoms that may be related to CF by as yet unidentified mechanisms. When

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considered in aggregate, the neurological complications of CF affect a significant number of patients, and the CF specialist needs to be familiar with the presentation and potential impact of these conditions. Children with CF are at high risk for malnutrition with deficiencies of fat, protein, fat-soluble vitamins, and many micronutrients. Malnutrition in infancy and childhood can cause cognitive delay, behavior problems, poor school performance, and lower IQ levels (39). The Wisconsin Cystic Fibrosis Neonatal Screening Project evaluated the impact of neonatal screening for CF on malnutrition clinical outcome (40). The subjects who were identified by newborn screening and a control group who were diagnosed by symptoms were assessed for nutritional status at diagnosis, including vitamin E levels, and cognitive function during childhood (aged 7–17 years). Children with lower vitamin E levels at diagnosis had lower scores on the Cognitive Skills Index and cognitive factor scores. The control subjects were diagnosed later because of symptoms. Those who had vitamin E deficiency at diagnosis had lower cognitive functioning than the screened subjects, regardless of the screened subjects’ original vitamin E levels. This suggests that early vitamin E deficiency, if not corrected rapidly, can cause long-term reduction in cognitive abilities (41). Reports of neurologic sequelae resulting from chronic, severe vitamin E deficiency in older patients with untreated or undertreated CF demonstrate possible consequences of malnutrition. Elias and Muller reported two patients aged 18 and 19 years who presented with ophthalmoplegia, peripheral neuropathy, and cerebellar dysfunction (42). Both patients had CF and undetectable vitamin E levels with some improvement of symptoms after supplementation. Vitamin K deficiency also occurs in patients with CF and untreated pancreatic insufficiency. Vitamin K deficiency can cause coagulopathy and bruising and bleeding diatheses. There are two reports of CF patients presenting with intracranial hemorrhage in infancy (43–45). One child presented at two and a half months of age with a history of poor weight gain, abnormal stools, and bruising but no diagnosis of CF. After neurologic symptoms developed, an intracerebral hemorrhage was discovered as well as a coagulopathy and vitamin K deficiency. CF was subsequently diagnosed by sweat testing. At age 12, she is reported to have persistent neurologic deficits (45). The other reported subject had no symptoms suggestive of CF until he presented at 3.5 months of age with lethargy and an intracerebral hemorrhage on CT scan (43). These two cases illustrate rare but devastating complications from nutrient deficiency in undiagnosed CF. Vitamin A deficiency can result in neurologic disease as well. Hypovitaminosis A can cause increased intracranial pressure, possibly by decreased cerebrospinal fluid (CSF) absorption (46). Infants with vitamin A deficiency have presented with bulging fontanels and signs of increased intracranial pressure as their initial presentation of CF (47,48). In addition, vitamin A deficiency can cause facial nerve palsy, and there is a report of CF presenting in an infant with bilateral facial nerve palsy as the only complaint (49). Hypovitaminosis A leading to increased intracranial pressure is a phenomenon that is not restricted to infants. Lucidi et al. reported a case of a six-year-old girl not previously diagnosed with CF, who had, at diagnosis, papilledema and decreased visual acuity in one eye (50). The patient reportedly had a history of abnormal stools for two years with “slight recurrent chest infections.” She had recurrent frontal headaches for one year before diagnosis and visual changes for six months. Sweat testing confirmed

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the diagnosis of CF, and a CT scan and lumbar puncture confirmed the diagnosis of pseudotumor cerebri. She responded to pancreatic enzyme replacement, vitamin supplementation, and corticosteroids, but her visual deficit remained. Paradoxically, increased intracranial pressure has been observed in infants newly diagnosed with CF who have started treatment for pancreatic insufficiency (46,51–53). The mechanism of this is unclear; it has been hypothesized to relate to “catch-up growth” of the brain (53), but the rapid development of symptoms in some patients appears to argue against this. In one case report of a five-month-old with CF who developed increased intracranial pressure when pancreatic enzyme therapy was started, the symptoms resolved after the enzymes were temporarily halted and returned when they were restarted on two other occasions (54). Again, the time course and lability in symptoms in this case argue against brain growth as the cause of the increased intracranial pressure. Patients with CF are prone to several neurologic complications due to therapy for the disease. Patients with implanted, long-term central venous access catheters are at an increased risk for thromboembolic disease. If there is shunting at the atrial level, stroke or transient ischemic attacks can occur (55,56). Patients receiving high doses of quinolone antibiotics, especially if they are also taking theophylline, may be prone to seizures or acute hemiparesis (57,58). Most patients with CF experience a chronic cough or recurring coughing paroxysms at some time. Neurologic symptoms, including syncope, can be associated with severe coughing paroxysms (59). Stern et al. reported that 50% of their patients with CF and coughing paroxysms experienced neurologic symptoms. The most common symptoms were lightheadedness and headaches, found in almost all of those patients with neurologic symptoms. Less common were visual disturbances (24% of those with paroxysms), paresthesias (11%), tremors (16%), and syncope (1.6%) (60). Hemiplegic migraine induced by cough has also been reported in patients with CF (61). The mechanism for cough-related neurologic symptoms is unclear, but may result from decreased cerebral perfusion during coughing or hypoxemia (59,61). Chiari type I malformation is characterized by herniation of the cerebellar tonsils through the foramen magnum. Chiari malformation can cause a variety of neurologic symptoms including headache, weakness, or syncope; or it could be entirely asymptomatic. Chiari malformation has been described in patients with CF and may have a higher prevalence than in the general population (62–65). Patients with CF and Chiari malformation have had complaints of numbness, dysphagia, vomiting, headaches, amenorrhea, and syncope (62–65). Two infants with Chiari malformation were diagnosed postmortem with CF (63). Both infants had respiratory symptoms with poor weight gain as well as neurologic symptoms attributed to their Chiari malformation. The mechanism of an association between Chiari malformation and CF is not established. It has been hypothesized that CFTR plays a role in CNS development or that electrolyte imbalances in the CSF lead to displacement of the cerebellar tonsils (63). Alternatively, it has also been suggested that the rise in intracranial pressure with vitamin A deficiency or its replacement could lead to Chiari malformation (62). Regardless of mechanism, the neurologic symptoms of patients with CF and Chiari malformation impact negatively on their care. Patients with headaches were noted to perform fewer airway clearance treatments than prescribed, and patients with swallowing dysfunction had poor weight gain and nutrition (62). The diagnosis of Chiari

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malformation is confirmed by head and brainstem MRI and should be considered in patients with CF with recurrent unexplained neurologic symptoms. In summary, there are numerous neurologic complaints that may be relevant in the care of a patient with CF. Many neurologic symptoms can impact on the CF care regimen or cause significant morbidity themselves. Some symptoms, such as headaches, can represent vitamin A deficiency, Chiari malformation, sinus disease, or unrelated benign phenomena. All options must be considered to avoid delaying diagnosis of a potentially significant complication.

V. Cardiac Complications The effect of severe lung disease in CF on the heart in the form of pulmonary hypertension and cor pulmonale has been recognized since 1946 (66,67). The development of bronchiectasis leads to parenchymal abnormalities and a reduction in arterial oxygen tension. This, in turn, results in elevation of pulmonary artery pressure with increased strain on the right ventricle and the eventual development of right-heart failure (68,69). The development of pulmonary hypertension has a strong, inverse, correlation with the arterial oxygen saturation in adult CF patients with advanced lung disease (70). In severe cases with advanced lung disease, dysfunction of the left ventricle has also been demonstrated (71). Heart disease in CF patients appears to be secondary to their lung disease. Although CFTR expression has been reported in cardiac cells (72,73), there have been no reports to date of a direct effect on the heart from CFTR dysfunction. Measures of severity of lung disease including pulmonary function testing and chest radiographs have not proven to be sensitive in the diagnosis of pulmonary hypertension and cardiac dysfunction (66). Clinical signs of right-heart failure can be present in advanced cardiac dysfunction, but have not been proven to be helpful in detecting early or mild disease (74,75). Electrocardiography (ECG) can also be normal in patients with CF and right ventricular hypertrophy. The interference by hyperinflation and lung disease renders the ECG a tool with limited utility (69), since the increased air interposed between the heart and surface electrodes dampens the amplitude of the electrical signal. Echocardiography, therefore, has become an essential part of monitoring CF patients with advanced lung disease for signs of pulmonary hypertension and cardiac dysfunction (69). Echocardiography, however, can be technically difficult to perform in CF patients. One report had to reject 15% of all studies because of inadequate imaging of the right heart (71). Measurements of the right ventricle and right ventricle wall thickness have been difficult to standardize in CF patients, although right-ventricle thickness is reportedly increased and may be an indirect marker of pulmonary hypertension (66). The main therapy for prevention and treatment of pulmonary hypertension and cor pulmonale is supplemental oxygen (69). The use of oxygen in patients with hypoxemia while awake or during sleep is intended to reduce pulmonary artery pressure, prevent the development or progression of cor pulmonale, and improve the quality of life of patients with CF (66). While initial reports and small studies have been positive (76), and oxygen is commonly accepted as important therapy for patients with chronic hypoxemia, there is a paucity of long-term data regarding oxygen as a treatment for or prevention of pulmonary hypertension in CF. There have been brief reports of use of other vasodilators, such as sildenafil (77), for treatment of patients with CF and advanced lung disease, but their use is still considered preliminary.

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As with other aspects of CF care, the key to managing and preventing CF-related heart disease is the prevention of decline of lung function. Aggressive CF care coupled with a high index of suspicion for developing pulmonary hypertension in patients with significant lung disease is critical to diagnosis and management of this potentially fatal complication.

VI.

Sleep Disruption

Several studies have looked at breathing during sleep in CF, focusing mainly on identifying hypoxemia and the underlying mechanisms of gas exchange aberrations. More recently, a few studies have evaluated the relationships of sleep quality and daytime function with underlying CF disease severity. There have been, however, no longitudinal studies examining the associations among nocturnal gas exchange, sleep quality, and disease progression in CF (78). In the general population, sleep loss or disruption can impair immune, metabolic, endocrine, as well as cardiovascular function (79–83). Increases in inflammatory cytokines occur during sleep loss and are believed to be important in the development of cardiovascular disease (84,85). Recurrent partial sleep restriction has been associated with alterations in glucose metabolism in young healthy adults (86), while others who had difficulty maintaining sleep showed a higher risk of type 2 diabetes (87). Furthermore, sleep disruption, such as that experienced by adults with obstructive sleep apnea, significantly impairs daytime functioning and health-related quality of life (88). Sleep, as a mediator of disease outcome in CF, has yet to be fully evaluated. Patients with CF are at risk for chronic sleep disruption and, therefore, are vulnerable to its potential deleterious consequences. Adult CF patients with severe lung disease [forced expiratory volume in one second (FEV1) < 40% predicted and daytime hypoxemia] demonstrated poor sleep and impaired neurocognitive function compared with healthy nonCF controls (89). The CF patients showed reduced total sleep time due to prolonged awakenings after sleep onset and significant hypoxemia during sleep without evidence of hypoventilation or obstructive apnea. Sleep disruption and sleep loss were attributed to cough, hypoxemia, increased work of breathing, and possibly medication side effects. Decreased sleep efficiency correlated with the degree of hypoxemia. CF patients in this study did not report feeling sleepy during the day; however, they did show greater levels of fatigue and lower levels of activation and happiness on mood profile scales compared with controls. The CF patients also showed significant deficits in neurocognitive performance (simple addition and subtraction, word conflict, and reaction time) that were not related to the degree of hypoxemia during sleep. The authors of this study concluded that the deficits experienced by CF patients may be more related to long-term sleep deprivation than to acute sleep loss (89). They also speculated that adult CF patients with severe lung disease are chronically sleep deprived. Adult CF patients with mild-moderate disease at baseline, when experiencing a pulmonary exacerbation, also exhibited poor sleep quality and neurobehavioral performance compared with age-matched CF controls with equivalent lung function who were not experiencing an exacerbation (90). Following treatment in the pulmonary exacerbation group, there were improvements in sleep and neurobehavioral performance, while the control group’s performance and sleep quality stayed the same (90). Pulmonary exacerbations adversely affect sleep in adult CF patients by being associated with decreased rapid eye movement (REM) sleep, decreased slow wave sleep,

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and increased total arousal index, regardless of underlying disease severity (90). Decreased REM sleep during an acute infection may be the result of the production of somnogenic acute phase cytokines such as interleukin 6 (IL-6), tumor necrosis factor a, or IL-1B; these have been shown to shift sleep from REM to non-REM (91), and such mediators are known to be elevated during CF exacerbations (92,93). In addition, cough results in sleep disruption and delays progression to delta sleep and REM sleep (91). Adult CF patients with even mild baseline lung disease can have fragmented sleep and poor perceived sleep quality. A study of 20 adults with stable, mild, CF lung disease and age-matched healthy controls showed that although total sleep time, sleep efficiency (amount of time asleep while in bed with lights out), and sleep latency (time to fall asleep once lights are out) were similar in both groups, sleep was significantly more fragmented and self-perceived sleep quality was poor in the CF group (94). The difference in sleep fragmentation was related to disease severity, as measured by FEV1, but not to resting room air saturations or body mass index (BMI). Increased sleep disruption in CF patients was attributed to coughing, medications, and increased work of breathing or sleep-disordered breathing in this study. Children with CF also experience sleep disruption. In a prospective study of 24 children (age 14  3.8 years) with stable disease, Naqvi found frequent sleep complaints and significant alterations in sleep architecture: 39% reported sleep maintenance problems, 30% were noted to snore at night, and 73% reported daytime sleepiness (95). Children with CF had significantly decreased sleep efficiency and decreased REM sleep compared with controls. The magnitude of sleep disruption correlated with the severity of lung disease as measured by FEV1, but not to minimum oxygen saturation or maximum end-tidal CO2 as measured during overnight polysomnography. There was no difference in the respiratory disturbance index between CF and control subjects. In a cross-sectional study, CF children with mild lung disease demonstrated lower sleep efficiency and more frequent nocturnal awakenings than did healthy age-matched controls (96). In this study, CF children slept on average 25 minutes less per night than healthy controls. As with CF adults, severity of sleep disturbance worsened with lower FEV1. Children with CF and their parents also reported more frequent awakenings due to cough. Previous experiments in humans and animals have demonstrated that cough is suppressed during sleep and occurs following arousal from sleep (97–99). Thus, it is possible that as pulmonary function declines in CF, different mechanisms lower the arousal threshold for awakening, thereby allowing nocturnal cough (96). Another important cause of nocturnal awakenings in CF children is increased nocturnal bathroom use (96). Interestingly, nocturnal bathroom use was found more often to be for defecation, a potentially treatable cause if this history is ascertained during routine CF follow-up care. Parents also reported that CF children awakened because of nocturnal sweating, although children did not report this (96). Children with CF and their parents also reported difficulty in initiating sleep and more frequent television watching at bedtime compared with control children (96). This might be the result of long routines involving chest physiotherapy and respiratory treatments near bedtime, with television used as a reward. In the long run, however, such behavior can foster negative sleep associations, lead to poor sleep habits, and cause insomnia in susceptible children. Thus, anticipatory guidance regarding appropriate sleep hygiene is important in CF patients. Finally, a survey of 3- to 14-year-old CF children with mild disease in our CF center found that they slept less than healthy children, with much of this difference

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resulting from an earlier wake time (L. Meltzer, personal communication). This was likely due, in part, to the daily demands of medical treatments for CF. Notably, children whose health had worsened over the past year had more bedtime resistance, shorter sleep duration, more sleep anxiety, and more sleep disruptions. In addition, sleep in caregivers of CF children is disrupted because of stress related to the child’s underlying health (100). Not only can changes in sleep patterns reflect important changes in underlying health status, but their identification can also serve as important places to intervene to improve outcomes in the progression of or consequences of CF lung disease. Obstructive sleep apnea in the CF population has been reported (101–105), but its incidence and prevalence are not known. Frank respiratory events such as central or obstructive apneas are rare, even in patients with moderate-to-severe lung disease (104). The most common respiratory events seen are hypopneas (104), which can be either obstructive or nonobstructive in nature. CF patients do not tend to have adenotonsillar hypertrophy or an elevated BMI. However, since CF patients can have chronic nasal congestion and nasal polyposis, screening for snoring and obstructive sleep apnea, as recommended by the American Academy of Pediatrics (AAP) (106) would be prudent in individuals with symptoms of snoring or restless sleep or in those who have had poor response to traditional treatment. Polysomnography is the gold standard to diagnose obstructive sleep apnea, as clinical history alone cannot distinguish obstructive sleep apnea from primary snoring (106,107).

VII.

Gas Exchange Complications During Sleep

It is well known that patients with advanced CF lung disease and low resting daytime oxyhemoglobin saturation develop hypoxemia and hypercapnia during sleep, particularly in REM sleep (91,108–111). Hypoxemia and hypercapnia can also occur during sleep in stable and nonhypoxemic CF patients with moderate-to-severe lung disease. This seems to be unrelated to pulmonary function, respiratory muscle strength, and nutritional status (111). During REM sleep, tidal volume falls due to reduced respiratory drive and decreased tonic activity of the intercostal muscles and diaphragm, leading to decreases in FRC, hypoventilation, ventilation perfusion mismatch, and hypoxemia (108–111). Hypoventilation, particularly during REM sleep, is the major contributing factor to sustained falls in oxygen saturation and rises in carbon dioxide that occur in patients with severe CF lung disease (78). Furthermore, these gas exchange abnormalities are poor prognostic signs (112) and may lead to the development of cor pulmonale (113). In addition, chronic or frequently repeated hypoxic insults can impact on factors such as pulmonary circulation (70), sleep quality (114), and lung inflammation (115). These issues have not been specifically studied in terms of their impact on the progression of CF lung disease. Children with even mild symptoms of CF lung disease can also suffer from hypoxemia during sleep. A study of stable CF children demonstrated that they had significantly lower mean overnight SpO2 (oxygen saturation as measured by pulse oximetry) compared with controls (116). Additionally, mildly symptomatic CF infants and children less than three years old showed unsuspected nocturnal hypoxemia compared with asymptomatic CF infants and healthy age-matched controls (117). The symptomatic subjects spent more of their total sleep time with SpO2 < 93% than controls, and their SpO2 nadir was 86%. The authors suggested that respiratory

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symptoms (even if mild) and elevated nocturnal respiratory rate are two risk factors for nocturnal oxyhemoglobin desaturation in CF infants and small children. It is unclear, however, what level and duration of oxygen desaturation is important in CF. Additionally, the definition of nocturnal hypoxemia is not uniformly agreed upon in children (118). Daytime oxyhemoglobin saturations of < 93% may be predictive of nocturnal hypoxemia in CF adults (119); however, the negative predictive value of this is low and other studies have not supported this association. Currently, it is estimated that 1% to 2% of CF children use long-term supplemental oxygen therapy at night (120); however, the prevalence of nocturnal hypoxemia in patients with CF, who have adequate daytime saturations, is not known. Long-term oxygen therapy is indicated for adults with chronic obstructive pulmonary disease (COPD) when resting PaO2 is < 55 mmHg or SpO2  88% (121–123). However, the recommendations for nocturnal oxygen supplementation in adults may not be appropriate for children. For example, infants with bronchopulmonary dysplasia grow best when SpO2 is > 92% (124,125). Results of a Cystic Fibrosis Consensus Conference suggest that CF patients with resting room air saturation < 92% should be screened for nocturnal hypoxemia (126), and a more recent Cystic Fibrosis Consensus Report suggests that adults with CF should use supplemental nocturnal oxygen if oxygen saturation is < 88% to 90% for 10% of total sleep time (127). According to a recent Cochrane review, there are no published data to guide the prescription of chronic oxygen supplementation for people with advanced CF lung disease (128). Short-term oxygen therapy during sleep improves oxygenation but is associated with modest hypercapnia (102,103,128,129). There is a need for larger, welldesigned clinical trials to assess the safety and benefits of long-term nocturnal oxygen therapy on disease progression, disease severity, and quality of life in people with CF. Nocturnal noninvasive bilevel ventilation (NIV) administered though a tight-fitting nasal mask has proven to be as effective as low-flow supplemental oxygen in relieving sleep-related hypoxemia (103,105,129) in CF patients with advanced lung disease. In addition, NIV has the benefit of relieving sleep-related hypercapnia (103,105,129), without significantly affecting sleep architecture or sleep efficiency (129). In three studies, hypercapnia worsened when patients were treated with supplemental oxygen alone (103,105,129), a finding similar to that of Spier et al. (102). Despite the benefits of NIV in alleviating sleep-related hypercapnia, however, patients tended to prefer using supplemental oxygen to NIV (105,129). In summary, sleep disruption and gas exchange abnormalities occur in patients with CF, even at a young age, and the degree of the abnormalities correlates with the severity of lung disease. The effects of chronic sleep disruption in the CF population are particularly relevant, as sleep disruption can have a significant impact on disease progression and quality of life. Recognition of sleep-disordered breathing or subtle gas exchange abnormalities in children with CF is important. The potential role of oxygen therapy or noninvasive support in improving disease severity or progression in children with CF warrants further study. Longitudinal studies evaluating sleep disruption, with special attention on the association with disease progression and quality of life, should be studied systematically in children and adults with CF. Certainly, sleep quality should be assessed clinically as it can provide insights into the condition of the patient, perhaps even before changes occur in pulmonary function, BMI, or daytime oxyhemoglobin saturations. In addition, sleep quality should be considered as an outcome measure in CF clinical trials.

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77. Montgomery GS, Sagel SD, Taylor AL, et al. Effects of sildenafil on pulmonary hypertension and exercise tolerance in severe cystic fibrosis-related lung disease. Pediatr Pulmonol 2006; 41:383–385. 78. Piper AJ, Bye PTP, Grunstein RR. Sleep and breathing in cystic fibrosis. Sleep Med Clin 2007; 2:87–97. 79. Spiegel K, Sheridan JF, Van Cauter E. Effect of sleep deprivation on response to immunization. JAMA 2002; 288:1471–1472. 80. Irwin M. Effects of sleep and sleep loss on immunity and cytokines. Brain Behav Immun 2002; 16:503–512. 81. Punjabi NM, Sorkin JD, Katzel LI, et al. Sleep-disordered breathing and insulin resistance in middle-aged and overweight men. Am J Respir Crit Care Med 2002; 165:677–682. 82. Spiegel K, Leproult R, Van Cauter E. Impact of sleep debt on metabolic and endocrine function. Lancet 1999; 354:1435–1439. 83. Van Cauter E, Copinschi G. Interrelationships between growth hormone and sleep. Growth Horm IGF Res 2000; 10 (suppl B):S57–S62. 84. Kokturk O, Ciftci TU, Mollarecep E, et al. Elevated C-reactive protein levels and increased cardiovascular risk in patients with obstructive sleep apnea syndrome. Int Heart J 2005; 46:801–809. 85. Miller MA, Cappuccio FP. Inflammation, sleep, obesity and cardiovascular disease. Curr Vasc Pharmacol 2007; 5:93–102. 86. Spiegel K, Knutson K, Leproult R, et al. Sleep loss: a novel risk factor for insulin resistance and Type 2 diabetes. J Appl Physiol 2005; 99:2008–2019. 87. Meisinger C, Heier M, Loewel H. Sleep disturbance as a predictor of type 2 diabetes mellitus in men and women from the general population. Diabetologia 2005; 48:235–241. 88. Moyer CA, Sonnad SS, Garetz SL, et al. Quality of life in obstructive sleep apnea: a systematic review of the literature. Sleep Med 2001; 2:477–491. 89. Dancey DR, Tullis ED, Heslegrave R, et al. Sleep quality and daytime function in adults with cystic fibrosis and severe lung disease. Eur Respir J 2002; 19:504–510. 90. Dobbin CJ, Bartlett D, Melehan K, et al. The effect of infective exacerbations on sleep and neurobehavioral function in cystic fibrosis. Am J Respir Crit Care Med 2005; 172:99–104. 91. Stokes DC, McBride JT, Wall MA, et al. Sleep hypoxemia in young adults with cystic fibrosis. Am J Dis Child 1980; 134:741–743. 92. Aris RM, Stephens AR, Ontjes DA, et al. Adverse alterations in bone metabolism are associated with lung infection in adults with cystic fibrosis. Am J Respir Crit Care Med 2000; 162:1674–1678. 93. Krueger JM, Majde JA. Humoral links between sleep and the immune system: research issues. Ann N Y Acad Sci 2003; 992:9–20. 94. Jankelowitz L, Reid KJ, Wolfe L, et al. Cystic fibrosis patients have poor sleep quality despite normal sleep latency and efficiency. Chest 2005; 127:1593–1599. 95. Naqvi SK, Sotelo C, Murry L, et al. Sleep architecture in children and adolescents with cystic fibrosis and the association with severity of lung disease. Sleep Breath 2008; 12:77–83. 96. Amin R, Bean J, Burklow K, et al. The relationship between sleep disturbance and pulmonary function in stable pediatric cystic fibrosis patients. Chest 2005; 128:1357–1363. 97. Sullivan CE, Kozar LF, Murphy E, et al. Arousal, ventilatory, and airway responses to bronchopulmonary stimulation in sleeping dogs. J Appl Physiol 1979; 47:17–25. 98. Anderson CA, Dick TE, Orem J. Respiratory responses to tracheobronchial stimulation during sleep and wakefulness in the adult cat. Sleep 1996; 19:472–478. 99. Power JT, Stewart IC, Connaughton JJ, et al. Nocturnal cough in patients with chronic bronchitis and emphysema. Am Rev Respir Dis 1984; 130:999–1001. 100. Meltzer LJ, Mindell JA. Impact of a child’s chronic illness on maternal sleep and daytime functioning. Arch Intern Med 2006; 166:1749–1755.

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124. Groothuis JR, Rosenberg AA. Home oxygen promotes weight gain in infants with bronchopulmonary dysplasia. Am J Dis Child 1987; 141:992–995. 125. Moyer-Mileur LJ, Nielson DW, Pfeffer KD, et al. Eliminating sleep-associated hypoxemia improves growth in infants with bronchopulmonary dysplasia. Pediatrics 1996; 98:779–783. 126. Schidlow DV, Taussig LM, Knowles MR. Cystic fibrosis foundation consensus conference report on pulmonary complications of cystic fibrosis. Pediatr Pulmonol 1993; 15:187–198. 127. Yankaskas JR, Marshall BC, Sufian B, et al. Cystic fibrosis adult care: consensus conference report. Chest 2004; 125:1S–39S. 128. Elphick HE, Mallory G. Oxygen therapy for cystic fibrosis. Cochrane Database Syst Rev 2009:CD003884. 129. Gozal D. Nocturnal ventilatory support in patients with cystic fibrosis: comparison with supplemental oxygen. Eur Respir J 1997; 10:1999–2003.

23 Chronic Respiratory Failure and the Roles of Noninvasive Ventilation and Lung Transplantation SAMUEL GOLDFARB and HOWARD B. PANITCH University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Progressive loss of lung function in cystic fibrosis (CF) ultimately leads to gas exchange failure, manifested by hypoxemia with or without hypercapnia. Concurrently, the increased load placed on the respiratory muscles from airway obstruction and parenchymal damage, together with pulmonary hyperinflation and poor nutrition, place the respiratory muscles at a disadvantage and predispose the patient with advanced CF lung disease to respiratory muscle fatigue and failure. Until recently, the focus of care for CF patients with advanced lung disease was to relieve dyspnea, as the inexorable progression of lung disease led to death (1). Early reports of mechanical ventilation for acute respiratory failure in CF infants and children uniformly emphasized poor survival during the acute event, and equally dismal outcomes over the ensuing months following recovery from the episode of respiratory failure (2,3). As the general care for CF patients and critical care techniques have improved over the years, however, the outcome for infants and young children with CF and acute respiratory failure has improved dramatically (4,5). Adults with acute on chronic respiratory failure still experience high mortality rates (6), tempering the universal use of mechanical ventilation. The prospect of lung transplantation, however, has changed the way patients with advanced CF lung disease are supported, making both acute and chronic noninvasive ventilation (NIV) an option for many (7–13). This chapter will review the pathophysiology of respiratory failure associated with advanced CF lung disease and the role of noninvasive chronic assisted ventilation and lung transplantation in its treatment.

II.

Pathophysiology of Respiratory Failure in CF

CF patients with advanced lung disease can present with hypoxemia alone or accompanied by hypercapnia. Hypoxemia can occur during sleep (14–18) or exercise (19,20), or with acute pulmonary exacerbations (21,22) before it is apparent at rest. The prevalence of hypoxemia in patients with CF is not well defined. This is in part due to imprecision of the diagnosis of hypoxemia and a reluctance to perform arterial blood gas sampling in children (23,24). It is estimated that 1% to 2% of all children with CF in the United Kingdom use long-term oxygen therapy (25). Hypercapnia can occur episodically during acute pulmonary exacerbations (22), during sleep (18,26,27), and with exercise (28,29), but persistent hypercapnia is typically a late event in CF and portends a poor outcome (30,31).

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A. Effects of Increased Respiratory Load

CF patients who have moderate to severe pulmonary obstruction develop a breathing pattern different from healthy individuals, and their responses to expiratory resistive loads differ from normal individuals as well (32). Among 7 patients with forced expiratory volume in 1 second (FEV1) values between 18% and 51% of predicted, the resting tidal volume and rate were greater than those of controls at rest, and were found to be similar to those of controls breathing against a resistive load of 5 to 10 cmH2O. Furthermore, in response to an additional imposed expiratory load, neither minute ventilation nor end-tidal carbon dioxide (ETCO2) measurements changed in the CF patients, whereas minute ventilation increased and ETCO2 levels decreased among controls. In a separate study of 32 CF patients with advanced disease (FEV1 12–49% of predicted), alterations in breathing pattern were found to correlate with the FEV1: those with more severe obstructive disease had adapted a rapid, shallow breathing pattern with little change in minute ventilation (33). This led to greater ventilatory inefficiency, and those with the lowest FEV1 were both hypoxemic and hypercapnic. Respiratory drive during hypercapnic challenge, as measured by the occlusion pressure at the mouth in the first 100 msec (P0.1), has been shown to be normal or slightly supranormal in CF patients, independent of the degree of airway obstruction (34,35). In contrast, the increase in minute ventilation or respiratory drive as defined by tidal volume/inspiratory time (Vt/Ti) compared with the ETCO2 was blunted in correlation with the degree of airway obstruction (34). Furthermore, at the extremes of a hypercapnic challenge, control subjects increased minute ventilation primarily by increasing tidal volume with only a small increase in respiratory rate; in contrast, CF patients increased breathing frequency rather than tidal volume (35). Together, these data suggest that as lung disease worsens, mechanical limitations preclude the CF patient from increasing ventilatory output, even in the setting of normal drive. The decline in FEV1 among CF patients with advanced disease is associated with an increased load on the respiratory pump (33). Both total and elastic work of breathing were shown to correlate inversely with the FEV1, largely as a result of a direct correlation between dynamic lung compliance (CL dyn) and FEV1. Although 29 of the 32 subjects studied had evidence of dynamic hyperinflation, intrinsic PEEP did not correlate with FEV1. The authors speculated that the reduction of CL dyn with advancing disease reflected a change in lung parenchymal properties as a result of ongoing inflammation and tissue destruction. In addition, as respiratory rate increased in those with the most severe disease, the frequency dependence of compliance (reflecting widely different rates of filling and emptying of lung units in a diseased lung with heterogeneous time constants) probably contributed to some reduction in CL dyn. Furthermore, as hyperinflation shifts the position of the lung upward on the pressurevolume curve, inspiratory work increases and the amount that tidal volume could increase under additional expiratory loads would be limited (32). The development of a rapid shallow breathing pattern would have the overall effect of decreasing the elastic work of breathing per breath. The respiratory pump itself may add to an increased load. Thoracic kyphosis (defined as a Cobb angle >408) has been described in 22% to 77% of CF patients, depending on age and sex (36–38). Vertebral body fractures associated with osteopenia are presumed to contribute to this change in chest shape (37,38). The effects of thoracic kyphosis on chest wall compliance or lung function have not been studied directly in

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patients with CF. In adult women with kyphosis from osteopenia, however, there is an associated reduction in lung volumes and lateral rib expansion (39). Thus, it has been speculated that kyphosis contributes to reduced chest wall compliance and inefficiency of the respiratory pump in CF patients, leading to increased work of breathing and a more rapid, shallow breathing pattern (40). B. Alterations in Respiratory Pump Function

The respiratory pump includes muscles of ventilation of the chest and anterior abdominal wall and the thoracic skeleton. While little investigation has been performed to determine how alterations in chest wall shape affect ventilatory function in CF, several studies on respiratory muscle function have been performed. Factors that could impact respiratory muscle function include the increased load against which the pump must work, nutritional status, lung hyperinflation, and the general effects of hypoxemia and hypercapnia on muscle function. Studies of respiratory muscle strength in CF patients have yielded conflicting results from decreased (41–46), to normal (26,47,48), to supranormal (49–51). The varied outcomes are in part explained by differences in the groups studied with respect to age and disease severity, methods of testing and measurement, and whether the presence of hyperinflation (and thus the lung volume at which the measurements were taken) was accounted for. Some studies related reduced respiratory muscle strength with poorer nutritional status (43,44), whereas others found no relationship between respiratory muscle strength and malnutrition (46,47,49). Those that found supranormal values for inspiratory muscle strength speculated that the constant work against an increased load acts as a training effect for the diaphragm, and so maintains inspiratory muscle function even when peripheral skeletal muscle function may be compromised (47,50). Strength, however, might not be the critical characteristic of respiratory muscle function that predisposes to respiratory failure. Rather, the tendency of the muscle to resist fatigue under conditions of increased work is likely to be a chief determinant of respiratory success or failure. The tension-time index of the respiratory muscles (TTmus) is a measure of the tendency for the respiratory muscles to become fatigued. Its components are described in chapter 11, but conceptually, if the respiratory muscles have to generate a greater percentage of their maximal force with each breath, or if they have to contract for longer periods of the total respiratory cycle, they will also be more likely to fatigue within a finite amount of time. Even in CF patients with only mild to moderate disease (FEV1 46–114% predicted), the ratio of mean inspiratory pressure to maximal inspiratory pressure was found to be higher than in healthy age-matched controls (52). Although no CF subject was close to the fatigue threshold, the group had a significantly higher mean TTmus value compared with the control group. Furthermore, TTmus was found to correlate with the percent predicted FRC and other measures of hyperinflation and air trapping, and negatively with lean body mass. It has been established that the inspiratory muscles of CF patients with advanced lung disease must do more work during quiet breathing than those of patients with less severe disease (33). In a recent study of 47 children and adults with CF, the TTmus correlated with severity of lung disease, and those who were hypercapnic had values just below the fatigue threshold (53). Similar to the results of Hayot et al. (52), the major difference between CF subjects and controls was in the mean inspiratory pressure/ maximal inspiratory pressure fraction. Furthermore, the P0.1, from which mean

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inspiratory pressure was calculated, correlated with parameters that reflected airway obstruction and hyperinflation. Thus, CF patients increase respiratory neural output in response to increasing loads. From studies of respiratory mechanics and pump function, it is clear that as CF lung disease progresses, the load against which the respiratory muscles function must increase. At the same time, alterations in the shape of the thoracic cage, including caudal depression of the diaphragm from hyperinflation, can impair respiratory muscle function. Respiratory work increases although efficiency of the respiratory muscles can become impaired. The increased energy demands can contribute to nutritional compromise, and further stresses to the respiratory system like acute exacerbations or ongoing progression of lung disease can tip the balance from respiratory adequacy to failure.

III.

Noninvasive Ventilation for Chronic Respiratory Failure

A role for assisted ventilation in hypercapnic respiratory failure was recognized in a CF Foundation Consensus Conference report for several groups of CF patients: infants, those patients with a reversible cause for their respiratory failure, those whose underlying disease had not been treated aggressively, and those patients accepted as lung transplant candidates (54). While the use of noninvasive methods of assisted ventilation was mentioned in the report for patients with chronic respiratory failure who were not considered as candidates for lung transplantation, the practice was felt unlikely to be of any benefit except in selected patients. Chronic respiratory failure in CF patients is often presaged by alterations of gas exchange during sleep, along with disruption of sleep architecture (55) (chap. 22). It is noteworthy that several studies have demonstrated improvement in hypoxemia and hypercapnia during sleep in CF patients with advanced lung disease who used nocturnal NIV compared with those with no intervention or low flow nasal cannula oxygen (17,18,27). In two of the studies, hypercapnia worsened when patients were treated with supplemental oxygen alone (18,27), whereas in the third there was no change in transcutaneous CO2 determinations (17). Other acute effects of NIV have been assessed during short-term or overnight studies. Within 20 minutes of application of NIV to stable adult CF patients with hypercapnic respiratory failure, improvement in blood gases occurred along with reductions in minute ventilation (suggesting better alveolar ventilation) and work of breathing (56). Other studies have also demonstrated improved ventilation (17,57,58), unloading of the respiratory muscles (57,58), and improved cough and sputum production during physiotherapy (59) associated with short-term NIV use. Extended use of nocturnal supplemental oxygen in CF patients with awake hypoxemia (PaO2 < 65 mmHg) did not alter mortality or morbidity as reflected in frequency of hospitalization, change in lung functions, or blood gas values (60). Patients who used supplemental oxygen, however, maintained school or work attendance whereas those who were untreated did not. Similarly, in a randomized controlled crossover study of six weeks of air, supplemental oxygen, or NIV administration in random sequence to eight CF adults with advanced disease and diurnal hypercapnia, there were no effects of either nocturnal supplemental oxygen or NIV on awake blood gases or pulmonary function (27). In contrast, during the six weeks of NIV patients had better CF-Quality of Life and Transitional Dyspnea Index scores, and exercise performance compared with their six weeks of breathing air.

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Nonrandomized reports of long-term NIV use can lend some insight into why patients receiving nocturnal NIV feel better. Four CF adults with hypercapnic respiratory failure treated with NIV for up to 18 months all demonstrated an increase in respiratory muscle strength, which the authors speculated might have resulted from ameliorating the deleterious effects of hypoxia and hypercarbia on diaphragm function (8). In contrast to the crossover study above (27), an earlier nonrandomized trial reported improved forced vital capacity (FVC) and arterial blood gases, as well as fewer inpatient days at 3 and 6 months after starting NIV compared with the 1 to 2 months before among 10 subjects with hypercapnic respiratory failure treated for 1 to 15 months (61). More recently, 20 CF patients were treated with NIV for up to 26 months after they developed hypercapnic respiratory failure following treatment with supplemental oxygen for hypoxemia (62). Within one month of initiating nocturnal NIV, daytime PaCO2 levels fell significantly below pretreatment levels and remained lower for weeks to months. Why some studies show nocturnal NIV results in improvement in blood gases and others do not is unclear, but may relate to differences in study size or design, bias introduced by selection of certain patients for lung transplantation after a period of NIV, and study duration or nocturnal duration of NIV. The availability of lung transplantation as a life-saving modality has redefined the role of chronic NIV among patients with CF. NIV not only avoids the complications associated with endotracheal intubation, but it can also be accomplished outside of the intensive care unit or hospital. There have been several case series describing the use of NIV as a “bridge to transplantation” among CF children and adults (7,9,10,63,64). Others have used NIV to treat acute on chronic respiratory failure, not only in those awaiting transplant, but also in those not considered eligible for lung transplantation (11). Efrati and coworkers described improved nutrition and daily activity among the nine patients treated with NIV while awaiting transplantation (64). Given that pretransplant nutritional status and ability to train physically are important indicators of post-transplant successful outcome, it is intriguing to consider how the use of NIV might be expanded as a conditioning modality for patients awaiting lung transplantation. Clearly, carefully controlled studies of NIV must be conducted to answer these and other critical questions regarding its role in the care of CF lung disease.

IV.

Lung Transplantation

Bilateral lung transplantation is considered when all other therapies have been exhausted. Lung transplantation is an elective procedure and is not indicated for all patients with advanced lung disease. Patients with CF account for 55% of all those transplanted between the ages of 6 and 11 years and for 69% of lung transplants between the ages of 12 and 17 (65). A. Timing of Referral for Transplantation

The general indication for bilateral lung transplantation in CF is a trajectory of illness that, despite optimal medical therapy, puts the individual at risk of dying without a lung transplant. Prior to May 2005, a patient’s chance to be chosen for lung transplantation was based on time accrued on a list and patient/donor blood type; the likelihood of surviving to transplantation was not considered in determining the listing order. Recognizing that many patients died waiting for a suitable donor, investigators have tried to

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develop methods that predict when the topic of lung transplantation should be discussed with patients and when patients should be placed on the transplant list in the disease course. Kerem et al. recommended that CF patients with an FEV1 < 30% predicted be referred for lung transplant since this severity of illness was associated with a two-year mortality rate of 50%. Furthermore, for a given FEV1 in this cohort, females and children under the age of 18 years had a higher 2-year mortality rate than their counterparts (66). Milla et al. suggested that the principal predictor of mortality was not the absolute value of FEV1, but instead was the rate of decline below an FEV1 of 30% (67). Liou published a five-year survivorship model using multiple parameters derived from CF registry data (68). Survival after transplantation was decreased among adults with Burkholderia cepacia, arthropathy or a five-year predicted survival of more than 50%. Furthermore, survival of pediatric patients did not improve with lung transplantation. Thus, the model suggested that adult patients with a low five-year predicted survival and without B. cepacia infection should receive priority for lung transplantation. Another multidimensional model was no better at predicting short-term mortality than using the criterion of less than 30% FEV1 (69). Both methods demonstrated a positive predictive value for two-year mortality in the range of only 50% (i.e., half of the patients predicted to die within 2 years would actually survive) (69). Since May 2005, the process of prioritizing patients for lung transplantation has been based on a scoring system known as the Lung Allocation Score (LAS). The score is calculated from estimates of survival probability while on the lung transplant waiting list and following transplantation. It reflects a comprehensive evaluation that takes into account age and diagnosis as well as several indicators of disease severity such as FEV1, BMI, serum creatinine, presence of diabetes, six-minute walk test score, increases in supplemental oxygen need, serum carbon dioxide levels (PCO2), need for assisted ventilation, functional status, and presence of pulmonary hypertension (70–72). The referral process to a transplant center should be initiated when a patient’s FEV1 is less than 30% predicted or sooner if there has been a rapid decline in pulmonary function. This, however, does not imply that the patient will be immediately listed. Other considerations for referral include recurrent hemoptysis that is not controlled by embolization or other means, recurrent or refractory pneumothorax, and frequent respiratory exacerbations requiring antibiotic therapy (70,73). Education of potential candidates regarding bilateral lung transplantation as a future treatment modality can occur as a patient’s lung disease worsens. Any patient considered for transplantation must have an adequate social support system and must be able to follow the prescribed medical regimen after transplant. Bilateral lung transplant is an elective procedure; therefore, potential recipients and their families must demonstrate a willingness and an ability to adhere to the rigorous therapy, daily monitoring and reevaluation schedule after transplant (74). B. Contraindications to Lung Transplantation

There are several absolute contraindications for bilateral lung transplant for patients with CF, which are similar to the contraindications for lung transplantation for all causes. These include malignancy within the last two years (some centers recommend a fiveyear disease-free period), infections that include sepsis, active tuberculosis, acquired immunodeficiency syndrome, and hepatitis B or C with histological liver disease, multiple organ dysfunction, severe neuromuscular disease, and documented refractory nonadherence to a medical regimen (70,74).

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Relative contraindications to bilateral lung transplant occur commonly in patients with CF and include renal insufficiency, markedly abnormal body mass index, osteoporosis, poorly controlled diabetes mellitus, and colonization with resistant organisms (74). Significant renal dysfunction pre-transplantation can disqualify a patient from the procedure because of the risk of worsening renal function post transplant. The use of aminoglycosides in patients with CF is associated with a decline in renal function (75). Following transplant there is an increased incidence of chronic renal insufficiency from use of immunosuppressive medications. One study demonstrated that in the first three months post-transplant patients had a 33% decline in glomerular filtration rate (76). These findings were in accord with International Society of Heart and Lung Transplantation (ISHLT) registry data that reported a prevalence of hypertension at one-year post transplant at 39% and renal dysfunction, as measured by changes in serum creatinine, during the same time frame at roughly 9% (77). Thus, judicious use of antibiotics in the pre-transplant period to spare renal function is very important. Nutritional status affects both pre- and post-transplant mortality. Wasting, defined as a weight of less than 85% ideal body weight, has been shown to be a predictor of mortality in the pre-transplant CF patient independent of lung function, arterial blood oxygen, or carbon dioxide levels (78). In addition, when evaluating all subjects awaiting transplantation including those with CF, lean body mass depletion has been associated with more severe hypoxemia, reduced six-minute walking distance and a higher mortality, even in the setting of a normal BMI (79). In contrast, in both CF and non-CF transplant candidates, severe obesity is a significant indicator of post-transplant mortality. A BMI exceeding 27 kg/m2 was the greatest indicator of mortality in the first 90 days post transplant, whereas in those with a BMI of less than 17 kg/m2, only a statistically nonsignificant trend was demonstrated toward increased mortality (80). The degree of preexisting bone demineralization represents a major risk factor for severe osteoporosis following lung transplantation (81). Corticosteroids, used as part of the immunosupression regimen following transplant, contribute to bone disease by hastening bone resorption and inhibiting bone formation. Ideally, steroid dosing is gradually reduced to minimize the complications of long-term use (82). The use of antiresoprtive therapy to improve bone mineral density has expanded to include lung transplant recipients. While the effects of biphosponates in adults with CF related bone disease shows promise, the long-term efficacy and safety of biphosphonate use in children and adolescents requires further investigation. Cystic fibrosis related diabetes (CFRD) is often associated with worsening lung disease (83,84), but improvements in recognition and control of CFRD have reduced its impact on overall mortality (85). In the post-transplant period, there is an increased association of drug-related new onset diabetes mellitus (DM) in the pediatric lung and heart/lung transplant population when compared to either pediatric heart transplant recipients or adult lung transplant recipients (86–88). Twenty-five percent of all transplanted patients develop DM within the first year post transplant (89). CF patients with CFRD prior to transplant have more complication-related admissions to hospital post transplant and a higher mortality rate than those without CFRD who undergo transplant (88). These findings underscore the importance of both pre- and post-transplant management of DM. Patients colonized with multi- or pan-resistant Pseudomonas aeruginosa have no survival disadvantage post transplant (90). However, patients with B. cepacia complex

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infection have poorer outcomes and represent the majority of those who die of sepsis. CF patients infected with genomovar III of the B. cepacia complex before transplantation have markedly increased B. cepacia complex–related early mortality after transplantation. In one center, post-transplant survival percentages were significantly lower in those infected with B. cepacia complex at 1, 3, and 5 years (91). Post-transplant mortality among CF patients infected with Burkholderia, however, varies by infecting species, and there are even differences within the B. cenocepacia strains: infection with either one of the two so-called epidemic strains common in the United States (strain PHDC and the Midwest clone) are associated with increased survival when compared to infection with nonepidemic strains (92). As host-organism interactions become better understood, more centers may consider lung transplantation a viable option in this patient population. C. Listing

As of May 2005 lung transplant candidates 12 years are all listed according to the new LAS scoring system. Patients less than 12 years receive organs based on time accrued on a waiting list. D. Transplant Surgery

Double lung bilateral sequential transplantation is the procedure of choice in the pediatric population. The survival rate of children undergoing single lung transplantation was significantly lower when compared to those receiving bilateral transplants and so the procedure has been abandoned in this population (65). Organs are matched by height, weight and blood type. HLA antibody screening is performed and specific antibodies can be avoided if the recipient has elevated levels. E.

Immunosuppressive Therapy

Most centers have adopted the immunosupression protocol set forth by the International Pediatric Lung Transplant Consortium. Triple therapy with or without induction therapy has become standard practice. Roughly 50% of the pediatric centers reporting to the ISHLT use induction therapy in the post-transplant period. The majority of patients are given tacrolimus along with either azathioprine or mycophenolate. All centers at 1 and 5 years report the use of oral steroids as part of immunosuppressive therapy (65). F.

Complications

Complications from lung transplantation are divided into early and late and are listed in Table 1. Several that bear special mention are discussed below: Acute Rejection

The risk of acute cellular rejection (ACR) is highest in the first few weeks following transplantation. The incidence of ACR is 18% to 50% among those who undergo induction therapy and 50% to 55% among those who do not (82,93,94). Acute rejection is an infrequent problem beyond one year post transplant and infants tend to have a lower frequency of acute rejection than do older children. Risk factors for ACR include HLA mismatching, community acquired viral infection, and the immunosuppressive regimen (95). Although acute rejection is often asymptomatic, fever, dyspnea, and

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Table 1 Complications of Lung Transplantation

Early l Hyperacute rejection l Acute rejection l

Demyelinating disease (due to tacrolimus or cyclosporine)

l

Seizure disorder

l

Ectopic atrial tachycardia (EAT)

l

Infection

8 8 8 8 l

Bacterial (including atypical mycobacterium) Viral (CMV, adenovirus, RSV) Fungal (Aspergillus) Other (PCP)

Anastomosis dehiscence

Late l Acute rejection l Chronic rejection/bronchiolitis obliterans l

Bronchiolitis obliterans syndrome

l

Infection

8 EBV 8 CMV 8 HHV6 l

Diabetes

l

Hypertension

l

Renal insufficiency/failure

l

Post-transplant lymphoproliferative disease Osteoporosis

l

hypoxemia can be present. Common findings on chest radiograph include parenchymal densities and bilateral pleural effusions. The FEV1 and FVC can be diminished. Distinguishing between rejection and infection on clinical grounds alone is often difficult. Therefore, appropriate evaluation includes bronchoscopy with bronchoalveolar lavage and transbronchial biopsy. Acute rejection is graded A0 (none) to A4 (severe); grade A2 acute rejection and above is treated with augmented immunosupression. The initial therapy is usually pulsed steroids. The few cases in which acute rejection persists usually respond favorably to additional augmented immunosupression, usually with a mono- or poly-clonal T-cell antibody. Infection

Up to 60% to 90% of lung transplant recipients experience at least one episode of infection after undergoing the procedure (96,97). The risk for serious infection starts in

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the perioperative period, and infection is the major cause of morbidity and mortality during the first six months after transplant surgery. Prophylactic antimicrobials against bacteria, viruses, and fungi are used in most transplant programs. Antimicrobial treatments should be given perioperatively on the basis of donor cultures and most common hospital pathogens. In the CF population, antibiotic therapy is directed by bacterial sputum colonization and the antibiogram. Pneumocystis jiroveci prophylaxis is a routine in almost all postoperative lung transplant regimens. Respiratory viral infections can be relatively mild to life threatening in the transplant recipient. RSV, influenza, human herpes viruses (particularly HHV6), Epstein–Barr virus, and adenovirus cause significant morbidity and mortality. Cytomegalovirus (CMV) remains the most commonly encountered serious viral infection. Onset of CMV pneumonitis after lung transplant is reported to have a strong correlation with subsequent development of bronchiolitis obliterans (BO), which in turn leads to graft dysfunction and death (98). The highest risk for CMV pneumonia is in seronegative patients who receive lungs from seropositive donors. This increased risk persists even when these patients receive aggressive antiviral prophylaxis (98). In the pediatric population, respiratory viral infections are associated with an increased mortality at one year (99). In this population, etiology of underlying disease other than CF, younger age, and absence of induction therapy are independently associated with risk of respiratory viral infections (99). While colonization with Aspergillus can be as high as 50% in post-transplant patients, invasive disease occurs in only 3% and only in patients colonized with A. fumigatus within the first six months post transplant (100). Antifungal therapy both before and after transplant is therefore an important component of care. Risk factors for pulmonary fungal infections in children include A2 rejection, repeated acute rejection, a CMV-positive donor, a tacrolimus-based regimen and pre-transplant Aspergillus colonization (101). Bronchiolitis Obliterans

BO is the histopathological correlate of chronic organ dysfunction. It is the leading cause of morbidity and late mortality one year after lung or heart-lung transplantation. BO is an inflammatory process, leading ultimately to occlusion of small airways by fibromyxoid tissue (102). Both BO and bronchiolitis obliterans syndrome (BOS), the clinical correlate of BO, are manifestations of chronic lung allograft rejection. BOS describes the otherwise unexplained development of an obstructive decrease in pulmonary function. The cardinal clinical feature of BOS is a reduction in FEV1 and/or the mid-expiratory flow rate (FEF25–75), which does not respond to bronchodilators (103). Roughly half of lung transplant recipients are diagnosed with BO by five years after transplant. More than 40% of deaths occurring beyond a year post transplant are a direct or indirect result of BO (65). The etiology of BO remains elusive in spite of a growing body of basic science and clinical research. Gastroesophageal reflux (GER) has been shown to have an increased association with BO. Furthermore, surgical correction of GER has, in some patients, either stopped or improved the decline in lung function (104–106). There is no consistently effective treatment strategy for BO. Augmentation of immunosupression is usually the initial intervention. Options include anti-thymocyte preparations, cyclophosphamide, methotrexate, photopheresis, and total lymphoid irradiation. All have shown benefit in some patients but none has proven to be uniformly

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beneficial (107). Aerosolized cyclosporine is an investigational agent with substantial promise that may provide a survival advantage to lung transplant recipients (108). The use of tacrolimus as a “rescue” therapy in patients originally treated with cyclosporine may stabilize the decline in FEV1 (109). Sirolimus has also been used as a rescue agent in BO with some success (110,111). Several studies involving azithromycin administration, for its anti-inflammatory effect, have shown either improvement or stabilization of lung function (112–114). A retrospective study of patients prescribed statins for hypercholesterolemia found a lower incidence of BO and a better long-term survival. Re-transplantation may be an option in select individuals. G. Life Expectancy

Outcomes for bilateral lung transplantation have improved since the early nineties when this procedure was first introduced. Survival is the chief indicator of success for this procedure. Survival of pediatric patients is similar to that of adults with a median survival of 4.3 years over the period of 1990–2006. The major cause of death continues to be BO. There is a survival advantage to children with lung disease of all causes transplanted from 1 to 10 years of age when compared to adolescents (115). There are several proposed hypotheses for this survival difference: younger patients have fewer episodes of acute rejection and a lower incidence of BO (116). Alternatively, the adolescent population is predominately composed of patients with CF. Studies have also raised concerns that this age group is more prone to decreased adherence to medical regimens and this in turn could affect long-term survival (117–119). As noted above, early death (within the first 30 days following transplant) attributed to infection was only elevated in patients colonized with B. cepacia complex, whereas no other organisms conferred a survival disadvantage (90). H. Other Considerations in the CF Population

In recent years, some groups have begun to question the practice of lung transplantation, particularly in the adolescent age group. Liou et al. concluded that lung transplantation does not prolong life in children with CF (120). These findings contradicted other studies that showed a positive benefit for bilateral lung transplant in CF patients (121). While many have refuted Liou’s conclusion, there are several issues that must be considered when performing lung transplantation in the adolescent CF patient (122). Risk factors for this patient group have not been specifically identified. Potential specific risk factors would include poor adherence to a medical regimen, mood and adjustment disorders, and transplantation in centers with limited resources to work with the CF adolescent patient population. Adherence to medical regimens for patients with any chronic disease can be difficult, especially among adolescents (123). While complications are similar between adults and children, medical nonadherence appears to be more common in pediatric lung transplant recipients (124). Adolescents with CF underestimate the severity of their illness when compared with assessments of their health care providers and parents (125). More adolescent females than males receive lung transplants (71), and adolescent girls have been shown to have more difficulty than boys with adherence to CF care regimens (126). This general pattern of suboptimal adherence does not necessarily change post transplant; adolescents who received other solid organs struggle with medical regimen adherence as well (127).

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Depression, anxiety, and adjustment disorders are all comorbidities associated with CF. The CF 2007 patient registry reports an incidence of these problems of 5% to 17% with increasing frequency in older teenagers (128). Furthermore, depression rates in general in patients pre-transplant can be as high as 20%. Treatment of depression is an important aspect of pre-transplant care, and mental health treatment should be provided. Transplantation is not curative for anxiety, adjustment, or depressive disorders, and there can be reoccurrence of these problems post transplant. Such disorders can still be present, but they generally improve post transplant (129). A poorly planned transition from a pediatric to an adult center or transitioning adolescents to adult centers poorly prepared to care for them comprehensively could significantly affect a patient’s short- and long-term health. The added stress of dealing with issues around organ transplantation highlight the need for carefully planned transition of adolescent patients to adult care, as inadequate patient preparation can exacerbate poor patient adherence (130,131) and lead to decreased graft or overall patient survival.

V. Conclusions Progressive lung disease is the cause of morbidity and mortality for the majority of patients with CF. NIV can provide palliation of symptoms and perhaps restore some physiologic functions to patients with advanced disease, and is increasingly being used to support patients while they await lung transplantation. Lung transplantation in CF patients is a growing field and allows a therapeutic option for patients with advanced lung disease. The goal of transplantation is to increase the life expectancy of the transplanted patient. Comprehensive studies are also under way to understand better how lung transplantation improves quality of life. The ability of lung transplantation to restore function and respiratory health to CF patients with advanced disease is occasionally perceived as a panacea, but the process has its own set of complications and demands. Patients, families, and caregivers must be made aware of the benefits, risks, and medical demands associated with lung transplantation to make their best-informed choices.

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96. Kramer MR, Marshall SE, Starnes VA, et al. Infectious complications in heart-lung transplantation. Analysis of 200 episodes. Arch Intern Med 1993; 153(17):2010–2016. 97. Kanj SS, Tapson V, Davis RD, et al. Infections in patients with cystic fibrosis following lung transplantation. Chest 1997; 112(4):924–930. 98. Metras D, Viard L, Kreitmann B, et al. Lung infections in pediatric lung transplantation: experience in 49 cases. Eur J Cardiothorac Surg 1999; 15(4):490–494; discussion 495. 99. Liu M, Worley S, Arrigain S, et al. Respiratory viral infections within one year after pediatric lung transplant. Transpl Infect Dis 2009; 11(4):304–312. 100. Cahill BC, Hibbs JR, Savik K, et al. Aspergillus airway colonization and invasive disease after lung transplantation. Chest 1997; 112(5):1160–1164. 101. Danziger-Isakov LA, Worley S, Arrigain S, et al. Increased mortality after pulmonary fungal infection within the first year after pediatric lung transplantation. J Heart Lung Transplant 2008; 27(6):655–661. 102. Boehler A, Estenne M. Post-transplant bronchiolitis obliterans. Eur Respir J 2003; 22(6): 1007–1018. 103. Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002; 21(3):297–310. 104. O’Halloran EK, Reynolds JD, Lau CL, et al. Laparoscopic Nissen fundoplication for treating reflux in lung transplant recipients. J Gastrointest Surg 2004; 8(1):132–137. 105. Davis RD Jr., Lau CL, Eubanks S, et al. Improved lung allograft function after fundoplication in patients with gastroesophageal reflux disease undergoing lung transplantation. J Thorac Cardiovasc Surg 2003; 125(3):533–542. 106. Palmer SM, Miralles AP, Howell DN, et al. Gastroesophageal reflux as a reversible cause of allograft dysfunction after lung transplantation. Chest 2000; 118(4):1214–1217. 107. Estenne M, Hertz MI. Bronchiolitis obliterans after human lung transplantation. Am J Respir Crit Care Med 2002; 166(4):440–444. 108. Iacono AT, Corcoran TE, Griffith BP, et al. Aerosol cyclosporin therapy in lung transplant recipients with bronchiolitis obliterans. Eur Respir J 2004; 23(3):384–390. 109. Sarahrudi K, Estenne M, Corris P, et al. International experience with conversion from cyclosporine to tacrolimus for acute and chronic lung allograft rejection. J Thorac Cardiovasc Surg 2004; 127(4):1126–1132. 110. Hernandez RL, Gil PU, Gallo CG, et al. Rapamycin in lung transplantation. Transplant Proc 2005; 37(9):3999–4000. 111. Snell GI, Westall GP. Immunosuppression for lung transplantation: evidence to date. Drugs 2007; 67(11):1531–1539. 112. Verleden GM, Dupont LJ. Azithromycin therapy for patients with bronchiolitis obliterans syndrome after lung transplantation. Transplantation 2004; 77(9):1465–1467. 113. Shitrit D, Bendayan D, Gidon S, et al. Long-term azithromycin use for treatment of bronchiolitis obliterans syndrome in lung transplant recipients. J Heart Lung Transplant 2005; 24(9):1440–1443. 114. Gottlieb J, Szangolies J, Koehnlein T, et al. Long-term azithromycin for bronchiolitis obliterans syndrome after lung transplantation. Transplantation 2008; 85(1):36–41. 115. Magee JC, Krishnan SM, Benfield MR, et al. Pediatric transplantation in the United States, 1997–2006. Am J Transplant 2008; 8(4 pt 2):935–945. 116. Ibrahim JE, Sweet SC, Flippin M, et al. Rejection is reduced in thoracic organ recipients when transplanted in the first year of life. J Heart Lung Transplant 2002; 21(3):311–318. 117. White T, Miller J, Smith GL, et al. Adherence and psychopathology in children and adolescents with cystic fibrosis. Eur Child Adolesc Psychiatry 2009; 18(2):96–104. 118. Wray J, Waters S, Radley-Smith R, et al. Adherence in adolescents and young adults following heart or heart-lung transplantation. Pediatr Transplant 2006; 10(6):694–700.

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119. De Geest S, Dobbels F, Fluri C, et al. Adherence to the therapeutic regimen in heart, lung, and heart-lung transplant recipients. J Cardiovasc Nurs 2005; 20(5 suppl):S88–S98. 120. Liou TG, Adler FR, Cox DR, et al. Lung transplantation and survival in children with cystic fibrosis. N Engl J Med 2007; 357(21):2143–2152. 121. Aurora P, Whitehead B, Wade A, et al. Lung transplantation and life extension in children with cystic fibrosis. Lancet 1999; 354(9190):1591–1593. 122. Sweet SC, Aurora P, Benden C, et al. Lung transplantation and survival in children with cystic fibrosis: solid statistics—flawed interpretation. Pediatr Transplant 2008; 12(2):129–136. 123. Bender BG. Risk taking, depression, adherence, and symptom control in adolescents and young adults with asthma. Am J Respir Crit Care Med 2006; 173(9):953–957. 124. Wells A, Faro A. Special considerations in pediatric lung transplantation. Semin Respir Crit Care Med 2006; 27(5):552–560. 125. Britto MT, Kotagal UR, Chenier T, et al. Differences between adolescents’ and parents’ reports of health-related quality of life in cystic fibrosis. Pediatr Pulmonol 2004; 37(2):165–171. 126. Patterson JM, Wall M, Berge J, et al. Gender differences in treatment adherence among youth with cystic fibrosis: development of a new questionnaire. J Cyst Fibros 2008; 7(2): 154–164. 127. Wray J, Radley-Smith R. Longitudinal assessment of psychological functioning in children after heart or heart-lung transplantation. J Heart Lung Transplant 2006; 25(3):345–352. 128. Cystic Fibrosis Foundation Patient Registry: 2007 Annual Data Report Bethesda, Maryland, 2008:1–24. 129. Wray J, Radley-Smith R. Depression in pediatric patients before and 1 year after heart or heart-lung transplantation. J Heart Lung Transplant 2004; 23(9):1103–1110. 130. Annunziato RA, Emre S, Shneider B, et al. Adherence and medical outcomes in pediatric liver transplant recipients who transition to adult services. Pediatr Transplant 2007; 11(6): 608–614. 131. Watson AR. Non-compliance and transfer from paediatric to adult transplant unit. Pediatr Nephrol 2000; 14(6):469–472.

24 Gene Repair: Past, Present, and Future CHRISTIAN MUELLER and TERENCE R. FLOTTE University of Massachusetts Medical Center, Worcester, Massachusetts, U.S.A.

I.

Issues for the Success of CF Gene Therapy

While there are an array of functions and conflicting theories have been put forth to explain the pathophysiology of cystic fibrosis (CF) as a result of the absence of the cystic fibrosis transmembrane regulator (CFTR), it is widely accepted that a predilection to infection and an exaggerated and sustained inflammatory response to these bacterial, viral, and fungal agents in the lung leading to progressive tissue damage is a main feature of the CF phenotype. Since the majority of CF related mortality has been associated with the lung, this has made the disease a prime candidate for pulmonary gene therapy by gene augmentation. The concept of gene therapy is a comparatively simple one: isolate the gene of interest and use a gene transfer agent (GTA) to express it in the appropriate target cells to normalize cellular function with protein synthesis from the therapeutic gene. For these reasons, following the discovery of the CFTR gene in 1989, gene therapy for CF was a natural fit; CF is a single gene recessive trait that is characterized by a loss of function of the CFTR gene. As a target organ, the lungs also seemed to offer ideal access since the epithelial cells interface with the environment and therefore are easily accessible. Thus, CF was one of the first diseases targeted by human gene therapy trials, including the very first trials to utilize recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) vectors in humans. Unfortunately, despite the initial demonstrations of successful gene transfer to cultured epithelial cells and small animal models, to date no dramatic therapeutic benefits have been observed in humans. However, a wealth of information has been gained from the preclinical and clinical studies performed. These data have led to the optimization of vector technology, and an appreciation of immune and mechanical barriers that have to be overcome for the successful delivery of modified genes. Importantly, several key points were identified through these studies: (i) which type or types of cell have to be corrected, (ii) what degree of correction is required, and (iii) how can immune and mechanical barriers be circumvented? A. Target Cells for CF Gene Therapy

The success and clinical benefit of gene therapy clearly depends on the ability to target and transduce the appropriate cells in the airway epithelium. Historically, CF is considered to affect the conducting airways and not the alveolar space, and the surface epithelium may be responsible for the initial trigger of disease. The larger bronchial

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airways are lined by pseudostratified columnar superficial epithelium and have submucosal glands. The smaller bronchiolar regions are generally devoid of glands and contain simple columnar epithelium. The optimal time and target cell for gene replacement therapy are not clear and remain controversial. Clinically relevant observations addressing this question include infants with CF typically presenting with bronchiolitis (small airway obstruction) as their first pulmonary abnormality. This target tissue favors the relatively easier topical administration by inhalation for delivering gene transfer vectors. The downside to targeting the bronchial epithelia is that these cells are terminally differentiated and their eventual cell death may limit the benefits of the GTA. In contrast, the CFTR gene is expressed at the highest levels in submucosal gland serous cells, and cell culture experiments have shown that there are abnormalities in the gland volume composition and the regulation of HCO3
associated with CFTR dysfunction (1). For these reasons, there is renewed interest and focus on the identification of pulmonary stem cells; gene transfer to such cells may have the potential to affect long-term CFTR expression in progeny cells. B. How Much Gene Replacement Is Enough?

This leads to the second question; what degree of correction is required for therapeutic benefits? Initial experiments designed to answer this question used monolayers of immortalized CF epithelial cells mixed at varying ratios with CFTR “corrected” cells. From these experiments it was suggested that between 6% and 10% of the cells had to be CFTR corrected to restore the normal Cl
transport of the cell monolayer (2). The issue is actually more complex as correction of a higher percentage of cells is necessary to normalize airway epithelial sodium absorption. Unlike normalization of Cl
transport that may rely on the movement of Cl
ions from noncorrected cells to corrected cells via gap junctions, Naþ transport is thought to be more local and involves CFTR protein interactions at the single cell level (3). This is further exemplified by the epithelial Naþ channel (ENaC)-overexpressing mouse model. Unlike the Cftr
/
mice, the ENaC-overexpressing mice do develop substantial airway disease, characterized by airway surface liquid (ASL) volume depletion, mucus obstruction, neutrophil accumulation, and poor bacterial clearance even in the presence of functional murine CFTR (4). Other in vitro data further suggest that close to 100% of CF epithelial cells must be transduced with a normal copy of the CFTR gene to reduce sodium hyperabsorption (5). This implies the possibility that, if hyperabsorption of Naþ across the surface epithelia plays a major role in the pathogenesis of CF lung disease, amelioration of airway disease with a positive clinical course, but not a complete cure, may result if gene transfer to the airway epithelium corrects Cl
but not Naþ transport. Further complicating the correlation between epithelial cell correction and amelioration of CF lung disease is the more recent appreciation of CFTR expression in immune cells such as macrophages, neutrophils, and lymphocytes. This expression of CFTR, which may in turn affect pulmonary infection and inflammation (6–10), could further influence the choice of optimal target cell for gene replacement. C. Barriers to Gene Therapy

Both mechanical and immune barriers became apparent after early gene transfer attempts. One of the most obvious limitations to transduction of the epithelial lung

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surface is the overlying mucus layer that, under normal circumstances, is designed to clear particles such as bacteria and viruses, and therefore also viral gene therapy vectors, from the airway. In the CF airway, while having decreased clearance, this layer is even thicker with a more complex mixture of abnormal, underhydrated mucus and products of inflammation (e.g., dead and dying neutrophils, actin, DNA). In addition, both viral and nonviral gene therapy approaches have revealed that the immune system poses a significant barrier to safe and effective gene transfer by either mounting cytotoxic T lymphocyte (CTL) responses clearing the transduced cells or creating neutralizing antibodies (NAbs) to viral vectors hampering the efforts of vector readministration. The innate arm of the immune system also orchestrates inflammatory responses via toll-like receptors (TLRs) to bacterially derived plasmid DNA delivered with nonviral vectors. These data have informed the design of new vectors to circumvent or evade some of the immune responses associated with pulmonary gene delivery, as is discussed in the following section.

II.

Past and Current Efforts in CF Gene Therapy

Delivery of CFTR is a daunting task; given the nature of the CFTR as an intrinsic membrane protein, direct transduction of a significant proportion of airway epithelial cells throughout both lungs is required. The past, present, and possible future efforts to treat CF lung disease with gene therapy are reviewed here. Because, in general, naked DNA-mediated gene transfer only achieved low levels of transfection, the field has focused on developing GTAs, such as viral and nonviral synthetic agents, to improve transduction and transfection rates. To date, more than 15 phase I/II clinical trials involving approximately 400 patients with CF have been carried out using a various viral and nonviral GTAs (Table 1). Most of the early trials were conducted on the nasal epithelium due to safety concerns, and once the agents were deemed safe and promising, direct lung administration was tested. A. Recombinant Adenoviral Vectors

Adenovirus (Ad) is an icosahedral nonenveloped DNA virus with a 36 to 40 kb genome that is trophic for the airway epithelia, and naturally causes upper respiratory illnesses. Ad capsids enter epithelial cells by interacting with the coxsackievirus adenovirus receptor (CAR) as an initial binding step followed by secondary engagement of the integrin coreceptors a5b1, anb3, anb5, and aMb2. After entering the cell, Ad escapes the endosome and enters the nucleus via microtubules using nuclear pore complexes to inject its genome. The pioneering Ad vectors were based on Ad serotypes 2 and 5, two closely related members of group C of human Ads. The most extensively tested “first-generation” vectors had a deletion in the E1 region of the virus. The E1 deletion served to create a replication deficient vector that had lower immunogenicity due to reduced viral protein production. E1 deletion also increased the vector’s available packaging capacity. Despite successful early indications in mouse models, the basolateral localization of the CAR proved to be a major limiting factor in the transduction of airways in large animals and humans (Table 1) (13,25,26). This severely limited the Ad vector’s entry into epithelia when applied to the apical surface. While several strategies have been devised to disrupt epithelial tight junctions and overcome this issue, including the use of

Bronchoscope instillation Phase I and aerosol/lung Instillation/nose or Phase I intrabronchial spray/lungs Aerosol/lung Phase I

Instillation/maxillary sinus

Instillation/nose or Phase I intrabronchial spray/lungs

Aerosol/lung

Aerosol/lung

Ad

AAV2

AAV2

AAV2

AAV2

AAV2

Phase II Phase I Phase II

Cationic liposome Aerosol/lung

Cationic polymer

12

10 Liposome 2 Placebo 8

Safe and dose-dependent gene DNA transfer; no mRNA detectable Safe; no change in sinusitis frequency or other endpoints Safe; humoral immune response observed. DNA detected, mRNA and ion channel correction detected in a follow ex vivo study. Safe; vector DNA detected. mRNA not detected; FEV1 at day 14 increase; IL-8 at day 14 decreased; no change in sputum bacterial cultures Safe and well tolerated; no significant improvement in FEV1 or IL-8. Safe; no mRNA or physiologic response detected Safely readministered three times; mRNA detected along with mild physiologic effects Acute innate response elicited. mRNA detected in 3/8 patients Safe: DNA detected in dose-dependent manner. Partial physiologic effect in 8/12 patients

Transient and inefficient expression; CTL responses detected in most subjects Transient and inefficient expression; systemic immune response Transient and inefficient expression; nonepithelial cell were also transduced Systemic responses at the high dose

Results

Konstan et al., 2004 (24)

Ruiz et al., 2001 (23)

Hyde et al., 2000 (22)

Noone et al., 2000 (21)

Moss et al., 2007 (20)

Moss et al., 2004 (19)

Wagner et al., 2002 (16) Flotte et al., 2003 and 2005 (17,18)

Perricone et al., 2001 (13) Harvey et al., 2002 (14) Aitken et al., 2001 (15)

Zuckerman et al., 1999 (11) Joseph et al., 2001 (12)

Reference

Abbreviations: Ad, adenovirus; CTL, cytotoxic T lymphocyte; AAV, adeno-associated virus; IL, interleukin; FEV1, forced expiratory volume in one second

Instillation/nose

20 AAV2 17 Placebo

25

23

12

34

14

36

11

Subjects

51 AAV2 51 Placebo Phase I-II 11

Phase II

Phase II

Phase II

Phase I

Cationic liposome Spray/nose

Cationic liposome Spray/nose

Ad

Ad

Phase I

Bronchoscope instillation/ lung Aerosol/lung

Ad

Phase

Route/target site

Vector

Table 1 Recent Representative Gene Therapy Clinical Trials for Cystic Fibrosis

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Ca2þ chelators EGTA (ethylene glycol tetraacetic acid) or EDTA (ethylene diamine tetraacetic acid) or to formulate the vector in a 0.1% a-lysophosphatidylcholine solution (27), these strategies may not be clinically relevant. Experiments using a-lysophosphatidylcholine solution aerosolized to the rabbit airway caused mild pneumonia and fever (27). Furthermore, the concept of epithelial disruption itself raises serious safety concerns for CF patients, as disruption of the epithelial barrier could allow dissemination of bacteria that are already present in the airway of these patients. First generation, E1-deleted Ad vectors also elicited Th1 and Th2 immune responses and caused dose-dependent inflammation and cytotoxicity (28,29). Most of the immunogenicity was attributed to the other viral genes present in the vector. To address this, second generation Ad vectors were modified to delete the E2A, E3, and E4 genes. Clinical trials using second generation Ad vectors expressing the full-length CFTR gene were conducted in six CF patients, where a dependent but transient (30 days) expression of CFTR was observed (30). Similarly in another trial involving eleven CF patients and second generation Ad vectors, CFTR expression was observed at low levels for 42 days (11). Unfortunately, readministration of the vector in the first trial did not achieve the original CFTR expression levels and no expression was observed with the third administration, strongly suggesting an immune response to the vector. In fact, in the latter study, Ad-specific CD8 T cells were recorded in the majority of the subjects (11) (Table 1). Despite these setbacks, significant experimental progress has been made with respect to Ad vector design. The latest generation of Ad vectors is termed “gutless” due to the complete absence of viral genes, and these vectors are essentially helper dependent, capable of packaging a 37 kb genetic payload. This increased packaging capacity can be used to create CFTR vectors under the control of more biologically relevant, epithelial promoters such as the human cytokeratin 18 promoter (31). Others have attempted to reduce the immunogenicity of Ad vectors by coating the capsids with polyethylene glycol (PEG). This “PEGylation” reduces the generation of NAbs and T-cell responses after intratracheal delivery (32). While these newer Ad-CFTR vectors have not been tested to date in human trials, Ad vectors have fallen out of favor for potential CF clinical use and have been superseded by rAAV vectors. However, there is renewed interest for Ad vectors in gene therapy approaches to lung and other cancers. B. rAAV Vectors

Adeno-associated viruses (AAVs) are parvoviruses with a linear 4.7 kb single-stranded DNA genome (33,34). Originally, AAV2 was discovered as a laboratory contaminant of Ad cultures (35,36), and was later found to be a frequent isolate among children experiencing an outbreak of Ad-induced diarrhea (37). AAV replicates only in the presence of Ad, herpes virus, or other helper viruses; these helpers supply a number of early functions required for AAV gene expression and replication (37,38). Subsequently, none of the AAV serotypes have been associated with human disease, despite the fact that 90% of adult humans are seropositive for one or more of the so far discovered serotypes (39). The AAV particle is a nonenveloped icosahedron with a diameter of approximately 25 nm. The AAV genome contains two genes, cap, which encodes the three overlapping coat proteins and rep, which encodes the four regulatory Rep proteins, flanked by two inverted terminal repeats (ITR’s) of 125 bases each (40). Naturally

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occurring variations of the cap gene account for the discovery of many different genomic variants and serotypes of wild-type (wt) AAV in recent years (41,42). In the absence of helper Ad, AAV enters a latent state where Rep inhibits viral gene expression and DNA replication, as well as promotes site-specific chromosomal integration (43). In the presence of helper virus, Rep promotes viral gene expression (44), DNA replication, and rescue from the integrated state (45). In AAV vectors, all viral genes are deleted and only the ITR sequences are retained; rAAV is thus a truly gutted vector (46). Variants in the cap gene and/or AAV serotype provide the flexibility of creating altered tropisms for vector transduction. Thus, it is common practice to transcapsidate (or pseudotype) AAV vector genomes with different AAV capsid serotypes by using different AAV packaging plasmids in which AAV2 rep genes are combined with capsid genes of the desired serotype (47–49). rAAV has had no detectable toxicities across a wide range of preclinical studies and in a limited number of clinical trials, even at the highest deliverable doses. Expression profiling of infected cells with microarray analysis indicates that host cell gene expression is remarkably unperturbed after rAAV infection. While some humoral immune responses to the rAAV capsid have been observed, cell-mediated immune responses to AAV are uncommon in the absence of Ad coinfection (50). The relatively low immune profile and lack of toxicity, as well as studies demonstrating gene transfer in nondividing cells and very efficient transduction of a wide range of terminally differentiated cells including neurons, retinal photoreceptor cells, myofibers, cells, hepatocytes, and bronchial epithelial cells, have placed rAAV at the top of the list for candidates for pulmonary gene therapy (43,51–54,55). The majority of preclinical (56,57) and clinical studies with rAAV were performed using first generation vectors with AAV serotype 2–based genomes and capsids. Because of the limited packaging capacity (*4.7 kb) of rAAV and the 4.44 kb coding sequence of CFTR, plus the mandatory 0.3 kb ITR sequence for packaging, these first generation vectors relied on the endogenous promoter elements within the ITRs to drive CFTR expression. Nonetheless, the preclinical studies have demonstrated both vector DNA transfer and mRNA expression for more than six months without any indication of inflammation, toxicity, or rescue of rAAV by wt AAV or wt Ad (56). These studies supported a series of clinical trials testing the delivery of the vector to the nose, maxillary sinus, isolated bronchus, and finally to the entire lower respiratory tract by aerosol delivery (17,19,58–62). The initial phase I clinical trial involving sequential intranasal and endobronchial instillation of rAAV2-CFTR in adult patients with CF demonstrated safety over a 7-log dose range (17,58). Although dose-dependent gene transfer to the airway epithelial cells was demonstrated in these trials, the longevity of gene transfer observed in animals was not evident in humans. However, subsequent studies suggested that there was a correlation between the presence of vector DNA sequences, mRNA expression, and electrophysiologic correction of cAMP-activated chloride flux in primary cells obtained during this trial (18). Clinical studies eventually progressed through phase IIb and were followed by phase II multicenter double-blinded, placebo-controlled trials focusing on repeated aerosol administration of rAAV-CFTR (20). However, significant limitations were noted due to the inconsistent ability to directly demonstrate expression in vivo, the lack of vector genome persistence, the absence of detectable improvement in pulmonary function as measured by forced expiratory volume in one second (FEV1), and finally the

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lack of efficacy of repeated dosing in the face of rising NAbs. While there was some indication of positive biological effects and DNA transfer, they were short in duration (30–60 days), and clinical studies with this particular vector were concluded after phase IIb studies, with over 100 subjects treated without a single vector-related significant adverse effect (Table 1). These clinical studies elucidated some of the limitations associated with rAAV vectors. These limitations were, in fact, very similar to those of Ad vectors, including paucity of the AAV2 receptors (heparan sulfate proteoglycan, aV-b5 integrins, and fibroblast growth factor receptor) on the apical surface of the airway epithelium. This inefficient delivery was likely compounded by the weak ITR promoter activity driving the CFTR gene and the development of neutralizing Abs that could effectively limit readministration of rAAV. Second generation rAAV vectors have tried to address these limitations. Use of the strong and constitutive CMV enhancer chicken b-actin hybrid promoter (CBA) may increase CFTR expression, but created a vector-packaging capacity conundrum. As previously mentioned, if the full-length CFTR is to be packaged in AAV there is little room offered for a promoter. To circumvent this, functional CFTR “mini genes” based on alternative mRNA splicing were defined with removal of the first 117 or 264 amino acids of CFTR (63). These mini-CFTRs were functional because only a small portion of the first transmembrane domain (TMD1) is crucial for CFTR stability and function as a chloride channel (64). One of the latest versions of the CFTR minigene termed “AAV-CBAD264CFTR” has been successfully delivered to CF cell lines where it corrected Cl
fluxes; furthermore, intratracheal delivery of this construct to Cftr
/
mice was shown to ameliorate the pathology and inflammation associated with pulmonary exposure to Pseudomonas-agarose beads (63). This vector construct has also been successfully used to partially correct a CFTR-dependent hyper-IgE phenotype modeled in CF mice (8). Preclinical studies after aerosol administration in rhesus monkeys demonstrated safe and efficient delivery to the lung (105 copies of CFTR/mg of DNA), with no signs of inflammation as compared with untreated controls (65). Recently this cassette has been modified to put back a small leader sequence composed of the first 26 amino acids of TMD1, which further enhances the cell membrane stability of the channel. The second generation rAAV vectors also capitalize on the divergent tropisms of newly isolated AAV serotypes (42,66). This allowed investigators to pseudotype rAAV vectors with capsids, such as those from AAV1, AAV5, and AAV6 that would bind receptors on the apical surface of the airway epithelium more efficiently (67–71). One promising candidate was rAAV5, which showed upwards of 10-fold increased efficiency of gene delivery in the mouse lung (72). Further studies in differentiated airway epithelia demonstrated disparate results between human airway epithelia and those observed in a variety of lower nonhuman primates and rodents (71,73). In particular, rAAV5-based vectors again performed very well in mice and in airway cultures from mouse and lower primate species; in contrast, rAAV1-based vectors performed significantly better in human airway cultures. The findings from the in vivo chimpanzee comparison are consistent with those obtained from human airway cell cultures and strongly suggest that rAAV1 has significant advantages, both in terms of overall higher efficiency and lower immunogenicity (data not published). Thus, it seems very likely that the next CF patient trial with AAV will use AAV1 as the indicated serotype to achieve more productive airway delivery.

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One fundamental issue that is not addressed by the latest vectors is the question of persistence of transgene expression. Persistence may inversely correlate with Nab responses since the less frequent the administration, the less robust the memory immune response will be. Persistence of rAAV genomes in transduced cells is primarily episomal, and the site-specific integration observed with wt AAV2 does not occur in the absence of Rep protein (74,75). For the purpose of treating CF, this is problematic since the natural turnover rate (2–3 months) of airway epithelia is up to four times faster in the CF lung. This more rapid cell division would eventually dilute out the therapeutic episomes. There are several promising avenues to overcome this issue. These include limiting the activity of DNA-dependent protein kinase (DNA-PKcs) that convert linear AAV genomes to circular forms, thereby inhibiting genome integration (76–78), and by including Rep in rAAV vectors to promote site-specific integration during transduction (65,79,80). These strategies to promote AAV genome integration, combined with recent data suggesting that rAAV1 and rAAV5 can transduce airway stem/progenitor cells (81), support the hypothesis that lifelong correction of the airway epithelium may be achievable. Recent technical advancements in packaging rAAV vectors that allow up to 8.9 kb of DNA to be packaged into rAAV1 and rAAV5 capsids (albeit at lower titers) (82) may enable future clinical trials to test vectors including the full-length CFTR gene along with a constitutive promoter and the site-specific integration system. Safe and sitespecific integration of full-length CFTR may help circumvent some of the complications associated with readministration, and may offer long-term therapeutic benefits to patients from a single vector administration. C. Lentiviral Vectors

A number of lentiviruses have been derivatized for development of gene transfer vectors for CF, including HIV. These vectors are potentially attractive because they have the capacity and ability to integrate into dividing and nondividing cells. Unfortunately, this virus has low tropism for the apical surface of the airway epithelium (83). However, pseudotyping of lentiviral vectors has been used to enhance gene transfer into the airway cells. One example is the creation of a hybrid vector with Ebola enveloped glycoprotein that increased the efficiency of transduction via the apical surface of the epithelium (84). Promising preclinical data with CFTR vectors have been achieved with cell culture, rodent, and nonhuman primates. Clinical studies have not yet been performed. Although a promising viral vector delivery system, these viral vectors are plagued by their high rate of random integration in the host genome that has led to two patients developing T-cell leukemia in human gene therapy trials for severe combined immune deficiency (SCID) (85). D. Nonviral Vectors

Historically, nonviral vectors including cationic liposomes (lipoplexes) or cationic polymers (polyplexes) have resulted in less efficient airway epithelial gene expression when compared with viral vectors. Transgene expression is generally transient using these systems. Furthermore, while in theory these vehicles should avoid adaptive immune responses, innate immune responses have turned out to be a major hindrance. Recent advances in these vectors and a more comprehensive understanding of the innate responses, have led to animal and clinical studies with promising results using both cationic liposomes and polymers.

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Cationic Liposomes

Cationic liposome delivery systems are composed of cationic lipids that are usually mixed with cholesterol and dioleoylphosphatidyl-ethanolamine (DOPE). The liposomes bind to plasmid DNA via electrostatic interactions, and the resulting complexes are positively charged particles of 100 to 500 nm in diameter that can fuse with cell membranes and enter cells. The DNA/lipid complexes are then resistant to nuclease degradation and, unlike viral vectors, have no limit to the size of DNA that can be delivered. This allows tissue-specific promoters and locus control regions to be delivered along with the gene of interest, making the gene expression of the therapeutic protein more biologically accurate. Cationic liposomes have demonstrated both in vitro and in vivo efficacy. Formulation GL-67 (Genzyme) had 100-fold greater efficiency over other liposomes in the mouse lung (86) (Table 1), but had in vivo toxicity associated with pulmonary infiltration of neutrophils and to a lesser extent macrophages and lymphocytes, as well as elevated lung levels of the proinflammatory cytokines IL-6, TNF-a, and IFN-g in BALB/C mice (87). In human studies, delivery of GL-67 cationic liposomes containing the CFTR gene by a nasal perfusion to CF patients resulted in partial correction of nasal potential differences (for discussion of this technique, see chap. 8). However, CFTR expression could not be directly verified by the detection of CFTR mRNA in these studies, and subjects in this study developed fever and increased levels of IL-6 (88). These side effects were attributed to innate inflammatory responses orchestrated by TLR 9 in response to the recognition of bacterially derived unmethylated CpG repeats in the plasmid DNA. The immune response to GL-67 was further characterized in another study by Ruiz et al. in which he performed a dose escalation with aerosolized delivery. Subjects who had GL-67 liposome/DNA complexes delivered by aerosol also had fever and increased serum levels of IL-6 as soon as one hour after gene administration, perhaps due to a synergistic response to plasmid DNA complexed with GL-67 (23). Strategies that have been employed to reduce inflammatory stimulation by these vectors include the reduction of nonessential CpG sequences or the use of chloroquine as an inhibitor of the CpG signaling pathway (89,90). Alternatively, methylation of the CpG motifs, while successful at reducing inflammation, was associated with a severe reduction in transgene expression (91). F.

Cationic Polymers

Cationic polymers, such as poly-L-lysine (polyK), polyethyleneimine (PEI), and polyamidoamine dedrimers have also been examined as nonviral vectors for CF gene therapy. In a fashion similar to cationic liposomes, these polymers are able to condense DNA into small nuclease-resistant particles. Because of their net positive charge, the polymers can bind to cells via electrostatic interactions with the negatively charged membrane. In addition, PEI’s endosomal release is facilitated because their branched amines act as proton sinks that cause endosomes to swell and burst (92). In mice, PEI vectors preferentially localize to the lung after intravenous delivery. Transfection efficiency was 1% to 5% and restricted to the distal and branched airways close to the alveoli, including pneumocytes and endothelial cells but few epithelial cells (93). In

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contrast, epithelial cells were primarily transduced after aerosol delivery to the mice lungs (94). Unfortunately, high molecular weight PEI complexes are not biodegradable and PEI’s transduction efficiency has been tied to endosomal rupture, which causes toxicity both in vitro and in vivo (95,96). For these and additional reasons, cationic polymer delivery has recently focused more on polymers of L-amino acids, such as lysine (polyK). These polymers can be conjugated with targeting peptides for cell-specific and enhanced uptake. Although manufacturing large quantities of peptides for large animals may be prohibitively costly, this approach has been tested in mice. Specifically, the human CFTR gene was delivered to mouse lungs using polyK conjugated with a ligand to the serpin-enzyme complex receptor (SecR). SecR is found on hepatocytes, macrophages, and the apical surface of airway epithelia where it usually binds and promotes internalization of protease inhibitors once they have bound to and inactivated their ligands. While vector expression was transient (*12 days), there was a partial correction in chloride secretion (97). Although immune responses to polyK were not observed, there was an antibody response to the SecR ligand, once again hindering the possibility of readministration. Similar to the strategy used to create “stealth” (or PEGylated) Ad vectors, PEG has been used to modify the side chains of polyK. This allowed the polyK complexes to remain stable in physiological (normal) saline instead of the usual 1 M NaCl solution, and diminished the CpG-mediated immune responses; this contributed to a lack of in vivo toxicity (98). Delivery of PEGylated polyK CFTR complexes to the nasal airways of 12 CF patients in 3 escalating doses led to nasal potential difference correction in 8 of the 12 patients for up to six days. No adverse events were associated with this treatment (24) (Table 1).

III.

Conclusions

The data reviewed in this chapter highlight the significant physical and immunological hurdles that have been encountered in the CF gene therapy field. Major barriers for effective viral gene therapy include viral pathogenicity and CTL clearance in the case of Ad vectors. Repeated administrations of viral vectors, which are crucial for a disease such as CF are currently being hindered by NAbs in both Ad and AAV delivery methods. Nonviral delivery systems have to surmount CpG-associated inflammation, cell toxicity, and low gene transfer efficiency. Importantly, the lessons learned from clinical trials have to be adapted to novel ideas from the bench to further evolve currently available gene delivery systems. In this aspect, newer versions of AAV vectors should incorporate strategies or elements that help overcome these hurdles, such as facilitating site-specific integration. This would not only help bring lifelong therapy within a realistic grasp, but could also be directly applied to cell-based therapy approaches for CF. Recent data also suggest that CF gene therapy must consider the correction of alternative nonepithelial cell types. While CFTR gene therapy has naturally focused on the correction of airway epithelia, recent studies have suggested that the CFTR defect may affect the ability of immune cells in the lung to clear pathogens (99). Also, intratracheal delivery of AAV5-CB-D264CFTR and CFTR correction in the lung was associated with a correction of aberrant cytokine secretion from splenocytes in Cftr-

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deficient mice (6). Thus, it is conceivable that the CFTR defect may also be playing an intrinsic role in the integrity, effectiveness, and homeostatic balance of the immune system. Nonepithelial CF gene therapy is a relatively untested hypothetical target for CFTR gene correction that may potentially offer new possibilities for cell-based therapies, and may compliment the new generation of gene therapy vectors currently in the clinical pipeline.

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25 Restoration of CFTR Function with Small-Molecule Modulators WYNTON HOOVER and JOHN P. CLANCY University of Alabama at Birmingham and Children’s Hospital of Alabama, Birmingham, Alabama, U.S.A.

I.

Introduction—Cystic Fibrosis Therapeutic Strategies That Target Disease Symptoms and Disease Causes Current therapies for CF address the symptoms of the disease, and their systematic use has lead to steady and remarkable improvements in patient longevity. The rationale for all approved therapies is rooted in reducing disease symptoms, despite differences in how and when they are applied (1). For example, mucus obstruction and airway plugging is addressed by airway clearance methodologies, such as daily chest physiotherapy, nebulization of rhDNase (Dornase alfa, to reduce sputum viscosity), and more recently the addition of nebulized hypertonic saline (to hydrate mucus) as part of daily treatment regimens (2). Bacterial infections are treated with pathogen-directed antibiotics, and the choice of delivery route and antibiotic class is based on the infectious agent and the nature of the infection. Intravenous antibiotics directed toward Pseudomonas aeruginosa during pulmonary exacerbations helps restore lung function. Alternatively, cycled aerosolized tobramycin in patients with chronic P. aeruginosa infection suppresses bacterial growth and stabilizes lung function (2). New symptom-based therapies under development will continue to allow CF healthcare providers and patients to choose from diverse and effective treatment options for many years to come. An exciting and novel strategy to treat CF is to target the underlying cause of the disease, providing sufficient expression and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) to CF-affected cells to reduce or reverse their diseaserelated phenotype. This can theoretically be accomplished by three general strategies, including (i) replacement of the disease-causing gene with a normal CFTR gene [i.e., gene transfer or gene therapy (3,4), see also chap. 24], (ii) use of small molecules to overcome the underlying cellular mechanism(s) responsible for manifestation of specific CF-causing mutations (5–15), and (iii) progenitor cell-based regeneration of the airway epithelium [i.e., stem cells (16–18)]. Small-molecule therapy is the focus of this chapter and has drawn directly from large amounts of in vitro data characterizing common and uncommon CFTR mutations. It has also benefited from powerful drug discovery technologies such as high throughput screening (HTS) of combinatorial drug libraries to identify lead compounds for further development (5,6,13,19–23). These screening programs have been coupled with elegant in vitro and in vivo model systems to test candidate compounds, including the isolation

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and culture of primary human airway tissues from CF patients that faithfully recapitulate disease features (24) and the development/use of transgenic mouse models with CF disease-causing mutations (14,25–27). CF is particularly well suited for this restorative approach since the underlying genetic defect produces a recessive loss of Cl
channel activity rather than a dominant defect that disrupts normal CFTR function. Therefore, small differences in CFTR activity are sufficient to produce significant and meaningful differences in disease manifestations. As an example, comparisons of clinical outcomes between CF patients with and without pancreatic sufficiency suggest that retained pancreatic function predicts a significantly milder clinical course compared with patients with exocrine pancreatic dysfunction (28–31). Additionally, patients with “CFTR-opathies” such as congenital bilateral absence of the vas deferens, recurrent pancreatitis, pancreaticsufficient CF (caused by “mild mutations” with residual function), and pancreaticinsufficient CF (caused by “severe mutations”) demonstrate a hierarchy that relates CFTR function to organ manifestations across these different disease entities (29–32). Parallel in vitro and in vivo studies suggest that the amount of total CFTR activity necessary to convert a severe phenotype to a mild phenotype may be as little as 5% to 15% (32–35). Thus, agents that only achieve partial restoration of CFTR function may be quite clinically beneficial, particularly in the context of minimal background CFTR activity (i.e., patients with CF caused by two severe mutations).

II.

Classes of Cystic Fibrosis–Causing Mutations: Different Problems Need Different Strategies As is the case for most genetic diseases, the clinical manifestations of CF result from a number of mutations within the source gene. The absolute number of potential mutations is nearly limitless, but thankfully the mutations can often be grouped together in terms of basic causative mechanisms. While these specific groupings can appear arbitrary, the members within a mutation class share important defective features and often lend themselves to common strategies for functional restoration. Figure 1 provides a summary of the six known mutation classes in CF. These include class I (defects in biosynthesis), class II (defective protein processing), class III (regulatory defects), class IV (reduced Cl
channel conductance), class V (reduced levels of CFTR transcripts), and class VI (increased cell membrane turnover). It is apparent from this segregation that distinctly different approaches are needed to address the fundamental problems responsible for the mutations included within different classes. However, it has also become clear that many mutations are not necessarily restricted to a single class (i.e., they may manifest defects across one or more mutation classes). Furthermore, strategies that are under development for members of one mutational class may be logically applied to mutations within a separate class. Thus, the mutational class system, while helpful for general purposes of categorization, has become “muddy” and is not fully descriptive of mutation manifestations. III.

Suppression of Premature Termination Codons: Translational Readthrough Produced by Small Molecules Base pair substitutions, insertions, or deletions that create premature termination codons (PTCs) are commonly found across most genetic disorders and have been identified in

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Figure 1 Mutation classes in CF—summary of cellular mechanisms underlying different CF-

associated mutations. The figure summarizes the different classes of CFTR mutations in relation to different cellular compartments and processes (far left). The cell to the left demonstrates defects that produce severe CFTR dysfunction, including class I mutations that lead to biosynthetic defects in CFTR expression [such as nonsense (i.e., G542X, R553X, R1162X, or W1282X CFTR) and frameshift mutations], class II mutations that lead to defective protein folding and maturation (including the common DF508 CFTR mutation, which is normally targeted to the proteosome for degradation), and class III mutations that disrupt normal regulation of CFTR (including G551D, G551S, and G1349D CFTR, all of which affect ATP binding and hydrolysis and subsequent CFTR Cl
channel gating). The cell to the right summarizes CFTR mutation classes that typically do not produce a severe reduction in CFTR activity, including class IV mutations that reduce single channel conductance (such as R117H, R334W, R347P, and R347H CFTR), class V mutations that depress the transcript levels of CFTR (such as 3849 þ 10 kb C ? T CFTR, wtCFTR in cis with 5T CFTR, promoter, and intron mutations that reduce transcript splicing efficiency), and class VI mutations that alter the normal membrane endosomal recycling of CFTR. These class designations are not mutually exclusive for many of the described CFTR mutations. For example, DF508 CFTR (which is typically considered a class II mutation) also demonstrates altered channel gating (class III) and reduced stability at the cell surface (class VI) when localized to the plasma membrane. The legend at the top of the figure identifies the different aspects of CFTR transcription, translation, and protein maturation. Abbreviations: TGN, trans-Golgi network, ER, endoplasmic reticulum, NMD, nonsense-mediated decay; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator. Source: Adapted from Ref. 36.

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over 1800 separate medical conditions (37–39). PTCs halt translation by ribosomes, resulting in the production of abnormally truncated proteins that are generally dysfunctional. In addition, genes with PTC mutations are often associated with low levels of mRNA transcripts. These mRNAs often are more rapidly degraded through a process called nonsense-mediated decay (NMD), resulting in further loss of protein activity (40). Our increased understanding of ribosomal function and the regulatory features of translation as well as the identification of small molecule interactions between the ribosome and mRNA have helped to identify agents that “suppress” PTCs. These small molecules allow the ribosomal translation machinery to occasionally “read through” a PTC resulting in translation of a measurable amount of full-length and functional protein product (39). From this basic understanding, novel CF treatment strategies to restore protein synthesis and function through PTC suppression have emerged. Aminoglycosides were the first agents demonstrated to suppress PTCs that were responsible for disease-causing mutations. Aminoglycosides possess antimicrobial activity against many gram-negative bacteria, select gram-positive bacteria, and nontuberculous mycobacteria by interfering with ribosomal translation (41). There are two major classes of aminoglycosides, discriminated by 4,5- and 4,6-disubstituted 2-deoxystreptamine residues connected to an amino sugar backbone, respectively (42). The largest group includes the 4,6-disubstituted 2-deoxystreptamine derivatives, gentamicin, tobramycin, and amikacin. These aminoglycosides interfere with normal translation via binding to the decoding site within an internal loop of the bacterial 16S rRNA (43). This reduces discrimination between cognate and near cognate tRNA-mRNA complexes, consequently reducing translational fidelity (44,45). The end result is the accumulation of dysfunctional and/or truncated bacterial proteins, which is ultimately bacteriocidal. Mammalian and bacterial ribosomes have fundamental differences in their nucleotide sequences within the segment of the small ribosomal subunit to which the aminoglycosides bind; this confers selective activity of aminoglycosides for their bacterial target (46). Despite these differences, aminoglycosides also have the capacity to bind the human 18S rRNA subunit, albeit with lower affinity. This loose interaction can reduce discrimination of near cognate tRNAs by the ribosome (47–49). While this interaction is less stable than that in bacteria, it is sufficient to occasionally disrupt the normal proofreading function of the ribosome and allow insertion of a near cognate aminoacyltRNA into the growing polypeptide chain at a PTC (39). The result is suppression of the PTC and production of a full-length protein. The first studies capitalizing on PTC suppression to restore function to a diseasecausing mutation in mammalian cells was reported by Howard and Bedwell (15,39,50). They examined the ability of geneticin (G418) to suppress PTCs in cells transiently expressing cDNAs containing the four most common disease-causing PTC or “X” mutations (G542X, R553X, R1162X, and W1282X) in the CFTR gene; these data for W1282X were later confirmed in the immortalized IB3-1 bronchiolar cell line isolated from a W1282X compound heterozygous CF patient. Additionally, gentamicin, a commonly used aminoglycoside in clinical care, was found to allow readthrough of X mutations in CFTR and lead to full-length, functional protein production. Two mouse models possessing PTCs in disease-causing genes, including the mdx mouse (a model of muscular dystrophy) and the transgenic G542X-hCFTR mouse (a model of cystic fibrosis) have been examined in preclinical in vivo studies. Suppression

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of the PTC within the dystrophin gene of the mdx mouse with systemically administered gentamicin lead to increased expression of full-length dystrophin protein, improved muscle strength, and resistance to stress-induced injury (12). Similarly, gentamicin increased full-length CFTR expression and function in the gut of G542X-hCFTR mice (14). These mice, which express the human G542X CFTR cDNA under regulatory control of the intestine-specific FABP promoter on a Cftr knockout background, also had a trend toward improved survival with gentamicin treatment. Later reports confirmed these effects with clinically relevant doses of both gentamicin and amikacin in this mouse model and other model systems, with amikacin demonstrating somewhat greater efficacy (26,51). Recently reported studies in the CF and mdx mouse models have described effective translational readthrough and protein rescue with the novel stop codon suppressive agent PTC124. PTC124 was developed by PTC Therapeutics through an HTS program, and is a 1,2,4-oxadiazole benzoic acid derivative. PTC124 interacts with mammalian ribosomes in a fashion distinct from that described for aminoglycosides and has no reported antimicrobial activity (19). Systemic dosing studies demonstrated that PTC124 rescued dystrophin localization to the cell membrane of skeletal and cardiac myotubules in the mdx mouse (19). In the G542X-hCFTR mouse, enteral and intraperitoneal administration led to detectable full-length CFTR at the apical cell membrane of gut glandular cells by immunohistochemical staining, and improved CFTR activity to approximately 20% to 30% of that seen in wild-type (wt) mice (14,27). These findings suggest that significant clinical benefits of this strategy may be recognized with partial restoration of protein function, thus potentially attenuating the most severe clinical manifestations of CF (32). Translational studies and clinical trials of PTC suppressors have tested both proof of principle and explored efficacy. Many, but not all of these studies have demonstrated biologic activity in humans, which has raised important questions regarding factors that influence individual responses. Wilschanski and colleagues in Israel initially reported that topical gentamicin applied to the nasal mucosa of CF patients with PTCs (most of whom possessed W1282X CFTR, which is highly prevalent in Ashkenazi Jews) improved CFTR activity and membrane localization as assayed by the nasal potential difference (NPD) (see chap. 8) (52). These effects were confirmed in a follow-up study, and were not seen in CF patients homozygous for the DF508 CFTR mutation (11). Improvements in CFTR activity were not seen universally across treated patients, and subsequent studies demonstrated that responders had measurably higher CFTR transcript levels relative to nonresponding study subjects (53). Sermet and colleagues confirmed these data in CF subjects (8 out of 9 subjects were homozygous for the Y122X mutation) using systemic gentamicin, demonstrating improvements in NPD measurements (and reduction in sweat chloride values). Their work also provided a hierarchy for response based on the specific PTC possessed by study subjects (54). A larger multicenter trial of topical (nasal) gentamicin and tobramycin across a more genetically diverse CF population (with several different PTCs in CFTR represented) did not, however, demonstrate improvements in the NPD (55). A number of potential reasons for these discrepant results have been postulated, such as variable activity of gentamicin to suppress different PTCs in CFTR, variability of the NPD outcome measure when used in multicenter trials, and potentially different NMD rates across the study populations. In contrast, a recent open label clinical trial of oral PTC124

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in CF patients possessing a number of different PTCs reported improvements in CFTR activity while on study drug (56). A summary of CF clinical trials investigating suppression of PTCs is provided in Table 1. Despite these inconsistencies in biologic efficacy across clinical studies, suppression of PTCs is emerging as an exciting strategy to treat CF and a number of different genetic disorders. This approach is built on a growing foundation of work examining basic mechanisms of PTC suppression and exploring suppressive activities of aminoglycosides and novel agents in the laboratory and the clinic. These studies have helped to define important additional questions, including what factors underlie variable results across different studies. Also highlighted is the need to establish and/or refine standardized CFTR biomarkers that are sensitive and specific for translational readthrough, including CFTR activity (NPD, sweat Cl
) and expression (quantification of CFTR transcripts and detection of mature, full-length protein). This will ensure that multicenter studies will provide fully interpretable data when there is a paucity of patients possessing these mutations (58).

Restoring Function to DF508 CFTR: Correction, Activation, and Stabilization Deletion of phenylalanine from position 508 of CFTR (DF508 CFTR) is the most common mutation that causes cystic fibrosis (1,36, 59–61). DF508 CFTR is found in approximately 70% of CF-causing chromosomes and at least one copy is found in nearly 90% of CF patients. Because of its severe dysfunction and high prevalence, it is the most logical target for restorative strategies. During the normal maturation of CFTR, the nascent protein goes through a series of folding and glycosylation steps as it traverses the cell’s protein production machinery and traffics to the apical epithelial membrane (59). Immature CFTR has core glycosylation, which is demonstrated biochemically as an approximately 140 to 150 kDa protein (“A band” CFTR). Later steps in maturation lead to N-glycosylation of distinct amino acids in extracellular loop 4 producing “B band” and finally fully mature “C band” CFTR (with molecular weights of *165 and 180 kDa, respectively). In contrast, DF508 CFTR is a temperature-sensitive mutation (62); at 378C, DF508 CFTR accumulates in the immature B band form and produces minimal (if any) C band CFTR and is degraded by the 26S proteosome (59,63,64). Reductions of the growth temperature lead to the appearance of fully mature C band DF508 CFTR. Chemical agents that allow DF508 CFTR to mature to the C band form are termed DF508 CFTR correctors. This refers to the belief that they correct the folding and trafficking defect inherent to the mutant protein and lead to increased amounts of mature DF508 CFTR at the cell membrane. These “corrective” agents may act directly on DF508 CFTR to normalize folding, on protein binding partners to encourage correct folding or ignore incorrect folding, or potentiate effects that block DF508 CFTR degradation. It is also possible that these agents could have effects on other aspects of CFTR biology and metabolism (such as plasma membrane stability, recycling, etc.). In the absence of structural information regarding CFTR, initial corrective strategies were nonspecific and included correction by growth in low-temperature or smallmolecule osmolytes such as glycerol (62,65). With more recent data suggesting that DF508 is found on an external surface of the nucleotide-binding domain (NBD)-1, and is IV.

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Table 1 CF Clinical Trials of PTC Suppression

Reference Trial design 52

Open label

Study subjects CF

Results l

l

11 with PTC l

11

57

PlaceboCF: l controlled l and blinded

Open label

l l

l

Improvements in CFTR activity by NPD (Cl
and Naþ transport) and immunofluorescence while on topical nasal gentamicin

l

NPD effects specific for PTC group

l

Majority of patients with W1282X

l

Improvements in CFTR activity by NPD (Cl
transport) while on topical nasal gentamicin

l

NPD effects specific for PTC group

l

Ex vivo gentamicin dose/response effects (improved CFTR trafficking and function) in primary nasal airway cells from PTC patients 3 different PTC mutations represented

19 with PTC 5 DF508/DF508

CF: 5 with PTC 5 without PTC 5 non-CF

l

54

Open label

CF: l

l

9 with PTC l

55

56

Double crossover, double blinded

CF:

Open label, dose ascending

CF:

l l

Hierarchy of treatment effect across mutations shown

l

No improvements seen in CFTR activity by NPD during treatment with either gentamicin or tobramycin 6 different PTC mutations represented across 5 study sites

11 with PTC 18 without PTC

l

l

23 with PTC (part I) 21 with PTC (from part I, included in II)

Improvements in CFTR activity by NPD (Cl
transport) and sweat Cl
while receiving gentamicin All subjects with Y122X

l

l

l

Improvements in CFTR activity by NPD while on topical nasal gentamicin Majority of patients with W1282X

l l l

Improvements in CFTR activity by NPD (Cl
transport) during treatment No clear dose/response No effect on sweat Cl
3 PTC mutations represented

Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; NPD, nasal potential difference; PTC, premature termination codons.

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believed to interrupt normal interactions between the cytoplasmic loops of membrane spanning domains with the NBD motifs, corrector development strategies based on detailed structural information are becoming possible (59,66,67). This approach is attractive as it targets the underlying defects in DF508 CFTR, and is perhaps less likely to have off-target effects. The first clinical study to demonstrate evidence of DF508 CFTR correction was published by Rubenstein and Zeitlin (68). In their study, they applied basic laboratory observations made about butyrate compounds to correct DF508 CFTR (69) developing and executing a proof of principle trial of an approved oral butyrate compound (4phenylbutyrate or 4-PBA) in DF508 CFTR homozygous CF patients. Their results indicated that CF patients treated with 4-PBA had detectable improvements in CFTR activity by the NPD relative to placebo-treated controls. While the study was not designed to address clinical outcome measures beyond safety, it provided important proof of concept information and also provided support for the concept that small molecules could overcome CFTR defects and that an in vivo biomarker of CFTR activity (NPD) was capable of detecting biologic activity of DF508 CFTR corrective agents. A separate multicenter study of a different putative DF508 CFTR-activating compound (8-cyclopentyl-1,3-dipropylxanthine or CPX) was published by McCarty and colleagues (70,71). This study was also derived from investigator-initiated, laboratorybased observations (72–76). While the study did not detect biologic effects of CPX, it did provide important information about the challenges and complexities of using CFTR biomarkers in multicenter clinical trials. Another approach to identifying agents that promote increased levels of DF508 CFTR at the cell membrane is to apply therapies developed for non-CF uses to CF building off described drug effects that may overlap with the cellular pathology characteristic of cells that express DF508 CFTR. Bortezomib (Velcade1) is a known proteosome inhibitor developed for the treatment of multiple myeloma and other cancers. In vitro studies have suggested that this may be a means to increase the cell membrane levels of DF508 CFTR by inhibiting its degradation and altering its interactions with binding partners in the protein-folding pathway (77). Other approved pharmacologic agents developed for non-CF clinical uses that correct DF508 CFTR activity include sildenafil [a phosphodiesterase (PDE)5 inhibitor] and miglustat (Zavesca or Nbutyldeoxynojirimycin, a treatment for Gaucher’s disease) (78–83). This “low-hanging fruit” approach is attractive as the agents have well-described dosing ranges and toxicity profiles in humans. CF-specific clinical trials of these approved agents require assessment of pharmacokinetics (due to the frequently altered drug absorption and metabolism seen in CF patients) and careful consideration of CFTR biomarkers to detect biologic effects. Finally, certain nutritional supplements may have DF508 CFTR corrective activity. One recent example of this is curcumin, a spice that binds to SERCA pumps (sarcoplasmic/endoplasmic reticulum calcium) in the ER and lowers intra-ER calcium levels. Since calcium is a common and necessary cofactor for a number of chaperone proteins within the ER that regulate protein maturation, Egan and colleagues examined whether curcumin had effects on DF508 CFTR maturation and activity in cell lines and in transgenic DF508 CFTR mice (84). Their results indicated that curcumin rescued DF508 CFTR activity in vitro and in vivo, with evidence of improved survival of mice possessing the DF508 CFTR mutation with curcumin treatment. While subsequent

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studies have demonstrated that curcumin is capable of activating cell membrane CFTR, other laboratories were not able to replicate the corrective effects of curcumin in CF mice, perhaps due to differences in the mouse models used or other unidentified factors (85–89). Nevertheless, the positive results lead to dose ranging and proof-of-concept clinical trials of curcumin in DF508 homozygous CF patients. Preliminary reports suggested that there may be some detectable bioactivity based on secondary NPD analysis. These findings were difficult to rectify, however, due to minimally detectable levels of curcumin in the CF patients and the variability of NPD responses (90). Whether these inconsistencies could represent the effect of curcumin metabolites or other curcumin isoforms found in the curcumin preparations is currently unknown, and poses a barrier when bringing nutritional supplements to CF clinical trials. Further preclinical studies are likely necessary to address these questions before additional clinical trials will be conducted in CF patients. Programs designed to identify novel small-molecule “correctors” of DF508 CFTR were initiated nearly 10 years ago. These programs used sensitive and specific fluorescent readouts in living cultured cells to allow HTS of large compound libraries. Primary hits were typically confirmed in vitro, and a process of compound optimization followed. This work has lead to the identification of a number of novel CFTR-active compounds including putative CFTR correctors, potentiators (discussed in later sections of this chapter), and blockers (5,10,20,21,24,91). While the primary defect in DF508 CFTR is due to misfolding and mistrafficking, additional defects have been described in DF508 CFTR following its localization to the cell membrane including defective channel gating. DF508 CFTR’s open probability has been reported to be less than 0.10 by Hwang and colleagues, compared with nearly 0.50 for wtCFTR following activation by ATP and protein kinase A (92,93). This low singlechannel open probability remains present after low-temperature correction (89,94). These single-channel defects manifest as reduced Cl
conductance across DF508 CFTR-expressing airway cell monolayers following low-temperature correction (95). Full DF508 CFTR activation typically requires stimulation with a cAMP-independent CFTR activator in vitro (such as the flavonoid genistein, 50 mM), and suggests that the vast majority of DF508 CFTR activity may not be detectable by standard CFTRactivating maneuvers. These results have relevance, as they suggest that current CFTR biomarkers (NPD) used in clinical trials of DF508 CFTR correctors may be insensitive to detect DF508 CFTR at the cell membrane (58,96,97). DF508 CFTR also has a reduced cell membrane half-life relative to wtCFTR (98). This may result from accelerated endocytosis and shuttling of DF508 CFTR to the endosomal compartment rather than reduced recycling back to the cell membrane. Interestingly, a report by Varga demonstrated that a chemical corrector of DF508 CFTR improved the cell membrane stability of DF508 CFTR after correction by low temperature (99). These results suggest that corrective agents may be able to improve DF508 CFTR function beyond its well-described maturation defect. The results of studies examining correction of DF508 CFTR, its activity at the cell membrane, and the mutant proteins’ membrane stability after correction collectively provide strong support to explore treatment strategies that address the DF508 CFTR defects in maturation, activation, and stabilization. While the amount of CFTR activity that is necessary to improve clinical parameters in CF patients may be relatively low, it

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seems unlikely that DF508 CFTR correctors alone will achieve non-CF functional levels. Thus, maximizing the activity of corrected DF508 CFTR will likely be necessary for full clinical benefit. Whether this can be accomplished by single agents or will require combinational therapies is unknown.

V. Potentiation of Mutant CFTR at the Cell Membrane: Primary and Supplemental Strategic Targets The final class of defects in mutant CFTR activity can broadly be described as defects in CFTR when the protein is present at the cell membrane. There are several different potential mechanisms that could account for this phenotype, including defects in CFTR regulation (e.g., G551D, G551S, and G1349D CFTR), defects in CFTR conductance (e. g., R117H, R334W, R347P, and R347H CFTR), defects in CFTR splicing and/or promoter regulation that lead to reduced amounts of CFTR mRNA transcripts (e.g., 3849 + 10 kb C ? T CFTR, wtCFTR in cis with 5T) (see chap. 1), and mutations that reduce the stability or half-life of CFTR at the cell membrane. As the number of CF patients who carry mutations that fall into each of these different classes is limited, it is not feasible to develop independent strategies that are specific for each mutation described. Thus, most approaches to “potentiate” the activity of mutant CFTR at the cell membrane have considered these defects collectively, in addition to secondary defects in CFTR activity that may become manifest following treatment of an underlying primary defect (e.g., the resultant CFTR molecules that are produced following suppression of PTCs, or the activity of DF508 CFTR following correction of its maturation defect). Whether potentiating strategies can be applied across the described surface localizing CFTR mutations is unknown and should be considered in the process of drug development and testing. Two classes of molecules were initially shown to potentiate the activity of mutant CFTR at the cell membrane. First, high concentrations of xanthine-based PDE inhibitors (e.g., IBMX) increased the chloride conductance of G551D CFTR and temperaturecorrected DF508 CFTR (100,101). The concentrations necessary for this potentiating effect (>1 mM) were not feasible for clinical investigation, and this effect on surfacelocalized mutant CFTR is not clearly related to its PDE inhibitory effect. A series of studies by Drumm and colleagues subsequently reported that milrinone, a nonxanthinebased PDE3 inhibitor was capable of activating DF508 CFTR in human airway cells and in transgenic CF mice (102–104). A follow-up NPD-based study of topical milrinone in human subjects demonstrated that it was sufficient to activate wtCFTR-dependent chloride conductance in non-CF subjects, but it had no detectable effect on chloride conductance in CF subjects possessing the G551D CFTR or two copies of the DF508 CFTR mutation (105). The second type of agent capable of potentiating CFTR activity includes flavonoids, a class of molecules that are commonly found in a variety of fruits and vegetables. Several members of this class can activate CFTR, with genistein demonstrating the most consistent activating effects on wt and mutant CFTRs (including G551D CFTR and DF508 CFTR at the cell membrane). The mechanism of activation appears to be independent of cAMP and R domain phosphorylation, and may involve direct interactions with the NBDs (92,93). NPD studies of topical flavonoids in non-CF and CF patients have been reported by Illek and colleagues, in which genistein (and another

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flavonoid—quercetin) were shown to activate wt and G551D CFTR-dependent Cl
transport when applied topically to the nasal mucosa (106). As expected, this agent was unable to activate Cl
transport in CF subjects homozygous for the DF508 CFTR mutation. In parallel to HTS programs designed to identify agents that correct DF508 CFTR processing, screening programs have identified novel compounds that potentiate wt and/ or mutant CFTR activity at the cell membrane (6,23). The mechanisms of action for many of these compounds are not fully described, but some, like genistein, appear to act independently of CFTR phosphorylation. The most logical initial target for examining potentiating compounds in human subjects is G551D CFTR; the second most common mutation that causes CF. The G551D mutation severely disrupts gating of the CFTR chloride channel. The processing and membrane stability of G551D CFTR, however, is normal, and thus it represents an available target for testing CFTR potentiating compounds. One agent under development by Vertex Pharmaceuticals (Cambridge, Massachusetts, U.S.), VX-770, has entered dose ranging and safety clinical trials in CF patients. These studies are forced to enroll a limited number of study subjects due to the relatively low number of CF patients that possess the mutation (estimated at less than 5% of the CF population). A phase IIA study of oral VX-770 in CF patients possessing at least one copy of the G551D CFTR mutation suggested that there were dosedependent improvements in CFTR activity (as assessed by sweat chloride and NPD) during two and four weeks of treatment. These improvements were sufficient to fall outside of the CF range for a number of the study subjects. The improvements in CFTR biomarker measures were accompanied by improvements in lung function (FEV1) and trending improvements in patient-reported outcomes (PROs), and together provide strong proof of principle to support the development of new therapeutics that target mutant CFTR (www.clinicaltrials.gov—NCT00457821) (107). The number of CF patients who might benefit from potentiator monotherapy is unknown but may be quite sizeable, with estimates as high as 30% of CF patients. These potential patients would include those with well-described, surface-localizing CFTR mutations that fall into classes III to VI (which cumulatively include *15–20% of CF patients). In addition, investigators have provided evidence that activity of DF508 CFTR can be detected by NPD and/or by ex vivo intestinal current measurements in rectal biopsy tissue from a subgroup of CF patients who are homozygous for the DF508 CFTR mutation (108–110). Standardization of CFTR biomarkers (such as sweat chloride and NPD performance) and the development of new sensitive biomarkers (such as protein biochemistry and functional assessment of rectal biopsy tissue) are warranted as these drugs-in-development progress from patients with clear targets (e.g., patients possessing G551D CFTR) to patient populations that may have variable or low amounts of mutant CFTR at the cell membrane (e.g., subsets of patients who are homozygous for the DF508 CFTR mutation, or CF patients possessing rare, poorly characterized CFTR mutations predicted to have mutant CFTR protein at the cell membrane). It is also easy to envision that future studies will combine potentiators of CFTR with agents that address other primary defects in CFTR (e.g., combinations with drugs that suppress PTCs and/or drugs that correct mutant CFTR processing). The potential complexity of studying these patients and interpreting CFTR biomarker readouts is clear, as variable and unpredictable responsiveness appears likely. These upcoming challenges provide further support to efforts directed at executing standardized clinical

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research methodology across study institutions. Standardization of these methodologies will be crucial to ensure that appropriate CF subpopulations gain rapid access to these exciting agents, and to allow clear interpretation of studies that combine agents directed at primary and secondary CFTR defects.

VI.

Conclusions

As our understanding of the mechanisms underlying how CF-associated mutations produce disease increases, we are entering a time in which treatment strategies that address the underlying cause of CF are on the horizon. Restoration of CFTR function through the use of drugs designed to overcome mutational barriers has entered clinical trials, and several of the trials have provided important results that demonstrate that CFTR is a logical and feasible target for drug development. These advances are the direct result of a firm understanding of CFTR pathology and provide CF patients and healthcare providers real hope for fundamental changes in how CF is treated and what we can expect from our interventions.

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33. Ramalho AS, Beck S, Meyer M, et al. Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis. Am J Respir Cell Mol Biol 2002; 27(5):619–627. 34. Johnson LG, Olsen JC, Sarkadi B, et al. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 1992; 2(1):21–25. 35. Jaron R, Yaakov Y, Rivlin J, et al. Nasal potential difference in non-classic cystic fibrosislong term follow up. Pediatr Pulmonol 2008; 43(6):545–549. 36. Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med 2005; 352(19):1992–2001. 37. Kellermayer R. Translational readthrough induction of pathogenic nonsense mutations. Eur J Med Genet 2006; 49(6):445–450. 38. Krawczak M, Ball EV, Fenton I, et al. Human gene mutation database-a biomedical information and research resource. Hum Mutat 2000; 15(1):45–51. 39. Linde L, Kerem B. Introducing sense into nonsense in treatments of human genetic diseases. Trends Genet 2008; 24(11):552–563. 40. Khajavi M, Inoue K, Lupski JR. Nonsense-mediated mRNA decay modulates clinical outcome of genetic disease. Eur J Hum Genet 2006; 14(10):1074–1081. 41. Hermann T. Aminoglycoside antibiotics: old drugs and new therapeutic approaches. Cell Mol Life Sci 2007; 64(14):1841–1852. 42. Hermann T. Drugs targeting the ribosome. Curr Opin Struct Biol 2005; 15(3):355–366. 43. Moazed D, Noller HF. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 1987; 327(6121):389–394. 44. Ogle JM, Ramakrishnan V. Structural insights into translational fidelity. Annu Rev Biochem 2005; 74:129–177. 45. Shandrick S, Zhao Q, Han Q, et al. Monitoring molecular recognition of the ribosomal decoding site. Angew Chem Int Ed Engl 2004; 43(24):3177–3182. 46. Francois B, Russell RJ, Murray JB, et al. Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: role of the number of rings and positive charges in the specific binding leading to miscoding. Nucleic Acids Res 2005; 33(17): 5677–5690. 47. Kondo J, Francois B, Urzhumtsev A, et al. Crystal structure of the Homo sapiens cytoplasmic ribosomal decoding site complexed with apramycin. Angew Chem Int Ed Engl 2006; 45(20):3310–3314. 48. Kondo J, Urzhumtsev A, Westhof E. Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site. Nucleic Acids Res 2006; 34(2):676–685. 49. Hermann T, Tereshko V, Skripkin E, et al. Apramycin recognition by the human ribosomal decoding site. Blood Cells Mol Dis 2007; 38(3):193–198. 50. Bedwell DM, Kaenjak A, Benos DJ, et al. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat Med 1997; 3(11):1280–1284. 51. Keeling KM, Bedwell DM. Clinically relevant aminoglycosides can suppress diseaseassociated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system. J Mol Med 2002; 80(6):367–376. 52. Wilschanski M, Famini C, Blau H, et al. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations. Am J Respir Crit Care Med 2000; 161(3 pt 1):860–865. 53. Linde L, Boelz S, Nissim-Rafinia M, et al. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest 2007; 117(3):683–692. 54. Sermet-Gaudelus I, Renouil M, Fajac A, et al. In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study. BMC Med 2007; 5:5.

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channel activity. J Biol Chem 2005; 280(7): 5221–5226. 88. Loo TW, Bartlett MC, Clarke DM. Thapsigargin or curcumin does not promote maturation of processing mutants of the ABC transporters, CFTR, and P-glycoprotein. Biochem Biophys Res Commun 2004; 325(2):580–585. 89. Wang W, Bernard K, Li G, et al. Curcumin opens cystic fibrosis transmembrane conductance regulator channels by a novel mechanism that requires neither ATP binding nor dimerization of the nucleotide-binding domains. J Biol Chem 2007; 282(7):4533–4544. 90. Goss CH, Genatossio A, Rowbotham R, et al. A phase I safety and dose finding study of orally adminstered curcuminoids in adult subjects with cystic fibrosis who are homozygous

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26 Quality Improvement in Cystic Fibrosis Care MICHAEL S. SCHECHTER Emory University and Children’s Healthcare of Atlanta, Atlanta, Georgia, U.S.A.

I.

Introduction

Quality improvement (QI) efforts and the evolution of expectations regarding cystic fibrosis (CF) care that has occurred in the last decade are best understood in the context of the overall health care delivery system. Wide variations in the processes and outcomes of care among patients being treated for the same health care problems in different locations and health care settings was first noted by Wennberg and Gittelsohn in the mid-1970s (1). This phenomenon was initially of interest to health economists, who focused on the magnitude of variations in health care expenditures across delivery sites, but it soon became clear that this variation in practice led to differences in patient outcomes. While some variation in an activity as complex as medical practice is inevitable, there is a challenge and opportunity to identify the variations that produce better outcomes. From this standpoint, failure to learn from the variation would be a far more serious indictment of the profession than the variation itself (2). The Institute for Health Care Improvement (IHI) was founded in 1991 on the premise that the theory and methods used to manage quality in industry might hold the same promise for health care. In 1999, the Institute of Medicine (IOM) published To Err is Human (3), which contended that tens of thousands of patients die each year from medical errors. This was followed in 2001 by the landmark IOM monograph entitled Crossing the Quality Chasm, which identified problems in the system of health care delivery rather than deficiencies in individual physicians’ practice as the major impediment to attaining quality health care for all Americans. The IOM noted that “[we have] a health care system that frequently falls short in its ability to translate knowledge into practice” and concluded famously that “between the health care we have and the care we could have lies not just a gap, but a chasm (4).”

II.

The Pivotal Role of the Cystic Fibrosis Foundation

Most of the medical care for people with CF in the United States is provided by centers accredited by the Cystic Fibrosis Foundation (CFF), and the evolution of thinking about CF outcomes and care has occurred largely due to the CFFs efforts. A commitment to high-quality care was an important component of the aims of the CFF at its founding in the mid-1950s, leading to the creation of the care center network (5). Currently, there are over 115 CF care centers and 50 affiliate programs in the United States. CFF accreditation requires an on-site evaluation to ensure the presence of a multidisciplinary

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provider team, as well as adequacy of microbiologic testing techniques, sweat chloride testing, and other care practices. As survival into adulthood has become commonplace, the CFFs advocacy for the creation of adult CF care centers to complement existing pediatric centers has resulted in the establishment of more than 90 approved adult care programs. The CFF has produced a number of clinical care guidelines since 1990 (6–9). In the last few years, a more formal and uniform process has developed, with expert panels working in association with consultants at the Johns Hopkins Evidence-Based Practice Center to produce evidence-based guidelines for chronic respiratory and nutritional management (10–12). The development of these recent guidelines includes a systematic review of the literature that synthesizes data to draw conclusions and make recommendations in a way that minimizes bias (13). However, given the limited evidence base in CF care, practice guidelines often must rely on a synthesis of a formal review of the literature with clinical expertise and experience. The CFF also supports the dissemination of knowledge regarding state-of-the-art care by organizing the annual North American CF (NACF) conference, which brings together health care providers and researchers from all disciplines to an annual assembly with strong international participation. The multidisciplinary “networking” facilitated by the NACF meeting leads to the rapid spread of innovative ideas for care; past examples include the adoption of high-fat diets in the 1970s, and of more aggressive treatment of Pseudomonas airway infection in the 1990s. These novel approaches, initially advocated by a small minority, were then rapidly adopted by the mainstream of CF care centers, as word of anecdotal successes spread among colleagues.

III.

The Role of Patient Registries

A national registry containing demographic and clinical data on patients attending accredited CF care centers in the United States was begun by Dr Warren Warwick in the mid-1960s, and was taken over by the CFF in the 1970s; its content and use have evolved over the years. The registry was initially used to generate basic descriptive data regarding the CF population, such as average age of diagnosis, survival, and airway microbiology, and in the 1990s, it began to be used increasingly for analyses by epidemiologists seeking to identify risk factors and generate hypotheses regarding disease pathogenesis. In its earliest form, the registry was used to show improvements in mortality rate among centers that had evolved a comprehensive, multidisciplinary treatment program for CF care, facilitating the spread of this approach (5), but until recently, comparisons of outcomes between care centers were de-emphasized. A second CF patient registry, the Epidemiologic Study of Cystic Fibrosis (ESCF) was begun in the mid-1990s by Genentech, Inc., as a phase IV postmarketing study of dornase alfa (Pulmozyme1). ESCF collected similar clinical data as the CFF registry from over 10,000 subjects at selected CF treatment sites in the United States and Canada (14) but also collected therapeutic data not included in the CFF registry. The “modern era” of QI in CF began in 1999 when CFF national registry reports began to show that disease outcomes such as pulmonary function and nutritional status varied considerably by care center. At about the same time, personalized reports from the ESCF that were distributed directly to CF clinicians showed how their own specific practices compared with others nationally. This approach to personalized reporting was

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Figure 1 Representative displays from the CFF registry report to center directors showing care processes and outcomes at CF centers. (A) Percent of children meeting recommended outpatient monitoring (four clinic visits, two assessments of pulmonary function, and one sputum culture). The upper part of the figure shows each center represented by an individual column; the average for all centers is shown by the column at the far right-hand side; the column representing the center receiving the report is shown in a contrasting color in the original. The lower part shows the five-year trend for average percentage for the entire care center network; the performance of the 10 best centers for that particular measure; the performance of the individual center receiving the report. (B) A similar display representing average FEV1 for patients ¼ 18 years of age. Abbreviations: CFF, Cystic Fibrosis Foundation; FEV1, forced expiratory volume in one second.

then adopted and refined by the CFF, which developed annual center-specific reports showing individual center performance measures within the distribution of care practices and outcomes of all accredited CF centers (Fig. 1 shows examples). In 2003, the registry moved to an Internet Web-based portal, PortCF, allowing encounter-based patient data entry and transforming the registry into an invaluable resource and tool for QI activities. CF care teams can now print out summary data about individual patients to assist in clinic visits and can also access and download overall center outcomes. Port CF also provides additional data support to care teams by providing a readily available repository of current care guidelines and educational materials.

IV.

Partnerships to Improve CF Care

Patient quality of life and survival have increased dramatically in the 70 years since CF was first described, changing the face of the disease in a relatively short period of time

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Figure 2 The changes in median predicted age of death (using life table analysis) for the CF

population since the disease was first described.

(Fig. 2). Nevertheless, CF registry data suggests that care practices are inconsistent and outcomes quite variable among care centers, providing considerable opportunities for improvement. In the mid-1990s, regional consortia began to form with the assistance of the CFF and pharmaceutical industry partners, with the goal of comparing practices and outcomes among different care center within the same geographical regions of the United States (15). In 2002, the CFF introduced a large-scale initiative, titled “Accelerating the Rate of Improvement in CF Care,” which sought to develop a more sophisticated methodology for improving quality of care by improving the function of the health care delivery system for CF patients (16). A strategic plan set out plans to identify and disseminate “best practices,” teach QI techniques, develop leadership within the CF community, seek participation by patients and families, and provide data support and tools to care center teams, and included articulation of “seven worthy goals” (Table 1) (17). The CFF has attempted to establish an infrastructure to promote the development and spread of QI methods within the CF community and to train centers in their application. Partnerships were developed with several national QI organizations, including the IHI and its pediatric spin-off organization, the National Initiative for Children’s Health Care Quality (NICHQ); The Institute for Health Care Delivery Research, an affiliate of Intermountain Health Care; and the Center for the Evaluative Clinical Sciences at Dartmouth Medical School.

V. Teaching the Essentials of Quality Improvement At traditional continuing medical education (CME) activities, didactic sessions are provided to a passive audience of physicians with the expectation that they will use the new knowledge they receive to improve their practice. Studies of the effectiveness of such efforts suggest that they rarely achieve their intended goal (18). Multifaceted, health care systems–oriented approaches to change the process of care delivery at multiple levels, adapted to local practice settings, appear to be more effective at improving outcomes than passive approaches (19,20). Thus, a series of “Learning and Leadership” collaborative projects was initiated by the CFF, involving face to face meetings as well as teleconferences with multidisciplinary care teams from around the country, to teach the requisite systems-oriented methods and skills. These collaboratives have used a variety of

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Table 1 Seven Worthy Goals of the CFF

1.

Patients and families are full partners with the CF care team in managing this chronic disease. Information and communication will be given in an open and trusting environment so that every patient/family will be able to be involved in care at the level they desire. Care will be respectful of individual patient preferences, needs, and values.

2.

Children and adolescents will have normal growth and nutrition. Adults’ nutrition will be maintained as near normal as possible. All patients will receive appropriate therapies for maintaining lung function and reducing acute episodes of infection. Pulmonary exacerbations will be detected early and treated aggressively to return patients to previous levels of lung function. Clinicians and patients will be well-informed partners in reducing acquisition of respiratory pathogens, particularly Pseudomonas aeruginosa and Burkholderia cepacia. Patients will be screened and managed aggressively for complications of CF, particularly, CF-related diabetes. Severely affected patients will be well supported by their CF team in facing decisions about transplantation and end-of-life care. Patients will have access to appropriate therapies, treatments, and supports regardless of race, age, education, or ability to pay.

3. 4. 5. 6. 7.

Abbreviation: CFF, Cystic Fibrosis Foundation.

approaches, including the IHI/NICHQ model of targeted interventions based on prescribed change packages and centralized measurement and the Dartmouth microsystems method emphasizing professional formation, meeting skills, cause-and-effect diagrams, and flowcharts (15). More recent collaboratives have attempted innovative approaches based upon a synthesis of these methods and more selective teaching of relevant systems-based approaches in the specific CF environment by CF clinicians trained in QI methodology. At this point, the most efficient and reliably effective approach still needs to be identified, but there are certain theoretical and methodological principles of QI (21) that provide the underpinnings of any approach. These are discussed in the next section.

VI.

Basic Tenets of Quality Improvement

A. An Appreciation of the Need to Optimize the System in Which Health Care Is Delivered

The archetype of traditional medicine, the individual physician who by force of intellect and will establishes the correct diagnosis and prescribes the appropriate therapy to cure a patient, is anachronistic and likely ill-suited to the contemporary realities of health care provision for patients with chronic disease. CF care providers depend on the functioning of a complex system in which multiple caregivers must communicate and integrate a complex set of longitudinal data and then prescribe therapy on the basis of the appropriate use of these and other data. Knowledgeable physicians and staff are necessary for this care process, but not sufficient. To ensure the consistent delivery of appropriate care and minimize errors, it is essential to develop a supportive system that makes the correct decisions and actions the default. Much of the variation in practices and outcomes across different CF Care Centers is likely due to variation in the different systems’ ability to provide this support in a consistent manner (4).

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Figure 3 Wagner’s chronic care model envisions provider teams and patients interacting in an

environment where community resources and policies exist alongside and foster a health care delivery system that provides self-management support, an efficient delivery system design, decision support for providers, and a clinical information system that allows tracking of health care data on the individual patient and also on the aggregate population served by the provider team.

The Chronic Care Model

To optimize the care of patients with chronic disease, like CF, it is useful to conceptualize and work toward instituting an idealized system of health care delivery that is composed of several interdependent components inside and outside the practice setting. Furthermore, care should be considered within a long-term continuum, and not as isolated and independent events. Wagner’s chronic illness care model provides a useful framework for such care (20), and considers the impact of the following components (Fig. 3)a: 1.

2.

a

Community resources: National and regional social policies that impact access to health care resources play an important role affecting care (22), as do organizations such as the CFF. At a local level, medical community organizations and primary care providers can often supply needed services to patients, providing important resources that CF center providers should understand and be able to access. Medical center health delivery system: CF centers are most likely to be successful when they operate within organizations with a permeating culture that promotes safe, high-quality care (23). There should be an open and systematic approach to reducing errors, with incentives to acknowledge and reward high-quality care. Care should be coordinated within and across organizations.

A more detailed explication of this model may be found at http://www.improvingchroniccare.org

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The “microsystem”, consisting of several components at the interface between patients and the health care–delivery system: l Patient and family self-management Patients and families have a right to be informed and empowered as partners in care. When they assume that role, they become an enormous resource for assessment, goal setting, and treatment planning (24). Also, patient and family input can facilitate the configuration of an optimal and accessible delivery system (25). l Delivery system design Delivery system design includes the structure and function of the clinic, from the telephone to the reception area to the examination room. Team members should have clearly defined roles and responsibilities, and ensure that clinic flow is optimized, patient visits are planned to accomplish specific goals, and appropriate follow-up is ensured (26). l Decision support Decision support promotes the application of well-thought-out and, when available, evidence-based care through the use of guidelines, algorithms, and other clinical tools. This can help to ensure that reliance on rote memory is minimized and intended care is actually prescribed. These resources should be available immediately and easily to providers during clinic visits (26). l Clinical information systems For individual patient care, the system should provide ready access to data relevant to care decisions (laboratory, imaging, etc.), provide timely reminders regarding routine interval care, and facilitate sharing of data to coordinate care. The system should also facilitate identification of relevant subpopulations for proactive care and allow providers to monitor overall performance of the practice team. It is the lack of the latter data that limits provider understanding of the true effectiveness of their care (27).

B. Strategies to Improve Health Care Systems

An effective organizational change strategy is an essential component of improvement work. Attempts at making immediate, dramatic changes often fail in their planning stage because they get bogged down in endless preparatory meetings or self-destruct during implementation because of the number of unanticipated problems encountered. In contrast, the Model for Improvement, developed by Associates in Process Improvement, is an example of a more effective approach (28) (Fig. 4). To start, the team should set specific, measurable aims that will allow it to objectively monitor the effectiveness of the planned improvement process, and then identify changes that are likely to lead to the desired improvement. Implementation is accomplished by the repeated use of “plan/do/study/act (PDSA) cycles” to test theories and to identify and evaluate effective methods that accomplish meaningful improvements in care. The essential key to this approach is the use of small changes that are easily accomplished, followed by the analysis of data to evaluate the impact of the intervention.

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Figure 4 The Model for Improvement (14) provides one example of an approach for accomplishing change that has been used in CF improvement collaboratives. See text for further details, as well as http://www.ihi.org/IHI/Topics/Improvement/ImprovementMethods/HowToImprove/ testingchanges.htm.

C. Quality of Care Can and Must Be Measured

Quality of care has been defined by the IOM as “the degree to which health care services for individuals and populations increase the likelihood of desired health outcomes and are consistent with current professional knowledge” (29). Quality measure can be used to quantify any of the following (30): l

Structure measures address “sufficiency of resources and proper system design,” including organizational characteristics. Structure data describe the capability of organizations or professionals rather than care provided or results achieved.

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l

l

l

Process measures assess how well specific services are provided by a system to those patients who should receive them. The assumption is that the processes required to achieve optimal outcomes are known and measurable. Process measures are more immediately modifiable than outcome measures, and their measurement can identify specific areas of care that may yield improvement. Process goals should be clearly articulated, sensitive to change, quickly implementable, readily measurable, and valid, effective approaches to care (31–33) Access measures assess the patient’s attainment of timely and appropriate health care. Barriers to access may include inability to pay for health care, difficulty traveling to health care facilities, and difficulties contacting or communicating with health care providers. Patient experience measures aggregate reports of patients about their observations of and participation in health care. These measures provide the patient perspective on quality of care. Outcome measures describe how the care delivered affects the patient’s health, health status, and function (e.g., functional status, quality of life, and mortality). Intermediate outcome measures are linked to endpoint outcomes, such as disability or death [e.g., forced expiratory volume in one second (FEV1), body mass index (BMI), hospitalization, HgbA1c].

Data collection and analysis are essential to recognize where opportunities for improvement exist, and to garner feedback on what changes truly result in improvement. Once an organization decides to implement specific actions for improvement, it needs to track the consistency with which those actions are taken must be tracked. Improved performance on structure, process, and access measures can be considered as a preliminary step to improvement in the patient experience and outcome measures that are the true goal of the work. Feedback must be provided promptly and on a regular basis, and data should be reported in a way that can be easily understood and used by members of the care team, as well as interested outsiders. D. Collaboration, Comparison, and Data Transparency

Data comparisons are an important motivator of change, but for these comparisons to be effective, clinicians need to accept that the data are truly representative and that the comparisons are valid. The validity of registry data is threatened if patient inclusion is biased or if measurements are unreliable (especially in a nonrandom way) (34). Comparisons of processes and outcomes across CF centers, which seem to reveal great variation, are clearly misleading if, for example, disease severity differs among care centers. The challenges of case-mix adjustment are great and are a subject of controversy and intense study by health services researchers (35,36). O’Connor et al. (37) used variables reported in the CFF registry to evaluate intrinsic (primarily demographic) predictors of mortality, and proposed a case-mix adjustment of center performance using these characteristics (gender, nonwhite race, Hispanic ethnicity, symptomatic presentation, cystic fibrosis transmembrane conductance regulator (CFTR) genotype, age at diagnosis, and median household income by zip code); this has been presented in CF registry reports, and suggests that center variation is minimally affected by case mix.

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While this adjustment does not take into consideration several characteristics that might impact on center outcomes, such as state/regional influences on health due to differences in community and social services support (38,39) or referral patterns, particularly for transplant centers, the adjustment has allowed a cautious acceptance of the validity of center-to-center comparisons. Data sharing (transparency) is an important component of improvement collaboratives. Scientific researchers are familiar with the synergy derived from investigative collaboration, and this strategy is equally effective for the development and spread of innovations for improvement in the delivery of health services. Any health care team that is actively striving to improve its outcomes will devise novel and potentially effective ideas for how to accomplish this. Collaboration among centers that are trying to accomplish similar goals is therefore an important and effective strategy to accelerate change. For many physicians, the sharing of performance data with other providers is an uncomfortable jump from the secretive environment within which the health care team has traditionally operated with regards to care outcomes. Public reporting represents an even greater challenge. The previously mentioned IOM report, “Crossing the Quality Chasm,” challenged health care providers to “make information available to patients and their families that allows them to make informed decisions when selecting a health plan, hospital, or clinical practice” (4). Current experience suggests that most health care consumers make limited use of this information because they do not understand or trust it, and it seems to make a small, although increasing, impact on their decision making. Overall, there is some evidence that the publication of performance data has been associated with an improvement in health outcomes (40,41). In 2006, the CFF began to publish center-specific data from all the accredited CF centers and affiliates on lung function and nutritional status in children and adults, screening for cystic fibrosis–related diabetes (CFRD), and adherence to clinical guidelines care (i.e., outpatient visits, pulmonary function tests, and sputum culture in children and adults). The anecdotal experience among CF patients and providers seems to match that of the general population—the public reporting of data has been noted and used by health care systems, and has been a spur to QI activities, but has had little impact on patient choice of care center (15).

VII.

Searching for Best Practice—Clinical Practice Guidelines and Benchmarking When strong scientific evidence suggests that a specific approach works best, then the goal is to ensure that all appropriate patients receive it. As noted earlier, the CFF has sponsored the development of clinical guidelines to establish an evidence-based foundation for CF clinical care. However, while randomized clinical trials represent the gold standard approach to determining best practice, it is not feasible to perform adequately powered clinical trials to test every possible nuance regarding CF treatment. An alternative method, made possible by using the CFF registry data to compare center activities and outcomes, is to ascertain and spread care practices used at centers that exhibit highlevel performance on specific disease outcomes. The term “benchmarking” refers to the identification of practices that are used at sites that attain superior outcomes. Benchmarking originated as a business tool but has

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recently become recognized as a means to identify and spread effective strategies for health care delivery (42), and has obvious applications in CF, given the limitations in the evidence base. The CFF has sponsored both a pediatric and adult program benchmarking team to visit centers of different size and geographic location identified as having either exceptional nutritional care or exceptional pulmonary care consistently over the previous five years. Program performance was case-mix adjusted as noted earlier, and adult program performance was adjusted for patient status at age 21 to control for pediatric program performance. While some centers had both pulmonary and nutritional outcomes in the top quintiles, more tended to excel in one area and not the other. A multidisciplinary core team of CF experts with training and knowledge of systems-oriented approaches to care then partnered with volunteer teams from CF care centers to visit high performing centers with the goal of learning novel and successful methods to improve care. Benchmarking visits included structured dialogue and discussion regarding approaches to care, their rationale, and their apparent consequences. These visits also included examinations of center-specific registry reports of process and outcome measures. The discussions set a framework for observations in clinic. Team members were paired by discipline to observe and discuss specific approaches and activities in the outpatient clinic, and hospital tours focused on the processes of inpatient care. When possible, visitors attended host team meetings to observe the planning of care and interactions among team members. As a follow-up, the visiting teams presented an evaluation of differences in methods and developed a plan to assimilate new approaches into their practice. Several themes that were noted both at the pediatric and adult program visits included (43,44): 1.

2.

3.

High-functioning teams that provide consistent care. Team members communicate and cooperate with each other and meet regularly. It was typical to find directive center leadership, a strong and autonomous nurse coordinator, and experienced and knowledgeable telephone and clinic support staff. The team members are all aware of clearly defined criteria for diagnosis and treatment, responsibilities are clear, and decisions are communicated clearly to patients who are engaged and understand their care (see point 4 below) High expectations for outcomes among providers and families. For example, high-nutrition centers assume and expect patients to be at or above the 50th percentile for BMI. High-pulmonary centers expect no or minimal symptoms, and little to no loss of pulmonary function. Patients and families are trained to expect the same. Early and aggressive management, with little reliance on “rescues.” At high-nutrition centers, small declines in weight/BMI percentile prompt attention and action, and patients of concern are followed up at more frequent intervals. There is typically close involvement of engaged subspecialists (endocrinology and gastroenterology) who actively seek out complications before they are obvious. Successful pulmonary centers maintain a low threshold for antibiotic use, quickly treat new respiratory symptoms with oral antibiotics, and start IV antibiotics when oral antibiotics

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do not produce prompt improvement. They tend to favor hospitalization over outpatient IV antibiotic therapy. The need for airway clearance augmentation is emphasized from an early age. Patients/families are engaged, empowered and proactive, and well informed on disease management and its rationale. They know when to contact the center, whom to speak with, and what to expect when they call or when they come for a visit. Their expectations provide additional assurance that they will receive the right treatment.

These apparent themes suggest potentially fruitful approaches to improving outcomes, but the benchmarking method is primarily useful for generating hypotheses. Where possible, these observations should be validated using quantitative techniques such as clinical trials, structured questionnaires, or other methods for comparing treatment approaches across the entire care center network.

VIII.

The Impact of QI on CF Outcomes

The overall impact of QI on CF outcomes is difficult to quantify because it is not possible to distinguish changes in outcomes that have occurred specifically due to the influence of formal QI interventions from those that would have occurred without them or as a result of the introduction of new therapies and interventions (detailed in other chapters). Care at all centers has been impacted by QI thinking, to a greater or lesser degree, so there is no “untreated” comparison group. This is typical of the difficulties involved in measuring the impact of QI initiatives, so analytic approaches to measuring the impact of QI usually compare trends before and after the introduction of the

Figure 5 Average FEV1 of patients aged 18 to 19 years in the CFF registry, 1990 to 2006 with

95% confidence intervals. The slope of improvement over time was 0.52% predicted/yr from 1990 to 2000; the slope from 2000 to 2006 was 1.93% predicted/yr. Abbreviations: CFF, Cystic Fibrosis Foundation; FEV1, forced expiratory volume in one second. Source: CFF registry data courtesy of Hebe Quinton.

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Figure 6 Average BMI of patients aged 18 to 19 years in the CFF registry, 1990 to 2006 with 95% confidence intervals. The slope of improvement over time was 0.86 kg/m2/yr from 1990 to 2000; the slope from 2000 to 2006 was 1.70 kg/m2/yr. Abbreviation: BMI, body mass index; CFF, Cystic Fibrosis Foundation. Source: CFF registry data courtesy of Hebe Quinton.

interventions of interest to evaluate whether an improvement is discernable. Conventional statistical methodologies can be used, but it is often more appropriate to use statistical control methods that were pioneered in the manufacturing industry to distinguish normal variation from “special” variation resulting from the introduction of a new influence (45). Even so, if a number of secular influences develop or are implemented simultaneously, it is not possible to distinguish which has the greatest impact on outcomes of interest. Unfortunately, there are no publications detailing rigorous evaluations of the impact of QI on CF outcomes. Nevertheless, the trends in CF outcomes seem to indicate a significant acceleration in improvement since 2000, coincident with the initiation of the CFF QI program. As noted, a number of new CF therapies became available during this time as well, likely leading to an additive or synergistic effect as the appropriate and consistent use of these new therapies has been an emphasis of QI programs of many centers. Figures 5 and 6 show the mean FEV1 and BMI of patients 18 to 19 years of age between the years 1990 and 2006, derived from the CFF patient registry. These data suggest that the slope of increase in both of these measures has increased greatly between 2000 and 2006 compared with the years 1990 to 2000.

IX.

Summary

To date 84 CF centers, caring for over 17,000 patients, have been trained in a systemsoriented approach to QI, and data from these centers suggest the success of this initiative. CF QI projects have utilized the IHI/NICHQ model of a series of targeted interventions based on prescribed change packages and centralized measurement and the microsystems approach emphasizing professional formation, meeting skills,

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cause-and-effect diagrams, and flowcharts (15). More recent collaboratives have attempted innovative approaches on the basis of synthesis of these methods and more selective teaching of relevant systems-based approaches in the specific CF environment. At this point, the most efficient and reliably effective approach still needs to be identified. Key tasks for the future are to narrow and shift the center of the “bell curve” (46) of center performance for the nation and the world as a whole, and to find the most efficient way to do so. With the abundance of new therapies currently entering the therapeutic pipeline, it is critically important to prepare the CF community and care centers for their optimal use and to close the “chasm between the health care we have and the care we could have” (4).

References 1. Wennberg J, Gittelsohn. Small area variations in health care delivery. Science 1973; 182(117):1102–1108. 2. Blumenthal D. Quality of health care. Part 4: the origins of the quality-of-care debate. N Engl J Med 1996; 335(15):1146–1149. 3. Kohn L, Corrigan J, Donaldson M, eds. To Err is Human: Building a Safer Health System. Institute of Medicine Report on Medical Errors. Washington, DC: National Academy Press, 1999. 4. Institute of Medicine Committee on Quality Health Care in America. Crossing the Quality Chasm: A New Health System for the 21st Century. Washington, DC: National Academy Press, 2001. 5. Doershuk CF. Growth of the foundation’s medical program. In: Doershuk CF, ed. Cystic Fibrosis in the 20th Century. Cleveland, OH: AM Publishing, Ltd, 2001:218–238. 6. Cystic Fibrosis Foundation Center Committee and Guidelines Subcommittee. Cystic Fibrosis Foundation guidelines for patient services, evaluation, and monitoring in cystic fibrosis centers. Am J Dis Child 1990; 144(12):1311–1312. 7. Borowitz D, Baker RD, Stallings V. Consensus report on nutrition for pediatric patients with cystic fibrosis. J Pediatr Gastroenterol Nutr 2002; 35(3):246–259. 8. Yankaskas JR, Marshall BC, Sufian B, et al. Cystic fibrosis adult care: consensus conference report. Chest 2004; 125(1 suppl):1S–39S. 9. Aris RM, Merkel PA, Bachrach LK, et al. Guide to bone health and disease in cystic fibrosis. J Clin Endocrinol Metab 2005; 90(3):1888–1896. 10. Flume PA, O’Sullivan BP, Robinson KA, et al. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Am J Respir Crit Care Med 2007; 176(10): 957–969. 11. Flume PA, Robinson KA, O’sullivan BP, et al. Cystic fibrosis pulmonary guidelines: airway clearance therapies. Respir Care 2009; 54(4):522–537. 12. Stallings VA, Stark LJ, Robinson KA, et al. Evidence-based practice recommendations for nutrition-related management of children and adults with cystic fibrosis and pancreatic insufficiency: results of a systematic review. J Am Diet Assoc 2008; 108(5):832–839. 13. Cook DJ, Mulrow CD, Haynes RB. Systematic reviews: synthesis of best evidence for clinical decisions. Ann Intern Med 1997; 126(5):376–380. 14. Morgan WJ, Butler SM, Johnson CA, et al. Epidemiologic study of cystic fibrosis: design and implementation of a prospective, multicenter, observational study of patients with cystic fibrosis in the U.S. and Canada. Pediatr Pulmonol 1999; 28(4):231–241. 15. Quinton HB, O’Connor GT. Current issues in quality improvement in cystic fibrosis. Clin Chest Med 2007; 28(2):459–472. 16. Quality of Care and Outcomes Research in CVD and Stroke Working Groups. Measuring and improving quality of care: a report from the American Heart Association/American College

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Schechter of Cardiology First Scientific Forum on Assessment of Healthcare Quality in Cardiovascular Disease and Stroke. Circulation 2000; 101(12):1483–1493. Cystic Fibrosis Foundation. Accelerating the Rate of Improvement in CF Care. Available at: https://www.portcf.org/Resources/Quality%20Initiative/QI%20Plan%20%26%207%20Worthy%20Goals%20%28June%202005%29.pdf. Accessed December 16, 2009. Davis D, O’Brien MA, Freemantle N, et al. Impact of formal continuing medical education: do conferences, workshops, rounds, and other traditional continuing education activities change physician behavior or health care outcomes? JAMA 1999; 282(9):867–874. Margolis PA, Lannon CM, Stuart JM, et al. Practice based education to improve delivery systems for prevention in primary care: randomised trial. BMJ 2004; 328(7436):388. Wagner EH, Glasgow RE, Davis C, et al. Quality improvement in chronic illness care: a collaborative approach. Jt Comm J Qual Improv 2001; 27(2):63–80. Deming WE. Out of Crisis. Cambridge, MA: MIT Center for Advanced Engineering Study, 1986. Marmot M. Social determinants of health: from observation to policy. Med J Aust 2000; 172(8):379–382. O’Connor PJ, Sperl-Hillen JM, Pronk NP, et al. Primary care clinic-based chronic disease care: features of successful programs. Disease Management & Health Outcomes 2001; 9(12):691–698. Effing T, Monninkhof EM, van der Valk PD, et al. Self-management education for patients with chronic obstructive pulmonary disease. Cochrane Database Syst Rev 2007; (4): CD002990. Dunst CJ, Trivette CM, Hamby DW. Meta-analysis of family-centered help giving practices research. Ment Retard Dev Disabil Res Rev 2007; 13(4):370–378. Wasson JH, Godfrey MM, Nelson EC, et al. Microsystems in health care: part 4. Planning patient-centered care. Jt Comm J Qual Saf 2003; 29(5):227–237. Nelson EC, Batalden PB, Homa K, et al. Microsystems in health care: part 2. Creating a rich information environment. Jt Comm J Qual Saf 2003; 29(1):5–15. Langley G, Nolan K, Nolan T, et al. The Improvement Guide: A Practical Approach to Enhancing Organizational Performance. San Francisco: Jossey-Bass, 1996. Institute of Medicine. Medicare: A Strategy for Quality Assurance. Washington, DC: National Academy Press, 1990. National Quality Measures Clearinghouse. Using measures. In: Agency for Healthcare Research and Quality, 2009. Available at: http://www.qualitymeasures.ahrq.gov/resources/ measure_use.aspx. Accessed December 16, 2009. Lohr KN. Rating the strength of scientific evidence: relevance for quality improvement programs. Int J Qual Health Care 2004; 16(1):9–18. Nelson EC, Splaine ME, Godfrey MM, et al. Using data to improve medical practice by measuring processes and outcomes of care. Jt Comm J Qual Improv 2000; 26(12):667–685. Nelson EC, Splaine ME, Plume SK, et al. Good measurement for good improvement work. Qual Manag Health Care 2004; 13(1):1–16. Schechter MS. Patient registry analyses: seize the data, but caveat lector. J Pediatr 2008; 153(6):733–735. Ash AS, Shwartz M, Pekoz EA. Comparing outcomes across providers. In: Iezzoni LI, ed. Risk Adjustment for Measuring Healthcare Outcomes. 3rd ed. Chicago: Health Administration Press, 2003:297–333. Krumholz HM, Brindis RG, Brush JE, et al. Standards for statistical models used for public reporting of health outcomes: an American heart association scientific statement from the quality of care and outcomes research interdisciplinary writing group: cosponsored by the council on epidemiology and prevention and the stroke council. Endorsed by the American College of Cardiology Foundation. Circulation 2006; 113(3):456–462.

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37. O’Connor GT, Quinton HB, Kahn R, et al. Case-mix adjustment for evaluation of mortality in cystic fibrosis. Pediatr Pulmonol 2002; 33(2):99–105. 38. Goldhagen J, Remo R, Bryant T III, et al. The health status of southern children: a neglected regional disparity. Pediatrics 2005; 116(6):e746–e753. 39. Guagliardo MF, Ronzio CR. Is region of country a useful variable for child health studies? Pediatrics 2005; 116(6):1542–1545. 40. Fung CH, Lim YW, Mattke S, et al. Systematic review: the evidence that publishing patient care performance data improves quality of care. Ann Intern Med 2008; 148(2):111–123. 41. Marshall MN, Shekelle PG, Leatherman S, et al. The public release of performance data: what do we expect to gain? A review of the evidence. JAMA 2000; 283(14):1866–1874. 42. Nelson EC, Batalden PB, Huber TP, et al. Microsystems in health care: part 1. Learning from high-performing front-line clinical units. Jt Comm J Qual Improv 2002; 28(9):472–493. 43. Schechter MS, Leonard A, Nash J, et al. Benchmarking: signature themes. Pediatr Pulmonol 2006; (suppl 29):122–123. 44. Swarup AL, Sabadosa KA, Quinton HB, et al. Insights from the adult CF benchmarking questionnaire. Pediatr Pulmonol 2008; (suppl 31):409. 45. Ogrinc G, Mooney SE, Estrada C, et al. The SQUIRE (Standards for QUality Improvement Reporting Excellence) guidelines for quality improvement reporting: explanation and elaboration. Qual Saf Health Care 2008; 17(suppl 1):i13–i32. 46. Gawande A. The Bell Curve. The New Yorker 2004:82–91.

27 Cystic Fibrosis and Infection Control SUSAN RETTIG The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

SUSAN COFFIN University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Infection prevention and control practices for cystic fibrosis (CF) patients are unique to this patient population; more expansive than transmission-based precautions; and pose challenges for hospital staff, families, and patients. In 2003, the consensus document, “Infection control recommendations for patients with cystic fibrosis: microbiology, important pathogens, and infection control practices to prevent patient-to-patient transmission,” was published (1). Experts in the field of CF and infection prevention and control developed these evidenced-based recommendations that are aimed at reducing the transmission of infectious organisms to and between CF patients. These guidelines give detailed recommendations about interrupting transmission of pathogenic organisms to CF patients from other patients, contaminated equipment, or the hospital environment. These recommendations address risks associated with multiple environments and stress compliance with existing infection control guidelines from the Centers for Disease Control and Prevention (CDC) and Hospital Infection Control Practices Advisory Committee (HICPAC) as essential to prevention of cross-transmission of potential pathogens between patients, and form the basis for much of this chapter.

II.

Rationale for Infection Control Practices

Most individuals with CF harbor transmissible pathogens in their respiratory secretions. These organisms can infect other patients with CF through direct patient-to-patient contact via the contaminated hands of health care workers (HCWs) or through indirect contact with the contaminated environment or respiratory equipment. Molecular epidemiological studies have provided evidence of cross-infection of potentially harmful pathogens and suggest that the mechanisms of transmission can be varied. LiPuma demonstrated that Burkholderia cepacia complex (Bcc) could be transmitted between CF patients and that with strict adherence to infection control practices, including careful adherence to standard and transmission-based precautions, new acquisition decreased (2). In a prospective surveillance study, however, Jones noted that close attention to infection control measures in the absence of patient segregation did not prevent cross-infection of Pseudomonas aeruginosa (3). Finally, Speert studied the transmission of P. aeruginosa among CF patients in Vancouver CF clinics and was unable to find evidence of patient-to-patient spread without close social contact such as 440

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that which occurs with siblings (4). These observations support the use of at least modified infection control measures even if a CF patient is not known to be colonized with a multidrug-resistant organism, because patients may harbor resistant organisms before they are detected by laboratory methods. (5).

III.

General Principles for Health Care Settings

Multiple existing evidence-based infection prevention and control guidelines, used for the care of all hospitalized, ambulatory, and home care patients, are also applicable to CF patients. Compliance with hand hygiene remains the mainstay of infection prevention and control in most health care settings because most health care acquired infections are transmitted via the hands of HCWs. The Guideline for Hand Hygiene in Healthcare Settings discusses the appropriate use of hand hygiene products, for example, alcohol hand rub, soap and water, skin care, fingernail specifics, and monitoring compliance with hand hygiene (6). In addition to hand hygiene, standard precautions also apply to all patients regardless of their known or suspected infection status. Recommendations for standard precautions (as well as transmission-based precautions) are found in Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Health Care Settings (7). Besides hand hygiene, standard precautions include the use of personal protective equipment, for example, gloves, gowns, mask, and eye shields if contact with blood or body substances is anticipated. Other HICPAC guidelines that are critical to the safe care of CF patients include the Guideline for Disinfection and Sterilization, which details steps needed to adequately reprocess respiratory equipment (8); the Guideline for Environmental Infection Control in Health Care Facilities (9); the Guideline for Healthcare Acquired Pneumonia (10); Management of Multidrug-Resistant Organisms in Healthcare, which applies to many CF patients (11); and Guideline for Infection Control in Healthcare Personnel (12). These evidence-based recommendations deserve careful review and consideration by HCWs in the field of infection prevention and control, infectious diseases, pulmonary, pharmacy, environmental services, occupational health, respiratory therapy, and central processing. Transmission-based precautions are used in addition to standard precautions when additional measures are needed to prevent the transmission of potentially infectious organisms among patients and staff. Table 1 lists the types of transmission-based precautions used with epidemiologically important pathogens (13). These measures include the use of personal protective equipment specific to the route of transmission of a particular pathogen as well as the segregation of patients from each other. Common indications for transmissionbased precautions for hospitalized CF patients include the prior detection of resistant organisms [e.g., methicillin resistant Staphylococcus aureus (MRSA), Bcc, P. aeruginosa] in their respiratory secretions or acute infection with a respiratory virus (e.g., influenza, respiratory syncytial virus, adenovirus). Because transmission of CF pathogens can occur via the droplet and/or contact route, specific strategies are designed to interrupt these mechanisms of transmission (5). To interrupt contact transmission (usually by a HCW’s hands), gloves and a gown are recommended. Transmission of organisms spreads by coughing, sneezing, or talking and is interrupted by the use of a tight-fitting surgical mask. An alternative strategy to limit transmission is the practice of some form of segregation with all CF patients, that is, keeping a spatial distance of 3 ft between patients. This is sometimes referred to as “the 3-foot rule” and is based on the observation that large, respiratory droplets that can transmit organisms typically settle due to gravity within 3 ft of the source.

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Table 1 Transmission-Based Precautions to Prevent the Spread of Epidemiologically Important Pathogens in CF Patients

Type of precaution

Potential pathogen

Standard

Applicable to all CF patients including those infected with: NTM Pseudomonas aeruginosa (not multidrug resistant) Staphylococcus aureus (not MRSA) Multidrug-resistant organisms MRSA Burkholderia cepacia complex Multidrug-resistant P. aeruginosa Stenotrophomonas maltophilia Respiratory syncytial virus Parainfluenza virus Influenza virus Adenovirus Mycobacterium tuberculosis No data supporting the use of positive-pressure ventilation and 12 air exchanges for CF patients with lung transplantation; may consider in the setting of suspected transmission of Aspergillus spp. within transplant centers

Contact

Droplet viruses Airborne Protective environment

Abbreviations: CF, cystic fibrosis; NTM, non-tuberculous mycobacterium; MRSA, multidrug-resistant Staphylococcus aureus. Source: Adapted from Ref. 13.

Reprocessing of medical equipment with a special emphasis on respiratory equipment is another important component of a CF infection control plan. Equipment labeled as single patient use, such as spacers or spirometry mouthpieces, is not to be shared among patients. While it is prudent to follow manufacturers’ guidelines for reprocessing respiratory therapy and pulmonary function testing equipment, the respiratory therapy department’s cleaning and disinfection policies should be written in consultation with the central processing and infection control department to ensure patient safety. Because contaminated nebulizers can be a source of bacterial infection of the lower airways (14), nebulizers used in the hospital for a CF patient should be cleaned, disinfected, and rinsed after each use as per the CF Foundation’s guidelines (1). Usually respiratory therapists or nurses administer aerosolized medications to CF patients in a hospital setting; if there is insufficient time for appropriate reprocessing between patient use, then nebulizers must be discarded.

IV.

Surveillance for CF Pathogens

Respiratory tract culture should be performed on CF patients at least quarterly, during exacerbations, with a change in clinical stays, when hospitalized, and when epidemiologically indicated (1). Target pathogens should include Bcc, MRSA, and all P. aeruginosa, and CF centers should decide which organisms should be deemed important enough to calculate prevalence and incidence rates. The CF and infection control teams should work together on surveillance activities and share data on incidence and prevalence of select organisms.

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V. Infection Control Recommendations for Inpatient Settings A. Room Placement

Strict adherence to standard and transmission-based precautions, thorough medical equipment disinfection, and environmental cleaning may not be sufficient to prevent cross-transmission of potential pathogens between CF patients. CF sputum cultures processed by the microbiology laboratory do not always yield all of the potential pathogens an individual is harboring. As described above, molecular analysis had documented transmission of organisms, such as P. aeruginosa or Bcc, prior to recognition that the index patient is colonized. Thus, to prevent inadvertent transmission, some form of segregation of CF patients is warranted in most clinical settings. CF patients colonized or infected with Bcc, MRSA, vancomycin resistant enterococcus, or a multidrug-resistant gram-negative rod should be placed in a private room and contact precautions should be followed. While it is preferable to admit all CF patients to a private room, it may not be possible; if multibed rooms must be used for CF patients, then they should not share a room with another CF patient (1). Some CF centers have chosen to segregate patients with Bcc from those without evidence of colonization or infection by placing them in a different unit. Other hospitals have developed policies that limit nursing and respiratory staff to only care for one CF patient per shift to minimize indirect contact transmission. It is important to recognize that these practices would be unnecessary if all staff adhered to standard and transmission-based precautions, with meticulous attention to hand hygiene. Siblings with CF sometimes require hospitalization at the same time. Individual CF centers have developed their own strategies to care for families with more than one CF patient. Some CF centers allow siblings to reside in the same room, while others permit room sharing only if the siblings are documented to have similar colonization patterns. Finally, some CF centers make a distinction on the basis of whether the patients share a room at home. If siblings share a hospital room, some centers recommend that the privacy curtain be drawn to interrupt possible droplet transmission during respiratory therapy treatments and chest physiotherapy, although there are no well-designed studies to support this practice. B. Activity Outside the Patient’s Room

CF patients on transmission-based precautions should only be allowed to travel outside of their room for essential tests, procedures, and therapies. The hospital policy for patients colonized with multidrug-resistant organisms should be applied to all CF patients and provide guidance for CF clinicians and infection preventionists. Some patients require specific therapeutic programs that can easily be performed in the patient’s room; however, other patients may require therapies that will provide more benefit to the patient if administered in a specialized setting, such as the physical therapy gym. All CF patients, if developmentally able, should be instructed to perform hand hygiene, preferably with soap and water followed by alcohol hand rub, before exiting their room. Mask use by CF patients when outside of their rooms remains an unresolved issue (1). However, if a center opts for mask use, this practice should be consistently applied and all patients able to do so should be required to wear a mask when outside of their rooms. Caregivers may perceive this practice as beneficial to protecting their child

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from cross-transmission of CF pathogens and common respiratory viruses and inconsistencies in practice contribute to feelings of confusion and anger among patients and families. Each CF center will have to decide how best to segregate hospitalized CF patients to prevent cross-transmission of CF pathogens. Many academic children’s hospitals with a CF center have an inpatient unit at least partially dedicated to CF patients. These units may include a playroom, teen room, classroom, family lounge, and shared kitchen. Many hospitals also have off-unit family resource rooms, gift shops, and eatery establishments. Some hospitals even have outside playgrounds. A CF center’s infection control policy should include specific guidelines concerning patient travel to locations outside the patient’s room that are not deemed essential for their care. Additionally, it is important for the CF multidisciplinary team, infection prevention and control, child life, teachers, and other providers to agree and adhere to these recommendations. Inconsistent application of such policies is likely to generate confusion. Some CF centers may take a very conservative approach and not allow any CF inpatient’s travel to sites not deemed essential for their care. Others may allow visits to common areas provided only one CF patient is present at any given time. Staff supervision should be available if a CF center decides, preferably on the basis of low-prevalence rates, to allow more than one CF patient in a common area at a time. Areas frequented by CF patients should be cleaned on a regular basis and whenever contamination with respiratory secretions has occurred. C. Respiratory Therapy

Respiratory therapy usually involves the administration of aerosolized medications via a nebulizer followed by chest physiotherapy. These procedures should always be performed in the patient’s room. The staff who performs these procedures should adhere to standard precautions, especially since these procedures may cause the patient to cough. Thus, staffs will most likely need to don masks, gowns, gloves, and possibly eyewear when administering respiratory therapy, given the probability of cough and direct contact with respiratory secretions. Resistant organisms transmitted from respiratory secretions will usually not induce illness in an immunocompetent host. HCWs, however, can acquire transient colonization of their nares or hands, or contamination of their clothing, and could then transmit these organisms to another CF or immunocompromised patient. However, staff may have difficulty wearing a mask the whole time they are performing chest physiotherapy, so education of staff and patients is important to support this practice. In addition, patients should be reminded to practice respiratory etiquette as outlined by the CDC’s Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings (7). These practices include coughing into tissues or elbow, disposing of tissues properly, and performing hand hygiene. Nebulizers should be cleaned and disinfected in-between patient use and should only be used for a single patient. These devices can easily become contaminated with respiratory secretions. If not adequately cleaned after every use, the bacterial organisms contaminating the nebulizer equipment can then be aerosolized during subsequent use (13). A multistep process is needed for adequate disinfection. Cleaning is accomplished with a hospital-approved detergent and is performed as soon as possible after the

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completion of an aerosolized treatment. This step must be performed before disinfection and removes visible debris and reduces bioburden, which is necessary to allow for adequate disinfection. Disinfection can be achieved through either a three- to fiveminute soak of 70% alcohol or 1:50 dilution of sodium hypochlorite. Disinfection is followed by a sterile water rinse, air-dry equipment, and stored in a manner to reduce contamination. At some CF centers there are respiratory therapists dedicated to CF patients and these patients are admitted to an assigned unit. CF patients may need to be admitted to the intensive care unit, or possibly be housed on other units when the hospital is filled to capacity. If a CF center decides to reprocess nebulizers in-between patient use, then a clear, concise, and easy-to-locate policy should be available to all staff charged with this task.

VI.

Ambulatory Settings

A. Clinic Logistics

The CF Foundation guidelines stress the importance of keeping CF patients apart from each other as a way to prevent cross-transmission with possible pathogens. This poses a challenge for staff when a multidisciplinary team needs to see multiple CF patients during a single clinic session. Experienced providers should know approximately how much time a new and follow-up appointment will take and schedule accordingly. The ideal action would be to place patients in an exam room upon arrival to the clinic. Even when this cannot be accomplished, clinic staff responsible for patient flow through the clinic should be attuned to what is happening in the waiting room. Frontline staff should also be aware of which patients on the schedule have a documented history of multidrugresistant organisms in their sputum. These patients may travel to other areas of the hospital for studies; it is prudent to relay this information to the receiving departments so that appropriate precautions can be followed. CF centers with small waiting rooms may need to implement creative strategies to keep CF patients segregated. One suggestion is to ask families to wait in large common areas of the hospital and contact them by pager or cellular phone when an exam room is available. B. Waiting Room Practices

Alcohol hand rubs should be readily available in CF clinic waiting rooms for use by patients and families. Ideally, all ambulatory settings would have hand hygiene products accessible to patients and caregivers, either in the form of alcohol hand rub or disposable hand cleansing towelettes. Patients must be educated to maintain at least 3 ft of separation if interacting with other CF patients and to always avoid physical contact. Some CF centers that have included masks as precautions in their infection control policy make them available to patients upon arrival to clinic. This strategy is not evidence-based but is used in an attempt to interrupt droplet transmission between CF patients within the clinic setting. This strategy is now possible even with very young CF patients because of the availability of pediatric size masks. Many a time, non-CF patients share the waiting room with CF families, and they may have unfounded fears regarding mask use. Hospital staff not attending to CF patients should be able to articulate to families the reasons for mask use (while maintaining patient privacy) and assure families that this group of patients does not pose an infection risk to a non-CF child. CF parents and patients should also be provided with education in regards to

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expected waiting room behaviors. These instructions should be reinforced on a regular basis, through either newsletters or parent education nights. Toys, computers, books, and magazines should not be allowed in waiting rooms since they cannot be cleaned between use by patients. C. Precautions for Patients with Multidrug-Resistant Organisms

Patients known to be colonized with multidrug-resistant organisms may need to be managed with transmission-based precautions based on the organism and isolated from other patients. The CF Foundation guidelines recommend using contact and standard precautions if patients are coughing and are infected with Bcc, MRSA, multidrugresistant P. aeruginosa, or other epidemiologically important pathogens (1). CF centers need to consider and incorporate into their CF infection control policy the most efficient way to adhere to this recommendation. Some centers have found it easier to manage all patients known to be colonized with significant pathogens with standard and contact precautions. At a minimum, patients with a history of Bcc in their respiratory secretions should always be segregated from other CF patients, even those already colonized with Bcc; studies have demonstrated that a patient becomes ill after acquisition of a new genomovar of Bcc (15).

VII.

Environmental Infection Control

A. General Sterilization and Disinfection Measures

Staff assigned with the task of reprocessing medical devices should adhere to existing published guidelines for sterilization and disinfection and maintain annual certification of this practice (8). Cleaning and disinfection of nebulizers after use in a hospital setting has been discussed above. Nebulizers are never shared between patients although other devices can be safely reprocessed and used on multiple patients (e.g., dental instruments, endoscopes, and surgical instruments). Respiratory therapy equipment that comes into contact with mucus membranes requires high-level disinfection after every use. Some hospitals allow the respiratory therapy department to reprocess these items while others have all reprocessing performed by the central processing department. Regardless of who performs this important component of infection prevention or where it is performed, written policies should be in place and adhered to at all times. When hospitalized, routine cleaning and disinfection of the patient’s surrounding environment, for example, bathrooms, sinks, or bedrails should occur on a daily basis, which is the usual standard for all hospitalized patients. Room cleaning upon discharge should include all items not cleaned on a daily basis, for example, monitors and blood pressure cuffs. Privacy curtains may need to be changed if visibly soiled. Other areas that a CF patient visits (e.g., playroom, classroom, etc.) should be cleaned afterwards, especially if contamination with respiratory secretions has occurred. B. Pulmonary Function Testing Equipment

Special attention should be given to any environment or equipment that can easily become contaminated with respiratory secretions, such as the pulmonary function testing area. Pulmonary function testing frequently induces coughing, therefore the equipment and surrounding furniture is likely to become contaminated. Respiratory therapists

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should be instructed to clean and disinfect the environment and outside surfaces of the equipment upon completion of testing of every CF patient, regardless of their colonization status. Disinfection should include the use of the hospital-approved low-level disinfectant, which is usually a quaternary ammonium compound. Disposable disinfectant towelettes are available for ease of cleaning and to decrease staff exposure to aerosolized chemicals. Only disposable mouthpieces with filters should be used on pulmonary function testing equipment. C. Disinfection in an Ambulatory Care Setting

High-touch surfaces, such as sinks, door knobs, and exam tables in an ambulatory CF setting need to be cleaned more frequently than in other outpatient practice settings. Viable P.aeruginosa has been recovered from environmental surfaces contaminated more than a week earlier by the respiratory secretions from a colonized CF patient (16). Even though paper on the exam table is changed between patients, the surface of examination tables should be cleaned in between use in case there has been contamination with respiratory secretions. The ideal would be to have a dedicated housekeeper on site during CF clinic. When this is not practical, clinic staff needs to perform routine cleaning and disinfection tasks. The hospital’s CF infection control policy should detail housekeeping chores and frequency of performance.

VIII.

Infection Control in Non–Health Care Settings

A. Multipatient Families

Households with more than one family member with CF face difficult challenges in regards to infection control because contact density (i.e., multiple family members colonized with pathogenic organisms) contributes to cross-transmission. However, it is impracticable to ask siblings to maintain a 3-foot rule and avoid physical contact. Therefore, education should be concentrated on not sharing respiratory equipment, performing chest physiotherapy with only one child in the room, and practicing scrupulous hand hygiene and respiratory etiquette. B. Care of Respiratory Therapy Equipment in the Home

Cleaning and disinfection of nebulizers is essential for infection prevention in the home, but current practices in the home vary (17). Review of actual practices in the household setting has revealed that many patients do not clean and disinfect nebulizers and the devices are also overused (14). Manual cleaning with soap (e.g., dish detergent) and hot water should precede disinfection and be performed soon after completion of therapy. If there are time constraints, the device should be soaked in soap and water until adequate cleaning can be accomplished. Disinfection methods are outlined in Table 2 and will be dependent on manufacturer’s recommendations (13). Acetic acid (vinegar) is no longer recommended for nebulizer disinfection. If nebulizers are chemically disinfected they must be rinsed with sterile or filtered water rinse. Distilled water should not be used because it may contain organisms that are pathogenic to CF patients. This additional rinsing step may make other disinfection options (e.g., boiling in water, dishwasher, or microwave) more feasible for some families. Nebulizer care should be discussed and reviewed with families and patients during clinic visits and hospitalizations. CF centers should promote published guidelines and assist families in deciding which disinfection

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Table 2 Effective Methods to Disinfect Respiratory Equipment at Home

Disinfection method

Recommended duration a

Immerse in one of the following: 1:50 dilution of 5.25–6.15% sodium hypochlorite (household bleach) 70–90% ethyl or isopropyl alcohol 3% hydrogen peroxide

3 min 5 min 30 min

or Boil in water

5 min

or Use a standard-cycle dishwasher (>1588F or 708C)

30 min

or Use a home microwave (2.45 GHz)

5 min

Must be permissible by the manufacturer. a These preparations lose activity with time, but the optimal storage time is unknown. For example, chlorine preparations have a 50% reduction in activity after 30 days. Source: Adapted from Ref. 13.

method is most feasible for them. Nebulizers and associated equipment should be changed regularly as recommended by the specific manufacturer. C. Schools

Children with CF may attend the same school as non-CF children, but it is preferable that they not be in the same classroom or activity simultaneously. If that is unavoidable, they should adhere to the 3-foot spatial distance rule at all times. D. Camps

Current infection control guidelines recommend that all CF camps and overnight activities be discontinued (1). This difficult and unpopular decision was based on studies that demonstrated that summer camp attendance was a risk factor for Bcc acquisition. Subsequent analyses have suggested that acquisition may also have been influenced by the prevalence of Bcc at the camp, the extent of camper contact, the duration of camp attendance, and broad environmental contamination (18). European studies of crosstransmission at camps have produced conflicting data on whether cross-transmission occurs, and some CF centers in Europe have been reluctant to disband camps for all patients (19). Although prohibiting camp with peers also living with CF may seem like a draconian measure, CF-specific infection control measures (e.g., 3-foot rule, respiratory etiquette, and frequent disinfection) could not be adhered to in a camp setting. E.

Patient Gatherings

Patient support groups and education events are common within communities where individuals share the same disease entity. They offer psychosocial support, networking opportunities, and a chance to increase knowledge related to a particular disease.

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Although the potential benefits of patient gatherings are obvious, the CF Foundation infection control guidelines recommend excluding individuals with Bcc from these events due to the significant risk of transmission of the organism (1). Audiovisual and computer technology advances may provide a means for these individuals to feel connected to the larger group. Each CF center will have to decide whether or not to sponsor or encourage events where CF patients will gather. At this point, many young teenagers have grown up with the 2003 CF Foundation infection guidelines and are more easily able to adhere to the 3-foot rule, hand hygiene, and respiratory etiquette.

IX.

Psychosocial Issues Related to Infection Control

Because cross-transmission can occur between CF patients in health care settings even when staff adheres to evidence-based infection control guidelines, patient segregation has become a recognized component of CF-specific guidelines. This strategy is unique to the CF patient population, extends beyond the health care setting, and will persist throughout life. Segregation during hospitalization may cause patients to feel lonely or bored and acceptance may depend on maturity and previous experiences (20). Staff, such as child life specialists, teachers, nursing, and social work can facilitate and coordinate alternative activities for hospitalized CF patients confined to their room. Internet and chat rooms may help patients feel more connected to others but caution should be exercised since these are also sources of misinformation (21). Parents and patients provided with education about segregation and updated on a regular basis are more likely to be supportive of this measure (22). Allow parents and patients to verbalize their fears and concerns regarding crossinfection and segregation while at the same time acknowledging that the benefits of segregation outweigh the risk of cross-infection and clarify at each hospital admission expectations for isolation requirements (20).

References 1. Saiman L, Siegel J. Infection control recommendations for patients with cystic fibrosis: microbiology, important pathogens, and infection control practices to prevent patient-topatient transmission. Infect Control Hosp Epidemiol 2003; 24:S6–S52. 2. LiPuma J. Update on the Burkholderia cepacia complex. Curr Opin Pulm Med 2005; 11: 528–533. 3. Jones A, Dodd M, Govan J, et al. Prospective surveillance for Pseudomonas aeruginosa cross-infection at a cystic fibrosis center. Am J Respir Crit Care Med 2005; 171:257–260. 4. Speert D, Campbell M, Henry D, et al. Epidemiology of Pseudomonas aeruginosa in cystic fibrosis in British Columbia, Canada. Am J Respir Crit Care Med 2002; 166:988–993. 5. Gibson R, Burns J, Ramsey W. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Crit Care Med 2003; 168:918–951. 6. Boyce J, Pittet D. Guidelines for hand hygiene in health-care settings. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/ APIC/IDSA Hand Hygiene Task Force. MMWR Recomm Rep 2002; 51:1–45. 7. Siegel J, Rhinehart E, Jackson M, et al. Guideline for isolation precautions: preventing transmission of infectious agents in health care settings. Am J Infect Control and Hosp Epidemiol 2007; 35:S65–S164. 8. Rutala W, Weber D, and the Healthcare Infection Control Practices Advisory Committee. Guidelines for disinfection and sterilization in healthcare facilities. Centers for Disease Control and Prevention, Atlanta, 2008.

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9. Sehulster L, Chinn R. Guidelines for environmental infection control in healthcare facilities: recommendations of the CDC and Healthcare Infection Control Practices Advisory Committee. Centers for Disease Control and Prevention, Atlanta, 2003. 10. Tablan O, Anderson L, Besser R, et al. Guidelines for prevention of healthcare associated pneumonia: recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee. MMWR Recomm Rep 2004; 53(RR03):1–36. 11. Siegel J, Rhinehart E, Jackson M, et al. Guidelines for the management of multi-drug resistant organisms in healthcare settings, 2006. Am J Infect Control and Hosp Epidemiol 2007; 35:S165-S193. 12. Bolyard E, Tablan O, Williams W, et al. Guideline for infection control in healthcare personnel. Infect Control Hosp Epidemiol 1998; 19:407–463. 13. Saiman L, Siegel J. Infection control in cystic fibrosis. Clin Microbiol Rev 2004; 17:57–71. 14. Lester M, Flume P, Gray S, et al. Nebulizer use and maintenance by cystic fibrosis patients. Respir Care 2004; 49:1504–1508. 15. Jones M, Webb A. Recent advances in cross-infection in cystic fibrosis: B. cepacia complex, P. aeruginosa, MRSA, and Pandoraea. J R Soc Med 2003; 43:66–72. 16. Bush A. Decision facing the CF clinician at first isolation of P. aeruginosa. Paediatr Respir Rev 2002; 3:82–88. 17. Reychler G, Aarab K, Van Ossel C, et al. In vitro evaluation of efficacy of 5 methods of disinfection on mouthpieces and facemasks contaminated by strains of CF patients. J Cyst Fibros 2005; 4:183–187. 18. Mychalak-Walsh N, Casano A, Managan L, et al. Risk factors for B. cepacia colonization and infection among patients with cystic fibrosis. J Pediatr 2002; 141:512–517. 19. Brimicombe R, Dijkshoorn L, van der Reijden T, et al. Transmission of P. aeruginosa in children with cystic fibrosis attending summer camps in the Netherlands. J Cyst Fibros 2008; 7:30–36. 20. Russo K, Donnelly M, Reid A. Segregation—the perspectives of young patients and their parents. J Cyst Fibros 2006; 5:93–99. 21. Duff A. Psychological consequences of segregation resulting from chronic B. cepacia infection in adults with cystic fibrosis. Thorax 2002; 57:756–758. 22. Griffiths A, Armstrong D, Carzino R, et al. Cystic fibrosis patients and families support cross-infection measures. Eur Respir J 2004; 24:449–452.

28 Transition to Adult Care SHERSTIN G. TRUITT and JAMES R. YANKASKAS University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A.

I.

Introduction

Investments in cystic fibrosis (CF) research and care have produced marked improvements in available treatments, the quality of life, and survival. These changes have progressively increased the number of adults with CF, and have challenged the CF community to deal with the medical and social issues related to maturation and adulthood. Perceptive caregivers at Dartmouth College, the University of Pittsburgh, the University of North Carolina, and other institutions recognized these needs and started the first adult CF programs in the early 1980s. Issues associated with transition from pediatric to adult health care were identified and addressed on the basis of local needs and resources. Transition tasks and strategies have been addressed in symposia, workshops, short courses, and consultation clinics at every North American CF Conference since 1987. A 1997 meeting of CF center directors defined four adult CF program models, and mandated that all CF centers with more than 40 adults have an accredited adult CF program by 2000. The CF Foundation has fostered this mandate through administrative funding for adult care, workshops, mentoring programs, research and clinical fellowships, and support for faculty development through the Program for Adult Care Excellence (PACE) grants. This chapter summarizes the current goals, knowledge, and methods for planning effective transition by discussing its timing, challenges, and current models of execution. The progress in these important tasks provides a sound basis for ongoing improvement. CF provides a model for health care transition in diseases that were once limited to childhood, but now include large numbers of patients who have significant or normal adult life spans.

II.

Timing of Transition

A well-planned transition should be purposeful and expected by the patient. The process must begin early in life and be fostered during adolescence by asking patients to accept increasing responsibility in disease management as they mature. Interview time without parents (1,2), speaking directly to a patient instead of parents, and asking the teenagers’ opinion regarding treatment plans help young people gain understanding and knowledge of adult medical expectations. There should be some flexibility regarding the actual date of transition, but suggesting a target transfer age at least a year in advance gives both patients and staff more concrete timelines (3) and the opportunity to ask questions while adjusting to the idea of working with new providers.

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Occasionally, transition is not a mutually agreed-upon process by the patient and caregivers, but instead occurs as a result of patient behavior or limitations associated with licensing guidelines. Medical noncompliance, pregnancy, criminal activity, and suicide attempts can usher in rapid change that can be perceived as a punishment (2,4). Patients may have to move to an adult team because some hospitals cannot admit patients over age 21 years, some pediatric nurse practitioners are not permitted to treat individuals over the age of 25, and many consulting physicians are not comfortable caring for young adults (4).

III.

Challenges of Transition

Adolescence is a time to develop more mature relationships, become more confident in social roles, achieve emotional independence from parents, and gain responsibility in preparation for future employment and economic independence. Young adulthood is the period in which children leave home, adapt to living with a roommate or spouse, engage socially, obtain employment, manage a home, and participate in civic responsibilities (5) (Table 1). It is our responsibility to help patients achieve these milestones so they can successfully engage in society and more responsibly manage their health needs. This is a daunting task as not only must these teenagers manage the social pressures of adolescence but also must incorporate regular management of aerosol therapies, take multiple daily medications, and find time for frequent doctor appointments. Therefore, several governing bodies have recommended that adolescent care be paid special attention and given more focus. For example, in United Kingdom, the House of Commons Select Committee on Health, fifth report concluded in 1997 that “the transfer of young people, particularly those with special health needs, from child to adult services requires specific attention” (2,7). In the United States, “the Maternal and Child Health Bureau identified transition as 1 of 6 core outcomes that, when achieved, will indicate successful progress toward the goal of a community-based system of services for children with special health care needs” (8,9). Pediatric patients are often relatively healthy, but as they become older their disease status also progresses, creating a perception that adulthood heralds illness. These fears are perpetuated as teens notice older patients having more difficulties. The adverse effects of poor nutrition and airway colonization with Pseudomonas may increase in tandem with transition time. Patients may notice increased frequency of pulmonary exacerbations, have more difficulty with sinusitis and nasal polyps, be diagnosed with CF-related diabetes (about 5%), suffer from distal intestinal Table 1 Tasks of Late Adolescence (18 to 21 Years)

Shows physical, cognitive, emotional, social, and moral competency Supports a healthy lifestyle Create supportive relationships with others Positively interacts with society at large Displays self-confidence Effectively manages life stressors Demonstrates independent decision making Source: Adapted from Ref. 6.

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Table 2 Early Adult CF Complications

Increased number of exacerbations Worsening sinusitis CF-related diabetes DIOS, Liver disease Bone loss Hypertrophic osteoarthropathy Abbreviations: CF, cystic fibrosis; DIOS, distal intestinal obstruction syndrome.

obstruction syndrome and/or liver disease, be diagnosed with osteopenia or osteoporosis, and/or first experience joint pain due to hypertrophic osteoarthropathy (1,10) (Table 2). Adulthood is thus often viewed as leading to health complications, the need for lung transplantation, and ultimately death (2,10). An adult team must understand that these new disease manifestations may be perceived as poor adult care (2). The movement of care from pediatrics to adult medicine is thus an important and multifaceted change extending far beyond the transfer of medical records. Practitioners must be prepared to deal with delays in development, disease progression, and family anxiety in the context of typical adolescent experiences such as social adjustments, sexuality, new behaviors, and economic changes. For example, CF patients are often delayed in their tasks of adolescence due to vigilant parental involvement in diet, school activities, social venues, and health care. Parents may be reluctant to concede care to their children, concerned that it will not be done adequately. Thus, despite being older teenagers, some patients lack significant life planning experience, leaving them less able and less confident in management skills than their peers (5). In the midst of some of these health changes, patients find themselves being treated from a different stylistic perspective. There tends to be a stronger family focus in pediatrics with staff being more available for support. Adult medicine expects more independent relationships and visits are disease and treatment plan focused (4). Patients perceive these differences as a lack of personal interest, and parents often feel alienated from their children’s care (10). As disease worsens, new practitioners may give advice that seems to contradict the pediatric team, leading to mistrust and potential conflict until a new rapport is developed (3). Patients and families are also concerned that an adult provider will not offer the same quality of care as their pediatrician (Table 3). Despite the increasing number of accredited adult CF centers, many patients must still be transitioned to general pulmonologists who often lack specific training regarding the various manifestations and course of CF (1). Adolescents worry that their new physician will not be as accessible as their pediatrician (4,9). They would prefer to be treated by someone familiar with their unique patient experience and not in a clinic, potentially exposing them to new pathogens (10). Thus, there is little motivation to leave the pediatric relationships behind (2,9). Aside from establishing interpersonal relationships, an essential part of any medical care transition is the timely and complete communication of medical records. Checklists from the receiving center help referring practices understand what information is helpful. For example, summaries from pediatric subspecialists, including physicians, nurse coordinators, social workers, nutritionists, and physical therapists, give

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Table 3 Transition Concerns

Patient concerns

l l l l

Pediatrician concerns

l l

Caregiver concerns

l l

Adequate provider accessibility Exposure to new pathogens Working with strangers Need for lung transplantation Adult provider competence Paucity of adult programs Dependability of their child Alienation from their child’s care

adult providers a better understanding of a patient’s previous course and interaction with the health care system (2). Specific culture results including Pseudomonas and Burkholderia spp., presence of MRSA or atypical mycobacteria, and the antibiotic sensitivities are paramount. Records of weight and pulmonary function, in addition to dates of recent courses of IV or oral antibiotics, are very helpful. The ordering of ageappropriate screening tests prior to transfer (such as a DEXA scan, an oral glucose tolerance test, and a liver ultrasound) help the accepting provider better prepare for CFassociated morbidities, in addition to decreasing patient perceptions that the new physician is primarily focused on tests (10).

IV.

Development of Transition

Centers depend on family member support to achieve the goals of transition, and the process ideally begins prior to adolescence (4). Parents must lay the groundwork for independence and allow their children to perform tasks of daily living, assist with their medical self-care, and take increasing responsibility in asking for more urgent medical attention (4,9). During medical visits, teens should be asked about current activities, plans for higher education and employment as well as health care topics. Interest in CF patients’ futures is vital as many children are acutely aware of having never been asked about their future and understand this as limiting their attainable dreams (1). Social topics, such as drug and alcohol use and sexuality, must be addressed. Although difficult to discuss especially with limited interview time, they are exceptionally important in adolescent development and have clear impact on overall health status. The need for this education is clear as demonstrated in a recent study that reported only 57% of CF males had learned about infertility by age 20 years (11) Furthermore, surveys from teenagers demonstrate a desire to have physicians involved with their contraception decisions, fertility issues, and to address relationship difficulties (9,10). Given that many CF patients do not visit a primary care provider (PCP) regularly and adolescence spans both pediatric and adult care, each team must be comfortable raising these topics. In the midst of developing new social roles, teenagers must also learn the mechanics of interacting with their health care system. These include transportation to medical visits, obtaining medications, and understanding insurance coverage. Social work intervention and education is paramount to uncovering what needs are present at emancipation and informing patients of services for which they qualify. For example, at age 18, children are often dropped from their parents’ coverage unless they have

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enrolled in continuing education (1,4). Also, state funding from Title V Children with Special Health Care Needs programs typically ends at 21 years. Some patients, however lose their Title V coverage at age 18 as the Supplemental Security Income qualifications become more strict (4). Medicaid patients receive less coverage and fewer services once they turn 21 years (4) or may lose coverage altogether should they choose to marry.

V. Models of Transition Pediatricians and internists often differ in their philosophy of care. The gap between these models of medical practice must be bridged if patients are successful in gaining independence and parents are comfortable supporting this. Over the past several years, models have been suggested to meet this need: the transition clinic, the adolescent clinic, the primary care clinic, or the lack of transfer. The transition clinic is one in which patients are cared for by both pediatric and adult physicians. This allows for patients to be seen over several visits by either an adult or pediatric provider, or to continue seeing their pediatric pulmonologist with visits from an adult physician (1,5). Patients are able to develop a relationship with their future provider and become more accustomed to the style of questions asked by adult physicians. Personal involvement in the adolescent’s care plan familiarizes the adult team with the unique experiences of each case. The adolescent clinic is a separate clinic designed for teenagers. Younger patients are not treated during that time and the staff focuses on adolescent health concerns as well as the general needs of patients with chronic disease (5,12). The provider is a specialist in adolescent medicine. As such, he or she is trained to expect more independent thought from the patients and to fill the paucity of guidance regarding substance use and sexuality. The primary care model uses a PCP as the coordinator of general care and leaves the subspecialist to provide focused expertise (9,12). As general practitioners are often more focused on family needs, their exams may ensure that many needs are identified and addressed. Unfortunately, many PCPs do not have the most current medical knowledge regarding CF care or the ancillary resources to address the specific social needs discussed above. Some providers continue to advocate for patients to remain with their pediatrician throughout adulthood (5). This is not an optimal choice as there are a multitude of social and medical issues that extend beyond the expertise of pediatricians, especially as CF patients are living past their fourth decade. The multitude of CF comorbid conditions underscores the need for a comprehensive care clinic. In some areas of the United States a highly qualified adult provider is not available. Patients who are near death may not accrue sufficient benefits from adult care to justify reducing their interactions with a skilled pediatrician. Thus, in a few circumstances, transition to adult care may not be the better choice.

VI.

Summary

The movement of patients from pediatric to adult medicine is both exciting and anxiety provoking. Providing educational materials, personally introducing the new provider, printing cards with team contact numbers, sending news bulletins, having updated Web

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sites, offering support groups, and providing tours of adult facilities often abate many concerns (1,2,10). The multitude and variety of issues involved is great and many challenges remain. Many CF patients receive their primary care from subspecialists who are not trained in developmental tasks and whose clinics are not equipped to address adolescent-specific issues. Institutions that lack formalized transition programs can allow important needs to be overlooked. Medical insurance and adequate financial reimbursement for comprehensive care are essential (9). Many models have been proposed to address these topics. Whichever one is chosen, however, must be well defined. A distinct plan with rigorous attention to detail yields clear expectations for patients and families while guiding health care teams through the multitude of concerns needing to be addressed. Obviously, this is a complicated maneuver, but, if done well, helps launch CF patients into a lifetime of exemplary comprehensive and self-care.

References 1. Landau LI. Cystic fibrosis: transition from paediatric to adult physician’s care. Thorax 1995; 50(10):1031–1032. 2. Viner R. Barriers and good practice in transition from paediatric to adult care. J R Soc Med 2001; 94(suppl 40):2–4. 3. Por J, Golberg B, Lennox V, et al. Transition of care: health care professionals’ view. J Nurs Manag 2004; 12(5):354–361. 4. Reiss JG, Gibson RW, Walker LR. Health care transition: youth, family, and provider perspectives. Pediatrics 2005; 115(1):112–120. 5. Bryon M, Madge S. Transition from paediatric to adult care: psychological principles. J R Soc Med 2001; 94(suppl 40):5–7. 6. Hagan JF, Shaw SJ, Dunca PM, eds. Bright Futures: Guidelines for Health Supervision of Infants, Children, and Adolescents. 3rd ed. Elk Grove Village, IL: American Academy of Pediatrics, 2008. 7. House of Commons Select Committee on Health. Fifth Report: Hospital Services for Children and Young People. London: HMSO, 1997. 8. Maternal Child Health Bureau. Achieving Success for All: Children and Youth with Special Health Care Needs. A 10-Year Action Plan to Accompany Healthy People 2010. Washington, DC: Maternal and Child Health Bureau, 2001. 9. Scal P. Transition for youth with chronic conditions: primary care physicians’ approaches. Pediatrics 2002; 110(6 pt 2):1315–1321. 10. Webb AK, Jones AW, Dodd ME. Transition from paediatric to adult care: problems that arise in the adult cystic fibrosis clinic. J R Soc Med 2001; 94(suppl 40):8–11. 11. Fair A, Griffiths K, Osman LM. Attitudes to fertility issues among adults with cystic fibrosis in Scotland. The Collaborative Group of Scottish Adult CF Centres. Thorax 2000; 55(8): 672–677. 12. Callahan ST, Winitzer RF, Keenan P. Transition from pediatric to adult-oriented health care: a challenge for patients with chronic disease. Curr Opin Pediatr 2001; 13(4):310–316.

29 Reproduction, Sexuality, and Fertility LISA K. TUCHMAN Children’s National Medical Center, Division of Adolescent and Young Adult Medicine, Children’s Research Institute, Center for Clinical and Community Research, Washington, D.C., U.S.A.

IOANNA K. GISONE The Children’s Hospital of Philadelphia, Craig-Dalsimer Division of Adolescent Medicine, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

In 2007, more than 45% of people with cystic fibrosis (CF) were over age 18 and expectations are that individuals born after 2000 will survive into their fifth decade; thus CF-related reproductive health has become increasingly relevant for patients and families. Adolescents and young adults with CF have similar age of onset of sexual interest and activity as their healthy adolescent peers (1,2) and are in need of disease-specific sexual and reproductive health information that goes beyond what a primary pediatrician or gynecologist can be expected to provide. There are no formal guidelines about timing and content of discussions for CF providers although there have been several published recommendations in the academic literature (2–4). Adolescents and young adults with CF are more likely to be the initiators of reproductive health discussions than their health care providers (5). However, 87% of adolescents with CF report having never discussed sexual or reproductive health with their CF provider (6). Nonetheless, the majority identified their CF provider and parents as the preferred source of reproductive health information (6–8). This chapter addresses CF-specific reproductive pathophysiology, adolescence, gender-specific issues, reproductive health, pregnancy, fertility assistance, and behavioral aspects of sexuality and living with CF.

II.

Reproductive Pathophysiology

A. Structural Abnormalities

Almost all men with CF (99%) have congenital bilateral absence of the vas deferens (CBAVD). The prevalence of CABVD is 1% to 2% among infertile males without CF (9) with 80% positive for CFTR mutations (10). Thus, it seems that a normal amount of functioning CFTR protein is needed for embryologic development of the vas deferens. Although the exact mechanism is unknown, the detection of CFTR mRNA in the human fetal reproductive tract suggests that CFTR protein acts in the development of the reproductive tissues in men (11). In contrast, women with CF typically have structurally normal reproductive tracts. There is an increased prevalence of CFTR mutations among non-CF women with congenital absence of the uterus and vagina (CAUV), but given the

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low prevalence of CAUV among women with CF, it is unlikely that the mechanism of agenesis is the same for both genders. B. Puberty

Delayed puberty and menarche are common, even in healthy, well-nourished female adolescents with CF (12). While the mechanism is not well understood, it is known that multiple sites within the female reproductive tract are affected by mutations in CFTR. The hypothalamic pituitary axis controls the onset and rate of puberty. Gonadotropin-releasing hormone (GnRH) is secreted from the hypothalamus inducing luteinizing hormone (LH) and follicle stimulating hormone (FSH) production and release from the anterior pituitary gland. Fluctuations in LH and FSH are critical to ovulation by stimulating development of a primary ovarian follicle and causing the primary follicle to be released (ovulation). Expression of CFTR in the rat brain and the human hypothalamus has been documented (13–15). Thus, it has been speculated that altered CFTR expression in the hypothalamus results in abnormal secretion of GnRH that in turn leads to dysregulatory neuroendocrine secretion of hormones and thereby delays sexual maturation (12). Much of the female reproductive tract is lined by epithelial cells and is therefore also affected by mutations in CFTR (16). In addition to known ovulatory disturbances and tenacious cervical mucous in CF, there are changes in uterine bicarbonate secretion (12,17,18). An alternative theory to explain delayed puberty in females with CF is increased energy consumption [resting energy expenditure (REE)]. When compared with males with CF and control females, females with CF have higher REE (19). However, a recent study showed that raised REE appears to be independent of menarchal status (pre- or post-menarche), and independent of phase of menstrual cycle based on date of last menstrual period (LMP) (20). Additionally, in healthy populations, it is well established by using ultrasensitive recombinant cell assays for estrogen that prepubertal girls have 8-fold higher estrogen levels than prepubertal boys (21). In non-CF children, this may contribute to girls’ greater rate of skeletal maturation, earlier onset of puberty, and earlier cessation of growth when compared with boys. This prepubertal increase in estrogen also provides a potential explanation for why females with CF aged 2 to 20 years have had significantly poorer survival than male patients (22), given concerns about estrogen’s effect on net worsening of airway surface dehydration and decreased mucocilliary clearance (23). Data examining the longitudinal relationship between timing of menarche with disease symptoms and progression are sparse.

III.

Adolescence

Adolescents with CF must navigate through a dynamic physical, biological, social, and psychological transition while managing chronic illness and effects of CF on social and reproductive development and function. Many CF teens have problems with body image and are keenly aware of how they may look different than their peers. Accurate CF sexual and reproductive health information should be routinely offered so that parents and individuals with CF can anticipate how CF may affect physical, emotional, and interpersonal development. Much like their healthy peers, an ongoing age appropriate dialogue regarding reproductive health is critical in minimizing

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anxiety about how CF affects reproductive function and opens the door for continued discussions as concerns or issues arise. It is recommended that all females aged 13 to 15 years have their first gynecological or adolescent medicine visit for preventive healthcare services, including ageappropriate education and guidance (24). It is important to recognize that routine reproductive and sexual health screenings may not address the CF-specific reproductive health needs. The development of adolescent sexuality is an important complex phenomenon whose success significantly contributes to adaptation into adulthood. Adolescents with CF can expect normal sexual function and to have the same risk as healthy peers to be exposed to sexually transmitted infections (STIs). Although the prevalence of bacterial STIs in the CF population is unknown, it is likely to be very low given the frequent use of antibiotics. Viral STI prevalence (human papillomavirus, genital herpes) in CF populations is also unknown, but can be assumed to be similar to the general population of sexually active individuals. All people with CF should be advised they need to use condoms or barrier protections to reduce the risk of genital STI transmission. It has been well established that rates of cervical dysplasia are increased in immune-suppressed patients (25,26). Therefore, women with CF, particularly lung transplant patients, should get yearly cervical cancer screening. A. Risk Taking

Some risk-taking behavior is normal during adolescence and teens with CF are no different, although consequences can be great. While reported rates of risky behaviors (tobacco use, sexual intercourse) are lower among teens with CF than healthy peers, still 20% of teens with CF report smoking and 28% report sexual intercourse (27). Similarly, many youth with chronic health conditions experiment with adherence-related risk taking. A positive approach to maintaining health is generally more effective than focusing on negative behaviors. Thus, we recommend an open dialogue and nonjudgmental approach to guiding teens and young adults with CF toward healthy behaviors. B. Sources of Information

A recent study revealed that little disease-specific reproductive health information is required for pediatric pulmonology board certification despite the growing adolescent and young adult population with CF (28). Perhaps this stems from insufficient scientific evidence or unclear expectations about whose responsibility it is to provide CF-related reproductive health. Although adolescents may initially acquire sexual and reproductive health information from their parents, the Internet and/or other health care providers, pediatric, and adult pulmonologists along with their CF teams can greatly influence their patients’ reproductive decision-making. CF providers have the opportunity to address myths or concerns regarding inaccurate information seen on the Internet, especially given concerns regarding adherence to AMA guidelines (29) of CF informational Web sites (30). Understanding the role of reproductive health in CF disease as well as informing safer reproductive health care for the growing adolescent and adult CF population should be a priority area of future research. Guidelines for CF providers caring for teens and young adults are included in Figure 1 along with essential CF-specific reproductive health resources in Figure 2.

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A positive approach to maintaining health is generally more effective than focusing on negative behaviors. Thus, we recommend an open dialogue and nonjudgmental approach. Ask about and document hormonal contraception use, menstrual history, pubertal development, and encourage routine gynecologic care. Keep in mind:

 Men with CF should be n

informed that they are not impotent, but likely unable to conceive without fertility medical intervention during early adolescence, n offered semen analysis during adolescence, n advised to use condoms to prevent STI transmission, n offered hope regarding the potential for fatherhood.  Women with CF should be n informed that they are fertile, n offered contraceptive choices during adolescence, n advised to use condoms to prevent STI transmission, n monitored for vaginal yeast infections and incontinence, n screened for diabetes during and after pregnancy.

Figure 1 Tips for CF providers caring for teens and young adults.

1. 2. 3. 4.

Sawyer S, Roseby C. “What They Don’t Tell You: A Young Person’s Guide to Sexual and Reproductive Health Issues in Cystic Fibrosis”. Melbourne: Centre for Adolescents Health, 2001. ISBN 1740560043 “Sexuality and Cystic Fibrosis: Information for Adolescents” available in PORT CF and at: http://www.cysticfibrosis.ca/pdf/AdolescentSexuality2008E.pdf “Sexuality and Cystic Fibrosis: Information for Adults” available in PORT CF and at: http://www.cysticfibrosis.ca/pdf/SexualityAdultE.pdf “Guidelines for the management of pregnancy in women with CF” (53)

Figure 2 Essential Reproductive Health Resources for Adolescents and Young Adults with CF.

IV.

Women

A. Estrogen and CFTR

In women without CF, estrogen is known to increase the water content of cervical mucous; these changes are not observed in women with CF (31). One recent study reported that during phases of the menstrual cycle with increased estrogen levels, women experience decreased calcium-mediated chloride secretion, resulting in a net worsening of airway surface dehydration and decreased mucocilliary clearance (23). It is not known how these epithelial changes relate to clinical status, but it raises both concerns about exogenous estrogen administration and suggesting hormone-based therapeutic possibilities, highlighting the historical gender differences in CF pulmonary disease.

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B. Menses

Empirically, patients with CF report worsening of respiratory symptoms prior to and during menstruation. There is one small study published investigating lung function changes in relation to the menstrual cycle in twelve females with CF (32). Participants kept daily records during three menstrual cycles that included lung function, sputum quality, and need for IV antibiotics. Airway symptoms were compared at three time points: (i) ovulation: with high estrogen, low progesterone; (ii) luteal phase: high progesterone, low estrogen; and (iii) menses: low progesterone, low estrogen. Pulmonary function was significantly higher with increased forced expiratory volume in one second (FEV1) during the luteal phase compared to ovulatory and menstruation (p < 0.01) possibly due progesterone’s role in inducing smooth muscle relaxation affecting airway smooth muscle. It is unclear whether these differences in lung function and symptoms represent a worsening of pulmonary function because of elevated levels of estrogen during the follicular phase, an improvement because of elevated progesterone levels in the luteal phase, or some other process not yet elucidated. Another study reported higher nasal potential difference (NPD) during the luteal than follicular phase suggesting ion transport is altered by female hormones in vivo (33). Further supporting female sex hormone involvement in mediating pulmonary function is that cyclical changes in symptoms and lung function have only been observed in females with CF, not males (34). C. Models in Other Diseases (Asthma)

There is a well-described subset of female asthmatics, ranging from 33% to 52% reported prevalence, with worsening of asthmatic symptoms during the perimenstrual phase of the menstrual cycle (35–37). Almost half of women admitted to the hospital for an asthma exacerbation in one study were identified as perimenstrual (38). Numerous studies suggest that the use of combination estrogen and progesterone hormonal contraceptive methods reduce endogenous hormonal fluctuation and thereby significantly improve premenstrual asthma and reduce steroid dependence (39–41). Investigators speculate that the role of estrogen and progesterone in premenstrual asthma is related to reduced contractility and increasing relaxation of bronchial smooth muscle (42–44). Further, estrogen withdrawal is known to increase bronchial contractility (45). D. Prostaglandins, Menses, and CF

The sequential stimulation of the endometrium by estrogen (follicular phase) followed by progesterone (luteal phase) results in a large increase in endometrial stores of arachidonic acid, a precursor to prostaglandin production. During menstruation the arachidonic acid is converted to prostaglandin F2-alpha (PGF2), prostaglandin E2 (PGE2), and leukotrienes, which play a major role in inducing uterine contractions during menses. In CF, pulmonary exacerbations are characterized by increased oxidative stress and sputum concentrations of bioactive lipid mediators, including prostaglandins (46). A recent Cochrane meta-analysis concluded that high-dose ibuprofen can slow the progression of lung disease in people with CF, especially in children, suggesting that strategies to modulate lung inflammation can be beneficial for individuals with CF (47). Whether prostaglandins produced monthly during menses affects short- or long-term lung function via increased acute or cumulative inflammation is unclear.

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Fertility

The main hypothesized mechanism for decreased fertility among CF women is tenacious cervical mucous that is impermeable to sperm. Malnutrition, inflammation, and other described ovulatory disturbances may play a role (48). F.

Sexual and Reproductive Health Issues

Vulvovaginal candidiasis, stress incontinence, and vaginal dryness are common complaints of women with CF. Gynecology or adolescent medicine referral is appropriate to address these concerns. Recommending aerosol therapy prior to intercourse can reduce coughing and fatigue that may impact intimacy. G. Contraception

Women with CF absolutely need contraception to prevent pregnancy. The same options that exist for non-CF women are appropriate for women with CF. No clinical studies have addressed whether progestin-only methods [progestin only pills, depot medroxy progesterone acetate (intramuscular contraceptive injection), progestin-impregnated intrauterine device (IUD)] adversely affect bone health in CF, but can be assumed to have the same effects as in non-CF women. Pills, patch, and ring (combined estrogen and progesterone hormonal methods) are appropriate but it is unknown how exogenous estrogen may affect ion transport as reported in vivo. Among women with CF, prevalence of contraceptive use is estimated to be around 60% (49), although contraceptive information is not recorded in the CFF registry. Issues such as malabsorption of oral contraceptive pills (50), risk of venous thrombosis, liver dysfunction affecting metabolism of exogenous hormones, pulmonary hypertension (51), and possibility of decreased effectiveness of oral contraceptives with certain medications must be taken into consideration. Condom or barrier protection use should always be encouraged, both for back-up contraception and to reduce risk of STI transmission. H. Pregnancy

Empirically, women with CF report that pregnancy is the time they feel the best, both emotionally and physically. The number of pregnancies and live births reported has increased yearly in the CFF Registry. Encouragingly, the 10-year-survival rate of women with CF who have conceived and delivered children (while controlling for disease severity) was higher than those who never had children (52). Pregnancy and motherhood is a realistic possibility for many women with CF. It should be noted that pregnancy is contraindicated if a woman has preexisting pulmonary hypertension or cor pulmonale (53). There are also concerns of fetal hypoxia and growth retardation in women with severely impaired lung function that requires oxygen supplementation (53). Health optimization is critical prior to conception, although women with CF can expect to choose pregnancy and carry out a term delivery safely, mostly likely without negative long-term health consequences. Importantly, increased medical care around conception, pregnancy, and delivery is essential, especially CF-related diabetes screening and treatment (54,55). Breast-feeding is possible, but needs to be carefully weighed with nutritional demands and crossover of medications into breast milk.

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V. Men A. Fertility

Although obstructive azoospermia is the primary mechanism of infertility in men, reduced spermatogenesis, sperm and semen volume, as well as low semen pH have all been described and may contribute to reduced fertility (56). B. Sexual and Reproductive Health Issues

Both genders may experience coughing and fatigue impacting intimacy. These symptoms may be reduced by aerosol therapy prior to intercourse. It should be emphasized that while men with CF are unable to conceive naturally, they are not impotent. Men with CF have normal sexual function including arousal and ejaculation. Testosterone levels in men with CF are commonly lower than healthy controls and may impact libido, mood, bone health, and muscular function. Therefore, endocrine referral and routine testosterone level monitoring with intramuscular repletion is appropriate. While it remains unclear whether testosterone repletion affects respiratory muscle function and pulmonary outcomes (57), it likely improves mood and quality of life of men with CF. C. Semen Analysis

Males with CF may be unaware of their fertility status and many report not been offered sperm analysis despite wanting fertility testing (2,3,8). Discussions with CF males should ensure that impotence is not confused with infertility and include specific information regarding the percentage of infertile men with CF, the likelihood of normal spermatogenesis, abnormalities in semen parameters (4,58), and the option of sperm aspiration and intracytoplasmic sperm injection (ICSI) along with genetic counseling to help achieve pregnancy. To make informed decisions about their sexual and reproductive health, all men should be offered semen analysis to assess for fertility (46). Males with CF report preferring to receive information about fertility at an earlier age (14.4 years) than when their provider-patient discussions actually occurred (16.4 years) (59,60). Revisiting semen analysis throughout puberty and young adulthood is appropriate, as relevance is variable depending on maturity and developmental stage.

VI.

Assisted Reproductive Technologies

Advancements in reproductive technologies make conception and pregnancy easier and more successful for couples where one or both partners have CF. While almost all men with CF have abnormalities in semen parameters, biologic paternity can be achieved through surgical techniques that allow for the procurement of viable sperm via microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal or testicular sperm aspiration (PESA, TESA) in combination with ICSI and in vitro fertilization (IVF). Men with CF and their female partners may also choose IVF via donor sperm. Women with CF, while usually fertile, can use intrauterine insemination of washed sperm (61) or IVF to help achieve pregnancy. If ovulation induction is considered, risks such as ovarian hyperstimulation and increased risk of ovarian torsion must be considered.

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VII.

Parenting with CF

With increased life expectancy comes a greater desire for parenthood in women and men with CF (53). The newborn period is often an exhausting and emotional time in which parents with CF will require close medical monitoring and support. Balancing the care of a newborn with the demands of their own self-care can be overwhelming and requires a dedicated partner and family support.

VIII.

Genetic Screening

The probability of conceiving a child with CF is based on the carrier rate in the population and whether one or both partners have CF. A complete review of prenatal genetic-testing options coupled with prenatal counseling early on in planned conception should be offered. Pan-ethnic CF screening has been recommended by the American College of Medical Genetics and the American College of Obstetricians and Gynecologists for all couples interested in parenthood and should include ethnic-specific mutations that may be present within the diverse population of the United States (62). The National Institutes of Health strongly recommends genetic testing for couples when the male partner (or sperm donor) is positive for a CF mutation and has either CF or obstructive azoospermia, and the couple is taking steps to use assisted reproductive technologies (63). However, genetic screens do not yet detect all CF carriers given that there are more than 1400 different CF gene mutations. Therefore, even with a negative genetic screen, there is still a slight possibility that a partner may be a CF carrier. Currently, screening practices vary between cities, states, and countries, and routine screening of both partners may be unavailable or cost prohibitive depending on insurance status. Offering the option of preimplantation genetic diagnosis to allow women with CF and/or their partners to make reproductive choices prior to conception should be considered.

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57. Barry PJ, Waterhouse DF, Reilly CM, et al. Androgens, exercise capacity, and muscle function in cystic fibrosis. Chest 2008; 134(6):1258–1264. 58. Lewis-Jones DI, Gazvani MR, Mountford R. Cystic fibrosis in infertility: screening before assisted reproduction: opinion. Hum Reprod 2000; 15(11):2415–2417. 59. Sawyer SM, Farrant B, Cerritelli B, et al. A survey of sexual and reproductive health in men with cystic fibrosis: new challenges for adolescent and adult services. Thorax 2005; 60(4):326–330. 60. Sawyer SM, Tully MA, Colin AA. Reproductive and sexual health in males with cystic fibrosis: a case for health professional education and training. J Adolesc Health 2001; 28(1):36–40. 61. Kredentser JV, Pokrant C, McCoshen JA. Intrauterine insemination for infertility due to cystic fibrosis. Feril Steril 1986; 45(3):425–426. 62. Sugarman EA, Rohlfs EM, Silverman LM, et al. CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations. Genet Med 2004; 6(5):392–399. 63. Sokol RZ. Infertility in men with cystic fibrosis. Curr Opin Pulm Med 2001; 7(6):421–426.

30 A Biopsychosocial Model of Cystic Fibrosis: Social and Emotional Functioning, Adherence, and Quality of Life DAVID BARKER and ALEXANDRA L. QUITTNER University of Miami, Coral Gables, Florida, U.S.A.

I.

Introduction

Over the last two decades, advances in the diagnosis and treatment of cystic fibrosis (CF) have led to significant improvements in health outcomes and life expectancy. Median life expectancy in the United States is now 36.7 years (1) and the majority of pediatric patients diagnosed today are expected to live into adulthood. These improvements in life expectancy have shifted the focus of CF research and clinical care toward the complex interplay among behavioral, psychosocial, and health outcomes. This change in focus is epitomized by the growing prominence of patient-reported outcomes (PROs), such as health-related quality of life (HRQOL), and new efforts to address behavioral and emotional challenges, such as adherence and depression (2). This chapter introduces a developmental biopsychosocial model that highlights the bidirectional influences of age-related development on disease management and progression, and presents current research on these influences.

II.

A Developmental, Biopsychosocial Model of CF

Across the lifespan, physical, social, and emotional development occurs in the context of multiple interacting systems. Children are members of families, schools, communities, and cultures. All of these systems interact dynamically with each other to influence development (3) (Fig. 1). The presence of CF affects these systems in important ways: it increases family stress, changes family functioning and peer relations, and introduces new systems to the family (4) (health care providers, hospitals, etc.). CF also directly affects children’s physical development (i.e., delayed puberty, short stature) as well as their goals and plans for the future. These systems, in turn, affect the course of the illness and its management (5). The dynamic interplay of these systems often creates transition periods marked by rapid change (6). The most notable and predictable transition periods for patients and families with CF are (i) time of diagnosis, (ii) entrance into school, (iii) development of autonomy during adolescence, and (iv) transition into adult roles. Each transition is typically marked by major changes in one or more social, family, or medical contexts/ systems, requiring the others to adjust. For example, as children enter school, parents must coordinate CF treatments with school personnel, and children must decide how

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Figure 1 Systems and contexts that influence patient health. This figure depicts the most salient

systems and contexts that influence patient’s health. The white boxes represent systems and the gray boxes represent contexts. Overlapping boxes illustrate how patients and families manage CF in a number of contexts. Abbreviation: CF, cystic fibrosis.

much to disclose their illness to peers. Attending school and developing peer relationships are new contexts that influence how patients and families manage CF (7). Eventually, families adjust to these changes and settle into more predictable patterns of interactions and behaviors. These new patterns, however, may be very different from those preceding the transition. For example, a child who was adherent to his or her treatment regimen prior to entering school may be less adherent following this transition because school activities and homework compete with the time needed to do treatments. Conversely, parents may benefit from the structure school provides (e.g., returning to work) and adherence may improve because of reduced family stress (8). Identifying these transitions is important because even minimal interventions at these critical points may have large and lasting effects for patients and families. For example, children with CF may have difficulty getting enough snacks during the day at school. Facilitating collaboration between parents and schools to provide these snacks may improve the child’s nutritional status and prevent a decrease in weight gain and growth (9). Thus, transitions present critical opportunities for intervention. In the remainder of this chapter, we will briefly review the literature on social and emotional functioning, adherence, and quality of life. First, the impact of CF on each of these outcomes will be discussed, followed by research on their interrelationships during major developmental periods (infancy, middle childhood, adolescence, and young adulthood).

III.

Social and Emotional Functioning, Adherence, and Quality of Life in CF

A. Social and Family Functioning

Because it is present at birth, CF fundamentally changes many aspects of social functioning both within and outside the family. Following diagnosis, families must not only learn about a new and often frightening illness, but also rapidly shift their roles to accommodate caregiving. This leads to significant changes in parenting roles and plans for the future (8,10–12). The advent of newborn screening, which has now been

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implemented in a majority of states, is likely to improve long-term health outcomes, but has also been shown to contribute to parental distress (13). The illness continues to influence family functioning throughout development. A focus on diet, ingestion of enzymes, and several hours of treatment each day often generate difficulties with mealtimes, behavior problems related to adherence, and sleep disruptions during childhood (14–16). During adolescence, conflict between parents and teens escalates, primarily in relation to adherence, increasing the challenge of balancing the adolescent’s need for independence with adequate parental supervision (17,18). CF also affects social functioning outside of the family. The complex and timeconsuming treatment regimen, frequent hospitalizations, and infection control policies have changed how patients with CF interact with their peers (19,20). This is particularly true for adolescents, who may choose not to disclose their diagnosis. They may forgo social events to do their treatments or may skip treatments to hide their illness from friends (21,22). Moreover, infection control guidelines prohibit all face-to-face contact with other individuals who have CF, which limits the unique type of support these peers provide given their shared experiences (23,24). In other chronic conditions (e.g., pediatric cancer, diabetes, asthma, sickle cell disease) patients can meet in groups to discuss illness-related issues or attend illness-specific camps, which are generally believed to improve emotional well-being and disease management (25). Technology-based innovations, such as online social networks and web-enabled cell phones, may provide opportunities for safe social interactions among peers with CF (26,27). B. Emotional Functioning

Chronic illnesses, such as CF, increase the amount of individual and family distress, often leading to elevated symptoms of depression and anxiety. In CF, rates of depression using standardized screening measures have ranged from 11% to 29% in children and adolescents, 29% to 64% in caregivers, and 29% to 46% in adults (2,28,29). Higher rates of anxiety have also been found (30,31). With the exception of two studies (32,33), evidence of increased symptomatology is highly consistent (2) and has been linked to poorer health outcomes and quality of life (28). An international epidemiological study is currently underway to provide population-based estimates of depression and anxiety, and to examine their associations with health outcomes (tides-CF.org) (2). C. Treatment Adherence

Medications and treatments can only benefit patients if they are taken or completed. Estimates of adherence in CF vary by method of measurement, type of treatment, and age. In one of the few studies examining adherence to several treatment components using multiple methods of assessment, Modi and colleagues reported adherence rates for children ranging from 22% to 71% for objective measures and 67% to 100% for selfreport measures (34). This study also found great variability between treatments, a finding that is consistent with the larger literature on adherence. In general, adherence tends to be better for simple treatments that do not require lifestyle changes (pill taking) than for complex, time-intensive treatments (airway clearance, dietary recommendations, daily inhaled medications, etc.) (35–37). Adherence rates also vary by the patient’s developmental stage; they tend to be better in younger children but drop significantly during adolescence (38).

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Measuring adherence in CF is difficult because it is multicomponent and complex (e.g., dietary changes, inhaled medications, airway clearance, etc.). Although adherence behaviors cannot be observed in a practical sense, several indirect measures of adherence are available (39). The simplest, least expensive methods are standardized selfreport questionnaires or interviews to ask patients directly about their adherence behaviors. This method can be adapted to all aspects of the treatment regimen (diet, enzyme and vitamin usage, and airway clearance); however, it has been shown to be highly influenced by the patients’ desire to appear adherent, and thus, is biased toward inflation of these behaviors (39). Daily diaries help to reduce this social desirability bias by asking about daily activities over the past 24 hours in an unobtrusive manner, with few prompts about treatment behaviors. The Daily Phone Diary (DPD) (40) has been used extensively in CF and was judged “well established” in a recent review of evidence-based adherence measures (39). It has now been adapted for asthma, HIV-AIDS, diabetes, and traumatic brain injury. The DPD provides rich information about the social context surrounding treatment regimens (e.g., extent of parental supervision); however, it requires a trained telephone interviewer (34,39,40). Electronic monitors (electronic bottle caps, aerosol counters, etc.) provide a detailed and accurate measure of the date and time of treatment behaviors, but are restricted to treatments that can be monitored electronically (oral medications, nebulized medications, etc.) and are costly to purchase. Finally, pharmacy refill data provide accurate information about whether the patient fills a prescription, but only indirectly indicates whether the medication was taken (41,42). Given the strengths and weaknesses of these various measures, the use of multiple methods is recommended (39). However, there is currently little guidance on how to combine these different measures into a reliable and valid index of adherence (43). D. Patient-Reported Outcomes

Over the past 20 years, tremendous progress has been made in defining and measuring PROs with growing recognition of their importance in health outcomes research and clinical care (44). A PRO has been defined as any measure of a patient’s health status, elicited directly from the patient, that assesses how the patient “feels or functions with respect to his or her health condition” (45). This may include observable events, behaviors, or feelings (e.g., ability to run and exercise, lack of appetite, etc.) or unobservable outcomes known only to the patient (e.g., perceptions of pain, increased chest congestion, feeling sad, etc.). PROs range from single-item symptom ratings to complex, multidimensional HRQOL measures that can be used to evaluate new pharmaceutical, behavioral, and surgical interventions and aid in clinical decision making (44,46–48). PROs used to evaluate interventions must meet rigorous psychometric criteria, including a welldefined conceptual framework and strong evidence of reliability and validity (49,50). If these criteria are met, the Food and Drug Administration (FDA) now accepts PROs as primary or secondary outcomes in drug registration trials (45,51). Although generic measures of HRQOL have been used in CF (52), a substantial body of literature now suggests that disease-specific measures are more sensitive to change and provide information that is more relevant for clinical interventions (50). Efforts to develop reliable and valid PROs for CF have been very successful, resulting in

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Figure 2 CFQ-R respiratory scale. Abbreviation: CFQ-R, Cystic Fibrosis Questionnaire–Revised.

three disease-specific instruments and a growing base of empirical and clinical evidence (53). Currently, the Cystic Fibrosis Questionnaire–Revised (CFQ-R) (Fig. 2) (54) is the most widely used PRO for CF and has been deemed well established in reviews of evidence-based measures (53,55). The CFQ-R has three developmental versions: one for children aged 6 to 13, a parent report measure for the same ages, and a version for adolescents and adults aged 14 and older (56). A pictorial, preschool version is now being validated for young children with CF, aged three to six, and their parents (53). Evidence of reliability for the CFQ-R is strong, and several studies have demonstrated that it is sensitive to lung functioning (construct validity) and responsive to clinically relevant changes (responsivity). To date, scores on the CFQ-R have been associated with significant improvements in lung function in patients treated for pulmonary exacerbations (57), with independent ratings of pain (55), and with adults who have elevated symptoms of depression (28). Scores on this PRO have successfully differentiated patients by age (e.g., older patients reporting worse HRQOL), disease severity (normal, mild, moderate, and severe lung disease), gender, and socioeconomic and minority status (58). Further, the minimal clinically important difference (MCID) score was recently established using both anchor- and distribution-based methods (59). A 4-point change (out of 100) or greater on the CFQ-R respiratory scale indicates a clinically significant shift in patients’ symptoms. The CFQ-R has been used as a primary and secondary end point in several phase 2 and phase 3 clinical trials (51,60,61) and has been translated into 30 languages, facilitating its use in international studies. Next steps

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include publication of normative data on CFQ-R scores across ages and levels of disease severity to facilitate their use during routine practice to improve the quality of care.

IV.

Relationships Among Outcomes

The biopsychosocial model presented in this chapter suggests that social and emotional functioning, adherence, health outcomes, and quality of life interact dynamically across the life span. Many of the links between these developmental systems are empirically based. The following discussion organizes these research findings by developmental period. A. Early Childhood

Diagnosis of CF typically occurs within the first year of life. As mentioned previously, this creates a period of rapid change and adjustment for families, with high levels of parenting stress and depression commonly reported (2,11). Performing daily treatments is time consuming and requires a parent to be present throughout, which can interfere with parents’ social functioning and work roles (8). Ensuring that children receive the recommended number of pancreatic enzymes and fat-soluble vitamins can also be difficult if children have not learned to swallow pills (62). Programs have been developed to teach young children to swallow pills, which may increase adherence and promote weight gain and growth (63). Managing the disease increases parental distress and is associated with symptoms of depression, poor sleep quality, and marital strain in both mothers and fathers of young children with CF (11,64–66). The consequences of parental depression on children’s development are well established in the general literature and include increased risk for emotional, social, and behavioral difficulties later in life (67). Less is known, however, about how parental stress and depression affect health outcomes in CF. These links have only recently been examined. In a prospective study, Quittner and colleagues identified maternal depression as an important predictor of poor enzyme adherence in children aged 1 to 12 and, furthermore, demonstrated short-term causal effects on weight gain and growth (68). Another study found significant associations among parental stress and depression, child eating and sleeping difficulties, and poor adherence to nebulized medications during the preschool years (14). These studies suggest that annual screening of depression in caregivers and adequate support during early childhood could improve children’s health outcomes. B. Middle Childhood

Middle childhood is often more stable than other developmental periods because children and families have acquired a fair amount of knowledge about CF, are more comfortable in their relationships with CF team members, and have developed daily treatment routines. The major transition during this period is entry into school. Schools present a new system parents must negotiate, including coordinating extra snacks, highcalorie lunches, enzyme administration, and academic progress despite absences from school (7). Families may need to get up earlier to fit in treatments before school, and treatments after school must also be accommodated. Similar to early childhood, emotional and family functioning influence adherence during middle childhood. A recent study demonstrated a link between parent-child

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relationships and adherence, showing that higher quality relationships were associated with better adherence. This study also demonstrated that more depressive symptoms in children were related to worse adherence. Finally, there is emerging evidence that parents may experience “burnout” in their caregiving role near the end of childhood and in the beginning of adolescence. Interestingly, two studies have now shown that better adherence to airway clearance is associated with more depression in caregivers, which may reflect this phenomena of burnout. These findings highlight the importance of monitoring children and parents’ psychological distress and providing additional support to parent caregivers (29,69). Older school-age children may begin to resist doing their treatments. In one of the few studies to assess barriers to adherence, Modi and colleagues found that the most common barriers were behavior problems/treatment refusal and forgetting to do treatments (62). Identifying these barriers is essential to understanding how families manage CF; unfortunately, barriers are rarely systematically evaluated in studies or in clinical practice. More research is needed to understand the interrelationships among social, emotional, and health outcomes during this period. Interventions during late childhood may prevent the considerable challenges that await families during adolescence. C. Adolescence

Adolescence is a period of intense physical and pubertal development, characterized by rapid emotional and social changes and a drive toward autonomy. This developmental period is challenging for healthy teens and their parents, but presents unique difficulties for teens with CF in the areas of adherence, sexual maturation, and development of peer and romantic relationships. Adolescents are also at risk in terms of health outcomes, not only because of decreased adherence (38) but also because of increased pulmonary exacerbations, more frequent hospitalizations, and substantial declines in lung function. Poor adherence has been linked to time spent with peers outside of the home and away from parental monitoring, increased conflict with parents about responsibilities for medical care, and a heightened desire to be similar to healthy peers (17,18,70,71). Similar to healthy adolescents, teens with CF spend more time in social activities outside of the home, making it more difficult to schedule their treatments. They may also be reluctant to disclose their illness to peers, which may lead to worse adherence (72). Parental supervision has recently been identified as playing a key role in adherence, even among older adolescents. Using daily phone diaries, Modi and colleagues found that simply the presence of a parent in the room increased the likelihood that a treatment would be completed (18). The benefit of parental supervision, however, may be dependent on the quality of the parent-teen relationship (71). Family conflict has been identified as a consistent predictor of poor adherence during adolescence. Parents and teens struggle to find a balance between greater autonomy for the adolescent and assurance that daily treatments will be completed as prescribed (17,71,73). The optimal strategy is to provide adolescents with increasing responsibility for disease management, predicated on their ability to organize their time and perform these tasks. If parents transfer responsibility for disease management too quickly, the adolescent may become overwhelmed and nonadherent. If parents are reluctant to transfer responsibility, teens will not learn the necessary skills to independently manage their disease. They may also rebel against

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parental controls, resulting in worse adherence (71,73). Family-based interventions addressing these challenges have been successful in adolescents with CF and other chronic illnesses (17,74). Little is known about how peers and close friends influence adherence. In general, social support has been found to increase adherence across ages and illnesses (75). In CF, only two studies have examined social support during adolescence and, unfortunately, neither measured adherence (21,76). These studies did, however, describe the types of support provided by families and friends. Consistent with the broader literature on social support, friends tended to provide more emotional support and a sense of belonging, while family members provided more tangible assistance with treatments. These studies also found that families and friends were both sources of positive (appropriate reminders) and negative (drawing attention to the illness, nagging, etc.) behaviors. Greatz and colleagues linked negative behaviors to more emotional distress in these youth. To move this area of research forward, a disease-specific measure of social support is needed (76).

D. Adulthood

As patients make a transition from adolescence to adulthood, there are unique challenges that influence their disease management and quality of life. Adults continue to extend their social networks beyond their family and leave home to begin college, a job, or a long-term relationship. It can be difficult for adults with CF to achieve autonomy because of increasing symptoms, lengthy treatment times, and the need for health insurance (77,78). Finding a job can also be challenging because of the need for sick leave, which often requires disclosure (79–81). As patients with CF live longer, healthier lives, they participate in a broader array of activities. A recent survey of 865 adults with CF found that most were in a committed relationship, involved in higher education, employed, and lived away from home (82). However, finding the correct balance between treatments and daily activities is a significant challenge (78,83). Importantly, families continue to provide significant treatment-related support during adulthood (84). Adulthood also brings a transition from pediatric to adult care. This transition is important because adult teams focus on broader lifestyle questions, such as fertility, drug and alcohol use, independent self-care, transplantation, and end-of-life issues (85,86). A smooth transition likely depends on the transfer of responsibility from parents to patients during adolescence and an appropriate transition plan that focuses on self-care responsibility (87–90). The knowledge and skills required to manage this complex disease should be taught beginning in early childhood. Similar to younger patients, symptoms of depression and anxiety are related to adherence and HRQOL in adults. Higher rates of depression have been linked to poorer adherence, worse pulmonary functioning, and lower quality-of-life scores (28). In contrast, higher levels of anxiety have been associated with better treatment adherence (91). Patients who are concerned about their health and well-being may both report slightly higher levels of anxiety and be more conscientious about their treatments. Too much anxiety, however, may interfere with broader functioning. This “paradoxical” effect of anxiety is a promising area for future research.

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V. Conclusion Because CF is present at birth, it affects many aspects of social, emotional, and behavioral development, which in turn influence health outcomes and quality of life. There is growing evidence that these developmental processes are interrelated and importantly connected to health. A developmental, biopsychosocial model provides a unifying framework for both cutting-edge research and new interventions to improve clinical care. The model presented in this chapter emphasizes the importance of considering relationships among multiple systems (family, peers, school, health care team) as well as identifying key periods of transition that require heightened monitoring and targeted interventions. Several recommendations follow from this review. First, we recommend yearly depression and anxiety screening for both caregivers and patients. Screening for caregivers should begin shortly following diagnosis, and screening for patients at age seven (the youngest age covered by validated screening measures). There are a number of well-validated, brief, readily available measures for this purpose (2,92). This will necessitate the development of referral sources for caregivers and patients who may need both pharmacological and cognitive behavioral interventions (2). Second, because adherence is central to long-term health outcomes, we recommend that evidence-based interventions which improve adherence be integrated into routine clinical care. A number of interventions have been developed to help patients and families improve disease management, with several promising translational studies in process. For example, the iCARE (I Change Adherence and Raise Expectations) study identifies key barriers to adherence and engages teens, parents, and health care providers in brief problem-solving sessions (93). In addition, a randomized trial is underway to evaluate the effectiveness of a web-enabled cell phone (CFFONE) for improving adherence and providing safe peer support (27). Third, we suggest that well-validated PROs be included at annual visits to assess patient functioning across a variety of domains (e.g., social and emotional functioning, treatment burden, etc.), and that patients’ respiratory symptoms be assessed using standardized PRO-based instruments during routine clinic visits. This would provide clinicians with reliable information about symptom change, aiding in early identification of pulmonary exacerbations. Patients’ increased life expectancy and involvement in typical adult activities represent powerful evidence of improved clinical care in CF. Building on this success will require continued innovation and consideration of social and emotional functioning and promotion of adherence in clinical science and clinical care. Such efforts will ultimately lead to improvements in patients’ health and quality of life.

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46. Revivki DA, Erickson PA, Sloan JA, et al. Interpreting and reporting results based on patient reported outcomes. Value Health 2007; 10:S116–S124. 47. Detmar SB, Muller MJ, Schornagel JH, et al. Health-related quality of life assessments and patient-physician communication: a randomised controlled trial. JAMA 2002; 288. 48. Feeny D. Preference-based measures: utility and quality-adjusted life years. In: Fayers P, Hays R, eds. Assessing Quality of Life in Clinical Trials. Oxford: Oxford University Press, 2005:405–430. 49. Eiser C, Morse R. Quality-of-life measures in chronic diseases of childhood. Health Technol Assess 2001; 5:1–157. 50. Goss CH, Quittner AL. Patient-reported outcomes in cystic fibrosis. Proc Am Thorac Soc 2007; 4:378–386. 51. Retsch-Bogart G, Quittner AL, Gibson RL, et al. Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis. Chest 2009; 135:1223–1232. 52. Quittner AL. Measurement of quality of life in cystic fibrosis. Curr Opin Pulm Med 1998; 4:326–331. 53. Quittner AL, Modi AC, Cruz I. Systematic review of health-related quality of life measures for children with respiratory conditions. Pediatric Respir Rev 2008; 9:220–232. 54. Quittner AL, Buu A, Messer MA, et al. Development and validation of the cystic fibrosis questionnaire in the United States: a health-related quality-of-life measure for cystic fibrosis. Chest 2005; 128:2347–2354. 55. Palermo TM, Long AC, Lewandowski AS, et al. Evidence-based assessment of health-related quality of life and functional impairment in pediatric psychology. J Pediatr Psychol 2008; 33:983–996. 56. Modi AC, Quittner AL. Validation of a disease-specific measure of health-related quality of life for children with cystic fibrosis. J Pediatr Psychol 2003; 28:535–546. 57. Dobbin CJ, Bartlett D, Melehan K, et al. The effect of infective exacerbations on sleep and neurobehavioral function in cystic fibrosis. Am J Respir Crit Care Med 2005; 172:99–104. 58. Quittner AL, Schechter MS, Rasouliyan L, et al. Impact of socioeconomic status, race, and ethnicity on quality of life in patients with cystic fibrosis. Chest (in press). 59. Quittner AL, Modi AC, Wainwright C, et al. Determination of the minimal clinically important difference (MCID) score for the cystic fibrosis questionnaire-revised (CFQ-R) respiratory symptoms scale in two populations of patients with cf and chronic pseudomonas aeruginosa airway infection. Chest 2009; 135:1610–1618. 60. McCoy KS, Quittner AL, Oermann C, et al. Inhaled aztreonam lysine for chronic airway pseudomonas aeruginosa in cystic fibrosis. Am J Respir Crit Care Med 2008; 178:921–928. 61. Dupont LJ, Minic P, Fustik S, et al. A randomized placebo-controlled study of nebulized liposomal amikacin (ARIKACETM) in the treatment of cystic fibrosis patients with chronic Pseudomonas aeruginosa lung infection. Pediatr Pulmonol 2008; S31:301. 62. Modi AC, Quittner AL. Barriers to treatment adherence for children with cystic fibrosis and asthma: what gets in the way? J Pediatr Psychol 2006; 31:846–858. 63. Marciel KK, Cruz I, Barker DH, et al. Effectiveness of a pill swallowing program for young children with cystic fibrosis. Pediatr Pulmonol 2008; S31:458. 64. Glasscoe C, Lancaster GA, Smyth RL, et al. Parental depression following the early diagnosis of cystic fibrosis: a matched, prospective study. J Pediatr 2007; 150:185–191. 65. Yilmaz O, Sogut A, Gulle S, et al. Sleep quality and depression-anxiety in mothers of children with two chronic respiratory diseases: asthma and cystic fibrosis. J Cyst Fibros 2008; 7:495–500. 66. Meltzer LJ, Mindell JA. Impact of a child’s chronic illness on maternal sleep and daytime functioning. Arch Intern Med 2006; 166:1749–1755. 67. Halligan SL, Murray L, Martins C, et al. Maternal depression and psychiatric outcomes in adolescent offspring: a 13-year longitudinal study. J Affect Disord 2007; 97:145–154.

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68. Quittner AL, Barker DH, Geller D, et al. Effects of maternal depression on electronically monitored enzyme adherence and changes in weight for children with CF. J Cyst Fibros 2007; 6:77. 69. Snell C, Barker DH, Marciel KK, et al. Paradoxical relationships between parental depression and pulmonary functioning in cystic fibrosis: the role of treatment burden and daily mood. Pediatr Pulmonol 2008; S31:454. 70. Badlan K. Young people living with cystic fibrosis: an insight into their subjective experience. Health Soc Care Community 2006; 14:264–270. 71. Smith BA, Wood BL. Psychological factors affecting disease activity in children and adolescents with cystic fibrosis: medical adherence as a mediator. Curr Opin Pediatr 2007; 19:553–558. 72. D’Auria JP, Christian BJ, Richardson LF. Through the looking glass: children’s perceptions of growing up with cystic fibrosis. Can J Nurs Res 1997; 4:99–112. 73. Fiese BH, Everhart RS. Medical adherence and childhood chronic illness: family daily management skills and emotional climate as emerging contributors. Curr Opin Pediatr 2006; 18:551–557. 74. Wysocki T, Harris MA, Buckloh LM, et al. Effects of behavioral family systems therapy for diabetes on adolescents’ family relationships, treatment adherence, and metabolic control. J Pediatr Psychol 2006; 31:928–938. 75. DiMatteo MR. Social support and patient adherence to medical treatment: a meta-analysis. Health Psychol 2004; 23:207–218. 76. Barker DH, Cohen LM, Driscoll KA, et al. It takes a village: what family, friends and teachers do to facilitate disease management in adolescents with cystic fibrosis. Pediatr Pulmonol 2008; S31:452. 77. Palmer ML, Boisen LS. Cystic fibrosis and the transition to adulthood. Soc Work Health Care 2002; 36:45–58. 78. Pfeffer PE, Pfeffer JM, Hodson ME. The psychosocial and psychiatric side of cystic fibrosis in adolescents and adults. J Cyst Fibros 2003; 2:61–68. 79. Hogg M, Braithwaite M, Bailey M, et al. Work disability in adults with cystic fibrosis and its relationship to quality of life. J Cyst Fibros 2007; 6:223–227. 80. Burker EJ, Sedway J, Carone S. Psychological and educational factors: better predictors of work status than FEV1 in adults with cystic fibrosis. Pediatr Pulmonol 2004; 38:413–418. 81. Berge JM, Patterson JM, Goetz D, et al. Gender differences in young adults’ perceptions of living with cystic fibrosis during the transition to adulthood: a qualitative investigation. Fam Syst Health 2007; 25:190–203. 82. Quittner AL, Modi AC, Boyle MP. The role of independence in daily functioning for adults with cystic fibrosis: results from the adult data for understanding lifestyle and transitions (ADULT) survey. Pediatr Pulmonol 2008; S31:445. 83. Myers LB, Horn SA. Adherence to chest physiotherapy in adults with cystic fibrosis. Journal of Health Psychol 2006; 11:915–926. 84. McGuffie K, Sellers DE, Sawicki GS, et al. Self-reported involvement of family members in the care of adults with CF. J Cyst Fibros 2008; 7:95–101. 85. Chapman E, Landy A, Lyon A, et al. End of life care for adult cystic fibrosis patients: facilitating a good enough death. J Cyst Fibros 2005; 4:249–257. 86. Brumfield K, Lansbury G. Experiences of adolescents with cystic fibrosis during their transition from paediatric to adult health care: a qualitative study of young Australian adults. Disabil Rehabil 2004; 26:223–234. 87. Parker HW. Transition and transfer of patients who have cystic fibrosis to adult care. Clin Chest Med 2007; 28:423–432. 88. Patton SR, Graham JL, Varlotta L, et al. Measuring self-care independence in children with cystic fibrosis: the self-care independence scale (SCIS). Pediatr Pulmonol 2003; 36:123–130.

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89. Blum RW. Transition to adult health care: setting the stage. J Adolesc Health 1995; 17:3–5. 90. Schidlow DV. Transition in cystic fibrosis: much ado about nothing? a pediatrician’s view. Pediatr Pulmonol 2002; 33:325–326. 91. White T, Miller J, Smith GL, et al. Adherence and psychopathology in children and adolescents with cystic fibrosis. Eur Child Adolesc Psychiatry 2008; 18:96–104. 92. Cruz I, Marciel KK, Quittner AL, et al. Anxiety and depression in CF. Semin Respir Crit Care Med 2009; 30:569–578. 93. Quittner AL. Controlled trial of two adherence promotion interventions for cystic fibrosis. In: Health Economics & Outcomes Group, Novartis Pharmaceuticals, 2008.

31 Palliative and End-of-Life Care in Cystic Fibrosis WALTER M. ROBINSON Vanderbilt Children’s Hospital, Center for Biomedical Ethics and Society, Vanderbilt University, Nashville, Tennessee, U.S.A.

JEFFREY C. KLICK University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, U.S.A.

I.

Introduction

Throughout its history, cystic fibrosis (CF) has been identified in the public imagination as a fatal disease. Readers with even a passing familiarity with CF will recognize this as an outdated characterization: in the 21st century, CF is a manageable chronic illness, still with a limited life span, but with an increasingly healthy childhood population and an enlarging cohort of adults (1). Given the changing epidemiology of those living with CF, our goal in this chapter is twofold: first, to describe state-of-the-art palliative care throughout the lifespan of the child and adult with CF, and second, to describe highquality CF end-of-life care, which for the purposes of this chapter is defined as care for the family and patient during the last two months of life and bereavement care following the death of the patient. Because of confusion about the nature of palliative care, we begin with some definitions. In practice, “palliative care” is used to describe a clinical practice, a conceptual approach to care, and a specific set of interventions all aimed to enhance quality of life in the face of a life-threatening condition. Palliative care is often pursued in conjunction with interventions of curative intent and includes any intervention that focuses on reducing the severity of illness, slowing the progression of disease, improving quality of life, and ensuring that families are able to remain functional and intact (2). Hospice or “end-of-life” care is a subset of palliative care and refers to the end stages of care more focused on supportive care than life-sustaining therapies. Hospice services can be utilized simultaneously with measures of life-sustaining intent; a do-notresuscitate (DNR) order is not required as a condition of enrollment, and patients may disenroll from hospice and return to hospice care at a later date. Hospice agencies are valuable resources, providing 24-hour availability for in-home management of distressing symptoms; psychosocial, spiritual, and decision-making support for the patient and family; and ongoing interventions for grief and bereavement care for the family. Quality palliative care begins with effective symptom assessment. Recent studies in adults (3) and children (4) suggest that the symptom range in CF is quite broad, even in those with relatively mild disease. In addition, the most distressing symptoms may be less directly related to the pathophysiology of CF than to the difficulty of managing life with a chronic, unrelenting illness (5). Psychosocial symptoms such as anxiety and 482

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depression (6,7), as well as functional symptoms such as sleep disturbance and fatigue (8) are more common in CF than previously recognized. Some symptoms related to CF are emerging only currently as clinicians perform more systematic assessments; one recent example is urinary incontinence in women related to coughing (9). There have been few studies regarding the treatment of pain and suffering symptoms specific to the CF population. This lack of evidenced-based literature, however, should not limit the treatment of suffering in these children and adults. Instead, the guiding principle for treating any symptom in a child or adult with CF should be a trial of the medication or therapy, including multiple routes of administration, while assessing for efficacy and side effects. If the treatment proves effective for that patient, it should be continued. If it is ineffective or with too great a side-effect profile, a different treatment should be tried until success is achieved.

II.

Assessment and Management of Symptoms

There are four principles in assessing pain and suffering. First, the patient’s self-report is the gold standard of reporting. If the patient is unable to report—due to age, severity of illness, or cognitive impairment—the next of kin or care provider becomes the most reliable reporter. Second, assessment of pain and suffering must take into account the developmental understanding of the patient. Most adults are able to relate pain and suffering to experiences in the past (e.g., “the pain is now a 2/10 but was a 6/10 prior to receiving the morphine you prescribed”). Most young children, usually under seven years of age, only report pain in the moment with no historical context. Third, pain and suffering can change from minute to minute requiring frequent reassessment of symptoms and efficacy of therapy. The most efficient times to reassess symptoms are at any times the patient is at increased risk for pain, including after procedures, when medications are wearing off, and at the peak effect of initiated therapy if an appropriate dose is insufficient to relieve symptoms. Finally, physiological pain is often accompanied by nonorganic factors that can intensify suffering. Pain is not just physiological but also has spiritual, psychological, and social components (10). These aspects of pain are often unrecognized. CF psychosocial team members or a palliative care specialist can help recognize sources of distress and provide management options in these situations. A. Assessment and Management of CF-Related Pain

Headache, abdominal pain, and chest pain are commonly reported by children and adults with CF, even those with mild disease (11,12). Headaches should be characterized by location, frequency, quality of pain, and intensity. Headaches associated with sinus disease in CF may not express the typical localizing symptoms, and surveillance for the presence of polyps and sinus disease should be part of routine care (13,14). Morning headaches may indicate sleeping gas exchange abnormalities that can be diagnosed by means of careful history and/or a sleep study and treated by means of nocturnal oxygen or noninvasive ventilation. The most common causes of chest pain include frequent coughing episodes leading to musculoskeletal spasm and rib fractures, especially in those with existing osteoporosis (15); vertebral compression fractures (16); and musculoskeletal complications of increasing thoracic volume due to obstructive lung disease (17,18). Careful evaluation by physical therapists can lead to a diagnosis treatable by exercise or postural

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improvements (19,20). Pharmacological intervention using the pain ladder approach outlined below is also appropriate. Abdominal pain can be described as dull, aching, or cramping. Patients may also describe the burning pain associated with gastroesophageal reflux. Bowel distention and urgency can be associated with ineffective functioning of pancreatic replacement enzymes. Cramping and dull or aching pain can be associated with constipation and distal intestinal obstruction syndrome (DIOS) (21). If pain is acute and increasing, it may suggest appendicitis (22).

B. Opioid Use for Acute and Chronic Pain in CF

Mild pain is often effectively managed with nonopioid medications, such as acetaminophen or ibuprofen. If these medications prove ineffective, opioids become the foundation of good pain control. While there are many different formulations of opioids, it is an effective practice to utilize and gain comfort in a single type. Once comfortable with that medication, the practitioner can then better understand the benefits of other opioids, such as fentanyl for its rapid onset and metabolism or methadone for its very long duration of action. For this chapter, we will propose utilizing morphine as an effective initial opioid to base care decisions. Aside from the predictable though manageable side effects of opioid use (23), there are two specific issues that should be considered in using opioids in CF. The first is the increased risk of constipation. In all CF patients, even in those without a history of constipation or DIOS, lactulose should be started once opioids have been used for more than two days. Monitoring of bowel movement frequency and increased vigilance for constipation is appropriate, given the relative ease of preventing constipation compared to relieving it once it has occurred. The second issue regards the concern that opioids will cause respiratory depression and/or tolerance. Pain can cause alterations in breathing patterns that, when relieved, give the mistaken perception of decreased respiratory drive. Respiratory depression is very rare when opioids are dosed appropriately. It is preceded by sleepiness, decreased consciousness, decreased respiratory rate, and, finally, apnea. Once identified, this side effect is best managed by providing respiratory support, decreasing the dose, and waiting for resolution. An opioid antagonist such as naloxone should only be used in the rare case when all the symptoms of respiratory depression are present, especially since it can cause life-threatening withdrawal if used in the setting of chronic opioid therapy. Occasionally, dose-related side effects limit the use of opioids. In these circumstances, improved pain control with fewer side effects may be accomplished by switching to a different opioid (24). Patients exposed to an opioid for a long period of time can develop tolerance, requiring a higher dose to accomplish the same pain effect. When transitioned to another opioid, they may have incomplete cross-tolerance to the new opioid, meaning they may require a relatively lower dose of the new opioid. Because of this incomplete cross-tolerance, the new opioid dose should be decreased by 30% to 60% from a direct conversion. When initiating pain management, the clinician must consider which medication to use, the goals of therapy, the proper initial dose and route of administration, and a plan to manage breakthrough pain (pain that “breaks through” scheduled doses of an analgesic requiring rescue doses of the analgesic). Table 1 describes a thorough

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Table 1 Acute and Chronic Pain-Management Algorithm

Ladder

Degree of pain

Step 1

Mild

Step 2

Moderate

Frequency and route

Trigger to advance to next step

Start a nonopioid analgesic (NSAID or acetaminophen)

If no effective benefit or worsening or persistent symptoms advance to step 2

Rescue therapy Morphine oral immediate release preparation Children: 0.2–0.5 mg/kg/dose q 3–4 hr prn Adults: 5–30 mg every 3–4 hr prn Moderate- Sustained therapy: oral persistent Morphine sustained release preparation Children/adults: total daily dose divided b.i.d. Rescue therapy: oral Morphine immediate release preparation Children: 0.2–0.5 mg/kg every 3–4 hr prn Adults: 5–30 mg every 3–4 hr prn

If patient has frequent utilization of rescue medication should advance to step 3 to introduce more sustained and rescue therapy

Step 4

Severe

If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued If patient has frequent utilization of rescue medications should advance to step 5 to introduce more sustained and rescue therapy

Step 5

SevereConsider PCA: morphine persistent Opioid exposed Basal rate: daily morphine dose/24 hr Demand dose: 50–100% of the hourly dose Lockout interval: 5–10 min Opioid naive Children: Basal infusion: 0–0.02 mg/kg/hr Demand dose: 0.015–0.02 mg/kg/dose Lockout interval: 5–10 min Adult: Basal rate: 5–35 mg/hr or 0.07–0.5 mg/kg/hr Demand dose: 0.5–2.5 mg Lockout interval: 5–10 min

Step 3

Rescue therapy: IV, SQ Morphine: Children < 6 mos: 0.05–0.1 mg/kg every 3–4 hr Children > 6 mos: 0.1–0.2 mg/kg every 3–4 hr Adult: 2.5–10 mg every 2–6 hr

If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued If no effective benefit, frequent use of rescue doses, or worsening or persistent symptoms advance to step 4

If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued Sustained therapy dosing best determined using either the acute severe pain-management pathway or by determining amount required in 24 hr in step 4. If no effective benefit, frequent use of rescue doses, or worsening or persistent symptoms continue escalation of dosage until the patient is free of pain

Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; b.i.d., twice a day; IV, intravenous; SQ, subcutaneous; PCA, patient controlled analgesia.

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pain-management approach adapted from the World Health Organization’s recommendation for the treatment of pain (25). The goal of any pain therapy should be declared prior to initiating therapy. Simply improving pain and suffering scores is not sufficient. Goals should include a dramatic decrease in pain (e.g., decrease pain to zero to three level on numerical scales) and improvement in function (e.g., help the patient walk or tolerate airway clearance modalities). Medications designed to enhance the pharmacological effect of the opioids should then be targeted at the specific type of pain (e.g., gabapentin for neuropathic pain or lorazepam for pain associated with significant anxiety). C. Assessment and Management of Cough

Cough is a part of life with CF. Coughing as a means of deliberate airway clearance should be encouraged, and discussion of airway clearance techniques should take into account the patient’s desire to manage coughing episodes so as to avoid interrupting daily activities (26). Coughing in excess of routine airway clearance, however, can be disturbing and disruptive. A search for the etiology of the troubling cough beyond treatment of pulmonary exacerbations, including investigation of allergies, asthma, or gastroesophageal reflux (27) is essential. Chronic cough may also lead to other symptoms, such as urinary incontinence (9,28). While the use of cough suppressants is discouraged for its negative effect on airway clearance, the rare patient with debilitating cough may temporarily need such medications, particularly at night. Spasmodic and prolonged cough can be a feature of end-stage CF. In this type of cough, a short, sharp inspiration is followed by a prolonged forced exhalation with ineffective airway clearance. These prolonged periods of exhalation can lead to severe headache, syncope, and seizures, as well as dramatic changes in thoracic blood flow with cardiac repercussions (29). In some cases thinning of secretions with inhaled alfa dornase or hypertonic saline may improve the symptoms, but in others the cough may worsen; a monitored trial of efficacy of the therapy is appropriate (30). Utilizing opioids to dull the need to cough is appropriate when the cough is ineffective and distressing to the patient. The management of cough is similar to that for dyspnea (Table 2). D. Assessment and Management of Dyspnea

Dyspnea is a complex physical and psychological symptom (31). In CF, dyspnea can result from a host of interacting factors, including upper airway obstruction, sinus disease, gastroesophageal reflux, chest wall mechanical abnormalities, asthma, allergic responses, allergic bronchopulmonary aspergillosis, gas exchange abnormalities, reduced cardiac function, and physical deconditioning. Initially, symptoms of dyspnea are likely to be related to the level of exertion (32), and many patients will respond by restricting their activity, so that dyspnea may be pronounced when it is finally reported to the clinician. Pulmonary function tests and blood gas analysis are appropriate, but may have no correlation with the degree of experienced dyspnea. In this setting, the patient should simply rate his or her breathlessness on a numerical scale, similar to a pain scale. Management of chronic dyspnea is a complex medical and psychosocial process. For mild dyspnea, nonpharmacological treatments should be tried (33). Assistance with activities of daily living can reduce the time spent feeling breathless per day. Alterations

Nonpharmacological modalities

Consider changes in airway clearance techniques or timing. Initiate support with ADLs

Escalate support with ADLs After examination, consider initiating nocturnal supplemental oxygen and or NIV

Consider initiating nocturnal NIV Initiate fan blowing on the patient’s face (activates trigeminal sensory pathways)

Escalate support with ADLs Consider escalating NIV Continue fan on patient’s face

Degree of dyspnea

Mild

Moderate

Moderate-persistent

Severe

Table 2 Management Algorithm for Dyspnea

Pulmonary rehabilitation or respiratory muscle conditioning If no effect, advance to moderate pathway

Bronchodilators, if evidence of reversible obstruction Additional mucolytic therapy, with consideration of change in frequency of administration Rescue therapy: oral Morphine immediate release preparation Children: 0.2–0.5 mg/kg every 3–4 hr prn Adults: 5–30 mg every 3–4 hr prn Sustained therapy: oral Morphine sustained release preparation Children/adults: total daily dose divided b.i.d. to t.i.d. Rescue therapy: oral Morphine immediate release preparation Children: 0.2–0.5 mg/kg every 3–4 hr prn Adults: 5–30 mg every 3–4 hr prn. Consider adding lorazepam (see dosing below) Escalate rescue therapy: IV, SQ Morphine < 6 mos: 0.05–0.1 mg/kg every 3–4 hr > 6 mos: 0.1–0.2 mg/kg every 3–4 hr Adult: 2.5–10 mg every 2–6 hr (Continued)

If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued

If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued If minimal or insufficient relief, advance to severe pathway

If using multiple doses per day, consider chronic level of therapy If minimal or insufficient relief, advance to severe pathway

Additional therapy

Medication modalities

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Escalate support with ADLs Consider escalating NIV Continue fan blowing on the patients face

Severe-persistent

Additional therapy If the patient is receiving opioid therapy at presentation, an equivalent or greater dose should be continued If patient is alert, consider PCA

Medication modalities Initiate PCA or infusion with rescues Opioid exposed: morphine Basal infusion: daily dose divided per hour Rescue dose: 50–100% of the hourly dose Lockout interval: 5–10 min Opioid naive: morphine Children: Basal infusion: 0–0.02 mg/kg/hr Rescue dose: 0.015–0.02 mg/kg/dose Lockout interval: 5–10 min Adult: Basal infusion: 5–35 mg/hr Rescue dose: 0.5–2.5 mg Lockout interval: 5–10 min

Abbreviations: ADL, activities of daily living; NIV, noninvasive ventilation; b.i.d., twice a day; t.i.d., three times a day.

Nonpharmacological modalities

Degree of dyspnea

Table 2 Management Algorithm for Dyspnea (Continued )

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in airway clearance therapies may be appropriate, especially if the use of these therapies results in prolonged coughing episodes, exhaustion, and more dyspnea. Treatment should also always include assessment of exercise capacity, as improvement of conditioning can improve breathlessness (34). As respiratory function worsens, intermittent use of opioids or benzodiazepines can allow the patient to manage his or her daily activities. Although the use of inhaled opioids has been suggested (35,36), direct studies on their effects have yet to be persuasive (37) while oral and intravenous opioids can substantially reduce the sensation of dyspnea. Appropriate dosing of these agents will not cause respiratory depression (38). As symptoms of dyspnea progress, many patients will experience an added sense of anxiety. In these patients, benzodiazepines can be an effective adjuvant to opioid therapy. Near the end of life in CF, dyspnea is likely to be the most distressing symptom (39). At the end stage, even substantially somnolent patients may be struggling for breath. In this situation, the patient’s subjective sensation or the best assessment of the patient made by caring and compassionate observers should continue to drive the use of opioids to relieve breathlessness. This state of rapidly changing symptoms requires clear and ongoing discussion regarding close and careful monitoring as well as a planned response to any changes in suffering (38). Specific treatment options, including an algorithm for managing the evolution of symptoms for dyspnea is included in Table 2. E.

Assessment and Management of Life-Ending Hemoptysis

Massive hemoptysis with exsanguination is extremely distressing to the patient as well as to those at the bedside (40). (Management of less severe and chronic hemoptysis is discussed in chap. 16, as are the various treatment strategies for non-life-threatening hemoptysis.) A history of substantial hemoptysis (more than 240 cc in 24 hours or recurrent bleeding of more than 100 cc/day over several days) is associated with a high likelihood of recurrence (41). Unfortunately, sedation is the only way to reduce the substantial distress of a patient experiencing end-stage hemoptysis, though the use of dark towels or other dark-colored material to reduce the appearance of bright red blood is effective in reducing the anxiety of both the patient and those in attendance. Effective relief of the distress of life-ending hemoptysis in an outpatient setting is extremely difficult (42) and prediction of the timing of life-ending hemoptysis is virtually impossible. Forthright discussions with the patient and family regarding the possibility of life-ending hemoptysis are essential. F.

Assessment and Management of Constipation/Obstipation

Management of chronic constipation, including bowel regimens, are discussed in chapter 17. In the sicker CF patient, constipation can be exacerbated by medications, decreased fluid intake, slowed intestinal motility, and decreased physical activity. The use of opioids to manage pain and dyspnea near the end of life will increase the risk of constipation or obstipation. In general, stool management therapies should be a part of the regimen for all CF patients on opioids, as well as those patients at risk for dehydration or a history of DIOS. Table 3 suggests an approach to prevention and management of constipation.

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Table 3 Suggested Algorithm for the Management of Constipation

Step

Assessment and treatment options

Step 1: Prevention

Prophylactic lactulose, adequate enzyme intake with/or without enzyme enhancers, and monitoring of stool output and early contact with clinician Assessment, with physical examination and abdominal X rays. Consider osmotic enema and/or oral electrolyte regimen Evaluate obstruction/impaction and consider surgical referral

Step 2: Escalation and treatment

Step 3: Treatment failure

G. Assessment and Management of Anorexia

An adequate diet with sufficient caloric intake is an essential aspect of management of CF. Yet there are several specific issues that make eating onerous or even painful for patients. For example, chronic sinus disease can reduce the aroma, taste, and attractiveness of food. Remodeling of the thoracic cage can exert downward pressure on the stomach, so that even a moderately full stomach triggers respiratory discomfort. Gastroesophageal reflux disease can make eating uncomfortable, as can cramping associated with ineffective enzyme use. CF-related diabetes can limit or restrict food choices. Economic concerns may limit the patient’s choices of a variety of enticing meals. Finally, fatigue, loneliness, and depression can make the preparing of meals less attractive so that patients will default to more easily prepared but less nutritionally appropriate meals. A comprehensive approach to the assessment of eating strategies will focus not only on caloric intake but also on eating as an element within other activities of daily life. For children, behavioral interventions have shown improvements in caloric intake (43), and similar results can be achieved with comprehensive assessment and intervention in adults (44). H. Assessment and Management of Anxiety and Depression

Recent studies have suggested a higher incidence of depression among CF patients than previously suspected, and ongoing national studies may reveal the appropriateness of depression screening as a routine part of CF care (6). As with other aspects of quality of life, simple and short screening tools are available for depression. Treatment of depression in CF can involve multiple strategies, many of which will not be unique to CF. Optimal use of psychosocial clinicians coupled with referral outside the standard care team, when appropriate, is essential. Acute and chronic anxiety occurs commonly in children and adults with lifethreatening illnesses and can complicate the management of other symptoms. Recent reports suggest that anxiety and worrying are perceived as some of the most distressing symptoms in adults with CF (5). Simple screening tools are available and can be easily integrated into routine care (45). As patients may be anxious about disease progression, simple reassurance and good communication can be helpful. Treating pain, fatigue, dyspnea, cough, and nausea, as well as providing physical aids and nursing help with activities of daily life can facilitate participation in the care plan and reduce anxiety. In persistent or severe cases, medications may be used including oral or intravenous benzodiazepines.

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Prognostication in CF

Short- and long-term prognostication in CF remains an art (46,47). There is evidence that FEV1 less than 24% and BMI less than 18 are associated with death before discharge from the ICU (48). Children with CF admitted to the ICU for respiratory reasons do relatively well (49), but sicker adults in the ICU do worse than healthier adults (50,51). The need for mechanical ventilation in children prior to lung transplantation is associated with decreased survival (52), but that is not necessarily the case in adults (53). There are also some commonsense indications of an increased likelihood of death in an individual patient within six months: failure of a pulmonary exacerbation to respond to aggressive culture-based antibiotic therapy, weight loss unresponsive to maximum nutritional intervention, substantial (>240 cc/24 hr) hemoptysis unresponsive to embolization therapy, recurrent serious pneumothorax, and increasing daytime arterial PaCO2 levels unresponsive to therapy. Yet their absence is no guarantee of survival; regardless of the severity of existing pulmonary disease, death may be triggered by an acute event such as viral pneumonia or acquisition of a particularly resistant organism. The inability to clearly predict the end stage means that restorative and palliative care should remain intimately intertwined in CF (54). Yet specific palliative care interventions, including therapies directed at managing symptoms, are often not discussed until the patient is in dire straits, thus missing opportunities to improve quality of life and allow for advanced care planning. Much of the cultural anxiety with palliative care (e.g., “giving up” on a patient or “withdrawing therapy”) is exacerbated when such care is first mentioned only when the patient is extremely ill, especially when reasoned discussion of symptom assessment and management could have occurred earlier (55). J.

Lung Transplantation and End-of-Life Care

Lung transplantation (discussed in detail in chap. 23) influences end-of-life care for all patients with CF, even for those who do not pursue transplant. Patients awaiting transplant are more likely to die in the ICU, receive assisted ventilation, remain intubated on the last day of life, and have discussion about terminal care delayed so that they are unable to participate (56,57). While the provision of more aggressive care to those awaiting a transplant is appropriate, some argue that clinicians are opting for aggressive care without considering the realistic odds of survival to transplantation (57). More importantly, by providing an uncertain rescue from respiratory failure near the end of life, lung transplantation has altered the expected trajectory of life with CF, and complicated decisionmaking, advance care planning, symptom management, and end-of-life care for patients, families, and clinicians. K. Advanced Care Planning

Most adults with CF have considered—and discussed with family members—the type of care they would want to have if they became unable to decide for themselves (58). However, only one-third of them have in fact discussed advanced care planning with any member of the CF clinical team, suggesting that many clinicians are reluctant to discuss advanced care planning with CF adults. In minor children, advanced care planning should involve the parents and the child in a developmentally appropriate way. In teenagers, clinicians must not overlook the preferences of the adolescent regarding end-of-life care (59). As only the very rare

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adolescent has given no thought to the life-limiting nature of the disease (60), they should be allowed to participate in decision-making, allowing a transition from assent to autonomy, and maintaining the patient’s own body image and vision of independence as much as possible. Long-term relationships with the CF care team can facilitate multiple avenues of communication (61). Existing advanced care planning documents are generally based on an oncology model that may not fit CF, given the different trajectory of disease and the less toxic therapies utilized. In the attempt to improve advanced care planning, some CF centers have developed CF-specific living wills or have adapted programs such as Five Wishes (62) for use in CF. L.

Do Not Attempt Resuscitation and Do Not Intubate

Many clinicians see do not attempt resuscitation (DNaR) and do not intubate (DNI) orders as having practical use in advanced planning in CF. Many patients do not have a DNaR order in their chart until the last day or so of life (57,63). In practice, DNaR and DNI orders do not suggest a certain set of comfort care measures. Instead, these orders should be one part of a more comprehensive discussion of the goals of care. M. Psychosocial Support for Children and Adults with CF

Despite improvement in survival, some children and most adults with CF will die earlier than their unaffected peers. The possibility of death during childhood will come as a shock to most parents and feelings of grief and anger are to be expected. The presence of psychosocial clinicians on the CF care team is especially useful in helping patients and families navigate the stressors of a chronic disease and the potential for end-of-life issues. The cultural association of CF with premature mortality is a common theme in the public descriptions of CF in books (64), movies (65), or on the Internet (66). Adolescents and young adults may well have developed certain fantasies or beliefs about their lifespan that can have a direct influence on their planning for the future, adherence to the daily regimen, and quality of life. Time should be taken to discuss out-of-date or inaccurate representations of CF and provide more useful resources. Children with chronic life-threatening illnesses often know that they may die despite never being told directly (67). They will have concerns about impending death and separation from loved ones. Adolescents usually struggle to maintain independence in the face of increasingly severe disease (68). They may also grieve the loss of function, the lack of interaction with their family and peers, and changes in body image, and may decrease communication with the care team in an attempt to minimize the intrusive effects of medical care (69). Adults with CF will have had years to develop coping strategies for the challenges brought by life with a complex chronic illness (70). Most patients will continue to utilize these coping strategies as they face the acceleration of their disease, some inviting their parents back into their support networks (71,72). Each child and adult with CF will approach death differently, some pursuing aggressive lifesustaining measures, others focusing more on quality of life. Expecting each patient to pass through a standard series of prescribed stages is unrealistic. Most patients will want to pursue some mixture of restorative and palliative approaches tailored to their specific clinical and psychosocial circumstances.

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N. Grief and Bereavement

Immediately following the death of a loved one, clinicians can best support the family by simply being present. The clinician should remain in the background but be available to the family. Conversations should focus on the sadness of the loss rather than the medical details of the illness. Grief is often experienced during actual, anticipated, or perceived loss, including the time of diagnosis, loss of function, hospitalizations, or any event the patient or family sees tied to the disease. Siblings, especially those with CF, experience grief, although they may be overlooked in this setting (73). Experienced social workers, child-life therapists, and psychologists can be helpful in educating the families about how to provide appropriate support to siblings. In general, families do not “get over” the death of a child or adult, instead they incorporate memories of the deceased family member into their lives. Helping the family find meaning in the life and death of the child or adult is an important act in the resolution of grief. Expressions of sorrow and support in the form of cards or letters from the care team can be a great comfort to the family, as can participation in funerals and memorials. After the death of a patient, many clinicians experience a sense of loss, sadness, and even failure. Clinicians should be aware of their reactions to the death of a patient and should be open to discussion of the death with colleagues. Appropriate recognition and management of grief maintains balance and longevity in clinical practice and allows the clinician to support patients and families in the future. O. Palliative Care Specialists

As the death of patients with CF becomes less common, specialty and primary care providers can lose familiarity with comprehensive palliative and end-of-life care of the CF patient and family. This expertise can be found in pediatric and adult palliative care teams. Consultation with these teams is strongly suggested. Early consultation can allow the palliative care team to help the patient, family, and the CF team navigate the difficult decisions that are unfortunately still part of life with CF.

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35. Cohen SP, Dawson TC. Nebulized morphine as a treatment for dyspnea in a child with cystic fibrosis. Pediatrics 2002; 110:e38. 36. Graff GR, Stark JM, Grueber R. Nebulized fentanyl for palliation of dyspnea in a cystic fibrosis patient. Respiration 2004; 71:646–649. 37. Brown SJ, Eichner SF, Jones JR. Nebulized morphine for relief of dyspnea due to chronic lung disease. Ann Pharmacother 2005; 39:1088–1092. 38. Currow DC, Ward AM, Abernethy AP. Advances in the pharmacological management of breathlessness. Curr Opin Support Palliat Care 2009; 3:103–106. 39. Robinson W. Palliative care in cystic fibrosis. J Palliat Med 2000; 3:187–192. 40. Holsclaw DS, Grand RJ, Shwachman H. Massive hemoptysis in cystic fibrosis. J Pediatr 1970; 76:829–838. 41. Flume PA, Strange C, Ye X, et al. Pneumothorax in cystic fibrosis. Chest 2005; 128:720–728. 42. Savage R. Prone position as a life-saving measure for acute pulmonary haemorrhage in a young adult with cystic fibrosis. Anaesth Intensive Care 2002; 30:223–225. 43. Nasr SZ. An 8-week behavioral intervention can improve weight gain in children with cystic fibrosis. J Pediatr 2006; 148:700–701. 44. Watson H, Bilton D, Truby H. A randomized controlled trial of a new behavioral home-based nutrition education program, “eat well with CF,” in adults with cystic fibrosis. J Am Diet Assoc 2008; 108:847–852. 45. Riekert KA, Bartlett SJ, Boyle MP, et al. The association between depression, lung function, and health-related quality of life among adults with cystic fibrosis. Chest 2007; 132:231–237. 46. Konstan MW, Wagener JS, VanDevanter DR. Characterizing aggressiveness and predicting future progression of CF lung disease. J Cyst Fibros 2009; 8(suppl 1):S15–S19. 47. Liou TG, Adler FR, Fitzsimmons SC, et al. Predictive 5-year survivorship model of cystic fibrosis. Am J Epidemiol 2001; 153:345–352. 48. Vedam H, Moriarty C, Torzillo PJ, et al. Improved outcomes of patients with cystic fibrosis admitted to the intensive care unit. J Cyst Fibros 2004; 3:8–14. 49. Berlinski A, Fan LL, Kozinetz CA, et al. Invasive mechanical ventilation for acute respiratory failure in children with cystic fibrosis: outcome analysis and case-control study. Pediatr Pulmonol 2002; 34:297–303. 50. Slieker MG, van Gestel JP, Heijerman HG, et al. Outcome of assisted ventilation for acute respiratory failure in cystic fibrosis. Intensive Care Med 2006; 32:754–758. 51. Kremer TM, Zwerdling RG, Michelson PH, et al. Intensive care management of the patient with cystic fibrosis. J Intensive Care Med 2008; 23:159–177. 52. Elizur A, Sweet SC, Huddleston CB, et al. Pre-transplant mechanical ventilation increases short-term morbidity and mortality in pediatric patients with cystic fibrosis. J Heart Lung Transplant 2007; 26:127–131. 53. Bartz RR, Love RB, Leverson GE, et al. Pre-transplant mechanical ventilation and outcome in patients with cystic fibrosis. J Heart Lung Transplant 2003; 22:433–438. 54. Bourke S, Doe S, Gascoigne A, et al. An integrated model of provision of palliative care to patients with cystic fibrosis. Palliat Med 2009; 23(6):512–517. 55. Docherty S, Miles M, Brandon D. Searching for “the dying point:” providers’ experiences with palliative care in pediatric acute care. Pediatr Nurs 2007; 33:335–341. 56. Dellon EP, Leigh MW, Yankaskas JR, et al. Effects of lung transplantation on inpatient end of life care in cystic fibrosis. J Cyst Fibros 2007; 6:396–402. 57. Ford D, Flume PA. Impact of lung transplantation on site of death in cystic fibrosis. J Cyst Fibros 2007; 6:391–395. 58. Sawicki GS, Dill EJ, Asher D, et al. Advance care planning in adults with cystic fibrosis. J Palliat Med 2008; 11:1135–1141. 59. Edwards J. A model of palliative care for the adolescent with cancer. Int J Palliat Nurs 2001; 7:485–488.

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60. Birnkrant DJ, Hen J Jr., Stern RC. The adolescent with cystic fibrosis. Adolesc Med 1994; 5:249–258. 61. Freyer D, Kuperberg A, Sterken D, et al. Multidisciplinary care of the dying adolescent. Child Adolesc Psychiatr Clin North Am 2006; 15:693–715. 62. McCollum AT, Schwartz AH. Social work and the mourning parent. Soc Work 1972; 17: 25–36. 63. Robinson WM, Ravilly S, Berde C, et al. End-of-life care in cystic fibrosis. Pediatrics 1997; 100:205–209. 64. Merullo R. A Little Love Story. New York, NY: Vintage Books, 2005. 65. Parisse V, Stebbings P. Jack and Jill vs the World. 2008. 66. Anselmo MA, Lash KM, Stieb ES, et al. Cystic fibrosis on the Internet: a survey of site adherence to AMA guidelines. Pediatrics 2004; 114:100–103. 67. Waechter EH. Children’s awareness of fatal illness. Am J Nurs 1971; 7:1168–1172. 68. Segal TY. Adolescence: what the cystic fibrosis team needs to know. J R Soc Med 2008; 101(suppl 1):S15–S27. 69. Smith BA, Wood BL. Psychological factors affecting disease activity in children and adolescents with cystic fibrosis: medical adherence as a mediator. Curr Opin Pediatr 2007; 19:553–558. 70. Pinkerton P, Trauer T, Duncan F, et al. Cystic fibrosis in adult life: a study of coping patterns. Lancet 1985; 2:761–763. 71. Abbott J, Dodd M, Gee L, et al. Ways of coping with cystic fibrosis: implications for treatment adherence. Disabil Rehabil 2001; 23:315–324. 72. McGuffie K, Sellers DE, Sawicki GS, et al. Self-reported involvement of family members in the care of adults with CF. J Cyst Fibros 2008; 7:95–101. 73. Fanos J. Sibling Loss. Mahwah, NJ: Lawrence Erlbaum, 1996.

Index

AAP. See American Academy of Pediatrics (AAP) AAV2, 394 AAV-CBA-D264CFTR, 396 ABC. See ATP-binding cassette (ABC) ABPA. See Allergic bronchopulmonary aspergillosis (ABPA) Acapella1, 244 ACBT. See Active cycle of breathing technique (ACBT) Access measure. See Quality measure ACE. See Angiotensin converting enzyme (ACE) Acetaminophen, 484 Acetic acid (vinegar), nebulizer and, 447 Achromobacter xylosoxidans, 44 Acid-fast bacilli (AFB), 37 ACR. See Acute cellular rejection (ACR) Acrodermatitis enteropathica, 322 CFNDD and, 356 ACT. See Airway clearance therapy (ACT) Active cycle of breathing technique (ACBT), 242 Acute cellular rejection (ACR), 379–380 AD. See Autogenic drainage (AD) Ad. See Adenovirus (Ad) ADA. See American Diabetes Association (ADA) Adenovirus (Ad), 381, 392 rAd vectors, for CF gene therapy, 392–394 Adolescence asthma in, 202 pulmonary presentations and, differential diagnosis of, 204 tasks of late, 452, 453

Adolescents, with CF, 458–460, 474–475 adherence rates in, 470 aerosol therapy, regular management of, 452 CFQ-R PRO for, 472 clinic, 455 depression rate in, 470 discussion on social topics with, 454 family conflict, 474–475 governing bodies for, 452 parental supervision, 474 peer relationship, 474, 475 reproductive health information for, sources of, 459–460 risk-taking behaviors in, 459 Adults GI imaging and, 191, 193 hemoptysis in, 206 pneumothorax in, 204–205 pulmonary presentations and, differential diagnosis of, 204 respiratory infections in, 203 Adults, with CF, 475 CFQ-R PRO for, 472 complications in, 452–453 depression rate in, 470 psychosocial support for, 492 Advanced care planning, 491–492 Aerosol therapy, 444–445 AFB. See Acid-fast bacilli (AFB) Age factors in pulmonary exacerbations, 254–255 Air trapping, 185–186 computerized analysis of, 187 Airway clearance in coughing, 486

497

498 [Airway clearance] depression associated with, 474 in dyspnea, 489 need for, 434 treatment adherence and, 470–471 Airway clearance therapy (ACT), 239 cough and, 240, 241 huff and, 240, 241 physiology of, 240–241 techniques ACBT, 242 AD, 243 exercise, 245 HFCC, 244–245 IPV, 245 OscPEP, 243–244 PD&P, 241–242 PEP, 243 selection of, 245–246 Airway conductance (GAW), 163–164 Airway disease, 198 upper, 202 Airway modifiers refinement of, 85 Airway obstruction, measurements of airway resistance (RAW), 163–164 forced oscillometry, 164 gas washout, 164–166 spirometry, 161–163 Airway opening pressure (Pinit), 154 Airway reactivity. See Airway responsiveness Airway resistance (RAW). See Airway obstruction Airway responsiveness BHR, 169–170 bronchodilator responsiveness, 169 Airway surface liquid (ASL), 17, 24, 60, 237 in cystic fibrosis, 29–31 Airway surface liquid hydration therapy, 237 hypertonic saline, 238–239 Alcohol hand rub. See Hand hygiene Alkalinization, 285, 286 Allergens and pulmonary exacerbation, 251, 252

Index Allergic bronchopulmonary aspergillosis (ABPA), 45, 205, 255 classical, 209 defined, 208 diagnosis of, 209, 210 management of, 210–211 pathophysiology, 209 prevalence of, 208–209 prognosis, 211 screening, 210 stages of, 211 a-1 antitrypsin deficiency, 291 a1-antitrypsin (SERPINA1), 86 a-Fetoprotein, 295 a-linolenic acid (ALA), 317 a-Tocopherol, 321 Alveolar pressure (Palv), 154 Ambulatory settings clinic logistics, 445 precautions for patients with multidrugresistant organisms, 446 waiting room practices, 445–446 American Academy of Pediatrics (AAP), 364 American College of Medical Genetics, 464 American College of Obstetricians and Gynecologists, 464 American Diabetes Association (ADA), 341 American Thoracic Society and European Respiratory Society (ATS/ERS), 148 American Thoracic Society (ATS), 161 Amiloride-dependent sodium absorption, 111 Aminoglycosides, 212 Anesthesia, 213–214 Angiotensin converting enzyme (ACE), 291 Anorexia, assessment and management of, 490 Antenatal pulmonary presentations, 201 Antibiotic allergy, 212 Antibiotic choices, 258 Antibiotic resistance in microorganisms, 251, 258 Antibiotic susceptibility, 251, 256, 257, 258 Antibiotic therapy, 251, 252, 254, 257, 258, 259, 260 nephrotoxicity of, 257, 258 ototoxicity of, 257, 258

Index Anticholinergic agents pulmonary health in CF and, maintenance of, 231 Anti-inflammatory therapy implication for, 69–71 Antimicrobial susceptibility testing, 256–257 Antisaccharomyces cerevisiae antibody (ASCA), 275 Anxiety, 470 assessment and management of, 490 Aquagenic wrinkling of the palms (AWP), 357 chloride content in sweat and, 357 etiology of, 357 Aquaporin, 286, 287 Arachidonic acid, 461 metabolism, 63–64 Arterial oxygen, pulmonary hypertension and, 361 ASCA. See Antisaccharomyces cerevisiae antibody (ASCA) ASL. See Airway surface liquid (ASL) Aspergillus, 381 Aspergillus fumigatus, 205, 208, 209, 210 Asthma in adolescence, 202 in adults, 203 in childhood, 202 cough variant, 202 female with CF and, 461 ATP-binding cassette (ABC), 1 ATS. See American Thoracic Society (ATS) ATS/ERS. See American Thoracic Society and European Respiratory Society (ATS/ERS) Autogenic drainage (AD), 242, 243 versus PD&P, 243 AWP. See Aquagenic wrinkling of the palms (AWP)

Back-extrapolated volume (VBE), 149 calculation of, 150 Bacterial cultures, 256–257 BAL. See Bronchoalveolar lavage (BAL)

499 BALF. See Bronchoalveolar lavage fluid (BALF) BC. See Breathing control (BC) Behavioral Pediatrics Feeding Assessment Scale, 317 “benchmarking” in cystic fibrosis care, 433–434 Benzodiazepines, 489 Bereavement and grief, 493 b-Adrenergic stimulation in sweat duct, 16 b-Agonists pulmonary health in CF and, maintenance of, 231 b-cells, pancreatic, 341 b-Lactam antibiotics, 152, 257 efficacy of, 252 BHR. See Bronchial hyperreactivity (BHR) Bicarbonate ion, 285, 286, 287, 288 Bilateral lung transplantation. See Lung transplantation Bile acids for CFALD, 299 Bile formation, 285–287 alternate pathways of, 286–287 CFTR and, 285–287 Bile secretory unit, 285 cholangiocytes, 285, 286, 288, 299 hepatocytes, 285, 286, 292 Biliary manifestations of CFALD cholelithiasis, 294 microgallbladder, 294 Biliary secretion, 286, 299 Biofilms, 251–252, 258 susceptibility testing, 258 Biomarkers of pulmonary exacerbation, 257 Bishop–Koop ileostomy, 270 Bisphosphonates and BMD, 335–336 BMC. See Bone mineral content (BMC) BMI. See Body mass index (BMI) BO. See Bronchiolitis obliterans (BO) Body mass index (BMI), 329, 330, 349 CFRD and, 349–350 and cystic fibrosis in children, 316–317 in females, 316, 317 in males, 316, 317

500 Bone densitometry, 329 mass, 329, 330, 332, 336 mineralization, 328, 330 resorption, 331, 333, 335 Bone diseases biochemical markers of, 333 alkaline phosphatase as, 333 calcium deposition as, 333 osteocalcin as, 333 clinical manifestations of, 328–330 BMD and, 328–330 fractures and, 330 diagnosis of, 328, 333. See also Dual energy X-ray absorptiometry (DXA) histopathology of, 333 management of, 333–337 bisphosphonates in, 335–336 growth hormone (GH) therapy in, 336 vitamin supplementation, 333–335. See also Vitamin D therapy pathophysiology of, 330–332 calcium and, 331 CFTR protein and, 332 glucocorticoids and, 331–332 infection and, 331 LBM and, 332 malnutrition and, 330 vitamin D and, 330 vitamin K and, 331 Bone mineral content (BMC), 328, 329, 332, 336 measurement of, 328, 329. See also Dual energy X-ray absorptiometry (DXA) Bone mineral density (BMD), 328–330, 331, 332, 335, 336 age factors in, 329–330 body mass index (BMI) and, 329, 330 fractures and, 329 measurement of, 328, 329 z scores and, 329, 330 Bortezomib (Velcade1), 413 BOS. See Bronchiolitis obliterans syndrome (BOS) Boyle’s law, 163, 167 Breathing control (BC) cycle of, 242

Index Bronchial hyperreactivity (BHR), 169–170 Bronchiectasis, 57, 202 Bronchiolitis obliterans (BO), 381–382 Bronchiolitis obliterans syndrome (BOS), 381 Bronchoalveolar lavage (BAL), 58, 141, 183, 256 Bronchoalveolar lavage fluid (BALF), 198 Bronchodilator responsiveness, 169 Bronchoscopy, 198, 256 Burkholderia, 379 Burkholderia cepacia, 37, 40, 66, 200, 203, 206, 256, 377, 378–379

Calcidiol, 319 Calcitriol, 319 Calcium absorption, 330, 331 and bone diseases, 331, 336 Calcium bilirubinate, 294 Calmodulin kinase, 286 Calorie intake in cystic fibrosis, 314 cAMP, 286, 288, 299 Camps. See Non-health care settings, infection control in Candidate genes vs. genome-wide association studies, 84–85 Capacitant element (Xc), 151 CAR. See Coxsackievirus adenovirus receptor (CAR) Carbon monoxide (CO), 168–169 Cardiac complications, cystic fibrosis (CF) and, 361–362 cor pulmonale and, 361–362 pulmonary hypertension and, 361–362 Cardiopulmonary exercise testing (CPET), 170 Caregivers, depression rate in, 470. See also Parents “catch-up growth” of brain, 360 Cationic liposome delivery systems CF gene therapy and, 398 Cationic polymers for CF gene therapy, 398–399 Cationic trypsinogen (PRSS1), 270

Index CBAVD. See Congenital bilateral absence of the vas deferens (CBAVD) CDC. See Centers for Disease Control and Prevention (CDC) Celiac disease, 275 Centers for Disease Control and Prevention (CDC), 440 Cervical dysplasia, 459 CEV. See Cumulative expired volume (CEV) CF. See Cystic fibrosis (CF) CFA. See Coefficient of fat absorption (CFA) CFALD. See Cystic fibrosis–associated liver disease (CFALD) CFF. See Cystic Fibrosis Foundation (CFF) CFFONE, 476 CF Foundation Hepatobiliary Disease Consensus Guidelines, 295 CF gene genetic variation in, 79–80 CF gene therapy AAVs vectors for, 394–397 barriers to, 391–392 cationic liposomes for, 398 cationic polymers for, 398–399 gene replacement and, 391 lentiviral vectors for, 397 nonviral vectors for, 397 rAd vectors for, 392–394 target cells for, 390–391 CFNDD. See Cystic fibrosis nutrient deficiency dermatitis (CFNDD) CFQ-R. See Cystic Fibrosis Questionnaire– Revised (CFQ-R) CF-Quality of Life and Transitional Dyspnea Index scores, 375 CFRD. See Cystic fibrosis-related diabetes (CFRD) CFTR. See Cystic fibrosis transmembrane conductance regulator (CFTR); Cystic fibrosis transmembrane conductance regulator (CFTR) gene CFTR gene. See Cystic fibrosis transmembrane conductance regulator (CFTR) gene CFTR-opathies, 407

501 CGMS. See Continuous glucose monitoring system (CGMS) Chest physiotherapy, 214 radiography, 182–183 Chest X ray (CXR), 204 Childhood asthma in, 202 hemoptysis in, 206 pneumothorax in, 204 pulmonary presentations and, differential diagnosis of, 204 Children with CF, 315–317, 473–474 adherence rates for, 470 BMI in, 316–317 CFQ-R PRO for, 472 depression rate in, 470 early, 473 eating behavior in, 317 growth in, 315–317 lung function in, assessment of, 148–157 interrupter technique. See Interrupter technique specific airways resistance. See Specific airways resistance (sRaw) spirometry, modified. See Spirometry, modified tidal breathing, 156–157 medical interventions for, 317 MI and, 191 middle, 473–474 nutrition assessment of, 315–317 psychosocial support for, 492 spirometric findings in, 150 supplements for, 317, 322 weight-for-length stature in, 316, 317 Chloride channels, 285, 286, 287 Choke point (CP), 240 Cholangiocyte membrane, 285, 286, 288, 299 permeability, 285 receptor binding, 286, 287 transport, 285, 286, 287, 288, 291 Cholangiocytes, 285, 286, 288, 299 Cholecystectomy, 294 Cholera toxin (CT), 3

502 Choline deficiency in cystic fibrosis, 318–319 Chronic care model clinical information systems, 429 community resources, 428 decision support, 429 delivery system design, 429 medical center health delivery system, 428 patient and family self-management, 429 Cilia in cystic fibrosis, 31–32 Cisapride, 276 CL. See Lung compliance (CL) Clinic adolescent, 455 logistics, 445 primary care, 455 transition, 455 Clinical information systems. See Chronic care model Clostridium difficile, 274–275 CL-VI. See Collagen type VI (CL-VI) CME. See Continuing medical education (CME) CMV. See Cytomegalovirus (CMV) CNV. See Copy number variants (CNV) CO. See Carbon monoxide (CO) Cochrane Collaborative report, 231 Coefficient of fat absorption (CFA), 117, 315 Coherence function (g2), 153 Collagen type VI (CL-VI), 296 Community resources. See Chronic care model Compliance (Crs), 128 Computed tomography (CT), 141, 183 acquisition technique, 188 clinical utility, 183, 185–186 radiation-induced cancer, risk of, 189 research potential, 187 of sinuses, 190 surrogate endpoints, 187–188 Condoms, CF patients and, 459, 462 Congenital absence of the uterus and vagina (CAUV), 457–458 Congenital bilateral absence of the vas deferens (CBAVD), 7, 92, 457

Index Constipation, 277 assessment and management of, 489–490 opioids, side effects of, 484 Continuing medical education (CME), 426 Continuous glucose monitoring system (CGMS), 347–348 Contraception, 462 Copy number variants (CNV), 79 Corticosteroids used in lung transplantation, 378 Corticosteroid therapy pulmonary health in CF and, maintenance of, 232 Cough airway clearance with, 240, 241 assessment and management of, 486 variant asthma, 202 Coxsackievirus adenovirus receptor (CAR), 392 CP. See Choke point (CP) CPET. See Cardiopulmonary exercise testing (CPET) CPX. See 8-cyclopentyl-1,3dipropylxanthine (CPX) Crossing the Quality Chasm, 423 Crs. See Compliance (Crs) CT. See Cholera toxin (CT); Computed tomography (CT) CTL. See Cytotoxic T lymphocyte (CTL) CAUV. See Congenital absence of the uterus and vagina (CAUV) Cumulative expired volume (CEV), 164 Cutaneous complications, cystic fibrosis (CF) and, 356–358 aquagenic wrinkling of the palms (AWP) and, 357 categories of, 356 cystic fibrosis nutrient deficiency dermatitis (CFNDD), 356–357 CXR. See Chest X ray (CXR) 8-Cyclopentyl-1,3-dipropylxanthine (CPX), 413 Cystic fibrosis–associated liver disease (CFALD), 285 diagnosis of, 294–296 biochemical analysis in, 295. See also Serum liver enzymes

Index [Cystic fibrosis–associated liver disease (CFALD)] biomarkers in, 294–296 liver biopsy in, 296 physical examination in, 294 ultrasonography in, 296 genetic aspects of, 290 genetic liver disease and, 291 immune response and, 291 meconium ileus and, 290 sex factors in, 290 manifestations of, 285 biliary, 285. See also Biliary manifestations of CFALD liver, 285. See also Liver manifestations of CFALD pathophysiology of, 288–290 abnormal CFTR protein and, 289 abnormalities in bile secretion and, 289 abnormalities in mucin secretion and, 289 activation of stellate cell and, 288–290 bile acid toxicology and, 289 hepatic infections and, 290 prevalence of, 291–292 treatment of, 296–299. See also Future therapeutics for CFALD diet in, 297 liver transplantation, 299 nutrition therapy in, 296–297 pancreatic enzyme replacement therapy, 297 ursodeoxycholic acid, 297–298 vitamin supplementation in, 297 Cystic fibrosis (CF), 1 ACT for. See Airway clearance therapy (ACT) in adolescents, 474–475 in adults, 475 airway surface liquid hydration therapy for, 237 hypertonic saline, 238–239 mannitol, 239 airway surface liquid in, 29–31 bacterial STIs in, 459 in children. See Children with CF cilia in, 31–32

503 [Cystic fibrosis (CF)] classic and emerging pathogens in, 37 diagnosis of, 90–94 diagnostic algorithm for, 93–94 emotional functioning in, 470 gastrointestinal complications of Clostridium difficile infection, 274–275 DIOS, 271 dysmotility, 276–277 FC, 277 GER, 271–274 HP infection, 275 intussusception, 276 malabsorption, 266–267 malignancy, 277–278 MI, 270 pancreas, 267–269 pancreatitis, 269–270 rectal prolapse, 276 SBBO, 274 gastrointestinal (GI) imaging in neonate and young child, 191 older children and adults, 191, 193 prenatal, 190–191 incidence of, 1–3 infant lung function in, measures of, 141 mucolytic therapy for goals of, 236 NAC, 236 mutation classes in, 407, 408 nutritional aspects of, 308–322 dietary intake and, 314, 317, 319 nutritional supplements, 312, 313, 317, 320, 321 undernutrition, 317 vitamin A and, 320–321 vitamin D and, 319–320 vitamin E and, 321 zinc in, 322 and pancreatic insufficiency. See Cystic fibrosis related pancreatic insufficiency parenting with, 464, 473 pathogens in, 40–48 peer relationships in school and, 469 pneumothorax in. See Pneumothorax

504 [Cystic fibrosis (CF)] practice guidelines and benefits and limitations of, 228 PROs in, 471–473 pulmonary complications of ABPA. See Allergic bronchopulmonary aspergillosis (ABPA) antibiotic allergy and desensitization, 212 hemoptysis. See Hemoptysis lobar collapse, 211–212 pneumothorax. See Pneumothorax pulmonary exacerbations and, 252 pulmonary health in anticholinergic agents, 231 approaches to maintenance of, 224–227 b-agonists, 231 CFTR and, 233 corticosteroid therapy, 232 exercise and, 231 goals of treatment to maintain, 224, 225 ibuprofen therapy, 232 ion channel therapies, 232–233 macrolides, 232 mucociliary clearance and, 231 therapies for maintenance of, 231–233 signs and symptoms of, 91 sinus Imaging in, 190 sleep quality, cystic fibrosis and, 362–365 social and family functioning in, 469–470 systems, affected, 468–469 time frame of appearance of, 92 treatment adherence in, 470–471 viral STI in, 459 young children with lung function assessment in, 148–157 Cystic fibrosis (CF), complications in cardiac, 361–362 lung diseases and, 361–362 cutaneous, 356–358 allergic response of drugs and, 357 CFTR mutation and, 357 nutritional deficiencies and, 356–357 vasculitis and, 357 metabolic, 358 alkalosis and, 358 neurologic, 358–361 neurologic symptoms and, 360–361

Index [Cystic fibrosis (CF), complications in neurologic] nutritional deficiencies in, 359–360 therapeutics in, 360 sleep, 362–365 disruption, 362–364 gas exchange abnormalities and, 364–365 lung diseases and, 363 oxyhemoglobin saturation and, 364, 365 Cystic fibrosis (CF) care improvement, partnerships for, 425–426 Cystic fibrosis (CF) outcomes impact of QI on, 434–435 Cystic fibrosis (CF) pulmonary disease dornase alfa, 228 infection in preventive and maintenance treatment of, 228–231 pathophysiologic cascade of, 226 practice guidelines and benefits and limitations of, 228 principals of maintenance therapy for, 224–227 stages of, 225, 226 thoracic imaging in chest radiography, 182–183 CT. See Computed tomography (CT) MRI, 189–190 nuclear medicine, 189–190 Cystic fibrosis (CF) symptoms, assessment and management of anorexia, 490 anxiety and depression, 490 constipation/obstipation, 489–490 cough, 486 dyspnea, 486–489 hemoptysis, 489 pain, 483–484 opioid use for, 484–486 Cystic Fibrosis Foundation (CFF), 186, 256, 260, 315 benchmarking and, 432–433 clinical practice guidelines by, 432–434 collaboration, comparison, data transparency and, 431–432 goals of, 426 pivotal role of, 423–424

Index Cystic Fibrosis Foundation Hepatobiliary Disease Consensus Group, 296, 298 Cystic Fibrosis Foundation Patient Registry Annual Report, 348 Cystic fibrosis lung, inflammation in characteristics of, 57–59 disordered intracellular signaling as, 64–67 dysregulated arachidonic acid metabolism, 63–64 inflammatory mediators in, 60–63 overview, 57 pathologic consequences of neutrophil, 67–69 role of airway epithelia in, 59–60 Cystic fibrosis nutrient deficiency dermatitis (CFNDD), 356–357 acrodermatitis enteropathica and, 356 nutritional therapy for, 357 sweat test in, 357 Cystic Fibrosis Questionnaire–Revised (CFQ-R), 472 Cystic fibrosis-related diabetes (CFRD), 78, 341–352, 378, 490 diagnosis for, 342–343 hormone secretion and, 344–345 insulin, 344 pathophysiology of, 343–344 b-cell destruction and, 343–344 immunohistochemical studies for, 343–344 prevalence of, 348–349 gender difference in, 349 pulmonary function in, 350 screening for, 345–348 casual blood glucose testing in, 347 continuous glucose monitoring system (CGMS) in, 346–347 HbA1c in, 346 oral glucose tolerance tests (OGTTs) in, 346–347 symptoms of, 345 T1DM and, 342 T2DM and, 342 treatment of, 350–352 insulin therapy in, 350–351 nutritional therapy in, 352

505 Cystic fibrosis related pancreatic insufficiency fat malabsorption and, 314, 319, 321, 322 treatment of, 315 pancreatic enzyme replacement therapy (PERT), 315 Cystic fibrosis transmembrane conductance regulator (CFTR), 190, 199, 224, 267–268, 269–270, 285–289, 291, 332, 333, 342, 356, 390 abnormal, 266 in bile formation, 285–287 in development of reproductive tissues, 457 in female patients, 460 in liver physiology, 285–287 mutation of, 286, 289, 290, 291 pulmonary health in CF and, maintenance of, 233 in rat brain, 458 Cystic fibrosis transmembrane conductance regulator (CFTR) gene, 1, 11, 78, 103, 391 clinical assessment of, 116–118 diagnosis of, 106–107 diagnostic challenges, 107–110 in adulthood, 107–108 diagnostic tests ion channel measurements, 110–114 discovery of, 390 diseases of, 104–106 functions of, 104–106 genetic variation in, 79–80 genotype/phenotype correlation, 6–7 genotyping, 114–116 heterozygote carriers of, 8 ion transport, abnormal conditions, 15–19 ion transport, normal conditions, 12–15 mutations and bone diseases, 332 classes of, 3–6 classification of, 105 nomenclature for, 103–104 overview, 103 recomendation of, 118 Cytomegalovirus (CMV), 381 Cytotoxic T lymphocyte (CTL), 392

506 Daily Phone Diary (DPD), 471 Data sharing (transparency), improvement collaboratives and, 432 Decision support. See Chronic care model Dehydration, 212, 214 7-Dehydrocholesterol, 319 Delayed gastric emptying (DGE), 276 Delayed puberty and bone diseases, 332 Delivery system design. See Chronic care model Denufosol tetrasodium, 33, 232–233 Depression assessment and management of, 490 in parents, 473 rate, in CF, 470 Dermatologic complications in cystic fibrosis. See Cutaneous complications, cystic fibrosis (CF) and Developmental, biopsychosocial model of, 468–469 DGE. See Delayed gastric emptying (DGE) Diabetes and bone diseases, 332 Diabetes mellitus, classification of, 341 Diagnostic algorithm CF based on newborn screening, 94 European Union Diagnostic Working Group, 93 Diet and CFALD, 297 fat intake, 297 protein intake, 297 Dietary intake in cystic fibrosis, 314 Dietary Reference Intake (DRI), 319 Diffusion limitation to carbon monoxide (DLCO), 168–169 1,25-Dihydroxyvitamin D [1,25(OH)2D], 319. See also Calcitriol Dioleoylphosphatidyl-ethanolamine (DOPE), 398 DIOS. See Distal intestinal obstruction syndrome (DIOS) Disinfection in ambulatory care setting. See Environmental infection control measures and general sterilization. See Environmental infection control of nebulizers, 444–445

Index Disordered intracellular signaling cause of excessive inflammation, 64–67 Distal intestinal obstruction syndrome (DIOS), 86, 190, 191, 214, 266, 484 Dizygous twins, 80 DLCO. See Diffusion limitation to carbon monoxide (DLCO) DNA-dependent protein kinase (DNA-PKcs), 397 DNA-PKcs. See DNA-dependent protein kinase (DNA-PKcs) DNaR. See Do not attempt resuscitation (DNaR) DNI. See Do not intubate (DNI) Do not attempt resuscitation (DNaR), 492 Do not intubate (DNI), 492 DOPE. See Dioleoylphosphatidylethanolamine (DOPE) Dornase alfa, 228 treatment of CF with, 237 DPD. See Daily Phone Diary (DPD) DRI. See Dietary Reference Intake (DRI) Drug synergism, 257 Dual energy X-ray absorptiometry (DXA), 328, 329, 333, 335 in bone diseases, 328, 333 Duodenogastric bile reflux, 272 Duty cycle, 172 DXA. See Dual energy X-ray absorptiometry (DXA) Dysmotility, 274, 276–277 Dyspnea, assessment and management of, 486–489

ECG. See Electrocardiography (ECG) EFA. See Essential fatty acids (EFA) EFAD. See EFA deficiency (EFAD) EFA deficiency (EFAD) in cystic fibrosis, 318 EGG. See Electrogastrography (EGG) EIB. See Exercise-induced bronchospasm (EIB) Electrocardiography (ECG), 361 Electrogastrography (EGG), 276 Electronic monitors, 471 Emotional functioning, in CF, 470

Index Endobronchial bacteria, 251–252 Endobronchial pathogens, 251–252 End-of-life care defined, 482 lung transplantation and, 491 Endoplasmic reticulum (ER), 344 End-stage liver disease, 285 End-tidal carbon dioxide (ETCO2) measurements, 373 Energy intake in cystic fibrosis, 314, 317 eNO. See NO in exhaled breath (eNO) Enteral nutrition in cystic fibrosis, 317 Environmental infection control disinfection in ambulatory care setting, 447 general sterilization and disinfection measures, 446 pulmonary function testing equipment, 446–447 Enzymatic digestion, 314 Epidemiologic Study of Cystic Fibrosis (ESCF), 424 Epidemiology of Pulmonary Exacerbations, 254–255 Epithelial cells, 60 Epithelial sodium channel (ENaC), 18, 201 Epstein–Barr virus, 381 ER. See Endoplasmic reticulum (ER) ERS. See European Respiratory Society (ERS) ERV. See Expiratory reserve volume (ERV) Erythematous papules, 356 ESCF. See Epidemiologic Study of Cystic Fibrosis (ESCF) Escherichia coli, 3 Esophagitis, 272–273 Essential fatty acids (EFA), 292, 314, 317–318 Estrogen, in CF patients, 458, 460 ETCO2. See End-tidal carbon dioxide (ETCO2) European Respiratory Society (ERS), 161 Evidence-based management of pulmonary exacerbations, 257–260

507 Exercise for airway clearance, 245 and bone health, 332, 336 pulmonary health in CF and, maintenance of, 231 respiratory system and, 170–171 Exercise-induced bronchospasm (EIB), 170 Expiratory reserve volume (ERV), 167 Extracellular matrix proteins, 296 collagen type VI (CL-VI), 296 hyaluronic acid (HA), 296 procollagen III polypeptide (PIIIP), 296 prolyl hydroxylase (PH), 296 Extracellular polysaccharide matrix, 251 Eye shields. See Standard precautions

False-positive or false-negative sweat test conditions associated with, 96 Fat intake in cystic fibrosis, 314 malabsorption, 314, 319, 321, 322. See also Steatorrhea in stool, 314 Fatty acids defficiency of, 63–64 FC. See Fibrosing colonopathy (FC) DF508 CFTR restoring function to, 411, 413–415 FDA. See Food and Drug Administration (FDA) FDG-PET. See Fluorodeoxyglucose positronemission tomography (FDG-PET) FEF. See Forced expiratory flow (FEF) Females, with CF asthma, 461 estrogen and CFTR, 460 fertility, 462 menses, 458, 461 pregnancy, 462 prostaglandins, 461 sexual and reproductive health issues, 462 survival of, 458 Fentanyl, 484 Fertility in females with CF, 462 in males with CF, 463

508 Fibrocystic disease, 12 Fibrosing colonopathy (FC), 267, 277 Fingernail specifics. See Hand hygiene “First-generation” vectors, 392 Fluorodeoxyglucose positron-emission tomography (FDG-PET), 189 Flutter, 244 fMLP. See N-formyl-methionylleucyl-phenylalanine (fMLP) FO. See Forced oscillometry (FO) Follicle stimulating hormone (FSH), 458 Food and Drug Administration (FDA), 252, 471 Forced oscillometry (FO), 151–154, 164 quality control criteria for, 153 Forced vital capacity (FVC), 129, 149, 162, 256, 376 N-formyl-methionyl-leucyl-phenylalanine (fMLP), 62 Fractures and cystic fibrosis, 330 FRC. See Functional residual capacity (FRC) Frequency dependence, of resistance, 152 FSH. See Follicle stimulating hormone (FSH) Functional residual capacity (FRC), 124, 129, 132, 161, 240 Future therapeutics for CFALD, 299–300 alternative secretory pathways and, 299–300 bile acids as, 299 hepatic stellate cells in, 299–300 FVC. See Forced vital capacity (FVC)

Gabapentin, 486 g2. See Coherence function (g2) g-Glutamyltransferase, 295 g-Tocopherol, 321 Gas dilution, for PFT, 129, 130–131 helium, 168 nitrogen washout, 168 Gastric acid, 266 Gastric inhibitory polypeptide (GIP), 344–345 Gastroesophageal reflux disease (GERD), 271

Index Gastroesophageal reflux (GER), 191, 202, 266, 271–274, 381 hiatal hernias and, 272 Gastrointestinal complications Clostridium difficile infection, 274–275 DIOS, 271 dysmotility, 276–277 FC, 277 GER, 271–274 HP infection, 275 intussusception, 276 malabsorption, 266–267 malignancy, 277–278 MI, 270 pancreas, 267–269 pancreatitis, 269–270 rectal prolapse, 276 SBBO, 274 Gastrointestinal (GI) imaging neonate and young child, 191 older children and adults, 191, 193 prenatal, 190–191 Gastrostomy, 297 GAW. See Airway conductance (GAW) G551D CFTR, 415–416 Gene replacement and CF gene therapy, 391 Gene therapy. See CF gene therapy Genetic carriers, 291 Geneticin (G418) PTCs suppression and, 409 Genetic polymorphisms, 291 Genetic screening, 464 Gene transfer agent (GTA), 390 GER. See Gastroesophageal reflux (GER) GERD. See Gastroesophageal reflux disease (GERD) GI imaging. See Gastrointestinal (GI) imaging GIP. See Gastric inhibitory polypeptide (GIP) GL. See Lung conductance (GL) GL-67 (Genzyme), 398 Gloves. See Standard precautions GLP-1. See Glucagon-like peptide-1 (GLP-1) Glucagon-like peptide-1 (GLP-1), 344–345 Glycogenolysis, 341

Index GM-CSF. See Granulocyte macrophage colony-stimulating factor (GM-CSF) GnRH, Gonadotropin–releasing hormone (GnRH) Gonadotropin-releasing hormone (GnRH), 458 Gowns. See Standard precautions Granulocyte macrophage colonystimulating factor (GM-CSF), 60 Grief and bereavement, 493 Growth hormone (GH) therapy in bone diseases, 335 GTA. See Gene transfer agent (GTA) Guideline for Disinfection and Sterilization, 441 Guideline for Environmental Infection Control in Health Care Facilities, 441 Guideline for Hand Hygiene in Healthcare Settings, 441 Guideline for Healthcare Acquired Pneumonia, 441 Guideline for Infection Control in Healthcare Personnel, 441 Guidelines for Isolation Precautions, 441, 444 Guidelines for reprocessing respiratory therapy and pulmonary function testing equipment, 442 “Gutless,” 394 G542X-hCFTR mouse model, 409–410

Haemophilus influenzae, 40, 41, 59, 204 Hand hygiene alcohol hand rub, 441, 443, 445–446 fingernail specifics, 441 HCWs and, 441 skin care, 441 soap and water, 441, 443 Hardy–Weinberg proportions, 82 HbA1c. See Hemoglobin A1c (HbA1c) HCW. See Health care worker (HCW) He. See Helium (He) Health care system optimizing, need to, 427–429 quality of care and, 430–431 strategies to improve, 429–430

509 Health care workers (HCW), 440 Health-related quality of life (HRQOL), 468 Helicobacter pylori (HP), 275 Helium (He) dilution, 168 Hemochromatosis, 291 CFALD and, 291 genetic aspects of, 291 Hemoglobin A1c (HbA1c), 346 Hemoptysis adults, prevalence in, 206 assessment and management of, 489 children, prevalence in, 206 diagnosis of, 206 management of, 207 pathophysiology, 206 prognosis, 207 spirometry and, 207 Hepatobiliary disease, 290 Hepatocytes, 285 Herring–Breuer reflex, 128 HFCC. See High-frequency chest compression (HFCC) HFCWO. See High-frequency chest wall oscillation (HFCWO) HFO. See High-frequency oscillation (HFO) Hiatal hernias and GER, 272 HICPAC. See Hospital Infection Control Practices Advisory Committee (HICPAC) High-frequency chest compression (HFCC), 244–245 High-frequency chest wall oscillation (HFCWO), 244–245 High-frequency oscillation (HFO), 243–244 High-mobility group box 1 (HMGB1), 62 High pressure positive expiratory pressure (HiPEP), 243 High-resolution computed tomography (HRCT), 164, 198 advantage of, 188 High throughput screening (HTS), 406 HiPEP. See High pressure positive expiratory pressure (HiPEP) HLA. See Human leukocyte antigen (HLA)

510 HMGB1. See High-mobility group box 1 (HMGB1) Homeostasis, 251 Hormone replacement therapy, 336 Hormones estrogen, 458 FSH, 458 GnRH, 458 LH, 458 prostaglandins, 461 Hospice. See End-of-life care Hospital Infection Control Practices Advisory Committee (HICPAC), 440 House of Commons Select Committee on Health, 452 HP. See Helicobacter pylori (HP) HRCT. See High-resolution computed tomography (HRCT) HRQOL. See Health-related quality of life (HRQOL) HTS. See High throughput screening (HTS) Huff airway clearance with, 240, 241 Human leukocyte antigen (HLA), 290 25-Hydroxy vitamin D (25-OHD), 319, 320, 331, 333. See also Calcidiol Hypercapnia, 372 Hyperinflation, 168 Hypertonic saline, 231 mucociliary clearance and, 238–239 Hypogonadism and bone diseases, 332 Hypoxemia, 372

IBD. See Inflammatory bowel disease (IBD) Ibuprofen, 484 oral administration of, 59 Ibuprofen therapy pulmonary health in CF and, maintenance of, 232 %IBW. See Percent ideal body weight (%IBW) %IBW and cystic fibrosis, 315, 316 ICAM-1. See Intercellular adhesion molecule 1 (ICAM-1)

Index iCARE. See I Change Adherence and Raise Expectations study (iCARE) I Change Adherence and Raise Expectations study (iCARE), 476 ICM. See Ion channel measurements (ICM) ICS. See Inhaled corticosteroids (ICS) ICSI. See Intracytoplasmic sperm injection (ICSI) ICU. See Intensive care unit (ICU) IFRD1. See Interferon-related developmental regulator 1 (IFRD1) IGT. See Impaired glucose tolerance (IGT) IHI. See Institute for Health Care Improvement (IHI) Immunoreactive trypsinogen (IRT), 98 Immunosuppressive therapy lung transplantation and, 379 Impaired glucose tolerance (IGT), 343 Inertive element (Xi), 151 Infants CF in, pulmonary manifestations of, 124–125 clinical management of, role of PFT in, 142 lung function in CF, measures of, 141 lung function testing in, 123–143 with MI, 270 PFT in. See Pulmonary function testing (PFT) pulmonary presentations and, differential diagnosis of, 203 respiratory infections in, 201–202 respiratory mechanics in, 124 studying, challenges of, 123–124 Infection inflammation vs., 198–199 lung transplantation and, 380–381 Infection control, 260 environmental, 446–447 in non–health care settings, 447–449 practices, rationale for, 441–442 psychosocial issues related to, 449 recommendations for inpatient settings activity outside the patient’s room, 443–444 respiratory therapy, 444–445 room placement, 443

Index Inflammation role of airway epithelia in, 59–60 vs. infection, 198–199 Inflammatory bowel disease (IBD), 275 Inhaled corticosteroids (ICS), 70, 232 Institute for Health Care Improvement (IHI), 423 Institute of Medicine (IOM), 423 Insulin-dependent diabetes mellitus. See Type 1 diabetes mellitus (T1DM) Insulin pump, 351 Insulin resistance cystic fibrosis and, 345 defined, 341 Insulin secretion, 341 Intensive care unit (ICU), 214–215 Intercellular adhesion molecule 1 (ICAM-1), 62 Interferon-related developmental regulator 1 (IFRD1), 199 Interleukin (IL)-8, 59 International Pediatric Lung Transplant Consortium, 379 International Society of Heart and Lung Transplantation (ISHLT), 378 Interrupter technique, 154–155 gas mixing, 155 Intestinal cells, 314 Intestinal obstructions (MI), 86 Intestine ion transport, 16–17 Intracytoplasmic sperm injection (ICSI), 463 Intrapulmonary percussive ventilation (IPV), 245 Intravenous antibiotics, 254, 257– 260 anti-Pseudomonas antibiotics, 258 Intussusception, 276 Inverted terminal repeats (ITR’s) sequences, 394–395 IOM. See Institute of Medicine (IOM) Ion channel measurements (ICM), 104, 110–114 Ion channel therapies pulmonary health in CF and, maintenance of, 232–233

511 Ion exchange, 285, 287 Ion transport in abnormal CFTR gene, 15–19 in gene, 12 in intestine, 16–17 in liver, 17 in lungs, 17–19 in normal CFTR gene, 12–15 overview, 11 in pancreas, 16 physiology in vitro, 18 in reproductive tract, 17 roll of, 11–12 in sweat gland, 15–16 IPV. See Intrapulmonary percussive ventilation (IPV) IRT. See Immunoreactive trypsinogen (IRT) ISHLT. See International Society of Heart and Lung Transplantation (ISHLT) ITR’s sequences. See Inverted terminal repeats (ITR’s) sequences

KEAP1. See Kelch-like ECH-associated protein 1 (KEAP1) Kelch-like ECH-associated protein 1 (KEAP1), 67 Kyphosis and cystic fibrosis, 330

Lactulose, 484 LAS. See Lung Allocation Score (LAS) Last menstrual period (LMP), 458 Lateral chest radiograph, 182, 184 LBM and bone diseases, 329, 330, 332 LCI. See Lung clearance index (LCI) Lean body mass (LBM), 329, 330, 332, 336 “Learning and leadership” collaborative projects, 426–427 Lentiviral vectors for CF gene therapy, 397 LES. See Lower esophageal sphincter (LES) Leukotriene (LT), 60, 198 LH. See Luteinizing hormone (LH)

512 Life expectancy lung transplantation and, 382 median, in United States, 468 Lipase, 314, 315 Lipoplexes, 397 Lipopolysaccharide (LPS), 61 Liposomes, cationic for CF gene therapy, 398 Lipoxin A4 (LXA4), 64 Liver ion transport, 17 Liver biopsy, 296 complications of, 296 Liver manifestations of CFALD focal biliary cirrhosis, 292–293 hepatic steatosis, 292 multilobular cirrhosis, 292, 293 LMP. See Last menstrual period (LMP) Lobar collapse, 211–212 Lobectomy, 207 Logistic regression models, 254 Lorazepam, 486 Lower esophageal sphincter (LES), 272 “low-hanging fruit” approach, 413 LPS. See Lipopolysaccharide (LPS) LT. See Leukotriene (LT) Lungs air travel and, 212–213 altitude and, 212–213 anesthesia. See Anesthesia ICU. See Intensive care unit (ICU) ion transport, 17–19 Lung Allocation Score (LAS), 377 Lung clearance index (LCI), 129, 130, 155, 164 Lung compliance (CL), 142 Lung conductance (GL), 142 Lung disease pathophysiology of airway disease, early, 198 inflammation vs. infection, 198–199 pulmonary clinical presentations antenatal, 201 overview, 200–201

Index Lung function assessment of, in young children, 148–157 interrupter technique. See Interrupter technique specific airways resistance. See Specific airways resistance (sRaw) spirometry, modified. See Spirometry, modified tidal breathing, 156–157 assessment of, in older children, 161 during menstrual cycles, 461 Lung function testing, infants obstacles during, 123, 124 PFT, techniques for infant. See Pulmonary function testing (PFT) Lung transplantation in CF population, 382–383 complications acute cellular rejection (ACR), 379–380 bronchiolitis obliterans (BO), 381–382 early, 380 infection, 380–381 late, 380 contraindications to, 377–379 end-of-life care and, 491 immunosuppressive therapy, 379 life expectancy, 382 listing, 379 timing of referral for, 376–377 transplant surgery, 379 Lung volumes fractional, 166 measurement of static, 166 gas dilution, 168 plethysmography, 167–168 Luteinizing hormone (LH), 458 LXA4. See Lipoxin A4 (LXA4)

Macroduct, 95 Macrolides pulmonary health in CF and, maintenance of, 232 Magnetic resonance imaging (MRI) nuclear medicine and, 189–190 Malabsorption, 266–267

Index Males, with CF fertility, 463 semen analysis, 463 sexual and reproductive health issues, 463 Malignancy, 277–278 Malnutrition and bone diseases, 330 Management of Multidrug-Resistant Organisms in Healthcare, 441 Mannitol, 239 Mannose-binding lectin-2 (MBL2), 83 Mask. See Standard precautions Maternal and Child Health Bureau, 452 Matrix metalloproteinase (MMP), 62 Maximal expiratory pressure (MEP), 171 Maximal inspiratory pressure (MIP), 171 MBL2. See Mannose-binding lectin-2 (MBL2) MBW. See Multiple-breath washout (MBW) MCID. See Minimal clinically important difference (MCID) MDR. See Multiple drug resistance protein (MDR) mdx mouse model, 409–410 Meconium ileus (MI), 78, 190, 266, 270 infants with, 270 neonate and, 191 young children with CF and, 191 Medical center health delivery system. See Chronic care model Medical equipment reprocessing of, 442 for single patient, 442 nebulizers and, 442, 444 spacers and, 442 spirometry mouthpieces and, 442 Membrane permeability, 285 Menses, 458, 461 MEP. See Maximal expiratory pressure (MEP) MESA. See Microsurgical epididymal sperm aspiration (MESA) Metabolic alkalosis, cystic fibrosis and, 358 chloride loss in, 358 treatment of, 358 Metabolic complications, cystic fibrosis (CF) and, 358

513 Methacholine, 169 Methadone, 484 Methicillin resistant Staphylococcus aureus (MRSA), 43–44, 258, 441 MI. See Intestinal obstructions (MI); Meconium ileus (MI) Microbiology, CF detection and identification of pathogens, 36–39 overview, 36 pathogens in, 40–48 Achromobacter xylosoxidans, 44 Burkholderia spp., 42–43 communities of, 46–48 culture independent techniques, 46–48 Haemophilus influenzae, 41 molds, 45–46 MRSA, 43–44 nontuberculous mycobacteria, 44–45 Pseudomonas aeruginosa, 41–42 Staphylococcus aureus, 40–41 Stenotrophomonas maltophilia, 44 yeasts, 45–46 susceptibility testing, 39 synergy testing, 39–40 Microsurgical epididymal sperm aspiration (MESA), 463 Microvascular complications in CFRD, 350 Miglustat, 413 Minimal clinically important difference (MCID), 472 MIP. See Maximal inspiratory pressure (MIP) MMP. See Matrix metalloproteinase (MMP) Model for improvement, 429–430 Modifier genes assessing the validity of, 83 of CF pulmonary disease, 83–84 identifying disease, 81–82 modify CF, 80–81 of non-airway phenotypes, 85–86 refinement of airway, 85 variation of clinical characteristics, 78–79 sources of, 79 Monofrequency signals, 151 Morphine, 484

514 Mortality in CFRD, 349 Mosaic attenuation, 185–186 MOT. See Multiple occlusion technique (MOT) Mouth pressure (Pmo), 154 MRI. See Magnetic resonance imaging (MRI) MRSA. See Methicillin resistant Staphylococcus aureus (MRSA) MUC5AC, 25 MUC5B, 25 Mucins, 24 Mucociliary clearance, 28–29 airway surface liquid hydration therapy for, 237–239 hypertonic saline, 238–239 overview, 24 pulmonary health in CF and, maintenance of, 231 Mucolytic therapy dornase alfa, 237 goals of, 236 NAC, 236 Mucus layer, 24–26 overview, 24 Mucus plugging, 125 Multidisciplinary care team and, frequent monitoring by, 227 Multidrug-resistant organisms, precautions for patients with, 447 Multifrequency signals, 151 Multipatient families. See Non-health care settings Multiple-breath washout (MBW), 125, 129, 130–131, 133, 155 Multiple drug resistance protein (MDR), 1 Multiple occlusion technique (MOT), 128 Mutant CFTR potentiation of, at cell membrane, 415–417 Mutation classes, in CF, 407, 408 Mycobacteria, 209 Mycobacteria abscessus, 37

NAbs. See Neutralizing antibodies (NAbs) NAC. See N-acetylcysteine (NAC) N-acetylcysteine (NAC), 236

Index Naloxone, 484 Nasal intermittent positive pressure ventilation (NIPPV), 205 Nasal potential difference (NPD), 94, 109, 461 Nasal resistance, infants, 124 National Initiative for Children’s Health Care Quality (NICHQ), 426 National Institutes of Health, 464 NBD. See Nucleotide-binding domains (NBD) NBD-1. See Nucleotide-binding domain (NBD)-1 NBS babies. See New born screening (NBS) babies Nebulizers. See Medical equipment Neonate MI and, 191 Neurologic complications in, cystic fibrosis (CF), 358–361 pancreatic enzyme therapy and, 360 vitamin A deficiency and, 359 vitamin K deficiency and, 359 Neutralizing antibodies (NAbs), 392 Newborn screening, 97–99, 317 New born screening (NBS) babies, 198, 200 NGT. See Normal glucose tolerance (NGT) NICHQ. See National Initiative for Children’s Health Care Quality (NICHQ) NIPPV. See Nasal intermittent positive pressure ventilation (NIPPV) Nitrogen, washout, 168 NIV. See Non-invasive bilevel ventilation (NIV); Non-invasive ventilation (NIV) NMD. See Nonsense-mediated decay (NMD) NO in exhaled breath (eNO), 66 Non–health care settings, infection control in camps, 448 multipatient families, 447 patient gatherings, 448–449 respiratory therapy equipment in home, care of, 447–448 schools, 448

Index

515

Non-insulin-dependent diabetes mellitus. See Type 2 diabetes mellitus (T2DM) Noninvasive bilevel ventilation (NIV), noctural, 365 Noninvasive positive pressure ventilation, 259 Noninvasive ventilation (NIV), 215 for chronic respiratory failure, 375–376 Nonsense-mediated decay (NMD), 409 Nonsteroidal anti-inflammatory drugs (NSAID), 70, 296 Nontuberculous mycobacteria (NTM), 37, 44–45, 203 Nonviral vectors for CF gene therapy, 397 Normal glucose tolerance (NGT), 343 North American CF (NACF) conference, 424 North American Epidemiologic Study of Cystic Fibrosis, 227 NPD. See Nasal potential difference (NPD) Nrf2. See Nuclear factor E2–related factor 2 (Nrf2) NSAID. See Nonsteroidal antiinflammatory drugs (NSAID) NTM. See Nontuberculous mycobacteria (NTM) Nuclear factor E2–related factor 2 (Nrf2), 64 Nuclear medicine MRI and, 189–190 Nucleotide-binding domain (NBD), 1, 411, 413 Nutritional care in cyctic fibrosis, 315, 317 Nutritional management in cystic fibrosis, 315, 318 Nutritional supplements in cystic fibrosis, 312, 313, 317, 320, 321 Nutrition recommendations for cystic fibrosis, 308, 314, 315 energy intake, 314, 317 fat intake, 314 Nutrition screening in cystic fibrosis, 315

[Occlusion techniques, for PFT] Herring–Breuer reflex, 128 MOT, 128 SOT, 128 OGTT. See Oral glucose tolerance tests (OGTT) 1,25(OH)2D. See 1,25-dihydroxyvitamin D [1,25(OH)2D] 25-OHD. See 25-hydroxy vitamin D (25-OHD) Opiates, 214 Opioids fentanyl, 484 methadone, 484 morphine, 484 naloxone, 484 side effects of, 484 Opsonophagocytosis, 69 Oral glucose tolerance tests (OGTT), 342, 346–347 Oral hypoglycemics in CFRD, 351 Oropharyngeal swabs, 256 Oscillating positive expiratory pressure (OscPEP), 243–244, 245 OscPEP. See Oscillating positive expiratory pressure (OscPEP) Osteoblasts, 331, 332, 333 Osteoclasts, 332, 335 Osteoid, 328 Osteopenia, 328, 329, 332 t score and, 328 Osteoporosis, 328, 329, 332, 336 risk factors for, 378 t score and, 328 Outcome measure. See Quality measures Oxygen therapy for cor pulmonale, 361 for pulmonary hypertension, 361 Oxyhemoglobin desaturation elevated respiratory rate and, 365 respiratory symptoms and, 364–365 Oxyhemoglobin saturation and sleep quality, 364, 365

Obstipation, assessment and management of, 489–490 Occlusion techniques, for PFT

PA. See Pseudomonas aeruginosa (PA) PACE. See Program for Adult Care Excellence (PACE)

516 PA chest radiograph. See Posterior-anterior (PA) chest radiograph Pain, CF related, 483–486 Palliative care defined, 482 specialists, 493 Palv. See Alveolar pressure (Palv) Pamidronate and bone diseases, 335–336 Pancreas ion transport, 16 Pancreatic elastase, 268–269 Pancreatic enzyme replacement therapy (PERT), 267, 268–269, 297, 315, 317, 319, 322 FC and, 277 Pancreatic enzymes, 314–315 in digestion, 314 Pancreatic insufficiency (PI), 79, 175, 266, 267–269, 289, 360 Pancreatic sufficient (PS), 266, 267–269 Pancreatic transplantation, 269 Pancreatitis, 269–270 Pandoraea apista, 44 Panton–Valentine leukocidin (PVL), 44 Parathyroid hormone (PTH) on bone, 332, 335–336 Parenting, with CF, 464 Parents depression in, 473 Pathogens, surveillance for, 442 Pathogens transmission by coughing, 441 HCW’s hands and, 441–442 interruption of contact, 441 mechanisms of, 441, 442 by nebulizers, 442 segregation to limit, 441 by sneezing, 441 by spirometry mouthpieces, 442 by talking, 441 Patient and family self-management. See Chronic care model Patient experience measure. See Quality measures Patient gatherings. See Non-health care settings Patient registries, role of, 424–425

Index Patient-reported outcomes (PRO), 252, 416, 468 CFQ-R, 472 defined, 471 FDA and, 471 HRQOL measures and, 471 Patients activity outside the room of, 443–444 infection prevention and control guidelines for, 441–442 room placement, 443 segregation, 441. See also Infection control 4-PBA. See 4-phenylbutyrate (4-PBA) PCD. See Primary ciliary dyskinesia (PCD) PCL. See Periciliary layer (PCL) PCP. See Pneumocystis pneumonia (PCP); Primary care provider (PCP) PDE 5 inhibitor. See Phosphodiesterase (PDE) 5 inhibitor Pdif. See Slower pressure change (Pdif) PD&P. See Postural drainage and percussion (PD&P) PEG. See Polyethylene glycol (PEG) PEGylation, 394 PEI. See Polyethyleneimine (PEI) Pel. See Plateau (elastic recoil pressure) PEP. See Positive expiratory pressure (PEP) Percent ideal body weight (%IBW), 315, 316 Percutaneous epididymal sperm aspiration (PESA), 463 Periciliary layer (PCL), 24, 26–28 Peroxisome proliferator-activated receptors (PPAR), 64, 318 PERT. See Pancreatic enzyme replacement therapy (PERT) PESA. See Percutaneous epididymal sperm aspiration (PESA) PFT. See Pulmonary function testing (PFT) PGP. See Proline-glycine-proline (PGP) PH. See Prolyl hydroxylase (PH) 4-Phenylbutyrate (4-PBA), 413 Phosphodiesterase (PDE) 5 inhibitor, 413 Phosphorylation of CFTR, 15 PI. See Pancreatic insufficiency (PI) Picosiemens (pS), 13

Index PIIIP. See Procollagen III polypeptide (PIIIP) Pilocarpine iontophoresis, 94 Pilogel, 95 Pinit. See Airway opening pressure (Pinit) Piperacillin, 212 PKA. See Protein kinase A (PKA) “plan/do/study/act (PDSA) cycles,” 429 Planktonic bacteria in pulmonary exacerbations, 252 Plateau (elastic recoil pressure), 154 Plethysmography, 125, 128, 167–168 Pmo. See Mouth pressure (Pmo) Pneumocystis jirovecii, 203 prophylaxis, 381 Pneumocystis pneumonia (PCP), 203 Pneumothorax, 213 adults, prevalence in, 204–205 children, prevalence in, 204 diagnosis of, 205 management of, 205 pathophysiology, 205 prognosis, 205 Pollutants and pulmonary exacerbation, 251, 255 Polyamidoamine for CF gene therapy, 398 Polyethylene glycol (PEG), 271, 394 Polyethyleneimine (PEI) for CF gene therapy, 398–399 polyK. See Poly-L-lysine (polyK) poly-L-lysine (polyK) for CF gene therapy, 398–399 Polypeptide chain, 12 Polyplexes, 397 Polysomnography, 363, 364 Polyunsaturated fatty acid (PUFA), 317, 318, 319, 321 Portal hypertension, 291, 293, 296, 297, 298, 299 Port CF, 425 Positive expiratory pressure (PEP), 243, 245 oscillating. See Oscillating positive expiratory pressure (OscPEP) Posterior-anterior (PA) chest radiograph, 182, 184 Postural drainage and percussion (PD&P), 241–242 AD versus, 243

517 PPAR. See Peroxisome proliferator– activated receptor (PPAR) Pregnancy, CF patients and, 462 Premature termination codons (PTCs) suppression of, 407, 409–411 CF clinical trials, 412 Prenatal GI imaging in CF, 190–191 Preschool children modified spirometry and, 148–151 Preschool PFT techniques, 157 Pressure-time tracing, 154 Preventing Transmission of Infectious Agents in Health Care Settings, 441 Primary care clinic, 455 Primary care provider (PCP), 454, 455 Primary ciliary dyskinesia (PCD), 28, 201, 203 Primary sclerosing cholangitis (PSC) CFALD and, 291 PRO. See Patient-reported outcomes (PRO) Process measures. See Quality measures Procollagen III polypeptide (PIIIP), 296 Productive-sounding cough, 201 Prognostication, in CF, 491 Program for Adult Care Excellence (PACE) grants, 451 Proline-glycine-proline (PGP), 62 Prolyl hydroxylase (PH), 296 Prostaglandins in CF patients, 461 menses and, 461 Protease-anti-protease imbalance, 68–69 Protein. See also Hormones CFTR, 457 Protein kinase A (PKA), 1 Protein kinase C (PKC), 1 PRSS1. See Cationic trypsinogen (PRSS1) PS. See Pancreatic sufficient (PS) pS. See Picosiemens (pS) Pseudomonas aeruginosa (PA), 31, 36, 41–42, 59, 90, 108, 200, 206, 208, 251, 252, 255, 256, 257, 378–379, 441 airway infection, 424 detection of, 256 prevention of, 229 PTC124, 410

518 PTCs. See Premature termination codons (PTCs) PTH. See Parathyroid hormone (PTH) Puberty, in CF patients, 458 PUFA. See Polyunsaturated fatty acid (PUFA) PUFA abnormalities in cystic fibrosis, 317–319 docasahexaenoic acid (DHA) deficiency, 318 linoleic acid (LA) deficiency, 318 Pulmonary clinical presentations. See Pulmonary presentations Pulmonary exacerbations age factors in, 254–255 definition of, 252–254 diagnosis of antimicrobial susceptibility testing in, 256–257 biomarkers in, 257 chest tomography (CT), 257 pulmonary function in, 256 epidemiology of, 254–255 infection control of, 260 management of, 257–260 mortality in, 254 outcome in, 252, 255, 257–259 pathophysiology of, 251–252 allergens and, 251–252 irritants and, 251–252 pollutants and, 251–252, 255 viral respiratory infections and, 251–252, 255 prevention of, 260 inhaled corticosteroids in, 260 risk factors of, 254, 255 symptoms of. See Symptoms of pulmonary exacerbations treatment of, 257–260 aminoglycoside, 257, 258 anti-inflammatory agents in, 259 b-lactam in, 252, 257 clinical trials in, 252, 256, 258, 259, 260 cost effectiveness in, 259 efficacy of, 152, 257, 258, 259 hospital care versus home medical care in, 259 intravenous antibiotics in, 254, 257– 260 tobramycin in, 257, 258, 260

Index Pulmonary function, 256, 258, 259. See also Forced vital capacity (FVC) CFRD and, 350 Pulmonary function testing equipment. See Environmental infection control Pulmonary Function testing (PFT), 123, 148, 161 application of, 172–173 future directions, 142–143 preschool, 157 research studies employing infant interventional, 141–142 observational, 131–133, 141 pulmonary disease, measures of, 141 summary of, 134–140 role of, in clinical management of infants, 142 standards for performing, 173 techniques for infant, 124 devices, preparation of, 125 forced expiratory flow, 128–129 gas dilution, 129, 130–131 occlusion techniques. See Occlusion techniques, for PFT plethysmography, 125, 128 RVRTC, 128–129, 130 summary of, 126–127 Pulmonary hypertension, oxygen therapy for, 361 Pulmonary manifestations of, in Infants, 124–125 Pulmonary presentations adulthood, 203 antenatal, 201 childhood and adolescence, 202 differential diagnosis of adulthood, 204 childhood and adolescence, 204 infants, 203 infants, 201–202 overview, 200–201 Purinergic (P2) receptors, 286, 287 PVL. See Panton–Valentine leukocidin (PVL) P2Y receptors, 71

QI. See Quality improvement (QI) Quality improvement (QI), 423

Index [Quality improvement (QI)] basic tenets of, 427–432 chronic care model and, 428–429 model for improvement and, 429–430 essentials of, teaching, 426–427 impact of, 434–435 Quality measures access, 431 outcome, 431 patient experience, 431 process, 431 structure, 430 Quality of life, 251 Quorum sensing, 251

rAAV. See Recombinant adeno-associated virus (rAAV) rAAV2-CFTR, 395 rAd. See Recombinant adenovirus (rAd) Radiation-induced cancer risk of, CT and, 189 Radioallergosorbent test (RAST), 208 RAEs. See Retinol activity equivalents (RAEs) Raised volume rapid thoracoabdominal compression (RVRTC) technique, 128–129, 130, 132, 198 Randomized controlled trial (RCT), 205 RANTES. See Regulated on activation normal T cell expressed and secreted (RANTES) Rapid eye movement (REM), 362–363, 364 RAST. See Radioallergosorbent test (RAST) Rat, CFTR in, 458 RAW. See Airway obstruction RC Cornet1, 244 RCT. See Randomized controlled trial (RCT) R-domain of CFTR, 15 Receptor binding, 286, 287 Recombinant adeno-associated virus (rAAV), 390 AAV2, 394 vectors, for CF gene therapy, 394–397

519 Recombinant adenovirus (rAd) vectors, for CF gene therapy, 392–394 Recombinant human deoxyribonuclease I (rhDNase), 33 Recombinant human deoxyribonuclease (rhDNase), 188 Rectal prolapse, 276 REE. See Resting energy expenditure (REE) Regulated on activation normal T cell expressed and secreted (RANTES), 60 REM. See Rapid eye movement (REM) Rep proteins, 394, 396, 397 Reproductive pathophysiology, in CF patients puberty, 458 structural abnormalities, 457–458 Reproductive technologies, 463 Reproductive tract ion transport, 17 REs. See Retinyl esters (REs) Residual volume (RV), 129, 161 Resistance (Rrs), 128 frequency dependence, 152 Respiratory depression, 484 Respiratory failure, in CF lung transplantation for. See Lung transplantation noninvasive ventilation (NIV) for, 375–376 pathophysiology, 372 increased respiratory load, effects of, 373–374 respiratory pump function, alterations in, 374–375 Respiratory inductance plethysmography (RIP), 156 Respiratory muscle strength measurement of MIP/MEP, 171 TTI, 171–172 Respiratory pump functional alterations in, 374–375 Respiratory syncytial virus (RSV), 201 Respiratory system exercise and, 170–171

520 Respiratory therapy, 444–445 equipment in home, care of. See Non-health care settings Respiratory tract irritants and pulmonary exacerbation, 251 specimens, 38 Resting energy expenditure (REE), 458 Resuscitation, 207 Retinoids, 320 Retinol activity equivalents (RAEs), 320 Retinyl esters (REs), 320 rhDNase. See Recombinant human deoxyribonuclease I (rhDNase); Recombinant human deoxyribonuclease (rhDNase) RIP. See Respiratory inductance plethysmography (RIP) Risk factors for pulmonary exacerbations, 254, 255 Room placement for infection control, 443 Rrs. See Resistance (Rrs) RSV. See Respiratory syncytial virus (RSV) RV. See Residual volume (RV) RVRTC technique. See Raised volume rapid thoracoabdominal compression (RVRTC) technique

Salt hyper-absorption hypotheses, 19 SBBO. See Small bowel bacterial overgrowth (SBBO) SCC. See Staphylococcal chromosomal cassette (SCC) Scedosporium apiospermum, 208 Schools. See also Non-health care settings activities and homework, 469 collaboration between parents and, 469, 473 peer relationships in, 469 SCID. See Severe combined immune deficiency (SCID) SCV. See Small colony variants (SCV) SecR. See Serpin-enzyme complex receptor (SecR) Secretin, 286, 287, 299 in bile formation, 286, 287

Index Secretory leukocyte protease inhibitor (SLPI), 69 Sedation, 124 Semen analysis, 463 Serine protease inhibitor Kazal 1 (SPINK1), 270 Serpin-enzyme complex receptor (SecR), 399 Serum liver enzymes, 295 alanine aminotransferase, 295 alkaline phosphatase, 295 aspartate aminotransferase, 295 bilirubin, 295 in diagnosis of CFALD, 295 Serum trypsinogen, 117 Severe, Persistent, Unusual, Recurrent infections (SPUR), 200 Severe combined immune deficiency (SCID), 397 Sexual and reproductive health issues in females with CF, 462 in males with CF, 463 Sexually transmitted infections (STI), 459 Sexual maturation and bone diseases, 332 sGaw. See Specific airway conductance (sGaw) Shwachman–Diamond syndrome, 117 Shwachman’s syndrome, 203 Signal transducers and activators of transcription (STAT), 64 Sildenafil, 413 Single-breath occlusion technique (SOT), 128 Single-nucleotide polymorphisms (SNP), 79 Single-photon emission computed tomography (SPECT), 189 Sinuses CT imaging of, 190 Sirolimus, 382 Six-minute walk test, 170 Skin care. See Hand hygiene Sleep complications, cystic fibrosis (CF) and, 362–365 in children, 363–364 hypercapnia and, 364–365 hypoxemia and, 362, 364, 365 obstructive sleep apnea and, 364

Index Slight recurrent chest infections, 359 Slower pressure change (Pdif), 154 SLPI. See Secretory leukocyte protease inhibitor (SLPI) Small bowel bacterial overgrowth (SBBO), 266, 274 Small colony variants (SCV), 40 Small-molecule therapy, 406 DF508 CFTR, restoring function to, 411, 413–415 mutation classes in CF, 407, 408 potentiation of mutant CFTR at cell membrane, 415–417 suppression of PTCs, 407, 409–411 SMIP. See Sustained maximal inspiratory pressure (SMIP) SNP. See Single-nucleotide polymorphisms (SNP) Soap and water. See Hand hygiene Social and family functioning, in CF, 469–470 Socioeconomic factors and pulmonary exacerbations, 255 Sodium channel blockers, 33 SOT. See Single-breath occlusion technique (SOT) SP. See Surfactant protein (SP) Spacers. See Medical equipment Specific airway conductance (sGaw), 131 Specific airways resistance (sRaw), 128, 155–156 SPECT. See Single-photon emission computed tomography (SPECT) SPINK1. See Serine protease inhibitor Kazal 1 (SPINK1) Spirometry, 148–151, 161–163, 204, 256, 257, 258, 259 forced oscillometry, 151–154 hemoptysis and, 207 modified, 148–151 mouthpieces. See Medical equipment quality control criteria used for, 149 SPUR. See Severe, Persistent, Unusual, Recurrent infections (SPUR)

521 sRaw. See Specific airways resistance (sRaw) Standard precautions eye shields, 441, 444 gloves, 441, 444 gowns, 441, 444 mask, 441, 443, 444 Staphylococcal chromosomal cassette (SCC), 43–44 Staphylococcus aureus, 36, 40–41, 90, 108, 200, 207, 229 STAT. See Signal transducers and activators of transcription (STAT) Steatorrhea, 314 Stenotrophomonas maltophilia, 44, 174, 208 Sterilization and disinfection measures. See Environmental infection control STI. See Sexually transmitted infections (STI) Stool fats, 314 Streptococcus milleri, 46 Stress, 468. See also Depression Structural abnormalities in reproductive tracts of CF patients, 457–458 Structure measures. See Quality measures Surfactant protein (SP), 201 Surrogate endpoints, 187–188 Susceptibility testing, role of in CF, 39 Sustained maximal inspiratory pressure (SMIP), 172 Sweat chloride test, 94, 110 variability of, 112 Sweat gland ion transport, 15–16 Sweat testing, 94–97 in young infants, 95 Symptom-based therapies, 406–407 Symptoms of pulmonary exacerbations cough, 252–254 exercise intolerance, 252–254 hemoptysis, 252–254 reduced appetite, 252–254 sputum production, 252–254 weight loss, 252–254 Synergy testing, role of, 39–40

522 TAA. See Thoracoabdominal asynchrony (TAA) Tacrolimus, 382 Target cells for CF gene therapy, 390–391 Tauroursodeoxycholic acid (TUDCA), 299 T cells, 62 T1DM. See Type 1 diabetes mellitus (T1DM) T2DM. See Type 2 diabetes mellitus (T2DM) TEE. See Thoracic expansion exercises (TEE) Tension-time index of the respiratory muscles (TTmus), 374 Tension time index (TTI), 171–172 Terminal restriction length polymorphism (T-RFLP), 46 TESA. See Testicular epididymal sperm aspiration (TESA) Testicular epididymal sperm aspiration (TESA), 463 TGFb1. See Transforming growth factor b1 (TGFb1) TGV. See Thoracic gas volume (TGV) TH-17 cells, 62 “The 3-foot rule,” 441 Thoracic expansion exercises (TEE), 242 Thoracic gas volume (TGV), 125 Thoracoabdominal asynchrony (TAA), 156 Tidal breathing, 156–157 Time constant of the respiratory system (Trs), 128 Time to peak expiratory flow over total expiratory time (Tpef/Te), 156 TIPS. See Transjugular portosystemic shunts (TIPS) TIS. See Tobramycin inhalation solution (TIS) TLC. See Total lung capacity (TLC) TLRs. See Toll-like receptors (TLRs) Tobramycin, 39 Tobramycin inhalation solution (TIS), 230 To Err is Human, 423 Toll-like receptors (TLRs), 392 Total lung capacity (TLC), 124, 161

Index Tpef/Te. See Time to peak expiratory flow over total expiratory time (Tpef/Te) Transforming growth factor b1 (TGFb1), 83 Transition challenges of, 452–454 clinic, 455 concerns, 453–454 development of, 454–455 House of Commons Select Committee on Health, 452 Maternal and Child Health Bureau, 452 models of, 455 North American CF Conference and, 451 periods, for patients and families, 468 periods for patients and families with, 468 timing of, 451–452 Transjugular portosystemic shunts (TIPS), 298 Transmission-based precautions, 441 Transmission of cystic fibrosis, 260 nosocomial transmission, 260 Transplant surgery lung transplantation and, 379 Treatment adherence barriers to, 474 in children, 470 daily diaries, 471 DPD, 471 interviews, 471 questionnaires, 471 T-RFLP. See Terminal restriction length polymorphism (T-RFLP) Trs. See Time constant of the respiratory system (Trs) t scores, 328, 329 TTI. See Tension time index (TTI) TTmus. See Tension-time index of the respiratory muscles (TTmus) TUDCA. See Tauroursodeoxycholic acid (TUDCA) Type 1 diabetes mellitus (T1DM), 341–342 b-cell destruction and, 341, 342 Type 2 diabetes mellitus (T2DM), 341, 342 insulin resistance and, 341, 342

Index UDCA. See Ursodeoxycholic acid (UDCA) Undernutrition and cystic fibrosis, 317 United States Cystic Fibrosis Foundation (USCFF), 106–107, 203, 206 Urinary incontinence coughing, related to, 483, 486 Ursodeoxycholic acid (UDCA), 289 USCFF. See United States Cystic Fibrosis Foundation (USCFF)

Variation, modifier genes sources of, 79 VBE. See Back-extrapolated volume (VBE) Vibrio cholera, 3 Vinegar (acetic acid), nebulizer and, 447 Viral colds, 201 Vitamin A supplementation, 321 Vitamin D and bone diseases, 330–331 Vitamin D supplements, 319–320 cholecalciferol (D3), 319–320 ergocalciferol (D2), 319–320 Vitamin D therapy, 333–335 Vitamin E deficiency in cystic fibrosis, 321 Vitamin K supplementation, 335 VX-770, 416

523 Waiting room practices, 445–446 Wasting, defined, 378 WBP. See Whole-body plethysmograph (WBP) Weight monitoring in cystic fibrosis, 316 Wheeze, 201–202 Whole-body plethysmograph (WBP), 155–156 Wilson disease, 291 CFALD and, 291 Wisconsin Cystic Fibrosis Neonatal Screening Project, 359 Wisconsin system, 182–183 wtCFTR, 414 W1282X CFTR, 409, 410

Xc. See Capacitant element (Xc) Xi. See Inertive element (Xi)

Zinc deficiency in cystic fibrosis, 322. See also Acrodermatitis enteropathica

Cystic Fibrosis About the book The median age of survival for those with cystic fibrosis has risen considerably in recent years. This text thoroughly examines the developments and breakthroughs which have led to this improvement in life expectancy. With a focus on the latest discoveries in the diagnosis and treatment of the disease, this book provides a comprehensive overview of the past, current and forthcoming advancements in cystic fibrosis research and clinical care. Key features of Cystic Fibrosis include: • Opening chapters that discuss the molecular basis of the disease from gene abnormalities to modifiers of disease • An examination of standard and novel diagnostic methods and techniques, such as sweat testing, gene studies, lung function testing across the age spectrum, and imaging studies • A focus on pulmonary manifestations and recently introduced therapies • A discussion of clinical treatment of the many extra-pulmonary manifestations of cystic fibrosis, such as nutrition, bone health, and cystic fibrosis related diabetes • A look at emerging developments in treatment like gene repair and pharmacologic therapies • Concluding chapters which address the significance of healthcare systems and psychological factors About the editors JULIAN L. ALLEN is chief of the Division of Pulmonary Medicine and Cystic Fibrosis Center at The Children’s Hospital of Philadelphia. He received his M.D. from the Columbia University College of Physicians and Surgeons. Dr. Allen is a Professor of Pediatrics at the University of Pennsylvania School of Medicine, and is the recipient of the Robert Gerard Morse Endowed Chair in Pulmonary Medicine. Dr. Allen’s research interests include developmental pulmonary physiology, and the pulmonary complications of sickle cell disease and cystic fibrosis. HOWARD B. PANITCH is the Clinical Director of the Division of Pulmonary Medicine at The Children’s Hospital of Philadelphia. He received his M.D. from the University of Pittsburgh School of Medicine. Dr. Panitch is also the director of the Pulmonary Medicine Fellowship Program and the medical director for the Technology Dependence Center. A Professor of Pediatrics at the University of Pennsylvania School of Medicine, Dr. Panitch received the Dean’s Award for excellence in Clinical Teaching in 2002 and a Master Clinician Award from the Department of Pediatrics in 2008. Among Dr. Panitch’s many research interests are the implications and barriers of transitioning technology dependent children to adult care. RONALD C. RUBENSTEIN is the Director of the Cystic Fibrosis Center at The Children’s Hospital of Philadelphia and the University of Pennsylvania. Dr. Rubenstein, who received his M.D. and Ph.D.in Pharmacology from the University of Texas Southwestern Medical School and Graduate School of the Biomedical Sciences, is an Associate Professor of Pediatrics at the University of Pennsylvania School of Medicine. Dr. Rubenstein’s laboratory and translational research program focuses on pharmacologic repair of mutant CFTR function in cystic fibrosis. Dr. Rubenstein serves as Chair of the Clinical Research Committee of the Cystic Fibrosis Foundation. He was named Faculty Teacher of the Year at The Children’s Hospital of Philadelphia in 2006.

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