Lyme Disease: An Evidence-Based Approach (Advances in Molecular and Cellular Biology Series)

Advances in Molecular and Cellular Microbiology 20

Lyme Disease An Evidence-based Approach

Edited by

John J. Halperin, MD Atlantic Neurosciences Institute, New Jersey and Mount Sinai School of Medicine, New York

Advances in Molecular and Cellular Microbiology

Through the application of molecular and cellular microbiology, we now recognise the diversity and dominance of microbial life forms on our planet that exist in all environments. These microbes have many important planetary roles, but for we humans, a major problem is their ability to colonize our tissues and cause disease. The same techniques of molecular and cellular microbiology have been applied to the problems of human and animal infection during the past two decades and have proved to be immensely powerful tools in elucidating how microorganisms cause human pathology. This series has the aim of providing information on the advances that have been made in the application of molecular and cellular microbiology to specific organisms and the diseases that they cause. The series is edited by researchers active in the application of molecular and cellular microbiology to human disease states. Each volume focuses on a particular aspect of infectious disease and will enable graduate students and researchers to keep up with the rapidly diversifying literature in current microbiological research.

Series Editors Professor Brian Henderson University College London Professor Michael Wilson University College London

Titles Available from CABI 17. Helicobacter pylori in the 21st Century Edited by Philip Sutton and Hazel M. Mitchell 18. Antimicrobial Peptides: Discovery, Design and Novel Therapeutic Strategies Edited by Guangshun Wang 19. Stress Response in Pathogenic Bacteria Edited by Stephen P. Kidd 20. Lyme Disease: an Evidence-based Approach Edited by John J. Halperin Titles Forthcoming from CABI Tuberculosis: Diagnosis and Treatment Edited by Timothy McHugh Microbial Metabolomics Edited by Silas Villas-Bôas and Katya Ruggiero Antimicrobial Drug Discovery: Emerging Strategies Edited by George Tegos and Eleftherios Mylonakis

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© CAB International 2011. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Lyme disease : an evidence based approach / edited by John J. Halperin. p. ; cm. -- (Advances in molecular and cellular microbiology, 20) Includes bibliographical references and index. ISBN 978-1-84593-804-8 (alk. paper) 1. Lyme disease. I. Halperin, John J. II. Series: Advances in molecular and cellular microbiology, 20. [DNLM: 1. Lyme Disease. WC 406] RC155.5.L943 2011 616.9’246--dc22 2011002109 ISBN-13: 978 1 84593 804 8

Commissioning editor: Rachel Cutts Editorial assistant: Alexandra Lainsbury Production editors: Tracy Head and Simon Hill Typeset by Columns Design XML Ltd, Reading, UK. Printed and bound by CPI Group (UK) Ltd, Croydon, CR0 4YY.

Contents

Contributors Introduction John J. Halperin

vii ix

Part I: Biological Substrate 1

Ticks: the Vectors of Lyme Disease Robert P. Smith

1

2

Borrelia: Biology of the Organism Alvaro Toledo and Jorge L. Benach

29

3

Borrelia: Interactions with the Host Immune System Raymond J. Dattwyler and Kirk Sperber

54

4

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection Barbara J.B. Johnson

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5

Persistence of Borrelia burgdorferi Infection after Antibiotic Treatment: What Can We Learn from Animal Models? Joppe W.R. Hovius and Gary P. Wormser

6

Global Epidemiology of Borrelia burgdorferi Infections Paul S. Mead

89 100

Part II: Clinical aspects 7

Antibiotic Therapy for Infection Caused by Borrelia burgdorferi Sensu Lato Gary P. Wormser

115

8

Lyme Borreliosis in the UK and Ireland Susan O’Connell

127

9

Lyme Borreliosis: the European Perspective Gerold Stanek and Franc Strle

140

v

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Contents

10

Erythema Migrans Robert B. Nadelman

154

11

Cardiac Involvement Joseph M. Harburger and Jonathan L. Halperin

179

12

Rheumatological Involvement Leonard H. Sigal

190

13

Nervous System Involvement John J. Halperin

208

14

Lyme Disease in Children Eugene D. Shapiro

221

15

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’ Afton L. Hassett and Leonard H. Sigal

232

16

Chronic Lyme Disease Adriana Marques

248

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Lyme Disease: the Great Controversy John J. Halperin, Phillip J. Baker and Gary P. Wormser

259

Index

271

Contributors

Phillip J. Baker, PhD, Executive Director, American Lyme Disease Foundation, PO Box 466, Lyme, CT 06371, USA. Jorge L. Benach, PhD, Distinguished University Professor, Chairman, Department of Molecular Genetics and Microbiology, Director, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York 11794-5120, USA. Raymond J. Dattwyler, MD, Professor of Medicine and Microbiology/Immunology, Chief Division of Allergy/Immunology and Rheumatology, New York Medical College, Valhalla, NY 10595, USA. John J. Halperin, MD, Department of Neurosciences, Overlook Hospital, Atlantic Neurosciences Institute, Summit, NJ 07902, USA and Professor of Neurology & Medicine, Mount Sinai School of Medicine, New York, NY, USA. Jonathan L. Halperin, MD, Robert and Harriet Heilbrunn Professor of Medicine (Cardiology), Mount Sinai School of Medicine and Director, Cardiology Clinical Services, The Zena and Michael A. Wiener Cardiovascular Institute, The Marie-Josée and Henry R. Kravis Center for Cardiovascular Health, Mount Sinai Medical Center, Fifth Avenue at 100th Street, New York, NY 10029-6574, USA. Joseph M. Harburger, MD, Department of Medicine, Division of Cardiology, Westchester Medical Center and New York Medical College, Valhalla, NY 10595, USA. Afton L. Hassett, PsyD, Associate Research Scientist, Department of Anesthesiology, Chronic Pain & Fatigue Research Center, University of Michigan Medical School, 24 Frank Lloyd Wright Drive, Ann Arbor, MI 48106, USA. Joppe W.R. Hovius, MD, PhD, Center of Experimental and Molecular Medicine (CEMM), Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands. Barbara J.B. Johnson, PhD, Bacterial Diseases Branch, Division of Vector-borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), 3156 Rampart Road, Ft Collins, CO 80521, USA. Adriana Marques, MD, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 10/11N234 10 Center Drive, Bethesda, MD 20892, USA. Paul S. Mead, MD, MPH, Bacterial Diseases Branch, Division of Vector-borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), 3150 Rampart Road, Ft Collins, CO 80521, USA. vii

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Contributors

Robert B. Nadelman, MD, Professor of Medicine, Department of Medicine, Division of Infectious Diseases, New York Medical College, Munger Pavilion 245, Valhalla, NY 10595, USA. Susan O’Connell, Consultant Medical Microbiologist, Head, Lyme Borreliosis Unit, Health Protection Agency Microbiology Laboratory, Southampton University Hospitals NHS Trust, Southampton SO16 6YD, UK. Eugene D. Shapiro, MD, Professor of Pediatrics, Epidemiology, and Investigative Medicine, Department of Pediatrics, Yale University School of Medicine, 333 Cedar Street, PO Box 208064, New Haven, CT 06520-8064, USA. Leonard H. Sigal, MD, FACP, FACR, Clinical Professor, Departments of Medicine and Pediatrics, Adjunct Professor, Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ, USA. Robert P. Smith, MD, MPH, Director, Infectious Disease Fellowship Program, Maine Medical Center, Portland, ME, USA and Co-Director, Vector-Borne Disease Laboratory, Maine Medical Center Research Institute, Portland, ME, USA and Clinical Professor of Medicine, Tufts University School of Medicine, Boston, MA, USA. Kirk Sperber, MD, Associate Professor of Medicine, Division of Allergy/Immunology and Rheumatology, New York Medical College, Valhalla, NY 10595, USA. Gerold Stanek, MD, Medical University of Vienna, Institute of Hygiene and Applied Immunology, Kinderspitalgasse 15, 1095, Vienna, Austria. Franc Strle, MD, PhD, Department of Infectious Diseases, University Medical Center Ljubljana, Japljeva 2, 1525, Ljubljana, Slovenia. Alvaro Toledo, PhD, Research Scientist, Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, 5120 Centers for Molecular Medicine, Stony Brook, New York 11790-5120, USA. Gary P. Wormser, MD, New York Medical College, Division of Infectious Diseases, Munger Pavilion Room 245, Valhalla, NY 10595, USA.

Introduction

Spirochaetal infections somehow seem to take on larger-than-life roles. ‘The French disease’, a.k.a. syphilis, assumed almost mythic proportions. Initially brought back from the New World to the Old, perhaps as divine retribution for measles, smallpox and myriad other curses visited on North America’s aboriginal populations by European conquerors, neurosyphilis affected so many historic personalities as to give it a legitimate claim as a molder of history. Even early in the 20th century, a lack of understanding of the pathophysiology of many other diseases led to the attribution of all manner of disorders to this infection, often by default, because nobody could come up with a better explanation. Early in the history of Lyme disease, many latched on to the syphilis parallel, asserting that this new spirochaetosis, like its cousin, could masquerade as innumerable other ailments. However, the greatest similarity between the two has been the tendency to inappropriately attribute unrelated, but otherwise not readily explained, disorders to the unjustly accused spirochaete. The past decade or two has seen medicine move broadly and strongly towards the requirement for evidence-based support for its conclusions and actions. For those of us over a certain age, although this is certainly intellectually satisfying, it produces a distinct cognitive dissonance. When I was a resident, the truth was what the professors said and wrote in textbooks. The evidence basis at best consisted of case series and anecdotal observations. Unfortunately, even the most brilliant minds, individuals who led medicine for decades, were often proved wrong as new technologies – imaging, biochemical assays, DNA analysis or whatever – provided more powerful tools to answer questions. The Lyme disease ‘debate’ is this tension writ large. On the one hand, there is a group, consisting largely of family practitioners and other primary care providers, who see large numbers of patients suffering with medically unexplained symptoms. They struggle (quite legitimately) to understand the causes of these patients’ suffering, and draw on whatever technologies appear applicable – often accepting the test results uncritically. They then accept responsibility for treating these patients and actually try things! This earns them the sincere gratitude of patients who have been struggling both with chronic disabling symptoms and the inability of mainstream medicine to provide them with satisfying answers. Patients and treaters then provide each other with strong reinforcement. In contrast, other physicians, often with more advanced specialty-oriented training, have adopted the more rigorous, scientific approach. This group looks critically at all efforts to understand, diagnose and treat this disorder, and would rather say ‘I don’t know’ than make ix

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Introduction

assertions felt not to be based on sound science. They demand that all conclusions, diagnostic approaches and treatments meet current standards of evidence-based medicine. This culture clash fuels the debate – a debate that, if it were based just on scientific evidence, would have disappeared long ago due to the totally one-sided nature of the observations. However, the debate has not ended and many physicians in practice continue to see patients who are convinced that their symptoms are due to a chronic infection with Borrelia burgdorferi. Not only do these patients fear that they have a chronic, debilitating and difficult-to-treat infection, but they have been told that this infection will damage their nervous system and lead to inevitable irreparable brain damage. These frightened patients often come armed with reams of almost plausible-sounding material downloaded from the Internet, and are only too happy to debate the contents with their physicians. The goal of this book is to present the relevant evidence so that practising physicians can better understand the arguments being made and use the best information available to help their patients. The intent is to cover the areas most often identified as ‘controversial’ and to provide perspective, clarifying the debate. It is the hope of all the authors that this will help calm some of the anxiety (among both physicians and patients) about this disorder, and allow physicians to provide their patients with the most appropriate treatment. Before diving into the substance of the debate, it is essential that I acknowledge those who have made this work possible. First, I thank the patients who freely share not only their stories but also their insights and their fears, teaching me daily about the reality of their illnesses. Secondly, I am grateful to the numerous colleagues – many of whom graciously agreed to contribute to this volume – who have gone through the ‘Lyme wars’ with me over the years, educating me, debating with me, together contributing to the maturation of the knowledge base regarding this illness. Thirdly, I am forever grateful to the mentors of my formative years – the innumerable college and medical school faculty who taught me the importance of thinking critically and analytically about complex issues. At the apex of these were the three ultimate exemplars of what my son calls ‘eminence-based medicine’ – Raymond Adams, C. Miller Fisher and E.P. Richardson – three of the greats of 20th century neurology, with whom I had the privilege of working. Long before there was evidence-based medicine, these giants made it crystal clear that truth would be found not in the pronouncements of the giants but in a meticulous analysis of the data. I owe a very special thank you to the American Lyme Disease Foundation, which graciously provided funding to allow the publication of the colour illustrations in this volume. And finally, and most importantly, I thank my son, for being a never ending source of pride, joy, inquisitiveness and intellectual rigor, and my wonderful bride of three dozen years – who gamely puts up with my long hours, battle stories and innumerable imperfections, yet is always there as the supportive anchor of my universe, making it all possible. John J. Halperin

1

Ticks: the Vectors of Lyme Disease Robert P. Smith

1.1 Introduction Lyme disease, human granulocytic anaplasmosis and babesiosis are diseases transmitted in temperate zones around the northern hemisphere by Ixodes ticks. In Eurasia, the same ticks also transmit the agents of tick-borne encephalitis, while in North America they may carry a related flavivirus (Telford et al., 1997; Ebel et al., 2000). Most disease transmission to humans in North America and Eurasia is due to bites from four species of ticks (Ixodes scapularis, Ixodes pacificus, Ixodes ricinus and Ixodes persulcatus), which are grouped in the I. ricinus species complex (Kierans et al., 1999). These four species serve as bridge vectors of disease from ancient cycles in nature to humans. They are ‘generalist species’ that during their multi-year life cycles feed on diverse hosts, which may include species of mammals, birds or reptiles. Their importance as vectors of human disease varies with the disease agent, species of tick, vertebrate host community, geographical region and local ecological factors. As an example, I. scapularis (the black-legged tick, commonly known as the ‘deer tick’) is present throughout the eastern USA from Florida to Maine (Dennis et al., 1998; Diuk-Wasser et al., 2006). In northeastern US areas, it may account for the majority of human bites by ticks (Falco and

Fish, 1988; Rand et al., 2007). Nymphal and adult stages of the tick are often infected with Borrelia burgdorferi, the aetiological agent of Lyme disease. The infection may be carried by 10–30% of nymphs and 20–70% of adult ticks (Spielman et al., 1985; Tsao et al., 2004; Schulze et al., 2005a). The annual incidence of Lyme disease in some counties approaches 1% (CDC, 2008). In the southeastern USA, however, I. scapularis rarely parasitizes humans (Felz et al., 1996) and, as a result of differences in its preferred hosts, is infected with the agent of Lyme disease <2% of the time (Oliver et al., 2003). As a consequence, locally acquired Lyme disease in the southeastern USA is a rare event, despite the wide range of the potential vector (CDC, 2008). Lyme disease is transmitted by I. scapularis ticks in the eastern USA, and by I. pacificus (Western black-legged tick) in western North America. I. ricinus, the castor bean or sheep tick, transmits Lyme disease in Europe and far-western Asia, and I. persulcatus, the taiga tick, is its vector in eastern Europe and Asia (Fig. 1.1). In addition, other species of Ixodes tick maintain Lyme disease in ‘silent’ enzootic cycles but do not serve as a vector of the disease to humans. For example, in the western USA, Ixodes spinipalpis is a vector of enzootic Borrelia infection in woodrats in regions where Lyme disease is not endemic because of the absence of a

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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R.P. Smith

human biting (or ‘bridge’) vector (Maupin et al., 1994). The seabird tick, Ixodes uriae, which is not a member of the I. ricinus group, maintains enzootic Borrelia garinii infection in colonies of puffins, auks and kittiwakes in both the northern and southern hemispheres (Olsen et al., 1995). However, humans are rarely in contact with this tick, and although it occasionally bites seabird researchers, documented transmission of Lyme disease to humans has not been reported. Although these enzootic cycles between ticks and their hosts are ancient, the emergence of Lyme disease over the past 50 years in both North America and Europe highlights changes in the range and abundance of these ticks, the environment we share with them, and the activities that put us at risk. Despite great strides in our understanding of these factors, efforts to control these ticks and to prevent transmission of the diseases they carry have not yet had a major impact when viewed on a large regional scale. Interventions at the small scale of an individual lot or neighbourhood have had varying success. It is the purpose of this chapter to review the existing evidence for our current understanding of the evolution of these vectors, their means of dispersal and the reasons for range expansion, the

mechanisms they use to transmit disease, and the effectiveness of interventions to bring about their control.

1.2 Evolution and Historical Biogeography of Ixodes Ticks Ticks and insects last shared a common ancestor 490–550 million years ago (Douzery et al., 2004). Based on 16S rRNA gene phylogenies, hard ticks may have evolved during the radiation of bird taxa 50–100 million years ago (Black and Piesman, 1994). However, reconstruction of the evolutionary history of these invertebrates is based on a very limited fossil record (Klompen et al., 1996; de la Fuente, 2003). Phylogenetic analyses of existing I. ricinus complex ticks are delineating more recent evolutionary history. Using 16S ribosomal DNA of 11 species, Xu et al. (2003) provided evidence for four distinct clades within the I. ricinus group. One clade includes I. persulcatus, I. pacificus and four other tick species, the second includes I. ricinus, and I. scapularis is in a third. The fourth clade consists of two tick species not known to parasitize humans. Other ticks in these clades

I. persulcatus

I. pacificus

I. scapularis I. ricinus

Fig. 1.1. Geographical distribution of the primary vectors of Lyme borreliosis spirochaetes. (Designed by B. Kaye.)

Ticks: the Vectors of Lyme Disease

include several enzootic vectors that maintain ‘silent cycles’ of B. burgdorferi sensu lato (i.e. Ixodes minor in the southern USA), but do not parasitize humans (Xu et al., 2003). Not all important tick vectors of enzootic Borrelia in nature are in the I. ricinus complex. For example, Ixodes hexagonus, a major enzootic vector in Europe, is not closely related to ticks in the I. ricinus complex. That these vectors are not monophyletic is evidence that the ability to transmit and maintain borrelial spirochaetes has evolved multiple times. Population genetics of North American I. scapularis ticks indicate a bottleneck occurring at the time of the Pleistocene Ice Age around 12,000 years ago. According to Humphrey et al. (2010), ‘the mitochondria in both the midwestern and northeastern I. scapularis populations are derived from a single colonizing tick that originated in refugia to the south of the ice sheet.’ These studies, using 16S mitochondrial DNA, suggested that the midwestern population is younger than the northeastern population, and that both are more recent than southeastern populations, a conclusion also reached by others (Qiu et al., 2002; Rosenthal and Spielman, 2004). The three tick populations have distinct haplotype frequencies, with much more diversity present in the older southeastern group (Humphrey et al., 2010). Spielman et al. (1979) described the northern population of the black-legged or deer tick as a distinct species (i.e. Ixodes dammini) based on differences in nymphal tick morphology and questing behaviour (with associated differences in human parasitism). Subsequent genetic analyses differed on the separate species status of I. dammini, with differences in mitochondrial DNA (Caporale et al., 1995; Rich et al., 1995; Norris et al., 1996) but not nuclear ribosomal DNA (Wesson et al., 1993; Norris et al., 1996; McLain et al., 2001) evident between northern and southern populations. After assimilation of conflicting evidence, including assortative mating studies (Oliver et al., 1993b), I. dammini was relegated as a junior synonym of I. scapularis (Wesson et al., 1993; Norris et al., 1996; Rosenthal and Spielman, 2004). Whether a separate species or not, the vectors of most cases of Lyme disease in the

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eastern USA derive from a common postglacial founding population (Rosenthal and Spielman, 2004; Humphrey et al., 2010). European colonization of North America resulted in near extirpation of white-tailed deer by the end of the 19th century, creating another bottleneck for I. scapularis tick populations (McCabe and McCabe, 1997; Piesman, 2002). The current emergence of Lyme disease in the northeastern USA parallels the sharp increase in deer herd density and range (Spielman et al., 1993). Only a few remote or isolated refugia for deer remained in the northeastern USA by the early 20th century (Spielman et al., 1993; McCabe and McCabe, 1997; Piesman, 2002). One such site, Naushon Island, located off the coast of Massachusetts, maintained a deer herd in a private hunting reserve, and museum collections document I. scapularis ticks from areas near this location in the 1920s (Spielman et al., 1993). Although I. scapularis was first described in the USA in 1821 (Say, 1821), an authoritative review of Ixodes ticks (Cooley and Kohls et al., 1945) listed only 21 records of this tick, all in the southern USA, while citing a report of one isolated population in Cape Cod, MA (Piesman, 2002). Formalin-preserved 19th-century museum specimens of mice from this area reveal the presence of B. burgdorferi DNA (Marshall et al., 1994). The return of deer herds, sometimes to overabundance, presumably led to the recent expansion of the range of I. scapularis in the northern USA (Spielman et al., 1985, 1993; Piesman, 2002). In Europe, where the erythema migrans rash of Lyme disease was first described nearly a century ago, it is unclear whether similar population bottlenecks have occurred, as landscape features have been more stable, as has deer herd management (Zonneveld and Foreman, 1990). Nevertheless, deer herd densities are reported to have increased in many areas after World War II, parallel with increasing abundance of I. ricinus in some locations (Matuschka and Spielman, 1986; Rizzoli et al., 2009). However, documentation is limited, and, unlike I. scapularis, I. ricinus occurs in diverse ecological settings, sometimes in the absence of deer. This tick exists from coastal Western Europe and

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Scandinavia to central Asia (50–60° longitude) and south to the Atlas Mountains of North Africa (Gern and Humair, 2002). Habitats encompass moist coniferous forests and grasslands, as well as temperate deciduous forests (Gern and Humair, 2002). The ecological web supporting enzootic Lyme disease is also more complex in Eurasia, with three separate genospecies of Borrelia causing Lyme disease (Kurtenbach et al., 2002). Limited data exists on the phylogenetics of different populations of I. ricinus and I. persulcatus, and no evolutionary studies address post-glaciation events (McLain et al., 2001). It might be postulated that geographical evolutionary patterns in the tick vector would be reflected in the pathogen as well, as evidence suggests that B. burgdorferi, with the possible exception of one recently introduced subtype, has been present in North America for many thousands of years (Margos et al., 2008; Qiu et al., 2008; Gatewood Hoen et al., 2009). Reflecting the evolutionary history of its vector, Brisson et al. (2010) noted a greater degree of overlap of B. burgdorferi subtypes within the northeastern and midwestern tick populations when compared with subtypes from the southeastern USA. Gatewood Hoen et al. (2009) also documented ancient phylogenetic relatedness of northeastern and midwestern populations of B. burgdorferi, with evidence of more recent genetic divergence within each of these regions. The geographical distribution of B. burgdorferi subtypes suggests independent emergence in the northeast and midwest from separate relict foci, but with more rapid expansion of B. burgdorferi in the north-east (Gatewood Hoen et al., 2009). However, a phylogeographical comparison of the diversity of tick 16S mitochondrial haplotypes of I. scapularis in northeastern, midwestern and southeastern regions did not demonstrate similar patterns of genetic structure in populations of the vector ticks and populations of B. burgdorferi (Humphrey et al., 2010). While tick populations demonstrated genetic structure within each region, B. burgdorferi populations did not. Conversely, tick populations showed little genetic structure between regions, whereas

B. burgdorferi did. The authors noted that this lack of biogeographical and genetic concordance may occur due to differences in movement of the infected vertebrate host as opposed to the vector. However, other ecological factors, including differences in tick phenology, might account for the distribution of particular B. burgdorferi subtypes (Gatewood et al., 2009).

1.3 Life Cycles of I. ricinus Complex Ticks 1.3.1 Vertebrate hosts Although there is a great complexity of tick– host associations in this group, representing differing ecological communities and species histories, there are several features common to their life cycles. They are all ‘three-host ticks’ that take a blood meal once in each stage or instar (larva, nymph or adult) on a vertebrate host before moulting to the next stage (Kierans et al., 1999; Fig. 1.2). Mated female ticks lay a single mass of eggs (800– 3000) that hatch months later. The entire life cycle typically lasts 2 years for I. scapularis (range 2–4 years) and 3 years for I. pacificus, I. persulcatus and I. ricinus (range 2–6). Subadult ticks generally parasitize rodents or small mammals, birds and reptiles, but will also feed on large mammals including deer. There may be marked geographical differences in host choice for these subadults, even within a species, depending on the regional composition of the host community. For example, I. scapularis ticks in the southeastern USA feed preferentially on lizards, whereas northern and midwestern populations feed predominantly on small rodents (Piesman and Spielman, 1979; Spielman et al., 1985; Oliver et al., 1993a, 2003). This difference in host feeding results in the marked differences seen in tick infection by B. burgdorferi, Anaplasma phagocytophilum and Babesia microti, all of which are transmitted by infected rodents – but not reptiles – to I. scapularis ticks as they feed. In one island site with limited mammalian biodiversity (Nantucket Island, MA), Spielman concluded that over 90% of larval and nymphal

Ticks: the Vectors of Lyme Disease

I. scapularis ticks fed on white-footed mice (Spielman et al., 1985; Piesman, 2002). In Dutchess County, NY, however, while whitefooted mice hosted most larval ticks, eastern chipmunks hosted threefold more nymphs than mice (Schmidt et al., 1999). A similar pattern of host use was noted in Illinois (Slajchert et al., 1997). Recent blood meal molecular studies suggest that a greater proportion of ticks may feed on non-rodent hosts than earlier studies demonstrated (LoGiudice et al., 2003; Brisson et al., 2008). It is unclear whether fluctuations in rodent

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populations (i.e. mice) may also shift tick population host exposure to other mammals or birds, giving rise to temporal differences in pathogen prevalence in the same locale (Rand et al., 1998, Schmidt et al., 1999). Limited host choice may not limit establishment of this tick. On Monhegan Island, Maine, where most mammal species including mice are absent, subadult ticks feeding on Norway rats (and all stages feeding on deer) alone maintained the tick population until deer were removed from the island (Smith et al., 1993).

Larva feeds on host no. 1

Larvae seek new host Fully fed larva drops to ground Eggs hatch to larvae

Host no. 1 Larva moults to nymph

Eggs laid by female Fully fed female drops from host to ground

Host no. 3 Host no. 2

Female attaches and feeds on host no. 3

Nymph attaches and feeds on host no. 2

Nymph moults to adult * In western and southeastern USA ** In northeastern USA *** In parts of Europe

Fig. 1.2. Generalized development cycle for Ixodes pacificus, Ixodes persulcatus, Ixodes ricinus and Ixodes scapularis. Tick hosts include various rodents (exemplified by a mouse and a squirrel), lizards, birds, insectivors (hedgehog) and medium-sized (hare) and large (deer) mammals. The importance of these hosts for each life stage is indicated by the scale of depiction. (Designed by B. Kaye.)

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Adult ticks typically feed on mediumsized or larger mammals to the exclusion of rodents and birds (Eisen and Lane, 2002). Deer are the predominant definitive hosts for I. scapularis, although adult ticks will feed on a variety of mammals, including ungulates, black bear, coyotes, raccoons, skunks, opossums, dogs, cats, livestock and humans (Piesman and Spielman, 1979; Fish and Dowler, 1989; Piesman, 2002). I. scapularis tick populations are associated with areas with coexisting deer populations (Piesman and Spielman, 1979; Wilson et al., 1985, 1990). Adventitious importation of birds may lead to records of ticks from many areas where a tick population is not established (Smith et al., 1996; Ogden et al., 2008a). I. pacificus ticks parasitize over 100 vertebrate species (Castro and Wright, 2007). The primary hosts for subadult ticks are lizards (Western fence lizard, Southern alligator lizard), but many rodent and bird species are also parasitized (Eisen and Lane, 2002, Eisen et al., 2004). Infestation of rodents increased but did not surpass lizards in more moist habitats such as redwood/tanoak woodlands (Eisen et al., 2004). Western grey squirrels are frequently parasitized by this tick in oak woodland habitats (Lane et al., 2005). Similar to I. scapularis, the primary hosts for adult ticks in northern California are black-tailed deer, although they have been noted to feed on 29 mammal species (Westrom et al., 1985; Castro and Wright, 2007). However, unlike the single-vector cycle present in the northeastern USA, sylvatic maintenance of enzootic B. burgdorferi in the western states involves not only I. pacificus but also I. spinipalpis and perhaps Ixodes jellisoni (Brown et al., 2006). Of these three ticks, however, only I. pacificus is considered a bridge vector, transmitting infection to humans. Life cycles for I. ricinus ticks are more variable reflecting the heterogeneity of ecological situations in which they exist (Balashov, 1972; Gern and Humair, 2002). While they have been reported to feed on over 300 species of mammals and birds, subadult ticks preferentially feed on smaller mammals and birds, while adult ticks feed on larger mammals such as deer (Gray et al.,

1992). However, unlike I. scapularis, deer do not appear to be essential for perpetuation of this tick. In a heavily grazed sheep farming environment with a paucity of other mammals, sheep were the predominant hosts of all stages of the tick (Ogden et al., 1997). On an island off Sweden lacking large mammals, the I. ricinus population is maintained almost entirely by feeding on hares (Jaenson and Talleklint, 1996). The life history of I. persulcatus, which has a range in forests from the Far East to the Russian northwest, is similar in its diversity of hosts to I. ricinus (Balashov, 1972). However, while subadult I. persulcatus ticks may parasitize up to 50 species of small animals (Korenburg et al., 2002), there is a particular association of this tick with voles (Clethrionomys species) and shrews (Sorex species) across its range. Subadults may frequently also feed on chipmunks (in Russia) and red squirrels, and nymphs often feed on hooved animals (Korenberg et al., 2002). Birds are also frequently parasitized. Hares are notable because all three stages will feed on them, sometimes in large numbers (Korenberg et al., 2002). Adult ticks parasitize most larger mammals, whether wild or domestic. Korenberg notes that ‘in the southern taiga forests of the eastern East European Plain, cattle are parasitized by 52% of adult I. persulcatus ticks, hares 34%, elks 4%, and red foxes 2%’. A different situation exists in the Far East, where 62% fed on cattle and 24% on red deer (Korenberg et al., 2002). In Japan, mice (Apodemus species), voles and shrews, along with birds, are frequently parasitized by I. persulcatus, while adult ticks feed primarily on sika deer and red foxes (Miyamoto and Masuzawa, 2002). In contrast to I. scapularis and I. ricinus ticks, only the adult stages of I. persulcatus parasitize humans (Korenberg et al., 2001). To what extent vertebrate host species diversity determines the abundance of these ticks is an area of ongoing interest (Allan et al., 2003). I. scapularis abundance may be higher in environments with less mammalian biodiversity, provided that Peromyscus mice are present (Allan et al., 2003). In fact, due to high larval tick mortality associated with feeding on some hosts (i.e. opossums,

Ticks: the Vectors of Lyme Disease

squirrels), their removal from the host community increases tick abundance (Keesing et al., 2009). In general, increased feeding on competent reservoir hosts such as mice, chipmunks and shrews may lead not only to higher tick abundance but also to the highest levels of nymphal and adult tick infection with B. burgdorferi (LoGiudice et al., 2003; Brisson et al., 2008; Keesing et al., 2009). Given the diversity of hosts in any one site, and in different ecological settings, comprehensive field studies to determine host associations of these ticks are difficult and may require examination of many species of mammals and birds. Techniques for identification of the prior host from remnant blood in ticks provide a new means to quantify tick feeding history in a particular population, and to link this information to the presence of pathogen infection in the vector (Humair et al., 2007). Using reverse line blots to target host mitochondrial DNA in ticks, researchers in Switzerland documented different host exposures in different populations of field-collected I. ricinus on the northern and southern slopes of a single mountain (Cadenas et al., 2007). In an Irish research site, blood meal analysis revealed birds to be a major reservoir host for I. ricinus ticks (Pichon et al., 2005). As these techniques develop, it may be possible to recreate tick population host preferences with precision from samples of nymphal and adult ticks. 1.3.2 Questing behaviour These tick species all quest for hosts from vegetation, waiting for vibration, heat or other signals of the presence of a host as it passes (Balashov, 1972; Eisen and Lane, 2002). This activity occurs at a loss of energy and water, so ticks will descend to resorb water in the litter zone as needed. A requirement for tick survival is access to microhabitats with high humidity (>80–85%) at ground level (Stafford, 1994). To varying degrees, all species of this group have evolved behaviours to limit water loss and maximize questing success (Balashov, 1972). For I. scapularis, subadults often quest from the leaf litter or from low shrubs and grasses just above the

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ground, while adult ticks often quest 1 m or less from the ground in bushes and other forest understorey (Spielman et al., 1985). Similar questing behaviour has been reported for I. pacificus ticks in northwest California (Lane et al., 2007). However, unlike I. scapularis ticks, whose host-seeking behaviour varies with temperature and relative humidity (Clark, 1995; Vail and Smith, 1998, 2002), no such relationship was observed in the diurnal behaviour of I. pacificus nymphs (Lane et al., 2007). Prior experimental studies of adult I. pacificus, however, documented a positive association of questing activity with relative humidity, while another study reported lower nymph densities at mean daily temperatures >23°C (Loye and Lane, 1988; Eisen et al., 2002). Field studies suggest that the host-seeking activity of I. ricinus depends primarily on relative humidity and solar radiation (Jensen, 2000). Questing behaviour, and locomotor activities to move to a new site, may be decreased in dessicating environments (Balashov, 1972). Computer-assisted video tracking of I. ricinus ticks to measure locomotor activity under controlled climactic conditions documented regulation of this activity by water saturation deficit (Perret et al., 2003). Locomotor activity (i.e. movement from one site to another) was primarily nocturnal, serving also to reduce water loss. A similarly designed study of I. scapularis activity revealed diurnal locomotor activity only for autumn adults, with spring adults and nymphs exhibiting a unimodal pattern of activity peaking after dark (Madden and Madden, 2005). Interestingly, temperature changes exerted a more predictable response in activity than daylight (Madden and Madden, 2005). 1.3.3 Phenology (seasonal cycle development) While there are similarities in the life cycles of these ticks, there are differences in tick phenology (seasonal cycle development) that have an impact on transmission of enzootic pathogens. The bimodal seasonal inversion of subadult stages of I. scapularis, with nymphal population peaks (May–July) preceding

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larval peaks (August–September), increases infection transmission to hosts prior to larval contact (Spielman et al., 1985; Daniels et al., 1989). Regardless of the time of feeding, female I. scapularis ticks produce eggs in late spring leading to larval appearance in late summer (Yuval and Spielman, 1990). Based on field studies using confined ticks in their natural microhabitats, survival of different stages of unfed ticks could be determined, and progression to the next developmental stage could be timed in fed ticks (Yuval and Spielman, 1990). Adult ticks survived the winter, whether fed or not, but did not survive the following summer unless they fed by spring. Larvae that fed in September overwintered as nymphs, while later feeders overwintered as larvae. Unfed larvae survived less than 1 year. Fed nymphs moulted to adults that appeared in late autumn. Unfed nymphs may survive through two seasons, so that annual cohorts overlap (Yuval and Spielman, 1990). All stages of fed I. scapularis may enter diapause (dormancy), but only larvae and adults appear to survive the winter. This particular pattern is less evident in I. ricinus populations, however, although bimodal population peaks (spring and autumn) are observed. Eggs laid before midJuly may hatch in August, but many do not hatch until the following spring (Gray, 1981, 1991). Randolph et al. (2002) developed a quantitative framework for seasonal population dynamics of I. ricinus ticks that predicted tick demographic processes using a temperature-dependent model and measurements of tick fat content. This framework is consistent with the emergence of nymphs and adults in autumn followed by diapauses. Unfed ticks that feed in spring complete their development by autumn, along with the cohort of ticks that fed in the autumn. Although temperature differences may alter timing, the procession of a single cohort is maintained during these two periods. I. pacificus ticks exhibit an extended life cycle with active host seeking limited to the cooler season in late winter and spring (Padgett and Lane, 2001). Although eggs were laid in late winter or early spring by fed females, larvae hatching in late summer

remained in behavioural diapause until the next winter. Replete larvae moulted in midsummer, with nymphs also remaining in diapause until spring. Adults become active in late autumn and winter (Padgett and Lane, 2001). Based on these field studies in northwestern California, I. pacificus requires a minimum of 3 years to complete its life cycle (Padgett and Lane, 2001). 1.3.4 Ecological determinants of population dynamics Models of tick population dynamics in I. scapularis have attempted to provide insight into the drivers of tick abundance as well as determinants of B. burgdorferi infection (Ostfeld et al., 2006). Annual variation in precipitation, as a correlate for substrate moisture, and temperature, have been posited as determinants of black-legged tick population dynamics (Jones and Kitron, 2000), but have not predicted tick abundance over a span of 8–10 years in well-established areas (Ostfeld et al., 2006; Schulze et al., 2009). However, given the multiple variables involved, it is difficult to acquire adequate longitudinal data to provide robust conclusions. Assuming acceptable abiotic conditions (i.e. humidity, temperature), the debate continues on the relative role of different host species communities (i.e. host species type, diversity, abundance) in determining tick population dynamics. Once again, it appears that the relative importance of these variables may differ in different ecological settings. Ostfeld et al. (2006) provided evidence that small rodent population surges in response to acorn mast production predicted subsequent increases in I. scapularis numbers in a research site in New York State, and that climate and deer density were not predictive. However, others, working in habitats less dominated by oaks, have not observed a clear association of acorn mast and tick populations (Piesman and Spielman, 1979; Schulze and Jordan, 1996; Ginsberg et al., 2004). To complicate the overall prediction of tick density further, tick feeding on some hosts (i.e. opossums, squirrels) may result in increased tick mortality such that these hosts

Ticks: the Vectors of Lyme Disease

serve as ‘ecological traps’ to lower tick abundance (Keesing et al., 2009). Although the presence of a deer population is considered necessary for the maintenance of I. scapularis populations, substantial variations in deer herd density, if already high, may not be associated with marked changes in tick populations, perhaps due to aggregation of adult ticks on remaining deer or on medium-sized mammals (Jordan and Schulze, 2005; Ostfeld et al., 2006). However, in regions where deer populations are not already overabundant, deer herd density appears to be correlated to tick density (Wilson et al., 1985, 1990; Rand et al., 2003). As noted above, in the rare event of complete removal of deer, the deer tick life cycle may be disrupted, with a marked decline in tick numbers after 2–3 years (Wilson et al., 1988; Rand et al., 2004a). To date, no data strongly support a particular deer population threshold necessary either for colonization of ticks in a new area or for their disappearance with deer herd reduction. However, data from areas with wide ranges in deer abundance suggest that such a threshold, if it occurs at all, may occur only at relatively low densities of <7 deer/km2 (Wilson et al., 1988; Rand et al., 2004a).

1.4 Range Expansion and Projected Effects of Climate Change The rapid expansion of the range of I. scapularis into new areas throughout the northeastern USA, and to a lesser extent, the upper midwest, accounts, in part, for the remarkable rise of tick-borne disease attributable to this vector (Spielman et al., 1985, White et al., 1991; French et al., 1992; Pinger et al., 1996; Chen et al., 2005; Rand et al., 2007). Although systematic surveys do not exist, reports of I. scapularis ticks (Cooley and Kohls, 1945) from the first half of the 20th century suggest that this tick was not encountered frequently by humans, whether as tick bite victims or academic collectors, and its range was largely limited to the southeastern USA. Although widely distributed in the southeastern USA, this tick is not commonly found during drag sampling (or ‘flagging’) of vegetation, and rarely bites

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humans (Felz et al., 1996; Diuk-Wasser et al., 2006). In striking contrast to these observations, black-legged ticks are now the most abundant tick collected by flagging of vegetation in the northeastern and upper midwest USA (Diuk-Wasser et al., 2006). In endemic areas, they also may be the commonest tick parasitizing humans (Falco and Fish, 1988; Rand et al., 2007). Over the past four to five decades, I. scapularis ticks have become established, often with high population density, in suitable wooded or edge habitats throughout the northeastern USA (Dennis et al., 1998; DiukWasser et al., 2006), and are newly emergent in bordering provinces of Canada (Ogden et al., 2008a, 2010). In New York State and southern New England, I. scapularis distribution has moved progressively from coastal areas to more inland sites over a decade or less (White et al., 1991). During a 10-year span, larval abundance on rodents increased 30-fold in Westchester County, NY (Falco et al., 1995). Passive and active surveillance studies in Maine (1988–2006), at the northeastern edge of the tick’s current range, document initial recognition of the vector in the mid-1980s followed by range expansion along much of the coast and then to inland areas (Rand et al., 2007). Tick populations in southern coastal Maine, where I. scapularis first appeared in the late 1980s, have now reached a density comparable to highly endemic areas in the mid-Atlantic states (Diuk-Wasser et al., 2006). In the northeastern USA, cases of Lyme disease increased in incidence mirroring vector incursion and establishment, along with the appearance of anaplasmosis and, to a more geographically limited degree, babesiosis (Mather et al., 1996b; Herwaldt et al., 2003; Kogut et al., 2005; Chen et al., 2005). A recent large-scale standardized field study involving drag sampling for questing nymphal I. scapularis in areas in the USA east of the Mississippi revealed two significant highdensity clusters around endemic areas in the northeast and upper midwest and a lowdensity cluster in sites south of the 39th parallel, where nymphal ticks were collected in small numbers (Diuk-Wasser et al., 2006; Fig. 1.3).

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100th meridian

6.3 ticks/1000m2 (log scale)

N

0

250

500 Kilometers

I. scapularis A. americanum D. variabilis No ticks/other species

Fig 1.3. Density per 1000 m2 (log scale) of the most abundant species of ticks (nymphs and adults pooled) collected in each of the 95 study sites. The grid used to select the sampling sites is displayed in the background as well as the 100th meridian, the western limit of the study area. (Diuk-Wasser et al., 2008)

It is likely that the parallel increase in deer herd densities in these areas during the past century provided the host availability to promote successful colonization of new areas (Spielman et al., 1985, 1993; Wilson et al., 1985; Piesman, 2002). While small rodent hosts such as Peromyscus leucopus are ubiquitous, deer, which are the major hosts for adult ticks, were scarce in much of the USA at the start of the 20th century. By mid-century, over-population by deer had become an ecological problem in some areas (Leopold et al., 1947). In recent decades, deer herds have become superabundant in many coastal and some inland regions, with densities as high as 100 deer/km2, well above the optimal carrying capacity of the environment (Warren, 1991). In general, dense I. scapularis populations are associated with dense deer herds (Piesman, 2002; Rand et al., 2003). Despite this general relationship, differences of scale for the range

of vector populations, which may be aggregated in foci with a particular habitat over a small range, and their definitive adultstage host, which range over a much larger area, may complicate precise comparisons of the population density of the two species. Dispersal of ticks may occur within the range of deer (Madhav et al., 2004), but longdistance dispersal is facilitated by tick infestation of migratory songbirds (Anderson et al., 1986; Klich et al., 1996; Smith et al., 1996; Scott et al., 2001, Ogden et al., 2008a). Between 1 and 2% of migratory passerine birds carry I. scapularis ticks (predominantly nymphs) north during spring migration, with <1% of birds infested predominantly with larvae on autumn migration. Ticks aggregate on ground-foraging species as they move from one stopover habitat to another. Several hundred miles may be covered between stops, and feeding ticks will probably be

Ticks: the Vectors of Lyme Disease

transported from one suitable habitat to another. Estimates of tick importation derived from ticks removed from migratory birds at an isolated island site are 165–457 adult ticks/ hectare/year, and it is expected that these ticks will be aggregated at bird resting sites (Elias et al., 2011). Although tick importation by birds provides a compelling explanation for the establishment of discontinuous tick populations on mainland and island sites, the introduction of enzootic Lyme disease (and babesiosis) by this means is problematic, as transovarial transmission of Borrelia is rare (Spielman et al., 1985). Nymphs dispersed by spring migrants will subsequently feed on deer or other non-reservoir hosts as adults. However, larvae infected by spirochaetemic songbirds, or the birds themselves, could theoretically introduce B. burgdorferi to new sites, which is presumably the mechanism of enzootic introduction (Smith et al., 1996; Ginsberg et al., 2005; Ogden et al., 2008a, 2010). Babesia microti, however, does not infect birds, and is transmitted less effectively to rodents, which may be a barrier to the spread of this agent (Mather et al., 1990, 1996b). I. pacificus also feeds on birds, and has been removed in small numbers from migratory songbirds (Morshed et al., 2005; Slowik and Lane, 2001). It is unknown whether this tick species, which has been reported from Baja Mexico to British Columbia, has undergone a range expansion in recent decades. No genetic structuring is apparent in I. pacificus populations, although an isolated montane population in Utah lacked the expected mitochondrial DNA polymorphism, consistent with a postPleistocene founder event (Kain et al., 1999). Over 40 years ago, Hoogstraal and Kaiser (1965) documented the dispersal of I. ricinus ticks during bird migration, and subsequent reports from other European sites document the prevalence of these ticks as being as high as 20% on some ground-foraging species during migration (Mehl et al., 1984; Comstedt et al., 2006). Larvae more commonly infest migrating birds in autumn than spring (2.1% versus 0.9% of birds examined, respectively), while nymphal infestation is similar in the two seasons (2%). Tick abundance on birds (2.1–2.6 ticks per bird) was 20–30-fold less

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than infestation of rodents (Comstedt et al., 2006). Few I. persulcatus ticks were found on birds examined in Japan, where over 90% of ectoparasites on birds were Haemaphysalis tick species (Ishiguro et al., 2000). Models of range expansion and retraction of I. scapularis ticks with climate change predict eventual expansion of the vector into central midwestern areas of the USA and into southern Canada during this century with associated loss of these ticks in the southernmost areas of the USA (Brownstein et al., 2005a; Ogden et al., 2006, 2008b). Previous work using ticks in field enclosures and in climate-controlled laboratory experiments document a consistent thermal requirement for successful egg laying and subsequent larval development (Lindsay et al., 1995; Ogden et al., 2004; Rand et al., 2004b). However, based on these data, the current climate is not limiting for continued expansion into some northern areas of the USA and Canada (Rand et al., 2004b; Ogden et al., 2006). Other abiotic factors (i.e. relative humidity) are important to tick survival, as may be biotic factors such as habitat type or vegetation structure, host community and deer density. Large-scale maps of these predicted ranges do not incorporate smallerscale variables that may be necessary for successful colonization. For example, Newfoundland is included in one predicted expansion zone, but the absence of deer there might prevent establishment of the tick. In Europe, range expansion of I. ricinus to more northern latitudes and higher elevations is predicted with climate change (Lindgren et al., 2000). I. ricinus is already established in many northern areas of Europe, but there has been a range expansion in Sweden since the 1980s, which is postulated to be related to increases in the number of degree days with temperatures needed for tick survival (Gray et al., 2009). Increases in altitudinal distribution of I. ricinus in recent decades have also been well documented (Gray et al., 2009). In addition to range expansion of a vector, there may be temperature-dependent changes in vector density and phenology that increase or decrease pathogen transmission, depending on the seasonal synchrony of the tick life

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cycle (Randolph and Rogers, 2000). Socioeconomic or human activity changes with warmer temperatures may be the most important variable determining human disease incidence. While recent increases in cases of tick-borne encephalitis in central Europe were postulated to be a consequence of warmer temperatures, Randolph et al. (2008) provided evidence that increased disease incidence could be better attributed to changes in human activities during the warm weather rather than changes in vector distribution, abundance or infection prevalence. In Italy, where an upsurge of tick-borne encephalitis cases was also noted, changes in forest structure and increased density of roe deer best predicted increased disease risk rather than changes in climate (Rizzoli et al., 2009). As noted by Gray et al. (2009), given the multiple environmental and human components involved, ‘a complex chain of processes exists that makes the precise factors responsible for changes in disease incidence often difficult to determine’.

1.5 The Tick–Pathogen Interface During the millennia of association between I. ricinus complex ticks and B. burgdorferi sensu lato (as well as other Borrelia), this vector–pathogen interaction has been maintained in multiple ecological settings throughout temperate areas around the northern hemisphere. In fact, a guild (or group of species that use a common resource) of different vertebrate pathogens – (B. burgdorferi, A. phagocytophilum, Babesia species, flavivirus (i.e. tick-borne encephalits virus and Powassan virus lineage 2 or deer tick virus) – are transmitted by these ticks in both North America and Eurasia (Telford et al., 1997). Although elegant work delineates the orchestrated interaction between tick and Borrelia at times of vertebrate host feeding (Schwan et al., 1995), little is known about other interactions between these pathogens and their invertebrate hosts. The concept of vector competence for transmission of pathogenic agents to a particular host is sometimes incompletely understood. The presence of a known

pathogen in a tick does not indicate that the tick is a vector of this agent. For example, other biting flies or ticks may ingest microbes from an infected reservoir host that exist passively in this insect or tick, but these microbes are not subsequently transmitted to another host. To be a vector for a particular pathogen requires not only that the tick feeds on infectious vertebrates and acquires the pathogen through a blood meal, but also that it maintains it through one or more life stages (transstadial passage) and then transmits it to a new host when feeding again. Vectors that transmit pathogens from enzootic cycles to humans are considered ‘bridge vectors’, although they may be of minor importance in maintaining the cycle in nature. The long contact time of tick and host provides an opportunity for transmission of different disease agents that have evolved mechanisms to maximize successful infection of the new host during this interface. Tick adaptations to permit feeding on the host during this time may have an impact on pathogen transmission as well. I. ricinus complex ticks, which may feed on a host for 2–10 days, have evolved mechanisms to facilitate successful feeding, including an array of salivary proteins that provide anticoagulants (Salp14 and its paralogs), anti-complement proteins (Isac), antioxidants (Salp25) and a number of other immune-modulating proteins (Das et al., 2001; Ribeiro and Frachischetti, 2003; Narasimhan et al., 2004; Ramamoorthi et al., 2005). One of these proteins (Salp15) is of particular interest as it facilitates B. burgdorferi infection of the vertebrate host as well (Tyson et al., 2007), and its homologues are found in other I. ricinus group ticks (Hovius et al., 2008). Salp15 decreases interleukin-12 and T-cell activation at the site of tick feeding where it binds a lectin receptor and CD4 (Garg et al., 2006). It also binds outer-surface protein OspC, the surface protein of B. burgdorferi that is expressed at the time of tick feeding, with the effect of protecting the bacteria from antibody-mediated killing (Ramamoorthi et al., 2005). B. burgdorferi resides in the tick mid-gut, expressing a different outer-surface protein (OspA) until activated by the onset of a blood meal

Ticks: the Vectors of Lyme Disease

(Schwan et al., 1995). While in the tick midgut, Borrelia numbers are relatively low. The spirochaete binds to another protein (TROSPA), which binds the OspA surface protein (Pal et al., 2004). The induction of OspC 36–48 h into a blood meal initiates rapid bacterial multiplication, and transport via the haemocele to the salivary glands occurs before eventual invasion of the host (Schwan et al., 1995; Piesman et al., 2003). This delayed inoculation time varies somewhat between Ixodes species, and coincides with the time required for infection of the vertebrate host in animal models following tick attachment (Piesman and Dolan, 2002). During feeding, ticks ingest blood slowly at first while cuticular growth occurs in the tick mid-gut, with more rapid feeding at 12–36 h. The duration of feeding varies with stage and species, but ranges from 2–5 days for larvae to 6–11 days for adults (Balashov, 1972). Tick weight increases after repletion from 10–20-fold for larvae to up to 100-fold for adult females (Balashov, 1972; Eisen and Lane, 2002). As the tick feeds, with its injection of saliva, some vertebrate hosts may develop antibodies to tick proteins, leading to the possibility of decreased feeding success with subsequent bites, as was demonstrated with I. ricinus in a rabbit model (Bowessidjaou et al., 1977). These observations led to efforts to investigate the possibility of an ‘anti-tick’ vaccine that would interrupt tick feeding before transmission of pathogens could occur (Wikel et al., 1997; de la Fuente and Kocan, 2006). Recent identification of a highly conserved protein from I. scapularis involved in modulation of tick feeding and reproduction (subolesin) led to successful preliminary studies demonstrating effectiveness in cDNA or recombinant protein immunization experiments (Almazan et al., 2005). Other approaches using immunogenic proteins are under way. However, natural hosts with frequent exposure to ticks (i.e. mice, other small rodents, deer) may not readily mount an immune response to the tick salivary proteins (Randolph, 1979). The guild of pathogens common to ticks in the I. ricinus complex, while sharing the same vector and, in some cases, the same

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reservoir hosts, are maintained in different enzootic cycles (Telford et al., 1997; Telford and Goethert, 2004; Brown et al., 2009). There is little information on their possible coadaptations, although a recent analysis of multiple infections (i.e. B. microti, A. phagocyophilum, Bartonella species, cowpox virus) of field voles in Europe demonstrated interactions between some of these microbes in this rodent host (Telfer et al., 2010). However, prevalence surveys document that coinfection occurs at frequencies reflecting the individual pathogen prevalence in samples of ticks from the same areas (Steiner et al., 2008; Barbour et al., 2009). B. burgdorferi is more prevalent in I. scapularis ticks than Anaplasma or Babesia in most instances. In a comparison of infection and coinfection with these agents from sites in Indiana, Wisconsin, Pennsylvania and Maine, 55% of ticks harboured a single agent (Steiner et al., 2008). Coinfection with Anaplasma and B. burgdorferi occurred in 1–9%, with Babesia species (B. microti and Babesia odocoilei) and B. burgdorferi in 1–11% of ticks. In areas highly endemic for these pathogens, coinfection may be more prevalent, but even in the highest prevalence areas tick infection by Babesia or Anaplasma is usually 10% or less, whereas 40–60% of adult I. scapularis ticks host B. burgdorferi. In Rhode Island, B. microti infection of mice occurred only where deer tick densities were ‘moderate to high’, and an index of I. scapularis abundance predicted risk of human babesiosis (Mather et al., 1996b; Rodgers and Mather, 2007). Flavivirus infection of ticks appears less common. Like the viral agents of tick-borne encephalitis in Eurasia, the closely related flavivirus found in I. scapularis (Powassan virus lineage 2, or deer tick virus) is present in 1% or less of ticks (Telford et al., 1997; Ebel et al., 2000). Unlike tick-borne encephalitis viruses, however, deer tick virus has been implicated in only a few human cases of encephalitis to date (Tavakoli et al., 2009). I. ricinus ticks may also be infected with B. burgdorferi sensu lato, A. phagocytophilum and Babesia (i.e. Babesia divergens, B. microti, Babesia species EU1), with varying prevalence in different habitats and geographical areas (Wielinga et al., 2006; Brown et al., 2008; Gray

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et al., 2009). However, I. ricinus tick infection prevalence with Babesia species in Eurasia is low (1–2%), and human cases of babesiosis are rare (Becker et al., 2009; Gray et al., 2009). While I. ricinus ticks may serve as a bridge vector for anaplasmosis and babesiosis to humans in Europe, other ticks (i.e. Ixodes trianguliceps) may be more important for maintaining enzootic transmission (Brown et al., 2008). Both I. ricinus and I. persulcatus are the vectors of tick-borne encephalitis to humans over large regions of Eurasia, although geographical distribution of infected ticks is highly focal (Randolph and Rogers, 2000). I. persulcatus ticks may also be coinfected with B. burgdorferi and A. phagocytophilum, but little is known of their role as vectors of Babesia species (Masuzawa et al., 2008). In addition to known pathogens, these ticks carry other microbes of interest. In 1995, Fukunaga reported the presence of a relapsing fever group spirochaete in I. persulcatus (Fukunaga et al., 1995). Named after Dr K. Miyamoto, Borrelia miyamotoi was subsequently confirmed to be present in other tick species, including I. ricinus, I. scapularis and I. pacificus (Scoles et al., 2001; Richter et al., 2003; Mun et al., 2006). This agent, while not yet known to be a human pathogen, forms a monophyletic group with Borrelia theileri, the agent of bovine borreliosis. In I. scapularis nymphs from 11 northern states, the prevalence of infection with B. miyamotoi averages tenfold less (0.2 versus 0.02) than B. burgdorferi. Cultures of P. leucopus skin and blood revealed higher densities in mouse skin than in blood for B. burgdorferi, but the reverse was true for B. miyamotoi, where bacterial densities were higher in blood (Barbour et al., 2009). Also present in these ticks are Rickettsia, but they are considered an endosymbiont rather than a pathogen because, despite their high prevalence, they are not associated with known human disease (Magnarelli et al., 1991; Swanson and Norris, 2007; Steiner et al., 2008). I. ricinus also harbours Rickettsia helvetica, which has been associated with human disease in a small number of cases (Nilsson et al., 1999; Fournier et al., 2000) as well as other spotted fever group rickettsias

(Sprong et al., 2009). The microbial ecology of these ticks also includes a diversity of bacteria, the composition of which changes with the stage of the tick and with engorgement (Benson et al., 2004; Moreno et al., 2006).

1.6 The Tick–Human Interface and Vector Control Strategies Risk of tick-borne disease, and Lyme disease in particular, depends on the density of the vector ticks coupled with the prevalence of infection of the ticks, or entomological risk index, and also on human activities and behaviours that lead to contact with infected ticks (Mather et al., 1996a; Hayes and Piesman, 2007). For Lyme disease, the density of nymphal ticks, which is the stage responsible for most infections, is typically measured by drag sampling of vegetation (Daniels et al., 2000). This provides a measure of hostseeking ticks that might parasitize humans, but may not always correlate directly with nymphal populations as assessed on hosts (Schulze et al., 2009). Nymphal tick populations may fluctuate several-fold from year to year, and Lyme disease cases correlate with these fluctuations (Stafford et al., 1998; Falco et al., 1999; Ostfeld et al., 2006). In the northeastern USA, Lyme disease risk is considered primarily peridomestic, with individual exposure due to outdoor activities in habitats conducive to I. scapularis (Maupin et al., 1991). While the entomological risk index correlated with Lyme disease case rate when using data on the scale of towns, it did not correlate with human cases on the scale of individual residences in an endemic area (Mather et al., 1996a; Connally et al., 2006), presumably because human behaviour ultimately determines exposure to ticks in an area where ticks are prevalent. In studies of human outdoor activities in the northeastern USA, it is often difficult to identify specific high-risk behaviours, although gardening for >4 h/week and trail use for >5 h/week were correlated with risk in one study (Smith et al., 2001). However, exposure to the Western black-legged tick, which is less clearly peridomestic, was

Ticks: the Vectors of Lyme Disease

strongly correlated with prolonged contact on or near fallen logs on forest trails or with collecting firewood in forested areas during the spring or summer (Ley et al., 1995; Lane et al., 2004). In Europe, rural residence and outdoor recreational activities and forestry work are risk factors for I. ricinus exposure, although the recreational activities involving risk may include both urban parks and rural forested settings (Matuschka et al., 1996; O’Connell et al., 1998). Mapping geographical or ecological areas for risk of contact with I. scapularis ticks permits more targeted public education regarding protective measures. Habitat type and landscape features predict I. scapularis distribution at the large scale of a state or region (e.g. ‘north central USA’ or ‘Middle Atlantic region’), while the density of these ticks is associated with particular habitat types and landscape patterns at scales as fine as individual yards. On a large scale, using geographical information systems analysis, northern populations of I. scapularis in the Middle Atlantic region are positively associated with proximity to forest edge, sandy soils, vegetative cover and a moderate distance to water (Bunnell et al., 2003), and in the upper midwest, to deciduous, dry to mesic forests and sandy or loam soil types overlying sedimentary rock (Guerra et al., 2002). On a smaller scale, landscape features such as forest fragmentation (forest patch size <2 ha versus 2–8 ha) were associated with high nymphal tick densities (and entomological risk), but did not predict Lyme disease cases (Cromley et al., 1998; Allan et al., 2003; Brownstein et al., 2005b). Other models support the association of particular landscape features (clustered forest/herbaceous cover vs highly interspersed forest/herbaceous cover) with lower Lyme disease risk. Ecotone or ‘edge’ areas are important landscape features with regard to tick-borne illness, not because they present the highest entomological risk habitat, but because of high levels of human use (Maupin et al., 1991; Horobik et al., 2007). Although Lyme disease transmission is less clearly associated with peridomestic exposure in the western USA, geographical information systems modelling of higher-risk wooded habitats for I. pacificus

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exposure in Mendocino County, CA, correlated with Lyme disease incidence at the scale of residential zip codes (Eisen et al., 2004, 2006). Landscape design or modification may provide one mechanism for prevention of Lyme disease, either by decreasing human– tick contact or by lowering entomological risk (Jackson et al., 2006). Vegetation structure such as high shrub habitats rather than grassy or low shrub habitats, and wooded areas with understorey rather than open areas appear to be most conducive to the presence blacklegged ticks (Ginsberg and Ewing, 1989; Adler et al., 1992; Lubelczyk et al., 2004). Particular shrubs appear to be associated with dense tick populations in some areas. For example, in New England, invasive plants such as Japanese barberry, which occurs in areas with heavy deer browsing, is associated with high densities of all three stages of I. scapularis ticks (Lubelczyk et al., 2004; Elias et al., 2006; Williams et al., 2009). Removal of the barberry lowered the tick density in these areas (Williams et al., 2009). Landscape design measures to lower human exposure to questing ticks might include the placement of wood chip or other barriers to tick movement between lawns and shrubby or wooded areas (Piesman, 2006a; Dolan et al., 2009), or simple removal of shrub vegetation from a heavily used part of the yard to create more open areas. As these ticks are sensitive to desiccation, increased exposure to sun by removal of brush and leaf litter has lowered tick density (Schulze et al., 1995). In general, black-legged ticks are absent from or occur at low density on maintained lawns, fields and grasslands, as well some forested habitats such as northern coniferous forests with little understorey (Guerra et al., 2002; Lubelczyk et al., 2004; Brownstein et al., 2005b). Despite these observations, tick population correlations with particular habitats may vary by stage and year within study areas, and are also determined by other abiotic and biotic variables that may fluctuate, such as humidity of microhabitat, temperature, host prevalence and host movement (Ostfeld et al., 2006; Schulze et al., 2009). Developing consistent approaches towards testing the predictive

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value of these different factors that will be applicable in different ecological settings remains a challenge (Killilea et al., 2008). In addition to the landscape modifications described above, vector control to reduce human risk of exposure to ticks may include the application of acaricides to lawns or to tick hosts, biological tick control (i.e. fungal or bacterial tick control) and deer herd reduction. Spraying of acaricides (e.g. carbaryl, cyfluthrin, deltamethrin) in early spring on lawns lowers nymph tick populations by 68–100% (Stafford, 1991; Curran et al., 1993; Schulze et al., 2000). However, because of cost and concerns regarding pesticide impacts on non-target species, public acceptance of these interventions has been limited (Piesman, 2006b). The use of acaricides derived from botanicals provides another approach that is under investigation (Dolan et al., 2009; Rand et al., 2010). Innovative methods to target acaricides to ticks feeding on hosts (i.e. mice, deer) have had variable success when used as the sole intervention, but may provide a component to integrated tick management strategies. Provision of mice with cotton-based nesting material that contains permethrin has been effective in some environments but not in others, presumably due to differences in the tick host communities (Daniels et al., 1991; Stafford, 1992). A 3-year trial of mouse ‘bait boxes’ (Maxforce Tick Management System) designed to use topical fipronil to kill subadult ticks feeding on rodents demonstrated a reduction in mouse nymphal and larval tick infestations of 68 and 84%, and in questing nymphs of 50% (Dolan et al., 2004). An integrated tick management study that incorporated the initial use of these boxes and application of a barrier acaricide (deltamethrin) followed by continued use of a deertargeted topical acaricide (‘4-Posters’) demonstrated control of host-seeking nymphs and larvae (reduction of 85.9 and 89%) over 2 years (Schulze et al., 2008). Even the development of these protected, hosttargeted acaricides has caused concern regarding pesticide use, however, and has interfered with their more general application. Biological control of I. scapularis began in the 1930s with introduction of a parasitoid

wasp in an unsuccessful attempt to control tick numbers, but more recent efforts have focused on entomopathogenic fungi (Zhioua et al., 1997). Field trials of a native soil fungus (Metarhizium anisopliae) in nest boxes showed limited effectiveness on questing nymphs (Hornbostel et al., 2005), but the fungus has lethal effect on I. scapularis, and evaluations in other settings are ongoing. As deer herd density is correlated with I. scapularis density in some settings (Wilson et al., 1984, 1988; Rand et al., 2003), deer exclusion or deer herd control is of interest as a means to decrease entomological risk and the incidence of human Lyme disease. Several deer reduction or exclusion studies have demonstrated associated declines in hostseeking nymphal ticks (Wilson et al., 1984, 1988; Daniels et al., 1993; Deblinger et al., 1993; Stafford et al., 2003). On two island sites where deer were either extirpated or nearly extirpated, I. scapularis density declined markedly, to the point of tick extirpation at one site (Wilson et al., 1988; Rand et al., 2004a). However, in areas with dense deer populations, deer reduction may not significantly lower the entomological risk (Schulze et al., 2005b). Declines in entomological risk in these areas may require reductions in herd density to levels that are difficult to achieve and sustain (Wilson et al., 1984, 1988; Deblinger et al., 1993; Rand et al., 2004a). However, successful control of deer populations to limit density to 4 deer/km2 has been achieved in large rural areas (>160 km2) with controlled hunts (McDonald et al., 2007). Alternatives may include deer exclosure with fencing, but the minimum size of exclosure needed to provide protection is not known (Piesman, 2006b). In those areas where deer herd densities are not already high, and Lyme disease not yet endemic, it is possible that limitation of deer herd size might delay or preclude colonization by I. scapularis, but the threshold of deer herd density necessary for establishment of I. scapularis and enzootic B. burgdorferi is not known. Contact with ticks may also be prevented by personal measures such as avoidance of high tick density areas and the use of tick repellants on skin (e.g. DEET or picaridin) or clothing (permethrin) (Schreck et al., 1986).

Ticks: the Vectors of Lyme Disease

Educational programmes to increase knowledge regarding the use of repellants and avoidance of high-density areas demonstrate good knowledge levels but inconsistent application (Shadick et al., 1997; Malouin et al., 2003; Gould et al., 2008). Based upon a case–control study of personal protective measures, protective clothing and use of tick repellents all appeared to confer a degree of protection (Vásquez et al., 2008). In one controlled trial of an innovative education programme for prevention of tick exposure, a decrease in tick-borne illness was demonstrated during the following 2 months (Daltroy et al., 2007). ‘Tick checks’ (daily visual inspection for ticks) to prevent disease after tick exposure has a sound biological basis (Sood et al., 1997; Piesman and Dolan, 2002). Transmission of Lyme disease by I. scapularis requires at least 36 h once attachment has occurred, at which time the blood meal stimulates replication of B. burgdorferi and its migration from the tick mid-gut to the salivary glands prior to infecting a host (Piesman and Dolan, 2002). Risk of infection increases exponentially after 48–72 h of attachment to the host. Therefore, removal of attached ticks in this interval prevents infection (Sood et al., 1997). If an I. scapularis tick is already engorged, and removed within 72 h of its discovery, Lyme disease risk may be reduced from an average of 3.2 to 0.4% by treatment of the tick bite victim with a single (200 mg) dose of doxycyline, but this strategy is not approved for use in children under 8 years of age or pregnant women (Nadelman et al., 2001). New concepts for prevention of tickborne illness from I. scapularis include vaccination of rodent hosts against B. burgdorferi infection, a strategy that achieved modest success in a proof-of-principle field trial (Tsao et al., 2004). Challenges with this strategy include differences in reservoir host importance to enzootic maintenance of disease in different host communities and methods of delivery to rodent populations (Brisson et al., 2008). Vaccines to limit tick feeding (‘anti-tick vaccines’) for wildlife represent another strategy. These vaccines might employ antigens related to salivary proteins (‘exposed antigens’) or other

17

‘concealed antigens’, or a combination designed to immunize hosts to tick proteins. While this concept has been tested successfully with laboratory rodents, it may be more difficult in ‘natural’ hosts, who may fail to mount an immune response to repeated bites by ticks (Randolph, 1979; de la Fuente and Kocan, 2006). However, if successful, and sustained over a number of years, interruption of local tick life cycles appears feasible (Mount et al., 1997). A human ‘anti-tick’ vaccine might be a simpler approach, and the identification of immunogenic tick proteins may lead to novel vaccination strategies (Schuijt et al., 2011). However, while interrupted tick feeding might prevent infection with B. burgdorferi, it is less clear that it would provide protection against other tick-borne pathogens with shorter times required for disease transmission (e.g. flavivirus). While developing new approaches to prevention of tick-borne diseases, additional evaluation of combinations of methods already developed and tailored for particular communities where risk of tick-borne illness is high may show benefit (Piesman, 2006b). A logistical challenge for projects that integrate tested methods of vector control with interventions designed to limit tick-human contact is the demonstration not only of decreased entomological risk indices, but also of a measurable effect on the local incidence of Lyme disease and other tickborne illnesses. Given the difficulties of achieving adequate population size in a community trial to prove an impact on human disease incidence, it may be necessary to develop methods that better link entomological risk index reduction to predicted effects on human disease.

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Almazan, C., Blas-Machado, U., Kocan, K.M. Yoshioka, J.H., Blouin, E.F., Mangold, A.J. and de la Fuente, J. (2005) Characterization of three Ixodes scapularis cDNAs protective against tick infestations. Vaccine 23, 4403–4416. Anderson, J.F., Johnson, R.C., Magnerelli, L.A. and Hyde, F.W. (1986) Involvement of birds in the epidemiology of the Lyme disease agent Borrelia burgdorferi. Infection Immunology 51, 394–396. Balashov, Y.S. (1972) Bloodsucking ticks (Ixodoidea): vectors of diseases of man and animals. Miscellaneous Publications of the Entomologic Society of America 8, 161–376. Barbour, A.G., Bunikis, J., Travinsky, B., Gatewood Hoen, A., Diuk-Wasser, M.A., Fish, D. and Tsao, J.I. (2009) Niche partitioning of Borrelia burgdorferi and Borrelia miyamotoi in the same tick vector and mammalian reservoir species. American Journal of Tropical Medicine and Hygiene 81, 1120–1131. Becker, C.A.M., Bouju-Albert, A., Jouglin, M., Chauvin, A. and Malandrin, L. (2009) Natural transmission of zoonotic Babesia spp. by Ixodes ricinus ticks. Emerging Infectious Diseases 15, 320–322. Benson, M.J., Gawronski, J.D., Eveleigh, D.E. and Benson, D.R. (2004) Intracellular symbionts and other bacteria associated with deer ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts. Applied and Environmental Microbiology 70, 616–620. Black, W.C. IV and Piesman, J. (1994) Phylogeny of hard- and soft-tick taxa (Acari: Ixodidae) based on mitochondrial 16S rDNA sequences. Proceedings of the National Academy of Sciences USA 91, 10034–10038. Bowessidjaou, J., Brossard, M. and Aeschlimann, A. (1977) Effects and duration of resistance acquired by rabbits on feeding and egg laying in Ixodes ricinus L . Experientia 33, 528–530. Brisson, D., Dykhuizen, D.E. and Ostfeld, R.S. (2008) Conspicuous impacts of inconspicuous hosts on the Lyme disease epidemic. Proceedings of the Royal Society B 275, 227– 235. Brisson, D., Vandermause, M.F., Meece, J.K., Reed, K. and Dykhuizen, D.E. (2010) Evolution of northeastern and midwestern Borrelia burgdorferi, United States. Emerging Infectious Diseases 16, 911–917. Brown, K.J., Lambin, X., Telford, G.R., Ogden, N.H., Telfer, S.T., Woldehiwet, Z. and Birtles, R.J. (2008) Relative importance of Ixodes ricinus and Ixodes trianguliceps as vectors for Anaplasma phagocytophilum and Babesia microti in field vole (Microtus agrestis) populations. Applied and Environmental Microbiology 74, 7118–7125.

Brown, K.J., Lambin, X., Ogden, N.H., Begon, M., Telford, G., Woldehiwet, Z. and Birtles, R.J. (2009) Delineating Anaplasma phagocytophilum ecotypes in coexisting, discrete enzootic cycles. Emerging Infectious Diseases 15, 1948–1953. Brown, R.N., Peot, M.A. and Lane, R.S. (2006) Sylvatic maintenance of Borrelia burgdorferi (Spirochaetales) in northern California: untangling the web of transmission. Journal of Medical Entomology 43, 743–751. Brownstein, J.S., Holford, T.R. and Fish, D. (2005a) Effect of climate change on Lyme disease risk in North America. EcoHealth 2, 38–46. Brownstein, J.S., Skelly, D.K., Holford, T.R. and Fish, D. (2005b) Forest fragmentation predicts local heterogeneity of Lyme disease risk. Oecologia 146, 469–475. Bunnell, J.E., Price, S.D., Das, A., Shields, T.M. and Glass, G.E. (2003) Geographic information systems and spatial analyis of adult Ixodes scapularis (Acari: Ixodidae) in the Middle Atlantic region of the USA. Journal of Medical Entomology 40, 570–576. Cadenas, F.M., Rais, O., Humair, P.-F., Douet, V., Moret, J. and Gern, L. (2007) Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland). Journal of Medical Entomology 44, 1109–1117. Caporale, D.A., Rich, S.M., Spielman, A., Telford, S. and Kocher, T.D. (1995) Discriminating between Ixodes ticks by means of mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 4, 361–365. Castro, M.B. and Wright, S.A. (2007) Vertebrate hosts of Ixodes pacificus (Acari: Ixodidae) in California. Journal of Vector Ecology 32, 140– 149. CDC (2008) Surveillance for Lyme disease – United States 1992–2006. Morbidity and Mortality Weekly Report 57, 1–9. Chen, H.D., White, D.J., Caraco, T.B. and Stratton, H.H. (2005) Epidemic and spatial dynamics of Lyme disease in New York State, 1990–2000. Journal of Medical Entomology 42, 899–908. Clark, D.D. (1995) Lower temperature limits for activity of several ixodid ticks (Acari: Ixodidae): effects of body size and rate of temperature change. Journal of Medical Entomology 32, 449–452. Comstedt, P., Bergstom, S., Olsen, B., Garpmo, U., Marjavaara, L., Mejlon, H., Barbour, A. and Bunikis, J. (2006) Migratory passerine birds as reservoirs of Lyme borreliosis in Europe. Emerging Infectious Diseases 12, 1087–1095. Connally, N.P., Ginsberg, H.S. and Mather, T.N. (2006) Assessing peridomestic entomologic

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factors as predictors for Lyme disease. Journal of Vector Ecology 31, 364–370. Cooley, R.A. and Kohls, G.M. (1945) The genus Ixodes in North America. National Institute of Health Bulletin 184, 1–246. Cromley, E.K., Cartter, M.L., Mrozinski, R.D. and Ertel, S.H. (1998) Residential setting as a risk factor for Lyme disease in a hyperendemic region. American Journal of Epidemiology 147, 472–477. Curran, K.L., Fish, D. and Piesman, J. (1993) Reduction of nymphal Ixodes dammini (Acari: Ixodidae) in a residential suburban landscape. Journal of Medical Entomology 30, 107–113. Daltroy, L.H., Phillips, C., Lew, R., Wright, E., Shadick, N.A. and Liang, M.H. (2007) A controlled trial of a novel primary prevention program for Lyme disease and other tick-borne illnesses. Health Education Behavior 34, 531– 542. Daniels, T.J. and Fish, D. (1995) Effect of deer exclusion on the abundance of immature Ixodes scapularis (Acari: Ixodidae) parasitizing small and medium-sized rodents. Journal of Medical Entomology 32, 5–11. Daniels, T.J., Fish, D. and Falco, R.C. (1989) Seasonal activity and survival of adult Ixodes dammini (Acari: Ixodidae) in southern New York state. Journal of Medical Entomology 26, 610– 614. Daniels, T.J., Fish, D. and Falcon, R.C. (1991) Evaluation of a host-targeted acaricide for decreasing Lyme disease risk in southern New York State. Journal of Medical Entomology 28, 537–543. Daniels, T.J., Fish, D. and Schwartz, I. (1993) Reduced abundance of Ixodes scapularis (Acari: Ixodidae) and Lyme disease risk by deer exclusion. Journal of Medical Entomology 30, 1043–1049. Daniels, T.J., Falco, R.C. and Fish, D. (2000) Estimating population size and drag sampling efficiency for the blacklegged tick (Acari: Ixodidae). Journal of Medical Entomology 37, 357–363. Das, S., Banerjee, G., DePonte K., Marcantonia, N., Kantor, F.S. and Fikrig, E. (2001) Salp25D, and Ixodes scapularis antioxidant, is 1 of 14 immunodominant antigens in engorged tick saliva. Journal of Infectious Diseases 184, 1056–64. Deblinger, R.D., Wilson, M.L., Rimmer, D.W. and Spielman, A. (1993) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following incremental removal of deer. Journal of Medical Entomology 30, 144–150. de la Fuente, J. (2003) The fossil record and origin

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of ticks (Acari: Parasitiformes: Ixodida). Experimental and Applied Acarology 29, 331–344. de la Fuente, J. and Kocan, K.M. (2006) Strategies for development of vaccines for control of ixodid tick species. Parasitology Immunology 28, 275– 283. Dennis, D.T., Nakomoto, T.S., Victor, J.C., Paul, W.S. and Piesman, J. (1998) Reported distribution of Ixodes scapularis and Ixodes pacificus (Acari: Ixodidae) in the United States. Journal of Medical Entomology 35, 629–638. Diuk-Wasser, M.A., Gatewood, A.G., Cortinas, M.R., Yaremych-Hamer, S., Tsao, J., Kitron, U., Hickling, G., Brownstein, J.S., Walker, E., Piesman, J. and Fish, D. (2006) Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States. Journal of Medical Entomology 43, 166–176. Dolan, M.C., Maupin, G.O., Schneider, B.S., Denatale, C., Hamon, N., Cole, C., Zeidner, N.S. and Stafford, K.C. III (2004) Control of immature Ixodes scapularis (Acari: Ixodidae) on rodent reservoirs of Borrelia burgdorferi in a residential community of southeastern Connecticut. Journal of Medical Entomology 41, 1043–1054. Dolan, M.C., Jordan, R.A., Schulze, T.L., Schulze, C.J., Manning, M.C., Buffolo, D., Schmidt, J.P., Piesman, J. and Karches, J.J. (2009) Ability of two natural products, nootkatone and carvacrol, to suppress Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey. Journal of Medical Entomology 102, 2316–2324. Douzery, E.J.P., Snell, E.A., Bapteste, E., Delsuc, F. and Philippe, H. (2004) The timing of eukaryotic evolution: does a relaxed molecular clock reconcile proteins and fossils? Proceedings of the National Academy of Sciences USA 101, 15386–15391. Ebel, G.D., Campbell, E.N., Goethert, H.K., Spielman, A. and Telford, S.R. III (2000) Enzootic transmission of deer tick virus in New England and Wisconsin sites. American Journal of Tropical Medicine and Hygiene 63, 36–42. Elias, S.P., Lubelczyk, C.B., Rand, P.W., Lacombe, E.H., Holman, M.S. and Smith, R.P. Jr (2006) Deer browse resistant exotic-invasive understory: an indicator of elevated human risk of exposure to Ixodes scapularis (Acari: Ixodidae) in southern Maine coastal woodlands. Journal of Medical Entomology 43, 1142–1152. Elias, S.P., Smith, R.P. Jr, Morris, S.R., Rand, P.W., Lubelczyk, C.B. and Lacombe, E.H. (2011) Density of Ixodes scapularis Say on Monhegan Island after complete deer removal: a question of avian importation? Journal of Vector Ecology 36, 1–13.

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disease spirochete (Borrelia burgdorferi) and its shared history with the tick vector (Ixodes scapularis) in the northeastern United States. Genetics 160, 833–849. Qiu, W.-G., Bruno, J.F., McCrain W.D., Xu Y., Livey, I., Schriefer, M.E. and Luft, B.J. (2008) Wide distribution of a high-virulence Borrelia burgdorferi clone in Europe and North America. Emerging Infectious Diseases 14, 1097–1103. Ramamoorthi, N., Narasimhan, S., Pal, U., Bao, F., Yang, X.F., Fish, D., Anguita, J., Norgard, M.V., Kantor, F.S., Anderson, J.F., Koski, R.A. and Fikrig, E. (2005) The Lyme disease agent exploits a tick protein to infect the mammalian host. Nature 436, 573–577. Rand, P.W., Lacombe, E.H., Smith, R.P. Jr and Ficker, J. (1998) Participation of birds (Aves) in the emergence of Lyme disease in southern Maine. Journal of Medical Entomology 35, 270– 276. Rand, P.W., Lubleczyk, C., Lavigne, G.R., Elias, S., Holman, M.S., Lacombe, E.H. and Smith, R.P. Jr (2003) Deer density and the abundance of Ixodes scapularis (Acari: Ixodidae). Journal of Medical Entomology 40, 179–184. Rand, P.W., Lubelczyk, C., Holman, M.S., Lacombe, E.H. and Smith, R.P. Jr (2004a) Abundance of Ixodes scapularis (Acari: Ixodidae) after the complete removal of deer from an isolated offshore island, endemic for Lyme disease. Journal of Medical Entomology 41, 779–784. Rand, P.W., Holman, M.S., Lubelczyk, C., Lacombe, E.H., Degaetano, A.T. and Smith, R.P. Jr (2004b) Thermal accumulation and early development of Ixodes scapularis. Journal of Vector Ecology 29 ; 164–176. Rand, P.W., Lacombe, E.H., Dearborn, R., Cahill, B., Elias, S., Lubelczyk, C.B., Beckett, G.A. and Smith, R.P. Jr (2007) Passive surveillance in Maine, an area emergent for tick-borne diseases. Journal of Medical Entomology 44, 1118–1129. Rand, P.W., Lacombe, E.H., Elias, S.P., Lubelczyk, C.B., St Amand, T. and Smith, R.P. Jr (2010) Trial of a minimal-risk botanical compound to control the vector tick of Lyme disease. Journal of Medical Entomology 47, 695–698. Randolph, S.E. (1979) Population regulation in ticks: the role of acquired resistance in natural and unnatural hosts. Parasitology 79, 141–156. Randolph, S.E. and Rogers, D.J. (2000) Fragile transmission cycles of tick-borne encephalitis virus may be disrupted by predicted by climate change. Proceedings of the Royal Society B: Biological Sciences 267, 1741–1744. Randolph, S.E., Green, R.M., Hoodless, A.N. and Peacey, M.F. (2002) An empirical quantitative framework for the seasonal population dynamics

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of the tick Ixodes ricinus. International Journal of Parasitology 32, 979–989. Randolph, S.E., Asokliene, L., Avsic-Zupanc, T., Bormane, A., Burri, C., Gern, L., Golovljova, I., Hubalek, Z., Knap, N., Kondrusik, M., Kupca, A., Pejoch, M., Vasilenko, V. and Zygutiene, M. (2008) Variable spikes in tick-borne encephalitis incidence in 2006 independent of variable tick abundance but related to weather. Parasites and Vectors 1, 44. Ribeiro, J.M. and Frachischetti, I.B.M. (2003) Role or arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annual Reviews of Entomology 48, 73–78. Rich, S.M., Caporale, D.A., Telford, S.R. III, Kocher, T.D., Hartl, D.L. and Spielman, A. (1995) Distribution of the Ixodes ricinus-like ticks of eastern North America. Proceedings of the National Academy of Sciences USA 92, 6284– 6288. Richter, D., Schlee, D.B. and Matuschka, F.-R. (2003) Relapsing fever-like spirochetes infecting European vector tick of Lyme disease agent. Emerging Infectious Diseases 9, 697–701. Rizzoli, A., Hauffe, H.C., Tagliapietra, M., Neteler, M. and Rosa, R. (2009) Forest structure and roe deer abundance predict tick-borne encephalitis risk in Italy. PloS ONE 4, e4336. Rodgers, S.E. and Mather, T.N. (2007) Human Babesia microti incidence and Ixodes scapularis distribution, Rhode Island, 1998–2004. Emerging Infectious Diseases 13, 633–635. Rosenthal, B.M. and Spielman, A. (2004) Reduced variation among northern deer tick populations at an autosomal microsatellite locus. Journal of Vector Ecology 29, 227–235. Say, T. (1821) An account of the arachnides of the United States. Journal of the Academy of Natural Sciences of Philadelphia 2, 59–82. Schmidt, K.A., Ostfeld, R.S. and Schauber, E.M. (1999) Infestation of Peromyscus leucopus and Tamias striatus by Ixodes scapularis (Acari: Ixodidae) in relation to the abundance of hosts and parasites. Journal of Medical Entomology 36, 749–757. Schreck, C.E., Snoddy, E.L. and Spielman, A. (1986) Pressurized sprays of permethrin and DEET on military clothing for personal protection against Ixodes dammini (Acari: Ixodidae). Journal of Medical Entomology 23, 396–399. Schuijt, T.J., Hovius, J.W., van der Poll, T., van Dam, A.P. and Fikrig, E. (2011) Lyme borreliosis vaccination: the facts, the challenge, the future. Trends in Parasitology 27, 40–47. Schulze, T.L. and Jordan, R.A. (1996) Seasonal and long-term variation in abundance of adult Ixodes scapularis (Acari: Ixodidae) in different

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coastal plain habitats in New Jersey. Journal of Medical Entomology 33, 963–970. Schulze, T.L., Jordan, R.A. and Hung, R.W. (1995) Suppression of subadult Ixodes scapularis (Acari: Ixodidae) ticks following removal of leaf litter. Journal of Medical Entomology 23, 396– 399. Schulze, T.L., Jordan, R.A. and Hung, R.W. (2000) Effects of granular carbaryl application on sympatric populations of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) nymphs. Journal of Medical Entomology 37, 121–125. Schulze, T.L., Jordan, R.A., Schulze, C.J., Mixson, T. and Papero, M. (2005a) Relative encounter frequencies and prevalence of selected Borrelia, Ehrlichia, and Anaplasma infections in Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae) ticks from central New Jersey. Journal of Medical Entomology 42, 450–456. Schulze, T.L., Jordan, R.A. and Schulze, C.J. (2005b) Host associations of Ixodes scapularis (Acari: Ixodidae) in residential and natural settings in Lyme disease-endemic area in New Jersey. Journal of Medical Entomology 42, 966–973. Schulze, T.L., Jordan, R.A., Dolan, M.C., Dietrich, G., Healy, S. and Piesman, J. (2008) Ability of a 4-poster passive topical treatment devices for deer to sustain low population levels of Ixodes scapularis (Acari: Ixodidae) after integrated tick management in a residential community. Journal of Medical Entomology 45(5), 899–904. Schulze, T.L., Jordan, R.A., Schulze, C.J. and Hung, R.W. (2009) Precipitation and temperature as predictors of local abundance of Ixodes scapularis (Acari: Ixodidae) nymphs. Journal of Medical Entomology 46, 1025–1029. Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C. and Rosa, P.A. (1995) Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proceedings of the National Academy of Sciences USA 92, 2909–2913. Scoles, G.A., Papero, M., Beati, L. and Fish, D. (2001) A relapsing fever group spirochete transmitted by Ixodes scapularis ticks. Vectorborne and Zoonotic Diseases 1, 21–34. Scott, J.D., Fernando, K., Banerjee, S.N., Durden, L.A., Byrne, S.K., Banerjee, M., Mann, R.B. and Morshed, M.G. (2001). Birds disperse ixodid (Acari: Ixodidae) and Borrelia burgdorferi infected ticks in Canada. Journal of Medical Entomology 38, 493–500. Shadick, N.A., Daltroy, L.H., Phillips, C.B., Liang, U.S. and Liang, M.H. (1997) Determinants of tick-avoidance behaviors in an endemic area for Lyme disease. American Journal of Preventive Medicine 13, 265–270.

Slajchert, T., Kitron, U.D., Jones, C.J. and Manelli, A. (1997) Role of the eastern chipmunk (Tamias striatus) in the epizootiology of Lyme borreliosis in northwestern Illinois USA. Journal of Wildlife Diseases 33, 40–46. Slowik, T.J. and Lane, R.S. (2001) Birds and their ticks in northwestern California: minimal contribution to Borrelia burgdorferi enzootiology. Journal of Parasitology 87, 755–761. Smith, G., Wiley, E.P., Hopkins, R.B., Cherry, B.R. and Maher, J.P. (2001) Risk factors for Lyme disease in Chester County, Pennsylvania. Public Health Report 116, 146–156. Smith, R.P. Jr, Rand, P.W., Lacombe, E.H., Telford, S.R. III, Rich, S.M., Piesman, J. and Spielman, A. (1993) Norway rats as reservoir hosts for Lyme disease spirochetes on Monhegan Island, Maine. Journal of Infectious Diseases 168, 687– 691. Smith, R.P. Jr, Rand, P.W., Lacombe, E.H., Morris, S.R., Holmes, D.W. and Caporale, D.A. (1996) Role of bird migration in the long-distance dispersal of Ixodes dammini, the vector of Lyme disease. Journal of Infectious Diseases 174, 221–224. Sood, S.K., Salzman, M.B., Johnson, B.J.B., Happ, C.M., Feig, K., Carmody, L., Rubin, L.G., Hilton, E. and Piesman, J. (1997) Duration of tick attachment as a predictor of the risk of Lyme disease in an area in which Lyme disease is endemic. Journal of Infectious Diseases 175, 996–9. Spielman, A., Clifford, C.M., Piesman, J. and Corwin, M.D. (1979) Human babesiosis on Nantucket Island, USA: description of the vector, Ixodes (Ixodes) dammini, n. sp. (Acarina: Ixodidae). Journal of Medical Entomology 15, 218–234. Spielman, A., Wilson, M.L., Levine, J.F. and Piesman, J. (1985) Ecology of Ixodes damminiborne human babesiosis and Lyme disease. Annual Review of Entomology 30, 439–460. Spielman, A., Telford, S.R. III and Pollak, R.J. (1993) The origins and course of the recent outbreak of Lyme disease. In: Ginsberg, H.S. (ed.) Ecology and Environmental Management of Lyme Disease. Rutgers University Press, New Brunswick, NJ, pp. 83–96. Sprong, H., Wielinga, P.R., Fonville, M., Reusken, C., Brandenburg, A.H., Borgsteede, F., Gaasenbeek, C. and van der Giessen, J.W.B. (2009) Ixodes ricinus ticks are reservoir hosts for Rickettsia helvetica and potentially carry fleaborne Rickettsia species. Parasites and Vectors 2, 41–50. Steiner, F.E., Pinger, R.R., Vann, C.N., Grindle, N., Civitello, D., Clay, K. and Fuqua, C. (2008) Infection and co-infection rates of Anaplasma

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phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial symbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin. Journal of Medical Entomology 45, 289–297. Stafford, K.C. III (1991) Effectiveness of carbaryl applications for the control of Ixodes dammini in an endemic residential area. Journal of Medical Entomology 28, 32–36. Stafford, K.C. III (1992) Third-year evaluation of host targeted permethrin and control of Ixodes dammini (Acari: Ixodidae) in southeastern Connecticut. Journal of Medical Entomology 29, 717–720. Stafford, K.C. III (1994) Survival of immature Ixodes scapularis (Acari: Ixodidae) at different relative humidities. Journal of Medical Entomology 31, 310–314. Stafford, K.C. III, Cartter, M.L., Magnarelli, L.A., Ertel, S. and Mshar, P.A. (1998) Temporal correlation between tick abundance and prevalence and increasing incidence of Lyme disease. Journal of Clinical Microbiology 36, 1240–1246. Stafford, K.C. III, DiNicola A.J. and Kilpatrick, H.J. (2003) Reduced abundance of Ixodes scapularis (Acari: Ixodidae) and tick parasitoid Ixodiphagus hookeri (Heminoptera: Encrytidae) with reduction of white-tailed deer. Journal of Medical Entomology 40, 642–652. Swanson, K.I. and Norris, D.E. (2007) Co-circulating microorganisms in questing Ixodes scapularis nymphs in Maryland. Journal of Vector Ecology 32, 243–251. Tavakoli, N.P., Wang, H., Dupuis, M., Hull, R., Ebel, G.D., Gilmore, E.J. and Faust, P.L. (2009) Fatal case of deer tick virus encephalitis. New England Journal of Medicine 360, 2099–2107. Telfer, S., Lambin, X., Birtles, R., Beldomenico, P., Burthe, S., Paterson, S. and Begon, M. (2010) Species interactions in a parasite community drive infection risk in a wildlife population. Science 330, 243–246. Telford, S.R. III and Goethert, H.K. (2004) Emerging tick-borne infections: rediscovered and better characterized, or truly new? Parasitology 129, S1–S27. Telford, S.R., Armstrong, P.M., Katavolos, P., Foppa, I., Olmeda Garcia, S.A., Wilson, M.L. and Spielman, A. (1997) A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini. Emerging Infectious Diseases 3, 165–170. Tsao, J.I., Wootton, J.T., Bunikkis, J., Luna, M.G., Fish, D. and Barbour, A.G. (2004) An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in

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the Lyme disease cycle. Proceedings of the National Academy of Sciences USA 101, 18159–18164. Tyson, K., Elkins, C., Patterson, H., Fikrig, E. and de Silva, A. (2007) Biochemical and functional characterization of Salp20, an Ixodes scapularis tick salivary protein that inhibits the complement pathway. Insect Molecular Biology 16, 469–479. Vail, S.G. and Smith, G. (1998) Air temperature and relative humidity effects on behavioral activity of blacklegged tick (Acari: Ixodidae) nymphs in New Jersey. Journal of Medical Entomology 35, 1025–1028. Vail, S.G. and Smith, G. (2002) Vertical movement and posture of blacklegged ticks (Acari: Ixodidae) nymphs as a function of temperature and relative humidity in laboratory experiments. Journal of Medical Entomology 39, 842–846. Vásquez, M., Muehlenbein, C., Cartter, M., Hayes, E.B., Ertel, S. and Shapiro, E.D. (2008) Effectiveness of personal protective measures to prevent Lyme disease. Emerging Infectious Diseases 14, 210–216. Warren, R.J. (1991) Ecological justification for controlling deer populations in eastern national parks. Transactions of the. North American Wildlife and Natural Resources Conference 56, 56–66. Wesson, D.M., McLain D.K., Oliver, J.H., Piesman, J. and Collins, F.H. (1993) Investigation of the validity of the species status of Ixodes dammini (Acari: Ixodidae) using rDNA. Proceedings of the National Academy of Sciences USA 90, 10221– 10225. Westrom, D.R., Lane, R.S. and Anderson, J.R. (1985) Ixodes pacificus (Acari: Ixodidae): population dynamics and distribution on Columbian black-tailed deer (Odocoileus hemionus columbianus). Journal of Medical Entomology 22, 507–511. White, D.J., Chang, J.H.G., Benach, J.L., Bosler, E.M., Meldrum, S.C., Means, R.G., Debbie, J.G., Birkhead, G.S. and Morse, D.L. (1991) The geographical spread and temporal increase of the Lyme disease epidemic. Journal of the American Medical Association 266, 1230–1236. Wielinga, P.R., Gaasenbeek, C., Fonville, M., de Boer, A., de Vries, A., Dimmers, W., Jagers, G.A.O., Schouls, L.M., Borgsteede, F. and van der Giessen, J.W.B. (2006) Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in the Netherlands. Applied and Environmental Microbiology 72, 7594–7601. Wikel, S.K., Ramachandra, R.N., Bergman, D.K., Burkot, T.R. and Piesman, J. (1997) Infestation

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with pathogen free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks. Infection Immunology 65, 335–338. Williams, S.C., Ward, J.S., Worthley, T.E. and Stafford, K.C. III (2009) Managing Japanese barberry (Ranunculales: Berberidaceae) infestations reduces blacklegged tick (Acari: Ixodidae) abundance and infection prevalence with Borrelia burgdorferi (Spirochaetales: Spirochaetaceae). Environmental Entomology 38, 977–984. Wilson, M.L. and Childs, J.E. (1997) Vertebrate abundance and the epidemiology of zoonotic diseases. In: McShea, W.J., Underwood, H.B. and Rappole J.H. The Science of Overabundance: Deer Ecology and Population Management. Smithsonian Books, Washington, DC, pp 224– 248. Wilson, M.L., Levine, J.F. and Spielman, A. (1984) Effect of deer reduction on abundance of the deer tick. Yale Journal of Biology and Medicine 57, 697–705. Wilson, M.L., Adler, G.H. and Spielman, A. (1985) Correlation between deer abundance and that of the deer tick Ixodes dammini (Acari: Ixodidae). Annals of the Entomological Society of America 78, 172–176.

Wilson, M.L., Telford, S.A., Piesman, J. and Spielman, A. (1988) Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following the elimination of deer. Journal of Medical Entomology 25, 224–228. Wilson, M.L., Ducey, A.M., Litwin, T.S., Gavin, T.A. and Spielman, A. (1990) Microgeographic distribution of immature Ixodes dammini (Acari: Ixodidae) correlated with that of deer. Medical and Veterinary Entomology 4, 151–160. Xu, G., Fang, Q.Q., Kierans, J.E. and Durden, L.A. (2003) Molecular phylogenetic analyses indicate that the Ixodes ricinus complex is a paraphyletic group. Journal of Parasitology 89, 442–457. Yuval, B. and Spielman, A. (1990) Duration and regulation of the developmental cycle of Ixodes dammini. Journal of Medical Entomology 27, 196–201. Zhioua, E., Browning, M., Johnson, P.W., Ginsberg, H.S. and Lebrun, R.A. (1997) Pathogenicity of the entomopathogenic fungus Metarhizium anisopliae (Deuteromycetes) to Ixodes scapularis (Acari: Ixodidae). Journal of Parasitology 83, 815–818. Zonneveld, I. and Foreman, R. (1990) Changing Landscapes: an Ecological Perspective. Springer-Verlag, New York.

2

Borrelia: Biology of the Organism Alvaro Toledo and Jorge L. Benach

2.1 Introduction The spirochaete Borrelia burgdorferi, a tickborne bacterium, is the causative agent of Lyme disease (LD Borrelia) and is widely distributed through the northern hemisphere. Borrelia has a complicated enzootic cycle that involves a tick vector and different vertebrate hosts. In order to survive, the bacterium has to adapt to different environments in the tick and host. Interestingly, Borrelia lacks virulence factors common in other organisms, such as toxins, specialized secretion systems and lipopolysaccharides. On the other hand, the bacterium has a small chromosome but a large number of circular and linear plasmids that enable the spirochaete to complete its natural cycle. Throughout this chapter, we will describe the biology of Borrelia, emphasizing the bacterial components required for infection and the interaction with the immune system.

2.2 History of Lyme Disease The history of Lyme disease began when numerous cases of what appeared to be juvenile rheumatoid arthritis were reported to the Connecticut State Health Department by two mothers with affected children from Lyme (Connecticut). Soon after, a joint investigation by the Connecticut Health

Department and the Yale University School of Medicine evaluated the reports. Active surveillance in the affected towns of Lyme, Old Lyme and East Haddam (Connecticut) disclosed 39 children with recurrent attacks of swelling and pain affecting large joints (Steere et al., 1977a). In addition, 12 adults that lived in the same geographical area also developed similar signs and symptoms. This study concluded that cases were not juvenile rheumatoid arthritis and the disease was named Lyme arthritis. Among the symptoms described, onequarter of the patients developed a characteristic skin lesion, an erythematous papule that developed into an expanding red, annular lesion (Steere et al., 1977a). This manifestation was described previously in Sweden in 1909 by Arvid Afzelius who named it erythema migrans and associated the lesion with the bite of Ixodes ricinus ticks. Hence, Connecticut State Health Department included as part of the sentinel programme developed in the area the follow-up of patients with cutaneous lesions, which led to other findings. Many of these patients developed arthritis, while others had more severe manifestations such as myocardial conduction and neurological abnormalities (Steere et al., 1977b), underscoring the complex, multi-organ system disease that Lyme disease is. The established association between the I. ricinus tick and erythema migrans in Europe

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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led to investigations that showed the association between Ixodes species and Lyme disease. Two studies, on the incidence of Lyme disease and tick distribution, provided indirect evidence of a role of Ixodes scapularis and Ixodes pacificus in Lyme disease (Wallis et al., 1978; Steere and Malawista, 1979). However, initial attempts to isolate the agent were unsuccessful. Interestingly, human babesiosis played an important role in the discovery of the agent of Lyme disease. Babesiosis in humans is caused by the intraerythrocytic spiroplasm Babesia microti. The severity of the infection depends on the immunological status of the host, ranging from subclinical manifestations to death. The finding of transfusion-acquired cases of human babesiosis resulted in a ban of blood collections from endemic areas. Both Babesia and Borrelia share reservoirs and vectors, which leads to frequent coinfections in nature and occasional dual infections in humans. Therefore, a serosurvey for babesiosis (1978–1979) in Shelter Island, NY, also provided serum specimens from individuals diagnosed with Lyme disease. Subsequently, the spirochaete was cultured from ticks collected on Shelter Island in Kelly’s medium, which was designed for the cultivation of a relapsing fever spirochaete, Borrelia hermsii. The reaction of the sera with the newly cultured spirochaetes linked the disease with its causative agent (Burgdorfer et al., 1982). Shortly thereafter, spirochaetes were isolated from patients with Lyme disease (Benach et al., 1983; Steere et al., 1983), confirming the aetiology. Continuing field investigations led to the isolation of the spirochaete from the blood of white-footed mice (Bosler et al., 1983), identifying the main reservoir of the spirochaete. The finding was confirmed in several studies from other parts of the country (Burgdorfer et al., 1988). In addition, other vectors such as I. pacificus were confirmed by culturing the spirochaete (Burgdorfer et al., 1985). Years later, European researchers enlarged the number of B. burgdorferi genospecies when several isolates were proposed as new genospecies. This resulted in the naming of Borrelia afzelii and Borrelia garinii as

well as the designation of B. burgdorferi sensu stricto to the original isolate, with the entire complex being B. burgdorferi sensu lato (Baranton et al., 1992; Canica et al., 1993) (see section 2.3 for more information). Other notable breakthroughs in the field were the development of a murine model for Borrelia infection (Barthold et al., 1988), advances in genetic manipulation (Samuels et al., 1994; Samuels, 1995; Rosa et al., 1996) and sequence of the complete genome of B. burgdorferi (Fraser et al., 1997).

2.3 Taxonomy The diversity of Borrelia species gained the interest of the scientific community from the beginning. Genetic studies have been used as tools to identify the organism and to understand its evolution, the differences among genospecies and their adaptation to vectors and hosts. Molecular techniques used for the identification and typing of microorganisms can be categorized as either phenotypic, such as serotyping, or genetic, such as whole DNA–DNA hybridization (WDDH), on the basis of the macromolecular targets used for analysis. Molecular typing based on the genetic characteristics of microorganisms have been used extensively as they provide more precise information on the diversity of pathogenic bacteria. Although WDDH is considered the gold standard in taxonomy (Wayne, 1988), it is a time-consuming and labour-intensive technique. Moreover, this method is not applicable for bacteria that cannot be cultivated, and the data may not be reproducible (Stackebrandt et al., 2002). Therefore, alternative methods, especially DNA sequence analysis of some highly conserved gene loci, have largely been used. Traditionally, characterization of LD Borrelia has been reliant mainly on amplification and sequencing of certain genes such as rrs (Liveris et al., 1995), fla (Fukunaga and Koreki, 1995) and ospA (Norris et al., 1999) among others. However, the taxonomic value of a single-locus analysis is limited. Recently, it has been shown that multilocus sequence analysis is a useful tool to delineate species

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within the genus Borrelia and it correlates well with the WDDH technique (Richter et al., 2006). This technique has been used to clarify or delineate the taxonomic status of several Borrelia species (Rudenko et al., 2010; Toledo et al., 2010). The genus Borrelia is a tight phylogenetic cluster that is differentiated from other spirochaetal phylogenetic groups by genetic analyses (Paster et al., 1991) (Table 2.1). More than 30 species have been identified within the genus so far. The genus is usually categorized into two major groups, causing Lyme disease (LD Borrelia) and relapsing fever (RF Borrelia), on the basis of ecological and genetic characteristics. There are 17 LD Borrelia genospecies recognized today worldwide, including 11 strictly associated with Eurasia, four associated with North America and two present in both the New World and Old World (Table 2.2). All are transmitted by hard ticks of the genus Ixodes. The RF Borrelia are divided into the epidemic form, caused by the louse-borne Borrelia recurrentis, and the endemic form, caused by different species of Borrelia and transmitted by soft ticks of the genus Ornithodoros. Interestingly, there is a third group of spirochaetes genetically and microbiologically closer to the relapsing fever group but transmitted by hard ticks. Representative species of this group are Borrelia miyamotoi, Borrelia lonestari and Borrelia texasensis isolated respectively from Ixodes persulcatus, Amblyomma americanum and Dermacentor variabilis (Fukunaga et al., 1995; Armstrong et al., 1996; Lin et al., 2005). Different genetic methods have shown that B. burgdorferi has a clonal population structure (Boerlin et al., 1992; Dykhuizen et al., 1993) comprising several different genospecies (Dykhuizen and Baranton, 2001). The different genospecies are grouped under

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the term sensu lato. This grouping generated controversy as there was no evidence that differences were significant enough to warrant the division of Lyme disease spirochaetes into multiple species (Stevenson, 2001). Others maintain that in many cases this genetic classification is not always consistent with ecological differentiation, as a genospecies can comprise divergent ecotypes (Kurtenbach et al., 2002). Although the genospecies concept has limitations, it is commonly used and generally accepted so it is the term that will be used throughout this chapter.

2.4 Life Cycle B. burgdorferi has a complex enzootic cycle in which vectors are always hard ticks and the hosts are mammals and birds. The tick acquires the pathogen by feeding on an infected host. Transovarial transmission seems to be a rare event. On the other hand, the spirochaete is transstadially transmitted through the different tick stages, infecting new hosts every time the tick takes a blood meal. The main competent vectors for LD Borrelia are four tick species that belong to the I. ricinus species complex (Lane et al., 1991). In the USA, I. scapularis (Bosler et al., 1983) and I. pacificus (Burgdorfer et al., 1985) on the east and west coasts, respectively, are the vectors for LD Borrelia, whereas the vector in Europe and northern Africa is I. ricinus (Barbour et al., 1983) and I. persulcatus in Asia (Korenberg et al., 1987). Although their role in human transmission is less important, there are other vectors that participate in the maintenance of LD Borrelia in nature such as I. spinipalpis (Maupin et al., 1994) or I. hexagonus (Gern et al., 1991).

Table 2.1. Biological classification of B. burgdorferi sensu lato from phylum to genus. Phylum Class Order Family Genus

Spirochaetes phyl. nov. Spirochaetes Spirochaetales Spirochaetaceae Borrelia

32

A. Toledo and J.L. Benach

Table 2.2. Geographical origin and pathogenicity of B. burgdorferi genospecies. Geographical Pathogenica distribution

References

B. burgdorferi sensu stricto

Yes

North America

Johnson et al. (1984b)

Europe

Baranton et al. (1992)

B. garinii

Yes

Europe

Baranton et al. (1992)

B. afzelii

Yes

Europe

Baranton et al. (1992); Canica et al. (1993)

Asia

Canica et al. (1993)

B. japonica

No

Asia

Kawabata et al. (1993)

Genospecies

B. andersonii

No

North America

Marconi et al. (1995)

B. tanukii

No

Asia

Fukunaga et al. (1996)

B. turdii

No

Asia

Fukunaga et al. (1996)

B. valaisiana

No

Europe

Wang et al. (1997)

B. lusitaniae

No

Europe

Le Fleche et al. (1997)

B. bissettii

No

North America

Bissett and Hill (1987); Postic et al. (1998)

Europe

Strle et al. (1997)

Asia

Masuzawa et al. (2001)

B. sinica

No

B. turcica

No

Asia

Guner et al. (2004)

B. spielmanii B. carolinensis B. californiensis

Yes No No

Europe North America North America

van Dam et al. (1993); Richter et al. (2004) Rudenko et al. (2010) Postic et al. (2007)

B. bavariensis

No

Europe

Margos et al. (2009)

B. yangtze

No

Asia

Chu et al. (2008)

a Only

species regularly isolated from patients with Lyme disease are considered to be pathogenic.

A large number of vertebrates serve as hosts for B. burgdorferi sensu lato including mammals, birds and reptiles. However, some genospecies have a unique host range that suggests a specific association (Kurtenbach et al., 2002). For instance, it has been shown that birds but not small mammals are competent hosts for Borrelia valaisiana (Kurtenbach et al., 1998). In contrast, small mammals, especially rodents, maintain B. afzelii (Hanincova et al., 2003). Other genospecies such as B. burgdorferi sensu stricto can use both birds and small mammals as hosts (Anderson et al., 1990). Interestingly, lizards were considered zooprophylactic for almost all B. burgdorferi genospecies and act as dilution hosts in parts of North America (Lane and Loye, 1989; Salkeld and Lane, 2010), whereas in Europe, sand and common wall lizards serve as hosts for Borrelia lusitaniae (Richter and Matuschka, 2006).

The different LD Borrelia genospecies, and the large number of hosts and vectors, make the spirochaete life cycle complicated. Actually, more than a life cycle, it resembles a big network, connected by vectors that feed on different hosts, limited only by the host range of the genospecies. A simplified transmission cycle of B. burgdorferi sensu stricto is shown in Plate 1 (see colour plate section): larval ticks acquire the bacterium by feeding on infected hosts (small mammals or birds) by the end of spring and summer. The spirochaete colonizes the digestive tract of the ixodid tick, surviving throughout the molts to the nymphal and adult stages. The following spring, the nymphs feed on a wide range of mammals, from rodents to deer, and occasionally humans. In this stage, the tick can either transmit the spirochaete to the host or acquire it from an infected host if it was not already infected. In the autumn, adult ticks

Borrelia: Biology of the Organism

emerge and feed on hosts, giving the spirochaete an opportunity to infect a new host. Interestingly, the main host for the adult stage of I. scapularis is the white-tailed deer, which does not transmit the spirochaete to ticks (Telford et al., 1988). However, deer play an important role in the maintenance of tick populations (Stafford et al., 2003) and therefore in the presence of the spirochaete in nature and its impact on human health.

2.5 Cultivation of Spirochaetes The successful isolation and cultivation of unknown spirochaete species from ixodid ticks, vertebrate hosts and patients with Lyme disease was based on the efforts and discoveries made by other investigators beginning in the early 20th century. The first attempt to culture a spirochaete in vitro involved Treponema pallidum, the causative agent of syphilis. In 1909, Schereschewsky reported the cultivation of a treponeme in vitro, using a tissue containing the bacteria deep down in a high layer of gelatinized horse serum (Schereschewsky, 1909). However, he never succeeded in obtaining a pure culture of the organism. Mühlens obtained a first generation of a ‘pallida-type’ treponeme pure culture using Schereschewsky’s method to grow the organism for a few generations followed by Mühlens’s horse serum agar (Muhlens, 1909). In 1911, Noguchi reported a method for the pure cultivation of T. pallidum (Noguchi, 1911) and in 1912 he published the cultivation of T. pallidum using a fluid medium for the isolation and a solid medium after the adaptation had taken place (Noguchi, 1912a). Although the treponeme that was propagated by these authors was not T. pallidum, they established the basis for spirochaetal culture. To this day, T. pallidum cannot be cultured in vitro. In addition, Noguchi’s method was successfully used to propagate RF Borrelia and Leptospira (Noguchi, 1912b; Inada et al., 1916). The basis of his method included the use of rabbit kidney as the major source of nutrients, human ascitic fluid free of bile and a thickening agent such as gelatin (Noguchi, 1912b).

33

In 1922, Kligler and Robertson improved Noguchi’s medium for the culture of louseborne B. recurrentis by defining conditions to maintain and grow the spirochaete consistently (Kligler and Robertson, 1922). The major problem with Noguchi’s medium was its inconsistent results. Occasionally, good initial growth was obtained, but it was not always possible to passage the culture sucessfully, even when using the same medium. These authors found that human ascitic fluid from the original formula could be substituted for rabbit serum. However, the major discovery was the observation that growth could only occur between pH 7 and 8. The addition of buffers to the medium, such as peptone water and egg albumin, helped to keep the pH stabilized. They also confirmed that the organism was an aerobe and noted the importance of CO2. There were many other attempts to propagate Borrelia spirochaetes in vitro, most of them based on different formulations of animal sera and fluids identified previously. However, all these attempts failed to grow the bacterium in a continuous and reliable manner. It was not until 1971 that Kelly reported a big breakthrough in Borrelia cultivation. Kelly successfully cultured Borrelia hermsii for 8 months (36 passages), and the organism remained infectious (Kelly, 1971). Kelly’s medium basically had a buffer system, glucose, pyruvate, gelatin, sodium bicarbonate, rabbit serum, bovine albumin and – what seems to be most important – N-acetylglucosamine, the absence of which from the medium reduced growth by 90% (Kelly, 1971). We now know that Borrelia is a limited-genome organism that must obtain many of its biochemical building blocks, including N-acetylglucosamine, from external sources. Kelly’s medium represented a big advance in the field, providing an adequate in vitro environment for borreliae for the first time. However, this medium still had one serious problem, the rather low efficiency in cultivating the organism as it exists naturally in animals. The medium could not support the growth of a blood culture inoculate of less than 800 organisms (Stoenner, 1974). Some modification of the medium or culture

34

A. Toledo and J.L. Benach

procedure was necessary to improve its efficiency for isolating Borrelia from the blood of animals or patients. Stoenner enriched Kelly’s medium by the addition of CMRL tissue culture medium to a 5% concentration (without glutamine and sodium bicarbonate) and yeastolate to a 0.2% concentration to the complex medium, which was subsequently termed fortified Kelly’s medium (Stoenner et al., 1982). This medium was used to isolate and clone spirochaetes from I. scapularis, linking this organism to the disease after the demonstration of its reactivity with sera from patients with Lyme disease (Burgdorfer et al., 1982). In order to simplify the production and improve the medium, new changes were developed sequentially. BSK-I medium (Barbour et al., 1983) based on Stoenner’s medium and BSK-II medium, which differs from the original in the absence of glutamine from CMRL-1066 and the addition of yeastolate, were introduced (Barbour, 1984). Modifications of BSK medium have been reported and used routinely to grow Borrelia from different biological and geographical sources, including a modified Kelly’s medium (Preac-Mursic et al., 1986), and a standardized medium commercially available and termed BSK-H (Pollack et al., 1993) that has been widely used for the isolation and cultivation of B. burgdorferi from different sources. Basically, all the BSK formulations contain N-acetylglucosamine, yeast extract, amino acids, vitamins, nucleotides and serum, but there are differences in some other chemical components. It has been reported that growth, gene expression and infectivity vary depending on the formulation used. This could be due to lot variations in compounds such as bovine serum albumin or other components in the BSK formulation (Callister et al., 1990; Yang et al., 2001). Alternatively, this could be due to other factors including: (i) differential gene/protein expression (Yang et al., 2001; Wang et al., 2004) or (ii) selective overgrowth of clonal and less infectious populations. For instance, the loss of plasmid content during passages is a wellknown factor that leads to a heterogeneous population with different plasmid profiles (Schwan et al., 1988; Grimm et al., 2003)

decreasing the ability of B. burgdorferi to infect (Schwan et al., 1988; Purser and Norris, 2000). However, variations in the components of the medium also may have an attenuation effect on infectivity and pathogenicity of B. burgdorferi isolates independently of plasmid loss (Wang et al., 2004). Interestingly, this phenomenon is not shared by RF Borrelia species, which retain infectivity after serial passages (Kelly, 1971; Lopez et al., 2008). Although culturing borreliae has some undesirable collateral effects, it is necessary to grow B. burgdorferi isolates or clones for subsequent studies on infectivity and pathogenicity in laboratory animals. Also, culturing Borrelia has allowed for the genetic manipulation of the bacterium and has opened an entire new and challenging genetic field for Lyme disease.

2.6 The Preferred Anatomical Locations of the Borrelia LD Borrelia is a highly motile and invasive spirochaete that spreads out from the point of infection, the tick bite, to colonize selected tissues. This migration is possible as a result of the ability of the pathogen to cross the endothelial cell layer (Szczepanski et al., 1990). The invasion is also facilitated by a variety of outer-surface proteins. For instance, proteins such as OspA (Hu et al., 1995), OspC (Lagal et al., 2006) and OspErelated proteins (ERPs) (Brissette et al., 2009b) bind plasminogen, which is subsequently converted into plasmin that degrades the extracellular matrix, enhancing the dissemination of the spirochaete (Coleman et al., 1995, 1999). This is important for an invasive organism like Borrelia that lacks exoproteolitic activity and has to ‘borrow it’ from the host. Other proteins such as the complement regulator-acquiring surface proteins (CRASPs) bind components of the host complement leading to natural resistance to this innate immune defence. It is also known that Borrelia interacts with other host proteins such as glycosaminoglycans, integrins and fibronectin receptors. The spirochaete affects: (i) the skin, causing different cutaneous manifestations

Borrelia: Biology of the Organism

such as erythema migrans (EM), borrelial lymphocytoma (BL) and acrodermatitis chronica atrophicans (ACA), which were known as distinct skin entities long before they were linked to Lyme disease (Braathen et al., 1987); (ii) the nervous system, causing neuroborreliosis, which can be classified according to the location of the lesion within the neuraxis or according to the time course of the infection (Garcia-Monco and Benach, 1995); (iii) cardiac tissue, characterized by a self-limited conduction derangement, most commonly involving the atrioventricular node (Fish et al., 2008); and (iv) the joints, with arthritis as a late-stage manifestation of Lyme disease that usually occurs months after the onset of the disease. These topics are covered in greater detail in specific chapters in this book.

2.7 The Borrelia Genome The genome of Borrelia is complex and consists of a small linear chromosome of ~900 kb, which carries the vast majority of the housekeeping genes and is constant in gene content and organization across the genus. On the other hand, plasmids are much more variable and encode most of the differentially expressed genes. Interestingly, Borrelia has a variable number of circular and linear plasmids, typically 12 linear and nine circular plasmids in B. burgdorferi strain B31. The presence of linear plasmids is uncommon in bacteria; whether these represent an advantage to Borrelia is not known. The genome of Lyme disease spirochaetes reveals interesting features (Casjens et al., 2010). The small genome size of B. burgdorferi is associated with the absence of genes for the synthesis of amino acids, fatty acids, enzyme cofactors and nucleotides. The lack of biosynthetic pathways explains why B. burgdorferi is such a fastidious organism to culture and has strict nutrient requirements. This is also consistent with previous biochemical data indicating that Borrelia lack the ability to elongate long-chain fatty acids, such that the fatty-acid composition of Borrelia cells reflects that present in the growth medium (Fraser et al., 1997; Casjens, 2000).

35

The genome of the Lyme disease spirochaete provided a new starting point for the study of the pathogenesis, prevention and treatment of Lyme disease. This organism, with the exception of a small number of putative virulence genes, contains few recognizable genes involved in virulence or host–parasite interactions. Lipoproteins, which represent more than 8% of coding sequences, are tightly regulated and have been shown to trigger components of the mammalian immune system. Certain lipoproteins such as OspC and VlsE have been shown to be required for infection or persistence in the vertebrate host. Interestingly, these proteins display significant differences at the nucleotide and amino acid level (JaurisHeipke et al., 1993; Livey et al., 1995; Glockner et al., 2006). A large number of genes, more than 6% of the B. burgdorferi chromosome, arranged in eight operons (Fraser et al., 1997), code for proteins involved in motility and chemotaxis. Motility is essential for B. burgdorferi, whose unique flagellar structure confers the ability to move through viscous solutions. The flagellae are in the periplasmic space, inserted at each end of the cell, and extend towards the middle of the cell body. One of the most interesting features of the genome of B. burgdorferi is the presence of linear and circular plasmids. The plasmids are unusual, compared with most bacterial plasmids, in that they contain many paralogous sequences (two genes or clusters of genes at different chromosomal locations in the same organism, structurally derived by duplication from a common ancestral gene, that have since diverged from the parent copy, evolving new functions, by mutation and selection or drift), a large number of pseudogenes and, in some cases, essential genes (Fraser et al., 1997; Casjens et al., 2000). A number of plasmids, belonging to the family cp32, are prophages (Zhang and Marconi, 2005). The study of the function of endogenous plasmids is vital for understanding the pathogenesis of B. burgdorferi. Although the function of the product of many of the multi-copy plasmid-encoded genes is not yet known, some are involved with infection and virulence while others are related to overall fitness. Plasmids such as

36

A. Toledo and J.L. Benach

lp25 and lp28-1 are important for infection and virulence in the mammalian host (Purser and Norris, 2000; Grimm et al., 2004a). The lack of either of these plasmids results in noninfectious variants in mice, while restoration of plasmid lp25 or lp28-1 in non-infectious clones that naturally lack the corresponding plasmid re-establishes infectivity in mice (Grimm et al., 2004a). Plasmids lp25 and lp28-1 are unstable during in vitro propagation (Xu et al., 1996; Casjens et al., 2000; LabandeiraRey and Skare, 2001; Grimm et al., 2004a). Although the loss of either lp25 or lp28-1 renders the bacterium incapable of infecting mice, this does not affect the overall fitness of the bacterium in an in vitro environment. Both plasmids carry important virulence factors such as nicotinamidase (Purser et al., 2003) that are required for infection. In contrast, the linear plasmid lp54, which is present in all characterized B. burgdorferi isolates, is stable, is maintained during in vitro propagation and is critical for the overall fitness of the spirochaete.

2.8 The Morphology of Borrelia The factors involved in bacterial shape are varied and complex. In most cases, cell morphology is directly associated with the peptidoglycan layer. Several enzymes and proteins have complex associations and roles in morphology. However, not all bacteria have peptidoglycans. In members of the Archaea and Mycoplasmataceae, the shape is associated with an S-layer protein array. In others, such as the genus Spiroplasma, helically shaped bacteria that lack peptidoglycan, the factors that determine the morphology of the bacteria are unknown. Borrelia typically have a flat-wave morphology (Fig. 2.1). This is the result of a complex interaction between the periplasmic flagella and the cell cylinder. The spirochaete has a unique flagella arrangement, which consists of a variable number of seven to 11 helically shaped flagella (Barbour and Hayes, 1986) that are inserted at each end of the cell cylinder and extend towards the middle of the cell body. The flagella are involved in both the morphology and motility of the bacterium

(Motaleb et al., 2000). Inactivation of the flaB gene, which encodes the major periplasmic flagellar filament protein (FlaB), results in non-motile and rod-shaped mutants. Recently, Dombrowski et al. (2009) demonstrated, using a mathematical approach, that the mechanical coupling of the helical periplasmic flagella to the rod-shaped cell cylinder is sufficient to cause the flat-wave morphology of B. burgdorferi. The periplasmic space of B. burgdorferi is narrow but widens in the vicinity of the flagella. These periplasmic flagella form a tight-fitting ribbon that wraps around the protoplasmic cell cylinder in a right-handed sense (Charon et al., 2009). This configuration is more advantageous than a bundle for both swimming and forming the flat-wave morphology (Charon et al., 2009). The morphology of B. burgdorferi is implicitly connected to motility, and motility is likely to be essential for these organisms to cause disease (Sellek et al., 2002; Botkin et al., 2006). The arrangement of the periplasmic flagella confers two main advantages to Borrelia – the ability to swim in both low and high viscosity media, and the localization of the flagella in the perisplasmic space, underneath the membrane and therefore hidden from the host’s immune system.

2.9 Prominent Borrelia Antigens and Virulence Factors The protein profile of B. burgdorferi changes during the life cycle of the spirochaete. Some proteins play an important role in the adaptation and mid-gut colonization of the tick, whereas others are produced in response to a blood meal when the tick starts feeding. Two outer-surface proteins, OspA and OspB, are predominantly expressed by the spirochaete in the tick gut and in culture. Both OspA and OspB are abundant on the surface of bacteria in ticks, but are downregulated during tick feeding and in the subsequent transmission to a host (Schwan et al., 1995). Studies on the role of OspA and OspB in traffic through the tick suggest that these are adhesins that mediate the attachment and colonization of the tick midgut (Pal et al., 2000; Fikrig et al., 2004;

Borrelia: Biology of the Organism

(a)

(c)

37

(b)

(d)

Fig. 2.1. (a) Morphology of Borrelia. (b) Membrane damage resulting in flagella exposure and lost shape. (c, d) Detail of the Borrelia membrane displaying the outer membrane and the protoplasmic cylinder. Bars, 2 m (a, b); 100 nm (c); 500 nm (d).

Neelakanta et al., 2007). These proteins, together with BptA, a lipoprotein of unknown function (Revel et al., 2005), a putative Dps homologue (BB0690) (Li et al., 2007), the product of the gene BB0365 (Pal et al., 2008) and the immunogenic protein encoded by BB0323 (Zhang et al., 2009), among others, are essential for bacterial survival in ticks. The association of specific plasmid loss through culture passages with loss of infectivity (Johnson et al., 1984a; Schwan et al., 1988; Xu and Johnson, 1995; Purser and Norris, 2000; Labandeira-Rey and Skare, 2001) led to the initial efforts to identify genes required for infection. Plasmids lp25 and lp28-1 are critical for infectivity and encode virulence factors important in the pathogenesis of B. burgdorferi infection (Purser and Norris, 2000). Some of the genes required for mammalian infection play a physiological

role. For instance, PncA, a nicotinamidase required for the biosynthesis of NAD and encoded within lp25, is required for infection (Purser et al., 2003). Other factors such as OspC, which is only required in the early stages of infection (Grimm et al., 2004b; Tilly et al., 2006, 2009), are important for host colonization or innate immune evasion. Interestingly, OspC is required to establish infection in the host through a tick bite or needle inoculation, but B. burgdorferi lacking OspC can be transmitted to a naïve mouse by implanting tissue pieces of an infected mouse (Tilly et al., 2009). OspC binds plasminogen (Lagal et al., 2006), a trait shared by OspA and some ERPs (Fuchs et al., 1994; Brissette et al., 2009b). This binding probably facilitates infection and dissemination of B. burgdorferi (Coleman et al., 1995, 1997), but its specific role in infection is still unknown.

38

A. Toledo and J.L. Benach

VlsE is a protein known to be required for persistent infection and is synthesized around the same time that OspC production ceases (Glockner et al., 2006). The function of VlsE has not yet been defined and, interestingly, the full length VlsE is not required for survival in vivo (Coutte et al., 2009), suggesting that whatever the role of VlsE, it does not depend on its length. The lipoprotein has a refined system for variation (Zhang et al., 1997) that allows the spirochaete to express it on the bacterial surface during infection without being cleared by the host immune system. Recently, it has been shown that the recombination process of VlsE is mediated by the RuvAB complex (Dresser et al., 2009; Lin et al., 2009) and it is RecAindependent (Liveris et al., 2008). The spirochaete can also inhibit the action of complement by CRASPs that bind factor H and factor H-like proteins, blocking activation of the alternative pathway (reviewed by Bykowski et al., 2008). Factor H-coated organisms would presumably be protected from the action of complement, facilitating infection. Surprisingly, both wildtype and factor H-deficient mice can be infected by B. burgdorferi with no differences in terms of infection, suggesting that factor H binding does not confer any specific protection (Woodman et al., 2007). Other putative virulence factors are associated with the ability of LD Borrelia to interact with components of the extracellular matrix (ECM), potentially using the ECM tissue to hide from antibodies. There are a variety of proteins in LD Borrelia that interact with the ECM: decorin-binding proteins A and B (DbpA and DbpB) bind decorin, BBK32 and RevA bind fibronectin (Probert and Johnson, 1998; Brissette et al., 2009a), OspA binds proteoglycans (Rupprecht et al., 2006), P66 binds integrins (Defoe and Coburn, 2001) and Bgp is a glycosaminoglycan-binding protein (Parveen and Leong, 2000). Studies using mutants found that DbpA and DbpB are not essential for infection in mice but play a critical role in overall virulence (Shi et al., 2006, 2008; Weening et al., 2008). As mentioned above, B. burgdorferi is maintained in an enzootic cycle alternating between tick vectors and vertebrate hosts. In

the transition from the tick to the vertebrate milieu, the spirochaete undergoes a dramatic switch in gene expression. The transcriptional initiation is modulated by sigma factors (), which bind the RNA polymerase core to form an RNA polymerase holoenzyme, providing specificity recognition. The most common  factor among bacteria is 70, which in general controls housekeeping genes. Most bacterial species have alternative  factors that recognize specific promoter sequences, controlling the transcription of selected genes in response to certain environmental signals. The spirochaete upregulates a large number of genes in response to the host milieu (Tokarz et al., 2004) controlled by two alternative factors, RpoN (54) and RpoS (S), which constitute the 54-S cascade. Many genes are controlled by the 54-S cascades, most notably ospC, in response to environmental changes, which enable the spirochaetes to establish mammalian infection following tick inoculation (Hubner et al., 2001; Yang et al., 2003; Smith et al., 2007; Ouyang et al., 2008; Skare et al., 2010). Recently, BosR/Fur (BB0647) a novel DNA-binding protein in the Fur/PerR family of transcriptional regulators (Boylan et al., 2003; Katona et al., 2004) has been shown to be required for the induction of RpoS and its regulon (Hyde et al., 2009; Ouyang et al., 2009). This is consistent with expression of the bosR/fur gene being at its highest during mammalian infection (Medrano et al., 2007) and with BosR/Fur being required for infectivity (Hyde et al., 2009; Ouyang et al., 2009). In addition, BosR/Fur regulates 50 genes whose expression is independent of RpoS (Ouyang et al., 2009).

2.10 The Host Response to Borrelia 2.10.1 The role of antibodies Antibody generation plays a major role in the control of B. burgdorferi. Mice lacking antibody production – SCID (severe combined immunodeficiency) and rag–/– – develop persistent arthritis and carditis, underscoring the importance of the humoral response in Lyme disease resolution. Adoptive transfer

Borrelia: Biology of the Organism

experiments confirmed that B-cell and pathogen-specific antibodies are required to clear the pathogen and for disease regression. The first demonstration of the pivotal role of antibodies in defence against Borrelia infection in vivo was the finding that the passive transfer of immune sera protects against bacterial infection (Johnson et al., 1986). Subsequent studies further demonstrated the importance of antibodies as primary mediators, as passive transfer of immune sera protected SCID mice, which lack both B cells and T cells, against Borrelia (Schaible et al., 1990). The protective capacity of sera against specific Borrelia antigens and monoclonal antibodies derived from immune sera has been studied extensively (Schaible et al., 1990; Barthold and Bockenstedt, 1993; Fikrig et al., 1994; Barthold et al., 1997; Zhong et al., 1997;). The importance of antibodies in combating Borrelia infection has also been proven in immunization studies where OspA, OspC, P66 and DbpA, among other proteins, elicited a protective antibody response (Fikrig et al., 1992; Bockenstedt et al., 1997; Hanson et al., 1998; Exner et al., 2000). Phagocytes are capable of ingesting and killing spirochaetes (Benach et al., 1984) and one of the important functions of specific antibodies is their participation in macrophage-mediated control of spirochaetes, as coiling phagocytosis and generation of NO and O2– radicals are significantly enhanced by opsonization of spirochaetes with monoclonal antibodies (Rittig et al., 1992; Modolell et al., 1994; Connolly and Benach, 2005). In the big picture of the host response, antibodies work as the bridge between the innate and acquired immune responses by opsonizing Borrelia, which are subsequently removed by macrophages via Fc receptors on phagocytes. This seems to be the best method of removing Borrelia spirochaetes as the bacterium has the ability to evade the complement cascade. 2.10.2 Complement In order to establish infection in a mammalian host, LD Borrelia has to overcome the innate immune response. A rapid and powerful component of the innate immune response is

39

the complement system, which can be activated through three different pathways: classical, alternative and mannan-binding lectin. The pathways differ in their initial steps and method of recruitment, but produce similar results, which include opsonization, inflammatory cell recruitment and the formation of the membrane attack complex (MAC) (see Plate 2 in colour plate section). In the absence of specific antibodies, B. burgdorferi is resistant to the bactericidal action of complement, despite the capacity of the spirochaete to activate complement. Complement-mediated killing of B. burgdorferi requires the presence of anti-borrelial IgG (Kochi and Johnson, 1988). IgG includes antigen-binding (Fab) and crystallizable (Fc) fragments. The Fab fragment mediates B. burgdorferi killing by itself, as the complementactivating domain of IgG, the Fc fragment, is not required for killing the bacterium (Kochi et al., 1993). However, the killing efficiency of the Fab fragments is less than that of intact IgG. The antibody also alters the bacterial outer membrane to allow effective MAC formation (Kochi et al., 1991). There are numerous monoclonal antibodies and antisera to borreliae that depend on complement for bactericidal activity in vitro (reviewed by LaRocca and Benach, 2008), as would be expected in the host response to an extracellular pathogen. Interestingly, there are complementindependent immunoglobulins with bactericidal effects on both RF and LD Borrelia (Escudero et al., 1997; Connolly and Benach, 2001). The bactericidal action resides in the variable region of the antibody (LaRocca et al., 2008). In LD Borrelia, the antibody recognizes the OspB, binding and pulling it out, thus increasing the permeability of the outer membrane and resulting in an osmotic imbalance that kills the organism (LaRocca et al., 2009). The bactericidal effect of this antibody is not transferable to Escherichia coli expressing OspB (LaRocca et al., 2009). 2.10.3 B-cell responses B-cell populations can be divided into two sets, B1 and B2, each with two subsets. B1

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cells contain the B1a and B1b subsets, found mainly in the pleural and peritoneal cavities. These subsets are T-cell-independent and self-renewing, are responsible for natural IgM secretion and can be driven to expand and secrete specific IgM, assuming an important role in the early response to infection (Martin and Kearney, 2001). B2 cells contain follicular (FO) and marginal zone (MZ) subsets, which account for the majority of B cells in the host. FO B cells are T-celldependent, and generally depend on the T helper 2 (Th2) phenotype, whereas MZ B cells are T-cell-independent. In both Lyme disease and relapsing fever infections, T-celldependent and T-cell-independent responses are triggered. The importance of B-cell activation in the control of B. burgdorferi is well known. Studies on deficient SCID mice proved that passive transfer of sera from immunocompetent or T-cell-deficient mice reduced the spirochaete burden (McKisic and Barthold, 2000). CD40L-deficient mice are capable of producing protective antibodies, despite the inability of these mice to mediate T-celldependent immune responses (Fikrig et al., 1996). Therefore, T-cell-independent B-cell responses are sufficient to prevent infection and reduce the bacterial burden (Fikrig et al., 1996; McKisic and Barthold, 2000). B cells can produce different antibody subclasses. Among them, IgM antibodies have been shown to be sufficient to reduce the spirochaete load in ticks feeding on mice, whereas the IgG subclass had no effect (Belperron and Bockenstedt, 2001). IgG antibodies are bound in the tick’s haemolymph by immunoglobulin-binding proteins and are excreted into the host again through the tick’s saliva (Wang and Nuttall, 1999). MZ B cells encounter Borrelia in the marginal sinuses of the spleen in mice, resulting in their expansion (Malkiel et al., 2009). MZ B cells produce specific IgM against T-cell-independent blood-borne particulate antigens (Martin et al., 2001) and their depletion results in higher bacterial burdens and more severe arthritis (Belperron et al., 2007). MZ B cells bind and transport IgM immune complexes to follicular dendritic

cells, assisting in the priming of naïve T cells (Ferguson et al., 2004). Other B cells that produce IgM are B1 cell sets and FO B cells, which produce IgM prior to isotype switching after interaction with activated T cells. An adaptive immune response results in T-cell activation and FO B-cell production of switched antibodies to Borrelia. Although the production of IgG is not required for a protective response, it contributes to the course of the infection. Some antigens elicit a protective immune response after immunization that seems to be strain-dependent (Barthold et al., 1997). In a SCID mouse model, polyclonal antisera to arthritis-related proteins, passively transferred to infected mice, attenuated the disease by eliminating the bacterium in selective tissues (Barthold et al., 2006). 2.10.4 Innate immune response Immune-mediated host defence mechanisms play a pivotal role during the course of Borrelia infection. The innate immune response is the first defensive line against the infection; its recognition of the pathogen and activation are critical in spirochaete clearance. The innate immune response also plays an important role in the modulation of adaptive responses. The innate immune system recognizes the pathogen via several classes of pattern recognition receptors (PRRs), some of which have been suggested to be involved in the recognition of Borrelia: Toll-like receptors (TLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs). Once Borrelia has been recognized by PRRs, a signalling cascade is triggered leading to the production of pro-inflammatory cytokines. Among the TLRs receptors, TLR2 has been found to be the most important as it has been identified as the signal-transducing receptor for bacterial lipoproteins, in a CD14dependent manner, leading to nuclear translocation of the inflammatory transcription factor NF-κB, which provides a mechanism for the initiation and modification of inflammatory events associated with Lyme disease (Sellati et al., 1998; Aliprantis et al.,

Borrelia: Biology of the Organism

1999; Brightbill et al., 1999; Hirschfeld et al., 1999; Lien et al., 1999). However, experiments using mice deficient in CD14 (Benhnia et al., 2005), TLR2 (Wooten et al., 2002) and the TLR adapter protein myeloid differentiation factor 88 (MyD88) (Liu et al., 2004) suggested that LD Borrelia may use additional TLRindependent pathways to induce inflammation. Live spirochaetes also induce, within the phagolysosomes, TLR2-independent responses that are distinct from those generated by lipoproteins (Salazar et al., 2009). TLR2 is required for innate but not acquired host defences to LD Borrelia (Wooten et al., 2002). Recently, infections of peritoneal macrophages from mice deficient in receptorinteracting serine/threonine kinase (RICK) with LD Borrelia indicated that, in addition to the known TLR2, recognition of peptidoglycan receptor NOD2 plays a role in recognition of the spirochaete and in induction of cytokines (Oosting et al., 2010). Finally, the mannose receptor may play a role in this process, given that it binds to Borrelia, but uptake of spirochaetes through the mannose receptor has not been described (Cinco et al., 2001; Oosting et al., 2010). Recently, invariant (i) T-cell receptor- chain natural killer cells (iNKT cells) have been shown to be important in the immune response against LD Borrelia. Most of the iNKT cells are in the liver sinusoids but they can also be found in the spleen and lungs and in small amounts in the lymph nodes (Olson et al., 2009). The liver sinusoids are a narrow capillary network that serves as a filter to prevent the dissemination of pathogens via the blood to other organs or tissues such as the joints (Tupin et al., 2008). iNKT cells are activated by -galactosylceramide via CD1d, producing large amounts of gamma interferon (IFN-) and interleukin (IL)-4 (Geissmann et al., 2005). In the liver, Kupffer cells ingest the spirochaetes and present antigen via CD1d to iNKT and form clusters on Kupffer cells, which leads to the production of IFN- but only small amounts of IL-4 (Tupin et al., 2008; Lee et al., 2010). The iNKT cells have an important role in the clearance of spirochaetes from blood, therefore limiting

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their dissemination to organs and tissues such as joints and the heart (Tupin et al., 2008; Olson et al., 2009). 2.10.5 T-cell response The contribution of T cells in the immune response to Borrelia is not well defined. However, Th1/Th2 cytokine expression balance – and more recently Th17 – has been associated with the development and control of Lyme arthritis in mouse models. Studies on resistant BALB/c and susceptible C3H mice have suggested that CD4+ T cells are required for immunological control of spirochaete levels and affect the severity of arthritis compared with infected mice treated with a control monoclonal antibody. In contrast, the CD8+ T-cell compartment, particularly in susceptible C3H mice, appears to promote the disease process, as elimination of this subset in vivo leads to a reduction in both arthritis and spirochaete levels in joints and skin when compared with infected control mice (Keane-Myers and Nickell, 1995b). Studies on CD4+ Th1 and Th2 cytokines associated IFN- (Th1) with high spirochaete burden and increased inflammation, whereas IL-4 (Th2) was associated with milder disease (Keane-Myers and Nickell, 1995a; KeaneMyers et al., 1996). Interference with Th1-cell priming, with anti-IL-12 monoclonal antibodies, resulted in IFN- and IgG2a reduction and a decrease in the severity of the disease in C3H mice (Anguita et al., 1996), giving further support for a role for Th1 cells in disease severity. In contrast, a study blocking the B7/ CD28 pathway, which has been shown to influence the differentiation of Th-cell subsets, resulted in the elimination of IL-4 and upregulation of IFN- responses by B. burgdorferi-specific T cells and in the reduction of B. burgdorferi-specific IgG in a BALB/c mouse model. Despite the shift toward a Th1 cytokine pattern, no exacerbation of arthritis was seen. Subsequent studies using C3H IFN-–/–, DBA IL-4–/–, C57BL/6 and BALB/c mice deficient in IL-4 showed the same

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arthritis severity as in wild-type counterparts and in the disease-resistant 129/SvEv mouse (Brown and Reiner, 1999; Potter et al., 2000; Glickstein et al., 2001). Therefore, these studies suggest that susceptibility/resistance to Lyme arthritis in mice does not depend on a Th1/Th2 cytokine balance. Two other related T-cell subsets, Th17 and CD4+ CD25+Foxp3+ T regulatory (Treg) cells, have been associated with arthritis development in Lyme disease. These subsets have a common precursor and represent a distinct lineage from Th1 and Th2 cells (Harrington et al., 2006). Lyme disease-vaccinated B56BL/6 IFN–/– mice subsequently challenged with B. burgdorferi developed a prominent chronic arthritis. Treatment with anti-IL-17 not only delayed the onset of the swelling but also inhibited the development of arthritis (Burchill et al., 2003). This same mouse model has been used to show that IL-15 and IL-23, which are required for IL-17 production, are also required for induction of arthritis (Amlong et al., 2006; Kotloski et al., 2008) and to show the role of IL-6 and transforming growth factor-, which induce the production of IL-17, in the severity of the arthritis (Nardelli et al., 2008). Reduction of IL-17 has been associated with an increase of Treg cells; a balance between IL-17 and Treg cells could contribute to arthritis severity. In a CD28–/– mouse model that lacked Treg cells, the severity and duration of arthritis was greater compared with a control group (Shahinian et al., 1993; Iliopoulou et al., 2007). In both patients and the murine model, those with lower numbers of Treg cells seemed unable to resolve the arthritis (Iliopoulou et al., 2007; Shen et al., 2010). 2.10.6 Autoimmunity About 10% of treated patients develop a chronic arthritis than can last from months to years (Steere et al., 1994). This condition does not improve with antibiotics and the Lyme disease agent is not detectable in synovial fluid by PCR (Carlson et al., 1999). The putative autoimmune response could be

triggered by OspA within an inflammatory milieu, as treatment-resistant arthritis and treatment-responsive arthritis differ in the humoral and cellular response to OspA (Chen et al., 1999). Moreover, T cells in the synovium express an adhesion molecule, human lymphocyte function-associated antigen 1 (hLFA-1), which has a molecular resemblance to an immune-dominant T-cell epitope of B. burgdorferi OspA (OspA165–173) (Gross et al., 1998). On the other hand, this phenomenon also seems to depend on the genetic predisposition of the patient. Some clinical studies associate the human leukocyte antigen (HLA)-DR locus with the incidence and severity of chronic LA (Pfluger et al., 1989; Steere et al., 1990). HLA-DR4 has been associated with prolonged severe arthritis, and more recently HLA-DRB1*0401 or HLADRB1*0101 has been associated with chronic LA (Steere and Glickstein, 2004). To elucidate the role of HLA-DR alleles and the mechanisms that lead to chronic LA, a mouse model was developed recently (Iliopoulou et al., 2008; Iliopoulou et al., 2009). A CD28–/– mouse with DR4+/+ CD28–/– developed joint inflammation even after antibiotic therapy, in contrast to CD28–/–. Additional hallmarks suggested a Th1 pro-inflammatory response (Iliopoulou et al., 2008). A study on transgenic DR4 and DR11 mice indicated that DR11 is associated with a protective autoimmune response, whereas DR4 predisposed to a strong inflammatory response (Iliopoulou et al., 2009). Additionally, when the DR11 allele was introduced on to the CD28–/– background, the mice did not develop chronic LA (Iliopoulou et al., 2009). Although the possibility of an autoimmune component to Lyme disease cannot be dismissed based on these findings, it is also difficult to prove that autoimmunity is a major component of the pathogenesis of this spirochaetosis.

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Borrelia: Biology of the Organism

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Revel, A.T., Blevins, J.S., Almazan, C., Neil, L., Kocan, K.M., De La Fuente, J., Hagman, K.E. and Norgard, M.V. (2005) bptA (bbe16) is essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector. Proceedings of the National Academy of Sciences USA 102, 6972–6977. Richter, D. and Matuschka, F.R. (2006) Perpetuation of the Lyme disease spirochete Borrelia lusitaniae by lizards. Applied and Environmental Microbiology 72, 4627–4632. Richter, D., Schlee, D.B., Allgower, R. and Matuschka, F.R. (2004) Relationships of a novel Lyme disease spirochete, Borrelia spielmani sp. nov., with its hosts in Central Europe. Applied and Environmental Microbiology 70, 6414– 6419. Richter, D., Postic, D., Sertour, N., Livey, I., Matuschka, F.R. and Baranton, G. (2006) Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov. International Journal Systematic and Evolutionary Microbiology 56, 873–881. Rittig, M.G., Krause, A., Haupl, T., Schaible, U.E., Modolell, M., Kramer, M.D., Lutjen-Drecoll, E., Simon, M.M. and Burmester, G.R. (1992) Coiling phagocytosis is the preferential phagocytic mechanism for Borrelia burgdorferi. Infection and Immunity 60, 4205–4212. Rosa, P., Samuels, D.S., Hogan, D., Stevenson, B., Casjens, S. and Tilly, K. (1996) Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi. Journal of Bacteriology 178, 5946–5953. Rudenko, N., Golovchenko, M., Grubhoffer, L. and Oliver, J.H. Jr (2010) Borrelia carolinensis sp. nov., a new species of Borrelia burgdorferi sensu lato isolated from rodents and tick from the southeastern United States. International Journal Systematic and Evolutionary Microbiology 61, 381–383. Rupprecht, T.A., Koedel, U., Heimerl, C., Fingerle, V., Paul, R., Wilske, B. and Pfister, H.W. (2006) Adhesion of Borrelia garinii to neuronal cells is mediated by the interaction of OspA with proteoglycans. Journal of Neuroimmunology 175, 5–11. Salazar, J.C., Duhnam-Ems, S., La Vake, C., Cruz, A.R., Moore, M.W., Caimano, M.J., VelezCliment, L., Shupe, J., Krueger, W. and Radolf, J.D. (2009) Activation of human monocytes by live Borrelia burgdorferi generates TLR2dependent and -independent responses which include induction of IFN-. PLoS Pathogens 5, e1000444.

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fever Borrelia species isolated from patients. Journal of Clinical Microbiology 48, 2484–2489. Tupin, E., Benhnia, M.R., Kinjo, Y., Patsey, R., Lena, C.J., Haller, M.C., Caimano, M.J., Imamura, M., Wong, C.H., Crotty, S., Radolf, J.D., Sellati, T.J. and Kronenberg, M. (2008) NKT cells prevent chronic joint inflammation after infection with Borrelia burgdorferi. Proceedings of the National Academy of Sciences USA 105, 19863–8. van Dam, A.P., Kuiper, H., Vos, K., Widjojokusumo, A., De Jongh, B.M., Spanjaard, L., Ramselaar, A.C., Kramer, M.D. and Dankert, J. (1993) Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clinical Infectious Diseases 17, 708–717. Wallis, R.C., Brown, S.E., Kloter, K.O. and Main, A.J. Jr (1978) Erythema chronicum migrans and Lyme arthritis: field study of ticks. American Journal of Epidemiology 108, 322–327. Wang, G., van Dam, A.P., Le Fleche, A., Postic, D., Peter, O., Baranton, G., De Boer, R., Spanjaard, L. and Dankert, J. (1997) Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19). International Journal of Systematic Bacteriology 47, 926–932. Wang, G., Iyer, R., Bittker, S., Cooper, D., Small, J., Wormser, G.P. and Schwartz, I. (2004) Variations in Barbour–Stoenner–Kelly culture medium modulate infectivity and pathogenicity of Borrelia burgdorferi clinical isolates. Infection and Immunity 72, 6702–6706. Wang, H. and Nuttall, P.A. (1999) Immunoglobulinbinding proteins in ticks: new target for vaccine development against a blood-feeding parasite. Cellular and Molecular Life Sciences 56, 286– 295. Wayne, L.G. (1988) International Committee on Systematic Bacteriology: announcement of the report of the ad hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Zentralblatt fur Bakteriologie Mikrobiologie und Hygiene A, 268, 433–434. Weening, E.H., Parveen, N., Trzeciakowski, J.P., Leong, J.M., Hook, M. and Skare, J.T. (2008) Borrelia burgdorferi lacking DbpBA exhibits an early survival defect during experimental infection. Infection and Immunity 76, 5694– 5705. Woodman, M.E., Cooley, A.E., Miller, J.C., Lazarus, J.J., Tucker, K., Bykowski, T., Botto, M., Hellwage, J., Wooten, R.M. and Stevenson, B. (2007) Borrelia burgdorferi binding of host complement regulator factor H is not required for efficient mammalian infection. Infection and Immunity 75, 3131–3139. Wooten, R.M., Ma, Y., Yoder, R.A., Brown, J.P.,

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Zhang, H. and Marconi, R.T. (2005) Demonstration of cotranscription and 1-methyl-3-nitrosonitroguanidine induction of a 30-gene operon of Borrelia burgdorferi: evidence that the 32-kilobase circular plasmids are prophages. Journal of Bacteriology 187, 7985–7995. Zhang, J.R., Hardham, J.M., Barbour, A.G. and Norris, S.J. (1997) Antigenic variation in Lyme disease borreliae by promiscuous recombination of Vmp-like sequence cassettes. Cell 89, 275– 285. Zhang, X., Yang, X., Kumar, M. and Pal, U. (2009) BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle. Journal of Infectious Diseases 200, 1318–1330. Zhong, W., Gern, L., Kramer, M., Wallich, R. and Simon, M.M. (1997) T helper cell priming of mice to Borrelia burgdorferi OspA leads to induction of protective antibodies following experimental but not tick-borne infection. European Journal of Immunology 27, 2942– 2947.

3

Borrelia: Interactions with the Host Immune System Raymond J. Dattwyler and Kirk Sperber

3.1 Introduction In this chapter, we will link the scientific advances of recent decades with the clinical aspects of Lyme disease to put this illness in an appropriate scientific context. The illness that we know as Lyme disease has been recognized since the early part of the 20th century. None the less, virtually all of the scientific advances that led to the present views of this infectious process took place after Burgdorfer and colleagues identified a new Borrelia species in 1982 (Burgdorfer et al., 1982) and two groups, one led by Jorge Benach (Benach et al., 1983) and the other by Allen Steere (Steere et al., 1983) isolated this newly described spirochaete from patients with Lyme disease. This seminal work established this Borrelia species as the agent causing Lyme disease. The pathogen was named Borrelia burgdorferi after Wille Burgdorfer. With the establishment of B. burgdorferi as the etiological agent of Lyme disease, it became evident that an array of clinical syndromes described in Europe beginning in the early 20th century were manifestations of infection with B. burgdorferi senso lato species complex spirochaetes. Erythema migrans (EM), the skin lesion of early disease, was first described in 1909 by Afzelius (Afzelius, 1910), speaking to the Swedish Academy of Dermatology. He hypothesized that EM 54

resulted from the tick-borne transmission to humans of a zoonotic pathogen (Burgdorfer, 1986). In 1921, Lipshitz identified Ixodes ricinus as a vector (Burgdorfer, 1986). By the 1940s, European investigators had associated EM with different neurological, other dermatological and musculoskeletal disorders (Buchwald 1883; Herxheimer and Hartmann, 1902; Garin and Bujadoux, 1922; Bannwarth, 1941; Bafverstedt, 1943; Bannwarth, 1944). A decade later, Lenhoff, a Swedish pathologist, described spirochaetelike structures in skin biopsies, while others demonstrated that penicillin was effective in the treatment of EM (Hollstrom, 1951; Burgdorfer, 1986). Scrimenti reported the first documented case of EM acquired in the USA in 1970 (Scrimenti, 1970). EM was reported in the northeastern USA by Mast and Burrows who described a cluster of cases of EM in southeastern Connecticut in 1976 (Mast and Burrows, 1976). Approximately 1 year after those reports, Steere and his colleagues at Yale began investigating a cluster of cases of arthritis in patients in and around Old Lyme, Connecticut (Steere et al., 1977) and called this Lyme arthritis. The name was changed to Lyme disease after it became apparent that most arthritis patients had previously had EM, and that heart block, facial nerve palsy and meningitis were also associated with this disorder (Steere et al., 1977).

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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3.2 Etiological Agent The spirochaetes responsible for Lyme disease belong to the family Spirochaetaceae, genus Borrelia (Wang et al., 1999). Analysis of isolates from patients, ticks and reservoir animals has revealed that there are at least 13 genospecies and an unknown but large number of substrains. The B. burgdorferi species complex evolved by differentiation of lineages (asexually), and is consequently composed of an array of clones (Dykhuizen et al., 2008). There are both pathogenic and non-pathogenic genospecies in the B. burgdorferi sensu lato complex. They include, B. burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia japonica, Borrelia valaisiana, Borrelia lusitaniae, Borrelia A14S, Borrelia andersonii, Borrelia bissettii, Borrelia tanukii, Borrelia turdi, Borrelia sinica, Borrelia californiensis, and Borrelia spielmanii (Wang et al., 1999; Rauter et al., 2002; Schulte-Spechtel et al., 2006) and a large number of substrains (Wilske et al., 1992; Dykhuizen et al., 1993). Of this group of Borrelia, the number of species recognized as human pathogens has expanded from the original three, B. burgdorferi sensu stricto, B. garinii and B. afzelii, to include B. lusitaniae, Borrelia A14S, B. valaisiana, B. bissettii and B. spielmanii (Casati et al., 2004; Floris et al., 2007; Fingerle et al., 2008). Although all of the other members of the B. burgdorferi sensu lato complex have been isolated in Europe, most European infections are caused by B. burgdorferi sensu stricto, B. afzelii and B. garinii. In contrast, B. burgdorferi sensu stricto is the only humanpathogenic species found in North America. Particular clinical manifestations are associated with the various genospecies. This helps explain why certain manifestations that are common in Europe are absent or uncommon in North America. Because of this diversity, European studies cannot be expected to relate universally to North American patients. Although there is overlap in the clinical manifestations between Europe and North America, the particular genospecies of Borrelia causing infection will shape the clinical presentation (van Dam et al., 1993).

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In Europe, there are a minimum of seven outer-surface protein A (OspA) serotypes (Wilske et al., 1995). Skin isolates belong primarily to B. afzelii (Balmelli and Piffaretti, 1996; Ornstein et al., 2001), especially from patients with acrodermatitis chronicum atrophicans (ACA), a chronic skin disease not present in America. Isolates from cerebrospinal fluid (CSF) and ticks are heterogeneous, consisting chiefly of B. garinii (Rauter et al., 2002). The most frequent genomic groups in Europe, B. afzelii and B. garinii, occur across the continent (Derdakova and Lencakova, 2005). Strains can have extensive heterogeneity, even in small areas. Focal prevalence of species or subtypes is also observed. Mixed infections have been seen in ixodid ticks and in specimens from patients. The heterogeneity of Borrelia antigens complicates laboratory diagnosis. For important serodiagnostic antigens, interspecies amino acid sequence identities are only 40– 44% for decorin-binding protein A (DbpA) and 54–68% for OspC for representative strains of B. burgdorferi sensu stricto, B. afzelii and B. garinii (Wilske et al., 1993, 1995; SchulteSpechtel et al., 2006). Although highly heterogeneous proteins sometimes have relatively conserved immunogenic epitopes (e.g. the C6 peptide of the variable surface antigen VlsE and the pepC10 peptide derived from OspC), even with the C6 peptide there are significant sequence differences among genospecies (Gomes-Solecki et al., 2007). It was initially thought that B. burgdorferi contains lipopolysaccharide (LPS) and that this contributed to inflammation in Lyme disease (Beck et al., 1985). However, chemical analysis (Takayama et al., 1987) and genomic sequencing failed to provide evidence of this (Fraser et al., 1997). The lack of LPS has important clinical consequences as Lyme disease-infected patients do not manifest sepsis-like pathophysiology comparable to that seen in patients with Gram-negative bacteraemia (Steere, 2001; Smith et al., 2002; Wormser et al., 2005). In Gram-negative bacteria, lipoproteins are affixed to the inner aspect of the outer membrane and the cytoplasmic membrane (Narita et al., 2004). By contrast, lipoproteins are found on the borrelial surface. The surface lipoprotein

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composition of the B. burgdorferi outer membrane is shaped by environmental signals and immunological pressure.

3.3 Genome The complete sequence of the B. burgdorferi strain B31 chromosome and plasmid genomes were published separately in 1997 and 2000 (Fraser et al., 1997; Casjens et al., 2000). These studies highlighted some unique features of B. burgdorferi in comparison with other Gramnegative and Gram-positive bacterial pathogens. A large portion, 40% of the genome, is composed of plasmid DNA contained in 12 linear and nine circular plasmids, with pseudogenes and a large amount of redundancy in the plasmid sequences (Stewart et al., 2003; Grimm et al., 2004a). The genome does not contain orthologues of recognized virulence factors. The pseudogenes indicate that B. burgdorferi is in a rapid state of evolutionary flux. Eight of the nine circular plasmids are derivatives of the same plasmid family, and seven of these contain large, almost identical, stretches of DNA with lipoprotein-encoding hypervariable regions interspersed in islands (Eggers et al., 2000; Stevenson et al., 2000). The small chromosome explains the spirochaete’s limited biosynthetic and metabolic capacity and accounts for its fastidious growth requirements. B. burgdorferi does not contain genes for oxidative phosphorylation utilizing the Embden–Meyerhof glycolytic pathway as their primary energy source. Thus, it has no iron requirement (Posey and Gherardini, 2000), making it resistant to host defenses that limit the ability of pathogens to scavenge iron.

3.4 Ecology of the Zoonosis B. burgdorferi is adapted to live in two very different worlds – the tick and the mammalian host. Tick feeding triggers complex changes (Hagman et al., 2000; Cugini et al., 2003; Tokarz et al., 2004), a burst of replication (de Silva and Fikrig, 1995; Piesman et al., 2003) and the expression of new surface components

required for survival in the mammalian host (de Silva and Fikrig, 1995; Kraiczy et al., 2001; Kurtenbach et al., 2002; Grimm et al., 2004b; Tilly et al., 2006). In non-feeding ticks, spirochaetes are found primarily in the mid-gut. However, as the tick begins to feed, the spirochaetes multiply and migrate from the tick’s mid-gut to its salivary glands. As feeding continues, spirochaetes enter the tick’s saliva and then the skin of the mammal. In the resting tick, OspA is the primary protein expressed on the surface of the spirochaetes. With feeding and proliferation of the spirochaete, the ospA gene is downregulated and ospC expression is upregulated, resulting in a change of outer-surface proteins from OspA to OspC. In animal studies, at least 48 h of feeding is required to transmit B. burgdorferi sensu stricto to its host (Benach et al., 1987), a finding corroborated in human studies (Berger et al., 1995; Sood et al., 1997; Nadelman et al., 2001). B. burgdorferi species are transmitted exclusively by hard-shelled ticks of the genus Ixodes; cases are limited to areas where this genus is endemic. Ixodes ticks are born uninfected and must acquire B. burgdorferi by feeding on an infected animal. The Ixodes ticks have a 2-year life cycle with four stages: egg, larva, nymph and adult, and feed three times, one at each non-egg life stage (Anderson, 1989; Bosler, 1993). Neither hosts nor ticks transmit B. burgdorferi to their offspring (Lane et al., 1991). Lyme disease is maintained through a zoonotic cycle of larval ticks feeding on infected reservoir hosts, becoming infected, surviving to the nymphal stage (Lane et al., 1991) and transmitting the infection to uninfected hosts. In North America, Peromyscus leucopus, the white-footed mouse, is the primary source of infection for Ixodes scapularis. These mice are ubiquitous in forested environments and can remain infected with B. burgdorferi for life (Donahue et al., 1987). However, other small mammals and even some bird species, such as robins, may also serve as sources of infection. Although Lyme disease is the most common vector-borne infectious disease in North America and Europe, this zoonosis has a limited geographical distribution and is

Borrelia: Interactions with the Host Immune System

only present when B. burgdorferi, vertebrate reservoir hosts and Ixodes tick vectors are all present. North America offers a good example of the limited distribution of Lyme disease, with over 90% of Lyme disease cases being reported from New York, Connecticut, Rhode Island, Pennsylvania, Delaware, New Jersey, Maryland, Massachusetts and Wisconsin. Even within these states, the areas where the three necessary organisms coexist are limited. In New York, which has the largest number of cases, 80% of cases are reported from just five of 62 counties. The risk of being infected with B. burgdorferi is directly linked to the density of infected Ixodes nymphs (Falco et al., 1999; Piesman et al., 1999).

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Infection begins as an acute local infection at the tick bite site as B. burgdorferi is inoculated into the skin. With the establishment of infection, B. burgdorferi proliferates locally, spreading through the dermis. From the dermis, it disseminates hematogenously to seed various organ systems including the heart, nervous system, joints and remote areas of the skin (acute disseminated infection). During the time between initial dissemination and the onset of late disease, there is usually a symptomfree interval in which the infection can remain latent before late manifestations develop. It is important to realize that not everyone will develop late manifestations (Steere et al., 1987).

3.5 Clinical Manifestations 3.5.1 Dermatological manifestations To a large extent, the natural history of Lyme disease is based on observations made in the late 1970s and 1980s – observations in many ways different from the clinical picture of the disease today. The manifestations associated with acute disseminated and late disease are much less common today because of the increased recognition of early infection and the prompt use of the effective antibiotic regimens established in the mid- to late 1980s (Wormser et al., 2006). Importantly, Lyme arthritis, once described as the key manifestation of late North American Lyme disease, is now relatively rare. Despite this shift in clinical presentations, there remains too great an emphasis on the late manifestations of Lyme disease. Unfortunately, some have misinterpreted these old observations to foster an inaccurate view of this illness. The early hypothesis that Lyme disease consisted of three stages: stage 1, EM; stage 2, neurological or cardiac involvement; and stage 3, arthritis (Steere et al., 1987), is now obsolete. As more data regarding the clinical course of Lyme disease were generated, it became apparent that the clinical manifestations do not proceed from one stage to the next in an orderly fashion and stages are not pathophysiologically distinct. It is more logical to view B. burgdorferi infection as a progressive but variable infectious disease.

An annular erythematous skin lesion arising at the site of the tick bite, EM (see Nadelman, Chapter 10, this volume), is the earliest and most recognized manifestation of B. burgdorferi infection. Although once thought to develop in only about 60% of patients, it is now realized that EM develops at the tick bite site in approximately 90% of patients (Steere et al., 1998). It typically begins within a few days or weeks after the bite of an infected tick. Considering that tick bites are painless and nymphal ticks are small, it is not surprising that only 14–32% of patients with EM are aware of the tick bite (Nadelman et al., 1996; Smith et al., 2002) In the older literature, EM is described as an expanding target lesion, at least 5 cm in diameter, or an annular erythematous skin lesion with central clearing. It is now recognized that EM is considerably more variable. Berger established that expansion of the lesion is a prime characteristic of EM. He documented that EM typically expands at a rate of 20 cm2/day (Berger, 1993). Central clearing is no longer felt to be an essential characteristic, occurring in less than half of cases. The two largest North American studies of EM found that only 37% (Nadelman et al., 1996; Smith et al., 2002) and 9% (Smith et al., 2002) of patients had central clearing. The most common lesions are homogeneously

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erythematous. Less common variations include vesicular and purpuric lesions. EM may be confused with fungal dermatitis, but unlike fungal dermatitis there is no scaling at the erythematous border in EM (Berger, 1993). Although EM can be located anywhere, in adults it is most commonly found below the waist (Nadelman et al., 1996; Smith et al., 2002) Constitutional symptoms reported in association with EM range from none to mild and transitory malaise, fatigue, headache and low-grade fever or chills (Wormser et al., 2006). As with other aspects of the disease, there is a difference in the incidence of reported symptoms between American and European EM patients. Less than 50% of European patients have extracutaneous manifestations, while more than 75% of American patients have such manifestations (Wormser et al., 2006). Wormser found that in patients presenting with EM, approximately 45% were spirochaetaemic (Wormser et al., 2005). Of the spirochaetaemic individuals, 42% had multiple EM compared with 15% of nonspirochaetaemic patients. Secondary lesions were generally smaller than the primary lesion. In untreated patients, EM clears spontaneously within weeks to months. If inadequately treated, EM can relapse rarely. Patients with a prior history of EM are still susceptible to reinfection and to recurrent episodes. Two additional dermatological disorders, Borrelia lymphocytoma and ACA, are associated with Borrelia infection in Europe (Asbrink and Hovmark, 1988). Borrelia lymphocytoma appears as a solitary, bluishred nodule on the earlobes of children, and the nipple or genital areas of adults (Colli et al., 2004). It may appear in acute infection, or months later (Asbrink and Hovmark, 1988). The histological appearance of a dense lymphocytic infiltrate in the dermis or subcutaneous tissue can be difficult to differentiate from lymphoma (Colli et al., 2004). Both B. afzellii and B. garinii have been associated with lymphocytoma, but B. burgdorferi sensu stricto has not (Ghislain et al., 2003). ACA presents initially as diffuse oedema with varying erythema, similar in appearance

to the acute oedematous phase of scleroderma. Over time, the lesions evolve becoming atrophic or sclerotic. Typically, ACA involves the distal extremities and less commonly the trunk. The face, palms and soles are spared. ACA is found primarily in northern, central and eastern Europe. It occurs years after initial infection, typically developing 10 years after the onset of untreated infection. ACA is linked principally to infection with B. afzellii (Tazelaar et al., 1997) but never with B. burgdorferi sensu stricto. Extracutaneous manifestations including polyneuropathy, small joint arthritis with subluxation, arthritis of the large joints and periosteal thickening of the bones are frequently observed in the same extremity as ACA. 3.5.2 Nervous system manifestations B. burgdorferi can seed the nervous system as the organism enters the bloodstream. Early studies of North American EM patients found that 15% of untreated patients developed meningitis or cranial neuritis within the first 3 months of infection (Steere et al., 1983; Halperin et al., 1989) (see Halperin, Chapter 13, this volume). CSF abnormalities include a lymphocytic pleocytosis, moderate elevations of CSF protein and normal CSF glucose (Halperin et al., 1989; Halperin, 2008). In Europe, Bannwarth’s syndrome, a painful radiculoneuritis with inflammation of the nerve roots and lancinating radicular pain, is associated with the acute disseminated infection. CSF lymphocytic pleocytosis is commonly seen (Maida et al., 1986) and myelitis – and occasionally encephalitis – were initially reported to occur in more than 20% of affected patients. Long-term sequelae including spastic paraparesis and neurogenic bladder, which can persist even after appropriate therapy, have been reported (Kampner and Andersen, 1982). B. garinii is most commonly linked to Bannwarth’s syndrome, but it has been reported in association with other genospecies including B. burgdorferi sensu stricto. Although late-disseminated B. burgdorferi infection has been purported to be associated with a variety of nervous system

Borrelia: Interactions with the Host Immune System

abnormalities, the neurological entities recognized to be associated with late Lyme disease are limited to encephalomyelitis, peripheral neuropathy and encephalopathy (Halperin et al., 1989; Finkel et al., 1992), all of which are now rare. Only one case of encephalomyelitis, nine patients with peripheral neuropathy and seven patients with encephalopathy were seen by the panel members in the 5 years prior to the publication of the Infectious Diseases Society of America (IDSA) guidelines (Wormser et al., 2006). In the absence of clear objective manifestations associated with Lyme disease, there is no evidence that active B. burgdorferi infection is linked to vague non-specific symptoms such as fatigue or memory problems. Even in patients with a history of B. burgdorferi infection and fatigue or alteration of cognitive function, there is no evidence of active B. burgdorferi central nervous system infection. The peripheral nerve involvement of late disease is an axonopathy (Halperin et al., 1987, 1990; Logigian and Steere, 1992), and nerve conduction studies typically reveal abnormalities. In Europe, peripheral neuropathy can be found in association with ACA, but it rarely occurs in patients without ACA (Mygland et al., 2006). 3.5.3 Cardiac manifestations The incidence of symptomatic cardiac involvement has decreased dramatically like other aspects of acute disseminated Lyme disease (see Harburger and Halperin, Chapter 11, this volume). Early studies demonstrated that 4–10% of untreated EM patients developed Lyme carditis, typically manifested by the acute onset of variable atrioventricular heart block occurring proximal to the bundle of His. Myocarditis or pericarditis was observed in approximately 65% of these patients (Steere et al., 1980). By contrast, none of the 233 patients in the two prospective OspA vaccine trials who developed Lyme disease had any evidence of cardiac involvement (Sigal et al., 1998; Steere et al., 1998). Late cardiac manifestations are poorly defined. Chronic carditis and cardiomyopathy

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have been reported in Europe (Klein et al., 1991); analogous cases are not well documented in North America. Tachyarrhythmias and palpitations are not associated with Lyme carditis. 3.5.4 Lyme arthritis Lyme arthritis is the most common and most studied late manifestation of untreated B. burgdorferi sensu stricto infection (see Sigal, Chapter 12, this volume). Originally felt to be a North American phenomenon not associated with B. burgdorferi infection in Europe, Lyme arthritis is now recognized to occur in European patients (van Dam et al., 1993). Lyme arthritis is a large-joint monoarticular or oligoarticular arthritis characterized by episodes of joint inflammation with swelling, large effusions and minimal pain. The knee is the most commonly affected joint. In 55 patients who presented with EM and were observed without antibiotic treatment, 34 developed Lyme arthritis (Steere et al., 1987). Of these 34 patients, 28 experienced episodic arthritis that lasted from a few days to several months before spontaneously resolving – without antibiotic treatment. Over time, in these 28 patients, the interval between episodes increased gradually until the arthritis spontaneously remitted. The remaining six patients had a more chronic course that lasted a year or more. Patients that fall within this latter group with a more chronic course have generated considerable attention, especially those who subsequently failed to respond to antibiotics. These antibiotic-refractory cases have no evidence of ongoing infection, yet have prolonged unremitting arthritis. Spontaneous remissions occur in antibiotic-refractory patients. With the development of better antibiotic regimens for early Lyme disease in the late 1980s and early 1990s, there has been a marked decrease in the incidence of all late manifestations of Lyme disease including arthritis. The incidence of Lyme arthritis in studies carried out in the past 10–15 years has been 10% or less (Sigal et al., 1998; Steere et al., 1998). Because the overall incidence of frank arthritis has decreased dramatically, physicians must

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be more cautious in making a diagnosis of Lyme arthritis. Lyme arthritis is an inflammatory arthritis in which the cause is known. The hope was that studying this entity would provide insights and a better understanding of inflammatory arthritis in general. A few patients in the 1970s and 1980s with antibioticrefractory Lyme arthritis were reported to have developed joint erosions similar to those observed in rheumatoid arthritis (Steere et al., 1979). In untreated Lyme arthritis, synovial biopsy demonstrated hypertrophy with vascular proliferation, and mononuclear cell infiltration with a mixed infiltrate including T cells, B cells and follicular dendritic cells. mRNA for interleukin-1 (IL-1) and tumour necrosis factor  (TNF-) is expressed in the inflammatory infiltrate (Harjacek et al., 2000). In comparing patients with untreated Lyme arthritis with patients with antibioticrefractory Lyme arthritis, Lin et al. (2001) found that matrix metalloproteinase 1 (MMP1) and MMP-3 were present in the synovial fluid of untreated patients with B. burgdorferi DNA in their synovial fluid but not in treated patients with refractory arthritis (Lin et al., 2001). The patients with antibiotic-refractory Lyme arthritis were found to have MMP-8 and MMP-9 in their synovial fluid, associated with prominent inflammatory cellular responses in the fluid. The secretion of MMP-9 in human and murine macrophages can be induced by stimulation of CD14 and Toll-like receptor 2 (TLR2) by conserved pattern recognition receptors on B. burgdorferi (Gebbia et al., 2001). MMPs may play a role in the erosion of cartilage and bone. Active infection, retained B. burgdorferi antigens, post-infectious immune dysregulation and infection-induced autoimmunity have all been hypothesized as possible causes of antibiotic-refractory Lyme arthritis (Steere and Glickstein, 2004), but none has been proven.

1999). Furthermore, there is no evidence that B. burgdorferi persists in synovium. Synovial tissue samples obtained from 26 patients with antibiotic-refractory arthritis were probed with three primers targeting ospA, ospB and p41, and no spirochaetal DNA was detected in any of the tissue samples. A report from Germany that found four patients who had ongoing arthritis 8–10 weeks after initial antibiotic treatment with negative synovial fluid but detectable B. burgdorferi DNA in their synovial tissue has been used to argue for persistent infection. Notably, the arthritis resolved in all patients after another course of antibiotics (Priem et al., 1998).

Retained B. burgdorferi antigens It is possible that retained bacterial antigens persist, inducing continued inflammation even after microbiological cure. Borrelia surface proteins injected intra-articularly in rats induced joint inflammation, with antigens sticking to synovial membranes. However, no evidence could be found to support retained antigens as the cause of ongoing inflammation in vivo (Malawista, 2000).

Post-infectious immune dysregulation Defects in the ability to downregulate proinflammatory immune responses can be associated with ongoing inflammation. T regulatory (Treg) cells have been the subject of studies in autoimmune disease and are recognized as playing a critical role in regulating pro-inflammatory immune responses. FoxP3 Treg cells are decreased in the synovium of patients with rheumatoid arthritis. Similar decreases in the number or function of FoxP3 Treg cells have not been observed in antibiotic-resistant Lyme arthritis. Thus, there are no data to support this hypothesis.

Active infection Active infection is an unlikely explanation because B. burgdorferi DNA can be detected in synovial fluid before but is undetectable after treatment (Nocton et al., 1994; Carlson et al.,

Infection-induced autoimmunity Patients with antibiotic-resistant persistent Lyme arthritis have a higher incidence of

Borrelia: Interactions with the Host Immune System

human leukocyte antigen (HLA)-DRB1*0401, HLA-DRB1*0101 and HLA-DRB1*0404 allele expression (Steere and Glickstein, 2004). This observation, along with the finding that these patients commonly have high antibody titers and vigorous cellular responses to OspA, led Steere and colleagues to believe that refractory Lyme arthritis was probably associated with infection-induced autoimmunity linked to OspA. Ninety-three per cent of patients with antibiotic-refractory Lyme arthritis had cellular responses to OspA, while only 35% of patients with transient arthritis responded to OspA (Chen et al., 1999). A series of papers associated T-cell reactivity with human leukocyte function-associated antigen 1 (LFA-1) in antibiotic-refractory Lyme arthritis (Gross et al., 1998, 2001; Trollmo et al., 2001). The hypothesis was that the T-cell epitope YVLEGTLTA in OspA B31 (aa 165–173) induced T-cell cross-reactivity to YVIEGTSKQ in human LFA-1 (aa 332–340) producing T-cell autoimmunity and refractory Lyme arthritis. The link between OspA and the light chain of the human LFA-1 was based largely on the amino acid sequence of the two proteins (Gross et al., 1998, 2001; Trollmo et al., 2001). The fairly high sequence homology between these two proteins and the prediction that the LFA-1 sequence YVIEGTSKQ binds HLA-DRB1*0401 was felt to support the hypothesis that exposure to OspA could lead to T-cell autoimmunity to LFA-1 in genetically susceptible individuals. In essence, this suggested that antibiotic-refractory Lyme arthritis is an autoimmune disease caused by molecular mimicry between the pathogen and a host protein. The original report showing T-cell reactivity to LFA-1 was based on T cells from one antibiotic-refractory Lyme arthritis patient (Gross et al., 1998). Whether it was demonstrated in one patient or more, there is a major problem with this hypothesis. T-cell epitopes cannot be predicted by amino acid sequence (Benoist and Mathis, 2001): structure determines peptide binding to the T-cell receptor, not the amino acid sequence. In addition, because the average T-cell receptor can bind up to 105–106 different peptides, peptide binding to a particular T-cell receptor is not by itself predictive

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(Benoist and Mathis, 2001). Maier et al. (2000) concluded that T-cell cross-reactivity is a common phenomenon and that the presence of cross-reactive T-cell epitopes in a peptide does not predict molecular mimicry-induced autoimmune disease, after finding that 16 of 475 identified supertope-matching peptides stimulated one or more of seven B. burgdorferispecific HLA-DR4-restricted T cells. Benoist and Mathis (2001), reviewing infectioninduced autoimmune disease, highlighted the difficulty in proving that an infecting organism provokes clinically significant autoimmunity. They specifically stated that the reactivity of T-helper 1 cells to LFA-1 may merely reflect the promiscuous nature of the T-cell receptor.

3.6 Laboratory Diagnosis Unlike most bacterial illnesses, B. burgdorferi is difficult to diagnose by culture except during the early stages of the disease (Aguero-Rosenfeld et al., 2005). Culture is insensitive in the extracutaneous disease that characterizes the later stages. Culture remains a research tool with no place in the routine diagnosis of Lyme disease. Similarly, PCR has not found a place in the routine diagnosis of this infection. PCR is highly sensitive in detecting B. burgdorferi DNA from EM biopsies and synovial fluid in untreated Lyme arthritis (Aguero-Rosenfeld, 2008). In research studies, PCR has confirmed that B. burgdorferi was eradicated from the joint by antibiotic therapy (Nocton et al., 1994; Carlson et al., 1999). However, the quality of commercial PCR kits varies widely and may lack specificity (Aguero-Rosenfeld, 2008). The mainstay of the laboratory diagnosis of Lyme disease is serological assays (see Johnson, Chapter 4, this volume), although these are not without problems. At this time, no single test is considered to be adequate. The Centers for Disease Control and Prevention (CDC) currently recommends a two-step approach for the serodiagnosis of B. burgdorferi infection in which all positive firsttier tests, including ELISAs, immunofluorescence assays and lateral flow assays, be followed by a Western blot. Only if both

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the first-tier assay and the Western blot are positive should the patient be considered seropositive (CDC, 1995). This approach has a specificity of up to 99%. As with all laboratory tests, the positive predictive values of Lyme serologies are directly related to the pre-test likelihood of infection. IgM assays are less specific and are only recommended in the first month of infection. Although this approach is expensive and can delay diagnosis for a week or more, it is necessary due to the poor specificity of the most commonly used first-tier assays. Most of the current first-tier assays are based on whole B. burgdorferi or recombinant proteins. The sole exception is the C6 peptide assay (C6 Lyme ELISA, Immunetics Inc., www. immunetics.com), an assay utilizing a 26 amino acid peptide from the invariable region 6 (IR6) of the B. burgdorferi VlsE protein as its antigen. The C6 assay is the most specific of the first-tier assays and maintains a high degree of sensitivity for disseminated or late Lyme disease. Antigens from whole B. burgdorferi preparations or whole recombinant proteins have the advantage that they include multiple – both linear and conformational diagnostic – epitopes. However, these antigens have the disadvantage of containing epitopes that cross-react with homologous epitopes from other organisms. This has proved to be a major problem with anti-B. burgdorferi serological assays and was the reason that the two-tier approach was adopted. Despite extensive research, no recombinant protein-based assay has been developed that adequately addresses the problem of poor specificity (Bratton et al., 2008). The host immune response to B. burgdorferi is no different than the response to any bacteria. The IgM response develops first, followed by the IgG. IgM antibodies to B. burgdorferi are detectable within 1–2 weeks following the onset of infection, with IgG detectable a few days later. The earliest responses are to the 41 kDa flagellin B (FlaB) and OspC (25 kDa) antigens with responses to a number of additional antigens, such as VlsE, fibronectin-binding protein (BBK32), FlaA (37 kDa), BmpA (39 kDa) and DbpA, developing later as the infection progresses

(Engstrom et al., 1995; Bacon et al., 2003; Lahdenne et al., 2006; Nowalk et al., 2006). VlsE and its IR6 region are among the best-studied antigens in this latter group (Gomes-Solecki et al., 2007; Bratton, et al., 2008). VlsE is not expressed in the tick and is only expressed in the mammalian host after infection is established (Bykowski et al., 2006). Thus, in comparison with FlaB, OspC and other antigens expressed in the feeding tick, there is a delay in the IR6 being accessible to the immune system. Lahdenne et al. (2003) found that only 29/75 patients (39%) with EM for 7–14 days had IgG antibodies to the IR6 peptide antigen, while 65/75 of these patients (87%) had IgG antibodies to one or more variants of BBK32, an antigen expressed in the feeding tick (Lahdenne et al., 2003). Further complicating the use of the IR6 peptide to detect antibodies in early infection is that IR6 does not bind IgM very well (Embers et al., 2007). Embers and coworkers found that, in a group of 37 patients with early Lyme disease, only one developed significant levels of IgM against IR6; the other 36 failed to develop levels greater than the healthy controls. In a study comparing the development of anti-VlsE IgM responses with IgM responses to the OspC peptide pepC10, the sensitivity of the pepC10 ELISA was approximately ten times greater in patients who presented within 1 week after the onset of EM (Bacon et al., 2003). Responses to OspA and OspB only develop in a small percentage of patients after months of infection (Nowalk et al., 2006). New potential diagnostic target antigens have been suggested. Ideally, rather than depending on one or two antigens, an assay with five or six would offer better performance characteristics (Barbour et al., 2008). As with other infections, once an IgG response develops, IgG anti-B. burgdorferi antibodies remain positive for some time after the infection has been treated (Johnson et al., 1996).

3.7 Pathogenesis Ticks are attracted to heat and CO2. Humans are not part of the zoonotic cycle but are incidental targets for the questing tick. Once

Borrelia: Interactions with the Host Immune System

attached to the skin, the feeding process takes 72 h or more to complete. Only after 48 h of feeding does the risk of infection become significant (Wormser et al., 2006).The number of organisms introduced into the skin is probably fairly low (Piesman et al., 2001). Histological studies have demonstrated that B. burgdorferi has a predilection for vascular and perivascular connective tissue (Duray, 1989; Barthold et al., 1991). Utilizing cultured human umbilical and vein endothelial cells, Szczepanski et al. (1990) found that spirochaetes attach to vascular endothelium and move through intercellular junctions to attach themselves to the subendothelial matrix. Glycosaminoglycans, decorin, fibronectin and integrins have all been identified as receptors for B. burgdorferi (Coburn et al., 2005; Antonara et al., 2007). The interaction of B. burgdorferi with the host begins when the spirochaete is still in the tick’s gut. It must resist being killed by components in the blood meal. Studies of the interaction of B. burgdorferi with the alternative complement pathway have identified five surface lipoproteins that prevent activation of the complement cascade by binding factor H and/or factor H-like protein-1 (FHL-1) and plasma glycoproteins (Kraiczy et al., 2001, 2004; Alitalo et al., 2002; Kurtenbach et al., 2002; LaRocca and Benach, 2008). B. burgdorferi is not a toxin-producing bacterium. The genomic sequence does not contain toxin orthologues or the genetic components of a secretory apparatus required for toxin delivery (Fraser et al., 1997). Although unproven, it is felt that tissue damage and the clinical manifestations of Lyme disease are caused by the host response to B. burgdorferi (Wooten and Weis, 2001). In general, there has been a strong emphasis on Lyme arthritis in both human and animal studies, even though B. burgdorferi infection is a systemic infectious disease. The severity of arthritis in mice is genetically linked, and various inbred strains vary in the severity of arthritis they develop after experimental infection with B. burgdorferi: C57BL/6 mice develop mild arthritis (Barthold et al., 1990), BALB/c mice moderate arthritis and C3H/He mice severe arthritis. B.

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burgdorferi seeds the synovium and articular surfaces after murine infection (Steere and Glickstein, 2004) and induces the production of the pro-inflammatory cytokines TNF- and IL-6 and the expression of chemokines such as CXCL8 (IL-8), causing neutrophils to migrate into joint spaces (Dong et al., 1997). Unlike human infection, B. burgdorferi induces joint inflammation in the mouse model earlier, 10–14 days after initial infection. Initially neutrophils migrate into the affected joints, followed by infiltration of the synovium with mononuclear cells and synovial hypertrophy with pannus formation 3–5 weeks later. As in human Lyme arthritis, arthritis is self-limiting, with joint inflammation usually resolving within 8–10 weeks after the primary infection (McKisic and Barthold, 2000). With the establishment of infection, the many B. burgdorferi lipoproteins play a key role in inducing host innate immune responses. Unlipidated OspA is much less effective than lipidated OspA in the induction of a humoral response (Erdile et al., 1993). Dendritic cells and macrophages are stimulated through the interaction of borrelial lipoproteins with their pattern recognition receptors, TLR1 and TLR2. (Aliprantis et al., 1999; Brightbill et al., 1999; Hirschfeld et al., 1999; Lien et al., 1999; Ozinsky et al., 2000; Alexopoulou et al., 2002). The prototypic TLR, TLR4, plays no role in the response, as B. burgdorferi does not express its major ligand, LPS. Studies of mice with specific deficiencies of TLRs or pathways have allowed investigators to define the role that innate immune receptors play in the defence against B. burgdorferi infection. These mouse models demonstrate the importance that TLR1 and TLR2 signalling plays in B. burgdorferi infection. Mice deficient in TLR1 and TLR2 signaling pathways fail to respond to OspA (Singh and Girschick, 2006). Similarly alteration of TLR1 cell-surface expression is associated with non-responsiveness to OspA vaccination (Alexopoulou et al., 2002). TLR2-, CD14- or MyD88-deficient mice have higher spirochaete burdens and increased joint inflammation compared with wild-type mice (Wooten and Weis, 2001; Bolz et al., 2004; Liu

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et al., 2004; Wang et al., 2004; Behera et al., 2006). Macrophages from TLR2-deficient mice have a diminished response to live B. burgdorferi (Shin et al., 2008). In human monocytes, live spirochaetes induce stronger responses than bacterial lysates, and the enhanced responses are dependent on phagocytic uptake of live organisms (Moore et al., 2006; Cruz et al., 2008; Shin et al., 2008). TLR1 and TLR2 play an important role in the defence against B. burgdorferi; however, other components of the innate immune system play important roles. B. burgdorferi induces a prominent type I interferon (IFN) response in infected mice, pointing to the existence of borrelial pathogen-associated molecular patterns or PAMPs (i.e. LPS or Fla) other than lipoproteins, as TLR2 signalling does not result in stimulation of type I IFNs (Toshchakov et al., 2002). B. burgdorferi diacylglycerol glycolipid triggers a CD1ddependent natural killer T-cell response (Kinjo et al., 2006). Live but not lysed B. burgdorferi induced natural killer cells to produce IFN-, an effect that required dendritic cell uptake of the bacteria (Moore et al., 2006; Cruz et al., 2008). 3.7.1 Adaptive response There is both an antibody and a cellular immune response to B. burgdorferi. B. burgdorferi lipoproteins induce the production of bactericidal and non-bactericidal antibodies (Fikrig et al., 1997; Erdile et al., 1993). Only a few of the antibody responses against B. burgdorferi prevent infection. However, antibodies can play other roles, for instance, in rheumatic complications (Feng et al., 2000). In the mouse arthritis model, antiarthritis-related protein-1 (Arp1) antibodies reduce the severity of arthritis (Feng et al., 2000). B. burgdorferi-specific CD4+ and CD8+ T cells play a role in protection and pathogenesis. Severe combined immune deficient (SCID) mice exhibit increased severity of infection compared with wildtype mice (Keane-Myers and Nickell, 1995; Dong et al., 1997). The role of T cells is

complicated. Although they play an important role in protection against B. burgdorferi, in the absence of B cells, T cells can contribute to greater pathology. Adoptive transfer of T cells alone exacerbated arthritis and accelerated its onset in B. burgdorferiinfected C57BL/6 SCID mice, yet adoptive transfer of T and B cells reduced the severity of arthritis, suggesting a role for B cells in modifying joint inflammation (McKisic and Barthold, 2000). Skin biopsies from patients with EM reveal infiltrates containing increased numbers of T cells, monocytes/macrophages and dendritic cells and lower proportions of neutrophils, with virtually no B cells (Salazar et al., 2003). Neutrophil and macrophage expression of the activation markers CD14 and HLA-DR was increased, and T cells expressed an increased level of CD45RO, the low-molecular-mass form of CD45, and the costimulatory molecule CD27, a TNF receptor family member. Memory T cells tend to express CD45RO, but this can vary. Both primary and memory T cells express CD27. Dendritic cells, both the CD11c+ (monocytoid) and CD11c– (plasmacytoid) subsets, expressed activation/maturation surface markers and increased expression of TLR1, TLR2 and TLR4. The pro-inflammatory cytokines IL-6 and IFN- were the predominant cytokines in EM lesions. Patients with acute disseminated infection with multiple EM lesions had higher serum levels of IFN-, TNF- and IL-2, and their peripheral monocytes displayed greater surface expression of TLR1 and TLR2 than patients with a single EM. The CD11c+ dendritic cells of these multiple-EM patients showed increased expression of TLR2 and TLR4 (Salazar et al., 2003). There may be differences in immune responses to the different genospecies of B. burgdorferi. Comparing 19 EM patients infected with B. burgdorferi sensu stricto from New England with 37 Austrian EM patients infected with B. afzelii, Jones et al. (2008) found a higher level of chemokine CXCL1, CCL3, CCL4, CXCL9, CXCL10, and CXCL11 mRNA in the B. burgdorferi sensu strictoinfected patients.

Borrelia: Interactions with the Host Immune System

3.8 Conclusion As outlined here and elsewhere in this volume, the host immune response plays an essential role in Lyme disease, with specific interactions apparently contributing to the particular clinical phenomena that occur in this infection. An improved understanding of the host response should lead to significantly enhanced insights into the disease’s pathophysiology – insights that should inform future treatment, both of Lyme disease and potentially of other immune-mediated disorders.

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Liu, N., Montgomery, R.R., Barthold, S.W. and Bockenstedt, L.K. (2004) Myeloid differentiation antigen 88 deficiency impairs pathogen clearance but does not alter inflammation in Borrelia burgdorferi-infected mice. Infection and Immunity 72, 3195–3203. Logigian, E.L. and Steere, A.C. (1992) Clinical and electrophysiologic findings in chronic neuropathy of Lyme disease. Neurology 42, 303–311. Maida, E., Kristoferitsch, W. and Spiel, G. (1986) Cerebrospinal fluid changes in Garin– Bujadoux–Bannwarth meningoradiculitis. Nervenarzt 57, 149–152. Maier, B., Molinger, M., Cope, A.P., Fugger, L., Schneider-Mergener, J., Sønderstrup, G., Kamradt, T. and Kramer, A. (2000) Multiple cross-reactive self-ligands for Borrelia burgdorferi-specific HLA-DR4-restricted T cells. European Journal of Immunology, 30, 448–457. Malawista, S.E. (2000) Resolution of Lyme arthritis, acute or prolonged: a new look. Inflammation 24, 493–503. Mast, W.E. and Burrows, W.M. (1976) Eythema chronicum migrans in the USA. Journal of the American Medical Association 236, 859–860. McKisic, M.D. and Barthold, S.W. (2000) T-cellindependent responses to Borrelia burgdorferi are critical for protective immunity and resolution of Lyme disease. Infection and Immunity 68, 5190–5197. Moore, M.W., Cruz, A.R., LaVake, C.J., Marzo, A.L., Eggers, C.H., Salazar, J.C. and Radolf, J.D. (2006) Phagocytosis of Borrelia burgdorferi and Treponema pallidum potentiates innate immune activation and induces IFN- production. Infection and Immunity 75, 2046– 1062. Mygland, A., Skarpaas, T. and Ljostad, U. (2006) Chronic polyneuropathy and Lyme disease. European Journal of Neurology 13, 1213–1215. Nadelman, R.B., Nowakowski, J., Forseter, G., Goldberg, N.S., Bittker, S., Cooper, D., AgueroRosenfeld, M. and Wormser, G.P. (1996) The clinical spectrum of early Lyme borreliosis in patients with culture-confirmed erythema migrans. American Journal of Medicine 100, 502–508. Nadelman, R.B., Nowakowski, J., Fish, D., Falco, R.C., Freeman, K., McKenna, D., Welch, P., Marcus, R., Agüero-Rosenfeld, M.E., Dennis, D.T., Wormser, G.P. and the Tick Bite Study Group (2001) Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. New England Journal of Medicine 345, 79–84. Narita, S., Matsuyama, S. and Tokuda, H. (2004)

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Salazar, J.C., Pope, C.D., Sellati, T.J., Feder, H.M. Jr, Kiely, T.G., Dardick, K.R., Buckman, R.L., Moore, M.W., Caimano, M.J., Pope, J.G., Krause, P.J. and Radolf, J.D. (2003) Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans. Journal of Immunology 171, 2660–2670. Schulte-Spechtel, U., Fingerle, V., Goettner, G., Rogge, S. and Wilske, B. (2006) Molecular analysis of decorin-binding protein A (DbpA) reveals five major groups among European Borrelia burgdorferi sensu lato strains with impact for the development of serological assays and indicates lateral gene transfer of the dbpA gene. International Journal of Medical Microbiology 296 (Supplement) 40, 250–266. Scrimenti, R.J. (1970) Erythema chronicum migrans. Archives of Dermatology 236, 859– 860. Shin, O.S., Isberg, R.R., Akira, S., Uematsu, S., Behera, A.K. and Hu, L.T. (2008) Distinct roles for MyD88 and Toll-like receptors 2, 5, and 9 in phagocytosis of Borrelia burgdorferi and cytokine induction. Infection and Immunity 76, 2341–2351. Sigal, L.H., Zahradnik, J.M., Lavin, P., Patella, S.J., Bryant, G., Haselby, R., Hilton, E., Kunkel, M., Adler-Klein, D., Doherty, T., Evans, J., Molloy, P.J., Seidner, A.L., Sabetta, J.R., Simon, H.J., Klempner, M.S., Mays, J., Marks, D. and Malawista, S.E. (1998) A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. Recombinant Outer-Surface Protein A Lyme Disease Vaccine Study Consortium. New England Journal of Medicine 339, 216–222. Singh, S.K. and Girschick, H.J. (2006) Toll-like receptors in Borrelia burgdorferi-induced inflammation. Clinical Microbiology and Infection 12, 705–717. Smith, R.P., Schoen, R.T., Rahn, D.W., Sikand, V.K., Nowakowski, J., Parenti, D.L., Holman, M.S., Persing, D.H. and Steere, A.C. (2002) Clinical characteristics and treatment outcome of early Lyme disease in patients with microbiologically confirmed erythema migrans. Annals of Internal Medicine 136, 421–428. Sood, S.K., Salzman, M.B., Johnson, B.J., Happ, C.M., Feig, K., Carmody, L., Rubin, L.G., Hilton, E. and Piesman, J. (1997) Duration of tick attachment as a predictor of the risk of Lyme disease in an area in which Lyme disease is endemic. Journal of Infectious Diseases 175, 996–999. Steere, A.C. (2001) Lyme disease. New England Journal of Medicine 345, 115–125.

Borrelia: Interactions with the Host Immune System

Steere, A.C. and Glickstein, L. (2004) Elucidation of Lyme arthritis. Nature Reviews Immunology 4, 143–152. Steere, A.C., Malawista, S.E., Snydman, D.R., Shope, R.E., Andiman, W.A., Ross, M.R. and Steele, F.M. (1977) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis and Rheumatism 20, 7–17. Steere, A.C., Gibofsky, A., Patarroyo, M.E., Winchester, R.J., Hardin, J.A. and Malawista, S.E. (1979) Chronic Lyme arthritis. Clinical and immunogenetic differentiation from rheumatoid arthritis. Annals of Internal Medicine 90, 896– 901. Steere, A.C., Batsford, W.P., Weinberg, M., Alexander, J., Berger, H.J., Wolfson, S. and Malawista, S.E. (1980) Lyme carditis: cardiac abnormalities of Lyme disease. Annals of Internal Medicine 93, 8–16. Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E. and Malawista, S.E. (1983) The spirochetal etiology of Lyme disease. New England Journal of Medicine 308, 733–740. Steere, A.C., Schoen, R.T. and Taylor, E. (1987) The clinical evolution of Lyme arthritis. Annals of Internal Medicine 107, 725–731. Steere, A.C., Sikand, V.K., Meurice, F., Parenti, D.L., Fikrig, E., Schoen, R.T., Nowakowski, J., Schmid, C.H., Laukamp, S., Buscarino, C. and Krause, D.S. (1998) Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group. New England Journal of Medicine 339, 209–215. Stevenson, B., Zückert, W.R. and Akins, D.R. (2000) Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species. Journal of Molecular Microbiology and Biotechnology 2, 411–422. Stewart, P.E., Chaconas, G. and Rosa, P. (2003) Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi. Journal of Bacteriology 185, 3202– 3209. Szczepanski, A., Furie, M.B., Benach, J.L., Lane, B.P. and Fleit, H.B. (1990) Interaction between Borrelia burgdorferi and endothelium in vitro. Journal of Clinical Investigation 85, 1637–1647. Takayama, K., Rothenberg, R.J. and Barbour, A.G. (1987) Absence of lipopolysaccharide in the Lyme disease spirochete, Borrelia burgdorferi. Infection and Immunity 55, 2311–2313. Tazelaar, D.J., Molkenboer, M.J., Noordhoek, G.T., Plantinga, G., Schouls, L.M. and Schellekens, J.F. (1997) Detection of Borrelia afzelii, Borrelia

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4

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection1 Barbara J.B. Johnson

4.1 Introduction Serology is the only standardized type of laboratory testing available to support the clinical diagnosis of Lyme borreliosis (Lyme disease) in the USA. It is also the only type of diagnostic testing approved by the US Food and Drug Administration (FDA). Of the 77 devices cleared by the FDA for in vitro diagnostic use for Lyme disease, all are designed to detect immune responses to antigens of Borrelia burgdorferi sensu stricto, particularly IgM and IgG (FDA, 2010). Serological tests do not become positive until an infected individual has had time to develop antibodies. In Lyme disease, this means that early acute disease characterized by an expanding rash (erythema migrans or EM) at the site of a tick bite cannot be reliably diagnosed by serology. After a few weeks of infection, however, immunocompetent people will have made enough antibodies that serology is useful for confirming exposure to B. burgdorferi in all subsequent stages of Lyme disease. Antibody levels remain elevated for months to years after the infection is cured. A variety of direct tests for the agent of Lyme borreliosis have been developed. Direct

tests include culture of Borrelia from skin or blood and occasionally cerebrospinal fluid (CSF), and detection of genetic material by PCR in skin, blood, synovial fluid and CSF. These tests have specialized roles in research and in academic and reference laboratories but are not available for routine use. Culture and PCR each have distinct limitations that will be noted in this chapter. Diagnostic tests are of clinical value only if they are used appropriately. This has become particularly important in the field of diagnostic testing for Lyme disease, as both patients and doctors hear conflicting information about the risk of Lyme disease in various environments. Furthermore, patients are sometimes given laboratory diagnostic tests when they lack objective signs of Lyme disease and a history of potential exposure to infected vector ticks. A healthcare provider must estimate the pretest likelihood that a patient has Lyme disease in order to understand the positive and negative predictive values of tests for Lyme disease. Fortunately, there are resources available to assist providers in making this judgement. It is important to know that laboratories may offer ‘in-house’ testing for Lyme disease

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The findings and conclusions in this article are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention. © CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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that does not require review and approval by the FDA. Because some in-house tests have not been rigorously developed and validated, the Centers for Disease Control and Prevention (CDC) and FDA recommend that these tests only be used when their accuracy and clinical usefulness have been documented in peer-reviewed scientific literature (CDC, 2005). Unvalidated tests as of 2010 include capture assays for antigens in urine, immunofluorescence staining or cell sorting of cell wall-deficient or cystic forms of B. burgdorferi, lymphocyte transformation tests, quantitative CD57 lymphocyte assays, ‘reverse Western blots’ (Feder et al., 2007), in-house criteria for interpretation of immunoblots and measurements of antibodies in synovial fluid. This chapter considers the diagnostic testing for B. burgdorferi sensu stricto infection, the only organism established to cause Lyme disease in North America. Lyme disease also results from infection by Borrelia garinii or Borrelia afzelii in Europe and Asia, as well as by the recently described Borrelia spielmanii in Europe (Wang et al., 1999; Richter et al., 2006; Fingerle et al., 2008). Borrelia valaisiana and Borrelia lusitaniae have been associated anecdotally with Lyme disease in some parts of Europe (Crowder et al., 2010), particularly B. lusitaniae in Portugal (CollaresPereira et al., 2004). Borrelia bisettii has been cultured from a few patients in Europe (Strle et al., 1997), but has not been shown to cause human disease in North America. Diagnostic tests for B. burgdorferi sensu stricto will not necessarily perform well for infections by other genospecies of Lyme disease bacteria, although some do (e.g. assays based on the C6 peptide of the variable surface antigen (VlsE) or the whole VlsE protein). Guidelines for laboratory diagnosis of European Lyme borreliosis are available online in English (Health Protection Agency of the UK, 2010; German Society for Hygiene and Microbiology, 2000).

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4.2 Two-tiered Serology: the Current Standard for Serodiagnosis in North America The public health agencies of the USA and Canada advocate a two-step process for measuring antibodies in blood when Lyme disease is suspected. The CDC recommends two-tiered testing both for the evaluation of individual patients (CDC, 1995) and for epidemiological surveillance for Lyme disease (CDC, 1997). This recommendation was developed with the participation of the relevant major agencies of the USA, including the FDA, the National Institutes of Health, the Council of State and Territorial Epidemiologists, the Association of Public Health Laboratories and the Clinical Laboratory Standards Institute2 (ASTPHLD and CDC, 1995). The Canadian Public Health Laboratory Network (2007) guidelines also recommend two-tiered testing. The Infectious Diseases Society of America (IDSA) has endorsed two-tiered serology to support the diagnosis of Lyme disease in patients who have manifestations other than acute EM (Wormser et al., 2006). A schematic summarizing the features of two-tiered serology is shown in Fig. 4.1. The first tier consists of a sensitive initial serological test or tests that detect classspecific antibodies (IgM and IgG, either together or separately). First-tier tests are enzyme immunoassays (EIAs) such as ELISAs or, rarely today, indirect immunofluorescence assays (IFAs) as they require a skilled microscopist and cannot be scored objectively. If the result of first-tier testing is negative, the serum is reported to be negative for antibodies to B. burgdorferi and is not tested further. If the result is positive or indeterminate (a value that is sometimes called ‘equivocal’ or ‘borderline’), a second step should be performed. The indeterminate category is the range of test values that overlaps between Lyme disease patients and

The latter two were known at the time as the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the National Committee for Clinical Laboratory Standards (NCCLS), respectively.

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

controls and is specific to each test. Further information is needed from a second test in order to call the specimen positive or negative. The second tier consists of standardized immunoblotting, either by using Western blots or blots striped with diagnostically important purified antigens. When an IgG immunoblot is scored as positive (Dressler et al., 1993; CDC, 1995), two-tiered testing is reported as positive. When an IgM immunoblot is scored as positive (Engstrom et al., 1995; CDC, 1995), Lyme disease serology is reported as positive with the caveat that this finding is clinically relevant only in early disease, that is, in the first month of illness

(ASTPHLD and CDC, 1995; CDC, 1995). Immunoblots and the recommended criteria for scoring them are shown in Fig. 4.2. These scoring criteria have been validated for antibodies to B. burgdorferi sensu stricto, the agent of Lyme disease in North America, but not for immune responses to other genospecies of Borrelia. Two-tiered serology is considered positive only if the EIA (or IFA) and the immunoblot are both positive. Skipping either step increases the frequency of falsepositive results (see below). First-tier tests commonly use whole-cell antigens of B. burgdorferi grown in vitro. The immunodominant antigen VlsE also has been

Two-tiered serology (immunoblotting conditionally supplements EIAs)

Tier 1: IgG or IgM EIAs (combined or separate)

Positive or indeterminate

Negative

Reported as negative; two-tiered protocol complete

Tier 2: IgG and/or IgM immunoblots (separate)

Either blot positive

IgG-positive reported as positive IgM-positive reported as positive BUT clinically relevant only in early disease of less than 1 month duration Fig. 4.1. Two-tiered serology for Lyme disease.

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Negative

Reported as negative

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Fig. 4.2. Examples of conventional IgM (left panel) and IgG (right panel) immunoblots. Bands that are recommended for scoring are labelled. Two additional bands in the IgG blot are also labelled (OspA and OspB at 31 and 34 kDa, respectively; see text). Blots are considered to be positive if two of the three indicated IgM bands or five of the ten indicated IgG bands (excluding OspA and OspB) are present at an intensity equal to or greater than the calibration control. Left panel: IgM blot profiles for a patient with acute EM (lane 1) and for the same patient at convalescence (lane 2). Note the increase in the number and intensity of the bands at convalescence. Right panel: IgG blot profiles for eight patients with later manifestations of Lyme borreliosis (lanes 1–8). P, Positive-control serum; N, negative-control serum; C, calibration control (weak positive control). The molecular mass is indicated (kDa). The calibration controls (weak positive controls) have been digitally enhanced for greater clarity in reproduction.

approved by the FDA. One small portion of VlsE, a 26 amino acid peptide called C6 that reproduces the sixth constant region of the protein, was authorized by the FDA for commercial use (Immunetics) as a first-tier

test in 2001 (Liang et al., 1999; FDA, 2010). A diagnostic assay containing entire VlsE molecules expressed as recombinant proteins from both B. burgdorferi and B. garinii (Diasorin) became available as a first-tier test in 2007 (Ledue et al., 2008; FDA, 2010). C6and VlsE-based assays have the additional feature of detecting antibodies to Eurasian genospecies of Borrelia (i.e. B. garinii and B. azfelii) as well as B. burgdorferi sensu stricto. An extensive peer-reviewed scientific literature supports the rationale for and performance of two-tiered serological testing. This algorithm has been validated in both retrospective and prospective studies. The specificity of two-tiered testing is high – 99% or greater in diagnostic reference centres. The sensitivity is also high after the acute phase of EM. Patients with Lyme arthritis or late neuroborreliosis are nearly always seropositive (97–100%). The rate of seropositivity is lower in patients with acute-phase early neurological disease (80–100%, depending on the population studied). This stage of Lyme disease in particular is the subject of research to improve the sensitivity of serodiagnosis (Dressler et al., 1993; Engstrom et al., 1995; Johnson et al., 1996; Bacon et al., 2003; Peltomaa et al., 2004; Aguero-Rosenfeld et al., 2005; Steere et al., 2008; Branda et al., 2010; Wormser et al., 2011; and others). Patients often inform themselves about diagnostic testing for Lyme disease before visiting a physician. Unfortunately, the quality of information available on the Internet varies widely and some is not evidence-based (Cooper and Feder, 2004). Here are some questions that are commonly asked: 1. Aren’t ELISAs  insensitive and therefore unsuitable as first-tier tests? 2. Aren’t immunoblots  more sensitive than ELISAs? Shouldn’t they be used instead of two-tier testing? 3. Why do the recommended  blot scoring criteria ignore outer-surface protein A (OspA) and OspB? OspA was used as a vaccine, so why isn’t it scored in serology? 4. Why are you  disregarding my IgM test result just because I have had this illness for years?

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

5. How sensitive is  serology in late Lyme disease? How can you be sure, as seropositivity is part of the case definition for Lyme disease, except in patients with EM? Each of these questions will be addressed with reference to the scientific literature.

4.2.1 How sensitive are ELISAs? The sensitivity of first-tier tests varies by stage of Lyme disease. The antibody response to B. burgdorferi develops over the first few weeks after the spirochaete is introduced into the body, in a fashion similar to other bacterial infections. Patients with EM are often seronegative at the time of presentation, as EM can precede the development of a measurable antibody response. The probability of seroreactivity increases with duration of EM and with the development of signs of disseminated disease (AgueroRosenfeld et al., 1993, 1996; Johnson, 2006). Although 60% or less of EM patients test positive by ELISA during acute disease, by convalescence 80–90% of treated EM patients are seropositive (Aguero-Rosenfeld et al., 1993, 1996; Engstrom et al., 1995; Bacon et al., 2003; Johnson et al., 2004; Johnson, 2006). The well-known insensitivity of ELISAs in acute EM is the reason that the CDC and IDSA do not advocate serological testing of these patients. It is appropriate to treat patients who have rashes compatible with EM with antibiotics based on clinical presentation alone. The controversies about serological testing do not generally concern test performance in patients with EM, of course. Fortunately, after the first weeks of illness, the sensitivity of first-tier serology is excellent. Numerous published studies indicate that the sensitivity of whole-cell-lysate ELISAs is essentially 100% after the EM stage of illness (e.g. Dressler et al., 1993; Bacon et al., 2003; Johnson et al., 2004; Johnson, 2006). Antibody levels remain elevated for months to years following antibiotic therapy (Engstrom et al., 1995; Aguero-Rosenfeld et al., 1996; Kalish et al., 2001). Because IgM antibody levels may remain elevated after treatment, a single

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positive IgM ELISA test does not necessarily support the diagnosis of a new B. burgdorferi infection. How did the misconception arise that ELISAs are insensitive in stages of Lyme disease other than EM? Firstly, studies are often cited that describe tests that are obsolete and no longer used. For example, a study conducted in 1992–1994, before twotiered testing was recommended as a national standard, is commonly quoted (Bakken et al., 1997). Many early ELISAs were designed to be stand-alone tests. Some tests were insensitive in order to achieve better specificity using whole-cell lysates. Despite this, false-positive results with some serum samples from healthy donors approached 55% (Bakken et al., 1997). Of the 29 ELISAs approved by the FDA before 1993 (FDA, 2010), only three were used recently by a few laboratories (20/417) that participated in a College of American Pathologists (CAP) proficiency testing programme (CAP, 2009). Most ELISAs in current commercial use are sufficiently sensitive to perform well in a two-tiered testing scheme after the EM stage of illness (Aguero-Rosenfeld et al., 1993; Bacon et al., 2003; Johnson et al., 2004; Johnson, 2006). The excellent performance of ELISAs in proficiency tests can be reviewed by subscribers to the surveys carried out by CAP (2009), although it must be kept in mind that only a small number of samples were used in each evaluation. Secondly, the misconception that ELISAs are insensitive in later Lyme disease is supported by inappropriately applying data from EM patients to people with later manifestations of this illness. Online statements such as ‘The test misses 35% of culture-proven Lyme disease (only 65% sensitivity)’ (ILADS, 2010) fail to note that B. burgdorferi can be consistently cultured only from patients with acute EM, and not from patients with later disease (Aguero-Rosenfeld et al., 2005). It is incorrect to cite the performance of a serological test with samples from patients with EM, for whom serological testing is not recommended, and then claim that ELISAs are poor in diagnosing infections of longer duration.

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4.2.2 Why not skip the ELISA and go directly to immunoblots? It is important to appreciate that first- and second-tier tests are not independent indicators of exposure to B. burgdorferi (Wormser et al., 2000). ELISAs and immunoblots are usually constructed with the same antigens – whole-cell antigens of bacteria grown in culture – but they are processed differently. There is no a priori reason for immunoblots to be more sensitive than ELISAs. ELISAs provide an estimate of the magnitude of the IgG/IgM humoral antibody response to all of the antigens that are expressed under the culture conditions used to produce the whole-cell antigen or to the recombinant or peptide antigens used. ELISA results are objective and quantitative. They can be correlated with antibody titres. Immunoblotting techniques, in contrast, separate the many bacterial antigens spatially on a solid support so that the specificity and complexity of the antibody responses are revealed. Immunoblots are qualitative or, at best, semi-quantitative tests (Fig. 4.2). The rationale for determining IgM and IgG antibody profiles by immunoblotting is to learn whether a patient’s antibodies recognize proteins of B. burgdorferi that have been established to be more predictive of Lyme disease than other components of the bacteria (Dressler et al., 1993; Engstrom et al., 1995). Many antigens have similarities to those of other organisms, such as proteins involved in motility (e.g. flagellin) and responses to stress (e.g. ‘heat-shock’ proteins). Recognition of one or more antigens from this set by serum antibodies is not necessarily indicative of exposure to B. burgdorferi, although these reactions contribute to the signal strength measured in an ELISA. ELISAs for Lyme disease commonly may give false-positive results (up to ~55%) in patients with other spirochaetal diseases such as tick-borne relapsing fever, syphilis or leptospirosis (Johnson et al., 2004: Johnson, 2006), and cross-reactivity with Treponema denticola in patients with periodontal disease has been reported anecdotally. False-positive results also may occur in granulocytic anaplasmosis, although the frequency is

unclear because coinfection with B. burgdorferi may be present (Wormser et al., 1997). Nonspecific reactions due to polyclonal B-cell activation may occur in conditions such as Epstein–Barr virus infection or malaria (Magnarelli, 1995; Burkot et al., 1997). There are reports of false-positive reactions in Helicobacter pylori infections and bacterial endocarditis, although this has not been well studied (Kaell et al., 1993). In addition, noninfectious conditions within the differential diagnosis of Lyme disease yield false-positive rates of around 10%, depending on the patients studied. Cross-reactions are sometimes seen in serum from patients with antinuclear antibodies, rheumatoid factor, clinical rheumatoid arthritis or multiple sclerosis (Johnson et al., 2004; Johnson, 2006). Omitting an ELISA as a first-tier test and using immunoblot results alone decreases the specificity of serological testing. Decreased specificity has been observed both with serum samples from healthy blood donors from non-endemic areas and with samples from patients with other illnesses within the differential diagnosis of Lyme disease. For donors, the decrease in specificity was from 100% for two-tiered testing to 92% for blotting alone in a study by Engstrom et al. (1995) and from 100 to 98.5% in work by Johnson et al. (1996). In patients with other illnesses, there was a 4% decrease from 100% specificity for two-tiered testing to 96% for blotting alone (Johnson et al., 1996). Seemingly small changes in specificity have large public health impacts. The volume of laboratory diagnostic testing for Lyme disease has recently been evaluated. In 2008, more than 3.4 million tests for Lyme disease were performed in the USA (A. Hinckley, CDC, 2010, personal communication). Each 1% decrease in testing specificity would generate about 34,000 false-positive results per year. To put this number in context, 38,468 cases of Lyme disease (confirmed plus probable) were reported to the CDC as part of the US national system for surveillance of notifiable diseases in 2009 (Bacon et al., 2008; CDC, 2011). Why does specificity decrease if immunoblotting alone is used? The Clinical Laboratory Standards Institute identifies one

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

reason: ‘The erroneous scoring of a faint band is a common reason for false-positive readings…’ (NCCLS, 2000). IgM results are more affected by this problem than IgG blots. In general, IgM antibodies are more nonspecifically ‘sticky’ than IgG antibodies, in part because of their pentameric structure in serum compared with monomeric IgG. In addition, only two of three specified bands are required for an IgM blot to be reported as positive, whereas five of ten bands are necessary for an IgG blot to be positive by the recommended blot interpretation criteria (CDC, 1995; Fig. 4.2). Consequently, a single erroneously scored faint band will affect IgM results more readily than it will affect IgG results. Faint bands, particularly in IgM blots, may not be diagnostically significant even for so-called ‘specific’ antigens. If healthcare providers adhere to the recommendation to demonstrate that antibodies are present at a positive or indeterminate level by a first-tier test before ordering an immunoblot, the risk of an erroneously positive serology based on scoring faint bands is reduced but not eliminated. 4.2.3 Why don’t the scoring criteria for immunoblots include OspA and OspB? The bands at the 31 and 34 kDa positions of immunoblots are produced by OspA and OspB, respectively (Fig. 4.2). It has been recognized since the early 1990s that antibodies to OspA and OspB are infrequently detected and when they are observed, it is usually in patients with longstanding Lyme arthritis. Ma et al. (1992) wrote that ‘… antibodies against the 31- and 34-kDa proteins were rarely detected and, consequently, became less significant when compared with other protein bands in this study’. Steere’s laboratory reported in Dressler et al. (1993) that, although antibodies to OspA and OspB were detectable in some patients with Lyme arthritis or late neurological disease, the frequency of antibody responses to these polypeptides was not as high as to ten other antigens. Blot interpretation criteria that could best discriminate Lyme disease patients from controls therefore did not include scoring

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antibodies to OspA or OspB. When bands at 31 or 34 kDa are observed, they are virtually always in the context of a robust IgG response to a large number of scored antigens. Patients may inquire specifically about why OspA is not scored when it was the basis for an effective vaccine (ILADS, 2010). People naturally think of the usual way that vaccines work, neutralizing infection in a mammalian host, and expect a vaccine antigen to be a good diagnostic antigen. They may be unaware that the OspA vaccine works by killing B. burgdorferi in vector ticks as they feed (de Silva et al., 1996). OspA is well expressed by B. burgdorferi in unfed ticks and is a suitable target for antibodies that enter a tick during a blood meal from an OspAvaccinated host. When ticks are exposed to a blood meal and the body temperature of a mammal, B. burgdorferi stops expressing OspA (Schwan and Piesman, 2000). Another outer-surface protein, OspC, is expressed instead. Reciprocal expression of these two Osps has been demonstrated at the level of single cells (Srivastava and de Silva, 2008). It is not surprising, therefore, that antibody responses to OspC are diagnostically useful in early Lyme disease, but responses to OspA are lacking. In later manifestations of Lyme disease, especially Lyme arthritis, some people develop antibodies to OspA and/or OspB. OspA expression is upregulated in an inflammatory milieu such as an arthritic joint. OspA expression can be artificially upregulated in a controlled in vivo environment by exposure to zymosan, a yeast cell-wall extract that induces inflammation (Kalish et al., 1993; Crowley and Huber, 2003). Thus, it is no longer a paradox that B. burgdorferi expresses little or no OspA as it is transmitted to mammalian hosts, but that OspA can be produced late in the course of untreated Lyme disease. Some claim that patients should be judged seropositive based on finding immunoblot bands solely at the 31 or 34 kDa positions, even when their serum is negative by an ELISA that uses whole-cell antigens. However, B. burgdorferi grown in culture expresses OspA and OspB abundantly (Crowley and Huber, 2003) and ELISAs made

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from cultured whole cells contain these antigens. Thus, samples from patients who have diagnostically significant levels of antibodies to OspA or OspB will react in a whole-cell-lysate ELISA. When an ELISA is negative but an immunoblot of the same sample is scored positive, it is probable that faint immunoblot bands are being ‘overread’. 4.2.4 When is IgM testing clinically useful? IgM testing should be performed only in patients with early Lyme disease, defined by the CDC (1995) as within the first month of infection. Some investigators have suggested recently that IgM responses may have diagnostic utility for an additional 2 weeks (Branda et al., 2010, and personal communication). Whether the cut-off for IgM testing is best set at 4 or 6 weeks, IgM testing is appropriate only during a limited early time window. Recall also that serological testing is not useful in patients with EM, the earliest manifestation of Lyme disease, simply because antibodies have not yet had time to develop. This further restricts the clinical utility of IgM testing. Some physicians use IgM serology to assess patients with longstanding illness (many months to years). They point to the new IgM responses to OspB that have been observed to develop late in infection in patients with prolonged disease. This new IgM response, however, occurs in the context of a robust IgG response to a large number of the antigens in the recommended IgG scoring criteria (Kalish et al., 1993). The existence of a new IgM response in Lyme arthritis patients is not good evidence that IgM serology alone, and especially not IgM immunoblotting alone, can properly support the diagnosis of late Lyme disease. During the month or so after initial infection, antibodies rise in titre, recognize an increasing number of borrelial antigens and switch class from a predominantly IgM response to IgG. The evolution of the immune responses during early infection is illustrated

by the serological findings in patients with early neurological disease. In a study of patients with facial paralysis, 87% had diagnostic levels of IgM antibodies, 66% were IgG positive and all were seropositive for at least one antibody class (Peltomaa et al., 2004). This profile of antibody reactivity by class (i.e. a greater frequency of positive IgM responses than IgG, with many people seropositive for both classes) also is seen in patients with other manifestations of early neurological disease, typically meningitis and/or radiculoneuritis (Roux et al., 2007). In the event that a patient with a suspected early manifestation of Lyme disease is seronegative, CDC guidelines note that ‘serologic evidence of infection is best obtained by testing of paired acute- and convalescentphase samples’ (ASTPHLD and CDC, 1995) obtained several weeks apart. By the time patients develop later manifestations of Lyme disease, they are almost universally seropositive for IgG (Dressler et al., 1993; Kannian et al., 2007). Numerous studies with robust sample sizes have been published about the immune responses in Lyme arthritis. Patients with Lyme arthritis typically have high IgG titres, higher than those seen in any of the other various manifestations of Lyme disease, and waning IgM responses. Late neurological Lyme disease, presenting as encephalomyelitis, peripheral neuropathy or encephalopathy, is rare (Wormser et al., 2006; Halperin et al., 2007). It has been speculated that late neuroborreliosis has become rarer in recent years due to earlier diagnosis and treatment, preventing progression to late-stage manifestations. Serum IgG antibodies have been found consistently in patients who have been available for study (Dressler et al., 1993; Bacon et al., 2003). For these reasons, the CDC does not recommend the use of IgM responses in the absence of diagnostic levels of IgG antibodies to support the diagnosis of any manifestation of Lyme disease after 1 month of illness. Furthermore, as noted by Sivak et al. (1996), the predictive value of a positive IgM blot is ‘poor in patients with minimal clinical evidence of Lyme disease’.

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

4.2.5 How can you study the sensitivity of tests for Lyme disease when seropositivity is part of the definition of a case? The clinical signs and symptoms of Lyme disease after the first weeks of infection are not unique to this illness. Clinical findings are not specific enough to permit a confident diagnosis without laboratory testing. As noted by Steere et al. (2008), ‘It is problematic to determine the frequency of seroreactivity in patients with neurological, cardiac, or joint manifestations of Lyme disease, because serological confirmation is a part of the case definition.’ These considerations raise the important question of how to properly select serum samples for studying the performance of serological tests. To avoid circular reasoning, a previous positive serological result should not be the basis for inclusion of a specimen in such a study. However, independent assessment of infection status, for example by bacteriological culture, is routinely successful only in early disease (Aguero-Rosenfeld et al., 2005) and is generally performed only in research settings. To approach this problem, investigators look to the natural history of untreated Lyme disease. Patients with late disease frequently have a documented history of earlier signs and symptoms of Lyme disease that support the clinical diagnosis. For selection of ‘gold standard’ specimens for assessment of serological test performance in later Lyme disease, serum from patients with antecedent clinical findings compatible with earlier Lyme disease are used. Supplementary research tests such as PCR add additional confidence to the classification of some specimens (Bradley et al., 1994; Nocton et al., 1994). Patients with early neurological Lyme disease commonly have a history of recent EM. Lyme facial paralysis, for example, was associated with EM in 72–87% of patients, depending upon the study (Peltomaa et al., 2004). Patients with carditis, an uncommon presentation of Lyme disease that occurs in the early weeks of infection and manifests primarily as atrioventricular block, also typically have either previous or concurrent

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EM (>80%) or sometimes early neurological Lyme disease (Wormser et al., 2006). Patients with late neurological Lyme disease, a rare condition, generally have a history of other clinical manifestations of Lyme disease such as EM or Lyme arthritis. In a report by Bacon et al. (2003), 100% of 11 late neurological Lyme disease patients were seropositive. All of these patients had antecedent other clinical manifestations of Lyme disease that were the basis for including the serum samples in the study.

4.3 Newer Serological Tests Two-tiered serology has good performance characteristics, that is, high sensitivity and specificity after the first weeks of B. burgdorferi infection. Experienced laboratories with good-quality control and quality assurance programmes obtain consistent results (e.g. Bacon et al., 2003; Kannian et al., 2007; CAP, 2009). Nevertheless, there are limitations to two-tiered testing that are being addressed by newer testing methods. As noted, twotiered testing is insensitive in acute EM and may be negative in early neuroborreliosis. Other drawbacks are that the two-step procedure is complex, technically demanding and costly. Immunoblots are only semiquantitative. Traditional blots are hard to standardize, as reading them involves judgement about the significance of weak bands. Other difficulties with two-tiered serology are the need to know the date of disease onset to appropriately request IgM testing and the inconvenience of sometimes having to draw a second blood sample. The latter may occur if the second test is indicated and the first test was performed by a laboratory that does not offer immunoblotting. The research community is actively addressing these limitations, and a number of new testing approaches have been developed. The Public Health Service agencies have established the standard that new tests should meet or exceed the performance of two-tiered testing in order to be deemed suitable for clinical use (ASTPHLD and CDC, 1995). New approaches are either improvements in one of the steps of the two-step

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testing regime or a potential alternative to two-tiered testing. Striped blots with defined, purified antigens were FDA-approved in 2009 and are now commercially available (FDA, 2010). Viramed offers these immunoblots (Virablots) as an improvement over Western blots. Bands are striped at pre-defined positions so that calibration problems are avoided. They are read with a scanning densitometer to provide an objective measure of whether each band has sufficient colour density to be scored as a diagnostically significant reaction. Branda et al. (2010) have devised a two-tiered procedure consisting of whole-cell ELISAs and IgG Virablots that include a new band of VlsE. Only a VlsE band would be required for a positive reaction in early Lyme disease and five or more of 11 bands in the late disease (the bands in Fig. 4.2 plus VlsE). This approach provides sensitivity comparable to or higher than standard two-tiered testing in each stage of Lyme disease, while maintaining high specificity. If adopted, it would render IgM blots obsolete. The problems of falsepositive IgM blots due to over-reading of faint bands and the difficulty of knowing how long a patient has been infected would be avoided. A second approach, developed by Zeus Scientific, seeks to avoid immunoblotting altogether by using defined peptides in a multiplex microsphere assay on the Luminex diagnostic platform. This assay, called the AtheNA Multi-Lyte test system, has been FDA-approved as a first-tier test and also evaluated with favourable results as an alternative to immunoblotting when other approved assays are used as the first-tier test (FDA, 2010; Porwancher et al., 2011). Both the C6 peptide and whole VlsE assays have been approved as alternatives to whole-cell ELISAs as first-tier tests. In addition, the Immunetics C6 assay has recently been evaluated as an assay that could be used in place of both steps of two-tiered testing, that is, as a simple ‘stand-alone’ test. The C6 ELISA as a single step is significantly more sensitive in patients with EM than twotiered testing (66.5 versus 35.2%, P0.001; Wormser et al., 2011). Furthermore, the C6 assay performed comparably to two-tiered testing in sera from patients with early

neuroborreliosis or Lyme arthritis. The specificity of the C6 assay was slightly less than two-tiered testing (98.9 versus 99.5%, P0.05), however, which will be a key consideration when the assay is reviewed for approval as a stand-alone test. Various diagnostic testing approaches will offer value to clinicians. The general practitioner may prefer a simple, objective, less-costly one-step test. The specialist may prefer the added information that immunoblots provide to diagnose atypical cases. The type and number of reactive bands offer insights about the stage of Lyme disease. Expanding profiles of reactivity with paired samples may support suspicion of ongoing infection.

4.4 Direct Assays Two types of direct assay have been important in Lyme disease research and are useful in the laboratory diagnosis of some patients. These assays are culture of B. burgdorferi and detection of DNA by molecular methods (PCR or quantitative real-time PCR). Neither culture nor PCR are components of the routine evaluation of patients with suspected Lyme disease and no nationally standardized or FDA-approved tests are available. Both techniques have played important roles in understanding the pathogenesis of B. burgdorferi infections, however, and have assisted investigators in establishing serum banks from authenticated Lyme disease patients. Direct detection methods have been reviewed in detail by Aguero-Rosenfeld et al. (2005) and have not changed significantly since this work was published. B. burgdorferi can be recovered from skin biopsy samples of EM patients with 50% efficiency. Efficiency of recovery is inversely correlated to the duration of EM, indicating that spirochaetes are rapidly cleared from the region of skin inoculated by tick bite. In acute EM, spirochaetes also can be grown from blood, especially high-volume plasma cultures, with recovery rates of 40%. The period of haematogenous dissemination of borreliae, however, is brief (several weeks). In later stages of the disease, blood cultures are

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

generally negative. There are only anecdotal reports of B. burgdorferi cultured from synovial fluid, an apparently hostile environment, and CSF. The low sensitivity of culture after the EM stage of illness (which can be treated based on the appearance of the rash) and the length of time necessary to monitor cultures (3 weeks or longer, depending on the protocol) greatly limit the clinical usefulness of bacteriological culture. PCR is a sensitive method to detect B. burgdoferi DNA in skin biopsy and synovial fluid specimens (Dumler, 2001). AgueroRosenfeld et al. (2005) calculated median PCR sensitivities of 64% in skin biopsy samples from EM patients (four studies, range 59– 67%) and 83% in synovial fluid specimens (four studies, range 76–100%). PCR has been particularly useful diagnostically in evaluating patients with treatment-resistant Lyme arthritis (Nocton et al., 1994). DNA detection methods have been less helpful in evaluating patients with neurological signs. Reported PCR sensitivities in CSF have been low and highly variable. PCR tests were positive in 38% of early and 25% of late US neuroborreliosis patients (n = 60; Nocton et al., 1996). Urine is not a suitable sample for PCR testing (Rauter et al., 2005).

4.5 Appropriate Use of Diagnostic Tests Laboratory testing of patients without objective signs of Lyme disease or a history of potential exposure to infected vector ticks is not clinically useful. Laboratory diagnostic tests with excellent sensitivity and specificity will not have helpful predictive values if they are used inappropriately (Sackett et al., 1991). Predictive value is determined both by test characteristics (sensitivity and specificity) and, importantly, by the population in which it is used. The practice of testing patients with a low likelihood of Lyme disease can generate more false-positive results than true-positive results, resulting in misdiagnosis and thereby harming ill people (Seltzer and Shapiro, 1996; Tugwell et al., 1997). The positive predictive value is the probability that a patient who has a positive

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test result truly has Lyme borreliosis. Negative predictive value is the probability that a patient who has a negative test result does not have Lyme borreliosis. An assay with high diagnostic sensitivity improves negative predictive value; one with high diagnostic specificity improves positive predictive value. Serological testing is recommended only for patients who have appropriate pre-test probabilities of Lyme disease in order for the results to have useful predictive values. A position paper published by the American College of Physicians (ACP) concluded that laboratory testing should be requested only for patients who have an estimated pre-test probability of Lyme disease between 0.20 and 0.80 (Tugwell et al., 1997). The ACP panel members pointed out that patients who have only non-specific signs and symptoms of illness such as headache, fatigue and muscle or joint pains, even when they reside in a geographical area endemic for Lyme disease, have a pre-test probability of Lyme disease of less than 0.20, usually much less. Patients with non-specific findings and no risk of exposure to infected ticks will have an extremely low pre-test probability. When the pre-test probability of Lyme disease is greater than 0.80, laboratory evaluation adds little useful information (Tugwell et al., 1997). This situation only occurs in patients presenting with EM in an endemic area, as all of the other clinical manifestations of Lyme disease can be found in other conditions. The risk of Lyme disease is geographically focal. Of more than 300,000 cases reported to the CDC over the last 15 years, most occurred in ten states of the northeast and upper midwest. Maps of reported cases of Lyme disease by county and tables of incidence by state are updated annually by the CDC and published online (CDC, 2011)c. The mapped density of host-seeking Ixodes scapularis nymphs in the USA is consistent with the pattern of reported human cases (DiukWasser et al., 2006). A ‘Lyme disease tick map’ has recently become available as an iPhone application through the Apple iTunes store (American Lyme Disease Foundation, 2010). The concepts of positive and negative predictive value are well established and

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Table 4.1. Effect of disease prevalence on predictive values of diagnostic tests a Prevalence = 1% Test positive

Test negative

Total

Disease 10 0 No disease 20 970 Total 30 970 Predictive value of a negative result = 970/970 = 100% Predictive value of a positive result = 10/30 = 33% (67% false positives) Prevalence = 40% Test positive

Test negative

Total

Disease 392 8 No disease 12 588 Total 404 596 Predictive value of a negative result = 588/596 = 99% Predictive value of a positive result = 392/404 = 97% (3% false positives) aIllustration

10 990 1000

400 600 1000

assumes that test sensitivity and specificity are each 98%.

have been described carefully elsewhere (e.g. Sackett et al., 1991; Seltzer and Shapiro, 1996; Tugwell et al., 1997). They are briefly illustrated in Table 4.1 for two different clinical situations. In both cases, diagnostic tests with good performance characteristics are assumed: 98% sensitivity and 98% specificity. In the first situation, the true frequency of disease in the population to be tested (prevalence) is only 1%. This represents the pre-test likelihood of Lyme disease in a patient with non-specific symptoms and no objective physical signs of this illness who resides in an endemic area (CDC, 2011). For patients with no history of residence in or travel to an endemic area, the prevalence of Lyme disease is much less than 1%. In the second situation, the true frequency of Lyme disease is 40%. This prevalence (or higher) is

the approximate pre-test likelihood of Lyme arthritis in patients with pronounced knee swelling who reside in an endemic area (Tugwell et al., 1997). Good tests have markedly different predictive values depending on the setting of use (Table 4.1). When the pre-test probability is 40%, the predictive values of both negative and positive results are very high (99% and 97%, respectively). However, when the pretest probability is low, most positive test results are false positives (67%). Clinicians are currently ordering an extraordinary number of diagnostic tests for Lyme disease – more than 3.4 million tests annually, as noted above. It is critically important to the well-being of patients that tests only be used when the predictive value of a positive result is high (Fig. 4.3).

Where disease is rare Positives mostly deceive Even with good tests Paul Mead

Fig. 4.3. Haiku to diagnostic testing.

Laboratory Diagnostic Testing for Borrelia burgdorferi Infection

References Aguero-Rosenfeld, M.E., Nowakowski, J., McKenna, D.F., Carbonaro, C.A. and Wormser, G.P. (1993) Serodiagnosis in early Lyme disease. Journal of Clinical Microbiology 31, 3090–3095. Aguero-Rosenfeld, M.E., Nowakowski, J., Bittker, S., Cooper, D., Nadelman, R.B. and Wormser, G.P. (1996) Evolution of the serologic response to Borrelia burgdorferi in treated patients with culture-confirmed erythema migrans. Journal of Clinical Microbiology 34, 1–9. Aguero-Rosenfeld, M.E., Wang, G., Schwartz, I. and Wormser, G.P. (2005) Diagnosis of Lyme borreliosis. Clinical Microbiology Reviews 18, 484–509. American Lyme Disease Foundation (2010) Lyme disease tick map iPhone app . ASTPHLD and CDC (1995) Proceedings of the Second National Conference on Serologic Diagnosis of Lyme Disease, Dearborn, Michigan, 27–29 October 1994. Association of State and Territorial Public Health Laboratory Directors and the Centers for Disease Control and Prevention, Washington, DC. Bacon, R.M., Biggerstaff, B.J., Schriefer, M.E., Gilmore, R.D. Jr, Philipp, M.T., Steere, A.C., Wormser, G.P., Marques, A.R. and Johnson, B.J. (2003) Serodiagnosis of Lyme disease by kinetic enzyme-linked immunosorbent assay using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with 2-tiered testing using whole-cell lysates. Journal of Infectious Diseases 187, 1187–1199. Bacon, R.M., Kugeler, K.J. and Mead, P.S. (2008) Surveillance for Lyme disease – United States, 1992–2006. MMWR Surveillance Summaries 57, 1–9. Bakken, L.L., Callister, S.M., Wand, P.J. and Schell, R.F. (1997) Interlaboratory comparison of test results for detection of Lyme disease by 516 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists proficiency testing program. Journal of Clinical Microbiology 35, 537–543. Bradley, J.F., Johnson, R.C. and Goodman, J.L. (1994) The persistence of spirochetal nucleic acids in active Lyme arthritis. Annals of Internal Medicine 120, 487–489. Branda, J.A., Aguero-Rosenfeld, M.E., Ferraro, M.J., Johnson, B.J., Wormser, G.P. and Steere, A.C. (2010) 2-Tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a

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VlsE band as the second-tier test. Clinical Infectious Diseases 50, 20–26. Burkot, T.R., Schriefer, M.E. and Larsen, S.A. (1997) Cross-reactivity to Borrelia burgdorferi proteins in serum samples from residents of a tropical country nonendemic for Lyme disease. Journal of Infectious Diseases 175, 466–469. CAP (2009) Tick-transmitted Disease Survey TTDB. College of American Pathologists, Northfield, IL. Canadian Public Health Laboratory Network (2007) The laboratory diagnosis of Lyme borreliosis: guidelines from the Canadian Public Health Laboratory Network. Canadian Journal of Infectious Diseases and Medical Microbiology 18, 145–148. CDC (1995) Recommendations for test performance and interpretation from the second national conference on serologic diagnosis of Lyme disease. Morbidity and Mortality Weekly Report 44, 590–591. CDC (1997) Case definitions for infectious conditions under public health surveillance. Morbidity and Mortality Weekly Report. Recommendations and Reports 46, 1–55. CDC (2005) Notice to readers: caution regarding testing for Lyme disease. Morbidity and Mortality Weekly Report 54, 125–126. CDC (2011) Lyme disease data and statistics . Collares-Pereira, M., Couceiro, S., Franca, I., Kurtenbach, K., Schafer, S.M., Vitorino, L., Goncalves, L., Baptista, S., Vieira, M.L. and Cunha, C. (2004) First isolation of Borrelia lusitaniae from a human patient. Journal of Clinical Microbiology 42, 1316–1318. Cooper, J.D. and Feder, H.M. Jr (2004) Inaccurate information about Lyme disease on the internet. Pediatric Infectious Disease Journal 23, 1105– 1108. Crowder, C.D., Matthews, H.E., Schutzer, S., Rounds, M.A., Luft, B.J., Nolte, O., Campbell, S.R., Phillipson, C.A., Li, F., Sampath, R., Ecker, D.J. and Eshoo, M.W. (2010) Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe. PLoS One 5, e10650. Crowley, H. and Huber, B.T. (2003) Host-adapted Borrelia burgdorferi in mice expresses OspA during inflammation. Infection and Immunity 71, 4003–4010. de Silva, A.M., Telford, S.R. III, Brunet, L.R., Barthold, S.W. and Fikrig, E. (1996) Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine. Journal of Experimental Medicine 183, 271– 275.

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Diuk-Wasser, M.A., Gatewood, A.G., Cortinas, M.R., Yaremych-Hamer, S., Tsao, J., Kitron, U., Hickling, G., Brownstein, J.S., Walker, E., Piesman, J. and Fish, D. (2006) Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States. Journal of Medical Entomology 43, 166–176. Dressler, F., Whalen, J.A., Reinhardt, B.N. and Steere, A.C. (1993) Western blotting in the serodiagnosis of Lyme disease. Journal of Infectious Diseases 167, 392–400. Dumler, J.S. (2001) Molecular diagnosis of Lyme disease: review and meta-analysis. Molecular Diagnosis 6, 1–11. Engstrom, S.M., Shoop, E. and Johnson, R.C. (1995) Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. Journal of Clinical Microbiology 33, 419–427. FDA (2010) Medical devices: in vitro diagnostics/510(k) premarket notification ; product code LSR. Feder, H.M. Jr, Johnson, B.J., O’Connell, S., Shapiro, E.D., Steere, A.C., Wormser, G.P., Agger, W.A., Artsob, H., Auwaerter, P., Dumler, J.S., Bakken, J.S., Bockenstedt, L.K., Green, J., Dattwyler, R.J., Munoz, J., Nadelman, R.B., Schwartz, I., Draper, T., Mcsweegan, E., Halperin, J.J., Klempner, M.S., Krause, P.J., Mead, P., Morshed, M., Porwancher, R., Radolf, J.D., Smith, R.P. Jr, Sood, S., Weinstein, A., Wong, S.J. and Zemel, L. (2007) A critical appraisal of “chronic Lyme disease”. New England Journal of Medicine 357, 1422–1430. Fingerle, V., Schulte-Spechtel, U.C., Ruzic-Sabljic, E., Leonhard, S., Hofmann, H., Weber, K., Pfister, K., Strle, F. and Wilske, B. (2008) Epidemiological aspects and molecular characterization of Borrelia burgdorferi s.l. from southern Germany with special respect to the new species Borrelia spielmanii sp. nov. International Journal of Medical Microbiology 298, 279–290. German Society for Hygiene and Microbiology (2000) Quality standards for the microbiological diagnosis of infectious diseases: Lyme borreliosis . Halperin, J.J., Shapiro, E.D., Logigian, E., Belman, A.L., Dotevall, L., Wormser, G.P., Krupp, L., Gronseth, G. and Bever, C.T. Jr (2007) Practice parameter: treatment of nervous system Lyme disease (an evidence-based review): Report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 69, 91–102. Health Protection Agency of the UK (2010) Diagnosis and treatment of Lyme borreliosis

. ILADS (2010) Basic information about Lyme disease. International Lyme and Associated Diseases Society, Bethesda, MD . Johnson, B. (2006) Lyme disease: serologic assays for antibodies to Borrelia burgdorferi. In: Detrick, B., Hamilton, R. and Folds, J. (eds) Manual of Molecular and Clinical Laboratory Immunology, 7th edn. ASM Press, Washington, DC. Johnson, B.J., Robbins, K.E., Bailey, R.E., Cao, B.L., Sviat, S.L., Craven, R.B., Mayer, L.W. and Dennis, D.T. (1996) Serodiagnosis of Lyme disease: accuracy of a two-step approach using a flagella-based ELISA and immunoblotting. Journal of Infectious Diseases 174, 346–353. Johnson, B.J.B., Bacon, R.M. and Schriefer, M.E. (2004) Correspondence. Journal of Infectious Diseases 189, 1962–1964. Kaell, A.T., Redecha, P.R., Elkon, K.B., Golightly, M.G., Schulman, P.E., Dattwyler, R.J., Kaell, D.L., Inman, R.D., Christian, C.L. and Volkman, D.J. (1993) Occurrence of antibodies to Borrelia burgdorferi in patients with nonspirochetal subacute bacterial endocarditis. Annals of Internal Medicine 119, 1079–1083. Kalish, R.A., Leong, J.M. and Steere, A.C. (1993) Association of treatment-resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi. Infection and Immunity 61, 2774–2779. Kalish, R.A., Mchugh, G., Granquist, J., Shea, B., Ruthazer, R. and Steere, A.C. (2001) Persistence of immunoglobulin M or immunoglobulin G antibody responses to Borrelia burgdorferi 10–20 years after active Lyme disease. Clinical Infectious Diseases 33, 780–785. Kannian, P., McHugh, G., Johnson, B.J., Bacon, R.M., Glickstein, L.J. and Steere, A.C. (2007) Antibody responses to Borrelia burgdorferi in patients with antibiotic-refractory, antibiotic responsive, or non-antibiotic-treated Lyme arthritis. Arthritis and Rheumatism 56, 4216–25. Ledue, T.B., Collins, M.F., Young, J. and Schriefer, M.E. (2008) Evaluation of the recombinant VlsE-based Liaison chemiluminescence immunoassay for detection of Borrelia burgdorferi and diagnosis of Lyme disease. Clinical and Vaccine Immunology 15, 1796– 1804. Liang, F.T., Steere, A.C., Marques, A.R., Johnson, B.J., Miller, J.N. and Philipp, M.T. (1999) Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an

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immunodominant conserved region of Borrelia burgdorferi VlsE. Journal of Clinical Microbiology 37, 3990–3996. Ma, B., Christen, B., Leung, D. and Vigo-Pelfrey, C. (1992) Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. Journal of Clinical Microbiology 30, 370–376. Magnarelli, L.A. (1995) Current status of laboratory diagnosis for Lyme disease. American Journal of Medicine 98, S10–S12; discussion S12–S14. NCCLS (2000) Western blot assay for antibodies to Borrelia burgdorferi; approved guideline. National Committee for Clinical Laboratory Standards document M34-A, Vol. 20, No. 20. Nocton, J.J., Dressler, F., Rutledge, B.J., Rys, P.N., Persing, D.H. and Steere, A.C. (1994) Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis. New England Journal of Medicine 330, 229–234. Nocton, J.J., Bloom, B.J., Rutledge, B.J., Persing, D.H., Logigian, E.L., Schmid, C.H. and Steere, A.C. (1996) Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid in Lyme neuroborreliosis. Journal of Infectious Diseases 174, 623–627. Peltomaa, M., Mchugh, G. and Steere, A.C. (2004) The VlsE (IR6) peptide ELISA in the serodiagnosis of Lyme facial paralysis. Otology and Neurotology 25, 838–841. Porwancher, R.H., Hagerty, C.G., Fan, J., Landsberg, L., Johnson, B.J.B., Kopnitsky, M., Kulas, K., and Wong, S.J. (2011) Multiplex immunoassay for Lyme disease using VlsE1IgG and pepC10-IgM antibodies: improving test performance through bioinformatics. Clinical and Vaccine Immunology 18, 851–859. Rauter, C., Mueller, M., Diterich, I., Zeller, S., Hassler, D., Meergans, T. and Hartung, T. (2005) Critical evaluation of urine-based PCR assay for diagnosis of Lyme borreliosis. Clinical and Diagnostic Laboratory Immunology 12, 910–917. Richter, D., Postic, D., Sertour, N., Livey, I., Matuschka, F.R. and Baranton, G. (2006) Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov. International Journal of Systematic and Evolutionary Microbiology 56, 873–881. Roux, F., Boyer, E., Jaulhac, B., Dernis, E., ClossProphette, F. and Puechal, X. (2007) Lyme meningoradiculitis: prospective evaluation of biological diagnosis methods. European Journal of Clinical Microbiology and Infectious Diseases 26, 685–693.

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Sackett, D.H., Haynes, R.B., Guyatt, G.H., and Tugwell, P. (1991) Clinical Epidemiology: a Basic Science for Clinical Medicine. Little, Brown and Co., Boston, MA. Schwan, T.G. and Piesman, J. (2000) Temporal changes in outer surface proteins A and C of the Lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. Journal of Clinical Microbiology 38, 382–388. Seltzer, E.G. and Shapiro, E.D. (1996) Misdiagnosis of Lyme disease: when not to order serologic tests. Pediatric Infectious Disease Journal 15, 762–763. Sivak, S.L., Aguero-Rosenfeld, M.E., Nowakowski, J., Nadelman, R.B. and Wormser, G.P. (1996) Accuracy of IgM immunoblotting to confirm the clinical diagnosis of early Lyme disease. Archives of Internal Medicine 156, 2105–2109. Srivastava, S.Y. and de Silva, A.M. (2008) Reciprocal expression of OspA and OspC in single cells of Borrelia burgdorferi. Journal of Bacteriology 190, 3429–3433. Steere, A.C., McHugh, G., Damle, N. and Sikand, V.K. (2008) Prospective study of serologic tests for Lyme disease. Clinical Infectious Diseases 47, 188–195. Strle, F., Picken, R.N., Cheng, Y., Cimperman, J., Maraspin, V., Lotric-Furlan, S., Ruzic-Sablijic, E. and Picken, M.M. (1997) Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genomic and phenotypic similarities of strain 25015. Clinical Infectious Diseases 25, 273–280. Tugwell, P., Dennis, D.T., Weinstein, A., Wells, G., Shea, B., Nichol, G., Hayward, R., Lightfoot, R., Baker, P. and Steere, A.C. (1997) Laboratory evaluation in the diagnosis of Lyme disease. Annals of Internal Medicine 127, 1109–1123. Wang, G., Van Dam, A.P., Schwartz, I. and Dankert, J. (1999) Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clinical Microbiology Reviews 12, 633–653. Wormser, G.P., Horowitz, H.W., Nowakowski, J., Mckenna, D., Dumler, J.S., Varde, S., Schwartz, I., Carbonaro, C. and Aguero-Rosenfeld, M. (1997) Positive Lyme disease serology in patients with clinical and laboratory evidence of human granulocytic ehrlichiosis. American Journal of Clinical Pathology 107, 142–147. Wormser, G.P., Carbonaro, C., Miller, S., Nowakowski, J., Nadelman, R.B., Sivak, S. and Aguero-Rosenfeld, M.E. (2000) A limitation of 2-stage serological testing for Lyme disease:

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enzyme immunoassay and immunoblot assay are not independent tests. Clinical Infectious Diseases 30, 545–548. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S. and Nadelman, R.B. (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and

babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43, 1089–1134. Wormser, G., Schriefer, M., Aguero-Rosenfeld, M., Levin, A., Steere, A., Nadelman, R., Nowakowski, M., Marques, A., Johnson, B. and Dumler, J. (2011) Single-tier testing with the C6 peptide ELISA kit compared with two-tiered testing for Lyme disease. Journal of the American Medical Association (in press).

5

Persistence of Borrelia burgdorferi Infection after Antibiotic Treatment: What Can We Learn From Animal Models? Joppe W.R. Hovius and Gary P. Wormser

5.1 Introduction Lyme disease, or Lyme borreliosis, has become the most common tick-borne infection in parts of the northeastern USA and Europe (Steere et al., 2004). Lyme borreliosis is caused by spirochaetes of the Borrelia burgdorferi sensu lato group. In the USA, Borrelia burgdorferi sensu stricto, hereafter referred to as B. burgdorferi, is the causative agent, whereas in Europe B. burgdorferi also occurs, but Borrelia garinii and Borrelia afzelii predominate. B. burgdorferi is transmitted by the deer tick, Ixodes scapularis, whereas the European and Asian Borrelia species are principally transmitted by Ixodes ricinus (the sheep tick) and Ixodes persulcatus, respectively.

5.2 Human Lyme borreliosis When humans become infected with B. burgdorferi in the USA, asymptomatic infection seems to occur only in approximately 10% of infected individuals, whereas in Europe, this percentage is thought to be much higher (Steere et al., 1986; Fahrer et al., 1991; Kuiper et al., 1993; Vos et al., 1994; Steere et al., 2003). The most common manifestation of symptomatic infection is an expanding red skin lesion with a centrifugally migrating

outer margin, called erythema migrans (EM). In untreated individuals, spirochaetes can disseminate to other skin sites or affect various organs, including the joints, central and peripheral nervous systems, and heart. The course of the disease can be divided into an acute phase, encompassing early or localized disease (days to weeks), early disseminated disease (weeks to months) and a late or chronic phase (months to years) (Steere, 2001; Steere, 2006), as discussed in more detail throughout this book. In most infected patients, the immune response will eventually clear these clinical manifestations, even in the absence of antibiotic treatment (Steere et al., 1987). However, antibiotic therapy will greatly accelerate the rate of resolution for many of the clinical manifestations and prevent later ones from developing, (see Wormser, Chapter 7, this volume). In a minority of cases, accompanying non-specific symptoms such as fatigue or arthralgias will last for a long period of time despite recommended antibiotic treatment and resolution of the prior objective clinical manifestation. These symptoms are probably better referred to as post-Lyme borreliosis symptoms or syndrome rather than chronic Lyme borreliosis or chronic Lyme disease, because the latter terms are used in the lay literature to designate persisting B. burgdorferi infection.

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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No study has provided convincing evidence of persistent infection in such patients or provided evidence of an overall beneficial effect of retreatment with antibiotics (Klempner et al., 2001; Klempner, 2002; Kaplan et al., 2003; Krupp et al., 2003; Fallon et al., 2008).

5.3 Animal Data on Persistence of B. burgdorferi Infection Several studies using animal models have addressed the question of whether B. burgdorferi is capable of causing persistent infection in mammals, with or without antibiotic treatment, as reviewed previously by Hovius et al. (2009) and Wormser and Schwartz (2009). Animal models offer several opportunities for investigations not afforded by studies of patients. For example, while B. burgdorferi has been detected in patient material by culture or PCR, the sensitivity of such assays is relatively low (Barbour, 1984; Schwartz et al., 1992; Picken et al., 1997; Nadelman et al., 1999). In contrast, animal models allow the harvesting of multiple tissues during the course of infection and post mortem, facilitating detection of the spirochaete. In addition, experimental animal models allow investigation of the disease in a controlled fashion, often with known genetic information of both host and pathogen. Models also circumvent other confounding factors such as reinfection (Nadelman and Wormser, 2007) or coinfection with other tickborne pathogens. Animal models, however, also have a number of limitations, and their relevance to what happens clinically needs to be established rather than assumed. One limitation is that they may not adequately replicate the biological events found in human infection. With regard to treatment issues specifically, the role of antibiotic therapy may not even be assessable in certain animal systems. For example, the most common clinical manifestation of Lyme borreliosis in humans is EM, a manifestation not known to occur in either mice or dogs. A fundamental methodological concern in

animal studies is the often markedly different pharmacokinetic parameters of antibiotics in animals compared with humans. Insufficient attention to this issue will lead to antibiotic exposures in animals that do not faithfully reproduce, and usually underestimate, those that occur in humans. Finally, the selection of appropriate end points for judging antibiotic efficacy in animal systems is also an important consideration. Inappropriate end points that do not mirror those that are relevant to patient care might also lead to unjustifiable extrapolations to human infections. There has been a lack of attention to these issues in most of the published studies on the use of antibiotics for treatment of experimental Lyme borreliosis in animal systems, as will be discussed in more detail in this chapter. Lyme borreliosis has been examined using a variety of animal systems, including hamsters (Johnson et al., 1984; Schmitz et al., 1988), rats (Barthold et al., 1988), rabbits (Burgdorfer et al., 1982; Kornblatt et al., 1984; Pachner et al., 1994; Foley et al., 1995), mice (Barthold et al., 1990, 1991, 1992; Barthold, 1991; Barthold and de Souza, 1995), dogs (Appel et al., 1993; Straubinger et al., 1997a,b, 2000; Straubinger, 2000) and non-human primates (England et al., 1997; Pachner et al., 1995, 1998, 2001a,b,c; Philipp et al., 1993, 2001, 2005; Roberts et al., 1995, 1998b). In this chapter, we will discuss the most widely used models, that is, the murine, canine and nonhuman primate models for Lyme borreliosis, with a focus on studies investigating the persistence of B. burgdorferi in untreated and treated animals. 5.3.1 The murine model When interpreting data on the persistence of B. burgdorferi in antibiotic-treated or untreated laboratory mice, it is important to realize that inbred mice are closely related to their wildlife counterparts, which, in contrast to humans, are natural reservoir hosts for B. burgdorferi, in which long-term persistence of infection is a naturally occurring phenomenon. None the less, the murine model is used by most Lyme disease researchers (Weis, 2002). C3H/HeJ or C3H/

Persistence of Borrelia burgdorferi Infection after Antibiotic Treatment

HeN mouse strains develop acute carditis and subacute arthritis upon inoculation with B. burgdorferi (Barthold et al., 1990, 1991, 1992, 1993; Barthold, 1991; Barthold and de Souza, 1995; Weis, 2002) and can also be persistently infected with B. burgdorferi (Barthold et al., 1993). In C3H/HeJ mice that were intradermally inoculated with B. burgdorferi, viable spirochaetes could be detected by culture up to 1 year post-infection (Barthold et al., 1993). In addition, during later stages of infection, these mice showed mild intermittent episodes of both carditis and arthritis. In infected mice, a 5-day course of treatment with ceftriaxone reduces culture positivity but may not necessarily eliminate it (Malawista et al., 1994; Moody et al., 1994; Pavia et al., 2002; Yrjanainen et al., 2007), and borrelial DNA may still be detected by PCR (Malawista et al., 1994; Yrjanainen et al., 2007). Although mice treated with longer courses of ceftriaxone therapy may still be PCR positive, cultures are typically negative (Kazragis et al., 1996; Bockenstedt et al., 2002; Hodzic et al., 2008; Yrjanainen et al., 2010). One group of investigators has argued that mice treated with a 5-day course of ceftriaxone are more likely to be culture positive when immunosuppressed with antitumour necrosis factor  (anti-TNF-) (Yrjanainen et al., 2007), but the data presented in support of that contention are unconvincing, as we have discussed previously in more detail (Hovius et al., 2009; Wormser and Schwartz, 2009). Why inhibition of TNF- would plausibly play such a significant biological role in host defence for a nongranulomatous infection such as Lyme borreliosis is unclear. Treatment with corticosteroids has not resulted in culture positivity in the murine treatment system (Pavia et al., 2002). Similarly, Kazragis et al. (1996) observed negative cultures for B. burgdorferi in B. burgdorferi-infected severe combined immune deficient (SCID) mice treated with a 9-day course of ceftriaxone. In addition, the available clinical experience with the use of TNF- inhibitors does not suggest that they pose a risk for recrudescence of Lyme borreliosis. Steere and Angelis (2006) treated four patients who had antibiotic-refractory Lyme borreliosis-associated arthritis with

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infliximab, a TNF- inhibitor, following antibiotic therapy. Prior to treatment, all four patients were shown to have negative results by PCR for B. burgdorferi DNA in joint fluid samples. Three of the four patients experienced resolution of the arthritis with infliximab therapy. The patient with nonresponsive arthritis underwent arthroscopic synovectomy; PCR results for B. burgdorferi DNA were negative in both joint fluid and synovial tissue samples obtained during the procedure. Notably, none of the four patients had evidence of active infection during the period following antibiotic therapy, and residual infection was not brought out by treatment with a TNF- inhibitor. Bockenstedt et al. (2002) showed that, after treatment of B. burgdorferi strain N40infected C3H/HeJ mice with a 1-month course of ceftriaxone (subcutaneous administration) or oral doxycycline, low levels of spirochaete DNA could be detected by PCR in a subset of antibiotic-treated mice. However, in contrast to mock-treated mice, antibiotic-treated mice became culture negative and did not have histopathological evidence of tissue inflammation. Ticks feeding on antibiotictreated mice were able to acquire B. burgdorferi at 3 but not at 6 months after treatment. These ticks were unable to transmit the spirochaete to other C3H/HeJ mice. Bockenstedt and collaborators concluded that the few residual spirochaetes remaining after antibiotic treatment were avirulent and would eventually be eradicated by the host’s immune system. Although the antibiotics used by Bockenstedt et al. (2002) have shorter serum half-lives in mice, dosage adjustments were not made to replicate the antibiotic exposure in humans, as discussed in detail by Wormser and Schwartz (2009). In addition, potential variability in the blood levels of the antibiotics among individual mice was not assessed. The key pharmacodynamic property for antibiotic efficacy of ceftriaxone (and other -lactam antibiotics) for bacterial infections in general, as well as for Borrelia in particular, is the time that the blood level of drug exceeds the minimum inhibitory concentration (‘time over MIC’ or T/MIC) (Wormser et al., 2007). The mean serum halflife of ceftriaxone administered by the intra-

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peritoneal route in mice is only 0.47–1.1 h, compared with a half-life of 5.8–8.7 h in humans who received doses parenterally (Wang et al., 2005). Thus, in murine treatment studies, multiple daily doses of ceftriaxone would be required to simulate the T/MIC of ceftriaxone found in humans. For doxycycline, the pharmacodynamic property that appears to predict antimicrobial activity is the area under the curve (of free drug) over the MIC (AUC/MIC) (Ambrose et al., 2007), but data for borrelial infections specifically are limited (Lee and Wormser, 2008). Based on the data provided, the AUC of doxycycline in the study by Bockenstedt et al. (2002) fell short of that found in humans who are treated for Lyme borreliosis. The importance of doxycycline exposure in mice to treatment outcome is well illustrated by a study in which two different preparations of doxycycline were administered to mice shortly after infection with B. burgdorferi. A single dose of doxycycline was 43% effective in treating incubating B. burgdorferi infection when administered orally but 100% effective when administered by a single subcutaneous injection of a sustained-release preparation of the drug (Zeidner et al., 2004). In a study by Hodzic et al. (2008), it was shown that a 30-day course of ceftriaxone (administered intraperitoneally) cleared B. burgdorferi from infected immunocompetent mice, as detected by culture. However, low levels of B. burgdorferi DNA could occasionally be detected by PCR, especially when treatment was initiated 4 months after infection (compared with 3 weeks after infection). Persistence of B. burgdorferi DNA was not associated with tissue inflammation and, although ticks feeding on these mice could acquire B. burgdorferi and transmit the bacterium to immunocompromised (SCID) mice, the SCID mice did not develop histological evidence of disease, thereby not fulfilling Koch’s postulates. Whether these ticks were able to transmit the spirochaetes to immunocompetent mice was not reported. Furthermore, as emphasized previously by Wormser and Schwartz (2009), the number of residual spirochaetes after antibiotic treatment decreased with time. More recently, the same investigators performed a similar

study using two different dosage regimens of a new antibiotic, tigecycline (administered once daily by subcutaneous injection for 10 days), and showed that low levels of spirochaetal DNA could be detected 3 months after cessation of treatment in mice treated during the early and late phases of infection (Barthold et al., 2010). However, none of the antibiotic-treated mice was culture positive, although there was evidence of at least some B. burgdorferi gene transcription. Similar to the prior ceftriaxone study, ticks feeding on the antibiotic-treated mice were able to transmit the bacterium to SCID mice, which did not become culture positive or develop histological signs of disease. Similarly, SCID mice could be infected by transplantation of skin allografts from treated mice. The authors hypothesized that they were likely to have achieved tigecycline blood levels comparable to that found in humans, but no information on measurements of drug levels or degree of protein binding was provided in the paper to verify this. Furthermore, the efficacy of tigecycline for the treatment of B. burgdorferi sensu lato infection in humans is unknown, and this drug is unlikely ever to be used clinically for this indication because it needs to be administered intravenously twice daily and the price is over 400 times that of doxycycline. Together, the murine studies illustrate: (i) that B. burgdorferi, as well as clinical signs of Lyme borreliosis, can persist in untreated mice; (ii) that a sufficient dose/duration of antibiotics can lead to disappearance of both cultivable spirochaetes and clinical signs, even in animals that are highly immunocompromised; (iii) that spirochaetal DNA can persist after treatment, can be detected by xenodiagnosis and can be transmitted to SCID mice, but cannot cause objective evidence of disease in SCID mice; and (iv) that not all mice will be culture negative after short-term, probably inadequate, treatment with 5 days of ceftriaxone. 5.3.2 The canine model The course of Lyme borreliosis in experimentally infected dogs was first described by

Persistence of Borrelia burgdorferi Infection after Antibiotic Treatment

Appel et al. (1993). The authors showed that young dogs infected with B. burgdorferi through a tick bite developed lameness with fibrinopurulent arthritis 2–5 months after tick attachment. B. burgdorferi could be detected by PCR and culture for up to 1 year after tick attachment and in subsequent studies by Straubinger et al. (2000) for up to 500 days. Despite the persistence of infection, untreated dogs became asymptomatic (Appel et al., 1993). Thus, in the untreated canine model for Lyme borreliosis, there is evidence for persistent B. burgdorferi infection with cultivable spirochaetes. Straubinger et al. (1997b) have further shown that B. burgdorferi can be detected by PCR and occasionally by culture in infected dogs after antibiotic treatment. In their first study, they showed that three (25%) of 12 dogs had a positive culture following the completion of a 30-day course of antibiotic therapy with doxycycline (twice daily; two culture-positive dogs) or amoxicillin (two or three times a day; one culture-positive dog), but only on a single tissue sample out of numerous samples that were cultured (Straubinger et al., 1997b). For example, one dog treated with doxycycline was culture positive on a skin sample taken at 6 months after initiation of treatment, but was culture negative on all post-mortem samples and on multiple earlier skin samples. In a subsequent study (Straubinger et al., 2000), all of the doxycycline-treated dogs were culture negative (see below). Notably, the one amoxicillin-treated dog in the study that was culture positive was treated with twice daily rather than three times daily. Insufficient blood levels of the drug may have contributed to the culture positivity. In this study, the authors noted a late rise in antibody titres to B. burgdorferi of unclear significance. In the follow-up study, the antibody titres of the doxycycline-treated dogs did not appear to demonstrate the same pattern (Straubinger et al., 2000). Furthermore, these authors, in conjunction with other investigators, have reported dramatic falls in C6 antibody levels in ceftriaxone-treated dogs that remained seropositive by other assays, some of whom were PCR positive in tissues post-treatment

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(Straubinger et al., 2000; Philipp et al., 2001). C6 is an antigenic peptide that is used to detect antibodies against the immunodominant and conserved invariable region 6 (IR-6) of the B. burgdorferi protein VlsE. This protein is expressed by replicating spirochaetes in vivo. C6 test results were not reported for dogs treated with antibiotics other than ceftriaxone. In later experiments in which Straubinger and collaborators studied the course of Lyme borreliosis over a period of approximately 1.5 years, they demonstrated that 75% (9/12) of antibiotic-treated dogs remained PCR positive in several tissues (Straubinger et al., 2000). However, none of these animals was culture positive, including those dogs that were treated with doxycycline (Straubinger et al., 2000). In this study (Straubinger et al., 2000), the dogs were treated with doxycycline at a later time point post-infection and for unclear reasons also had higher blood levels of drug compared with the prior study (Straubinger et al., 1997b). In addition, when PCR-positive dogs were treated orally with prednisone at 2 mg/kg twice a day for 14 days, they remained asymptomatic and culture negative (Straubinger et al., 2000). By contrast, in control dogs that had not been treated with antibiotics, this transient immunosuppression resulted in painful and swollen joints in all four limbs. Whether or not untreated control dogs were transiently immunosuppressed, they were culture positive in multiple tissues during the course of infection and post mortem (Straubinger et al., 2000). These studies demonstrate that: (i) untreated dogs can be persistently infected with B. burgdorferi; (ii) antibiotic failure – as detected by isolation of viable spirochaetes by culture – is infrequent and inconsistent within and between studies and may be related to inadequate blood levels of antibiotics; (iii) antibiotic-treated culturenegative dogs may be PCR positive for a prolonged period extending to 455 days after the conclusion of antibiotic therapy; and (iv) that PCR-positive culture-negative dogs did not become culture positive or develop clinical illness when immunosuppressed with corticosteroids.

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5.3.3 The non-human primate model The course of infection in rhesus macaques (Macaca mulatta) has been studied by Pachner and colleagues and Philipp and collaborators (Roberts et al., 1995; England et al., 1997; Pachner et al., 2001c). These investigators, however, used rhesus macaques that originated from different geographical areas, which may account for some of the differences in the findings (a topic beyond the scope of this chapter). Moreover, these animals are outbred and thus have various genetic backgrounds. Besides developing arthritis and carditis, rhesus macaques develop EM when infected intradermally with B. burgdorferi, either naturally via ticks or by syringe inoculation (Pachner et al., 1995; England et al., 1997). The rhesus model is the only animal model to exhibit signs of Lyme neuroborreliosis of both the peripheral and central nervous systems (CNS). Neuroborreliosis of the peripheral nervous system has been evaluated both functionally and histologically (England et al., 1997; Roberts et al., 1998a). The functional evaluation consisted of nerve conduction studies of both motor and sensory nerves. Five of the eight B. burgdorferi-infected animals that were studied had a mononeuropathy or mononeuropathy multiplex pattern with axonal loss. Light microscopy showed axon loss, fibrosis and a decreased number of myelinated axons in sural nerve sections from two of the infected animals. Evidence that B. burgdorferi accesses the CNS of rhesus macaques was first provided by Pachner et al. (1995). This study included five animals, one of which was immunocompromised by dexamethasone injections. CNS invasion was documented by PCR in the cerebrospinal fluid (CSF) of all of the animals (rather than in brain parenchyma), and by culture in two, one of which was immunocompromised. An important feature of the model is the finding that both PCR positivity and CSF pleocytosis were detected for as long as 18 weeks postinoculation in all of the immunocompetent animals (Pachner et al., 1995). In a similar experiment involving four animals that had

been inoculated with a neurotropic strain of B. burgdorferi (Ramesh et al., 2003), one of the animals exhibited marked pleocytosis that lasted for several weeks. However, in most untreated immunocompetent animals (i.e. animals not transiently treated with corticosteroids), spirochaetes, as detected by culture, are eventually cleared (Pachner et al., 2001c); none the less mild carditis can persist for years in some immunocompetent macaques in association with low numbers of spirochaetes as detected by quantitative PCR (Cadavid et al., 2004). In animals that have been transiently immunosuppressed by treatment with corticosteroids, infection may persist for a longer period of time and involve a greater number of tissue sites (Cadavid et al., 2003; Pachner et al., 2001a,b). In a study in which infection of rhesus macaques with B. burgdorferi was confirmed by skin biopsy culture, animals were treated 3 months post-infection with 2 mg/kg of doxycycline, twice a day for 60 days (Philipp et al., 2001). The rationale for this dose was not described; the dose is similar to paediatric dosing in humans (4 mg/kg/day) but substantially lower than that used in the dog studies discussed above (Straubinger et al., 1997b, 2000). It is stated that a peak blood level of 2.1 g/ml was achieved with a trough level of 0.3 g/ml. The level of protein binding was not reported, and in general there is a paucity of information in the published literature on the pharmacokinetics of doxycycline and other antibiotics in primates (Vietri et al., 2006). The doxycycline serum levels in this primate study were lower than those found in humans, and the rapid reduction in drug levels over just 12 h implies that doxycycline has a much shorter half-life in this particular primate species compared with humans. So far, the culture and PCR results from treatment studies of primates with B. burgdorferi infection have not been reported. However, reported levels of anti-C6 serum antibodies wane rapidly posttreatment and become negative, in contrast to untreated animals, which remain strongly seropositive (Philipp et al., 2001).

Persistence of Borrelia burgdorferi Infection after Antibiotic Treatment

5.3.4 Concluding remarks on the available animal data Experiments performed in various animal models demonstrate that, when untreated, B. burgdorferi can cause persisting infection, based on the recovery of viable spirochaetes in culture. Indeed, in human studies, B. burgdorferi has been isolated by culture, albeit only sporadically, months to years after untreated infection (Steere et al., 1983; Asbrink and Hovmark, 1985; Stanek et al., 1990; Picken et al., 1997). Studies of antibiotic efficacy in murine and canine animal systems have shown evidence that is consistent with post-treatment persistence of small numbers of replicationdeficient spirochaetes, or at least their DNA, in certain tissues. It is debatable, however, whether any end point other than culture should be the primary microbiological outcome measure in animal systems investigating the efficacy of antibiotic treatment for experimental Lyme borreliosis. It can be argued that only culture positivity establishes that the spirochaetes are fully viable. The observations that the residual organisms cannot be cultured, do not divide or divide very slowly, produce only a limited array of mRNA, are not associated with inflammation or tissue injury, do not cause clinically objective disease manifestations, are disappearing over time and – in the case of the mouse model – cannot infect immunocompetent mice suggest that these spirochaetes are moribund and will ultimately be eliminated by the host’s immune system. Future studies should attempt to determine whether this is indeed the case. Future studies should also attempt to replicate the findings with other species of mice and with antibiotic regimens demonstrated to produce levels of antibiotic exposure commensurate with that in humans being treated for Lyme borreliosis.

5.4 Implications of Available Animal Data for Human Lyme borreliosis The relevance of these findings to human Lyme borreliosis in general, and to the

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pathogenesis of post-Lyme disease syndrome in particular, remains unclear. When interpreting data from animal studies on the persistence of B. burgdorferi after antibiotic treatment, it is of paramount importance to emphasize that, in the treatment of most infections, antimicrobial therapy per se does not eliminate every single microorganism from an infected host. Indeed, treatment with antibiotics that inhibit rather than kill microorganisms is highly effective in a wide range of infections (Pankey and Sabath, 2004). Thus, the role of antimicrobial therapy can be thought of in terms of ‘tipping the balance’ in favour of the host’s own defence mechanisms against a particular pathogen (Wormser and Schwartz, 2009). Therefore, resolution of objective disease manifestations is the most straightforward and logical way to assess treatment effect, and progression or relapse of objective manifestations is the most reasonable standard to assess failure of therapy. Whether a few spirochaetes or fragments of spirochaetal DNA persist in humans is irrelevant in judging the outcome of treatment for Lyme borreliosis, unless these residual organisms can be shown to cause tissue inflammation or objective clinical manifestations. It is also difficult to envision how non-dividing, non-cultivable residual spirochaetes could cause subjective nonspecific symptoms. B. burgdorferi is not known to produce systemic toxins, and in the animal systems discussed above, the systemic immune response appears to be waning (Wormser and Schwartz, 2009).

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model of Lyme neuroborreliosis: characterization by PCR, serology, and sequencing of the OspA gene from the brain. Neurology 44, 1938–1943. Pachner, A.R., Delaney, E., O’Neill, T. and Major, E. (1995) Inoculation of nonhuman primates with the N40 strain of Borrelia burgdorferi leads to a model of Lyme neuroborreliosis faithful to the human disease. Neurology 45, 165–172. Pachner, A.R., Schaefer, H., Amemiya, K., Cadavid, D., Zhang, W.F., Reddy, K. and O’Neill, T. (1998) Pathogenesis of neuroborreliosis – lessons from a monkey model. Wiener Klinische Wochenschrift 110, 870–873. Pachner, A.R., Amemiya, K., Bartlett, M., Schaefer, H., Reddy, K. and Zhang, W.F. (2001a) Lyme borreliosis in rhesus macaques: effects of corticosteroids on spirochetal load and isotype switching of anti-Borrelia burgdorferi antibody. Clinical and Diagnostic Laboratory Immunology 8, 225–232. Pachner, A.R., Cadavid, D., Shu, G., Dail, D., Pachner, S., Hodzic, E. and Barthold, S.W. (2001b) Central and peripheral nervous system infection, immunity, and inflammation in the NHP model of Lyme borreliosis. Annals of Neurology 50, 330–338. Pachner, A.R., Gelderblom, H. and Cadavid, D. (2001c) The rhesus model of Lyme neuroborreliosis. Immunology Reviews 183, 186–204. Pankey, G.A. and Sabath, L.D. (2004) Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Grampositive bacterial infections. Clinical Infectious Diseases 38, 864–870. Pavia, C., Inchiosa, M.A. Jr and Wormser, G.P. (2002) Efficacy of short-course ceftriaxone therapy for Borrelia burgdorferi infection in C3H mice. Antimicrobial Agents Chemotherapy 46, 132–134. Philipp, M.T., Aydintug, M.K., Bohm, R.P. Jr, Cogswell, F.B., Dennis, V.A., Lanners, H.N., Lowrie, R.C. Jr, Roberts, E.D., Conway, M.D., Karacorlu, M., Peyman, G.A., Gubler, D.J., Johnson, B.J., Piesman, J. and Gu, Y. (1993) Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans. Infection and Immunity 61, 3047–3059. Philipp, M.T., Bowers, L.C., Fawcett, P.T., Jacobs, M.B., Liang, F.T., Marques, A.R., Mitchell, P.D., Purcell, J.E., Ratterree, M.S. and Straubinger, R.K. (2001) Antibody response to IR6, a conserved immunodominant region of the VlsE lipoprotein, wanes rapidly after antibiotic treatment of Borrelia burgdorferi infection in experimental animals and in humans. Journal of Infectious Diseases 184, 870–878.

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Philipp, M.T., Wormser, G.P., Marques, A.R., Bittker, S., Martin, D.S., Nowakowski, J. and Dally, L.G. (2005) A decline in C6 antibody titer occurs in successfully treated patients with cultureconfirmed early localized or early disseminated Lyme borreliosis. Clinical and Diagnostic Laboratory Immunology 12, 1069–1074. Picken, M.M., Picken, R.N., Han, D., Cheng, Y., Ruzic-Sabljic, E., Cimperman, J., Maraspin, V., Lotric-Furlan, S. and Strle, F. (1997) A two year prospective study to compare culture and polymerase chain reaction amplification for the detection and diagnosis of Lyme borreliosis. Molecular Pathology 50, 186–193. Ramesh, G., Alvarez, A.L., Roberts, E.D., Dennis, V.A., Lasater, B.L., Alvarez, X. and Philipp, M.T. (2003) Pathogenesis of Lyme neuroborreliosis: Borrelia burgdorferi lipoproteins induce both proliferation and apoptosis in rhesus monkey astrocytes. European Journal of Immunology 33, 2539–2550. Roberts, E.D., Bohm, R.P. Jr, Cogswell, F.B., Lanners, H.N., Lowrie, R.C. Jr, Povinelli, L., Piesman, J. and Philipp, M.T. (1995) Chronic Lyme disease in the rhesus monkey. Laboratory Investigation 72, 146–160. Roberts, E.D., Bohm, R.P. Jr, Lowrie, R.C. Jr, Habicht, G., Katona, L., Piesman, J. and Philipp, M.T. (1998a) Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system. Journal of Infectious Diseases 178, 722–732. Roberts, E.D., Bohm, R.P. Jr, Lowrie, R.C. Jr, Habicht, G., Katona, L., Piesman, J. and Philipp, M.T. (1998b) Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system. Journal of Infectious Diseases 178, 722–732. Schmitz, J.L., Schell, R.F., Hejka, A., England, D.M. and Konick, L. (1988) Induction of Lyme arthritis in LSH hamsters. Infection and Immunity 56, 2336–2342. Schwartz, I., Wormser, G.P., Schwartz, J.J., Cooper, D., Weissensee, P., Gazumyan, A., Zimmermann, E., Goldberg, N.S., Bittker, S., Campbell, G.L. and Pavia, C.S. (1992) Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. Journal of Clinical Microbiology 30, 3082–3088. Stanek, G., Klein, J., Bittner, R. and Glogar, D. (1990) Isolation of Borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy. New England Journal of Medicine 322, 249–252.

Steere, A.C. (2001) Lyme disease. New England Journal of Medicine 345, 115–125. Steere, A.C. (2006) Lyme borreliosis in 2005, 30 years after initial observations in Lyme Connecticut. Wiener Klinische Wochenschrift 118, 625–633. Steere, A.C. and Angelis, S.M. (2006) Therapy for Lyme arthritis: strategies for the treatment of antibiotic-refractory arthritis. Arthritis and Rheumatism. 54, 3079–3086. Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E. and Malawista, S.E. (1983) The spirochetal etiology of Lyme disease. New England Journal of Medicine 308, 733–740. Steere, A.C., Taylor, E., Wilson, M.L., Levine, J.F. and Spielman, A. (1986) Longitudinal assessment of the clinical and epidemiological features of Lyme disease in a defined population. Journal of Infectious Diseases 154, 295–300. Steere, A.C., Schoen, R.T. and Taylor, E. (1987) The clinical evolution of Lyme arthritis. Annals of Internal Medicine 107, 725–731. Steere, A.C., Sikand, V.K., Schoen, R.T. and Nowakowski, J. (2003) Asymptomatic infection with Borrelia burgdorferi. Clinical Infectious Diseases 37, 528–532. Steere, A.C., Coburn, J. and Glickstein, L. (2004) The emergence of Lyme disease. Journal of Clinical Investigation 113, 1093–1101. Straubinger, R.K. (2000) PCR-based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-day postinfection period. Journal of Clinical Microbiology 38, 2191–2199. Straubinger, R.K., Straubinger, A.F., Harter, L., Jacobson, R.H., Chang, Y.F., Summers, B.A., Erb, H.N. and Appel, M.J. (1997a) Borrelia burgdorferi migrates into joint capsules and causes an up-regulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks. Infection and Immunity 65, 1273–1285. Straubinger, R.K., Summers, B.A., Chang, Y.F. and Appel, M.J. (1997b) Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. Journal of Clinical Microbiology 35, 111–116. Straubinger, R.K., Straubinger, A.F., Summers, B.A. and Jacobson, R.H. (2000) Status of Borrelia burgdorferi infection after antibiotic treatment and the effects of corticosteroids: an experimental study. Journal of Infectious Diseases 181, 1069–1081. Vietri, N.J., Purcell, B.K., Lawler, J.V., Leffel, E.K., Rico, P., Gamble, C.S., Twenhafel, N.A., Ivins, B.E., Heine, H.S., Sheeler, R., Wright, M.E. and

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Friedlander, A.M. (2006) Short-course postexposure antibiotic prophylaxis combined with vaccination protects against experimental inhalational anthrax. Proceedings National Academy of Sciences, USA 103, 7813–7816. Vos, K., van Dam, A.P., Kuiper, H., Bruins, H., Spanjaard, L. and Dankert, J. (1994) Seroconversion for Lyme borreliosis among Dutch military. Scandinavian Journal of Infectious Diseases 26, 427–434. Wang, E., Bergeron, Y. and Bergeron, M.G. (2005) Ceftriaxone pharmacokinetics in interleukin-10treated murine pneumococcal pneumonia. Journal of Antimicrobial Chemotherapy 55, 721–726. Weis, J.J. (2002) Host–pathogen interactions and the pathogenesis of murine Lyme disease. Current Opinion in Rheumatology 14, 399–403. Wormser, G.P. and Schwartz, I. (2009) Antibiotic treatment of animals infected with Borrelia burgdorferi. Clinical Microbiology Reviews 22, 387–395. Wormser, G.P., Barthold, S.W., Shapiro, E.D., Dattwyler, R.J., Bakken, J.S., Steere, A.C.,

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Bockenstedt, L.K. and Radolf, J.D. (2007) Antitumor necrosis factor- activation of Borrelia burgdorferi spirochetes in antibiotic-treated murine Lyme borreliosis: an unproven conclusion. Journal of Infectious Diseases 196, 1865–1866. Yrjanainen, H., Hytonen, J., Song, X.Y., Oksi, J., Hartiala, K. and Viljanen, M.K. (2007) Anti-tumor necrosis factor- treatment activates Borrelia burgdorferi spirochetes 4 weeks after ceftriaxone treatment in C3H/He mice. Journal of Infectious Diseases 195, 1489–1496. Yrjanainen, H., Hytonen, J., Hartiala, P., Oksi, J. and Viljanen, M.K. (2010) Persistence of borrelial DNA in the joints of Borrelia burgdorferiinfected mice after ceftriaxone treatment. Acta Pathologica, Microbiologica et Immunologica Scandinavica 118, 665–673. Zeidner, N.S., Brandt, K.S., Dadey, E., Dolan, M.C., Happ, C. and Piesman, J. (2004) Sustainedrelease formulation of doxycycline hyclate for prophylaxis of tick bite infection in a murine model of Lyme borreliosis. Antimicrobial Agents and Chemotherapy 48, 2697–2699.

6

Global Epidemiology of Borrelia burgdorferi Infections1 Paul S. Mead

6.1 Introduction Lyme borreliosis, or Lyme disease, is a multisystem tick-borne illness caused by several genospecies of the spirochaete Borrelia burgdorferi sensu lato (Steere et al., 2004). Clinical features of human infection include dermatological, rheumatological, neurological and cardiac abnormalities (Strle and Stantic-Pavlinic, 1996; Steere, 2001; Stanek and Strle, 2003; Nau et al., 2009). Transmission occurs through the bite of infected ticks of the Ixodes ricinus complex, which are found widely in temperate regions of the northern hemisphere (Piesman and Gern, 2004). Although not formally described until the mid-1970s, Lyme borreliosis is now recognized as the most common vector-borne disease in both Europe and North America. Many factors interact to determine the epidemiology of Lyme borreliosis. These include the genospecies of B. burgdorferi and their distribution in nature, the abundance and feeding habits of the various vector tick species, and the demographic and behavioural characteristics of the exposed human population. In addition, perceptions

of disease frequency and character are coloured by surveillance practices. Lack of surveillance obviously leads to underreporting of cases. Even where surveillance is in place, however, differences in methodology can systematically influence the apparent features of disease. For example, laboratory-based surveillance may disproportionately detect patients who are seropositive and therefore more likely to have later stages of illness. The interpretation of surveillance data is further complicated by the existence of clinically similar diseases (Wormser et al., 2005; Mantovani et al., 2007), the vagaries of serological testing (Hunfeld et al., 2002; Aguero-Rosenfeld, 2003; Ekerfelt et al., 2004), the potential for asymptomatic infections (Feder et al., 1995; Steere et al., 2003; Rojko et al., 2005) and uncertainty regarding the pathogenic potential of some B. burgdorferi genospecies (van Dam, 2002; da Franca et al., 2005). With these limitations in mind, this chapter attempts to provide an overview of the epidemiology of B. burgdorferi infections in various regions of the world, underscoring similarities and differences across regions.

1

The findings and conclusions in this article are those of the author and do not necessarily represent the views of the Centers for Disease Control and Prevention. The author thanks Ms Anna Perea for assistance with the illustrations.

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6.2 Agents, Vectors and Geographical Distribution At least 17 distinct genospecies of B. burgdorferi sensu lato have been described based on isolates from rodents, birds and ticks (Rudenko et al., 2010). Three of these cause the majority of human infections: Borrelia afzelii, Borrelia garinii and B. burgdorferi sensu stricto. These agents are transmitted to humans by four species of Ixodes ticks, in various combinations of vector and pathogen. The distribution of these tick species in Eurasia and North America generally defines the geography of Lyme borreliosis in humans (Fig. 6.1). Within this area, however, the risk of human infection varies widely due to differences in tick abundance and infection rates, which can range from 0 to 40% depending upon local enzootic cycles (Piesman and Gern, 2004). Other genospecies of B. burgdorferi sensu lato have occasionally been isolated from humans, including Borrelia spielmanii, Borrelia bavariensis, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissetii (Diza et al., 2004; Maraspin et al., 2006; de Carvalho et al., 2008;

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Rudenko et al., 2008; Rudenko et al., 2009). The clinical features, frequency and overall public health significance of these infections are poorly defined. I. ricinus is the principal vector of Lyme borreliosis in Europe and transmits all three major pathogenic genospecies. Populations of I. ricinus are found throughout western, central and eastern Europe (Fig. 6.1), generally at elevations below 1300 m (Piesman and Gern, 2004). Rates of infection in adult ticks tend to be higher in eastern than western Europe, and the relative frequency of infection with the different genospecies appears to vary across regions. Ticks collected in northern and eastern Europe (e.g. Scandinavia, Baltic states, Czech Republic, Slovakia, Croatia and Bulgaria) are most likely to carry B. afzelii, while those from western Europe (e.g. Austria, Switzerland and the UK) are more likely to be infected with B. garinii (Rauter and Hartung, 2005). The distribution of I. ricinus also extends into the northern reaches of Morocco, Algeria and Tunisia, where carriage of B. lusitaniae is more common (Piesman and Gern, 2004).

Ixodes Distribution I. pacificus • B. b. sensu stricto I. scapularis • B. b. sensu stricto I. ricinus • B. afzelii • B. garinii • B. b. sensu stricto I. persulcatus • B. afzelii • B. garinii

Fig. 6.1. Approximate global distribution of principal Ixodes vectors of Lyme borreliosis and associated genospecies of Borrelia burgdorferi sensu lato (Korenberg, 1994; Masuzawa, 2004; Postic et al., 1997; Hao et al., 2010).

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The distribution of Ixodes persulcatus, the principal vector in Asia, extends from western Russia, where it overlaps with I. ricinus, eastwards through Mongolia and China to the Pacific Ocean and Japan (Fig. 6.1). This species transmits B. afzelii and Asian and Eurasian variants of B. garinii; it is not known to transmit B. burgdorferi sensu stricto (Korenberg, 1994; Korenberg et al., 2002; Masuzawa, 2004). In North America, B. burgdorferi sensu stricto is the only identified cause of Lyme borreliosis in humans (Steere, 2001; Piesman and Gern, 2004). Transmission in northeastern and north-central states is by I. scapularis ticks, which are locally abundant and have infection rates of up 40%. Although I. scapularis ticks are also found in the southeastern USA (Fig. 6.1), they are much less abundant and are rarely infected with B. burgdorferi sensu stricto, possibly as a result of genetic and local ecological factors (Piesman and Gern, 2004). To date, the few B. burgdorferi sensu stricto isolates that have been identified in the southeastern USA have all been collected within a few miles of the Atlantic coast (Lin et al., 2003; Oliver et al., 2008). Distinct foci of infection occur in the western coastal USA extending northward into southern British Columbia. Transmission to humans in these areas is by Ixodes pacificus ticks, which, because of local transmission cycles, are rarely infected and account for relatively few cases (Piesman and Gern, 2004).

6.3 Incidence by Region 6.3.1 North America The USA accounts for nearly all Lyme disease cases reported in North America. Over 325,000 confirmed cases have been reported in the USA since Lyme disease was first designated as a nationally notifiable condition in 1991 (Bacon et al., 2008; CDC, 2008b, 2009, 2010). Within the USA, incidence is highest in the northeastern, mid-Atlantic and northcentral states. In 2009, 14 states (Connecticut, Delaware, Maine, Massachusetts, Maryland, Minnesota, New Hampshire, New Jersey,

New York, Pennsylvania, Rhode Island, Vermont, Virginia and Wisconsin) accounted for over 96% of confirmed cases nationwide (CDC, 2010). Verified cases reported from other states are usually associated with travel to highly endemic areas (Bacon et al., 2008). Exceptions occur along the West Coast in California, Oregon and Washington where I. pacificus is an established although rarely infected vector (Piesman and Gern, 2004). The lack of appreciable transmission in nonor low-endemic areas, as defined by human surveillance, is strongly supported by data on serological testing of domestic dogs. In a nationwide sample of nearly 1 million commercial assays performed by veterinarians, overall seroprevalence for anti-Borrelia antibodies was 10.7% among 435,537 dogs in the 14 highly endemic states listed above, 1.9% among 32,285 dogs in California, Oregon and Washington, and 0.5% among 514,514 dogs in the remaining non-endemic states (see Table 1 in Bowman et al., 2009). The 0.5% value is consistent with the frequency of travel-associated cases in dogs (Duncan et al., 2005) and with the manufacturer’s published range for false-positive results for the assay (IDEXX, 2010). Incidence rates for endemic US states typically range from 10 to 100 per 100,000 population (Table 6.1). The highest recorded state-wide incidence is 134 per 100,000, reported in Connecticut in 2002 following implementation of mandatory laboratorybased reporting (Bacon et al., 2008; Connecticut Department of Public Health, 2009). Areas of hyperendemicity with county-level rates in excess of 200 per 100,000 include Windham County in Connecticut, Dukes and Nantucket counties in Massachusetts, Hunterdon County in New Jersey, Columbia, Dutchess, Putnam and Greene counties in New York and Washburn County in Wisconsin (Bacon et al., 2008). In the northeastern states where many homes are situated in heavily tick-infested areas, exposure is thought to occur primarily in the peridomestic environment (Cromley et al., 1998). In the north-central USA, areas of highest risk are often lightly populated; infection in these states is more often related to outdoor recreational activities.

Global Epidemiology of Borrelia burgdorferi Infections

Table 6.1. Reported or estimated incidence of Lyme borreliosis per 100,000 for selected countries. Country/region Austria Belgium Bulgaria Canada Croatia Czech Republic Denmark Estonia Finland France Germany Great Britain England and Wales Scotland Hungary Iceland Ireland Italy Japan Latvia Lithuania Moldovia The Netherlands Norway Poland Portugal Russia European okrug Northwest okrug Urals okrug Siberian okrug Far East okrug Sverdlovsk/Jekaterinberg Tomsk Serbia and Montenegro Slovakia Slovenia Spain Sweden (southern) Switzerland Turkey Ukraine USA Connecticut Delaware Maine Maryland Massachuesetts Minesota New Hampshire New Jersey New York Pennsylvania Rhode Island Wisconsin Vermont Virginia

Incidence

Year/period

Reference

135a 16 13 0.1 5.9 36 1.1 133.0 27.5 9.4 36.5

2005 2005 2005 1995–2006 1993–2000 2005 2009 2009 2009 1999–2000 2006

Smith and Takkinen (2006) Smith and Takkinen (2006) Smith and Takkinen (2006) Ogden et al. (2008) Hubalek (2009) Smith and Takkinen (2006) EpiNorth (2010) EpiNorth (2010) EpiNorth (2010) Letrilliart et al. (2005) Fulop and Poggensee (2008)

1.7 1.7 12.8 0.6 0.6 0.1 0.1 31.8 108 0.7 103a 5.6 27.1 0.1

2008–2009 2002–2005 2001–2005 1999–2003 1995 2001–2005 2000–2005 2009 2009 2003–2005 2005 2009 2009 1999–2004

Smith and O’Connell (2010) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hashimoto et al. (2007) EpiNorth (2010) EpiNorth (2010) Hubalek (2009) Hofhuis et al. (2006) EpiNorth (2010) EpiNorth (2010) Hubalek (2009)

4.6 9.2 8.3 9.8 4.3 14.7 28 2.4 16 206 9.8 69 25.1 0.1 2.1

1999–2006 1999–2006 1999–2006 1999–2006 1999–2006 1999–2006 1993–1994 1988–1994 2005 2005

Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Hubalek (2009) Smith and Takkinen (2006) Smith and Takkinen (2006) Hubalek (2009) Berglund et al. (1996) Hubalek (2009) Hubalek (2009) EpiNorth (2010)

78.2 111.2 60.0 25.7 61.0 20.2 75.2 52.8 21.2 39.3 14.2 34.5 51.9 8.9

2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009 2009

1992 1988–1998 1990–2002 2009

Surveillance methods vary widely and values may not be directly comparable. a Estimated

CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010) CDC (2010)

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In the 18 years since implementation of national reporting, the number of confirmed cases in the USA has increased from approximately 9000 to nearly 30,000 annually (Bacon et al., 2008; CDC, 2010). Much of this increase can be attributed to greater recognition and enhanced surveillance. In Connecticut, for example, the total case number increased threefold following implementation of laboratory-based reporting, despite the number ascertained through physician reporting remaining stable (Connecticut Department of Public Health, 2009). Nevertheless, there is also evidence of true increases in disease incidence and geographic expansion in some areas. Incidence rates have increased disproportionately northward along the upper Hudson River Valley in New York and southward into Fairfax County, Virginia. During 1992–2006, the percentage of counties reporting at least one case increased from 33 to 74% in Minnesota and from 68 to 97% in Wisconsin (Bacon et al., 2008). In Canada, populations of I. scapularis have been identified along the US border in Manitoba, Ontario and Nova Scotia. I. pacificus ticks are established in coastal areas of southern British Columbia (Ogden et al., 2009). Human cases are identified through voluntary reporting and laboratory referrals; approximately 50% are related to travel to endemic areas in the USA and Europe. In British Columbia, three to seven locally acquired cases are reported each year, yielding an incidence of 0.5 per 100,000, comparable to that in neighbouring Washington State. During 1995–2006, ten to 30 locally acquired cases were reported each year from eastern Canada (Ogden et al., 2008). Although I. scapularis ticks are found in northeastern Mexico and I. pacificus are found in Baja California (Piesman and Gern, 2004; GordilloPerez et al., 2009), evidence for human Lyme borreliosis in Mexico is limited to a few case reports (Gordillo-Perez et al., 2007). 6.3.2 Europe Lyme borreliosis is widespread in Europe. Endemic foci may be found from Portugal

and the British Isles east to Turkey and north into Scandinavia and Russia (Fig. 6.1). Reporting practices vary widely and Lyme borreliosis is not a notifiable condition in many countries (EUCALB, 2010). Nevertheless, available data suggest that transmission is most intense in central and northeastern Europe. Reported incidence ranges from 20 to 80 per 100,000 in the Czech Republic, Germany, Latvia, The Netherlands, Poland, Switzerland and Sweden (Table 6.1) (Lindgren and Jaenson, 2006; Fulop and Poggensee, 2008; Hubalek, 2009; EUCALB, 2010). In Austria, Estonia, Lithuania and Slovenia, rates in excess of 100 per 100,000 have been reported (Hubalek, 2009; EpiNorth, 2010). The reported incidence generally decreases moving northward in Scandinavia, from east to west in central Europe, and southward in Spain, France, Italy and Greece (Lindgren and Jaenson, 2006). In the British Isles, rates average 1 per 100,000 population. Over the last decade, the incidence of reported cases has increased in Poland, eastern Germany, Slovenia, Bulgaria, Norway, Finland, Belgium, Great Britain and The Netherlands (Hofhuis et al., 2006; Smith and Takkinen, 2006; Fulop and Poggensee, 2008). As in North America, this increase may reflect a combination of both improved awareness and a true increase in transmission in some areas (Kampen et al., 2004; Hofhuis et al., 2006; Smith and Takkinen, 2006). 6.3.3 Asia The risk of Lyme borreliosis extends in a large swath across Eurasia, reaching from Japan to the western border of Russia (Fig. 6.1). In Russia, official records on Lyme borreliosis have been kept since 1992 (Korenberg, 1994). The reported incidence in endemic areas generally ranges from 5 to 10 per 100,000. However, considerably higher rates are reported in areas northeast of Moscow in Vologda oblast, in the Sverdlovsk (Urals) region and western Siberia (WHO, 1995; EpiNorth, 2010). Infected ticks are found through much of Mongolia, although information on human cases appears scarce. B. burgdorferi sensu lato strains have been

Global Epidemiology of Borrelia burgdorferi Infections

isolated from rodents and ticks in at least 20 provinces in China, including Heilongjiang in the north-east, Xinjiang in the northwest and Guizhou, Hunan and Zhejiang provinces in southern China (Ai et al., 1990; Chu et al., 2008; Hao et al., 2010; Zhang et al., 2010). B. garinii and B. afzelii are among the isolated strains, and human illness has been detailed among forestry workers in Heilongjiang province (Ai et al., 1988; Hao et al., 2010). Both B. garinii and B. afzelii have been isolated from patients in Japan; however, the overall incidence is 0.1 per 100,000. Most cases occur on Hokkaido Island in northern Japan or, less commonly, from exposures in subalpine forested areas in central Japan (Nakama et al., 1994; Hashimoto et al., 2007). Enzootic cycles are established in Korea and Taiwan, and B. garinii has been isolated in culture from at least one patient from northern Taiwan (Chao et al., 2010). 6.3.4 The Tropics and the southern hemisphere Illnesses resembling Lyme borreliosis have been reported periodically in tropical and southern hemisphere countries, including Australia (Russell, 1995), Brazil (Mantovani et al., 2007) and South Africa (Stanek et al., 1987b). In addition, serosurveys and diagnostic testing have occasionally detected antibodies reactive to B. burgdorferi sensu lato antigens among residents of tropical areas (Miranda et al., 2009; Santos et al., 2010). While the possibility of Borrelia-related illness with distinct enzootic cycles in these areas cannot be excluded, a great deal more information will be needed to determine the relationship, if any, between these reports and Lyme borreliosis as currently defined.

6.4 Seasonality Lyme borreliosis occurs most often in the warmer months, reflecting both the questing habits of ticks and the recreational tendencies of humans (Ai et al., 1990; Piesman and Gern, 2004; Bacon et al., 2008). For I. scapularis and I. ricinus, nymphal ticks are thought to play a

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particularly important role in transmission due in part to their minute size and relative abundance. Questing usually peaks in spring or early summer and, after allowing for an incubation period of one to several weeks, a similar peak follows in the onset of acute cases in humans (Falco et al., 1999). In the USA, onset peaks in June or July, with 56% of all cases having onset in these two months (Bacon et al., 2008). A slightly later onset peak in August has been reported in Estonia (Lindgren and Jaenson, 2006) and Sweden (Berglund et al., 1995), perhaps because of their more northern latitude. Questing behaviour is sensitive to meteorological factors (Alekseev and Dubinina, 2000; Eisen et al., 2002) and onset of human illness can vary from year to year based on climatic conditions (Mead et al., 2010). Due to longer and more variable incubation periods, later stages of the disease tend to peak slightly later in the year (Stanek et al., 1987a; Berglund et al., 1995) and show less seasonal fluctuation (Stanek et al., 1987a; Strle, 1999; Bacon et al., 2008). For example, 67% of US cases of erythema migrans (EM) had onset in June and July, compared with only 37% of arthritis cases (Bacon et al., 2008).

6.5 Age and Sex Data on age and sex distribution are often published as case counts rather than incidence rates, although this hinders comparisons; however, several trends are generally apparent. With respect to age, the distribution of Lyme borreliosis cases is most often bimodal. Rates peak among children between the ages of 5 and 15 years, decrease among 20–25-year-olds and peak again among adults, typically in those aged 50 years or older (Fig. 6.2a) (Bacon et al., 2008; Fulop and Poggensee, 2008; Hubalek, 2009). The absolute highest rate usually occurs among adults in Europe and among children in North America. These patterns probably reflect behaviour-related differences in rates of exposure across populations, although they may also be influenced by surveillance methods and age- and sex-specific differences in clinical illness. With respect to sex, females

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account for the majority of cases in most European countries. In studies from Austria, Czech Republic, Germany, Italy, Slovenia, Sweden, Switzerland and Poland, 51–60% of identified cases were among females. Where information on incidence is also available, these percentages correspond to higher incidence among females, but often only in adults (Fig. 6.2a). Despite the overall preponderance of female cases, incidence is actually higher for boys than girls in both Sweden and Germany (Fulop and Poggensee, 2008; Berglund et al., 1995).

The situation with respect to gender is appreciably different in North America (Fig. 6.2b). During 1992–2006, females accounted for only 47% of US cases, yielding an overall incidence of 5.4 per 100,000 for females, as compared with 6.3 per 100,000 for males (Bacon et al., 2008). Incidence was higher among males in nearly all age groups. Over time, incidence has increased disproportionately among males, shifting the overall sex ratio from 51% male in 1992 to 53% male in 2006 (Bacon et al., 2008). Although unexplained, this sex-specific increase has

(a) 14 Male

12

Female

10 8 6 4 2 0

0–4

10–14

20–24

30–39

50–59

70+

50–59

70+

Age (years)

(b)

14 12 10 8 6 4 2 0

0–4

10–14

20–24

30–39

Age (years) Fig. 6.2. Age- and sex-specific incidence (cases per 100,000 population per year) of Lyme borreliosis in six German states (a, redrawn from Fulop and Poggensee, 2008) and the USA (b) for 2002–2006.

Global Epidemiology of Borrelia burgdorferi Infections

been most pronounced among children. In a separate analysis of US data for 2001–2002, age and sex distribution were found to vary among cases reported in endemic and nonendemic areas (CDC, 2004). In 12 highly endemic states, the modal age was 6 years and 54% of cases were among males. In contrast, in non-endemic states, the modal age was 44 years and 47% of cases were among males. Barring fundamental differences in risk factors for infection, this discrepancy suggests that an appreciable proportion of illnesses reported as Lyme borreliosis in non-endemic areas are actually due to other conditions. This is consistent with a higher risk of misdiagnosis in nonendemic areas, a consequence of the relationship between prior probability of the disease and the predictive value of clinical and laboratory findings (Tugwell et al., 1997).

6.6 Clinical Features Clinical manifestations of Lyme borreliosis include EM, acrodermatitis chronica atrophicans (ACA), lymphocytoma, acute and chronic neuroborreliosis, arthritis and carditis. These are well described elsewhere (Steere, 2001; Stanek and Strle, 2003; Nau et al., 2009); the salient issue with respect to epidemiology is the frequency and distribution of these clinical forms among different populations. While all three major genospecies can cause dermatological, neurological or rheumatological illness, they appear to have differing proclivities. In European studies, isolates from patients with neuroborreliosis are most commonly B. garinii (Ruzic-Sabljic et al., 2001b), while those from patients with EM – and especially ACA – are predominantly B. afzelii (van Dam et al., 1993; Busch et al., 1996; Ornstein et al., 2001; Ruzic-Sabljic et al., 2001a). A similar affinity has been suggested for B. burgdorferi sensu stricto and arthritic manifestations (Steere, 2001; Steere and Glickstein, 2004). EM is universally the most common manifestation, accounting for 60–90% of cases in both North America and Eurasia. Carditis is consistently rare, generally accounting for less than 1% in most studies (Ai et al., 1988; Steere, 2001; Stanek and Strle, 2003). In the

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USA, where human infection is limited to B. burgdorferi sensu stricto, 32% of cases reported through national surveillance were associated with arthritis, while only 12% had neurological symptoms (usually facial palsy) (Bacon et al., 2008). Although the absolute percentages of these manifestations vary by state (probably as a result of varying degrees of reliance on laboratory-based reporting), the greater frequency of arthritis is consistent across states. In studies from Europe, arthritis is generally reported less commonly than neuroborreliosis, sometimes markedly so (Stanek et al., 1987a; Berglund et al., 1995; Letrilliart et al., 2005). Among 1471 Swedish patients, 16% had manifestations of neuroborreliosis while only 7% had arthritis (Berglund et al., 1995), and among 873 Austrian patients, 24% had neurological manifestations compared with only 2% with arthritis (Stanek et al., 1987a). The dermatological manifestations ACA and lymphocytoma are well known in Europe but extremely rare in the USA, a reflection of their particular association with B. afzelii infection (Busch et al., 1996). The absolute frequency of clinical features varies across countries and may be related to local differences in the prevailing genospecies (Saint Girons et al., 1998; Rauter and Hartung, 2005), although it is also likely to be influenced by the method of case ascertainment. Patient age and sex also appear to influence the clinical features of disease, although unifying patterns are difficult to discern. It is clear from European studies that children are more likely than adults to present with lymphocytoma or neuroborreliosis, especially facial palsy (Stanek et al., 1987a; Berglund et al., 1995; Huppertz et al., 1999; Hubalek, 2009; Henningsson et al., 2010). Conversely, ACA, with its insidious onset, is a condition of adults, particularly women, who outnumber men by a ratio of 2:1 or more among patients with this manifestation (Asbrink et al., 1986; Stanek et al., 1987a; WHO, 1995; Hubalek, 2009). As a general rule, dermatological features appear to be more common among women in Europe, while males account for the majority of neuroborreliosis cases (Fulop and Poggensee, 2008; Hubalek, 2009). The higher rates of

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Lyme borreliosis among adult women in central and northern Europe compared with the USA (Fig. 6.2) probably reflects the predominance in the region of B. afezelli, with its predilection to cause dermatological illness among females. In the USA, the frequency of clinical features is generally consistent across age groups, with the exception of arthritis, which is distinctly more common among children aged 5–15 years (Bacon et al., 2008), an observation also made in some European studies (Huppertz et al., 1999). A recent retrospective study of 125 patients found no difference in clinical presentation by sex in the USA (Schwarzwalder et al., 2010). Despite the high frequency of infection, very few deaths due to Lyme borreliosis have been reported in the medical literature (Marcus et al., 1985; Kirsch et al., 1988; Waniek et al., 1995; Tavora et al., 2008). A recent review of US death certificates for the years 1999– 2003 identified 23 records that listed Lyme disease as the underlying cause of death, 11 of which were improperly coded and only one of which listed a causal sequence possibly consistent with a prior case report (Kugeler et al., 2011).

6.7 Risk Factors and Transmission In the northeastern USA, B. burgdorferi infections are most often acquired from the peridomestic environment (Falco and Fish, 1988; Maupin et al., 1991; Klein et al., 1996). A series of studies has identified peridomestic risk factors for infection, including the presence of suitable tick habitat, landscaping practices, deer density and outdoor activities such as gardening (Ley et al., 1995; Orloski et al., 1998; Smith et al., 2001; Rand et al., 2003). Lyme borreliosis is also, however, an occupational risk. Increased risk of infection has been noted among forestry workers, farmers, hikers, soldiers, hunters and orienteers in studies from the USA (Schwartz and Goldstein, 1990), Asia (Ai et al., 1994; Nakama et al., 1994) and throughout Europe (Cinco et al., 2004; Kaya et al., 2008; Bilski, 2009; Buczek et al., 2009; Hubalek, 2009). Animal studies and clinical observations indicate that I. scapularis ticks require at least

36 h of attachment in order to transmit B. burgdorferi sensu stricto, supporting a possible preventative role for daily tick checks and showering after exposure (Vazquez et al., 2008; Connally et al., 2009). Unfortunately, similar studies have demonstrated that I. ricinus ticks, especially when infected with B. afzelii, can transmit infection efficiently after much shorter periods of attachment (Piesman and Gern, 2004). Observed patterns of Lyme borreliosis are thoroughly consistent with the wellestablished mechanism of transmission by Ixodes ticks. Nevertheless, alternative modes of transmission have been investigated. Inoculation of blood with laboratoryadapted strains of B. burgdorferi has demonstrated the organism’s ability to survive under blood-banking conditions, raising the spectre of transfusion-associated infection (Johnson et al., 1990). However, while transfusion-associated infection with less common Ixodes-transmitted pathogens (e.g. Babesia or Anaplasma) has been demonstrated repeatedly, no cases of transfusionassociated Lyme borreliosis have ever been documented (McQuiston et al., 2000; CDC, 2008a). Similarly, despite a series of studies in animals, there is no credible evidence of transmission through sexual contact, semen, urine or breast milk (Moody and Barthold, 1991; Woodrum and Oliver, 1999). Intrauterine infection has been documented in rare reports of miscarriage and stillbirth in women infected during pregnancy (Schlesinger et al., 1985). A causal relationship to the miscarriage has not been established, however, as B. burgdorferi has also been identified in placentas of women with normal pregnancy outcomes (Figueroa et al., 1996) and larger epidemiological studies have identified no definable pattern of teratogenicity (Markowitz et al., 1986; Walsh et al., 2007). Pregnant women who develop Lyme disease have good outcomes if they receive appropriate antimicrobial therapy (Walsh et al., 2007).

6.8 Conclusion Lyme borreliosis is both a local and a global problem. Areas of risk, though discrete in

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space and time, are found throughout the northern hemisphere. Etiological agents, principal vectors and clinical manifestations vary widely by region. A detailed knowledge of Lyme borreliosis epidemiology is both clinically relevant (Makhani et al., 2010) and essential for the development of effective prevention measures. Despite enormous gains in knowledge over the last two decades, a great deal remains to be learned about risk factors for infection, enzootic cycles, the role of other B. burgdorferi genospecies and, most importantly, how best to prevent human infection and morbidity.

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nov., a new species of Borrelia burgdorferi sensu lato isolated from rodents and tick from the southeastern United States. International Journal of Systematic and Evolutionary Microbiology 61, 381–383. Russell, R.C. (1995) Lyme disease in Australia– still to be proven! Emerging Infectious Diseases 1, 29–31. Ruzic-Sabljic, E., Arnez, M., Lotric-Furlan, S., Maraspin, V., Cimperman, J. and Strle, F. (2001a) Genotypic and phenotypic characterisation of Borrelia burgdorferi sensu lato strains isolated from human blood. Journal of Medical Microbiology 50, 896–901. Ruzic-Sabljic, E., Lotric-Furlan, S., Maraspin, V., Cimperman, J., Pleterski-Rigler, D. and Strle, F. (2001b) Analysis of Borrelia burgdorferi sensu lato isolated from cerebrospinal fluid. Acta Pathologica Microbiologica et Immunologica Scandinavica 109, 707–713. Saint Girons, I., Gern, L., Gray, J.S., Guy, E.C., Korenberg, E., Nuttall, P.A., Rijpkema, S.G., Schonberg, A., Stanek, G. and Postic, D. (1998) Identification of Borrelia burgdorferi sensu lato species in Europe. Zentralblatt für Bakteriologie 287, 190–195. Santos, M., Ribeiro-Rodrigues, R., Lobo, R. and Talhari, S. (2010) Antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients from the Brazilian Amazon region with skin diseases not related to Lyme disease. International Journal of Dermatology 49, 552–556. Schlesinger, P.A., Duray, P.H., Burke, B.A., Steere, A.C. and Stillman, M.T. (1985) Maternal–fetal transmission of the Lyme disease spirochete, Borrelia burgdorferi. Annals of Internal Medicine 103, 67–68. Schwartz, B.S. and Goldstein, M.D. (1990) Lyme disease in outdoor workers: risk factors, preventive measures, and tick removal methods. American Journal of Epidemiology 131, 877– 885. Schwarzwalder, A., Schneider, M.F., Lydecker, A. and Aucott, J.N. (2010) Sex differences in the clinical and serologic presentation of early Lyme disease: results from a retrospective review. Gender Medicine 7, 320–329. Smith, G., Wileyto, E.P., Hopkins, R.B., Cherry, B.R. and Maher, J.P. (2001) Risk factors for Lyme disease in Chester County, Pennsylvania. Public Health Reports 116 (Supplement 1), S146– S156. Smith, R. and O’Connell, S. (2010) Lyme borreliosis trends in England and Wales: 2005–2009. In: 12th International Conference on Lyme Borreliosis and other Tick-borne Diseases, 26–29 September 2010, Ljubljana, Slovenia.

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Austrian Society for Hygiene, Microbiology, and Preventive Medicine, Vienna, Austria. Smith, R. and Takkinen, J. (2006) Lyme borreliosis: Europe-wide coordinated surveillance and action needed? Eurosurveillance 11(25): pii–2977. Stanek, G. and Strle, F. (2003) Lyme borreliosis. Lancet 362, 1639–1647. Stanek, G., Flamm, H., Groh, V., Hirschl, A., Kristoferitsch, W., Neumann, R., Schmutzhard, E. and Wewalka, G. (1987a) Epidemiology of Borrelia infections in Austria. Zentralblatt für Bakteriologie Mikrobiologie und Hygiene A 263, 442–449. Stanek, G., Hirschl, A., Stemberger, H., Wewalka, G. and Wiedermann, G. (1987b) Does Lyme borreliosis also occur in tropical and subtropical areas? Zentralblatt für Bakteriologie Mikrobiologie und Hygiene A 263, 491–495. Steere, A.C. (2001) Lyme disease. New England Journal of Medicine 345, 115–125. Steere, A.C. and Glickstein, L. (2004) Elucidation of Lyme arthritis. Nature Reviews Immunology 4, 143–152. Steere, A.C., Sikand, V.K., Schoen, R.T. and Nowakowski, J. (2003) Asymptomatic infection with Borrelia burgdorferi. Clinical Infectious Diseases 37, 528–532. Steere, A.C., Coburn, J. and Glickstein, L. (2004) The emergence of Lyme disease. Journal of Clinical Investigation 113, 1093–1101. Strle, F. (1999) Lyme borreliosis in Slovenia. Zentralblatt für Bakteriologie 289, 643–652. Strle, F. and Stantic-Pavlinic, M. (1996) Lyme disease in Europe. New England Journal of Medicine 334, 803. Tavora, F., Burke, A., Li, L., Franks, T.J. and Virmani, R. (2008) Postmortem confirmation of Lyme carditis with polymerase chain reaction. Cardiovascular Pathology 17, 103–107. Tugwell, P., Dennis, D.T., Weinstein, A., Wells, G., Shea, B., Nichol, G., Hayward, R., Lightfoot, R., Baker, P. and Steere, A.C. (1997) Laboratory evaluation in the diagnosis of Lyme disease. Annals of Internal Medicine 127, 1109–1123. van Dam, A., Kuiper, H., Vos, K., Widjojokusumo, A., De Jongh, B., Spanjaard, L., Ramselaar, A., Kramer, M. and Dankert, J. (1993) Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clinical Infectious Diseases 17, 708–717. van Dam, A.P. (2002) Diversity of Ixodes-borne Borrelia species – clinical, pathogenetic, and diagnostic implications and impact on vaccine development. Vector-borne and Zoonotic Disease 2, 249–254. Vazquez, M., Muehlenbein, C., Cartter, M., Hayes,

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Woodrum, J.E. and Oliver, J.H. Jr (1999) Investigation of venereal, transplacental, and contact transmission of the Lyme disease spirochete, Borrelia burgdorferi, in Syrian hamsters. Journal of Parasitology 85, 426–430. Wormser, G.P., Masters, E., Nowakowski, J., McKenna, D., Holmgren, D., Ma, K., Ihde, L., Cavaliere, L.F. and Nadelman, R.B. (2005) Prospective clinical evaluation of patients from Missouri and New York with erythema migranslike skin lesions. Clinical Infectious Diseases 41, 958–965. Zhang, F., Gong, Z., Zhang, J. and Liu, Z. (2010) Prevalence of Borrelia burgdorferi sensu lato in rodents from Gansu, northwestern China. BMC Microbiology 10, 157.

7

Antibiotic Therapy for Infection Caused by Borrelia burgdorferi Sensu Lato Gary P. Wormser

7. 1 Introduction Lyme disease, or Lyme borreliosis, is the term usually applied to infection with Borrelia burgdorferi sensu lato (Bbsl). Because this term is now often used inaccurately to describe patients with a wide range of conditions but who have no evidence of Bbsl infection (Steere et al., 1993; Feder et al., 2007; Hassett et al., 2008, 2009), in this chapter the term Bbsl infection will be used instead. Human disease is principally caused by three genospecies of Bbsl – exclusively B. burgdorferi sensu stricto in North America, and predominantly Borrelia afzelii and Borrelia garinii in Europe (Stanek et al., 2011). Although each of the three major Bbsl species can cause erythema migrans (EM) and/or neurological manifestations, B. afzelii is most closely associated with skin manifestations, B. garinii appears to be the most neurotropic and B. burgdorferi sensu stricto is the most likely to cause arthritis (Stanek et al., 2011; Wormser, 2011). The objective clinical manifestations of Bbsl infection are thought to be due to an inflammatory reaction, presumably to live spirochaetes or their undegraded antigens (Malawista and Bockenstedt, 2007; Steere, 2010). Localized infection typically is manifest as a single focus of infection in the skin, EM. Systemic symptoms such as fatigue or arthralgias accompany EM in approximately

65% of US patients compared with about 35% of European patients (Tibbles and Edlow, 2007). Disseminated disease is usually characterized by multiple EM skin lesions or by an objective neurological, cardiac or musculoskeletal manifestation of Bbsl infection (Wormser et al., 2006). Clinical evidence of dissemination may appear within days of the appearance of the EM skin lesion, but arthritis, the skin condition known as acrodermatitis chronica atrophicans or certain rare late neurological manifestations typically only become apparent after months to years.

7.2 Antibiotic Susceptibility The preferred antibiotic for a bacterial infection is usually based on the organism’s sensitivity to it in vitro, taking into consideration the agent’s pharmacokinetics, pharmacodynamics, safety, ease of administration and cost. Unfortunately, in vitro studies of the sensitivity of Bbsl to antibiotics have lacked standardized methodologies and used a variety of end points (Nowakowski and Wormser, 1993; Terekhova et al., 2002), making interpretation challenging. Variations in minimum bactericidal concentrations (MBCs) have generally been greater than that for minimum inhibitory

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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concentrations (MICs). Therefore, inconsistencies among published studies in the reported MICs and MBCs for various antibiotics against Bbsl may be related more to differences in assay techniques than to true strain variations. In five different studies, MBC values of the B31 strain of B. burgdorferi sensu stricto to doxycycline have been reported to be 0.80 (Baradaran-Dilmaghani and Stanek, 1996), 4.0 (Levin et al., 1993), 8.0 (Sicklinger et al., 2003), 16 (Morgenstern et al., 2009) and 25 (Barthold et al., 2010), representing a >30-fold range of reported values. Alternatively, it may be that the studied strains were not actually all B31, as it is unclear whether the isolates were cloned before testing. Bbsl are susceptible to tetracyclines, most penicillins and many second- and thirdgeneration cephalosporins, but first-generation cephalosporins are not active in vitro or effective clinically (Nowakowski et al., 2000). Bbsl are also resistant to certain fluoroquinolones and rifampin in vitro (Wormser et al., 2006). Success rates for treatment of patients or laboratory animals with macrolide antibiotics has been less successful than in vitro testing might have predicted (Wormser et al., 2006; Wormser and O’Connell, 2011). Multidrug efflux pumps exist in Bbsl (Bunikis et al., 2008), as they do in virtually all Gram-negative bacteria. These pumps are believed to be biologically important and potentially involved in the processes of detoxification of intracellular metabolites, bacterial virulence, cell homeostasis and intercellular signal trafficking (Martinez et al., 2009). Tetracycline-specific efflux pumps, which confer resistance to this class of drugs, however, would not be expected and have not been demonstrated in Bbsl.

7.3 Prevention As with any infection, the best strategy is to avoid Bbsl infection – specifically by avoiding tick-infested environments or, when in such environments, covering bare skin and using tick repellents. Bathing within 2 h of tick exposure has been shown to decrease the risk of Bbsl infection (Connally et al., 2009).

Transmission of Bbsl requires 36 h of attachment (24 h for some European Bbsl species) (Kahl et al., 1998). A daily tick check, encompassing the entire skin surface (including scalp) with removal of any attached ticks may help to prevent infection. In highly endemic regions of the USA, fewer than 4% of individuals who find and remove an attached I. scapularis tick will become infected with Bbsl (Wormser, 2006; Warshafsky et al., 2010a). If the tick is not removed in a timely fashion or not found at all, the probability of infection appears to approach the infection rate in the regional tick population (typically 25% of nymphal stage I. scapularis ticks in highly endemic areas of the northeast and midwest USA) (Nadelman et al., 2001). No vaccine is currently available to prevent Bbsl infection in humans.

7.4 Treatment of Incubating Bbsl Infection (chemoprophylaxis) The relatively small number of spirochaetes present very early in Bbsl or other spirochaetal infections provides an opportunity to eradicate them with a much shorter course of treatment than otherwise needed, as demonstrated in rabbits experimentally infected with Treponema pallidum (Magnuson and Eagle, 1945; Eagle et al., 1950; Hollander et al., 1952). Successful short-course early post-exposure antibiotic treatment of spirochaetal diseases is well documented including single-dose procaine penicillin G for syphilis (Schroeter et al., 1971), a 4-day course of doxycycline for relapsing fever (Hasin et al., 2006) and a once-weekly 200 mg dose of doxycycline for leptospirosis in US military personnel (Takafuji et al., 1984). Similarly, the likelihood of developing Bbsl infection can be reduced by a single 200 mg dose of doxycycline given within 72 h of I. scapularis tick removal (Nadelman et al., 2001) – a strategy found to be 87% effective in preventing EM at the tick bite site. Studies have not been conducted on the efficacy of antibiotic prophylaxis for I. ricinus tick bites. The pharmacodynamics and pharmacokinetics of the specific antibiotic administered affect its efficacy in preventing

Antibiotic Therapy for Infection Caused by Borrelia burgdorferi Sensu Lato

Bbsl infection following a tick bite (Lee and Wormser, 2008). A single parenteral dose of a long-acting doxycycline preparation was 100% effective in eliminating B. burgdorferi sensu stricto from mice in two different studies (Zeidner et al., 2004, 2008), whereas a single oral dose of doxycycline was 43% effective in the original murine study (Zeidner et al., 2004). Although the 43% efficacy rate is less than the 87% efficacy rate observed in the human trial of single-dose doxycycline (Nadelman et al., 2001), a single dose of doxycycline given orally to mice was nevertheless significantly more effective than no antibiotic treatment (P = 0.02; Zeidner et al., 2004), thus providing proof of concept (Warshafsky et al., 2010b). The lower observed efficacy of a single dose of oral doxycycline in mice compared with humans is probably explained by the fact that the antibiotic exposure in the mouse species studied differed substantively from that in humans. Following a single 200 mg dose of doxycycline, the area under the curve of unbound doxycycline (fAUC0–∞) in humans was 2.25 times greater than that provided by the doxycycline dose used in the mouse study (Lee and Wormser, 2008). Interestingly, feeding mice doxycycline at the time of tick feeding was even more effective. Allowing five ticks infected with B. burgdorferi sensu stricto to feed to repletion (96 h) (Dolan et al., 2008) on mice consuming bait containing doxycycline resulted in none of the mice becoming infected. Remarkably, B. burgdorferi sensu stricto could no longer be cultured from the ticks that had fed on mice that received the higher of the two concentrations of doxycycline in the bait. Eradication of B. burgdorferi sensu stricto in the tick itself suggests how potent doxycycline is against this spirochaete (Wormser and O’Connell, 2011). Importantly, failure of antibiotic prophylaxis for spirochaetal infections has not been found to change the presentation of the disease or cause seronegative persistent infection (Magnuson and Eagle, 1945; Hollander et al., 1952; Korenberg et al., 1996; Nadelman et al., 2001). In one study (Hollander et al., 1952), rabbits that received penicillin for incubating syphilis were ‘either

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cured or subsequently developed clinically recognizable lesions’. Single subcurative doses of penicillin only prolonged the ‘incubation period of experimental syphilis… up to a limit of 30–40 days’. After lesions developed, all animals become seropositive.

7.5 Treatment of Localized Infection Clinical manifestations of Bbsl infection (Steere et al., 1987) usually eventually resolve without antibiotic treatment; treatment accelerates the rate of resolution and prevents later sequelae (Wormser et al., 2006). In the USA and Europe, oral doxycycline, amoxicillin and cefuroxime axetil are recommended for EM (Table 7.1) (Wormser et al., 2006). Phenoxymethylpenicillin is also used for this indication in Europe (Stanek and Strle, 2003; O’Connell, 2009). Macrolides are somewhat less effective than other oral antibiotics; hence, these agents are usually a second-line therapy (Wormser et al., 2006). Up to 15% of US patients with EM may experience an increase in the size or intensity of the erythema, with more intense systemic symptoms, within 24 h of starting antimicrobial therapy, which has been interpreted to represent a Jarisch–Herxheimer-like reaction. Contrary to the opinion of some (Oksi et al., 2007), however, such reactions do not occur at later times during treatment. Fever, if present, should resolve within 48 h and the skin lesion itself usually within 7–14 days (Wormser et al., 2006). Subjective symptoms, such as fatigue or arthralgia, tend to improve, but do not invariably resolve within this time frame, lasting for more than 3 months in approximately one-quarter of US patients (Wormser et al., 2003). Extending the initial course of treatment does not result in faster or more complete relief of symptoms (Wormser et al., 2003).

7.6 Oral Doxycycline for Nervous System Bbsl Infection Doxycycline is highly lipophilic, allowing ready entry into many tissues including the nervous system, and has a very high oral

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Table 7.1. Recommended therapy for adult patients with Bbsl infectiona (modified from Wormser et al. 2006). Manifestation

Duration daysb

Therapy

Erythema migrans (EM) Borrelial lymphocytoma Acrodermatitis chronica atrophicans Bbsl arthritis Bbsl carditis (mild) Cranial neuropathy

14 14 days 21 days 28 days 14 days 14 daysc

Doxycycline 100 mg PO BID or amoxicillin 500 mg PO TID or cefuroxime axetil 500 mg PO BID

Bbsl meningitis, cranial neuropathy or radiculoneuropathy in Europe and possibly in USA

14 days

Doxycycline 100 mg PO BID

Bbsl arthritis that failed oral therapy Late or severe neurological Bbsl infection Bbsl carditis requiring hospitalization Bbsl meningitis, cranial neuropathy or radiculoneuropathy in USA

14–28 days 14–28 days 14 days 14 days

Ceftriaxone 2 g IV daily

EM in a patient intolerant of doxycycline and -lactam antibiotics

6–10 days

Azithromycin 500 mg PO daily

PO, by mouth; IV, intravenous. aNote: Regardless of the clinical manifestations of Lyme disease, a complete response to treatment may be delayed beyond the treatment duration. Relapse may occur with any of these regimens; patients with objective signs of relapse may need another course of treatment. bA 10-day course of doxycycline is sufficient for EM. cAlthough any one of the first-line oral antibiotics appears to be effective in patients with cranial neuropathy, there is only limited experience in patients with a cranial neuropathy other than 7th nerve palsy or with agents other than doxycycline.

bioavailability (90%), such that blood concentrations are generally similar whether the drug is given intravenously or orally (Wormser and Halperin, 2008). Not surprisingly then, this agent demonstrated efficacy comparable to ceftriaxone in a double-blind, multicentre treatment trial in which 118 Norwegian patients were randomized to receive 14 days of oral doxycycline or intravenous ceftriaxone for presumed neurological Bbsl infection (Ljostad et al., 2008). Similar efficacy was shown at 4 and 12 months follow-up (Ljostad and Mygland, 2010). None of the patients required additional antibiotic treatment. These results are consistent with a previous meta-analysis of prior reports of European patients with early neurological Bbsl infection, which found the response rate to doxycycline to be comparable to that of parenteral penicillin or ceftriaxone (95% confidence interval 94.8– 102.5%; Halperin et al., 2007).

Thus, there is compelling evidence that European patients with early neurological Bbsl infection will respond as well, overall, to a 2-week course of oral doxycycline as to a 2-week course of ceftriaxone. Whether similar results could be attained with an oral -lactam agent, such as amoxicillin, with its less favourable pharmacokinetic profile, is unclear, but seems less likely (Wormser and O’Connell, 2011). Also unclear is whether the efficacy of oral doxycycline and parenteral -lactams would be comparable in North American nervous system Bbsl infection (Wormser and Halperin, 2008). Finally, as the effectiveness of oral doxycycline has not been established for patients with severe neurological manifestations, including parenchymal brain involvement, a parenteral antibiotic, such as ceftriaxone, remains the recommended agent in such exceptional cases (Wormser and Halperin, 2008).

1

2

Plate 1. Life cycle of I. scapularis. The tick has four stages in its 2-year life cycle: egg, larva, nymph and adult. Between stages, the tick needs a blood meal in order to mature. The infectious agent is transstadially transmitted from one stage to another. The size of the animals represents the preferred host for each tick stage. Plate 2. The complement system. Complement activation can occur through three pathways: the classical, alternative and the mannan-binding lectin pathways. The initial steps of each pathway are different, although the outcome of all three is similar. In the classical pathway, the C1 complex (C1q, C1r and C1s) binds the antibodies that have already bound the antigen. After being cleaved by C1r, C1s cleaves C2 and C4. In the mannan-binding lectin pathway, the mannan-binding lectin protein (MBL) recognizes carbohydrate patterns, such as mannose residues, on the surface of a large number of organisms. The binding of MBL to the pathogen’s surface results in activation of this pathway by the cleavage of C4 and C2 by the MBL-associate serine proteases (MASPs). From here, both follow the same pathway. In the alternative pathway, C3b binds the membrane of the pathogen. Factor B binds C3b allowing factor D to cut it. The C3 and C5 convertases in this pathway are slightly different compared with the MBL and classical pathways. Once complement activation occurs (C3 and C5 covertases formed), complement effector functions, common to all three pathways, can occur. The effector functions are: (i) formation of the MAC; (ii) recruitment of inflammatory cells (not shown); and (iii) opsonization (not shown).

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Plate 3. Seasonal variation in the frequency of the diagnosis of Lyme borreliosis, Slovenia 1993–2007. Data obtained from the website of the Institute of Public Health (www.ivz.si) in Ljubljana, Slovenia (Anon., 2009). Vertical axis represents cases/month.

4a

4b

5

6

7

Plate 4. Erythema migrans lesions with a raised punctum (arm) (a) or depressed punctum (leg) (b). Plate 5. A triangular erythema migrans lesion. (Reprinted by permission of Elsevier, Infectious Disease Clinics of North America.) Plate 6. A vesicular erythema migrans lesion. Plate 7. Multiple erythema migrans lesions, resulting from haematogenous dissemination of B.burgdorferi.

8

9b

9a

9c

Plate 8. A probable hypersensitivity reaction to a tick bite mimicking erythema migrans (EM). The rash (well over 5 cm and thus technically fulfilling CDC criteria for a diagnosis of EM) was noted at the time an adult I.scapularis tick was removed, a few hours prior to taking this photograph. The patient experienced intense pruritus at the site, which she had noted in the past with tick bites. There were no associated systemic symptoms. The rash resolved within approximately 48 h without treatment. The patient remained well, and serology for antibodies to B. burgdorferi, performed after approximately 3 months, was negative. Plate 9. Examples of a single erythrma migrans on the face (a), chest (b) and arm (c).

Antibiotic Therapy for Infection Caused by Borrelia burgdorferi Sensu Lato

7.7 Role of Parenteral Antimicrobial Therapy Oral antibiotics are recommended as the firstline treatment for the other cutaneous manifestations of Bbsl infection (Wormser et al., 2006). However, parenteral antibiotics are recommended for patients with certain neurological manifestations and initially for those with cardiac Bbsl infection during the time they are hospitalized for monitoring (Wormser et al., 2006). Parenteral antibiotics are often given to patients with Bbsl arthritis who have failed to respond to one or more courses of oral antibiotic treatment, although the risks and benefits of this treatment strategy have never been studied systematically (Wormser et al., 2006; Wormser and O’Connell, 2011). Ceftriaxone is the preferred parenteral agent because of its in vitro activity against Bbsl, its ability to readily cross the blood– brain barrier and its long serum half-life, allowing the convenience of once-daily administration (Wormser et al., 2006). Cefotaxime is similarly effective and does not cause biliary concretions or cholecystitis, recognized adverse effects of ceftriaxone, but does cause leukopenia. High-dose intravenous penicillin is a third alternative.

7.8. Treatment Duration Clinical experience with most infections indicates that treating until all symptoms resolve, until a cerebrospinal fluid pleocytosis disappears or until serological tests revert to negative is neither necessary nor rational. Prolonged courses of antibiotics substantially increase the risk of serious adverse events, increase costs and promote antibiotic resistance. Both a prospective, randomized, double-blind treatment trial of 180 US patients (Wormser et al., 2003) and a large retrospective cohort study of 607 US patients (Kowalski et al., 2010) have demonstrated that a 10-day course of doxycycline is just as effective as longer courses of treatment with this antibiotic for patients

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with EM. Fourteen days of an appropriate oral -lactam antibiotic also seems to be as effective as longer courses of treatment with these agents, although systematic comparisons are lacking (Wormser et al., 2006). Even shorter durations of treatment may well be effective for early Bbsl infection. In one German study of 73 patients with EM, 5 days of ceftriaxone treatment was just as effective as 12 days of oral penicillin (Weber et al., 1990). Moreover, prolonged courses of antibiotics have never been needed in other spirochaetal infections (Table 7.2) (Wormser, 1995). At least eight studies – including patients with early localized infection (e.g. single EM skin lesion), early disseminated infection (e.g. multiple EM skin lesions) and late infection – have compared different durations of treatment for Bbsl infection. None has shown a beneficial effect for protracted courses of therapy (Weber et al., 1990; Wormser et al., 2002, 2003, 2006; Dattwyler et al., 2005; Oksi et al., 2007; Kowalski et al., 2010).

7.9 Bbsl Arthritis Relatively long courses of oral antibiotics are recommended as the first-line treatment for Bbsl arthritis (Wormser et al., 2006). Patients with Bbsl arthritis with persistent joint swelling following completion of 28 days of oral antibiotics are often retreated either with another 28-day course of oral antibiotics or with 14–28 days of ceftriaxone – a recommendation based on expert opinion rather than randomized trials. Although patients treated following this protocol have been shown to have excellent outcomes (Tory et al., 2010), additional studies are warranted to determine the optimal treatment approach for such individuals. Although Bbsl arthritis typically responds to antibiotic treatment (often combined with non-steroidal anti-inflammatory drugs (NSAIDs); Wormser et al., 2006), approximately 10% of US patients do not respond clinically and are said to have antibiotic-refractory arthritis. This condition has been defined as synovitis persisting for at

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Table 7.2. Comparison of the recommended duration of treatment for certain manifestations of Bbsl infection based on IDSA treatment guidelines (Wormser et al., 2006) with that of other selected spirochaete infections. Bbsl infection manifestation

Treatment duration

Selected other spirochaete infections

Erythema migrans

10–14 days

Syphilitic chancre or secondary syphilis Relapsing fever (louse-borne) Relapsing fever (tick-borne)

Meningitis

14 days

Neurosyphilis Relapsing fever meningitis

Cardiac disease

14 days

Cardiovascular syphilis

least 2 months after completion of intravenous ceftriaxone (or 1 month after completion of two 4-week courses of oral antibiotics), in conjunction with negative PCR testing on synovial fluid and on synovial tissue if available (Steere and Angelis, 2006). In view of the negative PCR testing, these patients are no longer believed to be actively infected, and are treated with NSAIDs, intra-articular injections of corticosteroids or disease modifying anti-rheumatic drugs, rather than with additional courses of antimicrobial therapy (Wormser et al., 2006). If these modalities are ineffective, arthroscopic synovectomy may be successful.

7.10 Coinfection Ixodes ticks potentially carry additional pathogens such as Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA), Babesia species including Babesia microti, the primary cause of babesiosis in the USA, and tick-borne encephalitis virus (Swanson et al., 2006). Coinfection is generally uncommon, but depends on the particular species of Ixodes tick and on the geographical area. Coinfection should be considered, especially in patients who have high-grade fever for more than 48 h or develop recurrent fever during treatment of Bbsl infection

Duration Single dose of benzathine penicillin G or 14 days of oral doxycycline One dose of tetracycline 5–10 days of tetracycline or erythromycin 10–14 days of intravenous penicillin G 14 days of intravenous penicillin G, ceftriaxone or cefotaxime Three doses of benzathine penicillin G given at weekly intervals

and in those who have unexplained leukopenia, thrombocytopenia or anaemia. A. phagocytophilum/Bbsl coinfection is treated with doxycycline, as HGA does not respond to -lactam antibiotics. Patients coinfected with Babesia require additional treatment with azithromycin plus atovaquone, or clindamycin plus quinine (Wormser et al., 2006).

7.11 Post-treatment Persistent Subjective Symptoms As discussed elsewhere in this volume, there has been considerable concern about individuals in whom subjective symptoms persist following treatment that is usually microbiologically curative. Such subjective symptoms must be distinguished from those due to significant tissue damage occurring prior to treatment, with residual objective problems due to as yet incomplete healing following resolution of the infective process (Wormser and O’Connell, 2011). Active coinfection with a second Ixodes-transmitted pathogen (A. phagocytophilum or B. microti) has been investigated and generally excluded as the explanation for such persistent symptoms (Klempner et al., 2001). Estimates of the frequency and severity of these purely subjective symptoms

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following treated Bbsl infection are inconsistent, with symptoms persisting for 6 months after antibiotic treatment ranging from none to 40.8% (median 11.5%) in eight randomized treatment trials of US patients with EM, and from none to 23.4% in five European studies (median 15.4%) (summarized by Cerar et al., 2010). Patients with symptoms that are disabling and persistent for at least 6 months following treatment for Bbsl infection are sometimes referred to as having post-Lyme disease syndrome (PLDS) (Wormser et al., 2006) (see Hassett, Chapter 15, and Marques, Chapter 16, this volume). As similar symptoms occur not infrequently in the general population (Hassett et al., 2008, 2009), it is difficult to know if the incidence of PLDS exceeds that of a chance association; few hard data are available. Of interest, two recent prospective European treatment studies incorporated a control group without Bbsl infection (Skogman et al., 2008; Cerar et al., 2010) – one evaluated children with neurological Bbsl infection (Skogman et al., 2008) and the other adults with a single EM (Cerar et al., 2010). In the later study, the controls were also followed prospectively. In neither study did the frequency of subjective symptoms present at 6 months differ between treated patients and uninfected controls. Although it has been suggested that symptoms might be due to spirochaetes persisting despite antibiotic treatment (Cameron et al., 2004), carefully performed microbiological evaluations have failed to find any credible evidence supporting this hypothesis, including studies focusing on possible occult CNS infection (Klempner et al., 2001; Kaplan et al., 2003; Krupp et al., 2003; Fallon et al., 2008). Four National Institutes of Healthsponsored, randomized, placebo-controlled trials of intensive antibiotic retreatment of US patients with persisting symptoms following treatment for Bbsl infection (Klempner et al., 2001; Krupp et al., 2003; Fallon et al., 2008) failed to provide any evidence of a measurable benefit that outweighed treatment-associated risks. The investigators in these trials concluded that prolonged use of antibiotics is not in the best interest of these patients (Klempner et al., 2001; Kaplan et al., 2003;

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Krupp et al., 2003; Fallon et al., 2008). These results are therefore consistent with the negative microbiological findings. A fifth retreatment study carried out by a single physician (Cameron, 2008) was too flawed to interpret, as described elsewhere (Wormser et al., 2009). Therefore, symptomatic treatment is the recommended approach for such patients (Wormser et al., 2006).

7.12 Guidelines In 2006, the Infectious Diseases Society of America (IDSA) published updated guidelines for the diagnosis, treatment and prevention of Bbsl infection (Wormser et al., 2006). Following an unprecedented degree of external politicization after the guideline’s publication, the IDSA convened an independent panel, vetted by an ombudsman for potential conflicts of interest, to review these guidelines. The eight-member panel reviewed the 2006 guidelines and the supporting evidence in their entirety and concluded that the recommendations were medically and scientifically sound and that no changes were necessary (Lantos et al., 2010). Although the IDSA guidelines were intended for use in North America, they are remarkably similar to diagnostic and treatment guidelines prepared independently by specialist societies and expert groups in various European countries (O’Connell, 2009; Wormser and O’Connell, 2011). No evidencebased European guideline recommends prolonged or multiple courses of antibiotics for persistent symptoms following previously treated Bbsl infection.

7.13 What Constitutes Cure of an Infection? Fundamental to much of this ‘debate’ is an understanding of the appropriate standard by which to judge successful treatment of an infectious disease. Patients treated for pneumonia usually do not feel back to normal at the end of their course of antibiotic therapy and do not yet have clear chest X-rays. Patients treated for meningitis often still have cerebrospinal fluid abnormalities at the end

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of successful antibiotic treatment, with persisting headaches and malaise for quite some time. In most infections, treatment is judged successful based on the historic observation that patients receiving that course no longer worsen or relapse and in fact improve with time – a reasonable standard for therapeutic success (Wormser and O’Connell, 2011). From a microbiological perspective, similarly, it is probably unrealistic to expect that antimicrobial therapy will eradicate every last microorganism from an infected host; moreover, such an action is rarely, if ever, required for a successful outcome. Antimicrobial therapy can be thought to ‘tip the balance’ in favour of the host’s own defences in their fight against a pathogen (Wormser and Schwartz, 2009). For many infectious diseases, antibiotic treatment that only inhibits rather than kills a microorganism is highly effective (Pankey and Sabath, 2004; Wormser and Schwartz, 2009). The host’s immunological response against spirochaetal infections plays a crucial role – as evidenced by the observation that most of the objective clinical manifestations of Bbsl infection will eventually resolve even in the absence of antibiotic treatment (Steere et al., 1987). It has been suggested that the observation, exclusively in animal systems, of postantibiotic-treatment PCR positivity for Bbsl DNA – in the absence of culture positivity – could provide an explanation for PLDS (Hodzic et al., 2008). Bbsl cells remaining after treatment in these animal systems do not elicit a local inflammatory response (Wormser and Schwartz, 2009; Barthold et al., 2010). Antibody responses to Bbsl decline, suggesting a reduction in the overall immunological response to the spirochaete (Philipp et al., 2001). As Bbsl does not appear to produce a systemic toxin (Fraser et al., 1997), it is difficult to imagine how residual spirochaetes – in the absence of a detectable local or generalized immunological or inflammatory response by the host – could cause chronic subjective symptoms (Wormser and Schwartz, 2009). Certainly, latent infections with other microorganisms are generally clinically silent. Whether a few spirochaetes might persist is irrelevant in judging the outcome of

treatment, unless these residual organisms can be shown to cause objectively demonstrable disease. Recent interest in a ‘test of cure’ beyond that of clinical resolution of EM, carditis, meningitis or other neurological manifestations, or arthritis is arguably misdirected, and is inconsistent with the way treatment success is judged for almost every other infectious disease (Wormser and O’Connell, 2011).

7.14 Conclusions Treatment of Bbsl infection is usually successfully accomplished with 10–28 days of an appropriate oral or parenteral antibiotic. A course of therapy as brief as a single dose of doxycycline is effective if given during the incubation period within 72 h of inoculation of B. burgdorferi sensu stricto by a tick bite (Nadelman et al., 2001; Wormser et al., 2006). The objective clinical manifestations of Bbsl infection are thought to be due to an inflammatory reaction to live spirochaetes or to their undegraded antigens (Malawista and Bockenstedt, 2007; Steere, 2010). Post-treatment subjective symptoms may last for 3 or more months after initiation of antibiotic therapy and are unaffected by prolonging the initial course of treatment (Wormser et al., 2003, 2006). It remains unclear whether the frequency of subjective symptoms at 6 months after treatment exceeds the background rates of these symptoms in the general population. Clarification of this issue should be a research priority. Microbiological studies have failed to find evidence of Bbsl infection or of a coinfection in such patients, and they clearly are not benefited by additional courses of antibiotic treatment. Future research should address other potential explanations of posttreatment symptoms and alternative therapeutic approaches for their management.

Acknowledgements The author thanks Lisa Giarratano, Lenise Banwarie and Mary Cox for their assistance.

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8

Lyme Borreliosis in the UK and Ireland Susan O’Connell

8.1 Background

8.2 Ecology

The first case report of erythema migrans (EM) acquired in the UK appeared in 1977, the same year as the seminal description of Lyme arthritis by Steere and colleagues and a few years after Scrimenti’s report of EM in Wisconsin (Scrimenti, 1970; Obasi, 1977; Steere et al., 1977). EM and neurological conditions preceded by tick bites had long been recognized in many parts of Europe, and a tick-borne infectious cause had been suspected but never proven. The isolation of Borrelia burgdorferi by American workers and confirmation of its causative role in European as well as American infections provided a unifying concept of the disease and also led to the development of diagnostic tests (Benach et al., 1983; Asbrink and Hovmark, 1985). International attention to the emergence of Lyme disease stimulated British clinicians and scientists to seek evidence of its presence in the UK, and a series of publications describing various clinical manifestations and epidemiological data appeared throughout the 1980s (Muhlemann, 1984; Williams et al., 1986; Muhlemann and Wright, 1987; Bateman et al., 1988; Guy et al., 1989). Since then, our knowledge of Lyme borreliosis in the British Isles has expanded and has largely affirmed the findings of these and other valuable early UK-based studies.

Complex ecological, environmental, climatic and human behavioural factors affect the incidence of Lyme disease regionally (Gray et al., 2009; Lambin et al., 2010). They include factors affecting ixodid tick survival and the availability of mammalian and avian tickfeeding hosts that are also suitable borrelial reservoir hosts. Differences in genospecies of B. burgdorferi also influence disease incidence and clinical presentations. There is a significantly lower incidence of Lyme borreliosis in the UK and Ireland than in many other European countries. Ixodes ricinus ticks are present in many woodland areas, which also provide suitable habitats for the small mammals and groundfeeding birds that are potential reservoirs of B. burgdorferi sensu lato, but the British Isles are among the least-forested regions in Europe and this is a major factor affecting disease incidence (European Environment Agency, 2010a,b). Although ticks are also found on heathland and moorland in the British Isles, only a small proportion are likely to carry borreliae in these areas, because the major feeding hosts for all stages of the ticks’ lifecycle in these habitats are usually livestock such as cattle and sheep, which are not competent borrelial reservoir hosts (Gray,

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1998). Authors of a meta-analysis of European-based research published in 2005 suggested overall borrelial infection prevalence rates in ticks of 3.9–8.5% in the UK studies that fulfilled inclusion criteria (Rauter and Hartung, 2005). These are significantly lower than rates found in most other Lyme-endemic parts of Europe. The presence of Borrelia valaisiana as a major contributor to infected tick populations in the British Isles is another contributory factor to low disease prevalence rates in the UK and Ireland. This genospecies is essentially non-pathogenic. In some tick populations surveyed regionally, it accounts for about 50% of the borreliae carried by infected ticks, in contrast to the much lower rates of B. valaisiana carriage found in most surveys performed in mainland Europe (Rauter and Hartung, 2005). Borrelia garinii is the most frequently identified pathogenic genospecies in UK and Irish I. ricinus ticks and clinical samples (Kirstein et al., 1997; Robertson et al., 2000a). Borrelia afzelii has been identified in some tick population surveys and clinical isolates (Robertson et al., 1999). In the UK, B. burgdorferi sensu stricto occurs the least frequently of any major pathogenic genospecies, and has been detected mainly in Scottish Highland tick populations (Rauter and Hartung, 2005; Ling et al., 2000). An important ecological study in a woodland area of Dorset in the south of England underlined the importance of ground-feeding birds, including pheasants, as reservoir hosts of B. garinii and B. valaisiana (Kurtenbach et al., 1998). B. garinii is neurotropic in human infections and this study’s findings correlate well with epidemiological data from the area, where cases of neuroborreliosis occur regularly. The findings also have public health implications for other areas, as pheasant and other game bird rearing is an important contributor to the rural economy in many parts of the country.

8.3 Epidemiology Lyme borreliosis is not statutorily notifiable in England, Wales and Northern Ireland, but

a reference laboratory-based enhanced voluntary reporting system for seropositive cases in these countries has been in place since 2000, and a mandatory laboratory reporting scheme was introduced in 2010. Screening tests for antibodies to B. burgdorferi are widely available in local diagnostic laboratories, but the vast majority of immunoblot testing for the three countries is provided in a single reference laboratory at the Health Protection Agency (HPA). This enabled relatively complete reference laboratory reporting of seropositive cases for these countries, even before the recent changes in public health regulations. A similar situation exists in Scotland, where a single laboratory provides national reference facilities. Laboratory reporting of seropositive cases has been mandatory in Ireland since 2004 (P. McKeown, Dublin, 2010, personal communication). There are obvious limitations to data collected only on seropositive cases, principally exclusion of some seronegative or untested patients who have early infection, but the laboratory reporting systems in these countries have been stable for some years and are useful in assessing year-on-year trends. Data on neuroborreliosis are particularly valuable, as the clinical features and laboratory findings are robust diagnostic markers. They are used successfully for epidemiological monitoring in several other European countries, including Denmark and Norway. About 800–1200 seropositive cases of Lyme borreliosis have been reported in the UK annually since 2006. The HPA assessed the likely level of under-ascertainment associated with reliance on laboratory reports and estimated that there may be between 2000 and 3000 cases each year (HPA, 2011a). Findings of sentinel surveillance in endemicarea general practices and an incidence capture–recapture study in south-west England, which adjusted for cases of EM diagnosed on clinical grounds, suggest that this is a realistic estimate (Hoek et al., 2007). Provisional data for 2009 indicate that 973 seropositive cases from England and Wales were reported to the HPA’s Centre for Infections, a mean annual rate of 1.79 per

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100,000 of the population. At least 173 (18%) are known to have been acquired abroad, giving a likely incidence rate of 1.59 per 100,000 for indigenously acquired seropositive cases. There has been a steady increase in reported cases since 2001, when only 268 cases (0.64 per 100,000) were serologically confirmed (HPA, 2011a). A similar increase was noted in Scotland, where the annual rate was estimated at 5.9 per 100,000 in 2009 compared with 2.8 per 100,000 in 2006 (Health Protection Scotland, 2010). These national figures hide considerable variations in local incidence. A recent analysis of data from the south-west region of England, which includes rural areas of high endemicity as well as several large conurbations, indicates an annual regional incidence of 4.2 per 100,000 rising to 15.9 per 100,000 focally. The incidence in the Scottish Highlands, which has a relatively sparse and largely rural population, was estimated as 43.4 per 100,000 in 2009. Several factors are thought to have contributed to the rise in reported cases. These include greater health professional and public awareness of Lyme borreliosis following intensive educational efforts and media coverage, leading to better disease recognition. Tick populations have expanded in some parts of the country, associated with increased numbers and geographical range of deer, and cases have been reported from semi-rural and suburban areas where there has been recent extension of the deer range (Parliamentary Office on Science and Technology, 2009). A succession of mild winters allowed survival of larger numbers of ticks, and also affected their periods of feeding activity, so that some cases of Lyme borreliosis occurred during winter months. There is evidence that suburban and semirural residential developments in previously wooded areas in some parts of the country have led to new local high-incidence areas. Changes in land management, including farming and forestry practices, can also affect tick populations and Lyme borreliosis incidence in the future. In some areas, pine forest monoculture is being replaced by mixed broadleaved woodland, which is more suitable in bringing together all the features

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required for an optimum Lyme-permissive habitat (Forest Research, 2009; EUCALB, 2010). Information available from the enhanced surveillance scheme indicates that most cases of Lyme borreliosis in patients from England or Wales were associated with residential or recreational risk. About 15% of cases identified annually are known to have been acquired in other countries, mainly recreationally. The rise in popularity of outdoor activities and holidays in endemic areas of the UK, other European countries and the USA has contributed to the increased observed incidence. Another factor in recent years has been migration of people from highly endemic regions of eastern and central Europe who acquired infections in their own countries prior to immigration or during return visits to their home countries. Few cases of occupationally acquired Lyme borreliosis are reported each year, and these are mainly in forestry workers or deer handlers. Serological surveys have shown a low overall seroprevalence in UK agricultural workers and Irish park rangers, and negligible seroprevalence in healthy blood donors who are not resident in areas of Lyme-permissive habitat (Robertson et al., 1998; Thomas et al., 1998). A study on forestry workers in the New Forest, a well-recognized Lyme-endemic area, showed 25% IgG seroprevalence using enzyme immunoassays and Western blotting, but no participants had current illness or any previous history suggesting neuroborreliosis or Lyme arthritis, suggesting that asymptomatic or mild infection may be common in people with heavy tick exposure in endemic areas (Guy et al., 1989). A survey of healthy blood donors in the same area showed an IgG seroprevalence of 4% (O’Connell et al., 1992). More recent studies of similar occupational groups with heavy tick exposure in other parts of Europe have shown even higher IgG seroprevalence in some surveys (Cetin et al., 2006). People in risk groups such as these may maintain high levels of IgG antibodies through frequent reexposure to borreliae from infected tick bites. The major Lyme-endemic areas of the UK include popular recreational and tourist destinations such as the New Forest, Exmoor,

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the South Downs and Thetford Forest, all in the south of England, the Lake District, north Yorkshire moors and the Scottish Highlands and Islands, but foci of infection are also present in many other parts of the country, particularly in woodland areas of the southern counties. In Ireland most cases are reported from the Galway region and from West Cork and Kerry, all of which are also important tourist destinations. Ecological studies have shown foci of infected ticks that correlate well with the incidence of human disease in these areas, but also show evidence that infection risk from individual tick bites in affected areas is low (Robertson et al., 2000a). Demographic data from reports of seropositive patients in the UK show that the incidence in males and females is approximately equal, and is highest in the 45–64 years age group. Clinical data indicate that reports of EM with or without accompanying systemic symptoms have increased significantly in recent years, suggesting that there is greater recognition of this early presentation than in earlier years of data collection. Other skin manifestations such as borrelial lymphocytoma or acrodermatitis chronica atrophicans (ACA) are rarely reported. Between 10 and 15% of reported cases annually have neurological presentations, principally consistent with acute neuroborreliosis. Lyme arthritis is reported in 3% of cases, mainly in association with a history of tick exposure in the USA or Germany, although a few cases of UK-acquired Lyme arthritis have been ascertained. Cardiac complications are rarely reported. Analysis of clinical and demographic data from reports of seropositive patients in Ireland gives essentially similar findings (R.M.M. Smith, 2011, Zoonoses Surveillance Unit, UK, personal communication).

8.4 Clinical Presentations Clinical presentations of Lyme borreliosis in the British Isles are similar to those seen in other parts of Europe. The European Union Concerted Action on Lyme Borreliosis (EUCALB) case definitions, originally

published in 1996 and revised in 2010, have been valuable for clinical and epidemiological use throughout Europe and are used for both purposes in the UK (Stanek et al., 1996, 2011; www.hpa.org.uk). EM is by far the most common clinical manifestation. Other skin manifestations such as borrelial lymphocytoma and ACA are reported very uncommonly in UK patients. In most European studies, these unusual skin presentations have been associated mainly with B. afzelii infection, and their rare occurrence in the UK probably reflects the low overall local prevalence of this organism. Some cases of ACA in UK patients are known to have been acquired in Scandinavian countries, where B. afzelii is highly prevalent (Rauter and Hartung, 2005). No cases of ACA have been reported in children in the UK. Acute neuroborreliosis is the most commonly reported manifestation of disseminated Lyme borreliosis acquired in the British Isles. This is not surprising as B. garinii, the most frequently identified pathogenic genospecies in the UK and Ireland, is neurotropic. ‘The European Federation of Neurological Societies (EFNS; membership of which includes the Association of British Neurologists and the Irish Institute of Clinical Neuroscience) recently published guidelines for the diagnosis and treatment of neuroborreliosis in Europe, describing a variety of clinical manifestations (Mygland et al., 2010). The guidelines have gained wide acceptance among neurologists and other clinicians in the UK. The range of neuroborreliosis presentations in the British Isles is similar to that seen in other European countries, and shows marked seasonality, with most cases presenting in the summer and early autumn months, usually within about 4–12 weeks of infection. Some patients with acute neuroborreliosis have recent or concurrent EM (Bateman et al., 1988; Lovett et al., 2008; Elamin et al., 2010; www.hpa.org.uk). In adults, the most common features of neuroborreliosis are those of Garin– Bujadoux–Bannwarth syndrome (painful meningoradiculitis that may be accompanied by unilateral or bilateral facial palsy) (Bateman et al., 1988; Lovett et al., 2008). Over

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85% are positive in antibody tests for B. burgdorferi at the time of presentation, and seroconversion has usually occurred within several weeks of presentation in patients who were initially seronegative (data from the HPA Lyme Borreliosis Unit, UK). Some patients present with clinical signs affecting different anatomic sites, consistent with a mononeuritis multiplex. Other than facial palsy and occasionally other cranial nerve palsies, motor function is not usually seriously affected in most cases. There have been occasional reports of patients who have significant weakness of the muscles of the abdominal wall or limbs (Miller et al., 2006). A 59-year-old woman required ventilatory support for several weeks because of diaphragmatic paralysis due to bilateral phrenic nerve palsies. She also had lymphocytic meningitis and painful radiculopathy with weakness in one leg and sensory loss in the other. She eventually made a good recovery (Abbott et al., 2005). A few patients with clinical presentations resembling Guillain– Barré syndrome clinically, but with primarily axonal damage, have also been observed. Examination of their cerebrospinal fluid (CSF) showed lymphocytic pleiocytosis and intrathecal synthesis of antibodies to B. burgdorferi. Rare cases of myelitis have also been reported, with a good response to antibiotic treatment (Dryden et al., 1996). A smaller number of patients, mainly in older age groups, present at any time of the year with a more gradual onset of severely painful radiculopathy, and they usually have strongly positive results in borrelial antibody tests. Many of these patients retrospectively recognize having had a rash consistent with EM some months previously, usually on the area of the body subsequently affected by radicular pain. In addition to haematogenous spread of B. burgdorferi to nerve roots, it has been postulated that radiculopathy can also be associated with direct migration of organisms along a peripheral nerve to a nerve root, and this may account for the slower onset of symptoms in some cases (Rupprecht et al., 2008). Some UK patients with Lyme radiculopathy have been misdiagnosed initially with conditions such as renal or biliary colic,

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spinal cord compression or even suspected myocardial infarction, depending on the anatomic site affected. Some with severely painful radiculopathy have experienced significant weight loss, sleep disturbance and reactive depression, as also described in Hansen’s large study of Danish patients with neuroborreliosis (Hansen and Lebech, 1992). Many patients with radiculopathy require opiates for pain relief, but analgesia requirements usually reduce rapidly following commencement of appropriate antibiotic treatment. A similar spectrum of disease presentations has been reported from a series of patients from the Galway region of Ireland (Elamin et al., 2010). Late encephalomyelitis cases are rare, and have been diagnosed mainly following identification of CSF pleiocytosis or other unexpected findings in patients investigated for possible multiple sclerosis. Clinical features have included encephalomyelitis and spastic–ataxic gait disorders. Antibody testing of both serum and CSF in these patients was strongly positive, with evidence of intrathecal antibody synthesis. Antibiotic treatment, usually with ceftriaxone, has resulted in marked improvement in most patients, which continued over many months following completion of treatment, although recovery was incomplete in patients who had sustained severe neurological damage prior to treatment. In UK children, the most common presentations of neuroborreliosis are facial palsy with or without clinical or laboratory signs of meningitis (Bateman et al., 1988; Lovett et al., 2008). Cases of children with bilateral facial palsy and/or other cranial nerve palsies and meningitis or meningoencephalitis occur occasionally. Severe Lyme meningoencephalitis is rare. Several cases of raised intracranial pressure in association with previously untreated Lyme meningitis have been noted in children in the UK. All had high levels of antibodies to B. burgdorferi in serum and CSF, with CSF pleiocytosis and high protein levels. Following antibiotic treatment, these children recovered without residual damage. Lyme arthritis (mainly affecting the knee) and carditis are diagnosed uncommonly

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in adults and children in the UK and Ireland. Clinical presentations, diagnostic features and treatment responses are similar to those seen in other parts of Europe and North America. Each year, a significant number (usually about 15%) of UK patients with Lyme borreliosis are known to have acquired their infections in other countries, including the USA (www.hpa.org.uk). This has offered British clinicians an opportunity to compare the characteristics of US-acquired infections with those of European infections. Overall, US-acquired infections tend to have more acute features than UK-acquired disease, with systemic symptoms more likely to accompany EM. Antibody response also appears to develop more rapidly, and antibodies to US-acquired infections are readily detectable on European-derived test systems. Multiple EM rarely occurs in UK-acquired Lyme borreliosis, but has been seen regularly in patients in the UK who have US-acquired early disseminated disease. Acute presentations of US neuroborreliosis appear to be similar to those seen in UK-acquired infections, with facial palsies, lymphocytic meningitis and radiculopathies seen in affected adults, suggesting that Bannwarth’s syndrome is a significant feature of US neuroborreliosis (Halperin, 2008). No cases of more indolently presenting radiculopathies, ACA or borrelial lymphocytoma have been observed so far in this case series of US-acquired infections. Lyme arthritis is uncommon in UK patients: the majority of cases identified in the past 10 years have been acquired in other countries, predominantly in the USA or Germany.

8.5 Laboratory Diagnostic Tests In keeping with the EUCALB case definition recommendations and EFNS guidelines, laboratory supporting evidence is required to confirm a diagnosis of disseminated Lyme borreliosis in the UK and Ireland, as none of the clinical presentations of later-stage disease is unique to the infection. Direct detection tests for B. burgdorferi (principally borrelial DNA detection by PCR) are available

from reference laboratories but have limited use in clinical practice (see Johnson, Chapter 4, this volume). A two-tier antibody test system, similar to that recommended in the USA, is used in many parts of Europe, including the UK and Ireland (European Society of Clinical Micobiology and Infectious Diseases, 2004). The principles, applications and limitations of tests for antibodies to B. burgdorferi are extensively covered in Chapter 4 (this volume). They are generally applicable to the UK, Ireland and other parts of Europe, but several factors require some additional consideration because of the greater variety of pathogenic genospecies present in Europe. They include speed of immune response stimulation by different borrelial genospecies, which can affect the clinical sensitivity of antibody tests in early infection. Seroconversion in European-acquired infections caused by genospecies other than B. burgdorferi sensu stricto can be slower than in US-acquired infections, which are caused exclusively by B. burgdorferi sensu stricto (Strle et al., 1999). This organism causes the most acute disease presentations of any of the pathogenic European genospecies and relatively rapid immune stimulation. Seroconversion in B. garinii infections also tends to be brisker than in B. afzelii infections, reflecting the relatively more acutely pathogenic nature of B. garinii than B. afzelii (Logar et al., 2004). Variations in expression of borrelial antigens by different genospecies in vitro and in vivo also affect test performance parameters. Immunoblots based on whole-cell lysates of B. afzelii are widely used in Europe, including the UK, following several studies showing that they provide the best ‘catch-all’ sensitivity for general use (Hauser et al., 1997; Robertson et al., 2000b). The heterogeneity of pathogenic genospecies in Europe means that US criteria for B. burgdorferi sensu stricto immunoblot interpretation are less valuable in Europe than locally developed criteria for use on immunoblots derived from European genospecies (Wilske et al., 2007). Recent developments in recombinant and peptide-based antigens have enabled production of recombinant immunoblots,

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incorporating specific antigens derived from all European pathogenic genospecies. Hybrid blots using whole-cell lysates and additional stripes of recombinant antigens, including the variable surface antigen VlsE (an in vivoexpressed antigen) and outer-surface protein C (OspC) derived from other genospecies, are now commercially available. Both types of immunoblot give enhanced sensitivity compared with earlier-generation tests. An even more important application of recombinant and synthetic peptide antigens in Europe and North America is in the development of new-generation enzyme immunoassays, which have enhanced specificity compared with earlier-generation tests, without loss of sensitivity (Ledue et al., 2008). These are now used as screening tests in many British laboratories and their greater specificity should lead to a reduced requirement for immunoblot tests. The appropriate use of diagnostic tests for Lyme disease is discussed in detail in Chapter 4 (this volume) and the author’s comments are very pertinent to circumstances in the UK, where there has been a recent marked increase in requests for antibody tests on samples from patients with very low pre-test likelihood of Lyme borreliosis (see Fig. 8.1). It is likely that over 100,000 samples are tested for antibodies to B. burgdorferi in the UK each year. Currently, about 1200 seropositive patients are identified annually. This is a similar ratio of samples to seropositives as that seen in the USA. This indiscriminate testing practice should be discouraged, as the predictive value of a positive result in these circumstances is very low and can lead to misdiagnosis. Inappropriate use of IgM tests, including immunoblots is particularly problematic, as they are inherently more prone to falsepositive reactions than IgG tests.

8.6 Treatment Recommendations for the treatment of various manifestations of Lyme borreliosis in the UK and Ireland are similar to those provided by national authorities and specialist societies in other European countries and the USA, as summarized on the

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HPA website (HPA, 2010; see also Wormser et al., 2006; Lantos et al., 2010; Mygland et al., 2010; British Infection Association, 2011; NHS Clinical Knowledge Summaries, 2011; Wormser and O'Connell, 2011). Oral treatment, usually with doxycycline (100 mg twice daily for adults and children aged over 12 years, as doxycycline is not licensed in the UK for younger children) or amoxicillin (500 mg three times daily for adults, and 50 mg/kg/day in three divided doses for children aged less than 12 years, to a maximum of 500 mg/dose) is recommended as the first-line treatment for all nonneurological indications. Duration of treatment is usually 14 days for EM and other early non-neurological presentations, 21 days for ACA and 28 days for Lyme arthritis. Cefuroxime axetil (500 mg twice daily for adults; 30 mg/kg/day in children 12 years, in two divided doses, to a maximum of 500 mg/dose) is a useful alternative in patients for whom doxycycline or amoxicillin is contraindicated. Macrolides are regarded as third-line options as treatment failures are well documented, particularly with erythromycin. Azithromycin is the preferred macrolide for this use. The EFNS neuroborreliosis treatment guidelines are now widely used in the UK. For adult patients with definite or possible early neuroborreliosis with symptoms confined to the meninges, cranial nerves, nerve roots or peripheral nerves, the guidelines state that oral doxycycline (200 mg daily) and intravenous ceftriaxone (2 g daily) for 14 days are equally effective. Adults and children aged 12 years or over with definite or possible early neuroborreliosis with parenchymal central nervous system (CNS) manifestations (myelitis, encephalitis, vasculitis) should be treated with intravenous ceftriaxone 2 g daily for 14 days and patients with evidence of late neuroborreliosis with CNS manifestations should receive intravenous ceftriaxone 2 g daily for 21 days. The guidelines recommend that adults with peripheral neuropathy associated with ACA should be treated with doxycycline 200 mg daily or intravenous ceftriaxone 2 g daily for 21 days. Children under the age of 12 years who have isolated facial palsy are usually treated

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Fig. 8.1. Graph showing totals and trends for samples received for Borrelia burgdorferi antibody tests July 2004–Dec 2010 at a UK reference unit. Positive IgG samples are shown as a percentage of the total samples received. (Note that multiple samples are often received from seropositive patients; hence total positive sample numbers significantly exceed total reports of new LB cases). Data from the Lyme Borreliosis Unit, Health Protection Agency, January 2011, derived from routine workload statistics.

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with oral amoxicillin for 2 weeks. Ceftriaxone is the usual choice for children in this age group who have other presentations of neuroborreliosis (50–75 mg/kg/day in a single dose, to a maximum of 2 g daily).

specific symptoms in the general population (Cerar et al., 2010).

8.7 Outcomes

Other ixodid tick-transmitted infections are rarely reported in the UK and Ireland, although surveys of tick populations in several parts of the British Isles have shown evidence of Anaplasma phagocytophilum, Babesia divergens and louping ill virus, all of which can be pathogenic to animals. Redwater fever, a cattle disease caused by B. divergens, occurs focally in the UK and Ireland, and rare cases of human babesiosis have occurred in immunocompromised or splenectomized individuals, who are prone to overwhelming infections with this organism. Several cases of human anaplasmosis have been reported in patients who had acquired infection in other parts of Europe. Anaplasmosis has also been identified rarely as a coinfection in UK patients with atypical features of Lyme borreliosis. Louping ill virus, a flavivirus closely related to tick-borne encephalitis virus, causes significant disease in animals, principally sheep and grouse, but rarely causes human disease (HPA, 2011b). Rickettsiae, including Rickettsia helvetica, have also been identified in ticks in the UK, but further studies will be necessary to assess their potential to cause tick-transmitted human disease. Only rare cases of clinical infection caused by R. helvetica have been reported in other European countries (TjisseKlasen et al., 2011).

Long-term outcomes of treated Lyme borreliosis in the UK and Ireland are generally good, and are in keeping with findings of studies in other European countries and the USA. Most patients with uncomplicated EM have rapid resolution of the rash after starting antibiotic treatment. Facial palsies appear to resolve completely in the majority of cases, and severe residual paresis is uncommon. Radicular pain can take many months to resolve completely, particularly in older patients, although pain intensity usually reduces rapidly following commencement of antibiotic treatment. Some patients with severe degrees of tissue damage prior to treatment (mainly with late encephalomyelitis) have recovered incompletely.

8.8 Post-Lyme Syndrome Non-specific symptoms including fatigue, musculoskeletal pain and cognitive complaints can persist for some time in a minority of patients following appropriate treatment and resolution of objective findings. Patients with significant presentations of disseminated disease prior to treatment appear to be more susceptible to persisting symptoms than those who had uncomplicated EM. These symptoms usually resolve over several months, but a small proportion of patients continue to have more prolonged disabling fatigue without evidence of continuing infection, and are not helped by repeated courses of antibiotics. No prospective studies have been performed in the UK to assess the incidence and possible causes of subjective symptoms following treatment for Lyme borreliosis. This should be a research priority for the UK, and any study of treatment outcomes should incorporate an uninfected control group to assess the incidence of non-

8.9 Other Tick-Transmitted Infections in UK and Ireland

8.10 Prevention of Lyme borreliosis No vaccine is currently available for Lyme borreliosis and post-tick bite antibiotic prophylaxis is not routinely recommended in the UK or Ireland. Prevention relies mainly on personal protection measures including tick awareness, avoidance of tick bites through the use of appropriate clothing and insect repellents, early removal of attached ticks and thorough checks for attached ticks at the end of each day in a tick-infested area

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(HPA, 2011c). In early spring each year, public health authorities highlight the risk of ticktransmitted disease and methods of avoidance, particularly to people at recreational and occupational risk of tick exposure, using a variety of media approaches including television features, articles in newspapers and special interest magazines and newsletters. Councils in some high-prevalence areas also publicize disease-prevention measures locally. Educational programmes for health professionals to promote early recognition and appropriate management of Lyme borreliosis have also been implemented. A multidisciplinary research project funded by the Rural Economy and Land Use Programme has assessed perceptions and responses of individuals and organizations to the risks of Lyme borreliosis, including modelling of seasonal tick exposure risk and evaluation of precautionary information (Quine et al., 2011). It is hoped that its findings will lead to improvements in risk management of Lyme borreliosis and more effective presentation of information regarding precautionary measures and early recognition of disease (Marcu et al., 2010; British Infection Association, 2011).

8.11 Controversies in Lyme Disease Controversies associated with the definition, diagnosis and management of chronic Lyme disease have arisen in recent years in the UK and Ireland, driven largely by information on Internet sites, much of which is very misleading (Cooper and Feder, 2004). Patients with a variety of conditions, including fatigue syndromes, multiple sclerosis, motor neuron disease, autoimmune diseases and human immunodeficiency virus infection, have received diagnoses of chronic Lyme disease based on non-specific clinical findings and laboratory tests such as live blood microscopy, lymphocyte transformation tests, CD57 natural killer cell counts, and non-standard and inadequately validated immunoblot tests. Many have received prolonged courses of oral and parenteral antibiotics and other agents, causing serious adverse events in some cases. Detailed clinical assessments and

laboratory investigations by experts in infectious diseases and neurology have shown that the great majority of the UK patients diagnosed and managed in this unorthodox way have no evidence of current or past Lyme borreliosis. A report from the UK Department of Health in 2006 raised concerns about the use of unorthodox and unvalidated tests (Department of Health, 2006) and the UK’s Chief Medical Officer issued a further warning about the dangers of misdiagnosis and inappropriate treatment in 2009 (CMO update, 2009). The British Infection Association has developed an evidence-based position paper for health professionals on the diagnosis and management of Lyme borreliosis in the UK because of its members’ concerns regarding these potentially dangerous practices (British Infection Association, 2011).

References Abbott, R.A., Hammans, S., Margason, M. and Alji, B.M. (2005) Diaphragmatic paralysis and respiratory failure as a complication of Lyme disease. Journal of Neurology, Neurosurgery and Psychiatry 76, 1306–1307. Asbrink, E. and Hovmark, A. (1985) Successful cultivation of spirochetes from skin lesions of patients with erythema chronicum migrans Afzelius and acrodermatitis chronica atrophicans Herxheimer. Acta Pathologica, Microbiologica and Immunologica Scandinavica (B) 93, 161–163. Bateman, D.E., Lawton, N.F., White, J.E., Greenwood, R.J. and Wright, D.J.M. (1988) The neurological complications of Borrelia burgdorferi in the New Forest area of Hampshire. Journal of Neurology, Neurosurgery and Psychiatry 51, 699–703. Benach, J.A., Bosler, E.M., Hanrahan, J.P., Coleman, T.J., Bast, T.F., Habicht, G.S., Cameron, D.J., Ziegler, J.L., Burgdorfer, W., Barbour, A.G., Edelman, R. and Kaslow, R.A. (1983) Spirochetes isolated from the blood of two patients with Lyme disease. New England Journal of Medicine 398, 740–742. British Infection Association (2011) The epidemiology, prevention, investigation and treatment of Lyme borreliosis in United Kingdom patients: A position statement by the British Infection Association. The Journal of infection 62(5): 329–338. Cerar, D., Cerar, T., Ruzic-Sabljic, E., Wormser,

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G.P. and Strle, F. (2010) Subjective symptoms after treatment of early Lyme disease. American Journal of Medicine 123, 79–86. Cetin, E., Sotoudeh, M., Auer, H. and Stanek, G. (2006) Paradigm Burgenland: risk of Borrelia burgdorferi sensu lato infection indicated by variable seroprevalence rates in hunters. Wiener Klinische Wochenschrift 118, 677–681. CMO update (2009) Testing for Lyme disease. Issue 49, p. 4 . Cooper, J.D. and Feder, H.M. Jr (2004) Inaccurate information about Lyme disease on the internet. Pediatric Infectious Diseases Journal 23, 1105– 1108. Department of Health (2006) Unorthodox and unvalidated laboratory tests in the diagnosis of Lyme borreliosis and in relation to medically unexplained symptoms . Dryden, M.S., O’Connell S., Samuel, W. and Iannotti, F. (1996) Lyme myelitis mimicking neurological malignancy. Lancet 348, 624. Elamin, M., Monaghan, T., Mullins, G., CorbettFeeney, G., O’Connell S. and Counihan, T.J. (2010) The clinical spectrum of Lyme neuroborreliosis. Irish Medical Journal 103, 46–49. EUCALB (2010) European Concerted Action on Lyme borreliosis. . European Environment Agency (2010a) Ten Messages for 2010: No. 5 – Forest Ecosystems . European Environment Agency (2010b) Forest map of Europe . European Society of Clinical Micobiology and Infectious Diseases (2004) Guidelines for the diagnosis of tickborne bacterial diseases in Europe. Clinical Microbiology and Infection 10, 1108–1132. Forest Research (2009) Ecotype No. 47 . Gray, J. (1998) The ecology of ticks transmitting Lyme borreliosis. Experimental and Applied Acarology 22, 249–258. Gray, J.S., Dautel, H., Estrada-Pena, A. Kahl, O. and Lindgren, E. (2009) Effects of climate change on ticks and tick-borne diseases in Europe. Interdisciplinary Perspectives on Infectious Disease 2009, 593232.

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and Soti, V. (2010) Pathogenic landscapes: interactions between land, people, disease vectors, and their animal hosts. International Journal of Health Geographics 9, 54. Lantos, P.M., Charini, W.A. Medoff, G., Moro, M.H., Mushatt, D.M, Parsonnet, J., Sanders, J.W. and Baker, C.J. (2010) Final report of the Lyme Disease Review Panel of the Infectious Diseases Society of America. Clinical Infectious Disease 51, 1–5. Ledue, T.B., Collins, M.F., Young, J. and Schriefer, M.E. (2008) Evaluation of the recombinant VlsE-based Liaison chemiluminescent immunoassay for the detection of Borrelia burgdorferi and diagnosis of Lyme disease. Clinical and Vaccine Immunology 15, 1796– 1804. Ling, C.L., Joss, A.W.L., Davidson, M.M. and Ho-Yen, D.O. (2000) Identification of different Borrelia burgdorferi genomic groups from Scottish ticks. Journal of Clinical Pathology 53, 94–98. Logar, M., Ruzic-Sabljic, E., Maraspin, V., LotricFurlan, S., Cimperman, J., Jurca, T. and Strle, F. (2004) Comparison on erythema migrans caused by Borrelia afzelii and Borrelia garinii. Infection 32, 15–19. Lovett, J.K., Evans, P.H., O’Connell S. and Gutowski, N.J. (2008) Neuroborreliosis in the south-west of England. Epidemiology and Infection 136, 1707–1711. Marcu, A., Barnett, J., Uzzell, D. and O’Connell S. (2010) Lyme disease patients’ information needs and their preferences for precautionary measures. In: 12th International Conference on Lyme Borreliosis, Ljubljana, Slovenia, abstract P134. Miller, R.F., O’Connell S. and Manji, H. (2006) Reinfection with Lyme borreliosis presenting as a painful radiculopathy: Bannwarth’s, Beevor’s and Borrelia. Journal of Neurology, Neurosurgery and Psychiatry 77, 1293–1294. Muhlemann, M.F. (1984) Thirteen British cases of erythema chronicum migrans, a spirochaetal disease. British Journal of Dermatology 111, 335–339. Muhlemann, M.F. and Wright, D.J.M. (1987) Emerging pattern of Lyme disease in the United Kingdom and Irish Republic. Lancet 329, 260– 262. Mygland, A., Ljostad, U., Fingerle, F., Rupprecht, T., Schmutzhard, E. and Steiner, I. (2010) EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis. European Journal of Neurology 17, 8–16. NHS Clinical Knowledge Summaries (2011) Lyme disease – management .

Obasi, O. (1977) Erythema chronicum migrans. British Journal of Dermatology 97, 459. O’Connell S., Sorouri-Zanjani, R., White, J.E. and Guy, E.C. (1992) Lyme disease: experience in an endemic area. British Journal of Dermatology 127 (Supplement 40), 21. Parliamentary Office on Science and Technology (2009) Postnote No. 325: Wild Deer . Quine, C.P., Barnett, J. (2011). Frameworks for risk communication and disease management: the case of Lyme disease and countryside users. Philosopical transactions of the Royal Society of London. Series B, Biological sciences 366(1573): 2010–2022. Rauter, C. and Hartung, T. (2005) Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks in Europe: a meta-analysis. Applied and Environmental Microbiology 71, 7203–7216. Robertson, J., Gray, J.S., MacDonald, S. and Johnson, H. (1998) Seroprevalence to Borrelia burgdorferi sensu lato infection in blood donors and park rangers in relation to local habitat. Zentralblatt für Bakteriologie 288, 293–301. Robertson, J., Murdoch, S., Foster, L. and Green, S. (1999) Isolation and species typing of Lyme borreliosis spirochaetes from UK patients with erythema migrans. European Journal of Epidemiology 15, 499–500. Robertson, J.N., Gray, J.S. and Stewart, P. (2000a) Tick bite and Lyme borreliosis risk at a recreational site in England. European Journal of Epidemiology 16, 647–652. Robertson, J., Guy, E. and Andrews, N. (2000b) A European multicenter study of immunoblotting in the serodiagnosis of Lyme borreliosis. Journal of Clinical Microbiology 38, 2097–2102. Rupprecht, T., Koedel, U., Fingerle, V. and Pfister, H.-W. (2008) The pathogenesis of Lyme neuroborreliosis: from infection to inflammation. Molecular Medicine 14, 205–212. Rural Economy and Land Use Programme (2011) Assessing and Communicating Animal Disease Risks for Countryside Users . Scrimenti, R.J. (1970) Erythema chronicum migrans. Archives of Dermatology 102, 104– 105. Stanek, G., O’Connell S., Cimmino, M., Aberer, E., Kristoferitsch, W., Granstrom M, Guy E and Gray, J. (1996). European Union concerted action on risk assessment in Lyme borreliosis: clinical case definitions for Lyme borreliosis. Wiener Klinische Wochenschrift 108, 741– 747.

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Stanek, G., Fingerle, V., Hunfeld, K.-P., Jaulhac, B., Kaiser, R., Krause, A., Kristoferitsch, W., O’Connell, S., Ornstein, K., Strle, F. and Gray, J. (2011) Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clinical Microbiology and Infection 17, 69–79. Steere, A.C., Malawista, S.E., Snydman, D.R., Shope, R.E., Andiman, W.A., Ross, M.R. and Steele, F.W. (1977) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis and Rheumatism 20, 7–17. Strle, F., Nadelman, R.B., Cimperman, J., Nowakowski, J., Picken, R.N., Schwartz, I., Maraspin, V., Aguero-Rosenfeld, A.E., Varde, S., Lotric-Furlan, S. and Wormser, G.P. (1999) Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York state and by Borrelia afzelii in Slovenia. Annals of Internal Medicine 130, 32–36. Thomas, D.R., Sillis, M., Coleman, T.J., Kench, S.M., Ogden, N.H., Salmon, R.L., MorganCapner, P., Softley, P. and Meadow, D. (1998) Low rates of ehrlichiosis and Lyme borreliosis in English farmworkers. Epidemiology and Infection 121, 609–614.

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9

Lyme Borreliosis: the European Perspective Gerold Stanek and Franc Strle

9.1 Introduction Lyme borreliosis in Europe is caused by various strains of Borrelia burgdorferi sensu lato or Lyme borreliae (Ružić-Sabljić et al., 2008; Baranton and De Martino, 2009) and presents with a variety of clinical signs, symptoms and disease courses (Strle, 1999; Stanek and Strle 2003). Most of the clinical manifestations that are today known to be part of Lyme borreliosis were known in Europe long before the spirochaetal aetiology had been discovered. They were, however, not seen as a single nosological entity. As Lyme borreliosis, like several other diseases, presents with numerous clinical features while laboratory testing has some limitations, there is a temptation to be over-inclusive in attributing clinical findings to this infection. To provide physicians of various disciplines with sound information about existing knowledge, several European countries have developed national guidelines. Furthermore, clinical case definitions for diagnosis and management of the disease were developed by a European Union-supported project named the European Union Concerted Action on Lyme borreliosis (EUCALB), as well as by subsequent work (Stanek et al., 1996, 2011). Such information needs to be broadly communicated and accepted in clinical practice – not just used for managing unusual

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problems in Lyme borreliosis. This more restricted definition of the disease, developed by predominantly academic physicians, is challenged by some who hold the misconception that substantial numbers of patients with chronic non-specific symptoms such as arthralgia, myalgia, headache, fatigue and so on – symptoms quite frequently present in the general population – are suffering from ‘chronic Lyme borreliosis’, and that such ‘chronic Lyme borreliosis’ requires long-term treatment with antibiotics. The latter approach has been expanding fairly rapidly, not only in the USA but also in several countries in Europe (Strle and Stanek, 2009).

9.2 Clinical Manifestations Clinical manifestations of today’s Lyme borreliosis appear to have been present in Europe for several centuries. Table 9.1 lists selected authors and their important contributions to the clinical description of Lyme borreliosis. Detailed information about the history of Lyme borreliosis in Europe can be found in Aspects of Lyme Borreliosis (Burgdorfer, 1993; Weber and Pfister, 1993). The clinical differences between European and North American disease concern particular manifestations that are rarely if

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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ever observed in North America, such as the skin disorders acrodermatitis chronica atrophicans (ACA) and borrelial lymphocytoma (BL). What is also different is the presence of several pathogenic species of Lyme borreliae in Europe compared with only one in North America. Among the currently described 19 genomic species of Lyme borreliae, six were isolated from specimens from human patients with Lyme borreliosis and thus are considered pathogens in Europe – namely Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto (which we will refer to as B. burgdorferi), Borrelia spielmanii, Borrelia lusitaniae and Borrelia bissettii, while Borrelia valaisiana has been demonstrated by PCR. B. afzelii is most frequently isolated from skin biopsies of patients with erythema migrans (EM) and ACA; B. garinii is predominantly isolated from the cerebrospinal fluid (CSF) of patients suffering from Lyme neuroborreliosis (LNB). B. burgdorferi is only rarely cultivated from such specimens, and the remaining genomic species were detected only in single cases (Ružić-Sabljić et al., 2008). Whether the varying clinical manifestations and clinical courses are due to the responsible borrelial genospecies or are mostly dependent on the genotype and immune response of the infected individual (Wormser et al., 2008) remains to be further elucidated.

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9.2.1 Skin manifestations Lyme borreliosis skin involvement manifests as EM, BL and ACA. These manifestations were well known as distinct skin disorders long before the discovery of the causative agent (Herxheimer and Hartmann, 1902; Afzelius, 1910; Lipschütz, 1913; Bäfverstedt, 1943; Asbrink and Hovmark, 1988). It has been suggested that Lyme borreliae might additionally be associated with a subset of patients with scleroderma circumscripta, lichen sclerosus et atrophicus and cutaneous B-cell lymphoma. Erythema migrans EM is by far the most frequent manifestation of Lyme borreliosis in Europe. In epidemiological studies in Sweden and Germany, EM represents 77–89% of all presentations (Berglund et al., 1995; Huppertz et al., 1999). In Slovenia, where notification of Lyme borreliosis has been mandatory for more than 20 years, EM occurs in about 90% of registered cases (Anon., 2009). EM is defined as an expanding red or bluish-red patch at least 5 cm in diameter, with or without central clearing. The advancing edge is typically distinct and often intensely coloured but not markedly elevated. In cases with typical EM, the clinical diagnosis can be made without laboratory support. If

Table 9.1. First descriptions of clinical manifestations that were eventually identified as features of Lyme borreliosis, and treatment attempts. Year

Clinical manifestation and treatment attempts

Author(s)

1883 1902 1909 1913 1922 1941 1943 1949

Diffuse idiopathic skin atrophy Acrodermatitis chronica atrophicans Erythema migrans Erythema chronicum migrans ‘Paralysie par les tiques’ Chronic lymphocytic meningitis Lymphadenosis benigna cutis Penicillin treatment of acrodermatitis chronica atrophicans Penicillin treatment of erythema chronicum migrans Tick-borne meningopolyneuritis (Garin–Bujadoux– Bannwarth) Erythema chronicum migrans meningitis – a bacterial infectious disease?

Buchwald, A. Herxheimer, K. and Hartmann, K. Afzelius, A. Lipschütz, B. Garin, C. and Bujadoux, C.H. Bannwarth, A. Bäfverstedt, B. Thyresson, N.

1958 1973 1974

Hollström, E. Hörstrup, P. and Ackermann, R. Weber, K.

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the erythema is less than 5 cm in diameter, then a history of tick bite, a delay in appearance after the tick bite of at least 2 days and an expanding rash at the site of the tick bite is required. In uncertain cases, detection of B. burgdorferi sensu lato by culture and/or PCR from a skin biopsy may be supportive (Stanek et al., 2011). However, this support is only available from specialized laboratories. Secondary lesions also may occur such as multiple EM with the presence of two or more skin lesions, at least one of which must fulfil the size criteria for solitary EM given above. As the most frequently seen manifestation in Lyme borrelioses, EM can also represent a clue in the diagnosis of other manifestations of the disease. In Europe, EM is most often caused by B. afzelii, less frequently by B. garinii, rarely by B. burgdorferi and only exceptionally by other species such as B. bissettii, B. spielmanii and as yet unidentified species (Strle et al., 1997; Ornstein et al., 2001; Ružić-Sabljić et al., 2002; Foldvari et al., 2005). Simultaneous infection with two or more genospecies of Lyme borreliae also occurs, as indicated by PCR and culture (Ciceroni, et al., 2001; RužićSabljić et al., 2001a; Ružić-Sabljić et al., 2005; Cerar et al., 2008). EM may be accompanied by local symptoms such as mild itching, burning or pain in half of European patients. Systemic symptoms such as fatigue and malaise, headache, myalgia and arthralgia may occur in a smaller proportion of patients. Fever is, however, unusual in European patients with EM (Asbrink et al., 1986a; Strle et al., 1996, 2002) and the skin lesion is, as a rule, the only abnormality found on physical examination. In general, European patients with EM less often report systemic symptoms than patients in the USA (Strle et al., 1999; Tibbles and Edlow, 2007). Borrelial lymphocytoma BL is a rare manifestation of European Lyme borreliosis. It is defined as a painless bluishred nodule or plaque, usually on the earlobe, ear helix, nipple or scrotum. BL consists of a dense polyclonal lymphocytic infiltration of the cutis and subcutis predominated by B

lymphocytes and with germinal centres (Asbrink and Hovmark, 1988; Strle et al., 1992). It is more frequent in children, in whom it is typically located on the ear lobe, than in adults. The onset of BL is usually observed in the second half of the year. A long-term study revealed that BL was localized on the ear lobe in 47% of patients, on the breast in 42% and on the nose, arm, shoulder or scrotum in 11%. Patients with BL on the earlobe were younger than those with the lesion on the breast, with a median age of 12 versus 42 years (Strle et al., 1992). Serology is mostly positive, or seroconversion can be observed. In unclear cases, histology is required. A recent or concomitant EM may facilitate the diagnosis; direct detection of Borrelia by culture and/or PCR may yield positive results only in about 25% of cases and therefore the number of borrelial isolates from this skin disorder is limited. However, among the isolates from BL tissue, the genospecies B. afzelii is most frequently identified. B. garinii and B. burgdorferi have been isolated in single cases; the presence of B. bissettii was confirmed once (Picken et al., 1997; Strle et al., 1997; Maraspin et al., 2002a; Ružić-Sabljić et al., 2002). While systemic symptoms are rare and mild in earlobe BL, about 80% of patients with breast BL complain of constitutional symptoms and localized discomfort in the region of the areola mammae. Because of the differential diagnosis, histological examination is usually required in breast lymphocytoma and in lymphocytoma at locations other than the earlobe (Strle et al., 1992; Strle and Stanek, 2009). Acrodermatitis chronica atrophicans ACA is a chronic skin manifestation of European Lyme borreliosis. It is defined as a long-standing red or bluish-red lesion, located usually on the extensor surfaces of the extremities. Initially, it manifests as a doughy swelling. The lesions eventually become atrophic, and skin induration and fibroid nodules may develop over bony prominences. Initially, the lesion is usually unilateral; later, it may become bilateral and more or less symmetrical. Serum IgG

Lyme Borreliosis: the European Perspective

antibodies to Lyme borreliae are usually present in high concentrations. Lyme borreliae may also be demonstrated in biopsies of lesional skin by culture and/or PCR. ACA is predominantly caused by B. afzelii (Rijpkema et al., 1997; Maraspin et al., 2002b), but B. garinii and B. burgdorferi have also been isolated from ACA, indicating that B. afzelii is the predominant, but not exclusive, etiological agent of ACA (Picken et al., 1998; Ružić-Sabljić et al., 2002). Constant histological findings in active ACA lesions are telangiectasias and a lymphocytic infiltrate with plasma cells. The histopathological pattern is not diagnostic in itself, but is characteristic enough to alert the experienced pathologist. Unlike EM and BL, ACA does not disappear spontaneously (Asbrink and Hovmark, 1988). Patients do not usually recall a preceding EM, BL or other manifestation of Lyme borreliosis. Thus, ACA can be the first and only clinical sign of Lyme borreliosis. ACA is more often diagnosed in women than in men and occurs only very exceptionally in children. Patients are, as a rule, over 40 years old. Sclerotic lesions may develop in about 10% of patients with typical ACA (Asbrink and Hovmark, 1987). Some such lesions are clinically and histologically indistinguishable from localized scleroderma (morphea) or lichen sclerosus et atrophicus, suggesting a possible relationship between these two skin conditions. Peripheral neuropathy is associated with long-standing ACA (Kristoferitsch et al., 1988). Joints and bones may also become affected in the area of the skin lesion (Asbrink et al., 1986b,c). ACA on the lower extremities is often misinterpreted as vascular insufficiency (Müllegger, 2004; Strle and Stanek, 2009). Other skin manifestations of potential borrelial aetiology Results of attempts to isolate Lyme borreliae from lesional skin of patients suffering from scleroderma circumscripta (morphea) and lichen sclerosus et atrophicus (Breier et al., 1999; Müllegger, 2004) have been controversial. These observations might indicate either that a subset of these disorders could be of

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borrelial origin or that these patients in fact have ACA with sclerotic lesions, clinically and histologically indistinguishable from morphea or lichen sclerosus et atrophicus (Asbrink et al., 1986c). Only a well-designed, multicentre prospective clinical study would help to elucidate this question of aetiology. Cutaneous lymphoma Similarly, controversial observations have raised the possibility of an association between primary cutaneous B-cell lymphomas and infection with Lyme borreliae (Müllegger, 2004; Schöllkopf et al., 2008). European results differ from findings in the USA and Asia, where neither molecular nor epidemiological studies have demonstrated an aetiopathogenetic role for Lyme borreliae in cutaneous B-cell lymphoma. In response to these findings, the European Organization for Research and Treatment of Cancer and the International Society for Cutaneous Lymphoma recently published consensus recommendations on the management of cutaneous B-cell lymphomas. In this article, treatment with antibiotics is proposed for patients with primary cutaneous marginal zone lymphoma and evidence of B. burgdorferi sensu lato infection (Senff et al., 2008). Again, more scientific effort is required to obtain conclusive information regarding the possible association of Borrelia infection and cutaneous B-cell lymphoma. 9.2.2 Lyme neuroborreliosis LNB is defined as involvement of the central and/or peripheral nervous system in an infection with Lyme borreliae. With the exception of peripheral neuropathy in patients with ACA, involvement of the peripheral nervous system is as a rule associated with involvement of the central nervous system. In Europe, it is caused predominantly by B. garinii and in a small proportion of patients by B. afzelii (Busch et al., 1996; Peter et al., 1997; Ornstein et al., 2002; Ružić-Sabljić et al., 2001b, 2002; Strle et al., 2006). B. burgdorferi is rarely a cause of LNB in Europe, and other Borrelia species such as

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B. valaisiana (Peter et al., 1997; Ryffel et al., 1999), B. bissettii (Strle et al., 1997; Fingerle et al., 2008) and as yet unidentified species were found only in single cases (Lebech et al., 1998; Ružić-Sabljić et al., 2001b, 2002; Ornstein et al., 2002). Early European LNB typically appears during the first few weeks or months after the onset of infection and typically presents with lymphocytic meningitis and/or involvement of cranial and peripheral nerves (Kristoferitsch, 1991). The most pronounced clinical symptom of European LNB in adults is pain as a result of radiculoneuritis. This pain is usually severe, increasing in intensity during the night, as a result often causing severe sleep disorders. Involvement of motor nerves may lead to pareses, which are usually asymmetric and not always clinically prominent. If untreated, the neurological signs and symptoms will persist for many weeks (Kristoferitsch, 1991; Hansen, 1994). Patients with borrelial meningitis usually suffer from mild intermittent headache, but in single cases headache may be severe. There is usually no fever, nausea is mild or absent, and vomiting is frequently absent as are meningeal signs (Kristoferitsch, 1991; Hansen, 1994). The abnormal CSF findings consist of a lymphocytic pleocytosis of up to several hundred cells  106/l, normal or slightly to moderately elevated protein concentration, and normal or mildly decreased glucose concentration. The course of borrelial meningitis resembles a relatively mild but unusually protracted viral meningitis, with intermittent improvement and deterioration (Stanek and Strle, 2003). In early LNB, the facial nerve is most frequently involved, although any cranial nerve may be affected. Bilateral peripheral facial palsy is uncommon but is more indicative of early LNB than is unilateral involvement (Lotric-Furlan et al., 1999; Halperin, 2008). Lymphocytic pleocytosis is often also present in patients with borrelial peripheral facial palsy, even if patients do not show any sign or symptom of meningitis (Halperin, 2008). However, shortly after the onset of symptoms, CSF pleocytosis may be absent (mainly in children with facial palsy), and intrathecal production of borrelial antibodies may not be

detectable. In children, painful radiculoneuritis is rare, but isolated meningitis and peripheral facial palsy are more common than in adults (Stanek and Strle, 2003; Strle and Stanek, 2009). Although the outcome of borrelial facial palsy is said to be excellent (Halperin and Golightly, 1992; Halperin, 2008), results of some studies report a relatively high proportion of sequelae. In a Swedish study clinical and neurophysiological examination 3–5 years after peripheral facial palsy associated with LNB showed that mild sequelae were present in half of children (Bagger-Sjoback et al., 2005). Another study from Sweden revealed that one-fifth of children with acute facial palsy have permanent mild-to-moderate dysfunction of the facial nerve, but that other neurological symptoms or health problems do not accompany the incomplete recovery of the facial palsy, and that treatment of LNB seems to have no correlation with the clinical outcome of peripheral facial palsy (Skogman et al., 2003). Worth mentioning is that the clinical presentation of early LNB, as described above, is associated with B. garinii but not B. afzelii infection. The majority of patients from whose CSF B. afzelii has been isolated did not fulfil the diagnostic criteria for LNB in Europe (Strle et al., 2006). Late LNB appears to be very rare in Europe, as it is in the USA. An exception is peripheral neuritis, which is associated with long-lasting ACA and occurs in about half of these patients (Kristoferitsch et al., 1988). 9.2.3 Lyme carditis Acute cardiac involvement, which usually presents with acute onset of varying degrees of intermittent atrioventricular heart block and sometimes in association with clinical evidence of myopericarditis, is a rarely observed manifestation in Europe (Berglund et al., 1995). European Lyme carditis is similar to Lyme carditis in North America; it occurs most often either in the course of an EM or within a few weeks after onset of infection, and seems to be transient and self-limiting. The diagnosis of Lyme carditis requires the

Lyme Borreliosis: the European Perspective

exclusion of other explanations for cardiac abnormalities; the differential diagnosis in Lyme carditis is very broad (Strle and Stanek, 2009). 9.2.4 Joint involvement Lyme arthritis is considered a rare manifestation in Europe. However, a nationwide survey in Germany, based on responses to a questionnaire, suggested that borrelial arthritis may be more frequent in Europe than once thought (Priem et al., 2003). Arthritis may be preceded by other manifestations such as EM or may represent the initial manifestation of Lyme borreliosis and occur within several months of the initial borrelial infection. Most frequently involved is the knee (about 50% of all cases), followed by the ankle, wrist, elbow and rarely smaller joints (Herzer, 1991). It occurs predominantly in the fourth decade of life; when it occurs in children, older children are more often affected. The isolation rate of borreliae from joint fluid and synovia is very low. Information on the aetiology in Europe is limited. B. burgdorferi is the principal but not the only Borrelia species involved in Lyme arthritis in Europe; other genospecies have been detected in synovial specimens of patients (Eiffert et al., 1998; Vasiliu et al., 1998; Jaulhac et al., 2000; Marlovits et al., 2004). 9.2.5 Eye involvement Eye involvement in the course of Lyme borreliosis appears to occur very rarely and may often be associated with other signs of the illness (Mikkila et al., 2000; Strle and Stanek, 2009) such as EM, LNB or Lyme arthritis, but can be the sole manifestation of the disease. The diagnosis of borrelial ocular involvement is difficult and is more often presumed than confirmed. 9.2.6 Other potential rare manifestations of Lyme borreliosis Lyme borreliae have been suggested as a possible cause of scleroderma circumscripta,

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progressive facial hemiatrophia and eosinophilic fasciitis (Shulman syndrome) (Stanek et al., 1987; Granter et al., 1994; Hashimoto et al., 1996; Müllegger, 2004), myositis (MüllerFelber et al., 1993), dermatomyositis (Waton et al., 2007), nodular fasciitis (Schnarr et al., 2002), panniculitis (Hassler et al., 1992; Viljanen et al., 1992) and osteomyelitis (Oksi et al., 1994). There are also reports of effects on individual organs or organ systems such as the liver, lymphatic system, respiratory tract, urinary tract and genitalia. Proof of the existence of such involvement in humans is lacking.

9.3 Laboratory Diagnosis Laboratory testing should be performed only if there is evidence of a disease; without signs and/or symptoms, there is no disease. Specifically, there cannot be a diagnosis of Lyme borreliosis in the absence of any clinical manifestations. The only sign that enables a reliable clinical diagnosis of Lyme borreliosis in Europe is a typical EM. Ear lobe BL, meningoradiculoneuritis (Garin–Bujadoux– Bannwarth syndrome) and ACA are also highly suggestive of the diagnosis. The clinical case definitions for diagnosis and management of Lyme borreliosis in Europe were developed to support this approach (Stanek et al., 2011). Laboratory evidence is essential in most of the clinical manifestations and consists predominantly of serology. This is because other approaches, particularly the currently available methods for the direct detection of the pathogens are both more demanding and time-consuming, and have lower sensitivity (in manifestations other than EM) (Wilske et al., 2000). In brief, in typical EM, the diagnosis is clinical; serology is not essential. Early treatment can result in cure and the absence of a detectable specific antibody response. Serology is essential for the diagnosis of BL. Testing of paired blood samples, one taken at the first visit and a second 4–6 weeks later, will typically show either seroconversion or a significant change in specific IgM and/ or IgG titre. For the diagnosis of early LNB, the demonstration of a CSF pleocytosis is

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essential. Intrathecal anti-borrelial IgG antibody production is typically demonstrable, a determination that requires simultaneously drawn blood and CSF samples. However, the absence of intrathecal specific antibody synthesis does not exclude LNB in the case of short-duration symptoms. For the diagnosis of Lyme carditis, it is essential to demonstrate specific IgM and/or IgG antibodies or a significant change in the concentration of specific IgG antibody against Lyme borreliae in paired serum samples. For the diagnosis of Lyme arthritis, it is essential to demonstrate the presence of specific IgG antibodies, usually at high levels. The same is true for the diagnosis of ACA. An important observation in Lyme borreliosis is that peripheral blood clinical laboratory parameters indicative of a bacterial infectious disease are usually absent. Almost all patients have normal or only slightly elevated C-reactive protein values and white blood cell counts are usually normal (Strle, 1999; Steere, 2001).

9.4 Treatment and Prophylaxis Treatment with antibiotics is effective for all clinical manifestations; however, it has been most effective early in the course of the illness (Kristoferitsch et al., 1987; Strle, 1999; Stanek and Strle, 2003). In Europe, various antibiotics are used for the treatment of the different manifestations of Lyme borreliosis. Patients with solitary EM and BL are treated with doxycycline, amoxicillin, phenoxymethylpenicillin (penicillin V), cefuroxime axetil or azithromycin. The last is used predominantly for young children allergic to -lactam antibiotics. The usual duration of treatment is 14 days. Nervous system involvement and Lyme carditis are treated with intravenous ceftriaxone or penicillin G, usually for 2 weeks, or oral doxycycline. Oral doxycycline or amoxicillin, or intravenous ceftriaxone are used for the treatment of ACA and arthritis. The duration of treatment is usually 3–4 weeks for ACA and 4 weeks in the case of oral therapy for arthritis. In terms of prophylaxis, antibiotic treatment of a tick bite is not recommended

(Stanek and Kahl, 1999; Stanek and Strle 2003). Immunoprophylaxis of Lyme borreliosis for humans is currently unavailable in Europe. Removal of an attached tick as soon as possible – on the same day – will largely avoid transmission of Lyme borreliae. According to the results of experimental studies with gerbils, borrelial infection was demonstrable in up to 50% of animals 17 h after attachment. This study also demonstrated that the method by which ticks were removed (pulling out with forceps, or after 3 min of intensive squeezing, or after applying nail polish to ticks about 1 h before removal) did not significantly influence the risk of becoming infected with Lyme borreliae (Kahl et al., 1998).

9.5 Epidemiology The main vector of Lyme borreliae in Europe is Ixodes ricinus. Ixodes persulcatus is a vector in the northeastern parts of Europe. The principal vertebrate reservoirs for Lyme borreliae are small mammals, such as mice and voles, and certain species of birds. The host-seeking activity of I. ricinus nymphs is highest in late spring to early summer time (Fig. 9.1). However, questing ticks may be found even in wintertime, depending on the weather conditions. A website run by Tick-radar Ltd, established by German acarologists, informs readers about the activity of I. ricinus ticks in Germany (www. zeckenwetter.de). Additionally, it includes valuable information about ticks and management of tick bites. A study on the prevalence of questing nymphal and adult I. ricinus ticks in a region of western Germany revealed that this has increased significantly over the last 15 years while the pattern of habitat-specific infection prevalence did not change. The infection rate with B. burgdorferi sensu lato was about 13 and 21% in nymphs and adults, respectively. Interestingly, results of genotyping showed that B. valaisiana (43% of infected ticks) was detected most frequently, followed by B. garinii (32%), B. afzelii (12%) and B. burgdorferi (2%) (Kampen et al., 2004). The analysis of I. ricinus ticks collected

Lyme Borreliosis: the European Perspective

Nymphs

147

Larval ticks × 10

Adults

300

Number of ticks

250 200 150 100 50 0

J

F

M

A

M

J

J

A

S

O

N

D

Month Fig. 9.1. Seasonal activity of Ixodes ricinus. The number of larval ticks is ten times the number shown on the graph. Data from www.eucalb.com, section BIOLOGY: The Tick: Seasonality.

in central Germany revealed that overall 11% were infected with B. burgdorferi sensu lato but, in contrast to the results from western Germany, the genospecies B. garinii was most frequently identified (56%), followed by B. burgdorferi (32%), B. afzelii (18%) and B. valaisiana; dual infection was also observed (Hildebrandt et al., 2003). More information about the ecoepidemiology of B. burgdorferi sensu lato in Europe can be obtained from articles in the volume Lyme borreliosis: Biology, Epidemiology and Control (Gray et al., 2002). Tick bites occur in tick activity season (March/April to October/November), most frequently on weekends, usually peaking in the summer holiday season, when people are outside to relax in nature and thus have direct contact with vegetation and tick habitats. Tick bites in children are most frequently localized on the head, and in adults on the lower limbs and on the abdominal and gluteal region (Berglund et al., 1995). It is difficult to assess the frequency of different manifestations of Lyme borreliosis after a tick bite. Awareness of a previous tick bite in European patients with Lyme borreliosis varies. Among 1471 patients with

Lyme borreliosis from southern Sweden, 1157 (79%) were aware of a tick bite preceding the onset of symptoms (Berglund et al., 1995). Variation in the awareness of tick bites was observed in Slovenia. While 73% of adult patients diagnosed with typical EM at the Ljubljana Lyme borreliosis clinic in 1993 reported a tick bite at the site where the EM skin lesion expanded, only 53% of patients with EM reported a bite in the year 2000 (Strle et al., 2002). In general, European patients with EM report a tick bite more often than patients in the USA (Strle et al., 1999; Tibbles and Edlow, 2007). Lyme borreliosis has been reported to be the most frequent tick-borne infection throughout Europe. However, it is very difficult to assess the true incidence of Lyme borreliosis in Europe. Only a few countries have made it a mandatory reportable disease, and only a subset of these appears to have long-term experience with this. Even in these countries, the true number of cases per year may well be above the reported ones. It has also been reported that the incidence has increased markedly in recent years (Fig. 9.2) (Fülöp and Poggensee, 2008; Anon., 2009). Table 9.2 shows the results of prospective

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5000 4500 4000

Number of cases/year

3500 3000 2500 2000 1500 1000 500 0

1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Year

Fig. 9.2. Increase of the number of reported cases of Lyme borreliosis, Slovenia 1993–2007. Data obtained from the website of the Institute of Public Health (www.ivz.si) in Ljubljana, Slovenia (Anon., 2009).

clinical studies from Sweden (Berglund et al., 1995) and Germany (Huppertz et al., 1999) and displays data from Slovenia obtained by the Department of Infectious Diseases, University Medical Center Ljubljana (Strle and Stanek, 2009). According to a report from Germany, a bimodal age distribution can be observed. The incidence peaks in children aged 5–9 years and in adults aged 65–69 years; female patients are more frequently affected

than males (Fülöp and Poggensee, 2008). EM is the most frequently diagnosed clinical manifestation, accounting for about 90% of all manifestations (Anon., 2009). The incubation period of EM ranges from a few days to weeks with a median incubation period of 17 days (Strle et al., 1999). The diagnosis of EM is most frequently made in the early summer. About 70% of all cases of Lyme borreliosis occur between June and September (see Plate 3 in colour section).

Table 9.2. Frequency (%) of clinical manifestations of Lyme borreliosis in Europe as assessed in prospective clinical studies and by a long-term reporting system based on clinical case definitions. Manifestation

Sweden (1995)a

Germany (1999)b

Slovenia (2000)c

Erythema migrans Lyme neuroborreliosis Lyme arthritis Acrodermatitis chronic atrophicans Borrelial lymphocytoma Lyme carditis

78 13 5 2 2 1

89 3 5 1 2 1

82 9 3 5 1 1

aBerglund

et al. (1995). et al. (1999). cStrle and Stanek (2009). bHuppertz

Lyme Borreliosis: the European Perspective

Analysis of 806 isolates obtained in prospective studies from Slovenian patients with different manifestations of Lyme borreliosis (Table 9.3) indicates that three genomic species of Lyme borreliae – among the many occurring in Europe – are important human pathogens, namely B. afzelii, B. garinii and B. burgdorferi. Interestingly, the frequency

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distribution of isolates from patients does not match that found in ticks as outlined previously. B. garinii and B. burgdorferi sensu stricto are isolated relatively more frequently from ticks than B. afzelii, whereas B. afzelii is the most frequent isolate from the skin of patients with EM and ACA (Picken et al. 1996; Fingerle et al., 2008).

Table 9.3. Frequency (%) of various genospecies of borrelial isolates obtained in prospective clinical studies from skin (EM, BL and ACA) and cerebrospinal fluid (LNB) samples of Lyme borreliosis patients in Slovenia. Borrelia genospecies

Solitary EMa

B. afzelii B. garinii B. burgdorferi

Ružc´-Sabljic´ et al. (2002) (n = 488) 433 (88.7%) 53 (10.9%) 2 (0.4%)

BLb Stupica et al. (2010) (n = 137) 119 (86.9%) 11 (8.0%) 7 (5.1%)

Picken et al. (1997) (n = 5)e 4 (80%) 0 0

ACAc Ružc´-Sabljic´ et al. (2002) (n = 9) 9 (100%) 0 0

Picken et al. (1998) (n = 22) 17 (77.3%) 4 (18.2%) 1 (4.5%)

LNBd Ružc´-Sabljic´ et al. (2002) (n = 74) 66 (89.2%) 5 (6.8%) 3 (4%)

Ružc´-Sabljic´ et al. (2002) (n = 35) 8 (22.9%) 26 (74.3%) 1 (2.8%)

Strle et al. (2006) (n = 36) 10 (27.8%) 23 (63.9%) 3 (8.3%)

EM, erythema migrans; BL, borrelial lymphocytoma; ACA, acrodermatitis chronica atrophicans; LNB, Lyme neuroborreliosis. The number of borrelial isolates in each study is indicated. a–dDuring the last 20 years other Borrelia species were also isolated from single patients with Lyme borreliosis, including B. bissettiia,b,d, B. spielmaniia and untypable strainsa,c,d eOne strain was typed as B. bissettii.

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eosinophilia: borrelial fasciitis. Journal of the American Medical Association 272, 1283–1285. Gray, J.S., Kahl, O., Lane, R.S. and Stanek, G. (eds) (2002) Lyme Borreliosis: Biology, Epidemiology and Control. CABI Publishing, Wallingford, UK. Halperin, J.J. (2008) Nervous system Lyme disease. Infectious Disease Clinics of North America 22, 261–274. Halperin, J.J. and Golightly, M. (1992) Lyme borreliosis in Bell’s palsy. Long Island Neuroborreliosis Collaborative Study Group. Neurology 42, 1268–1270. Hansen, K. (1994) Lyme neuroborreliosis: improvements of the laboratory diagnosis and a survey of epidemiological and clinical features in Denmark 1985–1990. Acta Neurologica Scandinavia 89 (Supplement 151), 7–44. Hashimoto, Y., Takahashi, H., Matsuo, S., Hirai, K., Takemori, N., Nakao, M., Miyamoto, K. and Iizuka, H. (1996) Polymerase chain reaction of Borrelia burgdorferi flagellin gene in Shulman syndrome. Dermatology 192, 136–139. Hassler, D., Zorn, J., Zoller, L., Neuss, M., Weyand, C., Goronzy, J., Born, I.A. and Preac-Mursic, V. (1992) Nodular panniculitis: a manifestation of Lyme borreliosis? Hautarzt 43, 134–138. Herxheimer, K. and Hartmann, K. (1902) Über Acrodermatitis chronica atrophicans. Archives of Dermatology and Syphilis 61, 57–76. Herzer, P. (1991) Joint manifestations of Lyme borreliosis in Europe. Scandinavian Journal of Infectious Diseases 77, 55–63. Hildebrandt, A., Schmidt, K.H., Wilske, B., Dorn, W., Straube, E. and Fingerle, V. (2003) Prevalence of four species of Borrelia burgdorferi sensu lato and coinfection with Anaplasma phagocytophila in Ixodes ricinus ticks in central Germany. European Journal of Clinical Microbiology and Infectious Diseases 22, 364–367. Hollström, E. (1958) Penicillin treatment of erythema chronicum migrans Afzelius. Archiv Dermatologie and Venereologie 38, 285–289. Hörstrup, P. and Ackermann, R. (1973) Durch Zecken übertragene Meningopolyneuritis (Garin–Bujadoux, Bannwarth). Fortschritte der Neurologie und Psychiatrie 41, 583–606. Huppertz, H.I., Böhme, M., Standaert, S.M., Karch, H. and Plotkin, S.A. (1999) Incidence of Lyme borreliosis in the Würzburg region of Germany. European Journal of Clinical Microbiology and Infectious Diseases 18, 697–703. Jaulhac, B., Heller, R., Limbach, F.X., Hansmann, Y., Lipsker, D., Monteil, H., Sibilia, J. and Piemont, Y. (2000) Direct molecular typing of Borrelia burgdorferi sensu lato species in

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Marlovits, S., Khanakah, G., Striessnig, G., Vécsei, V. and Stanek, G. (2004) Emergence of Lyme arthritis after autologous chondrocyte transplantation. Arthritis and Rheumatism 50, 259–264. Mikkila, H.O., Seppala, I.J., Viljanen, M.K., Peltomaa, M.P. and Karma, A. (2000) The expanding clinical spectrum of ocular Lyme borreliosis. Ophthalmology 107, 581–587. Müllegger, R.R. (2004) Dermatological manifestations of Lyme borreliosis. European Journal of Dermatology 14, 296–309. Müller-Felber, W., Reimers, C.D., de Koning, J., Fischer, P., Pilz, A. and Pongratz, D.E. (1993) Myositis in Lyme borreliosis: an immunohistochemical study of seven patients. Journal of Neurological Sciences 118, 207–212. Oksi, J., Mertsola, J., Reunanen, M., Marjamaki, M. and Viljanen, M.K. (1994) Subacute multiplesite osteomyelitis caused by Borrelia burgdorferi. Clinical Infectious Diseases 19, 891–896. Ornstein, K., Berglund, J., Nilsson, I., Norrby, R. and Bergstrom, S. (2001) Characterization of Lyme borreliosis isolates from patients with erythema migrans and neuroborreliosis in southern Sweden. Journal of Clinical Microbiology 39, 1294–1298. Ornstein, K., Berglund, J., Bergstrom, S., Norrby, R. and Barbour, A.G. (2002) Three major Lyme Borrelia genospecies (Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii) identified by PCR in cerebrospinal fluid from patients with neuroborreliosis in Sweden. Scandinavian Journal of Infectious Diseases 34, 341–346. Peter, O., Bretz, A.G., Postic, D. and Dayer, E. (1997) Association of distinct species of Borrelia burgdorferi sensu lato with neuroborreliosis in Switzerland. Clinical Microbiology and Infection 3, 423–431. Picken, R.N., Cheng, Y., Strle, F., Cimperman, J., Maraspin, V., Lotricˇ -Furlan, S., Ružic´ -Sabljic´, E., Han, D., Nelson, J.A., Picken, M.M. and Trenholme, G.T. (1996) Molecular characterization of Borrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates. European Journal of Clinical Microbiology and Infectious Diseases 15, 313–323. Picken, R.N., Strle, F., Ružic´ -Sabljic´, E., Maraspin, V., Lotricˇ -Furlan, S., Cimperman, J., Cheng, Y. and Picken, M.M. (1997) Molecular subtyping of Borrelia burgdorferi sensu lato isolates from five patients with solitary lymphocytoma. Journal of Investigative Dermatology 108, 92–97. Picken, R.N., Strle, F., Picken, M.M., Ružic´ -Sabljic´, E., Maraspin, V., Lotricˇ -Furlan, S., and Cimperman, J. (1998) Identification of three

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species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) among isolates from acrodermatitis chronica atrophicans lesions. Journal of Investigative Dermatology 110, 211–214. Priem, S., Munkelt, K., Franz, J.K., Schneider, U., Werner, T., Burmester, G.R. and Krause, A. (2003) Epidemiology and therapy of Lyme arthritis and other manifestations of Lyme borreliosis in Germany: results of a nationwide survey. Zeitschrift für Rheumatologie 62, 450– 458. Rijpkema, S.G.T., Tazelaar, D.J., Molkenboer, M.J.C.H., Noordhoek, G.T., Plantinga, G., Schouls, L. and Schellekens, J.F.P. (1997) Detection of Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii and group VS116 by PCR in skin biopsies of patients with erythema migrans and acrodermatitis chronica atrophicans. Clinical Microbiology and Infection 3, 109–116. Ružic´ -Sabljic´, E., Arnez, M., Lotricˇ -Furlan, S., Maraspin, V., Cimperman, J. and Strle, F. (2001a) Genotypic and phenotypic characterisation of Borrelia burgdorferi sensu lato strains isolated from human blood. Journal of Medical Microbiology 50, 896–901. Ružic´ -Sabljic´, E., Lotricˇ -Furlan, S., Maraspin, V., Cimperman, J., Pleterski-Rigler, D. and Strle, F. (2001b) Analysis of Borrelia burgdorferi sensu lato isolated from cerebrospinal fluid. Acta Pathologica, Microbiologica et Immunologica Scandinavica 109, 707–713. Ružic´ -Sabljic´, E., Maraspin, V., Lotricˇ -Furlan, S., Jurca, T., Logar, M., Pikelj-Pecˇnik, A. and Strle, F. (2002) Characterization of Borrelia burgdorferi sensu lato strains isolated from human material in Slovenia. Wiener Klinische Wochenschrift 114, 544–550. Ružic´ -Sabljic´, E., Arnez, M., Logar, M., Maraspin, V., Lotricˇ -Furlan, S., Cimperman, J. and Strle, F. (2005) Comparison of Borrelia burgdorferi sensu lato strains isolated from specimens obtained simultaneously from two different sites of infection in individual patients. Journal of Clinical Microbiology 43, 2194–2200. Ružic´ -Sabljic´, E., Zore, A. and Strle, F. (2008) Characterization of Borrelia burgdorferi sensu lato isolates by pulsed-field gel electrophoresis after MluI restriction of genomic DNA. Research in Microbiology 159, 441-448. Ryffel, K., Peter, O., Rutti, B., Suard, A. and Dayer, E. (1999) Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B.

afzelii, and B. valaisiana in humans. Journal of Clinical Microbiology 37, 4086–4092. Schnarr, S., Wahl, A., Jurgens-Saathoff, B., Mengel, M., Kreipe, H.H. and Zeidler, H. (2002) Nodular fasciitis, erythema migrans, and oligoarthritis: manifestations of Lyme borreliosis caused by Borrelia afzelii. Scandinavian Journal of Rheumatology 31,184–186. Schöllkopf, C., Melbye, M., Munksgaard, L., Ekström-Smedby, K., Rostgaard, K., Glimelius, B., Chang, E.T., Roos, G., Hansen, M., Adami, H.O. and Hjalgrim, H. (2008) Borrelia infection and risk of non-Hodgkin lymphoma. Blood 111, 5524–5529. Senff, N.J., Noordijk, E.M., Youn, H., Kim, Y.H., Bagot, M., Berti, E., Cerroni, L., Dummer, R., Duvic, M., Hoppe, R.T., Pimpinelli, N., Rosen, S.T., Vermeer, M.H., Whittaker, S. and Willemze, R. (2008) European Organization for Research and Treatment of Cancer and International Society for Cutaneous Lymphoma consensus recommendations for the management of cutaneous B-cell lymphomas. Blood 112, 1600– 1609. Skogman, B.H., Croner, S. and Odkvist, L. (2003) Acute facial palsy in children – a 2-year follow-up study with focus on Lyme neuroborreliosis. International Journal of Pediatric Otorhinolaryngology 67, 597–602. Stanek, G. and Kahl, O. (1999) Chemoprophylaxis for Lyme borreliosis? Zentralblatt für Bakteriologie 289, 655–665. Stanek, G. and Strle, F. (2003) Lyme borreliosis. Lancet 362, 1639–1647. Stanek, G., Konrad, K., Jung, M. and Ehringer, H. (1987) Shulman syndrome, a scleroderma subtype caused by Borrelia burgdorferi? Lancet 329, 1490. Stanek, G., O’Connell, S., Cimmino, M., Aberer, E., Kristoferitsch, W., Granstrom, M., Guy, E. and Gray, J. (1996) European Union concerted action on risk assessment in Lyme borreliosis: clinical case definitions for Lyme borreliosis. Wiener Klinische Wochenschrift 108, 741–747. Stanek, G., Fingerle, V., Hunfeld, K.-P., Jaulhac, B., Kaiser, R., Krause, A., Kristoferitsch, W., O’Connell, S., Ornstein, K., Strle, F. and Gray, J. (2011) Lyme borreliosis: clinical case definitions for diagnosis and management in Europe. Clinical Microbiology and Infection 17, 69–79. Steere, A.C. (2001) Lyme disease. New England Journal of Medicine 345, 115–125. Strle, F. (1999) Principles of the diagnosis and antibiotic treatment of Lyme borreliosis. Wiener Klinische Wochenschrift 111, 911–915. Strle, F. and Stanek, G. (2009) Clinical manifest-

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ations and diagnosis of Lyme borreliosis. Current Problems in Dermatology 37, 51–110. Strle, F., Pleterski-Rigler, D., Stanek, G., PejovnikPustinek, A., Ruzic, E. and Cimperman, J. (1992) Solitary borrelial lymphocytoma: report of 36 cases. Infection 20, 201–206. Strle, F., Nelson, J.A., Ružic´ -Sabljic´, E., Cimperman, J., Maraspin, V., Lotricˇ -Furlan, S., Cheng, Y., Picken, M.M., Trenholme, G. and Picken, R.N. (1996) European Lyme borreliosis: 231 culture-confirmed cases involving patients with erythema migrans. Clinical Infectious Diseases 23, 61–65. Strle, F., Picken, R.N., Cheng, Y., Cimperman, J., Maraspin, V., Lotricˇ -Furlan, S., Ružic´ -Sabljic´, E. and Picken, M.M. (1997) Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities to strain 25015. Clinical Infectious Diseases 25, 273–280. Strle, F., Nadelman, R.B., Cimperman, J., Nowakowski, J., Picken, R.N., Schwartz, I., Maraspin, V., Aguero-Rosenfeld, M.E., Varde, S., Lotricˇ -Furlan, S. and Wormser, G.P. (1999) Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and Borrelia afzelii in Slovenia. Annals of Internal Medicine 130, 32–36. Strle, F., Videcnik, J., Zorman, P., Cimperman, J., Lotricˇ -Furlan, S. and Maraspin, V. (2002) Clinical and epidemiological findings for patients with erythema migrans. Comparison of cohorts from the years 1993 and 2000. Wiener Klinische Wochenschrift 114, 493–497. Strle, F., Ružic´ -Sabljic´, E., Cimperman, J., Lotricˇ -Furlan, S. and Maraspin, V. (2006) Comparison of findings for patients with Borrelia garinii and Borrelia afzelii isolated from cerebrospinal fluid. Clinical Infectious Diseases 43, 704–710. Stupica, D., Lusa, L., Cerar, T., Ružic´ -Sabljic´, E. and Strle, F. (2010) Comparison of post-Lyme borreliosis symptoms in erythema migrans patients with positive and negative Borrelia burgdorferi sensu lato skin culture. Vector-borne

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and Zoonotic Diseases (Epub ahead of print, 17 November 2010) doi: 10.1089/vbz.2010.0018 Thyresson, N. (1949) The penicillin treatment of acrodermatitis chronica atrophicans (Herxheimer). Acta Dermato-Venereologica 29, 572–621. Tibbles, C.D. and Edlow, J.A. (2007) Does this patient have erythema migrans? Journal of the American Medical Association 297, 2617–2627. Vasiliu, V., Herzer, P., Rossler, D., Lehnert, G. and Wilske, B. (1998) Heterogeneity of Borrelia burgdorferi sensu lato demonstrated by an ospA-type-specific PCR in synovial fluid from patients with Lyme arthritis. Medical Microbiology and Immunology 187, 97–102. Viljanen, M.K., Oksi, J., Salomaa, P., Skurnik, M., Peltonen, R. and Kalimo, H. (1992) Cultivation of Borrelia burgdorferi from the blood and a subcutaneous lesion of a patient with relapsing febrile nodular nonsuppurative panniculitis. Journal of Infectious Diseases 165, 596–597. Waton, J., Pinault, A.L., Pouaha, J. and Truchetet, F. (2007) [Lyme disease could mimic dermatomyositis]. La Revue de Medecine Interne 28, 343–345. Weber, K. (1974) Erythema chronicum migrans meningitis – a bacterial infectious disease? Münchner Medizinische Wochenschrift 116, 1993–1998. Weber, K. and Pfister, H.-W. (1993) History of Lyme borreliosis in Europe. In: Weber, K. and Burgdorfer, W. (eds) Aspects of Lyme Borreliosis. Springer Verlag, Berlin/Heidelberg, pp. 1–20. Wilske, B., Zöller, L., Brade, V., Eiffert, H., Göbel, U.B. and Stanek, G. (2000) Quality Standards for the Microbiological Diagnosis of Infectious Diseases, MIQ 12/2000 Lyme Borreliosis. Urban & Fischer Verlag, Munich/Jena. Wormser, G.P., Brisson, D., Liveris, D., Hanincová, K., Sandigursky, S., Nowakowski, J., Nadelman, R.B., Ludin, S. and Schwartz, I. (2008) Borrelia burgdorferi genotype predicts the capacity for hematogenous dissemination during early Lyme disease. Journal of Infectious Diseases 198, 1358–1364.

10

Erythema Migrans Robert B. Nadelman

10.1 Introduction

10.2 Clinical Diagnosis

Erythema migrans (EM) (previously known as erythema chronicum migrans) is an expanding erythematous rash that develops at the site of the bite of certain Ixodes ticks within days to weeks (Steere et al., 1983a, 1985; Ǻsbrink and Olsson, 1985; Nadelman and Wormser, 1995, 2002). It is the most common objective manifestation of Lyme disease, accounting for about 90% of cases (Gerber et al., 1996; Krause et al., 1996; Nadelman and Wormser, 1998, 2002). The dramatic and distinct ‘bull’s eye’ appearance of the rash and its occurrence in the late spring and summer enabled the recognition of Lyme disease as a vector-borne infection years before the discovery of the causative pathogen, Borrelia burgdorferi sensu lato, and the development of the first diagnostic laboratory assays. Nevertheless, it is now evident that the ‘classic’ EM presentation with central clearing accounts for a minority of cases of early Lyme disease in the USA (Nadelman et al., 1996; Nadelman and Wormser, 2002; Smith et al., 2002). Furthermore, the rash alone cannot be said to be pathognomonic for infection with Borrelia burgdorferi because of the virtually indistinguishable appearance of certain other entities, in particular, southern tick-associated rash illness (see below) (Kirkland et al., 1997; Masters et al., 1998; Wormser et al., 2005c).

EM is an expanding erythematous skin lesion, usually round or oval, that develops 7–14 days (range 1–36 days) following the detachment of certain Ixodes ticks at the site of inoculation of B. burgdorferi (Steere et al., 1983a; Berger, 1989; Malane et al., 1991; Nadelman and Wormser, 1995; Nadelman et al., 1996) (see Plates 4-7 in the colour plate section). EM must be distinguished from localized and transient inflammatory reactions to the bite of an arthropod that are not associated with infection (see Plate 8 in the colour plate section). The latter resolve spontaneously within a day or two (Feder and Whitaker, 1995; Nadelman and Wormser, 1995; Wormser, 2006; Wormser et al., 2006). In order to increase the specificity of the diagnosis of EM by limiting confusion with such localized reactions, the Centres for Disease Control and Prevention (CDC) has designated 5 cm at the largest diameter as a minimum size for EM lesions (Bacon et al., 2008). Although useful for increasing accuracy in the clinical diagnosis of Lyme disease, particularly in clinical and epidemiological studies, the size limitation should not be used alone to exclude the diagnosis of EM in individual patients with clinical and epidemiological features that are otherwise suggestive (Krause et al., 2006; Wormser et al., 2006; Bacon et al., 2008).

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10.2.1 Epidemiology More than 90% of the 20,000 cases of Lyme disease that are reported to the CDC in the USA each year originate from ten states in New England, the Middle Atlantic and North Central regions (Bacon et al., 2008). Although not required for diagnosis of EM in an endemic area (see Laboratory diagnosis below), isolation in culture of B. burgdorferi sensu lato from clinical specimens has confirmed the diagnosis in patients from endemic areas in the USA, as well as much of Europe and parts of Asia where Borrelia afzelii and Borrelia garinii are the most common aetiological genospecies (Kuiper et al., 1994; Hashimoto et al., 1995; Busch et al., 1996; Strle et al., 1996a, 1999, 2011; Ornstein et al., 2001; Antoni-Bach et al., 2002; Lipsker et al., 2002; Logar et al., 2004; Masuzawa, 2004; Cerar et al., 2010). Reports of Lyme disease associated with EM from non-endemic regions in the USA and elsewhere (Sharma et al., 2010) without culture isolation of B. burgdorferi sensu lato from human specimens or vector ticks should be viewed with some scepticism, as the clinical appearance of a rash and serological testing have a low positive predictive value for B. burgdorferi infection in this setting. EM has been reported in approximately 70% of patients with Lyme disease in the USA (Bacon et al., 2008), but this is likely to be an underestimate for several reasons. This skin lesion may go unrecognized when it occurs at body sites such as the buttocks that are not easily visualized, or when it is associated with minimal or no systemic or local symptoms (Nadelman and Wormser, 1995, 1998; Gerber et al., 1996; Krause et al., 1996; Wormser et al., 2006). In addition, case reporting is biased towards detecting later manifestations of Lyme disease such as arthritis. The reason for this is that serological tests are reportable and tabulated in some states if positive, as they often are in extracutaneous Lyme disease. In contrast, no such laboratory reporting occurs if the tests are negative, as they usually are in patients with EM (see Laboratory diagnosis below) (Aguero-Rosenfeld et al., 1993, 2005; Wormser et al., 2006).

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There are two peaks in the age distribution for EM, occurring at 5–14 years and 45–54 years. Almost all cases of EM occur in late spring or summer (Falco et al., 1999; Krause et al., 2006) following bites of nymphal (rather than adult) Ixodes scapularis ticks, which are most active from May to July (Fish, 1995; Falco et al., 1999). This immature tick stage is more numerous than adult stage ticks. Nymphs are also much smaller than adult ticks and thus less likely to be noticed and removed before transmission of infection can occur (Nadelman et al., 2001; Wormser et al., 2006). In addition, humans are more likely to come into contact with ticks with increased outdoor activity during the warmer months (Fish, 1995). 10.2.2 Characteristics of erythema migrans EM begins as a small macule or papule at the site of a bite by certain Ixodes ticks that have fed and detached a median of 7–14 days (range 1–36 days) previously (Steere et al., 1983a; Berger, 1989; Nadelman et al., 1996; Nadelman and Wormser, 1998). European patients with EM are much more likely than US patients to recall a prior tick bite (Strle et al., 1999, 2011), perhaps because of more intense local reaction to the bite or faster transmission of infection. Only a minority (14–32%) (Nadelman et al., 1996; Smith et al., 2002; Hayes and Piesman, 2003; Wormser et al., 2006) of US patients recall the bite that transmitted infection, in part because the vector nymphal-stage ticks are only about the size of a poppy seed, and their bites are not associated with significant pruritus or pain (Nadelman and Wormser, 1995; Nadelman et al., 2001; Wormser et al., 2006). In addition, tick bites that ultimately result in infection typically occur at body sites where the tick can feed unvisualized for days, such as the buttocks in adults or the hairline of children (Nadelman and Wormser, 1995; Nadelman et al., 1996; Tibbles and Edlow, 2007). The reason for this is that the transmission of B. burgdorferi requires at least 36 h during which time the spirochaete must move from the tick mid-gut to the salivary

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glands before it can be transmitted to the skin of the human host (Ribeiro et al., 1987). Selected features of EM, including the location of primary lesions in one study of 119 US patients whose infection was cultureconfirmed, are depicted in Table 10.1 (Strle et al., 1999). EM lesions do not occur on mucous membranes, palms or soles (Steere et al., 1983a). Within days of the appearance of the initial macule or papule, a slowly enlarging erythematous patch develops (Steere et al., 1983a; Berger, 1989; Malane et al., 1991; Habif, 2004), sometimes with a depressed or raised area (punctum) at the centre of the lesion at the site of tick detachment (Berger, 1989; Malane et al., 1991; Melski et al., 1993; Nadelman and Wormser, 2007) (see Plate 4 in colour plate section). An annular or ‘bull’s eye’ appearance may develop when central or paracentral clearing occurs as the lesion expands over days to weeks. The skin lesion remains flat, blanches with pressure and usually does not desquamate or vesiculate at the periphery (Steere et al., 1983a; Malane et al., 1991; Nadelman and Wormser, 1995; Nadelman et al., 1996; Smith et al., 2002; Habif, 2004). The median diameter in each of five studies comprising a total of more than 500 US patients was between 10 and 16 cm, but lesions may exceed 70 cm (Steere et al., 1983a; Malane et al., 1991; Nadelman et al., 1996; Strle et al., 1999; Smith et al., 2002; Nowakowski

et al., 2003). EM size usually appears to be a function of its duration (Ǻsbrink and Olsson, 1985; Berger, 1989; Nadelman et al., 1996; Strle et al., 1999), varying in a linear fashion with a correlation coefficient of 0.7 (Nadelman et al., 1996). Early EM lesions grow at a rate of 20 cm2/day presumably related to the migration of spirochaetes away from the inoculation site (Berger, 1989). B. burgdorferi can be isolated from the centre and leading margin of EM lesions, and from normal skin surrounding the lesion (Berger et al., 1992; Kuiper et al., 1994; Nadelman and Wormser, 1995; Nadelman et al., 1996; Jurca et al., 1998; Smith et al., 2002). It is incorrectly assumed by many practitioners and patients that EM usually has central clearing. This feature occurred in only 37 and 9% of cases, respectively, in two large studies conducted in the northeastern USA, involving nearly 200 patients with culture-confirmed EM (Nadelman et al., 1996; Smith et al., 2002). The reason for the discrepancy is related to early descriptions of Lyme disease from Europe and from the USA in the days before effective antibiotic treatment was recognized. As central clearing appears to be a function of the duration of EM (Ǻsbrink and Olsson, 1985; Berger, 1989; Strle et al., 1999) an annular appearance was emphasized in early descriptions of the longstanding rashes (i.e. erythema chronicum migrans (ECM)) that were seen in untreated

Table 10.1. Selected characteristics of 119 patients with cultureconfirmed erythema migrans seen in Westchester County, NY (Strle et al., 1999). Characteristic

Number (%)

Recall of prior tick bite at site Median duration of rash at presentation (days) Median size of primary lesion (cm) Multiple lesions Location of primary lesion

30 (25) 4 (range 1–39) 14 (range 5–73) 16 (13)

Trunk and abdomena Leg Arm and shoulder Head and neck Central clearing of primary lesion

60 (50) 40 (34) 17 (14) 2 (2) 36 (35)b

aIncludes bRash

axilla, flank and groin. characteristics were described for only 102 of the 119 patients.

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patients. In addition, European cases, which comprised many of the first reports of Lyme disease, are most often associated with B. afzelii, which is responsible for a somewhat different clinical course and appearance of EM than disease related to B. burgdorferi sensu stricto in North America (Ǻsbrink and Olsson, 1985; Berger et al., 1992; Kuiper et al., 1994; Strle et al., 1999; Lipsker et al., 2002; Logar et al., 2004). In one early Swedish study, 80% of cases had central clearing, associated with EM of 5–6 weeks’ duration (Ǻsbrink and Olsson, 1985). This contrasts with US experience over the past two decades, where EM has been diagnosed and treated within 1–2 weeks of onset and usually lacks central or paracentral clearing at the time of presentation (Nadelman and Wormser, 1995, 2002; Nadelman et al., 1996; Smith et al., 2002). However, it is likely that additional factors besides rash duration influence whether central clearing occurs. In one study, Slovenian patients infected with B. garinii were nearly twice as likely as US patients infected with B. burgdorferi to have EM with central clearing, despite similar durations of the rash (Strle et al., unpublished data). Furthermore, there was no difference in duration of EM associated with B. garinii in the European patients who had central clearing compared with those who did not (Strle et al., 2011). EM lesions are usually oval or circular, with the shape partially influenced by the pre-existing lines of skin tension (Berger, 1989; Malane et al., 1991; Melski et al., 1993; Nadelman and Wormser, 1995). For example, groin lesions are generally oval along the horizontal axis (Malane et al., 1991; Melski et al., 1993; Nadelman and Wormser, 1995). Unusual configurations such as triangles may occur (Berger, 1989; Malane et al., 1991) (see Plate 5 in the colour plate section). EM margins are usually regular and are not raised compared with the interior. Central vesicles were present in 8% of lesions in one study (see Plate 6 in the colour plate section) (Goldberg et al., 1992), which may lead to confusion with spider bites, contact dermatitis, or even herpes simplex or varicellazoster virus infection. Scaling is uncommon in EM lesions,

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occurring primarily at the tick-bite site (punctum), in fading rashes of long duration or after antimicrobial treatment (Nadelman and Wormser, 1995). Use of topical steroids may also lead to scaling, in addition to conferring an uncharacteristic pallor (Nadelman and Wormser, 1995). EM lesions display a degree of erythema from faint pink to dark red. Lesions on the lower extremities may acquire a bluish tint (Berger, 1989; Malane et al., 1991). Lesions are warmer than the surrounding normal-appearing skin. Pruritus or pain may be present at the site of EM but is almost always mild in severity (Ǻsbrink and Olsson, 1985; Malane et al., 1991; Nadelman et al., 1996; Strle et al., 1996a; Logar et al., 2004). A minority of patients, more often in Europe, experience transient numbness or tingling at the site of EM (Steere et al., 1983a; Ǻsbrink and Olsson, 1985; Malane et al., 1991; Kuiper et al., 1994; Nadelman et al., 1996; Smith et al., 2002; Logar et al., 2004). In some patients, secondary lesions may arise as the result of spirochaetaemia (see Multiple erythema migrans and spirochaetaemia below.) 10.2.3 Associated systemic symptoms As many as 80% of patients with EM in the USA have related systemic complaints that may precede, accompany or follow the resolution of EM (Steere et al., 1983a; Berger, 1989; Nadelman and Wormser, 1995). The most common systemic complaints in more than 600 US patients enrolled in four large prospective studies were malaise (10–80%), headache (28–64%), fever and chills (31–59%) and myalgias and arthralgias (35–48%), with nausea, anorexia, dizziness and difficulty concentrating reported less frequently (Steere et al., 1983a; Berger, 1989, Nadelman et al., 1996; Smith et al., 2002). Neither diarrhoea nor respiratory symptoms are characteristic of Lyme disease and, if present, should raise the possibility of a different diagnosis or an additional coexisting process that is unrelated. In European patients with EM, systemic symptoms are less frequent than in US patients, reported in 23–50% of more than 800 patients in representative prospective studies

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from five different European countries (Ǻsbrink and Olsson, 1985; Weber et al., 1988, 1990; Kuiper et al., 1994; Strle et al., 1996a, 1999; Lipsker et al., 2002, Logar et al., 2004). The disparity between US and European disease is probably largely attributable to the lower virulence of European genospecies compared with B. burgdorferi sensu stricto, the only genospecies that has been implicated as causing human disease in the USA (Nadelman and Wormser, 1998; Strle et al., 1999, 2011; Antoni-Bach et al., 2002; Logar et al., 2004; Cerar et al., 2010). B. afzelii (the major cause of EM in Europe) appears to be less virulent than B. garinii, a European genospecies that also appears to be the most neurotropic (Logar et al., 2004). Patients with EM caused by B. garinii tended to have more frequent myalgia and chills, more often had local symptoms and abnormal liver function tests, were more frequently seropositive and had a shorter incubation period and faster evolution of EM when compared with patients with EM associated with B. afzelii (Logar et al., 2004). EM caused by either of these species in Slovenian patients was associated with significantly less systemic symptomatology than occurs in US patients infected with B. burgdorferi sensu stricto (Strle et al., 1999, 2011). These differences may be partially attributable to a greater ability of B. burgdorferi sensu stricto to stimulate macrophages to secrete higher levels of chemokines and cytokines and to activate both innate and adaptive immune responses compared with European genospecies (Strle et al., 2009).

10.2.4 Associated findings on physical examination The most common objective physical findings at the time of diagnosis of EM in US patients are regional lymphadenopathy (23–41%), fever (14–31%) and pain on neck flexion (5–20%) (Steere et al., 1983a; Nadelman et al., 1996; Smith et al., 2002; Nowakowski et al., 2003). Between 1 and 6% of patients have concomitant cranial nerve palsies (usually the facial nerve) (Steere et al., 1983a; Nadelman et al., 1996; Smith et al., 2002; Nowakowski et al.,

2003). Abnormal physical findings were much more common in patients with EM from New York State infected with B. burgdorferi sensu stricto than in patients from Slovenia with either B. afzelii or B. garinii infection (Strle et al., 1999, 2011). Regional lymphadenopathy, found in 7.2 % of 316 patients from two prospective studies, was the most common finding in Slovenian patients (Strle et al., 1996a, 1999).

10.2.5 Multiple erythema migrans and spirochaetaemia Half of 314 patients with EM in an observational study conducted in Connecticut from 1976 to 1982 developed multiple annular secondary lesions (Steere et al., 1983a), with 13% of patients having more than 20 lesions, including two patients with more than 100 (see Plate 7 in colour plate section). Secondary lesions are similar in morphology to the initial (i.e. primary) lesion with which most patients present but are typically smaller (usually 2–3 cm) (Steere et al., 1983a; Malane et al., 1991; Melski et al., 1993). Like primary lesions, they are not present on mucous membranes, palms or soles. As secondary lesions are a direct consequence of spirochaetaemia rather than a tick bite, they lack a punctum, vesiculation, local pruritus and tenderness. Viable spirochaetes may be recovered from a biopsy of the lesion or from blood using special culture media (Melski et al., 1993; Wormser et al., 2005a). Secondary lesions may be fleeting, and may emerge and vanish suddenly during examination (Steere et al., 1983a). Such evanescent lesions were described in one series as a separate entity, appearing for several weeks in untreated patients after resolution of primary and secondary lesions (Steere et al., 1983a). Up to half of US patients with EM have detectable spirochaetaemia at the time of presentation when blood is cultured with high-volume (9mL) samples (Wormser et al., 2005a). The duration of spirochaetaemia is unknown, but in one study blood cultures were positive in one-third to one-half of untreated patients seen at presentation at various time intervals ranging from 1 to

Erythema Migrans

37 days after the appearance of EM (Wormser et al., 2005a). Systemic symptoms are more frequent in patients with spirochaetaemia than in those with negative blood cultures, and spirochaetaemic patients also have more symptoms as well as a higher cumulative symptom severity score (Wormser et al., 2005a). Chills (but not fever), headaches, stiff neck, multiple EM lesions (40%) and regional lymphadenopathy are significantly more likely to be present in this group (Wormser et al., 2005a). However, no single characteristic or combination of variables had enough specificity and sensitivity (80%) to predict spirochaetaemia (Wormser et al., 2005a).

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contribute to these symptoms. Preliminary results of a study of patients with EM indicated that patients infected with RST 1 strains had more symptoms and greater cytokine levels including gamma interferon (IFN-γ) and the IFN-γ-inducible chemokines CXCL9 and CXCL10 (Strle et al., 2010). In addition, in this report, peripheral blood mononuclear cells from healthy humans secreted significantly higher levels of IFN-α, IFN-γ and CXCL10 when stimulated with RST 1 isolates compared with RST 2 or RST 3 strains (Strle et al., 2010).

10.2.7 Differential diagnosis 10.2.6 Influence of strain differences on clinical manifestations Haematogenous dissemination of B. burgdorferi from the initial site of the primary EM lesion is believed to be responsible for the occurrence of multiple EM lesions and the objective extracutaneous manifestations of Lyme disease (e.g. facial nerve palsy, meningitis, carditis and arthritis). B. burgdorferi sensu stricto can be classified into subtypes, using restriction fragment length polymorphisms to determine the 16S–23S ribosome intergenic spacer type (RST) of B. burgdorferi (Liveris et al., 1999), or based on genotyping of the outer-surface protein C (ospC) gene (Seinost et al., 1999; Grimm, et al., 2004). Certain subtypes of B. burgdorferi have been linked to disseminated disease, while others appear less likely to circulate in the blood (Liveris et al., 1999; Seinost et al., 1999; Wormser et al., 2005a) perhaps accounting for the observation in one study that 20% of 55 untreated patients with EM remained symptom free after a median of 6 years (range 3–8 years) (Steere et al., 1987). In general, patients infected with RST 1, RST 2 and OspC types A, B, I or K are more likely to have multiple EM lesions and spirochaetaemia (Liveris et al., 1999; Seinost et al., 1999). However, some patients with solitary EM lesions and less-invasive subtypes may have significant systemic complaints, implying that other factors (e.g. host factors; (Wormser et al., 2005d) or cytokine production) may

The most important but too often ignored key to recognizing EM is to perform an examination of the entire body with all clothes removed in order to evaluate areas poorly visualized by the patient. It is not uncommon to identify EM previously unrecognized by a patient with an otherwise non-specific acute illness. The diagnosis of EM should be considered especially in patients from endemic areas who present with new unexplained complaints of headache, myalgia, arthralgia and fever during the late spring and summer, even if a rash is not reported initially (Nadelman and Wormser, 1995; Nadelman et al., 1997). The diagnosis of EM should also be considered in patients with unexplained atrioventricular heart block, as carditis due to B. burgdorferi has been reported in 2–9% of untreated patients with EM (the higher incidence was observed in early studies predating recognition of effective antimicrobial therapy for Lyme disease) (Steere et al., 1983a; Rubin et al., 1992; Haddad and Nadelman, 2003). One report investigated the diagnostic value of clinical history and physical examination in the assessment of rashes consistent with EM (Tibbles and Edlow, 2007). The authors reviewed more than 50 European and US studies that enrolled more than 8000 patients, but were unable to identify a single element in the history or physical examination that was alone highly sensitive for the diagnosis of EM. In view of the wide variability in the clinical

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presentation, the authors cited the need for an algorithm combining specific signs or symptoms to improve diagnostic sensitivity. EM as a manifestation of Lyme disease occurs only in areas where vector ticks (I. scapularis or Ixodes pacificus in the USA, Ixodes ricinus in Europe and Ixodes persulcatus in Eurasia) are infected with B. burgdorferi sensu lato. In other parts of the world where infected vectors are not present (Sharma et al., 2010) or infrequently bite humans (Felz et al., 1996), rashes that are target-like or have other features resembling EM are not likely to be associated with Lyme disease (i.e. B. burgdorferi infection). Instead, other entities should be considered. Perhaps the most important alternative diagnosis to consider is a hypersensitivity reaction to the bite of an arthropod. An erythematous lesion surrounding a bite site while a tick is still attached, or within 48 h of detachment, is most likely a hypersensitivity reaction to the tick bite, and is unassociated with infection (Feder and Whitaker, 1995; Nadelman and Wormser, 1995; Wormser,

2006; Wormser et al., 2006) (Table 10.2). Such a lesion may be associated with significant pruritus (atypical for EM), and generally fades spontaneously within 24–48 h. In contrast, an EM lesion typically increases progressively in size over this time frame. Although local bite reactions are usually less than 5 cm in the largest diameter, they may expand (usually over hours rather than days, in contrast to EM) to a much larger size before spontaneously fading. More than half of patients with EM seen in the USA also have accompanying systemic symptoms, unlike those with local tick-bite hypersensitivity reactions. In cases of uncertainty in the diagnosis, it may be helpful for the healthcare practitioner to mark the contours of the rash with ink and observe over 1–2 days without treatment. If the rash expands or systemic symptoms develop, antimicrobial treatment should be initiated, whereas if the rash resolves within 48 h, no treatment is necessary (Nadelman and Wormser, 1995; Wormser et al., 2006) (see Plate 8 in the colour plate section).

Table 10.2. Differentiating erythema migrans from hypersensitivity reaction to an arthropod bite. (Adapted from Nadelman and Wormser, 1995; reprinted by permission of Elsevier, Infectious Disease Clinics of North America.) Characteristic

Erythema migrans

Arthropod-bite hypersensitivity reaction

Recall of bite at site ~20% Tick present at time of rash No

Variable Yes (or detached within prior 24 h); also may occur after other arthropod (e.g. mosquito) bites

Time interval between bite and rash Location

Hours

Local symptoms Evolution Resolution Size Systemic symptoms Fever a

Median 7–10 days (range 1–36 days)a Intertriginous areas, border of tightfitting clothing Rare; minimal if present Expands over days to weeks Days to weeks (median 4 weeks if untreated)b 5 cm (can be smaller) Up to 80% 16% documented, 39% subjectivec

Same; can also occur on exposed areas such as face or forearm Pruritus Expands over hours Less than 48 h 5 cm (can be larger) Absent Absent

Steere et al. (1983b); Berger (1989); Nadelman and Wormser (1995); Nadelman, et al. (1996). Steere et al. (1983b). c Nadelman et al. (1996). b

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Although arthropod bites unassociated with EM occur during the late spring and summer when EM is most prevalent, other processes do not have a seasonal variation. Staphylococcal and streptococcal cellulitis tend to develop rapidly, evolving over hours with a band-like rather than oval or circular shape, and are usually painful. They are commonly associated with high fever, leukocytosis and often a toxic-appearing patient, all which are very rare or uncommon with EM. Cellulitis caused by pyogenic organisms usually occurs on the distal lower extremities, sometimes after trauma, and often in a person with underlying vascular disease (e.g. venous stasis) or with a history of prior surgery that adversely affected venous or lymphatic flow (e.g. saphenous vein harvesting for coronary artery bypass surgery or mastectomy) (Nadelman and Wormser, 1995). Conversely, the location of an erythematous rash at locations unusual for bacterial cellulitis (e.g. buttocks, groin, axilla, popliteal fossa) should significantly raise the suspicion for EM. Patients with EM lesions having vesicular centres often present with the complaint of an unwitnessed ‘spider bite’ (Plate 6 in the colour plate section). This scenario should raise the suspicion for atypical EM in most areas of the USA endemic for Lyme disease, as there is little overlap with the geographical range of the brown recluse spider (which extends from southeastern Nebraska to southern Ohio) (Vetter and Bush, 2002; Frithsen et al., 2007). EM lesions with vesicular centres may also be confused with herpes simplex or varicella-zoster virus infections, but, unlike the latter viral exanthems, lack a dermatomal distribution. Although vesicular EM lesions may be somewhat more tender than those without vesiculation, pain is very prominent in herpetic lesions. Tinea infection may resemble EM with an erythematous border and central clearing. However, tinea rashes evolve much more slowly than EM (weeks to months rather than days to weeks) and systemic symptoms are absent. Scaling and thin irregular raised borders should suggest tinea. Characteristics of some skin disorders that may be confused with EM are summarized in Table 10.3.

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10.2.8 Southern tick-associated rash infection A rash resembling EM that occurs in many patients residing in the southern USA must be distinguished from EM caused by B. burgdorferi (Kirkland et al., 1997; Masters et al., 1998; Wormser et al., 2005b,c). Similarities with EM include rash appearance (including occasional multiple lesions), peak incidence in summer and similar incubation period after a tick bite. However, in contrast to patients with Lyme disease, efforts to culture B. burgdorferi in Barbour–Stoenner–Kelly (BSK) medium from biopsied skin lesions from patients with EM-like lesions in the southern USA have been consistently unsuccessful (in contrast to EM associated with Lyme disease where biopsy cultures have been positive in 50–86% of US patients; Berger et al., 1992; Nowakowski et al., 2001). Acute and convalescent-phase serological assays are almost always negative for antibodies to B. burgdorferi in patients with EM-like rashes in the southern USA (Wormser et al., 2005b,c). In addition I. scapularis ticks, the usual vector for Lyme disease, are rarely infected with B. burgdorferi in the southern USA (0.5%) and infrequently bite humans (Felz et al., 1996). Moreover, the tick vector for this rash in patients in the south appears to be Amblyomma americanum, which is not believed to be a competent vector for B. burgdorferi (Piesman and Sinsky, 1988). Therefore, it has been concluded that this rash does not represent Lyme disease; instead it is known as southern tick-associated rash illness (STARI), or Masters’ disease (after a key investigator) (Masters et al., 1998, Wormser et al., 2005b,c). Although a new Borrelia genospecies, Borrelia lonestarii, was postulated to be the pathogen (Barbour et al., 1996), a subsequent study of 19 patients with STARI failed to detect this organism (Wormser et al., 2005b), and the aetiology remains unknown. Patients with STARI have clinical characteristics somewhat distinct from EM patients with B. burgdorferi infection (Wormser et al., 2005c). In a prospective clinical evaluation of patients from Missouri with STARI and patients from New York with EM, Missouri

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Table 10.3. Differential diagnosis of erythema migrans (EM). (Adapted from Feder and Whitaker, 1995; Tibbles and Edlow, 2007; reprinted by permission of Elsevier, Infectious Disease Clinics of North America, and the Journal of the American Medical Association.)a Appearance

Tinea (ringworm)

Ring shape, with Variable; exposed satellite lesions; skin scaling at periphery Homogenous erythema; Distal extremities; band-like appearance; site of prior warm and tender, trauma lymphangitic streaking; tender regional lymphadenopathy Shape related to Variable contact; vesicles and bullae may be present

Bacterial cellulitis

Contact dermatitis

Body site

Size

Progression

Seasonal tendency

Miscellaneous

1–10 cm

Days to weeks

No

Pruritus; pet exposure

Rarely large except on lower extremities

More rapid than EM (hours to days)

No

Pain, fever, leukocytosis; history of prior trauma, vascular disease or surgery

Variable

Variable (often slow progression)

No

Variable

Waxes and wanes No over hours Fixed in size No

Pruritus often severe; history of contact with inciting substance (e.g. poison ivy) Pruritus

Urticaria

Raised, multiple lesions

Variable

Fixed drug eruption

Deep, well-demarcated violaceous plaque Necrotic; red, white and blue sign

Fixed; often Variable involves genitals Extremities Variable

Vesicles on erythematous base

Dermatomal distri- Variable bution

Brown recluse spider bite Herpes simplex/ varicella-zoster virus aSee

Spreads centrifugally May progress rapidly (days)

Table 10.2 for distinguishing erythema migrans from a hypersensitivity reaction to an arthropod bite.

Yes (mates May– September) No

Burning May be painful; uncommon in northeastern USA Prodrome may occur; pain (sometimes severe); pruritus, fever

R.B. Nadelman

Diagnosis

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patients were significantly more likely to recall a tick bite and had a shorter time to onset of rash than New York patients. EMlike lesions in Missouri patients were more circular and smaller in size but more likely to have central clearing. However, New York patients were more likely to be symptomatic and were more likely to have multiple skin lesions. After antibiotic treatment, Missouri patients recovered more rapidly than New York patients (Wormser et al., 2005c).

10.2.9 Coinfection Patients with EM may also be coinfected with other tick-borne pathogens, as I. scapularis, the vector tick for B. burgdorferi, may transmit the protozoan Babesia microti causing babesiosis, a malaria-like infection (Krause et al., 1996; Steere et al., 2003; Wormser et al., 2006) and the bacterium Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis (HGA; formerly known as human granulocytic ehrlichiosis (HGE)) (Nadelman et al.; 1997; Belongia et al., 1999; Steere et al., 2003; Wormser et al., 2006). In Eurasia, I. ricinus and I. persulcatus, the vector ticks for B. burgdorferi sensu lato, may also transmit the flavivirus causing tick-borne encephalitis (Mansfield et al., 2009). As B. burgdorferi infection does not cause cytopenias, the occurrence of leukopenia, thrombocytopenia or anaemia in a patient with Lyme disease should suggest coinfection (Nadelman et al., 1997, 1999; Belongia et al., 1999). Abnormal transaminases and other liver enzymes may be present in patients with Lyme disease, but are particularly common in patients with HGA (Steere et al., 1983a; Nadelman et al., 1996). Coinfection should be strongly considered in a patient without a rapid improvement (48 h) after receiving either amoxicillin or cefuroxime axetil (which have no activity against HGA, unlike doxycycline), particularly if fever persists (Wormser, 2006; Wormser et al., 2006). A toxic appearance or an illness requiring intensive care in a patient with EM should initiate a prompt assessment for babesiosis (especially in an immunocompromised or asplenic patient (Krause et al., 2008)) or HGA

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(Bakken and Dumler, 2006), as these illnesses may be fatal. 10.2.10 Laboratory diagnosis The diagnosis of EM is made on clinical grounds based on the characteristic appearance of the skin lesion in a patient with the appropriate epidemiological and exposure history. Routine laboratory tests such as complete blood counts and liver enzyme assays may, if abnormal, point to coinfection with A. phagocytophilum or B. microti (see above), but are usually unremarkable, as is sedimentation rate. However, support for the clinical diagnosis can be made through specific laboratory testing, with isolation of B. burgdorferi in culture from skin and/or blood being the gold standard for accurate identification. Although the sensitivity of blood and skin biopsy cultures in EM (as high as 50% (Wormser et al., 2000) and 86% (Berger et al., 1992)), is actually greater than that for cellulitis caused by pyogenic bacteria (Sigurdsson and Gudmundsson, 1989), these techniques are of limited value in clinical practice. This is because of the invasive (although minimally so) biopsy procedure and special isolation media required, the delay in detecting growth of B. burgdorferi until an average of approximately 2 weeks, the added cost and, most importantly, the straightforward clinical diagnosis of EM in endemic areas, rendering laboratory testing superfluous in most cases. However, laboratory tests may help validate the diagnosis when a rash is atypical or the exposure history uncertain, especially in an investigational setting (i.e. treatment trials or epidemiological studies). Besides culture, the diagnosis of infection with B. burgdorferi may be supported by serology (acute and convalescent phase) and PCR (nested and quantitative RT-PCR from skin). These tests were compared in 47 patients with EM (Table 10.4; Nowakowski et al., 2001). The most practical laboratory method available to the clinician is serological testing for antibodies to B. burgdorferi using a twotier system (usually polyvalent ELISA

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Table 10.4. Comparison of diagnostic tests for 47 adult patients with erythema migrans (Nowakowski et al., 2001; reprinted by permission of the University of Chicago Press, Clinical Infectious Diseases). Diagnostic method

Number of positive results (%)

Skin culture Blood culture (18 ml) Any culture Nested PCR Quantitative PCR Any PCR Acute phase serology Convalescent-phase serology Any serology Any test positive All tests negative

24 (51.1) 21 (44.7) 31 (66.0) 30 (63.8) 38 (80.9) 38 (80.9) 19 (40.4) 31 (66.0) 32 (68.1) 44 (93.6) 3 (6.4)

followed by IgM and IgG immunoblots if the first step test is positive or equivocal; CDC, 1995; Wormser et al., 2006; Aguero-Rosenfeld et al., 2005). However, serology lacks sensitivity in early Lyme disease; half of patients with EM have negative results on initial antibody testing (Aguero-Rosenfeld et al., 1993, 2005; Nowakowski et al., 2001). The probability of seroreactivity increases significantly with increased duration of EM (Aguero-Rosenfeld et al., 1993, 2005). In one report, all 14 patients presenting with an EM duration of 2 weeks had a positive ELISA and IgM immunoblot at study entry (AgueroRosenfeld et al., 1993). A further increase in the sensitivity of serological testing can be accomplished by including convalescentphase testing (Aguero-Rosenfeld et al., 2005; Wormser et al., 2006). Recently, the use of a C6 ELISA, based on a peptide (C6) with the amino acid sequence of a conserved, immunodominant region of the VlsE protein of Borrelia burgdorferi, has been proposed to replace immunoblotting in two-tiered testing with little loss of sensitivity or specificity, especially in patients with EM (Branda et al., 2010). In summary, the routine use of laboratory testing is not presently recommended for patients with EM because the clinical identification of EM is usually clear-cut, and serology often yields false-negative results

(Wormser et al., 2006). Diagnostic testing in patients with EM should generally be reserved for problematic cases (e.g. difficulty in distinguishing between EM and a hypersensitivity reaction to an arthropod bite, or an EM-like rash in a non-endemic region), or for those in clinical trials or epidemiological studies for whom a definitive diagnosis is essential. Use of the laboratory in the diagnosis of Lyme disease is discussed in more detail in Chapter 4 of this volume.

10.3 Treatment 10.3.1 Long-term outcome of untreated patients Untreated EM resolves spontaneously, within a median of 4 weeks (Steere et al., 1983a, 1987). Prior to the recognition that antimicrobial treatment was effective in both hastening resolution of EM and associated symptoms and preventing extracutaneous complications, a group of 55 patients was followed prospectively for a median duration of 6 years without receiving antimicrobial therapy (Steere et al., 1987). All EM lesions resolved spontaneously, but after 1–14 months, 9% had experienced recurrent EM at the site of the primary lesion, 5% had recurrence of secondary lesions and 7% had recurrence of both. Evanescent lesions returned in 5% including two children whose frequent episodes occurred over more than 3 years. In 12 patients, other manifestations of Lyme disease accompanied the recurrent skin lesions (Steere et al., 1983a). Eighty per cent of those enrolled had joint symptoms ranging from arthralgias to intermittent episodes of arthritis, to chronic synovitis. Of these patients, 11% also developed neurological abnormalities and 4% had cardiac involvement. Fifty-one per cent of patients experienced intermittent attacks of monoarticular or oligoarticular arthritis of large joints (almost always involving the knee), beginning months after the initial infection (Steere et al., 1987). Many patients experienced repeated attacks of arthritis for years, but the number of recurrences decreased each year

Erythema Migrans

by 10–20% (Steere et al., 1987). The severity of symptoms at the onset of illness predicted the development of late disease (arthritis) (Steere et al., 1987). However, over a median of 6 years follow-up (range 3–8 years), 20% of the 55 patients originally enrolled with EM had no subsequent manifestations of Lyme disease. 10.3.2 Treatment trials The first randomized prospective trial in the USA to study treatment for EM compared 10day courses of tetracycline, penicillin or erythromycin in 112 patients (Steere et al., 1983b). EM and associated symptoms improved more rapidly in patients receiving penicillin or tetracycline compared with those receiving erythromycin. An intensification of fever, rash or pain in the first 24 h after initiation of antimicrobial therapy, experienced by 15%, was considered to constitute a Jarisch–Hexheimer-like reaction. Patients treated with tetracycline or penicillin were less likely than those receiving erythromycin to develop objective extracutaneous complications such as meningitis, carditis and arthritis (Steere et al., 1983b). No additional benefit was experienced by those who completed 20 as opposed to 10 days of tetracycline treatment (Steere et al., 1983b). In two subsequent smaller studies, rapid resolution of EM and associated symptoms and a satisfactory outcome at 6 months was observed in nearly all patients who were randomized to receive either doxycycline or amoxicillin (to which probenecid was added to increase drug levels) (Dattwyler et al., 1990; Massarotti et al., 1992). Patients receiving azithromycin in a third treatment arm in one of these studies had a similar favourable outcome (Massarotti et al., 1992). Oral cefuroxime axetil 500 mg twice daily and doxycycline 100 mg three times daily (rather than the usual twice daily dose) were compared in two randomized multicentre investigator-blinded prospective controlled studies including 364 patients (from New York, New Jersey and Connecticut) with EM (Nadelman et al., 1992; Luger et al., 1995). A satisfactory clinical outcome (defined

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as resolution of EM by day 5 post-treatment with resolution or improvement of associated signs and symptoms at 1 month posttreatment) was observed in 93 and 90% of the cefuroxime axetil groups and in 88 and 95% of the doxycycline groups in the two respective studies (Nadelman et al., 1992; Luger et al., 1995). Of those who were evaluable 1 year after treatment, satisfactory outcomes were observed in 90 and 95% of patients receiving cefuroxime axetil and in 92 and 100% of those treated with doxycycline in the two respective studies (Nadelman et al., 1992; Luger et al., 1995). Patients with unsatisfactory outcomes principally had subjective symptoms including musculoskeletal complaints, headache, paresthesias, malaise and fatigue; several patients developed objective arthritis, although this was in some cases considered by the investigators to be unrelated to B. burgdorferi infection (Nadelman, et al., 1992; Luger, et al., 1995). Patients receiving cefuroxime axetil more often experienced diarrhoea, while those treated with doxycycline were significantly more likely to experience photosensitivity reactions. Most adverse effects were mild and did not result in patients stopping treatment (Nadelman et al., 1992; Luger et al., 1995). In summary, cefuroxime axetil and doxycycline were equally well tolerated, and equally effective in treatment of early Lyme disease and prevention of extracutaneous disease at 1 year of follow-up (Nadelman et al., 1992; Luger et al., 1995). An additional prospective (but unblinded) controlled multicentre clinical trial evaluated the efficacy of oral doxycycline versus parenteral ceftriaxone for treatment of adult and paediatric patients with EM and disseminated Lyme disease (defined as two or more EM lesions, carditis manifested by heart block, neurological abnormalities (seventh-cranial-nerve palsy or radiculitis of less than 3 months’ duration) and acute largejoint arthritis; Dattwyler et al., 1997). Patients with meningitis were excluded from the study. Of 140 patients with EM and disseminated disease, 133 (95%) had multiple EM lesions at enrolment, nine (6%) had carditis, ten (7%) had facial nerve palsy and nine (6%) had joint swelling. Adult patients

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received either 21 days of oral doxycycline (100 mg twice daily) or 14 days of parenteral ceftriaxone (2 g daily, intravenously or intramuscularly, at the discretion of the treating physician). Doses for children were adjusted for weight. Resolution of symptoms and prevention of complications were comparable in the two treatment groups (85 and 88% cured in the ceftriaxone and doxycycline groups, respectively, with most of the remainder unevaluable due to inadequate follow-up or withdrawal from the study; Dattwyler et al., 1997). Only two patients were felt to have failed treatment, one with facial nerve palsy that persisted despite an additional 5-week course of ceftriaxone, and another who developed arthritis that ultimately resolved after treatment with a 3-week course of ceftriaxone. Azithromycin (500 mg daily for 7 days) was compared with amoxicillin (500 mg three times daily for 20 days) in a multicentre prospective controlled randomized trial enrolling 246 patients (from two areas of New York, Connecticut, Missouri, Wisconsin, New Jersey, Minnesota, California and Rhode Island; Luft et al., 1996; it is probable that the patients from Missouri, a non-endemic area for Lyme disease, had STARI rather than EM). Amoxicillin was significantly more effective than azithromycin in bringing about the resolution of EM and accompanying symptoms, and in preventing objective evidence of relapse at 6 months (Luft et al., 1996). It is unclear whether the worse outcomes associated with azithromycin were related to the relatively short duration of treatment, to low achievable levels in blood or other body compartments, or to other factors. Azithromycin appeared to be more effective in European studies of early Lyme disease. Azithromycin showed comparable efficacy to phenoxymethylpenicillin and to doxycycline, possibly resulting in more rapid resolution of symptoms in prospective randomized trials from Slovenia and Germany (Strle et al., 1992, 1996b; Weber et al., 1993). A Scandinavian clinical trial of patients with uncomplicated EM compared phenoxymethylpenicillin to roxithromycin, a semisynthetic macrolide with promising in vitro activity against B. burgdorferi (Hansen et al., 1992). The study had to be terminated

prematurely because of treatment failure in five out of 19 patients receiving roxithromycin, including one patient who developed neuroborreliosis and two patients whose persistent EM was confirmed through isolation in culture of B. burgdorferi sensu lato. This compared with no failures among ten patients randomized to receive phenoxymethylpenicillin (Hansen et al., 1992). Ten-day courses of tetracyclines have been shown to have comparable efficacy to longer courses (Steere et al., 1983b; Nowakowski et al., 1995; Wormser et al., 2003; Kowalski et al., 2010). A prospective randomized double-blind controlled trial in Westchester County, New York, compared 10 days of oral doxycycline twice daily, with or without a single 2 g intravenous dose of ceftriaxone, with 20 days of oral doyxycycline twice daily (Wormser et al., 2003). The rate of complete response was similar for the three treatment groups at all assessment times over 30 months. Regardless of the regimen, objective evidence of treatment failure was extremely rare (Wormser et al., 2003). It was concluded that extending the course of doxycycline from 10 to 20 days, or adding one dose of intravenous ceftriaxone at the beginning of a 10-day course of doxycycline did not enhance therapeutic efficacy in patients with EM (Wormser et al., 2003). A recent retrospective study of 607 adult patients with early Lyme disease from Wisconsin evaluated outcomes a mean of 2.9 years after initiation of treatment with either 10 days, 11–15 days or 16 days of antimicrobials (93% doxcycline, 4% amoxicillin and the remainder with other or unknown medication; Kowalski et al., 2010). Two-thirds of patients (404/607) had EM including 275 (45%) with single and 129 (21%) with multiple EM lesions. A small percentage (4%) of patients with EM received retreatment for ‘possible treatment failure’ related to subjective symptoms and/or positive serological tests. Only four patients (1%) with EM were considered to have had objective treatment failure. One of these patients developed facial nerve palsy on day 12 of doxycycline treatment. In two others who developed facial nerve palsy 1 and 3 years after treatment, reinfection could not be ruled out. The last patient developed facial nerve

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palsy and lymphocytic meningitis after being treated with cefadroxil, a first-generation cephalosporin without significant activity against B. burgdorferi (Agger et al., 1992). His illness promptly responded to doxycycline. In summary, the overall outcome, regardless of treatment duration, was excellent (Kowalski et al., 2010). 10.3.3 Treatment recommendations A low incidence of serious adverse effects has been observed in treatment trials for early Lyme disease. Doxycycline has the advantage of twice-daily dosing, and efficacy against A. phagocytophilum with which patients may be coinfected. However, doxycycline may cause photosensitivity, a serious concern, as EM usually occurs in late spring or summer. Patients receiving doxycycline should accordingly be counselled regarding avoiding strong sunlight and using sun block. In addition, as doxycycline has been associated with oesophagitis, patients should be advised

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to drink a full 8 oz of fluid with this medication, and should avoid a recumbent position for 1 h afterwards. Doxycycline is relatively contraindicated in children 8 years old and in pregnant or breastfeeding women. Amoxicillin and cefuroxime axetil have been associated with rash, diarrhoea and other adverse effects. Ceftriaxone has no advantage over oral agents in the treatment of EM and should be reserved for patients with EM associated with meningitis and advanced heart block (Wormser et al., 2006). Macrolides such as azithromycin should be reserved for patients who cannot tolerate other more effective agents (Wormser et al., 2006). First-generation cephalosporins (e.g. cephalexin and cefadroxil), fluoroquinolones, metronidazole and sulfonamides have no appreciable activity against B. burgdorferi and should not be used to treat patients with Lyme disease (Nowakowski et al., 2000; Wormser et al., 2006). Guidelines from the Infectious Diseases Society of America (IDSA) for the treatment of EM are summarized in Table 10.5.

Table 10.5. Infectious Diseases Society of America recommendations for treatment of patients with erythema migrans (Wormser et al., 2006; reprinted by permission of the University of Chicago Press, Clinical Infectious Diseases).a Drug

Dosage for adults

Dosage for children

Amoxicillin

500 mg three times per day

Doxycycline

100 mg twice per day. Relatively contraindicated in pregnant or lactating women

Cefuroxime axetil

500 mg twice per day

50 mg/kg/day in three divided doses (maximum 500 mg per dose) Not recommended for children <8 years. For children 8 years, 4 mg/kg/day, in two divided doses (maximum 100 mg per dose) 30 mg/kg/day in two divided doses (maximum 500 mg per dose)

Preferredb

Alternativec Azithromycin Clarithromycin

Erythromycin

500 mg per day for 7–10 days 500 mg twice per day for 14–21 days. Relatively contraindicated in pregnant women 500 mg four times per day for 14–21 days

10 mg/kg/day (maximum 500 mg per day) 7.5 mg/kg twice per day (maximum 500 mg per dose)

12.5 mg/kg four times per day (maximum 500 mg per dose)

aIn patients suspected of having coinfection with human granulocytic anaplasmosis (HGA), doxycycline is preferred if not contraindicated. bRecommended duration is 14 days (10–21 for doxycycline or 14–21 days for amoxicillin and cefuroxime axetil). cBecause of their lower efficacy, macrolides are reserved for patients who are unable to take or who are intolerant of tetracyclines, penicillins and cephalosporins; patients treated with macrolides should be observed closely to ensure resolution of clinical symptoms.

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10.3.4 Treatment of children Children 8 years of age should not take doxycycline because tetracyclines may stain developing teeth (Lochary et al., 1998). Two alternative antimicrobial agents were compared in a prospective randomized unblinded study of 43 children aged 6 months to 12 years who received one of two different dosing schedules of cefuroxime axetil (20 mg/kg/day or 30 mg/kg/day) or amoxicillin (50 mg/kg/ day) (Eppes and Childs, 2002). EM and associated symptoms resolved in all patients with no long-term problems attributable to Lyme disease, and minimal adverse effects were observed (Eppes and Childs, 2002). Both amoxicillin and cefuroxime axetil have been recommended as the preferred regimen for paediatric patients 8 years old (Wormser et al., 2006). Treatment recommendations for Lyme disease in children are summarized in Table 10.5 and discussed in detail in Chapter 14, this volume. 10.3.5 Long-term outcome after treatment The long-term prognosis for patients with EM who receive timely and appropriate therapy is excellent (Nadelman et al., 1992; Luger et al., 1995; Smith et al., 2002; Nowakowski et al., 2003; Wormser et al., 2003; Wormser, 2006; Cerar et al., 2010; Kowalski et al., 2010). Two US cohorts of cultureconfirmed cases offer the best opportunity to study outcome because of the certainty of the diagnosis of EM. A multicentre observational study conducted in ten endemic states evaluated 118 patients seen with cultureconfirmed EM (from the LYMErix® vaccine trial; Steere et al., 1998), almost all of whom were treated with oral doxycycline or amoxicillin (Smith et al., 2002). Associated extracutaneous signs and symptoms persisted for more than 30 days after treatment in 11% of patients, decreasing to 4% at 60 days (Smith et al., 2002). All of these patients had only subjective complaints (e.g. headache, fatigue and arthralgias) except for two patients who manifested seventh-cranialnerve palsy with residual neurological symptoms of facial numbness or weakness.

All but one patient had completely recovered by follow-up at 20 months (Smith et al., 2002). In another prospective study, 99 patients seen in Westchester County, NY, with cultureconfirmed EM were followed for a mean of 4.9±2.9 years (Nowakowski et al., 2003). After antimicrobial treatment, EM resolved within 3 weeks in all cases, and no patient developed objective extracutaneous manifestations of late Lyme disease. Almost all patients (90%) were enrolled in treatment trials; although a few patients received a 7-day course of azithromycin (9%) or a course of intravenous ceftriaxone, the overwhelming majority of patients received doxycycline, amoxicillin or cefuroxime axetil for 10–21 days. Improvement was documented in all but the two patients (2%) who failed to return after the baseline visit. From 3 months onwards, 84– 92% of patients were asymptomatic or had symptoms consistent with their health status prior to Lyme disease (Nowakowski et al., 2003). Some asymptomatic patients developed subjective symptoms at a subsequent visit. Only eight (10%) of the 81 cases followed for 1 year were symptomatic at their last visit, a mean of 5.6±2.6 years into follow-up. Their symptoms were usually mild and intermittent, with only three patients (4%) consistently symptomatic at each follow-up visit. Two patients were retreated for continuing symptoms, one at 3 and 6 months and the other at 6 years, but neither had a significant clinical response. Patients with a greater number and severity of symptoms at study enrolment, and those with multiple EM were more likely to report symptoms at follow-up visits (Nowakowski et al., 2003). Repeat tick bites were reported by 47% of patients, and repeated episodes of EM occurred in 15% patients (see 10.4 Reinfection), emphasizing the need for education on preventing tick bites. Patients with EM treated with currently recommended regimens (Wormser et al., 2006) almost never develop objective evidence of late disease (Nadelman et al., 1992; Luger et al., 1995; Luft et al., 1996; Dattwyler et al., 1997; Wormser et al., 2003; Cerar et al., 2010). Some patients who developed objective neurological disease after treatment for EM had, in retrospect, findings suggesting subtle central

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nervous system involvement at the time oral antimicrobials were initiated (Massarotti, et al., 1992). Approximately 10% of patients report subjective complaints such as fatigue, myalgias and arthralgias, and vague neurological symptoms after completion of treatment for EM (Nowakowski et al., 2003; Nadelman et al., 1992; Luger et al., 1995; Luft et al., 1996; Wormser et al., 2003; Feder et al., 2007). Persistent infection is unlikely to cause these symptoms; prolonged antimicrobial treatment for these patients has been ineffective in achieving sustained improvement and can be harmful (Nadelman et al., 1991; Ettestad et al., 1995; Patel et al., 2000; Klempner et al., 2001; Krupp et al., 2003; Wormser et al., 2006; Fallon et al., 2008; Halperin, 2008; Holzbauer et al., 2010). Subjective background complaints are quite common in the general ‘healthy’ population, and may account for some of the symptoms that patients experience after treatment (Barsky and Borus, 1999; Seltzer et al., 2000). Ninety per cent of the general population describes one or more somatic symptoms in a given 2–4-week period and 30% report current musculoskeletal symptoms (Barsky and Borus, 1999). Significant fatigue is experienced by 20% of adults, and more than 75% of healthy college students report at least one symptom in a 3-day period (Barsky and Borus, 1999). One recently published report assessed subjective complaints in Slovenian patients with solitary EM after treatment with either doxycycline or cefuroxime axetil for 15 days (Cerar et al., 2010). A novel feature of the study was the selection by the patient of a control at baseline consisting of a spouse, family member or friend who did not have a history of prior Lyme disease and was within 5 years of the patient’s age. Patients and controls were evaluated at baseline, 6 and 12 months using identical questionnaires to assess symptoms. At the final visit, patients were somewhat less likely than controls to have subjective complaints (5/230, or 2%, versus 21/224, or 9%; P = 0.002). None of the symptoms experienced by patients was of sufficient severity to be functionally disabling (Cerar et al., 2010). These findings suggest that some of the subjective complaints

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experienced after treatment may be unrelated to infection. However, this conclusion may be somewhat limited because the study excluded patients with early disseminated Lyme disease, including those with multiple EM skin lesions who might have a greater likelihood of developing post-Lyme disease symptoms. It is also uncertain whether similar findings would be applicable to disease in US patients who are infected with B. burgdorferi sensu stricto rather than B. afzelii. Excellent outcomes in European patients treated for EM have been observed at shortand long-term follow-up (up to 3 years) (Weber et al., 1990, 1993; Strle et al., 1992, 1996b; Kuiper et al., 1994; Hulshof et al., 1997; Lipsker et al., 2002, Cerar et al., 2010). European EM is discussed in more detail in Chapter 9 of this volume. 10.3.6 Outcome in special patient groups: pregnancy and immunocompromised hosts Epidemiological studies have failed to confirm an early report (Markowitz et al., 1986) of 19 patients with adverse pregnancy outcomes initially attributed to B. burgdorferi infection during pregnancy (Strobino et al., 1993, 1999; Williams et al., 1995). Furthermore, a survey of 162 paediatric neurologists in Connecticut (the state with the highest incidence of Lyme disease at that time; Bacon et al., 2008) failed to identify a single child with a neurological problem believed to be related to Lyme disease during pregnancy (Gerber and Zalneraitis, 1994). In a prospective Slovenian study, 93/105 pregnant women (88.6%) with EM had excellent outcomes after treatment with intravenous -lactam antimicrobials; adverse outcomes (abortion, pre-term birth, syndactyly and urological anomalies) were not clearly linked to Lyme disease (Maraspin et al., 1999b). There have been no treatment trials comparing intravenous and oral antibiotics for pregnant women with EM. However, there are no data to support treating pregnant patients with EM differently from non-pregnant patients with EM, aside from avoiding doxycycline (Wormser et al., 2006).

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Controlled studies comparing the presentation and outcome of immunocompromised patients with EM with those having normal immune function are rare (Maraspin et al., 1999a; Fürst et al., 2006). Early disseminated disease and objective and subjective ‘treatment failure’ prompting repeat courses of antimicrobials were reported to be more common in 67 Slovenian patients with a variety of causes of immunosuppression when compared with a control group (Maraspin et al., 1999a). However, favourable outcomes were seen in both groups at 1-year follow-up (Maraspin et al., 1999a). A retrospective study from Austria found that 33 immunosuppressed patients with EM had similar clinical presentations, rates of seropositivity and favourable response to therapy when compared with controls with EM who had normal immune function (Fürst et al., 2006). In a preliminary report, 35 Slovenian patients with haematological malignancies had similar clinical presentations compared with 70 immunocompetent patients, but were more likely to have leukocytosis or leukopenia, and were more often retreated (one patient each with subjective symptoms, persistence of EM or development of multiple EM versus no immunocompetent patients; Maraspin et al., 2010). In an uncontrolled observational report from Slovenia, six patients with a history of prior organ transplant had excellent outcomes after treatment for EM (Maraspin et al., 2006).

10.4 Reinfection Repeat episodes of EM are not uncommon in endemic areas. As many as 15% of patients with EM followed in several prospective studies in the USA had more than one episode, with repeat episodes averaging 1.2– 3.1% per year over 1–5 years (Smith et al., 2002; Nowakowski et al., 2003; Wormser et al., 2003). This incidence is actually 20–50 times that for Lyme disease in the general population living in the same community (Nadelman and Wormser, 2007; Nadelman et al., 2007). For many years, it has been recognized that some patients may experience a second and occasionally more episodes of

Lyme disease after the first episode has resolved (Hollström, 1951). These subsequent occurrences, nearly invariably associated with EM, are almost always the result of reinfection rather than relapse (Nadelman and Wormser, 2007). Reinfection may be defined as a new infection that occurs after successful antimicrobial treatment of a prior episode of Lyme disease (Nadelman and Wormser, 2007). The most likely explanation for recurrent EM is that repeat tick bites are quite common in endemic areas. In Westchester County, NY, in a study of doxycycline prophylaxis after a recognized tick bite, 17% of 335 subjects sustained new tick bites over the 6 weeks following enrolment, despite receiving specific oral and written instructions on ways to reduce the risk of tick bite (Nadelman et al., 2001). Many people in endemic areas acquire tick bites and Lyme disease on their own property (Falco and Fish, 1988). The normal human immune response is insufficiently protective against reinfection. One reason for this may be that are at least 17 ospC genotypes of B. burgdorferi causing clinical disease in the USA (Ivanova et al., 2009). Preliminary evidence that reinfection with a new strain of B. burgdorferi accounts in part for repeat episodes of EM was obtained from 13 patients who had 17 consecutive episodes of culture-confirmed EM occurring up to 10 years apart (Nadelman et al., 2010). No two consecutive episodes were caused by the same ospC genotype, suggesting that these were new infections rather than incompletely treated infections that relapsed. This conclusion is supported by an animal experiment (Probert et al., 1997). Mice were immune to challenge with an ospC genotype to which they had previously been immunized, but were susceptible to infection with a different ospC genotype (Probert et al., 1997). Published information is limited concerning the clinical manifestations of patients with repeat episodes of EM. None of 28 patients seen in Rhode Island with recurrent EM was believed to have an immunodeficiency, and almost all cases occurred in the summer, paralleling the peak questing period of I. scapularis nymphal ticks,

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consistent with reinfection rather than relapse (Krause et al., 2006). The signs, symptoms and demographics of patients with recurrent EM appear generally similar to those associated with the initial episode (Nadelman et al., 2002; Krause et al., 2006; Nadelman and Wormser, 2007). A Swedish finding that women are more likely than men to have a second bout of EM (Jarefors et al., 2006) differs from the experience reported in the USA to date where the gender distribution is similar at first and second episodes (Nadelman et al., 2002; Krause et al., 2006; Nadelman and Wormser, 2007). In our own preliminary experience in 22 patients, there appeared to be a trend (not statistically significant) for multiple EM lesions to occur less frequently in recurrences than in initial episodes (3/11, or 14% of the total, versus 7/11, or 32%; P = 0.15; Nadelman et al., 2002; Nadelman and Wormser, 2007). This observation, if confirmed in a larger study, might be compatible with the acquisition of partial immunity aborting haematogenous spread of spirochaetes during the second episode (Nadelman and Wormser, 2007). Consistent with this hypothesis is the finding in a separate report that spirochaetaemia was significantly less likely in patients with a previous history of Lyme disease compared with those experiencing their first episode (odds ratio = 2.5, confidence interval 1.1–5.7; P = 0.03; Wormser et al., 2005a). Relapse of B. burgdorferi infection due to persistence of the organism after recommended courses of treatment is not well documented in the USA (Krause et al., 2006; Nadelman and Wormser, 2007). However, relapse has been well documented in patients who were treated with antibiotics not recommended for Lyme disease (e.g. cephalexin; Nowakowski et al., 2000) and has been reported in patients receiving macrolides (Luft et al., 1996).

10.5 Summary EM, the most common objective manifestation of Lyme disease, is associated with infection with B. burgdorferi sensu stricto in the USA as well as other genospecies causing Lyme

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disease (often referred to as Lyme borreliosis) in Europe and Asia (Kuiper et al., 1994; Hashimoto et al., 1995; Busch et al., 1996; Strle et al., 1996a, 1999, 2011; Ornstein et al., 2001; Antoni-Bach et al., 2002; Lipsker et al., 2002; Logar et al., 2004). Despite their characteristic appearance, EM-like lesions should not be considered pathognomonic for Lyme disease, because other skin lesions may appear indistinguishable, including localized tick-bite reactions and STARI, neither of which is associated with B. burgdorferi infection (Masters et al., 1998; Nadelman and Wormser, 2002; Wormser et al., 2005b,c; Tibbles and Edlow, 2007). EM is associated with excellent outcome after appropriate treatment with oral antibiotics; objective treatment failures are exceedingly rare (Dattwyler et al., 1990, 1997; Massarotti et al., 1992; Nadelman et al., 1992; Strle et al., 1992, 1996b; Weber et al., 1993; Luger et al., 1995; Luft et al., 1996; Eppes and Childs, 2002; Smith et al., 2002; Wormser et al., 2003, 2006; Wormser, 2006; Cerar et al., 2010). Although B. burgdorferi may be isolated in culture from a biopsy taken from a sample of the skin lesion (Berger et al., 1992; Kuiper et al., 1994; Busch et al., 1996; Nadelman et al., 1996; Strle et al., 1996a,b, 1999; Antoni-Bach et al., 2002; Smith et al., 2002; Logar et al., 2004) or blood (Wormser et al., 2000, 2005a) laboratory diagnosis, including the use of serology, is generally neither helpful nor necessary (Aguero-Rosenfeld et al., 1993, 2005; CDC, 1995; Wormser et al., 2006). For the practitioner, EM remains a clinical diagnosis (Nadelman and Wormser, 1998, 2002; Wormser et al., 2006).

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11

Cardiac Involvement

Joseph M. Harburger and Jonathan L. Halperin

11.1 Introduction Cardiac involvement in Lyme disease was first described by Steere et al. (1980) before the discovery of the Ixodes tick vector and before the spirochete (Borrelia burgdorferi) was reported in 1982 (Burgdorfer et al., 1982). Today, Lyme carditis is recognized as an occasional complication that typically involves the conduction system, especially in the region of the atrioventricular node, and occasionally produces a more diffuse form of myocarditis or pericarditis. The diagnosis of atypical manifestations of cardiac pathology associated with serological or microbiological evidence of B. burgdorferi infection requires clinical vigilance and a high index of suspicion, particularly in geographical regions where the disease is endemic.

11.2 Epidemiology Most cases of Lyme carditis arise between June and December (Fish et al., 2008). Cardiac involvement is more frequent with Lyme disease in the USA, where the incidence is 4–10%, than in Europe, where carditis is identified in 0.3–4% of cases (Fish et al., 2008; Rostoff et al., 2009), perhaps related to geographical variations in B. burgdorferi species (Fish et al., 2008). It is less clear why males contract Lyme disease only slightly

more frequently than females, yet are three times as likely to develop carditis (Fish et al., 2008; Lelovas et al., 2008). Clinical manifestations of carditis occur less frequently in children than in adults (Costello et al., 2009). One study found electrocardiographic (EKG) abnormalities in 29% (7/24) of children with probable or definite Lyme disease but no other clinical evidence of cardiac involvement (Woolf et al., 1991). By comparison, the prevalence of abnormal EKGs was 12% in a healthy cohort with a median age of 17 years prior to participation in athletics (Pelliccia et al., 2007).

11.3 Pathogenesis 11.3.1 Murine models Experimental studies of the pathogenesis of Lyme carditis have been carried out predominantly in mice. Most strains of mice are natural hosts for B. burgdorferi and, despite antibody responses to inoculation, exhibit few clinical manifestations of Lyme disease (Tilly et al., 2008). Some strains are susceptible, however, and mouse models of Lyme carditis have been developed (Tilly et al., 2008), athough it is not clear whether these findings in mice can be translated to human manifestations of the disease. In mouse models of Lyme carditis,

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cardiac involvement appears to arise both as a direct manifestation of B. burgdorferi infection and as a result of a secondary immune response. ‘Molecular mimicry’ may play a role, as evidenced by a higher antibody response to B. burgdorferi infection in autoimmune New Zealand black mice than other strains, associated with more extensive myocardial inflammation (Raveche et al., 2005). A comparison of amino acid sequences of the B. burgdorferi outer-surface protein A (OspA) protein with the Streptococcus pyogenes M protein revealed homology, and both proteins had sequence homology to myosin. In addition, anti-B. burgdorferi IgM antibody displayed cross-reactivity with myosin and the S. pyogenes M protein. The S. pyogenes M protein is believed to play a role in the generation of antibodies responsible for rheumatic carditis (Gorton et al., 2009; Guilherme and Kalil, 2010), and myosin cross-reactivity of antibodies produced in response to B. burgdorferi infection may play a role in the pathogenesis of Lyme carditis. While autoimmunity may be an important mediator, immunodeficiency states have been associated with Lyme carditis in murine models. The results of several studies suggest links between deficiencies of T cells and the cytokine gamma interferon (IFN-) in the pathogenesis of Lyme carditis (Schaible et al., 1989; Barthold et al., 1992; Bockenstedt et al., 2001; Brown et al., 2006; Olson et al., 2009). Mice with Lyme carditis deficient in CD4+ Th1 cells are treated effectively when administered IFN-γ-producing CD4+ T-helper 1 (Th1) cells (Bockenstedt et al., 2001). Severe combined immunodeficiency (SCID) mice deficient in T and B cells display more severe manifestations of Lyme carditis than normal controls, while Lyme carditis can be overcome by mice with normal T cells and immature B cells incapable of antibody production (Schaible et al., 1989; Barthold et al., 1992). Inoculation of SCID mice with immune mouse serum containing mature B cells is ineffective in treating Lyme carditis (Bockenstedt et al., 2001). Activation of invariant natural killer T (iNKT) cells in mice results in the production of IFN-γ, promoting resolution of Lyme carditis; this provides additional evidence of the importance of

IFN-γ and suggests a role for iNKT cells in Lyme carditis (Olson et al., 2009). Mice deficient in Stat1, the major transcription factor produced in response to IFN-γ, are more susceptible to developing Lyme carditis. Macrophages and polymorphonuclear leucocytes (PMNs) also appear to play a role in the pathogenesis of Lyme carditis (Gueraude-Arellano et al., 2005; Montgomery et al., 2007). Mice deficient in CCR2 (the macrophage chemokine receptor) have similar degrees of carditis as those with CCR2, but cardiac inflammatory infiltrates differ in the two strains. Mice deficient in CCR2 have a greater prevalence of PMNs in cardiac tissue, while mice with CCR2 have a greater prevalence of macrophages (Montgomery et al., 2007). Mice unable to produce 2 integrins, adhesion molecules that cause leukocyte adhesion to endothelium allowing extravasation of inflammatory cells into tissue, develop more severe Lyme carditis than normal controls (Guerau-de-Arellano et al., 2005). Dendritic cells in mice unable to produce 2 integrins are upregulated to produce higher levels of monocyte/macrophage chemo-attractant protein 1, resulting in increased macrophage infiltration and inflammation in cardiac tissue. Distinct subtypes of North American B. burgdorferi cause Lyme carditis of different severities in mice (Wang et al., 2002). Although only North American isolates were analysed, this study supports the theory that the higher prevalence of human Lyme carditis in the USA than in Europe may be due to geographical differences in the distribution of B. burgdorferi species. 11.3.2 Human studies Human autopsy findings associated with Lyme carditis include diffuse inflammatory changes involving the endocardium, myocardium and epicardium (Tavora et al., 2008) with macrophage, lymphocyte and plasma cell infiltration and occasional eosinophils. The infiltrates are largely perivascular and interstitial with myocyte necrosis. Endocardial fibrosis, lymphocytic infiltration of the atrioventricular (AV) node and

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myocarditis prominently involving lymphocytes and macrophages have been described (Cary et al., 1990). Biopsies of tissue in patients with cardiac manifestations of Lyme disease have revealed inflammation of the endocardium, myocardium, epicardium, and pericardium. Lymphocyte and monocyte infiltration is common, and some studies report histiocyte, plasma cell and eosinophil infiltration (Klein et al., 1991; Hajjar and Kradin, 2002; Lelovas et al., 2008; Lalosevic et al., 2009). Both small and large vessel inflammation have been reported (Klein et al., 1991; Fish et al., 2008; Lelovas et al., 2008; Tavora et al., 2008), as well as myocardial necrosis (Hajjar and Kradin, 2002; Fish et al., 2008). Spirochetes have been described in biopsy specimens (Reznick et al., 1986; Bergler-Klein et al., 1993; Hajjar and Kradin, 2002; Lalosevic et al., 2009); similar findings have been described in adults and children with Lyme carditis (Costello et al., 2009).

11.4 Clinical Manifestations Cardiac manifestations typically arise during the second (early disseminated) phase of the disease, weeks to months after the inciting tick bite, at a mean of 21 days after the development of erythema migrans (EM) (Rostoff et al., 2009). Carditis may be concurrent with neurological and musculoskeletal symptoms, but has been reported as early as 4 days or as late as 7 months after tick bite or EM (Fish et al., 2008). Lyme carditis typically presents with symptoms of palpitations, lightheadedness, syncope, chest pain or dyspnea. In a surveillance study of Lyme disease in the USA, 69% (58/84) of patients with Lyme carditis reported to the Centers for Disease Control and Prevention reported palpitations (Ciesielski et al., 1989). In a paediatric cohort presenting to a quaternary children’s referral hospital, the incidence of palpitations was 18%, syncope 12%, near-syncope 6%, chest pain 15% and dyspnea 6% (Costello et al., 2009). All of these symptoms are non-specific and must be interpreted in the appropriate clinical context. Palpitations are reported in up to 16% of all persons presenting to medical providers

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(Summerton et al., 2001; Thompson, 2006; Thavendiranathan et al., 2009) for example, and syncope, chest pain and dyspnea occur in multiple cardiovascular and noncardiovascular disorders. Carditis is usually accompanied by other signs of Lyme disease (e.g. EM, arthritis or neurological manifestations), but may be the sole manifestation (Kimball et al., 1989; Panic et al., 2010). The cardiac manifestations of Lyme disease are summarized in Table 11.1 and discussed in detail below. 11.4.1 Conduction system disease Cardiac conduction abnormalities are the most common finding in Lyme carditis, usually in the form of AV block of varying degrees up to and including complete heart block (Nagi et al., 1996; Fish et al., 2008; Lelovas et al., 2008; Rostoff et al., 2009; HernandezMontfort et al., 2010). Of 52 cases of Lyme carditis reported in 1987, 87% (45/52) had heart block, of which 62% (28/45) were secondor third-degree block (McAlister et al., 1989). In another series of 105 cases of Lyme carditis, 49% of patients developed complete heart block, while 16% had second-degree AV block and 12% developed only first-degree AV block (van der Linde, 1991; Fish et al., 2008; Lelovas et al., 2008). In contrast, among 33 children with Lyme carditis, 42% presented with firstdegree AV block, 21% presented with seconddegree AV block and 15% presented with complete heart block, although 27% developed complete heart block during the course of their illness (Costello et al., 2009). Low-grade AV block in patients with Lyme disease is likely to be underdiagnosed, as EKGs are not invariably recorded in people with Lyme disease. In one study, 14% (2/14) of children with definite Lyme disease but no symptoms suggestive of carditis had first-degree AV block, while none had second- or third-degree heart block (Woolf et al., 1991). Patients with PR intervals of 300 ms and those with second-degree AV block face a relatively high risk of progression to complete heart block, which can occur rapidly; in contrast, those with PR intervals of 300 ms are at lower risk (Nagi et al., 1996; Fish et al.,

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Table 11.1. Clinical manifestations of Lyme carditis Established Conduction abnormalities: Atrioventricular block (most common manifestation) Prolonged QTc Bundle-branch block Ventricular tachycardia Fascicular tachycardia Supraventricular tachycardia Myocarditis Pericarditis Potential (further investigation needed) Chronic dilated cardiomyopathy Recurrent pericardial effusion Valvular disease Coronary artery aneurysms

2008; Lelovas et al., 2008; Costello et al., 2009). Heart block typically resolves with antibiotic therapy (Fish et al., 2008; Costello et al., 2009), so temporary pacemaker therapy is usually not required except when complete heart block develops without adequate ventricular escape rhythm (Reznick et al., 1986; Nagi and Thakur, 1995; Rosenfeld et al., 1999; Xanthos et al., 2006; Berger and McGillicuddy, 2009; Costello et al., 2009). In general, the indications for temporary and permanent pacing are the same as for other conditions associated with impaired cardiac conduction (Fish et al., 2008). Bundle-branch block and interventricular conduction delays have been reported in patients with Lyme carditis with or without AV block (van der Linde, 1991; Fish et al., 2008). Prolongation of the QTc interval typically resolves with therapy (Seslar et al., 2006; Costello et al., 2009), and the torsades de points type of ventricular tachycardia has not been reported with this disease. Other types of ventricular, fascicular and supraventricular tachycardias may occur in association with Lyme carditis, however (Vlay et al., 1991; Greenberg et al., 1997; Fish et al., 2008), either as a direct manifestation of myocardial involvement or as complication of pericarditis.

11.4.2 Myopericarditis Non-specific repolarization abnormalities are frequently seen on the EKG in patients with Lyme carditis, signifying myocardial and/or pericardial involvement (Steere et al., 1980; Nagi et al., 1996; Fish et al., 2008). In a series of 18 patients, 67% had changes in the morphology of ST segments or T waves (Steere et al., 1980). Patients with Lyme carditis may present with chest pain, STsegment elevation and elevated cardiac enzymes in serum (Horowitz and Belkin, 1995; Rostoff et al., 2009), mimicking acute myocardial infarction, but angiography may show no coronary obstruction and further investigation points to a diagnosis of myopericarditis associated with Lyme disease. Myocarditis can cause cardiac enlargement and depressed left or right ventricular systolic function that is usually transient, mild and reversible (Nagi et al., 1996; Hajjar and Kradin, 2002; Fish et al., 2008; Costello et al., 2009). In one study, 5% (4/84) of patients with Lyme carditis developed left ventricular (LV) systolic dysfunction (Ciesielski et al., 1989), and cardiogenic shock has been reported as well. In a series of 33 paediatric cases, four patients with Lyme carditis had depressed LV systolic function, three of whom developed cardiogenic shock. In these three patients, myocardial biopsies revealed diffuse myocarditis; all required temporary pacing and one required cardiopulmonary resuscitation and extracorporeal membrane oxygenation, but each recovered completely (Costello et al., 2009).

11.5 Less-validated Manifestations In addition to the established manifestations of Lyme carditis described above, several authors have proposed associations between Lyme disease and other cardiac findings. These findings have not been established as secondary to Lyme disease, and further investigation is necessary. Positive serological testing for B. burgdorferi frequently occurs in patients where Lyme disease is endemic, and may represent either prior exposure or

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infection (Steere et al., 1993; Marques, 2008). Over-diagnosis of Lyme disease is common and may result in inappropriate treatment (Sigal, 1990; Steere et al., 1993; Reid et al., 1998; Marques, 2008). Although a person may have signs of cardiac illness and positive serological markers for Lyme disease, causality cannot be confirmed until a sufficiently large number of persons with no alternative diagnoses are found to have Lyme disease associated with a particular cardiac manifestation. The entities described in the following discussion do not currently meet this criterion. 11.5.1 Chronic dilated cardiomyopathy Myocarditis associated with Lyme disease is a potential cause of chronic dilated cardiomyopathy. In one series of 175 patients with dilated cardiomyopathy, 14 (8%) were seropositive for B. burgdorferi (felt by the authors to be largely false positives; Sonnesyn et al., 1995). In a case reported in 1990, a man with a dilated cardiomyopathy presumed secondary to Lyme carditis presented with a febrile illness associated with cough, fatigue, arthralgias and headache 1 year before detection of biventricular dilated cardiomyopathy by echocardiography and radionuclide ventriculography (Stanek et al., 1990). Holter monitoring revealed frequent ventricular ectopy and brief episodes of nonsustained ventricular tachycardia. Cardiac catheterization and blood testing revealed no alternative aetiology of cardiomyopathy. Approximately 3 years after initial presentation, the patient developed clinical heart failure. Serological testing for Lyme disease was positive and endomyocardial biopsy yielded B. burgdorferi and histopathology characterized by mononuclear myocarditis and spirochetes. No improvement in LV function occurred following treatment with ceftriaxone. A subsequent study described a higher proportion of ELISA (without confirmatory Western blot) positivity for B. burgdorferi in patients with chronic heart failure and dilated cardiomyopathy (26.4%) than in patients with coronary disease (12.7%) or subjects without heart disease (8.2%) as evidence that

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Lyme disease may cause chronic heart failure (Stanek et al., 1991). In another, 32.7% of 54 patients with chronic heart failure were seropositive (by ELISA only) for B. burgdorferi (Klein et al., 1991). Later reports of patients with Lymeassociated dilated cardiomyopathy suggest a variable prognosis. In small series of patients with dilated cardiomyopathy and serological or biopsy evidence of B. burgdorferi myocarditis, treatment with intravenous ceftriaxone beginning at a mean of 3.7 months after detection of cardiac involvement (Gasser et al., 1992) or with angiotensin-converting enzyme inhibitors, digitalis and penicillin was usually followed by gradual resolution of LV dysfunction. In other reports, antibiotic therapy failed to improve LV function in a majority of patients with seropositive, culture-positive or biopsy-proven B. burgdorferi dilated cardiomyopathy (Bergler-Klein et al., 1992). The reasons for this variable course have not been fully elucidated. Antibiotic therapy seemed most effective in achieving resolution of cardiomyopathy when initiated within 6 months of the onset of LV dysfunction, beyond which less-specific treatment of cardiomyopathy may be more important. In some or all cases, Lyme disease may be incidental and another (e.g. viral) aetiology of myocarditis may be present that is unresponsive to antispirochetal therapy. Given the relatively large proportion of ELISA seropositivity that remains unconfirmed by Western blot analysis, the former assay is probably not sufficiently diagnostic of Lyme carditis in patients with cardiomyopathy in endemic areas. Favourable responses to antimicrobial therapy have been limited to Europe, suggesting that geographical differences in B. burgdorferi strains may have important therapeutic and prognostic implications (Fish et al., 2008). As the role of B. burgdorferi infection and the effectiveness of antimicrobial drugs in patients with dilated cardiomyopathy are uncertain, we recommend two-tier testing for B. burgdorferi in cryptogenic cases of cardiomyopathy if the clinical history suggests Lyme disease or exposure. In those with initial ELISA seropositivity, Western blot

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analysis is indicated. If this is confirmatory, a course of antibiotic therapy is reasonable, particularly when the onset of cardiomyopathy is recent. 11.5.2 Other potential manifestations Beyond conduction defects, arrhythmias, myocarditis and cardiomyopathy, associations with Lyme disease have been suggested for several other cardiac abnormalities, although proof of causality is lacking. Pericarditis with recurrent effusion was described in a seropositive patient with a clinical history suggestive of Lyme disease (Gasser et al., 1998). Repeated pericardiocentesis was required over more than a year yielding fluid antibody positive for B. burgdorferi. Serial echocardiography during the year following ceftriaxone therapy revealed no recurrence of pericardial effusion, but the literature is rife with reports of spontaneously resolving cases of idiopathic effusive pericarditis, making an aetiological relationship to Lyme disease speculative. Similarly, there are cases of progressive mitral regurgitation in patients with symptomatic carditis. In one such patient with B. burgdorferi infection confirmed by both ELISA and Western blotting (Canver et al., 2000), pericardial tamponade developed following mitral valve replacement surgery. The pericardium had histological features of lymphoplasmacytic pericarditis, although spirochetes were not visualized. Coronary artery aneurysms have also been described in seropositive patients (Gasser et al., 1994; Cuisset et al., 2008), noting similarities to syphilitic vasculopathy.

11.6 Diagnosis The diagnosis of Lyme disease begins with recognition of the typical clinical history, and carditis usually coincides with other features of early disseminated Lyme disease, including EM, neurological deficits and/or arthritis. There have been several reports of apparently isolated cardiac involvement without other manifestations of systemic Lyme disease

(Kimball et al., 1989; Panic et al., 2010), but patients may not recall antecedent tick bite or rash (Fish et al., 2008). Hence, two-tier serological testing should be considered whenever the diagnosis of Lyme carditis is suspected. The initial serological test should be the ELISA Lyme disease assay for detection of IgG and IgM antibodies to B. burgdorferi. If positive or borderline, a confirmatory Western blot analysis should be performed. Western blotting is more specifically diagnostic of serum antibodies against B. burgdorferi proteins. Given the latency involved in antibody development, serological testing may be unrevealing during the first weeks after infection, although seropositivity typically develops by the time carditis is manifest (Hajjar and Kradin, 2002; Fish et al., 2008; Lelovas et al., 2008). When the index of suspicion for Lyme carditis is high for a seronegative patient, however, the diagnosis should not be excluded and treatment for Lyme carditis should not be delayed. Echocardiography is usually performed in patients with suspected Lyme carditis to assess LV function and the pericardium and to exclude valvular vegetations suggestive of bacterial endocarditis as an alternative diagnosis. When a specific diagnosis is required, catheter-directed endomyocardial biopsy may reveal histological features of carditis, and spirochetes may be identified by microscopy. Myocardial biopsy is not routinely recommended in patients with Lyme carditis, however, given the small but tangible risk associated with the procedure, potentially focal distribution of inflammatory involvement (a negative biopsy does not exclude the diagnosis) and generally benign clinical course of the disease (Fish et al., 2008). Culture for the spirochetal pathogen responsible for Lyme disease is relatively insensitive and not routinely recommended (Lelovas et al., 2008). Gallium and indium111 anti-myosin antibody scintigraphy may reveal areas of increased uptake consistent with myocarditis (Jacobs et al., 1984; Reznick et al., 1986; Veluvolu et al., 1992; Bergler-Klein et al., 1993). Magnetic resonance imaging with gadolinium

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enhancement can identify zones of delayed enhancement (Globits et al., 1994; Munk et al., 2007; Naik et al., 2008). These typically resolve with successful antimicrobial therapy (Naik et al., 2008), but in some cases enhancement persists despite clinical improvement (Munk et al., 2007). No cardiac imaging findings are diagnostic of Lyme carditis, as similar abnormalities have been described in patients with myocarditis of other aetiologies.

11.7 Treatment 11.7.1 Antibiotic therapy Systemic administration of antibiotic medication is the mainstay of therapy for patients with cardiac manifestations of Lyme disease, although data from randomized trials are not available (Fish et al., 2008). In addition to anecdotal evidence of efficacy, patients with clinical features of extracardiac Lyme disease treated with antibiotics were no more likely to develop cardiovascular abnormalities than patients without Lyme disease (Sangha et al., 1998). Patients with Lyme disease and PR interval prolongation of 300 ms may be treated with oral antibiotic medication on an ambulatory basis. Common oral regimens include amoxicillin, 500 mg three or four times daily; doxycycline, 100 mg twice daily; or cefuroxime axetil for 4 weeks (Fish et al., 2008; Lelovas et al., 2008). Patients with PR intervals 300 ms or with second- or thirddegree AV block are at higher risk of progression to complete heart block, and hospitalization in a telemetry or coronary care unit is advised for monitoring during intravenous antibiotic therapy (Nagi and Thakur, 1995; Fish et al., 2008; Lelovas et al., 2008; Costello et al., 2009). Intravenous antibiotic regimens include ceftriaxone, 2 g daily or high-dose penicillin G. Once the PR interval shortens to 300 ms and there is no evidence of higher-grade AV block, an oral regimen may be substituted to complete a 4-week course of therapy on an outpatient basis (Fish et al., 2008). For patients with Lyme disease-associated dilated cardiomyopathy, the optimum treatment regimen

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has not been established, but the most favourable outcomes have been achieved with a 2-week course of ceftriaxone, 2 g intravenously twice daily (Gasser et al., 1992). Cardiac-specific studies are quite limited; most treatment trials, however, indicate that doses in excess of 2 g daily are unnecessary and carry a greater potential risk (see Wormser, Chapter 7, this volume). Shortly after initiation of antibiotic medication, approximately 10–15% of patients with Lyme disease develop a Jarisch– Herxheimer-like reaction (Rostoff et al., 2009). This phenomenon, characterized by fever, tachycardia and hypertension, has been attributed to release of antigens and/or cytokines following destruction of spirochetes (Maloy et al., 1998). Acute cardiovascular decompensation upon initiation of antibiotic therapy of Lyme carditis may develop as a result of this reaction. Effective preventive measures have not been defined; supportive care may include antipyretic or antiinflammatory measures and judicious hydration (Hajjar and Kradin, 2002). 11.7.2 Anti-inflammatory medication There have been a number of reports of corticosteroid treatment in patients with suspected Lyme carditis, but no evidence that this speeds the resolution of carditis (Fish et al., 2008; Lelovas et al., 2008). After withdrawal of steroid medication, joint or neurological symptoms of Lyme disease may recur or intensify (Cox and Krajden, 1991; Fish et al., 2008; Lelovas et al., 2008), and administration of corticosteroids is not routinely recommended for patients with cardiac manifestations of Lyme disease (Hajjar and Kradin, 2002). Use of salicylate compounds is similarly unfounded in evidence of efficacy (Fish et al., 2008). 11.7.3 Electronic pacing Indications for pacing in patients with conduction defects in the course of Lyme disease are the same as for other conditions (Fish et al., 2008). Implantation of a permanent

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pacemaker is rarely required, and even temporary pacing is usually unnecessary (Reznick et al., 1986; Nagi and Thakur, 1995; Rosenfeld et al., 1999; Xanthos et al., 2006; Berger and McGillicuddy, 2009; Costello et al., 2009).

11.8 Prognosis The prognosis of patients with Lyme carditis is generally excellent, with complete recovery in over 90% (Lelovas et al., 2008). Resolution of complete heart block typically occurs within a week, and lesser conduction delays typically resolve within 6 weeks (McAlister et al., 1989; Fish et al., 2008). Persistent conduction disturbances requiring permanent pacing are uncommon (Nagi and Thakur, 1995; Costello et al., 2009). There have been isolated case reports of fatality (Cary et al., 1990; Tavora et al., 2008); exact causes of death were not established, but in each case patients had AV block and cardiomegaly, and an inflammatory cardiac infiltrate was found at autopsy. In one of the cases (Tavora et al., 2008), Lyme carditis was diagnosed by means of positive ELISA and confirmatory Western blotting, and PCR confirmed the presence of B. burgdorferi DNA in the myocardium. In the other case (Cary et al., 1990), the presumed diagnosis of fatal Lyme carditis was more speculative, as the positive ELISA was not followed by confirmatory testing.

11.9 Summary and Conclusions Cardiac involvement is a complication of Lyme disease that typically develops during the early disseminated stage, weeks to months after exposure to the tick vector. The most common clinical features are AV conduction block of variable degree, although other conduction defects, QT-interval prolongation and arrhythmias, including supraventricular tachycardia and ventricular tachycardia, have been reported. Myocarditis and/or pericarditis may develop with or without associated electrocardiographic

changes, cardiac enzyme elevations or ventricular dysfunction. Lyme disease may cause dilated cardiomyopathy, but it remains unclear whether seropositive patients with dilated cardiomyopathy benefit from antimicrobial therapy. Recurrent pericardial effusion, valvular regurgitation and coronary aneurysms have occasionally been reported in patients with suspected Lyme carditis, but further investigation is needed to confirm causality. Patients with a clinical history suggestive of Lyme disease and acute cardiac conduction disorders, arrhythmias or myopericarditis should undergo serological testing using the ELISA assay to detect antibodies against B. burgdorferi. If positive or borderline, confirmatory testing should be carried out using Western blot analysis. Serological testing may be negative during the first weeks of infection, although serological tests are usually positive when clinical manifestations of Lyme carditis develop. When the clinical history strongly suggests Lyme carditis, treatment for Lyme disease should be provided even if serologic testing is not diagnostic. Imaging by echocardiography, gallium scanning, indium111 anti-myosin antibody scintigraphy or magnetic resonance imaging may reveal evidence of myocarditis in patients with Lyme disease, but the findings are not specific for Lyme carditis. Endomyocardial biopsy may confirm myocarditis and identify spirochetes, but should be reserved for selected cases because of the risks associated with the procedure and typically benign course of the disease. The prognosis of Lyme carditis is generally favourable, with full recovery in over 90% of cases. Patients with PR interval prolongation of 300 ms can be treated with oral antibiotics on an outpatient basis. Those with more severe conduction disturbances should be treated in hospital with intravenous antibiotics. Temporary pacing is infrequently necessary during the acute phase, but conduction disturbances due to Lyme disease almost always resolve so that permanent pacemaker implantation is rarely required.

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myopericarditis resulting from Lyme disease. American Heart Journal 130, 176–178. Jacobs, J.C., Rosen, J.M. and Szer, I.S. (1984) Lyme myocarditis diagnosed by gallium scan. Journal of Pediatrics 105, 950–952. Kimball, S.A., Janson, P.A. and LaRaia, P.J. (1989) Complete heart block as the sole presentation of Lyme disease. Archives of Internal Medicine 149, 1897–1898. Klein, J., Stanek, G., Bittner, R., Horvat, R., Holzinger, C. and Glogar, D. (1991) Lyme borreliosis as a cause of myocarditis and heart muscle disease. European Heart Journal 12 (Supplement D), 73–75. Lalosevic, D., Lalosevic, V., Stojsic-Milosavljevic, A. and Stojsic, D. (2009) Borrelia-like organism in heart capillaries of patient with Lyme-disease seen by electron microscopy. International Journal of Cardiology 145, e96–e98. Lelovas, P., Dontas, I., Bassiakou, E. and Xanthos, T. (2008) Cardiac implications of Lyme disease, diagnosis and therapeutic approach. International Journal of Cardiology 129, 15–21. Maloy, A.L., Black, R.D. and Segurola, R.J. Jr (1998) Lyme disease complicated by the Jarisch–Herxheimer reaction. Journal of Emergency Medicine 16, 437–438. Marques, A. (2008) Chronic Lyme disease: a review. Infectious Disease Clinics of North America 22, 341–360. McAlister, H.F., Klementowicz, P.T., Andrews, C., Fisher, J.D., Feld, M. and Furman, S. (1989) Lyme carditis: an important cause of reversible heart block. Annals of Internal Medicine 110, 339–345. Montgomery, R.R., Booth, C.J., Wang, X., Blaho, V.A., Malawista, S.E. and Brown, C.R. (2007) Recruitment of macrophages and polymorphonuclear leukocytes in Lyme carditis. Infection and Immunity 75, 613–620. Munk, P.S., Orn, S. and Larsen, A.I. (2007) Lyme carditis: persistent local delayed enhancement by cardiac magnetic resonance imaging. International Journal of Cardiology 115, e108– e110. Nagi, K.S. and Thakur, R.K. (1995) Lyme carditis: indications for cardiac pacing. Canadian Journal of Cardiology 11, 335–338. Nagi, K.S., Joshi, R. and Thakur, R.K. (1996) Cardiac manifestations of Lyme disease: a review. Canadian Journal of Cardiology 12, 503–506. Naik, M., Kim, D., O’Brien F., Axel, L. and Srichai, M.B. (2008) Images in cardiovascular medicine. Lyme carditis. Circulation 118, 1881–1884. Olson, C.M. Jr, Bates, T.C., Izadi, H., Radolf, J.D., Huber, S.A., Boyson, J.E. and Anguita, J. (2009)

Local production of IFN- by invariant NKT cells modulates acute Lyme carditis. Journal of Immunology 182, 3728–3734. Panic, G., Stanulovic, V. and Popov, T. (2010) Atrioventricular block as the first presentation of disseminated Lyme disease. International Journal of Cardiology (Epub ahead of print, 10 March 2010), doi: 10.1016/j.ijcard.2010.02.061. Pelliccia, A., Culasso, F., Di Paolo, F.M., Accettura, D., Cantore, R., Castagna, W., Ciacciarelli, A., Costini, G., Cuffari, B., Drago, E., Federici, V., Gribaudo, C.G., Iacovelli, G., Landolfi, L., Menichetti, G., Atzeni, U.O., Parisi, A., Pizzi, A.R., Rosa, M., Santelli, F., Santilio, F., Vagnini, A., Casasco, M. Di and Luigi, L. (2007) Prevalence of abnormal electrocardiograms in a large, unselected population undergoing preparticipation cardiovascular screening. European Heart Journal 28, 2006–2010. Raveche, E.S., Schutzer, S.E., Fernandes, H., Bateman, H., McCarthy B.A., Nickell, S.P. and Cunningham, M.W. (2005) Evidence of Borrelia autoimmunity-induced component of Lyme carditis and arthritis. Journal of Clinical Microbiology 43, 850–856. Reid, M.C., Schoen, R.T., Evans, J., Rosenberg, J.C. and Horwitz, R.I. (1998) The consequences of overdiagnosis and overtreatment of Lyme disease: an observational study. Annals of Internal Medicine 128, 354–362. Reznick, J.W., Braunstein, D.B., Walsh, R.L., Smith, C.R., Wolfson, P.M., Gierke, L.W., Gorelkin, L. and Chandler, F.W. (1986) Lyme carditis. Electrophysiologic and histopathologic study. American Journal of Medicine 81, 923–927. Rosenfeld, M.E., Beckerman, B., Ward, M.F. and Sama, A. (1999) Lyme carditis: complete AV dissociation with episodic asystole presenting as syncope in the emergency department. Journal of Emergency Medicine 17, 661–664. Rostoff, P., Gajos, G., Konduracka, E., Gackowski, A., Nessler, J. and Piwowarska, W. (2009) Lyme carditis: epidemiology, pathophysiology, and clinical features in endemic areas. International Journal of Cardiology 144, 328–333. Sangha, O., Phillips, C.B., Fleischmann, K.E., Wang, T.J., Fossel, A.H., Lew, R., Liang, M.H. and Shadick, N.A. (1998) Lack of cardiac manifestations among patients with previously treated Lyme disease. Annals of Internal Medicine 128, 346–353. Schaible, U.E., Kramer, M.D. Museteanu, C., Zimmer, G., Mossmann, H. and Simon, M.M. (1989) The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis. Journal of Experimental Medicine 170, 1427–1432.

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Seslar, S.P., Berul, C.I., Burklow, T.R., Cecchin, F. and Alexander, M.E. (2006) Transient prolonged corrected QT interval in Lyme disease. Journal of Pediatrics 148, 692–697. Sigal, L.H. (1990) Summary of the first 100 patients seen at a Lyme disease referral center. American Journal of Medicine 88, 577–581. Sonnesyn, S.W., Diehl, S.C., Johnson, R.C., Kubo, S.H. and Goodman, J.L. (1995) A prospective study of the seroprevalence of Borrelia burgdorferi infection in patients with severe heart failure. American Journal of Cardiology 76, 97–100. Stanek, G., Klein, J., Bittner, R. and Glogar, D. (1990) Isolation of Borrelia burgdorferi from the myocardium of a patient with longstanding cardiomyopathy. New England Journal of Medicine 322, 249–252. Stanek, G., Klein, J., Bittner, R. and Glogar, D. (1991) Borrelia burgdorferi as an etiologic agent in chronic heart failure? Scandinavian Journal of Infectious Diseases 77 (Supplement), 85–87. Steere, A.C., Batsford, W.P., Weinberg, M., Alexander, J., Berger, H.J., Wolfson, S. and Malawista, S.E. (1980) Lyme carditis: cardiac abnormalities of Lyme disease. Annals of Internal Medicine 93, 8–16. Steere, A.C., Taylor, E., McHugh, G.L. and Logigian, E.L. (1993) The overdiagnosis of Lyme disease. Journal of the American Medical Association 269, 1812–1816. Summerton, N., Mann, S., Rigby, A., Petkar, S. and Dhawan, J. (2001) New-onset palpitations in general practice: assessing the discriminant value of items within the clinical history. Family Practice 18, 383–392. Tavora, F., Burke, A., Li, L., Franks, T.J. and Virmani, R. (2008) Postmortem confirmation of Lyme carditis with polymerase chain reaction. Cardiovascular Pathology 17, 103–107.

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Thavendiranathan, P., Bagai, A., Khoo, C., Dorian, P. and Choudhry, N.K. (2009) Does this patient with palpitations have a cardiac arrhythmia? Journal of the American Medical Association 302, 2135–2143. Thompson, J. (2006) Psychological and physical etiologies of heart palpitations. Nurse Practitioner 31, 14-23. Tilly, K., Rosa, P.A. and Stewart P.E. (2008) Biology of infection with Borrelia burgdorferi. Infectious Disease Clinics of North America 22, 217-234. van der Linde, M.R. (1991) Lyme carditis: clinical characteristics of 105 cases. Scandinavian Journal of Infectious Diseases 77 (Supplement), 81–84. Veluvolu, P., Balian, A.A., Goldsmith, R., Gallant, T.E., Barthel, L., Vidaillet, H.J. and Melski, J.W. (1992) Lyme carditis. Evaluation by Ga-67 and MRI. Clinical Nuclear Medicine 17, 823. Vlay, S.C., Dervan, J.P., Elias, J., Kane, P.P. and Dattwyler, R. (1991) Ventricular tachycardia associated with Lyme carditis. American Heart Journal 121, 1558–1560. Wang, G., Ojaimi, C., Wu H., Saksenberg, V., Iyer, R., Liveris, D., McClain S.A., Wormser, G.P. and Schwartz, I. (2002) Disease severity in a murine model of Lyme borreliosis is associated with the genotype of the infecting Borrelia burgdorferi sensu stricto strain. Journal of Infectious Diseases 186, 782–791. Woolf, P.K., Lorsung, E.M., Edwards, K.S., Li, K.I., Kanengiser, S.J., Ruddy, R.M. and Gewitz, M.H. (1991) Electrocardiographic findings in children with Lyme disease. Pediatric Emergency Care 7, 334–336. Xanthos, T., Lelovas, P., Kantsos, H., Dontas, I., Perrea, D. and Kouskouni, E. (2006) Lyme carditis: complete atrioventricular dissociation with need for temporary pacing. Hellenic Journal of Cardiology 47, 313–316.

12

Rheumatological Involvement Leonard H. Sigal

12.1 Introduction The first description of the syndrome that came to be known as Lyme disease began with the investigation of a group of patients living in three towns along the Connecticut River (Steere et al., 1977b,c), who experienced monoarthritis resembling juvenile rheumatoid arthritis. As the researchers noted, juvenile rheumatoid arthritis does not occur in geographical, familial or temporal clusters. The initial seroepidemiological exploration identified no pathogen, although the pattern of case distribution suggested an arthropodborne infection (Steere et al., 1977a). Many patients recalled a preceding tick bite and/or an expanding erythematous rash, consistent with erythema chronicum migrans (ECM) (Steere et al., 1978) – a rash initially described in Sweden by Afzelius in 1909 (Afzelius, 1910) and first noted in the USA in 1970 by Scrimenti in Wisconsin (Scrimenti, 1970). Of note, Lyme arthritis may have been present on Eastern Long Island and along the New England coast for many years prior to the identification of Lyme disease; residents of Eastern Long Island recall cases of ‘Montauk knee’ occurring as early as the 19th century. Thus, arthritis (i.e. inflammation of one or more joints) was the first noted consequence of Lyme borreliosis in the USA. However, true arthritis is only one of the many musculoskeletal consequences of this 190

infection (Sigal, 1994; Steere, 2001; Puius and Kalish, 2008). Other clinical findings of Lyme disease were described (Steere et al., 1977a). ECM, now more commonly referred to as erythema migrans (EM), in Europe (Hollström, 1951) and the USA (Steere et al., 1980, 1983b), responded to antibiotics. A trial of intravenous penicillin for Lyme arthritis showed success in the majority of patients (Steere et al., 1985). A spirochaete, isolated from patient and tick sources, was named Borrelia burgdorferi (Steere et al., 1983a). European Lyme borreliosis and Lyme disease in the USA have many similarities, although differences in the causative agents and the ambient immunogenetics of those affected may contribute to differences in clinical features of infection (Sigal, 1988; Sigal, 1997).

12.2 Musculoskeletal Consequences of Lyme borreliosis Diagnosing a patient with possible Lyme disease requires that the clinician understand the proper use of serological studies: when to order serological tests in the first place; how to interpret the isotype (class) ELISA reactivities typically reported (IgG and IgM; some laboratories also offer IgA testing); when to obtain Western blot (immunoblot) confirmation of ELISA reactivity; and how to

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interpret IgG and IgM Western blot reactivity. It is very important to know when not to order serological tests. A positive test in a patient with a very low a priori likelihood of Lyme disease has a very low positive predictive value; in other words, the result is likely to be of no consequence and may cause confusion, anxiety and incorrect conclusions. Likewise, it is important to be wary of new testing technologies – all are potentially flawed by technique and/or interpretation, and none is as valuable in diagnosing Lyme arthritis as taking a detailed history and doing a complete physical examination. The term ‘Lyme disease test’ is a misnomer and is potentially dangerous. Serological tests merely detect antibodies that bind to B. burgdorferi. No test, in itself, is diagnostic of Lyme disease. When interpreted appropriately, used in a clinical setting suggestive of Lyme disease, these tests can offer substantiation of that clinical impression. Do not invest in a diagnosis of Lyme arthritis unless there is explicit evidence suggesting the diagnosis, including objective evidence on physical examination or specific historical features. Do not diagnose Lyme disease based merely on time of year, geographical location, family or neighbourhood history, local enthusiasm or whim. 12.2.1 Arthralgias/migratory polyarthralgias/myalgias The musculoskeletal system is commonly affected in early Lyme disease (Sigal, 1988) with symptoms including arthralgias – joint pain without inflammation – and true arthritis – joint inflammation, manifested by heat, warmth, erythema, pain, swelling and loss of function. The cause of the typically migratory polyarthralgias noted in many patients with early Lyme disease is unclear, but there is no evidence to suggest that there are live B. burgdorferi within affected joints. In the largest reported series of untreated patients with EM (patients accrued from 1976 to 1979) (Steere et al., 1987), 80% (44/55) developed some sort of musculoskeletal manifestation of Lyme disease. Noninflammatory musculoskeletal pain occurred

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in 18% (10/55), including arthralgias and myalgias, as well as pain in tendons, bursae, entheses (point of insertion of a ligament or tendon into bone) and bones. These were typically brief episodes of migratory mild to moderate pain with no signs of inflammation, usually beginning a mean of 2 weeks (range 1 day to 8 weeks) following the EM rash. Symptoms recurred for as long as 6 years, often with accompanying fatigue. Myalgias and arthralgias are common features of early Lyme disease. If they occur in the presence of EM or other findings explicitly suggestive of the diagnosis of Lyme disease, serological confirmation of the diagnosis may not be necessary. On the other hand, these non-specific complaints may be a relatively early finding of B. burgdorferi infection, often a harbinger of other clinical findings of Lyme disease, and may occur in the absence of EM or any other evidence of B. burgdorferi infection. Serological tests at this early stage are often negative; watchful waiting is in order rather than laboratory testing. A strongly positive serological test in such a patient would be unexpected, as this is early disease – such a result may be reactivity related to a prior infection or a false-positive test. Of greater concern is that the patient does not now have Lyme disease and the result is a false positive. Non-specific complaints in a patient from an endemic area may result in an incorrect diagnosis of Lyme disease, exposing the patient to the dual risks of unnecessary antibiotics and the possibility that another underlying disorder, perhaps one more serious than Lyme disease (perhaps life-threatening), will not be considered. Differential diagnosis Migratory polyarthralgias can occur in the setting of viral infections and early in the evolution of inflammatory rheumatological syndromes (e.g. systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis/polymyalgia rheumatica, seronegative spondyloarthropathies and sarcoidosis). It is crucial not to jump to the diagnosis of Lyme disease in the absence of objective evidence specifically supportive of this diagnosis.

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12.2.2 Monoarthritis/oligoarthritis The best-known inflammatory joint feature of Lyme disease is monoarthritis, usually affecting the knees. The knee is typically (although not always) quite swollen, with effusions of over 100 ml common. The knee is often more stiff and difficult to move than painful – pain may be absent, a pattern that should make one consider this diagnosis. Many adult patients with Lyme arthritis have no prior history of EM or other features of Lyme disease, but a history of prior findings suggestive of Lyme disease should be sought (e.g. EM, aseptic meningitis, carditis, or cranial or peripheral neuropathy). Prior or current ‘flu-like symptoms’ are not helpful as these are quite non-specific, usually only occurring in early disease, and most often are due to other processes. Physical examination to seek evidence of other features of Lyme disease is mandatory but often not fruitful. Laboratory evaluation focuses on blood and synovial fluid. The IgG anti-B. burgdorferi serology should be positive when Lyme arthritis is active – if the test is negative the diagnosis of Lyme arthritis should be very much in doubt. The corroborative Western blot should reveal a broad spectrum of reactivity, but no pattern is diagnostic, nor is there a pattern that predicts outcome. Antibody levels should not be expected to fall with antibiotic therapy and therefore levels should not be followed after therapy: persisting antibody levels are not a prognostic marker. There are three assays for which there is evidence of correlation with disease activity, although only one is currently commercially available. The latter is the C6 peptide assay, an ELISA based on a peptide derived from the invariable region 6 of VlsE, the antigenic variation protein of B. burgdorferi. The assay is both sensitive and specific for serologic confirmation of Lyme disease; one study showed changes in titers correlated with outcomes to antibiotic therapy in patients with Lyme disease (Philipp 2003). The second is a technique which detects serum immune complexes containing specific anti-B. burgdorferi antibodies and B. burgdorferi antigens (Brunner 1998, Brunner 2000, Brunner 2001), but is not available. Immune

complexes are formed only when B. burgdorferi is alive to liberate antigens to then be bound by antibodies. These complexes should not be confused with cryoglobulins found in Lyme disease patients, an immune phenomenon that correlated with the presence of arthritis (Hardin et al. 1979a and 1979b). The third test identifies borreliacidal antibodies, which bind OspC and kill the spirochetes by activation of complement (Rousselle 1998). A semi-automated flow cytometric assay provided sensitive and highly specific confirmation of the diagnosis (Callister 2002), but the test is not widely used because of the logistical complexity of flow cytometry requiring live spirochetes. The developers are exploring commercialization opportunities utilizing a peptide-based assay (Jobe 2008). There is no reason to believe that measuring the number of CD57+ natural killer (NK) cells in blood is of assistance in the diagnosis or management of Lyme disease (Marques 2009). Routine testing for the presence of antibodies to any of the organisms which can be “co-infections” with B. burgdorferi, e.g. Babesia microti, Anaplasma phagocytophila (previously known as “the human granulocytic Ehrlichia”), or Bartonella hensellae, is not warranted, unless clinically warranted. Synovial fluid should be analysed for cell count, chemistries, routine culture and crystal analysis (if warranted). Analysis of the fluid usually reveals a moderately elevated white blood cell count (a typical count is 20,000– 25,000 cells/l) with a neutrophilic predominance. The protein is elevated, the mean being 4.5 g/dl (Steere, 2001). There is nothing in the routine fluid analysis that is uniquely diagnostic or even suggestive of the specific diagnosis of Lyme disease. There is concentration of immune reactivity within the synovial space, both antigen-specific antibodies and mononuclear cells (Pachner et al., 1985; Sigal et al., 1986; Sigal, 1997). However, neither study is clinically useful, due to a lack of technique standardization (the former) and the need to get the fresh cells to the laboratory immediately (the latter). Culture of synovial fluid has a very low yield, and is not recommended in clinical practice, as anything but the extremely rare

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positive cannot be interpreted. Over 20 years ago, a novel growth medium based on Detroit city tap water was reported as growing B. burgdorferi in a very high proportion of samples; these results were never replicated and remain unexplained, but do not offer hope that culture can be a useful tool in clinical practice (Phillips et al., 1998). PCR is becoming more broadly available from reputable laboratories but is not recommended as a routine diagnostic tool. Clinical findings supplemented by judiciously obtained and interpreted serological results should be sufficient to make the diagnosis of Lyme arthritis. PCR can detect B. burgdorferi in a variety of clinical specimens, including synovial fluid and tissue (the latter is likely to have a better yield than the former). The yield in some studies has been as high as 80–85% (Nishio et al., 1993; Nocton et al., 1994). It has been suggested that PCR might be useful in determining whether sufficient antibiotic therapy has been given. PCR has been reported to become negative after receipt of 1–2 months of antibiotics (Carlson et al., 1999). However, while persisting PCR positivity may indicate ongoing infection, dead organisms can also give a positive PCR result (Sigal, 2001). Thus, PCR cannot be used as a ‘gold standard’; a positive test may not mean active infection – it could be due to the presence of dead or dying spirochaetes – and a negative test may not equate with cure, as, especially in inexperienced hands, the test may be falsely negative if there are very few organisms present in the sample. Differential diagnosis The immediate differential diagnosis of a monoarthritis is the triad of sepsis, crystalinduced and trauma. Although Lyme arthritis is an infection, at least early in its course its presentation is not as abrupt and its magnitude not as severe as arthritides caused by pyogenic organisms (e.g. Staphylococcus and Streptococcus species). Joint infections can also be caused by lessvirulent organisms, especially in immunocompromised patients, in whom the degree of inflammation from such infections may mimic Lyme arthritis. All fluids aspirated from a possibly infected joint must be sent for

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a Gram stain, cell count, chemistries and – always – culture. Infections typically cause a moderate to severely inflammatory fluid, with 20,000–100,000 cells/l, depending on the organism, with neutrophilic predominance; a Gram stain may reveal organisms. Clinical clues suggesting gonococcal infection can be obtained from a history (gonococcal monoarthritis may be preceded by migratory polyarthralgias) and physical examination (e.g. cutaneous pustules or other lesions, genital discharge). Gout (monosodium urate crystalinduced disease) and ‘pseudo-gout’ (calcium pyrophosphate dehydrate crystal-induced disease) usually present with an abrupt and more rapidly evolving severe monoarthritis, although occasionally the severity may mimic that of Lyme arthritis. Patients with gout are typically older males and may have a past history of such events or tophi and/or of features of the cardiac dysmetabolic syndrome. The joint is typically very swollen and invariably intensely painful. Desquamation over the affected joint can be seen in gout as the inflammation wanes, a sign never described in Lyme arthritis. Fluid analysis reveals moderately to severely inflammatory fluid, with 20,000 to 100,000 cells/l. Polarizing microscopy may reveal crystals within cells; finding a single crystal within a cell is diagnostic. Recent knee trauma may be blatant or subtle, and a history of even trivial trauma should be sought. Knee effusions may appear overnight in the setting of ‘internal knee derangement’ (e.g. torn meniscus or cartilage) and are due to a mechanical synovitis. The amount of intra-articular fluid build-up is usually less than seen in Lyme arthritis. Another difference is that mechanical synovitis is usually painful, especially on weight bearing and movements that stress the damaged structure. Synovial fluid analysis typically reveals at most mildly inflammatory fluid, with 500–3,000 cells/l. Finding blood in all tubes collected is evidence of a recently torn internal structure. Prior injury (e.g. damaged meniscus or ligaments) with chronic low level mechanical synovitis can also cause intermittent, although usually small, knee effusions with mildly inflammatory fluid.

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Osteoarthritis may be the cause of an acute bland effusion in older patients. A history and physical examination will be helpful in establishing this diagnosis, as there is often evidence of osteoarthritis elsewhere. However, the finding of a virtually ubiquitous disorder like osteoarthritis should not be taken as definitive evidence that the monoarthritis being evaluated is not Lyme disease – a proper evaluation should proceed to its logical outcome. Isolated monoarthritis or oligoarthritis may be the presenting feature of rheumatoid arthritis, reactive arthritis or psoriatic arthritis. The passage of time will reveal the evolution of these chronic inflammatory diseases. Asymmetric oligo- or polyarthritis may represent emerging psoriatic or reactive arthritis (the former affecting the upper more than the lower extremities, with the reverse pattern typical of the latter). Evidence of integumentary features of psoriasis (nails, extensor surface of the arms, scalp, periumbilicus and intergluteal fold), family history, eye inflammation, dactylitis and/or enthesitis and spondylitis may allow the diagnosis to be made definitively. The latter diagnosis is more likely with recent exposure to culpable enteric or genitourinary pathogens. The full differential diagnosis of oligoarthritis is beyond the scope of this chapter (the interested reader is referred to Sigal, 1994). Unless there is explicit evidence suggesting that the arthritis is due to Lyme disease (i.e. the a priori likelihood of the diagnosis is great), serological studies should not be done. In areas with endemic Lyme disease, prior – even inapparent – B. burgdorferi infection may be the cause of a persisting positive test that may be unrelated to the current arthritis. Plausible exposure should be considered; for example, a man who lives in an endemic area who takes walks with his dog in the fields and along deer paths is at risk, whereas an elderly woman who lives in an endemic area and never leaves her house except to get the mail and go shopping is not. Even in patients with no evidence of B. burgdorferi of any sort, falsepositive test results can lead to a false diagnosis, for, as Bayes said, if the a priori likelihood of a test is low, the positive

predictive value of a positive result will also be low. Polyarthritis (inflammation of multiple joints) is an extraordinarily rare feature of Lyme disease (the author has seen this twice in a long consultation career). Most patients with true polyarthritis after an episode of Lyme disease, even if the diagnosis of Lyme disease is correct (and misdiagnosis must be a consideration), have rheumatoid arthritis, lupus, psoriatic arthritis, viral arthritis (in this case, a transient polyarthritis) or some other cause. These and other possible causes of polyarthritis must be evaluated thoroughly. The diagnosis of Lyme disease in such a setting must be a diagnosis based on explicit evidence, but also one of exclusion. The exclusionary process must be pursued aggressively and without a priori bias in favour of the diagnosis of Lyme disease. Bursitis and tendonitis have been reported by many patients with Lyme disease, but there is no proof of the presence of the organism at the site of inflammation. There have been isolated cases reported of the biopsy-proven presence of B. burgdorferi in cases of fasciitis, myositis and dermatomyositis-like syndromes. 12.2.3 Patellofemoral joint dysfunction Osteoarthritis of the patellofemoral joint (also called by its histopathological name, chondromalacia patella) is a common cause of knee pain, regardless of age, and can cause small to moderate knee effusions of bland to mildly inflammatory fluid. Patellofemoral joint dysfunction is caused by malalignment of the patella in the femoral groove due to imbalance of the four muscle bellies that come together in the quadriceps femoris muscle (anterior thigh) and causes pain localized to the front of the knee or described as being immediately behind the patella. Any form of knee inflammation or serious damage, even of a relatively trivial nature, may cause reactive quadriceps muscle atrophy resulting in such imbalance. Patellofemoral joint dysfunction is quite common and should be considered as a cause of knee pain with modest effusion. Treatment is physical therapy to strengthen the muscles, to realign the patellofemoral joint. Even

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though the cause of the muscle atrophy may have been prior Lyme arthritis, in such a patient ongoing knee pain and effusion is due to patellofemoral joint dysfunction, not ongoing infection or inflammation. Correct diagnosis dictates proper therapy. 12.2.4 Persisting musculoskeletal pain following antibiotic therapy Following appropriate antibiotic therapy some patients with Lyme disease have persistence of some or all of their initial complaints, which may include musculoskeletal pain (Sigal, 1990). As long as the complaints are not increasing in severity or expanding in location and there are no new complaints, a pattern of slow but steady improvement is expected. Such persisting but slowly improving symptoms should not cause concern about ongoing B. burgdorferi infection. The appearance of new complaints, especially if accompanied by objective evidence of new inflammation or new organ system dysfunction, may represent ongoing infection, a new infection or the consequences of another independent pathological process, such as rheumatoid arthritis developing following (not because of) Lyme disease. Such a change should elicit a diligent search for objective evidence of infection or another process. As noted, patellofemoral joint pain may represent prior Lyme arthritis having induced muscle atrophy with subsequent mechanical damage causing symptoms and a mild synovitis. This should be treated with physical therapy, not further antibiotics. 12.2.5 Fibromyalgia-like syndromes Fibromyalgia is a relatively common noninflammatory musculoskeletal pain syndrome, often related to sleep disturbance/ deprivation and muscle deconditioning. Presenting complaints include widespread non-inflammatory pain, usually not localized solely to the joints; fatigue, with difficulty falling or staying asleep (these patients often need daytime naps); and cognitive, concentration and memory difficulties. Of note, some patients with early Lyme disease experience sleep disturbance, which can

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rarely persist even after adequate antibioticinduced eradication of infection. Patients presenting to Lyme disease clinics may have fibromyalgia, either full-blown or as a forme fruste (Sigal and Patella, 1992; Hsu et al., 1993; Lightfoot et al., 1993). Some may have been appropriately diagnosed and treated for Lyme disease, whereas in others that diagnosis was fallacious. In some, the musculoskeletal complaints of fibromyalgia have been mistaken for the polyarthralgia of early Lyme disease. In others, this noninflammatory process has been misclassified as polyarthritis and treated as if it was a manifestation of ongoing active infection. Many such diagnoses are based on speculation (by the patient or clinician) and/ or misuse of laboratory testing (either established or esoteric). Initial improvement of these vague complaints coincident with antibiotic therapy is often viewed (incorrectly) as proof of the diagnosis of Lyme disease. Inevitably, the antibiotics are ineffective and the patient suffers (Sigal, 1996). In whatever manner an incorrect diagnosis was made, many such patients have been subjected to repeated courses of inappropriate antibiotics. A review of recent developments in the pathogenesis and treatment of fibromyalgia is beyond the scope of this chapter. (The interested reader is referred to Sigal and Hassett, 2002).

12.3 Hypothesized Pathogenesis of the Clinical Manifestations Central to determining appropriate therapies for disease processes is identifying the pathogenesis of the disease’s various manifestations (Sigal, 1997). B. burgdorferi interacts with the host’s immune system in a number of different ways. In the mouse (and probably universally), B. burgdorferi does not produce or release toxins. The damage to infected organs (i.e. clinical disease) is determined by the host’s immune response to the organism, with release of pro-inflammatory molecules a part of that response. Peromyscus leucopus, the white footed field mouse, a reservoir host, does not get sick despite persistent carriage of the organism. This mouse has an inflammatory response that essentially ignores B. burgdorferi

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and there is no reason to believe the mouse is any the worse for this immunological lacuna. Any pathogenic organism elicits a range of both non-specific and antigen-specific immune responses, which can cause the variety of inflammatory manifestations occurring with that infection. Non-specific responses mediated by the innate immune system include complement fixation, with binding by C-reactive protein or other scavengers, engagement by Toll-like receptors (TLRs) or nucleotide-binding oligomerization domains (NODs), or being recognized by other innate immune receptors. Borrelial antigens are taken up by antigen-presenting cells (APCs), resulting in cytokine release and the initiation of antigen-specific T-cell activation. B cells, the other cells capable of producing an antigen-specific immune response, bind borrelial antigens without APC help. When activated, they are producers of anti-inflammatory cytokines and pro-bodies through antigen-specific immune activation. An infection may cause clinical signs and symptoms due to:  local inflammation due to the organism’s presence and the attendant immune response to it;  toxins released by the organism (some organisms produce toxins that cause damage, e.g. Clostridium difficile, Staphylococus aureus, but this is not the case with B. burgdorferi);  production of pro-inflammatory substances during the immune response to the organism;  a poorly regulated local immune reaction with a shift to an autonomous, independent and unregulated inflammatory condition;  a poorly regulated immune response to the organism resulting in autoimmune reactivity, e.g. molecular mimicry. This model, that ‘disease is due to the immune/inflammatory response to the organism’ (Sigal, 1997), provides a logical framework for understanding B. burgdorferi infection (Sigal, 1994), permits a better understanding of its various manifestations and informs the choice of potential appropriate therapies.

12.3.1 Local presence of the organism, dead or alive There is ample evidence to suggest that B. burgdorferi is present at a number of sites of inflammation and organ dysfunction: the skin (EM and acrodermatitis chronicum atrophicans), the heart (myocardium in cardiomyopathy, and the conduction system in conduction defects), the brain (meninges and spinal fluid in meningitis and brain encephalitis) and the joint (synovitis). It is reasonable to assume that the immune/ inflammatory reaction to the organism is the cause of organ dysfunction at each site. Thus, in each of these clinical circumstances one should expect that appropriate antibiotic therapy will eradicate the organism with resolution of the signs and symptoms resulting from local infection. No isolate of B. burgdorferi has ever been found to be resistant to the standard antibiotics recommended in the Infectious Disease Society of America (IDSA) guidelines (Hunfeld et al., 2005; Wormser et al., 2006). As noted, there is no evidence that this organism produces any toxins itself. The only toxic substances present at the site of inflammation are those produced by the host. Were there to be inflammation focused on debris of dead/dying organisms, one might expect a delay in the resolution of symptoms until the debris is cleared. This may be an explanation for the persistence of some symptoms in Lyme disease (Sigal, 1988; Sigal, 1994). The ultimate response will not be accelerated by further antibiotics. Some have theorized that live but dormant or some otherwise ‘alternative lifestyle’ B. burgdorferi persist within the synovial space, thereby causing ongoing inflammation. If a persisting organism is within a host cell, it is unlikely to be the source of ongoing inflammation. Intracellular organisms usually do their best to avoid immune activation, as inflammation will probably lead to the organism’s death. In the case of a dormant or hiding organism causing disease, the adaptive immune response would recognize it (the infection is not in an immunologically inaccessible state) and attack it, in concert with the antibiotics given to such patients. The lack of response and the usual lack of true inflammation in such cases

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make the claim of ongoing infection lose much of its plausibility. 12.3.2 Pro-inflammatory molecule release Many of the inflammatory and noninflammatory features of Lyme disease are due to the immune reaction to the organism (Sigal, 1997; Shin et al., 2007). This can include release of certain cytokines, such as interleukin (IL)-1 and tumour necrosis factor α (TNF-α) (which can cause fatigue, cognitive dysfunction and widespread musculoskeletal symptoms), as well as other pro-inflammatory molecules (Shin et al., 2007). Recent reports have documented the release of both type I and type II interferons, the former via interaction of B. burgdorferi with TLR7 and TLR9 (Petzke et al., 2009) and the latter possibly from NKT cells (Olson et al., 2009). The interaction of B. burgdorferi (more vigorous when using live rather than heatkilled organisms) with TLR7 and TLR9 also caused peripheral blood mononuclear cells to produce an array of NF-κB-associated cytokines and chemokines, including TNF-α, IL-1, IL-6, IL-8, IL-10,- and IL-12 (Petzke et al., 2009). A B. burgdorferi virulence factor, neutrophil-activating protein A (NapA) binds to TLR2 on monocytes, with release of IL-1, IL-6, IL-17, IL-23 and transforming growth factor β (TGF-β), suggesting that NapA is a potent activator of T-helper 17 (Th17) cells, which have been implicated in the chronic inflammation of rheumatoid arthritis. Th17 cells produce large amounts of IL-17 (thus their name), an inducer of cytokine release by stromal cells, synoviocytes, chondrocytes, fibroblasts and macrophages, and a potent recruiter and activator of neutrophils (Codolo et al., 2008). Thus, there is ample evidence of B. burgdorferi’s ability to activate innate immune mechanisms that effectively drive inflammation. It is clear that there are many components of B. burgdorferi that are immunogenic, eliciting antigen-specific immune responses. The organism is also capable of causing nonantigen-specific activation of many B cells, a phenomenon known as ‘polyclonal B-cell activation’ (Sigal et al., 1988). There is no evidence that this feature of B. burgdorferi is

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involved in the immunopathogenesis of disease. There is no evidence that B. burgdorferi contains a T-cell superantigen, which would cause non-antigen-specific activation of multiple T-cell families. The consequence of the resulting abrupt release of large quantities of T-cell-derived cytokines would be similar to toxic shock syndrome, caused by a staphylococcal superantigen – something never reported to occur as a manifestation of or in association with Lyme disease. The ‘flu-like syndrome’ of early Lyme disease is probably due to release of IL-1 and other inflammatory mediators, known to cause ‘flu-like symptoms’ in viral infections. The recurrent ‘flu-like symptoms’ reported by some later in the course of ‘chronic Lyme disease’ are likely to be non-specific and not of an immunological aetiology. As noted, production of these proinflammatory cytokines may also be due to stimulation of immune cells by debris derived from dead organisms, which could drive such an inflammatory process. The persistence within certain joints of non-viable organism-related material or live organisms may drive the inflammatory process in reactive arthritis (usually oligoarthritis following certain genitourinary or gastrointestinal infections; Petersel and Sigal, 2005). The fact that early antibiotic therapy may prevent the establishment of chronic synovitis is in keeping with the premise that early synovitis is due to active local infection, but later antibiotic-refractory synovitis may not represent ongoing infection. 12.3.3 Autonomous self-perpetuating immune/inflammatory reaction Early in the investigation of the immunopathogenesis of Lyme arthritis, studies suggested that the primary driver of the inflammation was a Th1 cell response to borrelial antigens, with gamma interferon (IFN-) the most prominent modulator of the process (Yssel et al., 1991). More recent work has called this view into question. Evidence is now pointing to a fundamental role for Th17 cells. One aspect of Th17 activation has already been described briefly in this chapter. A good summary of the potential role of Th17

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cells in the immunopathogenesis of Lyme arthritis is found in the review by Nardelli et al. (2008a). The autonomous activation of Th17 independent of further antigen-specific mechanisms may be an explanation for ongoing self-perpetuating inflammation independent of ongoing infection in antibiotic-refractory Lyme arthritis (Nardelli et al., 2008b; Nardelli and Schell, 2009), perhaps in a manner similar to, but different from, rheumatoid arthritis. IL-23 has also been implicated in the mouse model of Lyme arthritis (Kotloski et al., 2008). Dysfunction of CD4+ CD25+ T regulatory cells (Tregs) might be involved in the immunopathogenesis of Lyme arthritis (Nardelli et al., 2004, 2005). Shen et al. (2010) recently published a correlational study suggesting that a higher percentage of Tregs in the synovial fluid of patients with antibiotic-refractory Lyme arthritis is associated with a shorter time to resolution of the synovitis; the precise role of these cells in the antigen-specific T-cell response to B. burgdorferi remains to be fully defined. Singh and Girschick (2004) described means by which B. burgdorferi might cause a selfperpetuating disease. 12.3.4 Molecular mimicry Molecular mimicry is a condition in which an invading organism contains a molecule – an immunogen – that, as determined by the host’s immune system’s identifying skills, resembles a host molecule. When the organism-derived immunogen is recognized by the host’s immune system the pathogen is attacked, but so is the host molecule that the immune system recognizes as looking like that particular organism’s immunogen, a concept known as ‘crossreacting’. Recognition of the cross-reacting host molecule leads to subsequent damage to the host. Examples of this phenomenon include Chagas disease (Trypanosoma cruzi cross-reacts with human cardiac muscle and peripheral nerve, causing Chagasic cardiomyopathy and neuropathy) and rheumatic fever (the M proteins of certain group A -hemolytic streptococci cross-react with human cardiac myosin, causing rheumatic carditis). A similar breakdown of im-

munological tolerance can occur when antigens that have been sequestered (i.e. not available to the immune system previously) are suddenly liberated. The immune response to these antigens can then cause an organspecific autoimmune disease, such as posttraumatic sympathetic ophthalmitis and post-pericardiotomy (Dressler’s) syndrome. One proposed explanation for the development of antibiotic-refractory Lyme arthritis has been molecular mimicry, the presence of cross-reactivity between B. burgdorferi and synovial antigens. Steere and colleagues found that lymphocyte functionassociated antigen 1 (LFA-1; also known as αLβ2 integrin and CD11a/CD18) contained an epitope that cross-reacts with an epitope on outer-surface protein A (OspA) (Gross et al., 1998); both epitopes are presented by the same human leukocyte antigen (HLA)-DR molecule, HLA-DR4, on the surface of APCs. HLA-DR4 has been associated with antibioticrefractory Lyme arthritis and implicated as a risk factor. The same group found that antibiotic-refractory Lyme arthritis was associated with T-cell responses to certain epitopes of OspA (notably, not the same as the cross-reacting epitope noted above; Chen et al., 1999; Steere et al., 2003). This and further work was viewed as suggesting that molecular mimicry, leading to an organspecific autoimmunity, was at the root of antibiotic-refractory Lyme arthritis (Steere et al., 2001; Ball et al., 2009; Iliopoulou et al., 2009; Jones, 2009). Kalish et al. (2003) found no such association between clinical status and T-cell responses to OspA or LFA-1. Maier and colleagues found that Osp-A-specific T cells recognized many epitopes on many other human proteins, causing the authors to conclude that ‘…the existence of crossreactive epitopes alone does not imply molecular mimicry-mediated pathology and autoimmunity’ (Maier et al., 2000). LFA-1 is a ubiquitous protein; thus, autoimmunity would be expected to be a more systemic phenomenon were cross-reactivity to be aetiologically relevant (Sigal, 1997). Instead, there is a postulated autoimmune process affecting a single organ system, in fact a single joint. One might then speculate that the cross-reacting immune principle was active only in the joint, either because the specific LFA-1 epitope is available only in the

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synovium or because the immune reactivity is somehow isolated to the synovial space of a single joint. Neither of these theories is supportable by scientific evidence, and in fact the latter is illogical. An additional example of molecular mimicry is found between a linear epitope of the B. burgdorferi flagellin (p41) protein and another linear epitope of human heat-shock protein 60 (the latter epitope exposed only in cytoplasm and not mitochondrial HSP60) (Dai et al., 1993; Fikrig et al., 1993; Sigal and Tatum, 1988a,b; Sigal, 1993). A single monoclonal antibody to flagellin, H9724, binds to these two epitopes. Sera from patients with neurological manifestations of Lyme disease also bound these epitopes. When added to cultures of neuroblastoma cells, H9724 penetrated living neuroblastoma cells, profoundly suppressing the outgrowth of dendrites. Alteration of axonal/neuronal function has been suggested as a cause of the peripheral neuropathy of Lyme disease, but no clinical role for these cross-reacting antibodies has been proven. Thus, although there are two examples of molecular mimicry of theoretical interest in our understanding of the immunopathogenesis of different aspects of Lyme disease, neither has been demonstrated to be clinically relevant. In any event, were these mechanisms to be germane to the immunopathogenesis of antibiotic-refractory Lyme arthritis or neuropathy, there would be no role for further antibiotics. Archimedes is said to have noted that, given a long enough lever arm, he could move the world; using computerized searches, one can often find high-stringency cross-reactivity between a pathogen-related protein and many proteins in data banks, of human, mammalian and other sources. One must separate the wheat from the chaff. 12.3.5 The dulling of Ockham’s razor – the presence of a second pathogenetic process unrelated to B. burgdorferi infection From early in medical training we are drilled with the importance of Ockham’s razor, in the words of William of Ockham (William Seach, a 14th-century Franciscan friar) variously:

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Entia non sunt multiplicanda praeter necessitatem [Entities should not be multiplied unnecessarily]; numquam ponenda est pluralitas sine necessitate [Plurality must never be posited without necessity], frustra fit per plura quod potest fieri per pauciora [It is futile to do with more things that which can be done with fewer]. In the late 20th century, this was adapted directly from the Latin as, ‘Keep it simple, stupid’. However, sometimes the hoofbeats we hear are from an additional species. Some patients with prior established Lyme disease may be, develop or become symptomatic with other illnesses. One might predict the incidence of rheumatoid arthritis, ankylosing spondylitis, or other autoimmune and nonautoimmune disorders to be that of society at large, regardless of prior exposure to B. burgdorferi. Thus, a second, unrelated disorder can afflict a patient who has, or has previously been exposed to, Lyme disease. As noted, the second ailment might be related to Lyme disease without being directly attributable to ongoing infection per se (e.g. patellofemoral joint dysfunction); the treatment for such a second affliction is likely not to be antibiotics.

12.4 Proposed Treatments for these Clinical Consequences Many different forms of therapy have been proposed for Lyme disease. Antibiotics, intravenous or oral, depending on the manifestations of disease and prior treatment history, are effective in most patients with musculoskeletal features of Lyme disease. As noted, however, many patients with persisting musculoskeletal symptomatology do not have evidence of active infection with B. burgdorferi, and many have evidence suggesting the presence of another disease. It is far beyond the scope of this chapter to discuss therapies for the many non-Lyme disease causes of musculoskeletal symptoms in such patients, a list that includes fibromyalgia, patellofemoral joint dysfunction, rheumatoid arthritis, lupus, the seronegative spondyloarthropathies (ankylosing spondylitis, psoriatic arthritis, reactive arthritis), and osteoarthritis (Sigal, 1990; Sigal and Patella, 1992; Hsu et al., 1993; Lightfoot et al., 1993).

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Antibiotic therapy has been a topic of heated debate, with no sign of abatement. None the less, a recent review of the 2006 IDSA therapeutic guidelines by an independent panel, formed as a result of a lawsuit in Connecticut, unanimously found no reason to change these guidelines, based on the best evidence available. Thus, the antibiotic doses and durations suggested in this chapter are identical to those of the guidelines. Persisting symptoms, mistakenly interpreted as indicative of ongoing infection, have led some clinicians to innovative, albeit flawed, approaches to try to eradicate the suspected ongoing infection. The approach of ‘antibiotic treatment until cessation of symptoms’ is fundamentally flawed, potentially dangerous (both medically and psychologically) (Wormser et al., 2006) and has no place in informed, evidence-based practice. ‘Creative’ therapies have included some that might be considered bizarre:  In the 1970s and 1980s, patients were taken to a foreign country and given malaria, in an attempt to replicate a form of fever therapy used for syphilis (Heimlich, 1990); this form of therapy has never been subjected to scientific study, but apparently was ineffective.  Hyperbaric therapy was, and is, occasionally used, a therapeutic strategy apparently based on the aversion of B. burgdorferi for oxygen in vitro (Taylor and Simpson, 2005; Stricker, 2006;). Despite mention in these two reviews, this approach has never been subjected to scientific scrutiny and remains a highly speculative approach in the repertoire of certain ‘Lyme disease experts’ only. Its use is seemingly based more on the availability of the hyperbaric chamber than on proof of efficacy.  Cholestyramine has been suggested as a means of removing a theoretical toxin that certain clinicians believe is produced by B. burgdorferi; the claim is that this toxin causes some of the symptoms of chronic Lyme disease (Shoemaker et al., 2006). This toxin has never been identified and there is no evidence that it exists.  Hydroxychloroquine is an effective form

of treatment for the self-perpetuating synovitis seen in patients with rheumatoid arthritis or lupus, and may have an effect on the chronic synovitis of Lyme arthritis (Stricker, 2007). There is no clinical proof that hydroxychloroquine enhances the efficacy of antibiotics in the treatment of intracellular B. burgdorferi by enhancing entry of antibiotics into cells, thus increasing the likelihood of eradicating an occult site of infection, or by any innate antimicrobial activity.  Some clinicians continue to use very prolonged antibiotic therapy (in the author’s experience, 9 years of nearly continuous therapy is the record), combinations of oral and/or intravenous antibiotics, ‘cycling’ through various antibiotics and the addition of hydroxychloroquine to antibiotics (Sigal, 1990, 1994; Hunfeld et al., 2005). None of these therapies has established validity and thus these and other therapies discussed and rejected by the IDSA panel should be eschewed (Wormser et al., 2006). None of these therapies has ever been subjected to rigorous scientific study and found to be effective. In fact, Klempner et al. (2001) addressed the issue of long-term therapy and found it without merit. Scientifically valid studies supporting prolonged therapy have not been forthcoming. There have been claims that long-term therapy is of value (Stricker, 2007), but these assertions are not based on scientific study; in fact, there has never been a formal study of such prolonged antibiotic therapy by its advocates. None the less, some clinicians think that long-term therapy, until symptoms abate, is warranted. This is not evidence-based practice, but rather is based on speculation and hearsay, and does not serve the patients well, exposing them to the added risks of catheter infections, toxicities of the various agents used (minor and major) and expense.  Some clinicians prescribe diets, naturopathic therapies and neutraceuticals for their patients with ongoing musculoskeletal pains due to ‘chronic Lyme disease’. There is no evidence that any such approach has any effect.

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A word of caution: in treating Lyme disease, one must have patience. The organism does not die immediately. Debris from dead B. burgdorferi is not cleared immediately by phagocytes. Cytokine production does not decrease to baseline levels immediately. Early in the course of Lyme disease, when EM is present (or recently resolved) or other objective features of early Lyme disease are noted, musculoskeletal features are usually not the most prominent feature. None the less, in Steere’s classic description of the musculoskeletal features of Lyme disease (Steere et al., 1987), 62% of patients with early Lyme disease (34/55 patients with untreated early Lyme disease) experienced musculoskeletal complaints, including arthralgias and myalgias. The former may include pain in a single joint (monoarthralgia), pain in multiple joints (polyarthralgia), migratory joint pains (migratory polyarthralgia) or in periarticular structures (e.g. enthesis). Symptomatic therapy is in order, for example acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs) at recommended doses, in addition to the appropriate antibiotic therapy. True arthritis (i.e. joint inflammation) is uncommon in early Lyme disease. Its presence should suggest a second underlying disorder, as noted above. According to the IDSA guidelines (Wormser et al., 2006), the recommended antibiotic therapy for early Lyme disease is doxycycline (100 mg per os (PO;) twice a day), amoxicillin (500 mg PO three times a day) or cefuroxime axetil (500 mg PO twice a day) for 14 days (10– 21 days for doxycycline and 14–21 days for the latter two agents). First-generation cephalosporins are ineffective in the treatment of Lyme disease and therefore have no role. Other antibiotics are not of proven efficacy. Many patients experience persistence of non-specific complaints, often including arthralgias. Further therapy for these ongoing issues should be symptomatic, there being no evidence that further antibiotic therapy is warranted. As long as these complaints are not worsening and no new manifestations of Lyme disease occur, no further antibiotics should be given. It is crucial to evaluate all new complaints thoroughly. Thus, a patient with early Lyme disease and arthralgias

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given appropriate oral therapy who then develops central nervous system features of Lyme disease or true joint inflammation may require antibiotic therapy, whereas a patient whose joints continue to hurt, with the same or diminishing intensity, should complete the oral therapy with the initially prescribed dose and duration. Enthesitis is a common feature of the seronegative spondyloarthopathies, which include ankylosing spondylitis, psoriatic arthritis, reactive arthritis and inflammatory joint disease in the presence of inflammatory bowel disease. A single report by Weyand and Goronzy (1989) suggested that B. burgdorferi infection might be the cause of reactive arthritis, and there is a separate case report of sacroiliitis in a patient with Lyme disease (Kinigadner et al., 1991). There have been no further reports to substantiate this association and nothing to link any features of Lyme disease to HLA-B27, the genetic marker for susceptibility to reactive arthritis and ankylosing spondylitis. Thus, there is no proof that sacroiliitis and/or spondylitis are features of Lyme disease, regardless of the presence of HLA-B27. Patients with enthesitis and/or dactylitis as part of their ‘Lyme disease’ should be treated symptomatically, with antibiotics as appropriate for the primary features of their disease, and evaluated for an underlying seronegative spondyloarthopathy. Later in disease, arthritis (i.e. true inflammatory joint disease) may occur, most often as a monoarthritis or an asymmetric oligoarthritis. As noted, large knee effusions are common and an important part of therapy is removal of fluid to decrease the concentration of inflammatory cytokines perpetuating the synovitis and to decrease discomfort and prevent stretching of the surrounding connective tissues. Removal of synovial fluid from the knee (the most commonly affected joint), shoulder, ankle, elbow and some of the small joints of the hands and feet (the most commonly affected joints, in decreasing frequency) is an office procedure and can give instant and significant symptomatic relief. Fluid removal from the hip is a more difficult procedure, usually requiring radiographic targeting. Fluid should be sent for analysis as

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described earlier. Samples should not be sent to try to grow B. burgdorferi. Intra-articular steroids may give relatively rapid relief when injected into an evacuated joint. There is still speculation that such injections may predispose to persistence of inflammation in the injected joint. This is based entirely on post hoc analysis of patients with Lyme arthritis (Steere et al., 1985) and subsequent case reports. In all likelihood, the patients who received the intra-articular injections had more severe arthritis and a poorer prognosis than patients who did notthus, selection bias is probably at work and there is probably no contraindication to the judicious use of intra-articular steroids if the patient has been adequately treated with antibiotics. Antibiotic therapy for Lyme arthritis, as recommended by the IDSA (Wormser et al., 2006), is 28 days of doxycycline, amoxicillin or cefuroxime axetil, at the daily doses noted previously. Some clinicians give these oral agents for up to 6 weeks, but there is no scientific proof that longer therapy is any more efficacious. It is important to consider the possibility of central nervous system Lyme disease, as such patients would require intravenous antibiotics: ceftriaxone 2g IV qd (cefotaxime 3g intravenous twice a day or penicillin 18–24 million units daily in divided doses every 4 h) for 14 days (the duration varies from 10–21 days, depending upon clinician). In some patients, initial oral therapy for Lyme arthritis will be ineffective, in which case either a repeat course of oral therapy (recommended, which can be extended in duration – an approach not endorsed by the IDSA) or intravenous therapy for 21 days can be given. However, it is important to define ‘ineffective’ very carefully. As noted, inflammation due to initial B. burgdorferi infection may be slow to resolve totally. One should not judge slow but steady diminution in inflammation as a failure or ‘ineffective’ therapy. Clearly, if there is the appearance of arthritis in new joints, one must consider the possibility that the preceding therapy was ineffective. However, it is also possible that the initial diagnosis was incorrect. Outcomes in paediatric Lyme arthritis have been reviewed recently by Tory et al.

(2010). In reviewing 10 years of records at a tertiary care centre, 99 children with Lyme arthritis were identified, of whom 76 had full recovery in response to antibiotics. The authors could not predict response based on their clinical or laboratory data. A definition of joint involvement lasting at least 3 months after antibiotic therapy yielded 23 patients with antibiotic-refractory Lyme arthritis. Of these, the majority was successfully treated with NSAIDs (six patients), intra-articular injections of corticosteroids (four patients) or disease modifying anti-rheumatic drugs (two patients); five additional patients were lost to follow-up. None of the patients available for follow-up evaluation had chronic arthritis, joint deformities or recurrence of the infection. A lack of response to appropriate antibiotics should raise consideration that the diagnosis of Lyme arthritis was incorrect and should prompt referral to a rheumatologist with experience in the management of Lyme arthritis. Monoarthritis may represent psoriatic arthritis, atypical rheumatoid arthritis or another ‘idiopathic inflammatory joint disease’ unrelated to but coincident with or following Lyme disease. If this is the case, there is nothing to suggest causality and no immunopathogenetic mechanism to link B. burgdorferi with these inflammatory joint diseases (post hoc, ergo propter hoc does not make for good medical practice). In the rare case where Lyme arthritis does not resolve despite two or three adequate courses of appropriate antibiotics, one must reconsider the initial diagnosis of Lyme arthritis. If this diagnosis seems secure, and the synovitis has persisted for more than 6 months unabated, despite antibiotics and appropriate use of NSAIDs, therapeutic options are few and unproven. Arthroscopic synovectomy (Schoen et al., 1991) is often effective, although if the synovectomy is not complete (i.e. there is residual synovium remaining within the joint), there may be a return of inflammation (the synovium grows back and a selfperpetuating inflammatory process reemerges). In cases refractory to synovectomy or where synovectomy is not considered an option, therapies analogous to those used for rheumatoid arthritis have been adopted. These unstudied and therefore unapproved

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medical therapies arthritis include:

for

refractory

Lyme

 Hydroxychloroquine (200 mg PO twice a day or 400 mg PO once a day). This requires monitoring for retinal toxicity with retinal examination every 6 months and use of the Amsler grid for home monitoring of retinal toxicity. The serum creatine phosphokinase should be checked every 6 months. This drug can take 8 weeks or longer to start working. The dose should not be increased and, in fact, should be decreased for small patients, to no more than 7 mg/kg. The use of hydroxychloroquine is based on its efficacy in the chronic synovitis seen in rheumatoid arthritis or lupus, to treat what has become a self-perpetuating synovitis. This is in marked contrast to the unproven and purely speculative use of hydroxychloroquine to enhance entry of antibiotics into cells putatively infected with dormant or live B. burgdorferi.  Methotrexate, used for patients with rheumatoid arthritis, has been suggested for use in patients with the chronic, selfperpetuating synovitis, seen rarely after B. burgdorferi infection. Methotrexate is started at 7.5 or 10 mg given once a week. If an adequate response has not been attained after 6 weeks at a given dose level, the dose can be increased by 2.5 mg weekly until toxicity limits further increases. The maximum should be 25 mg once a week. The clinician must monitor for oral ulcers, bone marrow and hepatic toxicities and, rarely, interstitial pulmonary fibrosis, which presents as a dry, non-productive cough. Folic acid 1 mg/ day every day of the week is mandatory co-therapy to diminish the likelihood of toxicity related to folic acid deficiency – methotrexate inhibits the metabolism and activation of folic acid, so folic acid ‘prophylaxis’ is necessary to achieve healthy levels of the active metabolite tetrahydrofolate crucial in the synthesis of purines and pyrimidines. In addition, methotrexate forms polyglutamates that inhibit the enzyme 5-aminoimidazole4-carboxamidoribonucleotide (AICAR) transformylase. Excess AICAR, the substrate for this transformylase,

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accumulates as the result of this enzymatic blockade, leading to adenosine release extracellularly. Adenosine then binds to anti-inflammatory receptors on a variety of immune cells, e.g. lymphocytes and macrophages, leading to a lessening of inflammation.  The use of molecular biology therapies targeting TNF-α or IL-1 in patients with refractory Lyme arthritis has never been studied formally. Given the immune response to the organism, this sort of therapy has face validity but does not represent an approved use of these agents. Intra-articular infusion of an IL-1 antagonist into a single refractory Lyme arthritis joint has been discussed by some thought leaders informally, but has never been studied and is certainly not an approved use of any of the anti-IL-1 therapies currently available. Arthritis may cause overlying muscles to either atrophy or shorten. In either event, subsequent joint dysfunction may occur. Physical therapy is warranted for patients recovering from long-term synovitis, in order to assure a return to normal function. In the case of the knee, chronic arthritis often causes atrophy of the overlying quadriceps femoris mechanism, with subsequent patellofemoral joint dysfunction. This may cause pain, swelling and redness of the knee – in essence this is a mechanical synovitis, but it is not evidence of a recrudescence of the Borrelia as a newly reactivated infection and should not be treated as such. In such cases, aspiration of the fluid will serve two purposes: decrease pain and substantiate the diagnosis, as the fluid will be only minimally inflammatory (500–2,000 cells/l). Thus, it is imperative that the default diagnosis in a patient with the recurrence of knee arthritis after adequate antibiotic therapy not be recurrent Lyme arthritis. Some patients with prior documented B. burgdorferi infection (and some with an unsubstantiated diagnosis of Lyme disease) develop a syndrome compatible with the diagnosis of fibromyalgia; some may have features of ‘chronic fatigue syndrome’. There is no evidence that the fibromyalgia or chronic fatigue syndromes are in any way due to active infection with B. burgdorferi.

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Patients with these chronic and debilitating afflictions do not benefit from antibiotic therapy. In fact, we believe that the very insistence by their clinicians that they have ‘chronic Lyme disease’, a chronic, incurable infection that will never resolve, makes these patients worse. The sense of having no path forward, known as aporia, is very much part of the psychopathogenesis of these afflictions in these patients. It is crucial that these patients be disabused of the diagnosis of ‘chronic Lyme disease’ and be informed that such treatment is not in accord with evidencebased medical practice. Emotional support of these fragile individuals, listening to them and never dismissing their symptoms and suffering, is critical. They are often demanding and needy, but much of this is due to fear and anxiety and a sense of helplessness that medications do not cure (Sigal and Hassett, 2002). This issue and the therapeutic approaches that can be tried are dealt with in more detail by Hassett and Sigal, Chapter 15, this volume.

References Afzelius, A. (1910) Verhandlungen der dermatologischen Gesellschaft zu Stockholm. Archiv für Dermatologie und Syphilis 101, 104. Ball, R., Shadomy, S.V., Meyer, A., Huber, B.T., Leffell, M.S., Zachary, A., Belotto, M., Hilton, E., Bryant-Genevier, M., Schriefer, M.E., Miller, F.W. and Braun, M.M. (2009) HLA type and immune response to Borrelia burgdorferi outer surface A protein in people in whom arthritis developed after Lyme disease vaccination. Arthritis and Rheumatism 60, 1179–1186. Brunner, M. and Sigal, L.H. (2001) Use of serum immune complexes in a new test that accurately confirms early Lyme disease and active infection with Borrelia burgdorferi. Journal of Clinical Microbiology 39, 3213–3321. Brunner, M. and Sigal, L.H. (2000) Immune complexes from Lyme disease sera contain Borrelia burgdorferi antigen and antigenspecific antibodies: potential use for improved testing. Journal of Infectious Diseases 182, 534–539. Brunner, M., Stein, S., Mitchell, P.D. and Sigal, L.H. (1998) IgM capture assay for the serologic confirmation of early Lyme disease: analyzing immune complexes with biotinylated Borrelia

burgdorferi sonicate enhanced with flagellin peptide epitope. Journal of Clinical Microbiology 36, 1074–1080. Callister, S.M., Jobe, D.A., Agger, W.A., Schell, R.F., Kowalski, T.J., Lovrich, S.D. and Marks, J.A. (2002) Ability of borreliacidal antibody test to confirm Lyme disease in clinical practice. Clinical and Diagnostic Laboratory Immunology 9, 908-912. Carlson, D. Hernandez, J., Bloom, B.J., Coburn, J., Aversa, J.M. and Steere, A.C. (1999) Lack of Borrelia burgdorferi DNA in synovial fluid samples from patients with antibiotic-resistant Lyme arthritis. Arthritis and Rheumatism 42, 2705–2709. Chen, J., Field, J.A., Glickstein, L., Molloy, P.J., Huber, B.T. and Steere, A.C. (1999) Association of antibiotic treatment-resistant Lyme arthritis with T cell responses to dominant epitopes of outer surface protein A of Borrelia burgdorferi. Arthritis and Rheumatism 42, 1813–1822. Codolo, G., Amedei, A., Steere, A.C., Papinutto, E., Cappon, A., Polenghi, A., Benagiano, M., Paccani, S.R., Sambri, V., Del Prete, G., Baldari, C.T., Zanotti, G., Montecucco, C., D’Elios, M.M. and de Bernard, M. (2008) Borrelia burgdorferi NapA-driven Th17 cell inflammation in Lyme arthritis. Arthritis and Rheumatism 58, 3609– 3617. Dai, Z.Z., Lackland, H., Stein, S., Li, Q., Radziewicz, R., Williams, S. and Sigal, L.H. (1993) Molecular mimicry in Lyme disease: monoclonal antibody H9724 to Borrelia burgdorferi flagellin specifically detects chaperonin-HSP60. Biochimica et Biophysica Acta 1181, 97–100. Fikrig, E., Berland, R., Chen, M., Williams, S., Sigal, L.H. and Flavell, R. (1993) Fine mapping of the serologic response to the Borrelia burgdorferi flagellin demonstrates an epitope common to neural tissue. Proceedings of the National Academy of Sciences USA 90, 183– 187. Gross, D.M., Forsthuber, T., Tary-Lehmann, M., Etling, C., Ito, K., Nagy, Z.A., Field, J.A., Steere, A.C. and Huber, B.T. (1998) Identification of LFA-1 as a candidate autoantigen in treatmentresistant Lyme arthritis. Science 281, 703–706. Hardin, J.A., Steere, A.C. and Malawista, S.E. (1979a) Immune complexes and the evolution of Lyme arthritis. Dissemination and localization of abnormal C1q binding activity. New England Journal of Medicine 301, 1358–1363. Hardin, J.A., Walker, L.C., Steere, A.C., Trumble, T.C., Tung, K.S., Williams, R.C. Jr, Ruddy, S. and Malawista, S.E. (1979b) Circulating immune complexes in Lyme arthritis. Detection by the 125I-C1q binding, C1q solid phase, and Raji cell

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Schell, R.F. (2008) Interleukin-23 is required for development of arthritis in mice vaccinated and challenged with Borrelia species. Clinical Vaccine Immunology 15, 1199–1207. Lightfoot, R.W. Jr, Luft, B.J., Rahn, D.W., Steere, A.C., Sigal, L.H., Zoschke, D.C., Gardner, P., Britton, M.C. and Kaufman, R.L. (1993) Empiric parenteral antibiotic treatment of patients with fibromyalgia and fatigue and a positive serologic result for Lyme disease. A cost-effectiveness analysis. Annals of Internal Medicine 119, 503– 509. Maier, B., Molinger, M., Cope, A.P., Fugger, L., Schneider-Mergener, J., Sønderstrup, G., Kamradt, T. and Kramer, A. (2000) Multiple cross-reactive self-ligands for Borrelia burgdorferi-specific HLA-DR4-restricted T cells. European Journal of Immunology 30, 448–457. Marques, A., Brown, M.R. and Fleisher, T.A. (2009) Natural killer cell counts are not different between patients with post-Lyme disease syndrome and controls. Clinical Vaccine Immunology 16(8), 1249-50. Nardelli, D.T. and Schell, R.D. (2009) Expanded role for interleukin-17 in Lyme arthritis: comment on article by Codolo et al. Arthritis and Rheumatism 60, 1202. Nardelli, D.T., Burchill, M.A., England, D.M., Torrealba, J., Callister, S.M. and Schell, R.F. (2004) Association of CD4+ CD25+ T cells with prevention of severe destructive arthritis in Borrelia burgdorferi-vaccinated and challenged gamma interferon-deficient mice treated with anti-interleukin-17 antibody. Clinical and Diagnostic Laboratory Immunology 11, 1075– 1084. Nardelli, D.T., Cloute, J.P., Luk, K.H.K., Torrealba, J., Warner, T.F., Callister, S.M. and Schell, R.F. (2005) CD4+ CD25+ T cells prevent arthritis associated with Borrelia vaccination and infection. Clinical and Diagnostic Laboratory Immunology 12, 786–792. Nardelli, D.T., Callister, S.M. and Schell, R.F. (2008a) Lyme arthritis: current concepts and a change in paradigm. Clinical Vaccine Immunology 15, 21–34. Nardelli, D.T., Luk, K.H.K., Kotloski, N.J., Warner, T.F., Torrealba, J.R., Callister, S.M. and Schell, R.F. (2008b) Role of interleukin-17, transforming growth factor-, and IL-6 in the development of arthritis and production of anti-outer surface protein A borreliacidal antibodies in Borreliavaccinated and -challenged mice. FEMS Immunology and Medical Microbiology 53, 265– 274. Nishio, M.J., Liebling, M.R., Rodrigues, A., Sigal, L.H. and Louie, J.S. (1993) Identification of

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Borrelia burgdorferi using interrupted polymerase chain reaction. Arthritis and Rheumatism 36, 665–675. Nocton, J.J., Dressler, F., Rutledge, B.J., Rys, P.N., Persing, D.H. and Steere, A.C. (1994) Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis. New England Journal of Medicine 330, 229–234. Olson, C.M. Jr, Bates, T.C., Izadi, H., Radolf, J.D., Huber, S.A., Boyson, J.E. and Anguita, J. (2009) Local production of IFN- by invariant NKT cells modulates acute Lyme carditis. Journal of Immunology 182, 3728–3734. Pachner, A.R., Steere, A.C., Sigal, L.H., and Johnson, C.J. (1985) Antigen-specific proliferation of CSF lymphocytes in Lyme disease. Neurology 35, 1642–1644. Petersel, D. and Sigal, L.H. (2005) Reactive arthritis. Infectious Arthritis. Infectious Disease Clinics of North America 19, 863–883. Petzke, M.M., Brooks, A., Krupna, M.A., Mordue, D. and Schwartz, I. (2009) Recognition of Borrelia burgdorferi, the Lyme disease spirochete, by TLR7 and TLR9 induces a type I IFN response by human immune cells. Journal of Immunology 183, 5279–5292. Philipp, M.T., Marques, A.R., Fawcett, P.T., Dally, L.G. and Martin, D.S. (2003) C6 test as an indicator of therapy outcome for patients with localized or disseminated lyme borreliosis. Journal of Clinical Microbiology 41(11), 4955-60. Phillips, S.E., Mattman, L.H., Hulínská, D. and Moayad, H. (1998) A proposal for the reliable culture of Borrelia burgdorferi from patients with chronic Lyme disease, even from those previously aggressively treated. Infection 26, 364–367. Puius, Y.A. and Kalish, R.A. (2008) Lyme arthritis: pathogenesis, clinical presentation, and management. Infectious Disease Clinics of North America 22, 289–300. Rousselle, J.C., Callister, S.M., Schell, R.F., Lovrich, S.D., Jobe, D.A., Marks, J.A. and Wieneke, C.A. (1998) Borreliacidal antibody production against outer surface protein C of Borrelia burgdorferi. Journal of Infectious Diseases 178,733–41. Schoen, R.T., Aversa, J.M., Rahn, D.W. and Steere, A.C. (1991) Treatment of refractory chronic Lyme arthritis with arthroscopic synovectomy. Arthritis and Rheumatism 34, 1056–1060. Scrimenti, R.J. (1970) Erythema chronicum migrans. Archives of Dermatology 102, 104– 105. Shen, S., Shin, J.J., Strle, K., McHugh, G., Li, X., Glickstein, L.J., Drouin, E.E. and Steere, A.C. (2010) Treg cell numbers and function in

patients with antibiotic-refractory or antibioticresponsive Lyme arthritis. Arthritis and Rheumatism 62, 2127–2137. Shin, J.J., Glickstein, L.J. and Steere, A.C. (2007) High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory Lyme arthritis. Arthritis and Rheumatism 56, 1325–1335. Shoemaker, R.C., Hudnell, H.K., House, D.E., Van Kempen, A., Pakes, G.E. and the COL40155 Study Team (2006) Atovaquone plus cholestyramine in patients coinfected with Babesia microti and Borrelia burgdorferi refractory to other treatment. Advances in Therapy 23, 1–11. Sigal, L.H. (1988) Lyme disease: a worldwide borreliosis. Clinical and Experimental Rheumatology 6, 411–421. Sigal, L.H. (1990) Summary of the first one hundred patients seen at a Lyme disease referral center. American Journal of Medicine 88, 577–581. Sigal, L.H. (1993) The flagellin of Borrelia burgdorferi, the causative agent of Lyme disease, cross-reacts with a human axonal 64,000 molecular weight protein. Journal of Infectious Diseases 167, 1372–1378. Sigal, L.H. (1994) Persisting complaints attributed to Lyme disease: possible mechanisms and implications for management. American Journal of Medicine 96, 365–374. Sigal, L.H. (1996) The Lyme disease controversy: the social and financial costs of the mismanagement of Lyme disease. Archives of Internal Medicine 156, 1493–1500. Sigal, L.H. (1997) The immunology and potential mechanisms of immunopathogenesis of Lyme disease. Annual Review of Immunology 15, 63–92. Sigal, L.H. (2001) Synovial fluid polymerase chain reaction detection of pathogens: what does it really mean? Arthritis and Rheumatism 44, 2463–2467. Sigal, L.H. and Hassett, A.L. (2002) Contributions of societal and geographical environments to “Chronic Lyme disease”: the psychopathogenesis and aporology of a new “medically unexplained symptoms” syndrome. Environmental Health Perspectives 110 (Supplement 4), 607–611. Sigal, L.H. and Patella, S.J. (1992) Lyme arthritis as the incorrect diagnosis in fibromyalgia in children and adolescents. Pediatrics 90, 523– 528. Sigal, L.H. and Tatum, A.H. (1988a) Molecular mimicry in Lyme neurologic disease: crossreactivity between Borrelia burgdorferi and neuronal antigens. Neurology 38, 1439–1442. Sigal, L.H. and Tatum, A.H. (1988b) IgM in the

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serum of patients with Lyme neurologic disease binds to cross-reacting neuronal (NAg) and Borrelia burgdorferi (BAg) antigens. Annals of the New York Academy Sciences 539, 422–424. Sigal, L.H., Steere, A.C., Freeman, D.H. and Dwyer, J.M. (1986) Proliferative responses of mononuclear cells in Lyme disease: concentration of Borrelia burgdorferi-reactive cells in joint fluid. Arthritis and Rheumatism 29, 761–769. Sigal, L.H., Steere, A.C. and Dwyer, J.M. (1988) In vivo and in vitro evidence of B cell hyperactivity during Lyme disease. Journal of Rheumatology 15, 648–654. Singh, S.K. and Girschick, H.J. (2004) Lyme borreliosis: from infection to autoimmunity. Clinical Microbiology and Infection 10, 598–614. Steere, A.C. (2001) Lyme disease. New England Journal of Medicine 345, 115–125. Steere, A.C. and Angelis, S.M. (2006) Therapy for Lyme arthritis: strategies for the treatment of antibiotic-refractory arthritis. Arthritis and Rheumatism 54, 3079–3086. Steere, A.C., Hardin, J.A. and Malawista, S.E. (1977a) Erythema chronicum migrans and Lyme arthritis: cryoimmunoglobulins and clinical activity of skin and joints. Science 196, 1121– 1122. Steere, A.C., Malawista, S.E., Hardin, J.A., Ruddy, S., Askenase, W. and Andiman, W.A. (1977b) Erythema chronicum migrans and Lyme arthritis. The enlarging clinical spectrum. Annals of Internal Medicine 86, 685–698. Steere, A.C., Malawista, S.E., Snydman, D.R., Shope, R.E., Andiman, W.A., Ross, M.R. and Steele, F.M. (1977c) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis and Rheumatism 20, 7–17. Steere, A.C., Broderick, T.F. and Malawista, S.E. (1978) Erythema chronicum migrans and Lyme arthritis: epidemiologic evidence for a tick vector. American Journal of Epidemiology 108, 312–321. Steere, A.C., Hardin, J.A., Ruddy, S., Mummaw, J.G. and Malawista, S.E. (1979) Lyme arthritis: correlation of serum and cryoglobulin IgM with activity, and serum IgG with remission. Arthritis and Rheumatism 22, 471–483. Steere, A.C., Malawista, S.E., Newman, J.H., Spieler, P.N. and Bartenhagen, N.H. (1980) Antibiotic therapy in Lyme disease. Annals of Internal Medicine 93, 1–8. Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E. and Malawista, S.E. (1983a) The spirochetal etiology of Lyme disease. New England Journal of Medicine 308, 733–740.

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Steere, A.C., Hutchinson, G.J., Rahn, D.W., Sigal, L.H., Craft, J.E., DeSanna E.T. and Malawista, S.E. (1983b) Treatment of the early manifestations of Lyme disease. Annals of Internal Medicine 99, 22–26. Steere, A.C., Green, J., Schoen, R.T., Taylor, E., Hutchinson, G.J., Rahn, D.W. and Malawista, S.E. (1985) Successful parenteral penicillin therapy of established Lyme arthritis. New England Journal of Medicine 312, 869–874. Steere, A.C., Schoen, R.T. and Taylor, E. (1987) The clinical evolution of Lyme arthritis. Annals of Internal Medicine 107, 725–731. Steere, A.C., Gross, D., Meyer, A.L. and Huber, B.T. (2001) Autoimmune mechanisms in antibiotic treatment-resistant Lyme arthritis. Journal of Autoimmunity 16, 263–268. Steere, A.C., Falk, B., Drouin, E.E., Baxter-Lowe, L.E., Hammer, J. and Nepom, G.T. (2003) Binding of outer surface protein A and human lymphocyte function-associated antigen 1 peptides to HLA-DR molecules associated with antibiotic treatment-resistant Lyme arthritis. Arthritis and Rheumatism 48, 534–540. Stricker, R.B. (2007) Counterpoint: long-term antibiotic therapy improves persistent symptoms associated with Lyme disease. Clinical Infectious Diseases 45, 149–157. Stricker, R.B., Lautin, A. and Burrascano, J.J. (2006) Lyme disease: the quest for magic bullets. Chemotherapy 52, 53–59. Taylor, R.S. and Simpson, I.N. (2005) Review of treatment options for Lyme borreliosis. Journal of Chemotherapy 17 (Supplement 2), 3–16. Tory, H.O., Zurakowski, D. and Sundel, R.P. (2010) Outcomes of children treated for Lyme arthritis: results of a large pediatric cohort. Journal of Rheumatology 37, 1049–1055. Weyand, C.M. and Goronzy, J.J. (1989) Immune responses to Borrelia burgdorferi in patients with reactive arthritis. Arthritis and Rheumatism 32, 1057–1064. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S. and Nadelman, R.B. (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43, 1089–1134. Yssel, H., Shanafelt, M.C., Soderbreg, C., Schneider, P.V., Anzola, J. and Peltz, G. (1991) Borrelia burgdorferi activates a T helper cell type 1-like T cell subset in Lyme arthritis. Journal of Experimental Medicine 174, 593– 601.

13

Nervous System Involvement John J. Halperin

13.1 Introduction Lyme disease provides a fascinating window on the evolution of the very concept of a disease. The first descriptions of the clinical phenomenology – both in Europe and in the USA – focused on cutaneous abnormalities, primarily erythema migrans (Afzelius, 1910; Scrimenti, 1970). In the absence of information about the underlying pathogenesis, the subsequent European and American literature took very different paths. In Europe, the first recognition that this could be associated with systemic disease was Garin and Bujadoux’s 1922 report of the neurological triad – lymphocytic meningitis, cranial neuritis and painful radiculoneuritis (Garin and Bujadoux, 1922). These have been felt to constitute the essential elements of the systemic illness in Europe ever since. As a result, the European literature, and patient diagnosis and treatment, have dealt with this primarily as a neurological disorder, with management by physicians with expertise in nervous system diseases. In contrast, in the USA, the initial extra-cutaneous focus was on rheumatological aspects of the illness (Steere et al., 1977). Although neurological manifestations were soon identified (Reik et al., 1979; Pachner and Steere, 1984), joint manifestations have dominated the con-

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versation. Few of the leading voices in the field have had special expertise in the subtleties of what is – and is not – neurological disease. This notwithstanding, much of the debate about Lyme disease has involved symptoms that are primarily neurobehavioural. Logically, one might expect this to have been more of an issue in Europe, where nervous system involvement was heavily emphasized for half a century before the term ‘Lyme disease’ was even coined. However, lay concerns about possible nervous system involvement clearly originated in the USA. If this was not because nervous system disease is more prominent in the USA, this then begs the question of whether the responsibility for this focus rests with patients’ inaccurate perceptions, with the medical profession’s understanding and representation of the illness, or both. Many physicians consider neurological illness complex, if not incomprehensible; for many patients, it is simply terrifying. The goal of this chapter will be to clarify basic neurological concepts so that treating physicians can better understand what aspects of this infection truly are neurological. Hopefully this can then clarify the language of the conversation, resulting in lessened patient anxiety about this illness.

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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13.1.1 Phenomenology of neurological disease Virtually every behaviour that we exhibit is fundamentally dependent on the nervous system – a system that has evolved to allow us to interact successfully with our environment, responding to a broad range of both predictable and unpredictable internal and external stressors. Our behavioural responses to the environment are a combination of hard-wired functions and learned behaviours – dimensions that are remarkably intertwined, in actions ranging from the mechanics of walking to our emotional responses to baseball or opera. Nervous system functions are affected by the interplay of at least four different elements (Fig. 13.1): (i) changes in the structure, physiology and biochemistry of the nervous system itself; (ii) changes in its physiological milieu and (iii) changes in the broad range of external stimuli, all impacted by (iv) learned behavioural responses. Just as

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the host’s immune system’s response to external stimuli is impacted by its prior experience and learned responses, so too are neurobehavioural responses heavily dependent on past experiences and learned responses. As a matter of definition, disorders that we consider neurological affect – either macroscopically or microscopically – the structure of the nervous system. The physiological milieu is the province of general medicine. Behaviours not involving the first two dimensions are usually considered psychological. (Psychiatry encompasses both what are almost certainly biochemical abnormalities of synaptic function such as psychosis, and disorders considered psychological.) Although the diagnosis of a ‘psychological issue’ is viewed by many as pejorative and minimizing of its significance, in fact this means neither more nor less than the presence of a learned response to stressors that does not serve the subject well. This should no

Structure Biochemical – extracellular

Biochemical – cellular

Physiological – cellular

Physiological – extracellular

Learned response – physiological stimulus

Synaptic physiology/ chemistry Learned response – behavioural stimulus

Fig. 13.1. Elements affecting behaviour. Disorders due to abnormalities of nervous system structure, intracellular physiology or intracellular biochemistry (grey background) are generally considered neurological. Those affecting the extracellular milieu are generally medical (pale grey background). Those affecting learned responses to stimuli are generally considered psychological (white background). Psychiatric disorders include psychological and behavioural disorders due to abnormal synaptic physiology.

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more be considered a value judgement about neurobehavioural disorders than it would be when an aberrant immune response results in an autoimmune disease. This threefold division is important not just because it defines professional boundaries but because it defines the appropriate treatment and likely outcome of disease. Disorders that damage the structure of the nervous system are the most difficult to reverse, and therefore the most terrifying to affected patients. Changes in the physiological milieu are, at least from a neurological perspective, reversible, assuming the severity and duration of the abnormality is limited (e.g. hypoglycaemia or hypoxia). Problems that reflect unhelpful behaviours can be challenging to treat but generally are less daunting than severe neurodegenerative processes. Consequently, when considering a given individual’s problem, a key decision point is whether the issue is neurological, medical or behavioural. In most instances, differentiating among these is fairly straightforward. Acute focal changes in neurological function – such as hemiparesis, visual changes and radiculopathy – are typically fairly obvious. Similarly, significant causes of metabolic encephalopathies – such as hypoglycaemia, or renal or hepatic insufficiency – are fairly straightforward to diagnose. Where challenges typically arise is in more subtle neurobehavioural disorders in which patients exhibit non-focal changes in behaviour and cognitive function. Although these disorders are evidence of aberrant function of the nervous system, only rarely do they reflect underlying damage to the brain. These nonneurological disorders generally belong in one of two categories – encephalopathies and psychiatric disease. The term ‘encephalopathy’ is usually used to denote altered brain function in the absence of any underlying brain disease. Encephalopathic patients often have impairment of memory, orientation and other complex cognitive tasks. In contrast, patients with psychiatric disease – who can have remarkably similar subjective perceptions of their difficulties – typically have preservation of these functions but marked impairment of

concentration and mental focus. A minimental status examination is actually quite effective at differentiating between the two, provided the depressed patient can be cajoled into completing the task and not ‘let off the hook’ too easily with protestations that ‘I can’t do that’ – a comment that is actually remarkably uncommon among the neurologically impaired. 13.1.2 Pathoanatomy of nervous system disease Neurological disease is typically divided into disorders of the peripheral and central nervous systems. Diseases typically affect one or the other. Some systemic disorders, including inflammatory and infectious diseases, can affect both. The central nervous system (CNS) consists of the brain and spinal cord, while the peripheral nervous system (PNS) comprises the nerves that arise from the brainstem and spinal cord, the muscles and sensory end-organs, the peripherally occurring sympathetic, parasympathetic and sensory ganglia and the synaptic junctions that allow communication among these different elements. Medical disorders affecting the CNS can be divided into three broad groups. Parenchymal damage can be focal (e.g. stroke, trauma, encephalitis) or cellular (e.g. Alzheimer’s, Parkinson’s). Other processes can be extraparenchymal, involving either inflammation or infiltration of the lining of the brain (meningitis), or alterations in the flow of cerebrospinal fluid (CSF) (e.g. hydrocephalus, pseudotumour cerebri) but not the brain itself. Yet others can alter the function of the brain indirectly by biochemical or physiological effects. The latter is by far the most common mechanism, with significant alteration of brain function occurring frequently in non-nervous-system infections (e.g. pneumonia, sepsis, urinary tract infections), probably mediated directly by cytokines and indirectly by fever, as well as in numerous metabolic disorders. The PNS can similarly be affected in a myriad of disorders. Diabetes, the most

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common cause of neuropathy in the Western world, can cause either diffuse nerve damage or focal damage that can be thought of as ‘nerve strokes’. The commonest cause of neuropathy worldwide, leprosy, is an infection that causes multifocal nerve infiltration and inflammation. Metabolic disorders such as renal failure or hypovitaminoses cause fairly diffuse damage. Fluctuating biochemical abnormalities such as uncontrolled hyperglycaemia cause transient and reversible changes in nerve function, analogous to a metabolic encephalopathy. Differentiating neurological disease from the systemic effects of medical disorders starts with a careful clinical assessment, exploring the wide range of potential contributing medical and other elements. The neurological examination is designed to detect evidence of structural neurological disease. If there is a high index of suspicion for neurological disease, additional testing may be warranted, focusing on the part of the nervous system that appears to be involved. Neurophysiological testing (e.g. nerve conduction studies, electromyography) can be helpful if there is a strong suspicion of peripheral nerve damage. This procedure can differentiate demyelinating from axonal processes, can determine whether the disorder is focal, multifocal or diffuse, and can quantitate severity. Imaging of the relevant portion of the neuraxis, primarily with magnetic resonance imaging (MRI), can be extremely helpful if there is reason to suspect CNS involvement. However, as MRIs often demonstrate non-specific and irrelevant findings, performing this test in an individual with low a priori likelihood of disease typically leads only to additional testing, expense and patient stress, without obtaining any information that helps the patient. In inflammatory disorders of the CNS, examination of CSF can be helpful, primarily in providing evidence of specific infectious or, occasionally, neoplastic disorders, or at a minimum providing objective evidence of brain inflammation that is either acute or chronic. Importantly, bacterial infections of the CNS elicit a local inflammatory response that – virtually without exception – is evidenced in CSF abnormalities.

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13.2 Nervous System Disorders in Lyme Disease (neuroborreliosis) Nervous system involvement is reported to occur in 12% of confirmed cases of Lyme disease in the USA (Bacon et al., 2008). Although neuroborreliosis is sometimes described as protean or mimicking other diseases, this is no more true in neuroborreliosis than it is in any other systemic disorder causing multifocal neurological damage. Although cerebrovascular disease can affect right-arm strength, vision in the left eye, right facial sensation, gait coordination and a myriad of other functions, nobody would call stroke ‘the great imitator’. In a similar manner, Borrelia burgdorferi infection causes a small number of pathophysiological processes. These can affect the nervous system in different locations, but the underlying disease process is the same. Nervous system disorders caused by Lyme disease share one key element with virtually all other infectious diseases – involvement is due to infiltration of microorganisms into the nervous system and the presence of a significant inflammatory response to them. Early in infection, spirochaetes invade the subarachnoid space resulting in disseminated infection and inflammation of the meninges, i.e. Lyme meningitis (Garin and Bujadoux, 1922; Reik et al., 1979; Pachner and Steere, 1984). In all other instances, neuroborreliosis is due to parenchymal infection and inflammation of peripheral nerve or, very rarely, the CNS. Specific manifestations then depend only on the site of involvement. The only other form of altered nervous system function seen in Lyme disease is actually quite common but is not caused by nervous system infection or inflammation. Just as in many other systemic infectious or inflammatory diseases (e.g. any febrile illness), patients with active Lyme arthritis or other disseminated forms of infection commonly feel tired and cognitively slowed, probably as a result of the CNS actions of a number of circulating cytokines. This entity (Halperin et al., 1988, 1990a) was initially termed ‘Lyme encephalopathy’ to emphasize that it is analogous to most other

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encephalopathies; in other words, it is the remote effect of a systemic process and not evidence of direct CNS involvement. Unfortunately, this point appears not to have been made with sufficient clarity. These generalizations notwithstanding, there is one clinical syndrome that is quite characteristic of neuroborreliosis – the triad initially described by Garin and Bujadoux (1922) and subsequently by Reik et al. (1972). Lymphocytic meningitis, cranial neuritis and painful radiculoneuritis occur alone or in combination in up to 15% of infected individuals – a proportion that is comparable in Europe and the USA. In the USA, facial nerve palsy occurs in 8% of confirmed cases, radiculopathy in 3% and meningitis or encephalitis in 2% (Bacon et al., 2007). 13.2.1 Central nervous system involvement Lymphocytic meningitis occurs as an isolated clinical entity in approximately 2% of confirmed cases of Lyme disease (Bacon et al., 2007). This number is probably an underestimate as symptoms are highly variable. Although often presenting in a manner similar to other forms of ‘aseptic meningitis’ (i.e. with severe headache, photosensitivity, neck stiffness, fever and other systemic signs), patients with Lyme disease cranial neuritis often have a comparable CSF pleocytosis, without meningitis symptoms. There is good evidence that B. burgdorferi penetrates the CSF early (Keller et al., 1992; Logigian and Steere, 1992; Luft et al., 1992), not consistently accompanied by a vigorous pleocytosis. This suggests that infection of the meninges and subarachnoid space probably occurs more frequently than is clinically recognized or confirmed with a lumbar puncture. The clinical presentation and seasonal incidence of Lyme meningitis both overlap substantially with viral meningitis. Several algorithms have been published to help differentiate between these two entities (Shah et al., 2005; Garro et al., 2009; Tuerlinckx et al., 2009), which is important as one requires antibiotic treatment and the other does not. Patients with Lyme meningitis tend to take a

little longer to present for medical attention, presumably because the onset of symptoms is less dramatic and symptom severity is less. However, the strongest predictor is the simultaneous occurrence of facial nerve palsy, something that virtually never occurs in viral meningitis. Particularly among children, there appears to be an association with a pseudotumour cerebri-like (benign intracranial hypertension) syndrome (Jacobson and Frens, 1989; Kan et al., 1998; Zemel, 2000). Affected children develop headaches, raised intracranial pressure, optic disc swelling and potentially visual loss. Most reported cases have had either preceding or concurrent Lyme meningitis, making this more a matter of raised intracranial pressure associated with Lyme meningitis rather than what is usually considered pseudotumour cerebri. Semantic differences notwithstanding, it is important to recognize this entity, as visual damage and loss have been reported. In addition to treatment of the responsible infection, management of the raised intracranial pressure is important, and may include repeated lumbar puncture, corticosteroids (with and following appropriate antimicrobial therapy), carbonic anhydrase inhibitors, shunting and optic nerve sheath fenestration, as circumstances dictate. Parenchymal CNS infection is quite rare. Patients with European Lyme radiculitis sometimes develop segmental spinal cord involvement at the affected level (Mygland et al., 2010). Given the infrequent description of Lyme radiculitis in the USA, this issue has not been systematically addressed. Brain involvement is now rarely reported. In the late 1980s and 1990s, when diagnostic tools were still limited and as a result there were patients who went untreated for a significant period of time, rare cases of Lyme encephalitis were described. At the time, the incidence of Lyme encephalitis was estimated at one case per million of the population at risk per year in both Europe and the USA (Halperin et al., 1996). Since then, with widespread early treatment, the disorder is rarely if ever seen. In those rare patients with Lyme encephalitis, inflammation appears to affect white matter preferentially (Ackermann et al.,

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1988; Halperin et al., 1992), perhaps reflecting the demonstration in vitro of the spirochaete’s affinity for oligodendroglia and gangliosides (Garcia-Monco et al., 1989). As a result, the MRI can be confused with demyelinating disease, although the disorders can usually readily be differentiated both by clinical course and laboratory testing. Unfortunately, as a result, radiologists often persist in including Lyme disease in the differential diagnosis of any patient with the most trivial white matter abnormality – a point that is rarely helpful. Lyme encephalitis (Ackermann et al., 1988; Halperin et al., 1989; Logigian et al., 1990) can occur in either acute or late disseminated infection. Findings are usually typical of white matter disorders (e.g. spasticity, sensory findings, ataxia). Seizures are decidedly rare, as grey matter is rarely involved. Without treatment, the disease differs from multiple sclerosis in that Lyme disease encephalitis is a monophasic illness without relapses and remissions. Treatment with immunosuppressives in isolation can suppress symptoms, which then recur when treatment is stopped. Treatment with standard courses of antibiotics almost always cures the infection (Halperin et al., 2007; Mygland et al., 2010). Neurological residua may remain if there has been significant damage, but some degree of improvement is the norm and there should be no further loss of function. In the earliest descriptions of Lyme disease encephalopathy, a few patients were identified who had this as a manifestation of very mild Lyme disease encephalitis (Halperin et al., 1990a). However, this was a rare observation then, and in recent experience occurs extremely rarely, if ever. Diagnosis of CNS disease can sometimes be challenging. Lyme disease meningitis (as well as the often co-occurring cranial neuritis and radiculoneuritis) typically occurs very early in infection, occasionally even before there is a measurable antibody response. In such instances, if there has been probable exposure and the clinical picture is consistent with Lyme disease, empiric antibiotics can be started pending confirmatory follow-up serology.

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CSF findings in Lyme disease meningitis are similar to those in viral meningitis, with a modest lymphocyte-predominant CSF pleocytosis (typically hundreds to perhaps low thousands of cells/mm3), a mild increase in protein (100–300 mg/dl) and normal glucose. Culture, in the best of circumstances, is positive in no more than 10% of cases of Lyme disease meningitis; clinical laboratories rarely have the necessary specialized medium (BSK-2) or the ability to incubate for the requisite prolonged period of time at lowerthan-usual temperatures. Unfortunately, even the added test sensitivity of PCR-based techniques has not translated into improved clinical diagnostic sensitivity. It is likely that there are so few spirochaetes free in the CSF that any given aliquot may or may not contain one. On the other hand, the presence of organism-specific genomic material does not necessarily imply the presence of viable organisms – PCR positivity has been obtained with long-dead organisms (Rovery et al., 2005). Given the additional issue that some laboratories continue to have difficulty performing PCR with sufficient specificity to be certain a reported positive is meaningful, the positive and negative predictive values of this technique are very poor; this test is therefore neither clinically useful nor recommended. One test that can be useful is measurement of the production of specific anti-B. burgdorferi antibodies in the CSF (Henriksson et al., 1986; Wilske et al., 1986; Halperin et al., 1989; Steere et al., 1990). Importantly, this approach is only useful in the presence of CNS infection. CSF examination in patients with purely peripheral nerve disorders would only be expected to be abnormal in those who happened to have both PNS and CNS involvement. The CNS generally functions as an immunologically distinct compartment. Some immunoglobulin (normally <1%) normally filters in from serum. When an organism invades the CNS and remains for any period of time, reactive lymphocytes migrate in, mature and ultimately start producing specific antibodies locally. When chronic, this can be detected at a superficial level by the detection of oligoclonal bands

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and an overall increase in IgG synthesis within the CNS – something observed frequently in the European neuroborreliosis literature but less commonly in the US literature. This method can be used to help diagnose a specific infection by measuring the proportion of IgG in the CSF that is specific to the causative organism and comparing it with the corresponding proportion in serum. This can be performed by a number of different techniques. From a technical perspective, capture assays on serum and CSF inherently provide the relevant values (Hansen and Lebech, 1990; Steere et al., 1990). More intuitively understandable is to first measure CSF and serum IgG concentrations, dilute both fluids to the same final IgG concentrations and then perform conventional ELISAs for the organism in question, comparing the values (Halperin et al., 1989). Least reliable is to measure the IgG concentrations, perform CSF and serum ELISAs at standard concentrations, adjust the ELISA values mathematically for the differing IgG concentrations and calculate an index from those values. Regardless of the technique, it is important to understand that simply performing an anti-B. burgdorferi antibody ELISA in CSF is unhelpful: (i) if there is a significant amount of specific antibody in serum (which then filters into the CSF); or (ii) if there is any blood–brain-barrier breakdown, in which case the total concentration of all IgG in the CSF is greater than normal, making any measure of a specific antibody artificially high. There are three important limitations to this technique. Firstly, there is some immune cross-reactivity with other spirochaetes, potentially generating false positives. Fortunately, syphilis can usually be differentiated from Lyme disease by measuring reaginic antibodies (e.g. the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test), which are almost invariably present in syphilis and rarely present in Lyme disease. Also fortunately, other potentially cross-reacting organisms (such as the relapsing fevers) have little geographical overlap with Lyme disease. Secondly, the

ratio of CSF:serum antibody may remain elevated long after effective treatment (Hammers Berggren et al., 1993), presumably as antibody production in the two compartments slowly declines in parallel. Consequently a positive index is indicative of infection, past or present, not necessarily present. Fortunately, just as in neurosyphilis, non-specific markers of CNS inflammation (i.e. CSF cell count, protein) provide a reasonable measure of disease activity. Finally, although the specificity of the technique is high, estimates of sensitivity have varied widely, ranging from about 50% (Steere et al., 1990) to 90% (Blanc et al., 2007; Ljostad et al., 2007; Mygland et al., 2010). This is largely because there is no ‘gold standard’ diagnostic tool to determine whether or not CNS infection is present, and clinical case definitions in published series have varied widely. However, the technique should be nearly 100% sensitive and specific in patients with active CNS infection, particularly those with apparent B-cell stimulation as evidenced by overall increases in IgG synthesis and the presence of oligoclonal bands. If B-cell stimulation in these patients is in response to a specific infecting organism, there should be particularly elevated antibody targeted at that organism; thus, this technique should be particularly useful for differentiating between neuroborreliosis and multiple sclerosis. 13.2.2 Lyme encephalopathy The other entity often mistakenly thought to be due to CNS infection is the disorder referred to as ‘Lyme encephalopathy’. In early work with patients with untreated Lyme disease, it became apparent that many patients with active inflammatory arthritis and other non-CNS manifestations described difficulty with memory and information processing, abnormalities that could be confirmed with neuropsychological testing (Halperin et al., 1988, 1990a; Logigian et al., 1990; Krupp et al., 1991) and which improved after antimicrobial therapy. Subsequent investigations indicated that affected individuals could be divided into two very distinct groups. Starting with the

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assumption that active CNS Lyme disease – like every other known CNS bacterial infection including neurosyphilis – elicits a demonstrable local inflammatory response, a very small subset of these patients, with abnormal CSF (Halperin et al., 1990a; Logigian et al., 1990), was readily identifiable as actually having a very mild form of Lyme encephalomyelitis. These patients generally had abnormal brain MRI scans and significantly abnormal neurological examinations. In contrast, the vast majority of patients with Lyme disease and just these cognitive symptoms had normal neurological examinations (other than, variably, cognitive testing), normal imaging and normal CSF. It is this latter group who are most aptly characterized as having ‘Lyme encephalopathy’, a disorder comparable to the ‘toxicmetabolic’ encephalopathy seen in myriad other systemic (non-CNS) inflammatory disorders (e.g. viral infections, bacterial infections such as pneumonia, systemic lupus) and almost certainly mediated by the entry of cytokines and other neuro-immunomodulators into the CNS (Halperin and Heyes, 1992).

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Unfortunately this entity – named ‘Lyme encephalopathy’ specifically to differentiate it from a brain infection or encephalitis – has become part of the general folklore about Lyme disease. Some have felt it has specificity for this infection and treat patients with nothing but these non-specific symptoms with antibiotics for prolonged periods of time. Virtually every practising physician has seen this same phenomenon in innumerable other clinical contexts, rendering the inference that it is specific for Lyme disease untenable. Importantly, quality-of-life surveys show that, at any given time, one-third of the general population feels that they have discernable cognitive difficulty, with 2% of the population feeling that these symptoms are very severe (Luo et al., 2005) (Fig. 13.2). 13.2.3 Peripheral nervous system involvement The assertion that neuroborreliosis has ‘protean’ manifestations probably derives from the broad range of clinically different phenomena that has been reported. Extensive

Cognitive slowing, fatigue: 1/3 of population, severe in 2% (200/10,000)

Lyme disease 1/10,000

??? Medical illnesses (infectious, inflammatory, metabolic, etc.) Post-Lyme disease syndrome (1‒3% of Lyme disease paents, 0.02/10,000) Fig. 13.2. Is it Lyme disease encephalopathy? Subjective symptoms of cognitive slowing, all with objectively demonstrable abnormalities of memory and cognition (not to scale).

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neurophysiological evidence indicates that B. burgdorferi infection causes one phenomenon – a mononeuropathy multiplex (Halperin et al., 1990b; Logigian and Steere, 1992; Mygland et al., 2010). This type of disorder, often associated with vasculitic or vasculopathic disorders, causes multifocal peripheral nerve damage. Classically, this causes focal damage to one or several large individual nerves. At the other extreme, these disorders can affect innumerable small nerve fascicles, in aggregate mimicking a diffuse polyneuropathy, but termed a confluent mononeuropathy multiplex. Histologically, a mononeuropathy multiplex usually involves vascular changes, often inflammatory, with resultant patchy damage to nerve fascicles – precisely what has been seen in the few published biopsies of human nerves in this disorder (Halperin et al., 1987; Vallat et al., 1987; Elamin et al., 2009). Importantly, exactly the same neurophysiological and pathological changes have been found – with remarkably high frequency – in the only animal model of neuroborreliosis, the rhesus macaque monkey (Roberts et al., 1998).

Clinical phenomenology Cranial neuropathies are the most common peripheral nerve manifestation, occurring in about 8% of confirmed cases (Bacon et al., 2007). In about 80% of these cases, this involves the facial nerve. This can be bilateral in about 20% of these – something quite uncommon otherwise, with other aetiologies largely limited to Guillain–Barré syndrome, sarcoidosis, human immunodeficiency virus infection and other basilar meningitides. Other cranial nerves can be involved but much less frequently. Of these, nerves to the extra-ocular muscles (III, IV and VI), the trigeminal nerve (V) and the acousticovestibular nerve (VIII) have been reported with some frequency. Very rare cases of optic neuritis (II) have appeared, as have rare mentions of the lower cranial nerves. Other than the optic nerve (which is actually a CNS tract), these generally occur as part of a mononeuropathy multiplex. Although it would be reasonable to think that these are

caused by damage as the nerves pass through the subarachnoid space, only some have simultaneous meningeal inflammation, supporting the notion these are co-occurring but not causally linked phenomena. The other classically described – and probably significantly under-reported– disorder is the painful radiculoneuritis first described by Garin and Bujadoux (1922). This disorder, documented in about 3% of confirmed cases (Bacon et al., 2007), presents with symptoms largely indistinguishable from those of a mechanical radiculopathy, with severe dermatomal pain and sensorimotor and reflex changes. The European literature suggests that this occurs most often in the limb that was the site of the tick bite; no comparable data exist in the USA. Again, neurophysiological studies indicate that this is yet another manifestation of a mononeuropathy multiplex. Other PNS manifestations include brachial and lumbosacral plexopathies (Wendling et al., 2009), as well as other mononeuropathies. Some patients with longstanding untreated disease have a more diffuse picture, mimicking a diffuse polyneuropathy. Although there have been a few case reports of demyelinating neuropathies in association with Lyme disease (Muley and Parry, 2009), these are remarkably infrequent, and the causal relationship cannot be considered established at this point.

13.3 Pathophysiology The mechanism underlying these various manifestations remains unclear (Rupprecht et al., 2008). On the one hand, infection seems to play a key role, as antibiotic treatment is curative. On the other, it has been impossible to demonstrate viable spirochaetes – or even immunohistochemical or PCR evidence of their presence – in either human biopsies or in animal models. Immune amplification clearly plays a role in nerve and brain involvement; however, it is clear that Lyme disease-associated facial nerve palsy can occur before there has been a measurable antibody response. Molecular mimicry has been suggested, but specific targets are

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limited (Sigal, 1990; chapter 12 of this volume), disease seems far too restricted in anatomic distribution for such a non-focused mechanism, and both the rapid termination of disease with treatment as well as the onset before there are demonstrable antibodies render such a mechanism questionable. With regard to CNS disease, there is evidence that the immune stimulation associated with infection triggers the release of quinolinic acid (Halperin and Heyes, 1992) a tryptophan metabolite that can act as a glutamate receptor agonist, either affecting neuronal function or potentially damaging neurons. In vitro, B. burgdorferi outer-surface protein A (OspA) stimulates monkey astrocytes and microglia to produce tumour necrosis factor (TNF)- and interleukin (IL)-6 and IL-8, effects that are inhibited by doxycycline and minocycline (Bernardino et al., 2009). Inoculation of B. burgdorferi into the CSF of rhesus monkeys results in early intraCNS production of IL-6, IL-8, CCL2 and CXCL13 (markers of the innate immune response), weeks before there is discernible antibody production (Ramesh et al., 2009). In vitro exposure of rhesus brain slices to B. burgdorferi not only results in local production of many of the same cytokines but also triggers some neuronal and oligodendroglial apoptosis (Ramesh et al., 2008). Thus, there are many potential mechanisms by which the immune system and nervous system may be interacting to cause neuroborreliosis – mechanisms that may well inform our

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understanding of host immune responses in other nervous system infections (Table 13.1).

13.4 Treatment Although treatment is described in detail by Wormser in Chapter 7 of this volume, it is worthwhile emphasizing several aspects pertaining to nervous system infection. The original rationale for using both high-dose intravenous penicillin (Steere et al., 1983) and then ceftriaxone (Dattwyler et al., 1988) was to obtain good CNS penetration; however there have now been multiple European studies demonstrating the efficacy of oral doxycycline for Lyme disease meningitis, cranial neuritis and radiculoneuritis (Halperin et al., 2007; Mygland et al., 2010), making this an option offered in both US and European nervous system treatment guidelines. Although not proven to be effective in the USA, there is no obvious reason to think that the efficacy would be very different. A reasonable strategy now is to treat orally first, then follow up with parenteral ceftriaxone or another regimen if this is unsuccessful. As emphasized in Chapter 7 (this volume), treatment for 2–4 weeks with parenteral regimens is reasonable; treatment beyond this duration has not been shown to be efficacious and raises the risk of complications. There continues to be debate about the role of CSF examination, particularly in patients with facial nerve palsies (Halperin

Table 13.1. Neurological disorders in Lyme disease, grouped by mechanism. Peripheral nerve

Mononeuropathy multiplex

Cranial neuropathy Radiculitis Brachial plexopathy Lumbosacral plexopathy Diffuse polyneuropathy Motor neuropathy? Mononeuropathy ± mutiplex ‘Guillain–Barré-like’ (not demyelinating)

Central nervous system

Infection in subarachnoid space

Parenchymal infection Metabolic encephalopathy

Lymphocytic meningitis Pseudotumour-like presentation in children Encephalitis (‘MS-like’ but monophasic) Myelitis Encephalopathy

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et al., 1996, 2007; Wormser et al., 2006; Rupprecht and Pfister, 2009; Mygland et al., 2010). Although the European neuroborreliosis guideline recommends this, the logic is not clear cut. If oral treatment is almost always effective and will be tried first, the presence or absence of CNS inflammation becomes irrelevant to the treatment strategy and therefore of unclear importance. In contrast, CSF examination is clearly important in patients with obvious parenchymal CNS infection, both to prove that this is due to B. burgdorferi infection and to address other potential aetiologies. Finally, over the years there has been concern about the role of corticosteroids (Pachner et al., 2001). As steroids are now of demonstrated efficacy in facial nerve palsies (Sullivan et al., 2007) and have been shown to help in painful Lyme radiculitis (Pfister et al., 1988), this becomes an important point. Although there are no systematic studies addressing this definitively, evidence in other bacterial meningitides (Brouwer et al., 2010) and non-systematic evidence in patients with neuroborreliosis suggest that, as long as appropriate antibiotics are also administered, steroids do not worsen the prognosis and may be considered judiciously in appropriate circumstances.

13.5 Summary Lyme disease affects the nervous system in approximately 12–15% of untreated infected individuals. Patients can either get diffuse meningeal inflammation or multifocal inflammation in peripheral or cranial nerves (mononeuropathy multiplex) or, rarely, in the CNS. The disorder termed Lyme disease encephalopathy is typically an indirect, presumably cytokine-mediated, effect on the brain; rarely is there evidence of CNS infection in these individuals. Treatment with oral doxycycline is probably sufficient for most patients with nervous system Lyme disease not affecting the parenchyma of the brain or spinal cord. In those rare patients with parenchymal CNS infection, parenteral antibiotics are highly effective.

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Lyme disease. American Journal of Ophthalmology 107, 81–82. Kan, L., Sood, S.K. and Maytal, J. (1998) Pseudotumor cerebri in Lyme disease: a case report and literature review. Pediatric Neurology 18, 439–441. Keller, T.L., Halperin, J.J. and Whitman, M. (1992) PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients. Neurology 42, 32–42. Krupp, L.B., Masur, D., Schwartz, J., Coyle, P.K., Langenbach, L.J., Fernquist, S.K., Jandorf, L. and Halperin, J.J. (1991) Cognitive functioning in late Lyme borreliosis. Archives of Neurology 48, 1125–1129. Ljostad, U., Skarpaas, T. and Mygland, A. (2007) Clinical usefulness of intrathecal antibody testing in acute Lyme neuroborreliosis. European Journal of Neurology 14, 873–876. Logigian, E.L. and Steere, A.C. (1992) Clinical and electrophysiologic findings in chronic neuropathy of Lyme disease. Neurology 42, 303–311. Logigian, E.L., Kaplan, R.F. and Steere, A.C. (1990) Chronic neurologic manifestations of Lyme disease. New England Journal of Medicine 323, 1438–1444. Luft, B.J., Steinman, C.R., Neimark, H.C., Muralidhar, B., Rush, T., Finkel, M.F., Kunkel, M. and Dattwyler, R.J. (1992) Invasion of the central nervous system by Borrelia burgdorferi in acute disseminated infection. Journal of the American Medical Association 267, 1364–1367. Luo, N., Johnson, J., Shaw, J.W., Feeny, D. and Coons, S.J. (2005) Self-reported health status of the general adult U.S. population as assessed by the EQ-5D and Health Utilities Index. Medical Care 43, 1078–1086. Muley, S.A. and Parry, G.J. (2009) Antibiotic responsive demyelinating neuropathy related to Lyme disease. Neurology 72, 1786–1787. Mygland, A., Ljostad, U., Fingerle, V., Rupprecht, T., Schmutzhard, E., Steiner, I. and European Federation of Neurological Societies (2010) EFNS guidelines on the diagnosis and management of European Lyme neuroborreliosis. European Journal of Neurology 17, 8–16, e1–e4. Pachner, A.R. and Steere, A.C. (1984) Neurological findings of Lyme disease. Yale Journal of Biology and Medicine 57, 481–483. Pachner, A.R., Amemiya, K., Bartlett, M., Schaefer, H., Reddy, K. and Zhang, W.F. (2001) Lyme borreliosis in rhesus macaques: effects of corticosteroids on spirochetal load and isotype switching of anti-Borrelia burgdorferi antibody. Clinical and Diagnostic Laboratory Immunology 8, 225–232.

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Pfister, H.W., Einhäupl, K.M., Franz, P. and Garner, C. (1988) Corticosteroids for radicular pain in Bannwarth’s syndrome: a double blind, randomized, placebo controlled trial. Annals of the New York Academy Sciences 539, 485–487. Ramesh, G., Borda, J.T., Dufour, J., Kaushal, D., Ramamoorthy, R., Lackner, A.A. and Philipp, M.T. (2008) Interaction of the Lyme disease spirochete Borrelia burgdorferi with brain parenchyma elicits inflammatory mediators from glial cells as well as glial and neuronal apoptosis. American Journal of Pathology 173, 1415–1427. Ramesh, G., Borda, J.T., Gill, A., Ribka, E.P., Morici, L.A., Mottram, P., Martin, D.S., Jacobs, M.B., Didier, P.J. and Philipp, M.T. (2009) Possible role of glial cells in the onset and progression of Lyme neuroborreliosis. Journal of Neuroinflammation 6, 23. Reik, L., Steere, A.C., Bartenhagen, N.H., Shope, R.E. and Malawista, S.E. (1979) Neurologic abnormalities of Lyme disease. Medicine 58, 281–294. Roberts, E.D., Bohm, R.P. Jr, Lowrie, R.C. Jr, Habicht, G., Katona, L., Piesman, J. and Philipp, M.T. (1998) Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system. Journal of Infectious Diseases 178, 722–732. Rovery, C., Greub, G., Lepidi, H., Casalta, J.P., Habib, G., Collart, F. and Raoult, D. (2005) PCR detection of bacteria on cardiac valves of patients with treated bacterial endocarditis. Journal of Clinical Microbiology 43, 163–167. Rupprecht, T.A. and Pfister, H.W. (2009) What are the indications for lumbar puncture in patients with Lyme disease? Current Problems in Dermatology 37, 200–206. Rupprecht, T.A., Koedel, U., Fingerle, V. and Pfister, H.W. (2008) The pathogenesis of Lyme neuroborreliosis: from infection to inflammation. Molecular Medicine 14, 205–212. Scrimenti, R.J. (1970) Erythema chronicum migrans. Archives of Dermatology 102, 104– 105. Shah, S.S., Zaoutis, T.E., Turnquist, J., Hodinka, R.L. and Coffin, S.E. (2005) Early differentiation of Lyme from enteroviral meningitis. Pediatric Infectious Disease Journal 24, 542–545. Sigal, L.H. (1990) Molecular mimicry and Lyme borreliosis [letter]. Annals of Neurology 28, 195–196. Steere, A.C., Malawista, S.E., Hardin, J.A., Ruddy, S., Askenase, W. and Andiman, W.A. (1977)

Erythema chronicum migrans and Lyme arthritis. The enlarging clinical spectrum. Annals of Internal Medicine 86, 685–698. Steere, A.C., Pachner, A.R. and Malawista, S.E. (1983) Neurologic abnormalities of Lyme disease: successful treatment with high-dose intravenous penicillin. Annals of Internal Medicine 99, 767–772. Steere, A.C., Berardi, V.P., Weeks, K.E., Logigian, E.L. and Ackermann, R. (1990) Evaluation of the intrathecal antibody response to Borrelia burgdorferi as a diagnostic test for Lyme neuroborreliosis. Journal of Infectious Diseases 161, 1203–1209. Sullivan, F.M., Swan, I.R., Donnan, P.T., Morrison, J.M., Smith, B.H., McKinstry, B., Davenport, R.J., Vale, L.D., Clarkson, J.E., Hammersley, V., Hayavi, S., McAteer, A., Stewart, K. and Daly, F. (2007) Early treatment with prednisolone or acyclovir in Bell’s palsy. New England Journal of Medicine 357, 1598–1607. Tuerlinckx, D., Bodart, E., Jamart, J. and Glupczynski, Y. (2009) Prediction of Lyme meningitis based on a logistic regression model using clinical and cerebrospinal fluid analysis: a European study. Pediatric Infectious Disease Journal 28, 394–397. Vallat, J.M., Hugon, J., Lubeau, M., Leboutet, M.J., Dumas, M. and Desproges-Gotteron, R. (1987) Tick bite meningoradiculoneuritis. Neurology 37, 749–753. Wendling, D., Sevrin, P., Bouchaud-Chabot, A., Chabroux, A., Toussirot, E., Bardin, T. and Michel, F. (2009) Parsonage–Turner syndrome revealing Lyme borreliosis. Joint Bone Spine 76, 202–204. Wilske, B., Schierz, G., Preac-Mursic, V., von Busch, K., Kühbeck, R., Pfister, H.W. and Einhäupl, K. (1986) Intrathecal production of specific antibodies against Borrelia burgdorferi in patients with lymphocytic meningoradiculitis. Journal of Infectious Diseases 153, 304–314. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S. and Nadelman, R.B. (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43, 1089–1134. Zemel, L. (2000) Lyme disease and pseudotumor. Mayo Clinic Proceedings 75, 315.

14

Lyme Disease in Children Eugene D. Shapiro

14.1 Introduction Although we now know that a form of the illness, erythema migrans (EM), was recognized in Scandinavia in the early 20th century (Afzelius, 1921), modern Lyme disease first came to light when a cluster of children living on a small street in Lyme, Connecticut, with what originally was thought to be juvenile rheumatoid arthritis, was reported by their parents in the mid1970s. Investigation of this unexplained ‘epidemic’ of arthritis by Allen Steere and colleagues led to the first report of Lyme arthritis in 1977 (Steere et al., 1977a). The illness was characterized by recurrent attacks of asymmetric swelling and pain in a few large joints, particularly the knee. About a quarter of the patients noted an erythematous papule that developed into an expanding, red, annular lesion (now known to be EM), as much as 50 cm in diameter, that preceded development of the arthritis by weeks to months (Steere et al., 1977b). Most of the patients (children and adults) who first developed the rash also eventually developed arthritis. While the overall prevalence of the arthritis was 0.5% of residents of the area, 10% of the children living on four particular roads developed the illness. From these original observations, the saga of Lyme

disease emerged. Within several years, the bacterial aetiology of the illness (Burgdorfer et al., 1982; Steere et al., 1983), the fact that the illness is a zoonosis transmitted by ticks and the effectiveness of antimicrobial treatment of the illness were recognized (Steere et al., 1985).

14.2 Aetiology and Epidemiology The ecology and the epidemiology of Lyme disease are described in Chapters 1, 2 and 6. It should be noted that children have the highest incidence of Lyme disease; it is highest in children aged 5–9 years – nearly twice the incidence among adults (Bacon et al., 2008). Borrelia burgdorferi is transmitted by ixodid ticks – in the USA, primarily by Ixodes scapularis, the deer tick (Lane et al., 1991). Other vectors include Ixodes ricinus (the sheep tick), Ixodes persulcatus and Ixodes pacificus in Europe, Asia and the Pacific coast of the USA, respectively. Persons with occupational, recreational or residential exposure to tickinfested fields, yards or woodlands in endemic areas are at increased risk of developing Lyme disease. Children may be at increased risk because of their propensity to play in areas in which ticks are found.

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

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14.3 Clinical Manifestations

14.3.2 Early disseminated Lyme disease

The clinical manifestations of Lyme disease in children are similar to the manifestations in adults. The manifestations are classified into stages – early localized disease, early disseminated disease and late disease (Shapiro, 1995; Shapiro and Gerber, 2000). In the USA, EM (single or multiple) is found in about 90% of patients with Lyme disease (Gerber et al., 1996; Nadelman and Wormser, 1998; Steere et al., 1998).

Early disseminated Lyme disease is a result of dissemination of the bacteria to multiple sites throughout the body via the bloodstream. At this stage of the illness, non-specific systemic manifestations such as fever, headache, fatigue, myalgia and arthralgia are more common than with early localized disease. The most common manifestation of early disseminated Lyme disease among children in the USA is multiple EM (Fig. 14.1), which accounts for 20–25% of cases of Lyme disease overall (Gerber et al., 1996). The secondary skin lesions, which usually appear from 3–5 weeks after initial inoculation of the bacteria, consist of multiple annular erythematous lesions similar to, but usually smaller than, the primary lesion. Sometimes the larger primary lesion is apparent, but multiple EM may occur without any recognized primary skin lesion.

14.3.1 Early localized Lyme disease Single EM, the manifestation of early localized disease, appears at the site of the tick bite, 3–30 days (typically within 7–14 days) after the bite. The rash is a result of inflammation at the site of inoculation of the bacteria into the skin by the vector tick. EM begins as a red macule and expands, over days to weeks, to form a large, annular, erythematous lesion that is at least 5 cm and as much as 50 cm in diameter (median 15 cm) (see Plate 9 a–c in the colour plate section). Although EM commonly is thought of as appearing as a target (‘bull’s eye’) lesion, it is more common (about two-thirds of the time) for the rash to be uniformly erythematous (Tibbles and Edlow, 2007). However, it may appear as a target lesion with variable degrees of central clearing. It can vary greatly in shape, and occasionally may have vesicular or necrotic areas in the centre. EM can develop in any location, but often occurs in the groin or axillary regions. However, in children, EM occurs in the head and neck areas more frequently than it does in adults. EM most often is asymptomatic but may be pruritic or painful, and it may be accompanied by systemic findings such as fever, malaise, headache, regional lymphadenopathy, stiff neck, myalgia or arthralgia. Approximately two-thirds of cases of Lyme disease in children present with a single EM (Gerber et al., 1996).

Fig. 14.1. Example of multiple erythema migrans.

Lyme Disease in Children

Neurological involvement also occurs as a manifestation of early disseminated Lyme disease in children, the most common manifestation of which in the USA is cranial nerve palsy. Palsy of the seventh cranial nerve (facial nerve palsy), which occurs in about 3% of children with Lyme disease in the USA, is most common (Gerber et al., 1996). Involvement is usually unilateral, although it can be bilateral, in which case it is virtually pathognomonic for Lyme disease. About 1–2% of children with Lyme disease in the USA present with meningitis, which manifests like aseptic meningitis, although the duration of symptoms before the patients present is typically longer (days to weeks) than with enteroviral meningitis (Eppes et al., 1999; Tuerlinckx et al., 2003; Avery et al., 2006). In addition, unlike with most cases of viral meningitis, papilloedema and increased intracranial pressure occur in a substantial minority of children with Lyme meningitis (Rothermel et al., 2001; Moses et al., 2003). Indeed, headache and increased intracranial pressure, which can mimic the presentation of pseudotumour cerebri, can be a prominent feature of Lyme meningitis, and occasionally occurs even in the absence of pleocytosis (Moses et al., 2003). A rare presentation (1%) of early disseminated Lyme disease in children is carditis, which usually manifests as complete heart block (Costello et al., 2009). The heart block usually resolves spontaneously within 5–10 days so that only transient artificial pacing of the heart is necessary. Lesser degrees of heart block are more common but usually are asymptomatic. Although it occurs only rarely in the USA, borrelial lymphocytoma, an inflammatory infiltrate that typically occurs in the ear lobe or the areola of the breast, is seen with some frequency in patients with Lyme disease in Europe. Likewise, meningoradiculoneuritis (Bannwarth’s syndrome), a sometimes painful radiculopathy due to Lyme disease, is more commonly reported in Europe than in the USA (see Halperin, Chapter 13, this volume).

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14.3.3 Late Lyme disease The manifestation of late Lyme disease in children, which occurs weeks to months after the initial infection, is arthritis. The arthritis is usually monoarticular or oligoarticular and affects the large joints, particularly the knee. There is a wide spectrum in the mode of presentation of Lyme arthritis (Rose et al., 1994; Gerber et al., 1998; Thompson et al., 2009; Smith et al., 2011). Although the affected joint is typically swollen and somewhat tender, the intense pain associated with a septic arthritis is rarely present. However, Lyme arthritis can occasionally mimic acute septic arthritis. Most often the presentation is subacute. It is not unusual for the patient or the parent to attribute the swelling to an acute event, for example falling on the knee while playing. If the arthritis is not treated, it usually resolves over the course of 1–2 weeks (although it may persist for many weeks), only to recur in the same or a different joint. Encephalitis, encephalopathy and polyneuropathy are rare manifestations of late Lyme disease in adults, but they virtually never occur in children. Acrodermatitis chronica atrophicans, a chronic sclerosing dermatitis, is an uncommon manifestation of Lyme disease in Europe that has not been described among children in the USA.

14.4 Coinfections Ixodes ticks may transmit other pathogens in addition to B. burgdorferi, including Babesia, Anaplasma, other Borrelia species and viruses (Wormser et al., 2006). These agents may be transmitted either separately from or simultaneously with B. burgdorferi. However, the frequency with which coinfection occurs is unknown and its impact on both the clinical presentation and the response to treatment of Lyme disease is not well defined, especially in children.

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14.5 Diagnosis The diagnosis of Lyme disease, especially if the characteristic rash is not present, may be difficult, as the other clinical manifestations of Lyme disease are not specific. Even the diagnosis of EM may sometimes be difficult, as the rash initially may be confused with nummular eczema, granuloma annulare, an insect bite, ringworm or cellulitis. The relatively rapid and prolonged (untreated, it lasts for weeks) expansion of EM helps to distinguish it from these other conditions. The sensitivity of culture for B. burgdorferi is only moderate, and special media are required; moreover, it is necessary for patients to undergo an invasive procedure to obtain appropriate tissue or fluid for culture. Consequently, such tests are indicated only in rare circumstances. Likewise, diagnostic tests that are based on the identification of antigens of B. burgdorferi or its DNA, including PCR assays, generally are not sufficiently accurate to be clinically useful under non-experimental conditions. Although studies in research laboratories suggest that PCR may be a promising diagnostic test, contamination is a potential problem in commercial laboratories and an invasive procedure is still necessary to obtain material to test. Consequently, the laboratory confirmation of Lyme disease usually rests on the demonstration of antibodies to B. burgdorferi in the patient’s serum. The use of laboratory tests to diagnose Lyme disease is discussed by Johnson (Chapter 4, this volume). It is critically important to understand that the predictive value of antibody test results, even of very accurate tests, is highly dependent on the prevalence of the infection among patients who are tested. Consequently, tests for antibodies to B. burgdorferi should not be used as screening tests, especially as many of the non-specific symptoms (e.g. fatigue, headache, arthralgia) that may accompany objective signs of Lyme disease are widely prevalent in the community. Unfortunately, because many lay persons (as well as physicians) have the erroneous belief that chronic, non-specific symptoms, even in the absence of objective signs of Lyme disease such as EM, may be manifestations of Lyme

disease, parents of children with only nonspecific symptoms frequently demand that the child be tested for Lyme disease (and some physicians routinely order tests for Lyme disease on such patients). Even in highly endemic areas, Lyme disease will be the cause of the non-specific symptoms in very few such children, if any. However, because the specificity of even the best antibody tests for Lyme disease is nowhere near 100%, some of the test results in children without specific clinical evidence of Lyme disease will be positive; the vast majority of these (95%) will be false-positive results (Seltzer and Shapiro, 1996; Tugwell et al., 1997). Nevertheless, an erroneous diagnosis of Lyme disease, based on the results of these tests, is frequently made and such children are often treated unnecessarily with antimicrobials. Clinicians should realize that, even though a symptomatic patient has a positive serological test result for antibodies to B. burgdorferi, it is possible that Lyme disease may not be the cause of that patient’s symptoms. Firstly, it may be a false-positive result, or the patient may have been infected with B. burgdorferi previously and the patient’s current symptoms may be unrelated to that previous infection. Once serum antibodies to B. burgdorferi do develop, like any other antibodies, they may persist for many years despite adequate treatment and clinical cure of the illness (Feder et al., 1992; Kalish et al., 2001). In addition, because some people who have been infected with B. burgdorferi never develop symptoms, in endemic areas there will be a background rate of seropositivity among patients who have never had clinically apparent Lyme disease. Physicians should not routinely order antibody tests for Lyme disease either for patients who have not been in endemic areas or for patients with only non-specific symptoms. On the other hand, in patients with objective signs of early disseminated or late Lyme disease (e.g. facial nerve palsy or synovitis of the knee) and an epidemiological history suggestive of Lyme disease, the a priori probability of Lyme disease is substantial and the predictive value of a

Lyme Disease in Children

positive serological test result is high. By contrast, the results of arthrocentesis are usually not definitive for making a diagnosis of Lyme arthritis. Testing of synovial fluid for antibodies against B. burgdorferi is not indicated as results have not been shown to be useful. By contrast, testing of cerebrospinal fluid (CSF) for specific antibodies and calculation of a CSF index for these antibodies, which, if positive, suggests intrathecal production of specific antibodies against B. burgdorferi, may be useful (see Halperin, Chapter 13, this volume).

14.6 Treatment A panel of experts from the Infectious Diseases Society of America (IDSA) has made comprehensive recommendations in a publication that provides practice guidelines for the management of patients with Lyme disease, anaplasmosis and babesiosis (Wormser et al., 2006). These are available on the website of the society (www.idsociety. org) under Practice Guidelines. The American Academy of Neurology has also published evidence-based recommendations for treatment of neuroborreliosis (Halperin et al., 2007). Recommendations for the treatment of Lyme disease in children are shown in Table 14.1. There has been some uncertainty about the need to treat patients with cranial palsies or meningitis due to Lyme disease with intravenously administered antimicrobials. However, the evidence is that orally administered treatment of facial nerve palsy results in excellent outcomes, and clinical trials conducted in Europe have indicated that treatment of Lyme meningitis with orally administered doxycycline is not inferior to treatment with intravenously administered ceftriaxone (Shapiro and Gerber, 1997; Vazquez et al., 2003; Ljostad et al., 2008). Occasionally a Jarisch–Herxheimer reaction occurs in the first 24–48 h after treatment is begun. This reaction, usually manifest by increased fever and myalgia, is a response to the release of antigens from dying bacteria. Antimicrobial treatment should be continued, and the patient should be

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reassured and treated with non-steroidal anti-inflammatory medications; the reaction usually resolves within a day or two.

14.7 Outcomes 14.7.1 Lyme disease The long-term prognosis for children who are treated with appropriate antimicrobial therapy for Lyme disease, regardless of the stage of the illness, is excellent (Gerber et al., 1996; Seltzer et al., 2000; Vazquez et al., 2003; Wormser et al., 2006). The most common reason for a lack of response to appropriate antimicrobial therapy for Lyme disease is misdiagnosis, i.e. the patient actually does not have Lyme disease (Sigal and Patella, 1992; Steere et al., 1993; Seltzer and Shapiro, 1996; Reid et al., 1998; Qureshi et al., 2002). Non-specific symptoms such as fatigue, arthralgia or myalgia occasionally persist for several weeks, even in patients with early Lyme disease who are successfully treated; the presence of these non-specific symptoms should not be regarded as an indication for additional treatment with antimicrobials. Such symptoms usually respond to nonsteroidal anti-inflammatory agents. Within a few months of completing the initial course of antimicrobial therapy, these vague, nonspecific symptoms will usually resolve without additional antimicrobial therapy. Facial nerve palsy due to Lyme disease resolves completely except in rare instances when some degree of residual facial palsy may persist. There is no evidence of other residual neurological deficits following facial nerve palsy (Vazquez et al., 2003), even though some degree of associated inflammation of the central nervous system is common (Belman et al., 1997). Other manifestations of early disseminated Lyme disease in children, such as Lyme meningitis, also resolve completely (Skogman et al., 2008). There have been rare reports of patients with papillitis and meningitis in whom increased intracranial pressure persists despite adequate antimicrobial treatment. In such instances, treatment of the increased intracranial pressure is important both for

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Table 14.1. Treatment of Lyme disease in children. (a) Mode of administration Disease stage

Clinical manifestations

Early localized Erythema migrans Early disseminated Multiple erythema migrans Isolated cranial nerve palsy Meningoradiculoneuritis Meningitis

Late

Carditis  Ambulatory  Hospitalized Borrelial lymphocytoma Arthritis Recurrent arthritis after oral therapy Encephalitis Acrodermatitis chronica atrophicans

Treatment

Duration

Oral Oral Oral IV or oral

14–21 days 14–21 days 14–21 days 14–21 days (IV)/14– 21 days (oral) 14–21 days (IV)/14– 21 days (oral)

IV or oral

Oral IVa followed by oral Oral Oral Oral or IV IV Oral

14–21 days 14–21 days 14–21 days 28 days 28 days (oral)/14–28 days (IV) 14–28 days 14–28 days

Treatment

Adult dose

Paediatric doseb

Doxycycline (patients 8 years)

100 mg/dosec twice a day

Amoxicillin

500 mg/dose three times a day

Cefuroxime axetil

500 mg/dose twice a day

Ceftriaxone

2 g every 24 h

Cefotaxime

2 g every 8 h

Penicillin G

3–4 million units/dose every 4 h

4 mg/kg/dayc (up to 100 mg /dosec) in two divided doses 50 mg/kg/day (up to 500 mg/dose) in three divided doses 30 mg/kg/day (up to 500 mg/dose) in two divided doses 50–75 mg/kg/day (up to 2 g) every 24 h 150–200 mg/kg/day (up to 2g)/dose divided every 8h 200,000–400,000 U/kg divided every 4 h (up to 18–24 million U/day)

(b) Dosage of antimicrobials

Intravenous

a

At the time of discharge from hospital, the patient may receive oral medication to complete therapy. and maximum daily dosages and frequency of administration per day. c Up to 8 mg /kg or 200 mg/dose for neuroborreliosis. b Total

symptomatic relief and to avoid damage to cranial nerves such as the optic nerve (Rothermel et al., 2001). Likewise, the long-term outcome of children with Lyme arthritis also is excellent (Rose et al., 1994; Gerber et al., 1996; Wang et al., 1998). Indeed, long-term follow-up of the original children who developed Lyme arthritis before either the cause of the disease

or the appropriate antimicrobial treatment were known revealed that in the overwhelming majority of them symptoms and signs of Lyme disease eventually resolved even though they were not treated with antimicrobials (Szer et al., 1991). For the rare patients who have persistent symptoms more than 6 months after the completion of antimicrobial therapy, an

Lyme Disease in Children

attempt should be made to determine whether these symptoms are the result of a post-infectious phenomenon or of another illness. There is no convincing evidence that ‘chronic Lyme disease’ (meaning persistent active infection despite treatment with antimicrobials) exists. This issue is discussed by Marques in Chapter 16 (this volume).

14.8 Congenital Lyme Disease Because Lyme disease is caused by a spirochaetal bacterium, there naturally was concern about the possibility that, similar to congenital syphilis (caused by a different spirochaetal bacterium), infection of pregnant women with B. burgdorferi could lead to congenital disease. Extensive study has found little or no evidence that congenital Lyme disease is a clinical problem (Silver, 1997). Several studies, designed to assess the potential link between Lyme disease during pregnancy and congenital infection with B. burgdorferi, found no consistent pattern of disease and no clearly documented B. burgdorferi infections of either the fetus or the infant (Strobino et al., 1993; Williams et al., 1995; Strobino et al., 1999). In addition, the obstetric outcomes were similar among women who had documented Lyme disease during their pregnancies and those who did not. Moreover, in a survey of 162 neurologists practising in areas endemic for Lyme disease, the investigators were unable to identify any well-documented cases of prenatally acquired neuroborreliosis (Gerber and Zalneraitis, 1994). Although there has been a temporal relationship between Lyme disease during pregnancy and adverse outcomes, a causal relationship has not been established. There is no evidence of increased risk of abnormal outcomes with Lyme disease during pregnancy. Transmission of Lyme disease via breastfeeding has not been documented.

14.9 Prevention of Lyme Disease Reducing the risk of tick bites is one obvious strategy to prevent Lyme disease. In endemic areas, clearing brush and trees, removing leaf

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litter and woodpiles, and keeping grass mowed may reduce exposure to ticks. Application of pesticides to residential properties is effective in suppressing populations of ticks but may be harmful to other wildlife and to people. Tick and insect repellents that contain N,N-diethyl-meta-toluamide (DEET) applied to the skin provide additional protection (Vazquez et al., 2008), but frequent reapplication may be necessary for maximum effectiveness. Serious neurological complications have been reported in children from either frequent or excessive application of DEET-containing repellents, but they are very rare and the risk is low when these products are used according to the instructions on their labels. Use of products with concentrations of DEET greater than 30% is not necessary and increases the risk of adverse effects. DEET should be applied sparingly and only to exposed skin, but not to a child’s face, hands or skin that is either irritated or abraded. After the child returns indoors, skin that was treated should be washed with soap and water. Permethrin (a synthetic pyrethroid) is available in a spray for application to clothing only and is particularly effective because it kills ticks on contact. Because most people (~75%) who recognize that they were bitten by a tick remove the tick within 48 h, the risk of Lyme disease from recognized deer tick bites is low – approximately 1–3% in areas with a high incidence of Lyme disease. Indeed, the risk of Lyme disease is higher for unrecognized bites (as such ticks will feed for a longer time). Persons should inspect themselves and their children’s bodies and clothing daily after possible exposure to ixodid ticks. An attached tick should be grasped with medium-tipped tweezers as close to the skin as possible and removed by gently pulling the tick straight out. If some of the mouth parts remain embedded in the skin, they should be left alone, as they usually are extruded spontaneously. Additional attempts to remove them often result in unnecessary damage to tissue and may increase the risk of local bacterial infection. Analysis of ticks to determine whether they are infected is not

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indicated because the predictive value of results of such tests for the development of disease in humans is unknown. No vaccine for Lyme disease is available for humans. A study of antimicrobial prophylaxis for tick bites among persons 12 years of age found that a single 200 mg dose of doxycycline was 87% effective in preventing Lyme disease, although the 95% confidence interval around this estimate of efficacy was wide – the lower bound was 25% or less, depending on the method used (Nadelman et al., 2001; see Wormser, Chapter 7, this volume). In that study, the only people who developed Lyme disease had been bitten by nymphal-stage ticks that were at least partially engorged. The risk of Lyme disease in this group was 9.9% (among the recipients of a placebo), while it was 0% for bites by all larval and adult deer ticks. Unfortunately, the expertise to identify the species, stage and degree of engorgement of a tick, and thereby to assess the degree of risk, is rarely available to people who are bitten. The overall risk of Lyme disease after a recognized deer tick bite, even in highly endemic areas, is low (1–3%). In addition, the number of patients needing to be treated to prevent a single case of Lyme disease in highly endemic areas is high – 50 patients bitten by a tick need to be treated to prevent a single case of Lyme disease (Warshafsky et al., 2010). Furthermore, only doxycycline (which is not recommended for children 8 years of age) has been shown to be effective; one cannot assume that a single dose of amoxicillin would be effective in preventing Lyme disease. Treatment for Lyme disease, if it does develop, is very effective. Consequently, routine administration of antimicrobial prophylaxis for children is not recommended (Shapiro, 2001). Serological testing for Lyme disease after a recognized tick bite is also not recommended. Antibodies to B. burgdorferi that are present at the time that the tick is removed or in the ensuing month or two are probably due either to a false-positive test result or to an earlier infection with B. burgdorferi rather than to a new infection from the recent bite. In this setting, the predictive value of a positive result is very low.

14.10 Fear of Lyme Disease and ‘Chronic Lyme Disease’ A panel of experts that developed clinical guidelines for managing patients with Lyme disease for the IDSA concluded that there is no such diagnostic entity as ‘chronic Lyme disease’ (Wormser et al., 2006). This contention is supported by clinical evidence and by experts throughout the world (Feder et al., 2007). Nevertheless, there is an extremely media-savvy group of activists, supported by ‘Lyme-literate’ doctors (often reinforced by sensationalized stories in the lay press) who propound that virtually any symptom can be due to Lyme disease and that even serological test results that are negative in patients with chronic symptoms do not mean that the patient does not have an active infection with B. burgdorferi that requires long-term treatment with antimicrobials. Unvalidated anecdotes on the Internet have served to support these misguided notions (Cooper and Feder, 2004). Some parents whose children have chronic, intractable problems are easy prey for healthcare providers who may profit from these misconceptions. The non-specific symptoms sometimes attributed to Lyme disease are highly prevalent in the general population, can be caused by common ailments such as viral illnesses or may be manifestations of either anxiety or depression. In some instances, anxious (often misinformed) parents are driven by the fear that their child’s nonspecific complaints may be a manifestation of Lyme disease that, if not detected and treated, could lead to serious chronic disability. There is a large body of evidence that Lyme disease rarely causes long-term problems (Feder et al., 2007). Moreover, studies have indicated that the best predictor of the long-term outcome of Lyme disease is the psychological state of the patient before the infection (Solomon et al., 1998). There is a growing body of literature on ‘medically unexplained symptoms’ that is highly applicable to these situations (Hatcher and Arroll, 2008). There is absolutely no scientific evidence to support contentions that Lyme disease causes autism, attention-deficit/hyperactivity disorder, chronic fatigue syndrome, school

Lyme Disease in Children

phobia or any of the myriad of behavioural problems that some have claimed are caused by Lyme disease. Parents would be well advised to beware of physicians whose practices are focused primarily on treating patients for Lyme disease, as this is likely to be the diagnosis made regardless of the complaint. Physicians in referral centres who specialize in Lyme disease continue to be deluged by patients who either are thought to have (or who believe they have) chronic Lyme disease. Reports from such centres indicate that in the great majority of instances the patients either never had Lyme disease or the symptoms that led to the referral were not due to Lyme disease (Steere et al., 1993; Seltzer and Shapiro, 1996; Reid et al., 1998; Qureshi et al., 2002). The challenge for clinicians who are faced with such patients (or with the parents of such patients) is to be able to address their concerns without dismissing them. In most instances, the patients have (or the parents perceive there is) a problem. Sometimes, a parent’s anxiety about a child’s behaviour can be allayed by reassurance. In other instances, a parent may insist that the child is ill, even though objective signs of organic illness are not present. Helping such patients obtain the type of help they need without alienating both the child and the parents may be difficult. Sometimes this can best be accomplished by affirming the concerns of the parents and explaining that you want simultaneously to assess both possible organic and possible behavioural causes of the problems.

14.11 Summary We now have more than 30 years of solid, scientific research about Lyme disease, a relatively common, vector-borne illness in parts of the USA and Europe. Although there is still widespread misunderstanding of and misinformation about the disease among the lay public, its clinical manifestations as well as how to diagnose and treat it are now well understood. In the vast majority of cases of Lyme disease in children, simple treatment with a relatively short

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course of orally administered antimicrobials results in long-term cure with no adverse sequelae.

References Afzelius, A. (1921) Erythema chronicum migrans. Acta Dermatology and Venereology (Stockholm) 2, 120–125. Avery, R.A., Frank, G., Glutting, J.J. and Eppes, S.C. (2006) Prediction of Lyme meningitis in children from a Lyme disease-endemic region: a logistic-regression model using history, physical, and laboratory findings. Pediatrics 117, e1–e7. Bacon, R.M., Kugeler, K.J., Mead, P.S. and Centers for Disease Control and Prevention (CDC) (2008) Surveillance for Lyme disease– United States, 1992–2006. Morbidity and Mortality Weekly Report Surveillance Summaries 57, 1–9. Belman, A.L., Reynolds, L., Preston, T., Postels, D., Grimson, R. and Coyle, P.K. (1997) Cerebrospinal fluid findings in children with Lyme disease-associated facial nerve palsy. Archives of Pediatrics and Adolescent Medicine 151, 1224–1228. Burgdorfer, W., Barbour, A.G., Hayes, S.F., Benach, J.L., Grunwaldt, E. and Davis, J.P. (1982) Lyme disease: a tick-borne spirochetosis? Science 216, 1317–1319. Cooper, J.D. and Feder, H.M. Jr (2004) Inaccurate information about Lyme disease on the internet. Pediatric Infectious Disease Journal 23, 1105– 1108. Costello, J.M., Alexander, M.E., Greco, K.M., Perez-Atayde, A.R. and Laussen, P.C. (2009) Lyme carditis in children: presentation, predictive factors, and clinical course. Pediatrics 123, e835–e841. Eppes, S.C., Nelson, D.K., Lewis, L.L. and Klein, J.D. (1999) Characterization of Lyme meningitis and comparison with viral meningitis in children. Pediatrics 103, 957–960. Feder, H.M. Jr, Gerber, M.A., Luger, S.W. and Ryan, R.W. (1992) Persistence of serum antibodies to Borrelia burgdorferi in patients treated for Lyme disease. Clinical Infectious Diseases 15, 788– 793. Feder, H.M. Jr, Johnson, B.J., O’Connell, S., Shapiro, E.D., Steere, A.C., Wormser, G.P., Ad Hoc International Lyme Disease Group, Agger, W.A., Artsob, H., Auwaerter, P., Dumler, J.S., Bakken, J.S., Bockenstedt, L.K., Green, J., Dattwyler, R.J., Munoz, J., Nadelman, R.B.,

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Schwartz, I., Draper, T., McSweegan, E., Halperin, J.J., Klempner, M.S., Krause, P.J., Mead, P., Morshed, M., Porwancher, R., Radolf, J.D., Smith, R.P. Jr, Sood, S., Weinstein, A., Wong, S.J. and Zemel, L. (2007) A critical appraisal of “chronic Lyme disease”. New England Journal of Medicine 357, 1422–1430. Gerber, M.A. and Zalneraitis, E.L. (1994) Childhood neurologic disorders and Lyme disease during pregnancy. Pediatric Neurology 11, 41–43. Gerber, M.A., Shapiro, E.D., Burke, G.S., Parcells, V.J. and Bell, G.L. (1996) Lyme disease in children in southeastern Connecticut. Pediatric Lyme Disease Study Group. New England Journal of Medicine 335, 1270–1274. Gerber, M.A., Zemel, L.S. and Shapiro, E.D. (1998) Lyme arthritis in children: clinical epidemiology and long-term outcomes. Pediatrics 102, 905– 908. Halperin, J.J., Shapiro, E.D., Logigian, E., Belman, A.L., Dotevall, L., Wormser, G.P., Krupp, L., Gronseth, G., Bever, C.T. Jr and Quality Standards Subcommittee of the American Academy of Neurology (2007) Practice parameter: treatment of nervous system Lyme disease (an evidence-based review): report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 69, 91–102. Hatcher, S. and Arroll, B. (2008) Assessment and management of medically unexplained symptoms. British Medical Journal 336, 1124– 1128. Kalish, R.A., Kaplan, R.F., Taylor, E., JonesWoodward, L., Workman, K. and Steere, A.C. (2001) Evaluation of study patients with Lyme disease, 10–20-year follow-up. Journal of Infectious Diseases 183, 453–460. Lane, R.S., Piesman, J. and Burgdorfer, W. (1991) Lyme borreliosis: relation of its causative agent to its vectors and hosts in North America and Europe. Annual Review of Entomology 36, 587– 609. Ljostad, U., Skogvoll, E., Eikeland, R., Midgard, R., Skarpaas, T., Berg, A. and Mygland, A. (2008) Oral doxycycline versus intravenous ceftriaxone for European Lyme neuroborreliosis: a multicentre, non-inferiority, double-blind, randomised trial. Lancet Neurology 7, 690–695. Moses, J.M., Riseberg, R.S. and Mansbach, J.M. (2003) Lyme disease presenting with persistent headache. Pediatrics 112, no. 6 Pt 1, e477–9. Nadelman, R.B. and Wormser, G.P. (1998) Lyme borreliosis. Lancet 352, 557–565. Nadelman, R.B., Nowakowski, J., Fish, D., Falco, R.C., Freeman, K., McKenna, D., Welch, P.,

Marcus, R., Aguero-Rosenfeld, M.E., Dennis, D.T., Wormser, G.P. and Tick Bite Study Group (2001) Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. New England Journal of Medicine 345, 79–84. Qureshi, M.Z., New, D., Zulqarni, N.J. and Nachman, S. (2002) Overdiagnosis and overtreatment of Lyme disease in children. Pediatric Infectious Disease Journal 21, 12–14. Reid, M.C., Schoen, R.T., Evans, J., Rosenberg, J.C. and Horwitz, R.I. (1998) The consequences of overdiagnosis and overtreatment of Lyme disease: an observational study. Annals of Internal Medicine 128, 354–362. Rose, C.D., Fawcett, P.T., Eppes, S.C., Klein, J.D., Gibney, K. and Doughty, R.A. (1994) Pediatric Lyme arthritis: clinical spectrum and outcome. Journal of Pediatric Orthopedics 14, 238–241. Rothermel, H., Hedges, T.R. III and Steere, A.C. (2001) Optic neuropathy in children with Lyme disease. Pediatrics 108, 477–481. Seltzer, E.G. and Shapiro, E.D. (1996) Misdiagnosis of Lyme disease: when not to order serologic tests. Pediatric Infectious Disease Journal 15, 762–763. Seltzer, E.G., Gerber, M.A., Cartter, M.L., Freudigman, K. and Shapiro, E.D. (2000) Longterm outcomes of persons with Lyme disease. Journal of the American Medical Association 283, 609–616. Shapiro, E.D. (1995) Lyme disease in children. American Journal of Medicine 98, S69–S73. Shapiro, E.D. (2001) Doxycycline for tick bites – not for everyone. New England Journal of Medicine 345, 133–134. Shapiro, E.D. and Gerber, M.A. (1997) Lyme disease and facial nerve palsy: more questions than answers. Archives of Pediatrics and Adolescent Medicine 151, 1183–1184. Shapiro, E.D. and Gerber, M.A. (2000) Lyme disease. Clinical Infectious Diseases 31, 533– 542. Sigal, L.H. and Patella, S.J. (1992) Lyme arthritis as the incorrect diagnosis in pediatric and adolescent fibromyalgia. Pediatrics 90, 523– 528. Silver, H.M. (1997) Lyme disease during pregnancy. Infectious Disease Clinics of North America 11, 93–97. Skogman, B.H., Croner, S., Nordwall, M., Eknefelt, M., Ernerudh, J. and Forsberg, P. (2008) Lyme neuroborreliosis in children: a prospective study of clinical features, prognosis, and outcome. Pediatric Infectious Disease Journal 27, 1089– 1094.

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Smith, B.G., Cruz, A.I. Jr, Milewski, M.D. and Shapiro, E.D. (2011) Lyme disease and the orthopaedic implications of Lyme arthritis. Journal of the American Academy of Orthopedic Surgeons 19, 91–100. Solomon, S.P., Hilton, E., Weinschel, B.S., Pollack, S. and Grolnick, E. (1998) Psychological factors in the prediction of Lyme disease course. Arthritis Care and Research 11, 419–426. Steere, A.C., Malawista, S.E., Snydman, D.R., Shope, R.E., Andiman, W.A., Ross, M.R. and Steele, F.M. (1977a) Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis and Rheumatism 20, 7–17. Steere, A.C., Malawista, S.E., Hardin, J.A., Ruddy, S., Askenase, P.W. and Andiman, W.A. (1977b) Erythema chronicum migrans and Lyme arthritis. The enlarging clinical spectrum. Annals of Internal Medicine 86, 685–698. Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E. and Malawista, S.E. (1983) The spirochetal etiology of Lyme disease. New England Journal of Medicine 308, 733–740. Steere, A.C., Green, J., Schoen, R.T., Taylor, E., Hutchinson, G.J., Rahn, D.W. and Malawista, S.E. (1985) Successful parenteral penicillin therapy of established Lyme arthritis. New England Journal of Medicine 312, 869–874. Steere, A.C., Taylor, E., McHugh, G.L. and Logigian, E.L. (1993) The overdiagnosis of Lyme disease. Journal of the American Medical Association 269, 1812–1816. Steere, A.C., Sikand, V.K., Meurice, F., Parenti, D.L., Fikrig, E., Schoen, R.T., Nowakowski, J., Schmid, C.H., Laukamp, S., Buscarino, C. and Krause, D.S. (1998) Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme Disease Vaccine Study Group. New England Journal of Medicine 339, 209–215. Strobino, B.A., Williams, C.L., Abid, S., Chalson, R. and Spierling, P. (1993) Lyme disease and pregnancy outcome: a prospective study of two thousand prenatal patients. American Journal of Obstetrics and Gynecology 169, 367–374. Strobino, B., Abid, S. and Gewitz, M. (1999) Maternal Lyme disease and congenital heart disease: a case–control study in an endemic area. American Journal of Obstetrics and Gynecology 180, 711–716. Szer, I.S., Taylor, E. and Steere, A.C. (1991) The long-term course of Lyme arthritis in children. The New England Journal of Medicine 325, 159–163.

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Thompson, A., Mannix, R. and Bachur, R. (2009) Acute pediatric monoarticular arthritis: distinguishing Lyme arthritis from other etiologies. Pediatrics 123, 959–965. Tibbles, C.D. and Edlow, J.A. (2007) Does this patient have erythema migrans? Journal of the American Medical Association 297, 2617– 2627. Tuerlinckx, D., Bodart, E., Garrino, M.G. and de Bilderling, G. (2003) Clinical data and cerebrospinal fluid findings in Lyme meningitis versus aseptic meningitis. European Journal of Pediatrics 162, 150–153. Tugwell, P., Dennis, D.T., Weinstein, A., Wells, G., Shea, B., Nichol, G., Hayward, R., Lightfoot, R., Baker, P. and Steere, A.C. (1997) Laboratory evaluation in the diagnosis of Lyme disease. Annals of Internal Medicine 127, 1109–1123. Vazquez, M., Sparrow, S.S. and Shapiro, E.D. (2003) Long-term neuropsychologic and health outcomes of children with facial nerve palsy attributable to Lyme disease. Pediatrics 112, e93–e97. Vazquez, M., Muehlenbein, C., Cartter, M., Hayes, E.B., Ertel, S. and Shapiro, E.D. (2008) Effectiveness of personal protective measures to prevent Lyme disease. Emerging Infectious Diseases 14, 210–216. Wang, T.J., Sangha, O., Phillips, C.B., Wright, E.A., Lew, R.A., Fossel, A.H., Fossel, K., Shadick, N.A., Liang, M.H. and Sundel, R.P. (1998) Outcomes of children treated for Lyme disease. Journal of Rheumatology 25, 2249–2253. Warshafsky, S., Lee, D.H., Francois, L.K., Nowakowski, J., Nadelman, R.B. and Wormser, G.P. (2010) Efficacy of antibiotic prophylaxis for the prevention of Lyme disease: an updated systematic review and meta-analysis. Journal of Antimicrobial Chemotherapy 65, 1137–1144. Williams, C.L., Strobino, B., Weinstein, A., Spierling, P. and Medici, F. (1995) Maternal Lyme disease and congenital malformations: a cord blood serosurvey in endemic and control areas. Paediatric and Perinatal Epidemiology 9, 320– 330. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S. and Nadelman, R.B. (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43, 1089–1134.

15

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’ Afton L. Hassett and Leonard H. Sigal

15.1 Introduction Lyme disease is a symptomatic infection with the tick-borne organism Borrelia burgdorferi (Steere, 2001). It is extremely rare for patients treated with adequate antibiotic therapy to manifest objective evidence of ongoing inflammation or organ dysfunction, i.e. evidence of persisting infection (Wormser et al., 2006). Yet studies of adults with previously antibiotic-treated Lyme disease have found that 4–40% report post-antibiotic-therapy complaints including chronic physical, cognitive and/or emotional symptoms that they and their doctors attribute to Lyme disease (Asch et al., 1994; Shadick et al., 1994; Bujak et al., 1996; Dattwyler et al., 1997; Shadick et al., 1999; Seltzer et al., 2000; Smith et al., 2002; Wormser et al., 2003; Hassett et al., 2009). The patients often experience ongoing disability, with mounting medical bills and toxicities from chronic, often unproven, therapies. The most common complaints attributed to ‘chronic Lyme disease’ are ‘flu-like’ symptoms, which can include non-inflammatory musculoskeletal pain, fatigue, cognitive fogginess and mood disturbance (e.g. depression and anxiety). This cluster of symptoms is also consistent with symptoms observed in fibromyalgia, the common presence of which

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in the post-Lyme disease population was first described by our group two decades ago (Sigal, 1990). When depression and anxiety are observed in medical conditions, psychological explanations for these and other coincident symptoms are often sought. This is true for fibromyalgia and for the persistent symptoms attributed to ‘chronic Lyme disease’. Herein, we address the psychological aspects of symptom persistence in patients who attribute such symptoms to ‘chronic Lyme disease’. It is not our contention that these symptoms are psychiatric in nature. As a matter of fact, our own research has demonstrated that depression and anxiety are present in fewer than half of Lyme disease specialty centre patients and do not predict post-therapy symptom persistence (Hassett et al., 2009, 2010). Rather, there appears to be a complex interplay between biological, psychological and social factors, which might better illuminate the problem, help explain the pathogenesis of complaints and suggest potential approaches to treatment.

15.2 The Universe Divided into Three To lay the groundwork for exploring the relative contributions of psychological factors, our research has shown that patients

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’

who ascribe chronic symptoms to Lyme disease vary in terms of Lyme disease status and commitment to the diagnosis. We have found that one can broadly categorize patients with persistent complaints thought to be related to Lyme disease into three groups: 1. Patients who now  have Lyme disease (ongoing infection with B. burgdorferi: these patients may or may not have been treated previously with antibiotics). 2. Patients who once had  Lyme disease, but no longer have active infection with B. burgdorferi (e.g. post-Lyme disease syndrome; other unrelated medical conditions). 3. Patients who have never  had Lyme disease, but who believe, to varying degrees, that Lyme disease is the cause of their ongoing complaints (‘not Lyme’).

15.3 Current Infection and ‘AntibioticResistant’ Lyme Disease In the first and decidedly smallest group are patients afflicted with current or persistent infection. For some patients, the diagnosis of Lyme disease may have been missed in its earliest stages. These patients may present with later manifestations, often ascribed to different clinical conditions. Other patients may have been diagnosed correctly but treated inadequately; sometimes the antibiotic was incorrect, such as cephalexin, rather than the dependable amoxicillin, doxycycline or cefuroxime axetil. In some cases, the dosage or duration of an effective antibiotic was too small or short, while in other cases the therapy was not appropriate to the manifestations, such as oral antibiotics given for severe carditis, encephalitis or arthritis. Although we are unaware of any reports of an isolate of B. burgdorferi being antibiotic resistant, there is, of course, the possibility that treatment failures may occur, for example due to poor absorption of antibiotics, and of course some patients may be non-compliant. Although there are animal studies demonstrating that viable B. burgdorferi can persist after antibiotic therapy (Yrjanainen et

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al., 2007), there is no convincing evidence that live B. burgdorferi persists in humans after appropriate antibiotic therapy (Marques et al., 2000; Klempner, 2002; chapter 5 of this volume). A set of explanations for this phenomenon (persistence of complaints in patients with Lyme disease despite adequate antibiotic therapy) was proposed in 1994 (Table 15.1). Ongoing symptoms are often ascribed to persistent infection, with some medical practitioners hypothesizing antibiotic resistance of the organism or that the organism somehow evades the immune system, for example residing in a dormant and/or intracellular location. There is the theoretical possibility that B. burgdorferi might be resistant to the standard antibiotic agents in use, but we are unaware of any such isolates. If the organism is able to cause inflammation, it could not be strictly intracellular; thus, it would be available to induce an immune response, and ongoing seronegativity in an immunocompetent individual would be implausible. Furthermore, the absence of objective tissue damage or inflammation in almost all of these patients makes persistence of the organism even less likely to be the explanation for persistence of symptoms. Thus, no scientific argument can be made in favour of the premise that chronic infection is the cause of chronic symptoms.

15.4 Post-Lyme Disease Syndrome Post-Lyme disease syndrome (PLDS) refers to symptoms that continue for at least 6 months after initial diagnosis and appropriate treatment for proven Lyme disease (Wormser et al., 2006). Many patients will have a prompt response to antibiotics, although some may have lingering non-specific complaints for up to 6 months. Although most patients do improve following antibiotics, as many as 40% have chronic non-specific complaints that can last for months or years. Our prospective studies of newly infected patients followed over time have shown that between 13 and 32% of patients report ongoing symptoms ascribed to Lyme disease after treatment (Hassett et al., 2009, 2010). Often these subjective symptoms are not supported

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Table 15.1. Possible explanations for persistence of symptoms. Diagnosis

Explanation

Unrelated to Lyme disease Slowly resolving Lyme disease

Initial misdiagnosis, never was Lyme disease Prior infection, effectively treated, no live organism persists, a long recuperative phase Damage from prior infection and associated inflammation, if effectively treated; no persisting live organism Not specific for Lyme disease; if prior infection treated effectively, no persisting organism May never have been treated or was treated inadequately (wrong drug; right drug, inappropriate dose: too short a duration, too low a dose (oral rather than intravenous)) Ongoing inflammation not due to persisting infection Reactive phenomena/immune-mediated symptoms, central augmentation, anxiety/fear of chronic Lyme disease, adoption of a ‘chronic illness’ role

Permanent tissue damage

Factors related to chronic illness True persisting infection with B. burgdorferi

Sterile inflammation caused by persisting, poorly degraded dead organism Post-Lyme disease syndrome

Adapted from Sigal (1994).

by objective evidence, including findings on physical examination or neurocognitive testing (Shadick et al., 1999). In some cases, damage may have occurred that cannot resolve even after the infection is eradicated, such as the rare patients with unresolving facial nerve palsies or permanent heart block despite appropriate antibiotic treatment. Inflammation and organ dysfunction may continue after B. burgdorferi is killed by antibiotics, as host defences are activated by residual debris acting as a persisting focus of inflammation (Sigal, 1997). Until the organism is cleared from the site of infection and healing has subsided, there could be active inflammation and damage without active infection, misinterpreted as ‘active disease’. In far more chronically symptomatic patients, there is no evidence of inflammation or objective evidence of organ dysfunction; most such patients describe chronic musculoskeletal pain, fatigue, cognitive fogginess, depression and anxiety (Hassett et al., 2008, 2009). Although PLDS has been at the centre of much debate, to date there is no evidence of persistence of organisms and no immunopathogenic process has been identified (Sigal, 1997). There has been speculation that immunological cross-reactivity between the

organism and a host component may drive inflammation, but this has not been proven to be of clinical relevance (Sigal, 1997). Instead, about one-third of patients with PLDS meet the full criteria for fibromyalgia (Dinerman and Steere, 1992; Sigal and Patella, 1992; Hsu et al., 1993). Fibromyalgia and other similar chronic pain conditions are non-inflammatory in nature and do not respond to antibiotic therapy directed against Lyme disease (Hsu et al., 1993). None the less, many ‘chronic Lyme disease’ sufferers have been given antibiotics for months, even years, obtaining no lasting relief. A transient ‘response’ is often interpreted as proof of infection rather than evidence of a placebo effect or perhaps an unforeseen immunomodulatory effect of the antimicrobial agent being used, as has been summarized (Sigal, 1999). Oral and intravenous antibiotics, in combinations or sequential cycles, hyperbaric oxygen, even self-induced malaria (for a pyrexia-induced cure as was used many years ago for neurosyphilis) have been proposed and/or tried. No study has ever demonstrated that aggressive and/or long-term antibiotic therapy for PLDS of any greater intensity than is suggested by the Infectious Disease Society of America (IDSA) guidelines (Wormser et al., 2006) is of any proven efficacy

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’

for the long-term symptoms following Lyme disease. For example, a trial of long-term antibiotics funded by the National Institutes of Health was closed because it failed to show any effect on the symptoms of patients with ‘chronic Lyme disease’ (Klempner et al., 2001a). None the less, it is important to explore explanations for these refractory cases. The antibiotics used for long-term treatment have toxicities that are often worse than the patient’s original symptoms (Ettestad et al., 1995). Avoiding further iatrogenic damage is critical (Sigal, 1995, 1996).

15.5 Multiple Symptoms Ascribed to Lyme Disease (‘not Lyme’) The third group of ‘Lyme disease patients’ accounts for well over half of patients evaluated in academic Lyme disease referral centres (Sigal, 1990; Steere et al., 1993; Reid et al., 1998). These patients ascribe multiple diffuse symptoms to Lyme disease or ‘chronic Lyme disease’ but have no objective evidence of infection with B. burgdorferi at the time of assessment or ever in the past. Some have been misdiagnosed by physicians, while others are self-diagnosed (Sigal, 1990; Hsu et al., 1993; Klempner et al., 2001b). Many have undergone long-term and/or repeated antibiotic therapy, combination and/or cycling therapies and treatment with agents previously shown to be ineffective in Lyme disease (Sigal, 1990, 1996; Reid et al., 1998; Sigal and Hassett, 2005; Wormser et al., 2006). Many utilize considerable healthcare resources, experience adverse drug reactions and describe high rates of disability (Sigal, 2001). The symptoms are similar to those of PLDS – joint and muscle pain with no evidence of inflammation, fatigue, insomnia, cognitive complaints and mood disturbance (Reid et al., 1998; Sigal and Hassett, 2005); however, we have found that a number of these patients have readily identifiable medical conditions that may or may not have been addressed previously. In our large crosssectional study, we reported that 53 (22.1%) out of the 240 patients evaluated in our Lyme disease specialty centre had an identifiable medical condition, other than fibromyalgia,

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that accounted for their symptoms. Diagnoses ranged from the benign (e.g. age-related myalgias) to much more threatening conditions (e.g. multiple sclerosis, amyotrophic lateral sclerosis, Parkinson’s disease). Because we did not set out to study attributions, it is not clear how patients arrived at the notion that their symptoms were related to Lyme disease. We speculate that, in some cases, patients preferred a diagnosis of Lyme disease to other diagnoses, especially psychiatric diagnoses such as major depressive disorder, or more ominous medical diagnoses without good therapeutic options such as amyotrophic lateral sclerosis. In some cases, certain community physicians (the selfproclaimed ‘Lyme literate’) misdiagnosed patients and planted the seed of ‘chronic Lyme disease’ as the cause of their suffering.

15.6 Commitment to the Diagnosis of ‘Chronic Lyme Disease’ ‘Disease conviction’ has been described by Pilowsky and Spence (1983) as the belief that one has a physical illness despite repeated reassurances from physicians to the contrary. We have heard from many patients in our clinic that they are sure ongoing infection – active Lyme disease – accounts for their symptoms, despite all objective and scientific evidence to the contrary. When a patient has a high level of disease conviction, information is selectively processed: that which supports the belief is accepted, whereas information that refutes the belief is devalued or discarded. A common example of this is when numerous negative laboratory results are dismissed in favour of one equivocal or misinterpreted result (or a result from a nonreputable laboratory) that is taken as proof of active infection. Moreover, efforts to correct the initial mistaken belief often seem to harden the conviction of the patient and/or family that the diagnosis of Lyme disease is correct. The level of disease conviction varies among patients: some are reassured when another explanation is found for their symptoms, some are troubled that an incorrect diagnosis of a chronic illness was made, while others are incensed that the

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consulting physician has challenged the standing diagnosis of ‘chronic Lyme disease’. When disabused of the diagnosis of ‘chronic Lyme disease’, many can become beset by doubts, confusion and anxiety/fear of the unknown. All share one desire: to have an explanation for their symptoms and treatment to make the symptoms go away.

15.7 The Psychology of ‘Post-Lyme’ and ‘Not Lyme’ There are very few medical conditions where psychological variables play little or no role in the outcomes associated with the illness process. The purely biomedical model of chronic illness has generally been eschewed in favour of a biopsychosocial approach that takes into consideration the thoughts, affect and behaviour of the individual afflicted. In this context, factors such as hostility and depression are seen as important predictors of outcome in cardiovascular disease (Chida and Steptoe, 2009), while having high levels of positive affect (optimism) can serve to be protective in the case of the common cold (Cohen et al., 2003). Thus, evaluating the existing research for its role in ‘chronic Lyme disease’ is not meant to pathologize our patients, but rather to help chart the course for a more accurate and holistic approach to their care. 15.7.1 The role of psychiatric comorbidity Psychiatric comorbidity is common in nearly all chronic illnesses; having a medical condition that disrupts one’s ability to function physically and socially is depressing and/or anxiety-provoking in even the most resilient individuals. Reid et al. (1998) were perhaps the first to evaluate the presence of depression in patients presenting for treatment at a Lyme disease specialty clinic. They determined that in 60% of the 209 patients examined at the Yale University Lyme Disease Clinic there was no evidence of current or previous infection with B. burgdorferi. However, psychiatric comorbidity was common: 42% reported symptoms of

depression, while 16% were thought to have primary depression. Similarly, early studies in patients with PLDS found that they manifested higher rates of depression than patients who had recovered from Lyme disease (Bujak et al., 1996), as well as those with active Lyme disease (Reid et al., 1998). Rates of depression for PLDS have been estimated to be between 23 and 66% (Fallon et al., 1993; Reid et al., 1998). We found over 45% of the PLDS patients evaluated at our academic referral centre had a current major depressive episode, using the gold standard, the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders (Hassett et al., 2009). This is in contrast to the patients in our ‘not Lyme’ group who had depression at a rate of 26.2%. For context, a current major depressive episode occurs in the general population at a 1-year prevalence rate of about 6.7% (Kessler et al., 2005). We found that anxiety disorders occurred at a similar elevated rate, 29 and 25.6% respectively, for PLDS and ‘not Lyme’ patients. The 1-year prevalence for any anxiety disorder in the general population is estimated at approximately 18.1% (Kessler et al., 2005). Most striking about our findings was the observation that over 90% of the PLDS patients with one psychiatric disorder qualified for diagnosis of a second psychiatric disorder; depression and anxiety overlapped most frequently. Consistent with other medical populations, comorbid depression and anxiety were associated with worse outcomes in PLDS. For example, two separate studies reported that the affective symptoms of PLDS patients seemed to predispose them to the perception of cognitive impairment (Barr et al., 1999; Kaplan et al., 1999). Similarly, we found that the presence of clinical disorders such as anxiety and depression in patients seeking care for Lyme disease were predictive of worse functioning scores (Hassett et al., 2009). Based on the limited research thus far, one cannot say whether or not there is a direct causal relationship between psychiatric comorbidity and PLDS. Few studies have explored this question, and the findings have

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been contradictory. For example, Guadino et al. (1997) found that 26% of PLDS patients evaluated reported having been diagnosed with a psychiatric disorder before being diagnosed with Lyme disease. In contrast, findings from our prospective study, described in greater detail later, suggest that psychiatric comorbidity at baseline does not play a role in symptom persistence after treatment for Lyme disease infection (Hassett et al., 2010). 15.7.2 The contribution of other psychological factors A number of psychological factors can contribute to the behavioural manifestations of PLDS and potentially have a tremendous impact on the patients’ subjective experience of illness. For PLDS and ‘not Lyme’ patients, we found several psychological factors including catastrophizing, high negative affect and low positive affect to be associated with poor medical outcomes (Hassett et al., 2008, 2009). Furthermore, compared with patients with readily identifiable medical conditions, these psychological factors were more pronounced in PLDS and ‘not Lyme’, and were highly related to poor functioning for all patients (Hassett et al., 2009). However, because this particular study had a crosssectional design, it was not clear whether these factors preceded the Lyme disease, were the product of the experience of having a chronic illness or were a combination of both. These cognitive and affective factors seem to be important even in the absence of psychiatric comorbidity. One can have no discernable psychiatric disorder such as depression or anxiety but still manifest multiple psychological processes that put the patient at risk for poor outcomes. One study found that, although most of the PLDS group evaluated was not clinically depressed, their low levels of positive affect were associated with the severity of complaints of both physical and cognitive symptoms (Elkins et al., 1999). Positive affect refers to having general good feelings such as interest, enthusiasm and determination and is highly associated with good medical outcomes in

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other populations including lower levels of pain (Zautra et al., 2005) and resistance to viral infection (Cohen et al., 2003). Positive and negative affect are independent constructs; thus, having low negative affect (i.e. little or no depression, hostility or anxiety) alone is not sufficient for optimal health outcomes. The behaviour of some patients who present to Lyme disease specialty clinics has been the topic of research and editorials written by healthcare professionals (Sigal and Hassett, 2005; Feder et al., 2007; Halperin, 2008). Lamberg noted that personality disorders are often present in some of the most ‘difficult’ medical patients seen in many different settings (Lamberg, 2006). We explored this question and did not find antecedent personality disorders to be predictive of PLDS or ‘not Lyme’. Patients with PLDS did have slightly elevated rates of these disorders (29% compared with 21.1% for the medical comparison group), but the measurement of personality disorders is a particularly inexact science. Although we used an instrument considered the gold standard for assessment, the Millon Clinical Multiaxial Inventory (MCMI), and used a higher-than-recommended cut-off score, false positives can still be a problem (Guthrie and Mobley, 1994). This is evidenced by the higher-than-expected rate of personality disorders in our medical condition comparison group (21.1%); the estimated rate of personality disorder in the general population is approximately 9% (Lenzenweger et al., 2007). Nearly all the psychosocial risk factors noted above contribute to the general experience of psychological stress and such perceived stress appears to be common in PLDS patients. Reid et al. (1998) reported that 52% of their PLDS group reported high levels of perceived stress. Having a chronic illness can contribute to having high levels of perceived stress, but it is also possible that persistent stress could have preceded the infection with B. burgdorferi and be a risk factor for symptom persistence. Persistent and intensified activation of the stressresponse systems results in the dysfunctions of these systems. McEwen (2007) refers to

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such wear and tear on the body’s organs and systems as ‘allostatic load’ or the price the body pays for repeatedly responding to excessive stress and/or when the stress response fails to ‘turn off ’ when no longer needed. These changes can affect neuroendocrine processes and may account for the increased levels of pain mediators such as substance P, glutamate and nerve growth factor observed in chronic pain conditions like fibromyalgia (Clauw, 2009). Conversely, most PLDS and ‘not Lyme’ patients do not meet the criteria for psychiatric comorbidity, which is consistent with other reports describing subgroups of psychologically healthy fibromyalgia patients who in one study reported lower levels of pain despite increased pain sensitivity (Giesecke et al., 2003). It is quite possible that resilience factors such as positive affect could be better targets to explore. Prospective studies are required to address the role of such psychological risk and protective factors adequately. 15.7.3 Is there an aetiological role for psychological factors in post-Lyme disease syndrome? Whenever depression and anxiety are observed frequently in a medical condition, causality is speculated. However, most studies evaluating psychiatric comorbidity in PLDS and ‘not Lyme’ patients are crosssectional so causality cannot be inferred. Only prospective studies in PLDS can begin to answer questions about the true role of psychological factors in symptoms ascribed to Lyme disease. Solomon et al. (1998) explored the role of psychological factors in Lyme disease and found a strong association between a history of prior psychological trauma and chronic physical symptoms, which led them to hypothesize that antecedent traumatic psychological experiences may play an aetiological role in the persistence of PLDS symptoms. More recently, we tracked 99 patients with newly diagnosed Lyme disease to assess risk factors for persistent symptoms after treatment (Hassett et al., 2010). Based on the

findings of Bujak et al. (2000) who found that the number of symptoms and worse functional status at baseline more than triples the risk of developing chronic symptoms after antibiotic treatment, we assessed these same factors plus depression, anxiety, catastrophizing, somatosensory amplification and affective style (negative affect and positive affect). At 1 year, 32.4% reported chronic symptoms attributed to their previous infection with Lyme disease. Based on the literature and our own research, we predicted that functional status, depression and anxiety at baseline would be strong predictors of later symptomatic status. Much to our surprise, we found that only functional status and level of positive affect at baseline were predictive of persistent symptoms. Moreover, the level of positive affect was not related to functional status at baseline, which would be the case if symptom severity determined the level of positive feelings. Instead, high levels of positive affect predicted recovery regardless of symptom severity at baseline. We are not the first to find a protective role for positive affect in infectious disease – the cold studies conducted by Cohen and colleagues found positive affect to be an even better predictor of catching a cold and cold symptom severity than negative affect (Cohen et al., 2003; Cohen, 2005). 15.7.4 The ubiquitous presence of fibromyalgia In a review of the first 100 patients seen at the Lyme Disease Center at Robert Wood Johnson Medical School, New Jersey, only 37% of patients referred had current or preceding Lyme disease as the explanation for their complaints (Sigal, 1990). Instead, in patients where Lyme disease did not explain patient symptoms, close to 40% met the criteria for fibromyalgia. Interestingly, in most of the patients with fibromyalgia, the onset of fibromyalgia was subsequent to Lyme disease, as if Lyme disease had triggered the fibromyalgia. Steere et al. (1993) confirmed these findings; in the 788 patients evaluated at their clinic, only 23% had active Lyme disease. More than half of these patients

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’

appeared never to have had Lyme disease and many qualified for a diagnosis of chronic fatigue syndrome or fibromyalgia. Similarly, Reid et al. (1998) reported that 31% of patients presenting for evaluation at their academic Lyme disease specialty clinic did not have Lyme disease but had instead a fibromyalgialike ‘fatigue–arthralgia–myalgia syndrome’. More recently, we conducted two prospective studies assessing patients with active B. burgdorferi infection who received antibiotic treatment. In the first study of 46 patients treated in our own clinic, 13% later met American College of Rheumatology criteria for fibromyalgia with many more exhibiting a fibromyalgia-like illness (Hassett et al., 2009). In a separate and more rigorous study of 99 newly diagnosed Lyme disease patients treated and followed for 1 year, 32% developed a fibromyalgia-like syndrome characterized by chronic widespread pain, fatigue, mood disturbance and perceived cognitive dysfunction (Hassett et al., 2010). 15.7.5 Somatization disorders (it’s all in your head!) So what are the implications for a medical condition that has high rates of psychiatric comorbidity and overlap with a chronic pain condition like fibromyalgia? Some might argue that PLDS and ‘not Lyme’, as well as fibromyalgia, be considered somatization disorders. Escobar et al. (1998, p. 466) defined somatization ‘as the presentation of many symptoms suggestive of physical disease, but which remain unexplained after medical and laboratory assessments’. Somatization is thought to be the unconscious expression of unacceptable emotions through bodily symptoms, while somatoform disorders are psychiatric disorders described in the Diagnostic and Statistical Manual for Mental Disorders IV (American Psychiatric Association, 2000). Yet somatization in the classic sense artificially separates bodily and psychological symptoms that patients experience as a whole (Epstein et al., 1999) and that may have definable neurobiological relationships (Sternberg, 2000). There is overwhelming

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evidence supporting the hypothesis that humans experience emotions throughout their bodies (Pert, 1997). Neurotransmitters associated with thought and emotion are often the same as those associated with the immune system, stress-response systems and pain-transmission system to name but a few (Chrousos and Gold, 1992; Pert, 1997; Sternberg, 2000). Although reviewing the evidence for bidirectional relationships between psychological and physiological phenomena is beyond the scope of this chapter, consistent evidence across studies suggests that strong emotions, especially anxiety, hostility and depression, induce or aggravate somatic symptoms (Kellner, 1994). Thus, the psychoanalytic concept of the ‘psychopathogenesis’ of medical illness has increasing relevance today in light of findings from the fields of psychoneuroimmunology and neuroendocrinology. In some cases, psychological factors appear to play a particularly powerful role in the manifestation and mediation of medical illness. This seems especially true of chronic pain syndromes like fibromyalgia and perhaps PLDS and ‘not Lyme’. In each case, the aetiology and pathophysiology of the illness remain unclear, but high rates of clinical overlap between the disorders, including mood and anxiety disorders, suggest a shared pathophysiological basis (Korszun et al., 1998). 15.7.6 Central sensitivity syndromes (it’s all in your brain!) The term ‘central sensitivity syndromes’ refers to a group of disorders that share similar clusters of symptoms (e.g. chronic pain, fatigue, cognitive complaints, mood disturbance) and potentially pathophysiology (Yunus, 2007). Over the past decade, research related to the neuroscience of pain has contributed markedly to our understanding of the pathophysiology of chronic pain conditions like fibromyalgia, irritable bowel syndrome (IBS), interstitial cystitis, and temporomandibular disorder (TMD). This new understanding has greatly impacted how we conceptualize and treat these disorders.

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What has become increasingly clear is that pain can be characterized by its underlying mechanisms (Clauw, 2009). There are at least three distinct mechanisms – peripheral, neuropathic and central, – that can in isolation or combination account for the experience of pain. Peripheral pain is primarily caused by inflammation or mechanical damage in the periphery and is observed in conditions like rheumatoid arthritis, osteoarthritis and cancer, as well as physical injury. The second type of pain is neuropathic, which is usually the result of damage or entrapment of peripheral nerves. Examples of neuropathic pain include postherpetic neuralgia and diabetic neuropathy. Central or non-nociceptive pain is due to a disturbance in the central nervous system (CNS) that affects the processing of pain and other stimuli. Central pain is observed in conditions like fibromyalgia, TMD and IBS, and there is evidence that it also is present in peripheral pain conditions such as osteoarthritis (Clauw and Witter, 2009) and rheumatoid arthritis (Lee et al., 2009). Notably, as many as 25% of patients with autoimmune disease (e.g. systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis) also meet current criteria for the diagnosis of fibromyalgia (Clauw and Katz, 1995), a central pain disorder. Of the utmost importance, different pain mechanisms respond to different types of treatment; for example, peripheral pain responds well to non-steroidal antiinflammatory drugs and opioid medications, while central pain responds best to tricyclic antidepressants and other neuromodulators. Neuropathic pain responds to both types of treatment. Psychological and behavioural factors are almost always important in pain, but they appear to play the most prominent role in central pain conditions. CNS augmentation of the processing of pain and other sensory stimuli has been demonstrated reliably in fibromyalgia, IBS, interstitial cystitis, chronic low back pain and TMD (Naliboff et al., 2001; Gracely et al., 2002; Giesecke, J. et al., 2004; Giesecke, T. et al., 2004), as has attenuated activity of the descending analgesic pathways (Leffler et al., 2002; Julien et al., 2005). These findings have

resulted in the more accurate characterization of these conditions as central sensitivity syndromes (Yunus, 2007) or central pain syndromes. Augmented central processing has not been explored explicitly in PLDS, but the common presence of fibromyalgia suggests applicability to PLDS patients. Findings from twin studies suggest that approximately half of the risk of developing chronic widespread pain is genetic, while the other half is environmental (Kato et al., 2006). Central pain syndromes can be ‘triggered’ in approximately 5–10% of individuals who experience peripheral pain syndromes (e.g. osteoarthritis, rheumatoid arthritis), infections (e.g. parvovirus, Epstein–Barr virus, bacterial gastroenteritis, Lyme disease), physical trauma (e.g. automobile accidents) and psychological trauma/distress (Clauw, 2009). Thus, psychological factors can be the result of living with chronic illness, trigger central pain syndromes, influence and predict medical and functional outcomes, contribute to patient suffering and disability, and complicate the clinical picture for healthcare professionals. 15.7.7 The concept of ‘chronic Lyme disease’ – driving and maintaining forces It is one thing to understand the immunopathogenesis of an illness – its manifestations, how to diagnose it and how to treat it. It is quite another to understand an illness when it evolves into a movement – such as is the case with ‘chronic Lyme disease.’ In exploring the psychology of ‘not Lyme’, it is also important to understand the psychology of a movement that overtook and then supplanted the clinical entity: how anxiety, speculation and empiricism overcame logic, objective evidence and scientific study, respectively. 15.7.8 Pain and fatigue and the search for an explanation – the patient Seeking to understand and remedy suffering is part of human nature. Information from our environments and social interactions is processed using filters determined by belief

The Psychology of ‘Post-Lyme Disease Syndrome’ and ‘Not Lyme’

systems established in light of previous experiences. Causative models are then shaped and molded by societal norms, and then used to make sense of experiences including physical and emotional symptoms. Once an illness model has been established, one that explains the origins of the symptoms, a plan of action is developed that will serve to diminish or eliminate pain, suffering and disability. Symptoms of pain and fatigue are common complaints that can initiate diagnostic testing; however, ‘diagnostic testing’ is often not diagnostic. In the case of Lyme disease, a false-positive result can be interpreted incorrectly as proof of a diagnosis. This error is all too common in primary care, where a marginally positive antinuclear antibody test is wrongly interpreted as proof of systemic lupus erythematosus. Clinical evaluation by a rheumatologist is usually sufficient to reverse the wrong diagnosis of lupus. The disabusing clinician is not seen as having an ulterior motive, and the patient and physician making the initial misdiagnosis usually abandon the diagnosis (and the test), neither physician having any personal interest in a specific diagnosis, merely wanting to help the patient as efficiently as possible. However, this is often not how the wrong diagnosis is greeted in the case of ‘chronic Lyme disease’. How then did we come to a circumstance where anger (occasionally quite venomous) is the response to such disabuse of the ‘chronic Lyme disease’ diagnosis?

15.8 Ranging from Benevolent to Malevolent – the Physician’s Role The practice of medicine in the 21st century should be ‘evidence based’. This means that diagnostic and therapeutic decisions should be based on facts, derived from scientific study and logical thinking, not speculation and supposition. Diagnostic criteria should be described and substantiated. Laboratory tests should be validated as accurate and their limitations acknowledged. Therapies should be proven effective. Physicians who fail at these tasks are failing the holy trust

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their patients place in them. ‘Lyme disease is a clinical diagnosis’ is a mantra that has attracted many devotees; the phrase’s original intent was to remind physicians that the diagnosis of Lyme disease was not to be made merely based on a blood test result but should be based on objective clinical criteria. This phrase now means that if Lyme disease is suggested by historical or clinical findings, even the most tenuous, the diagnosis is assured. Alternative criteria are applied in the interpretation of validated laboratory tests, unvalidated tests are used, and many eschew laboratory tests entirely, alleging that all are inaccurate and thereby worthless. The duration, dose and drug of therapeutic choice, some quite novel and dangerous, are based on hearsay and misapplication of scientific facts gleaned from other diseases. In traditional practice, it is up to the responsible physician to help patients interpret the science and medical jargon so the patient can be a part of the decisionmaking process. In the current Lyme disease climate, some physicians are ill-prepared to take on this responsibility. Some are not well informed and willingly remedy this, but others have adopted practices outside the standard of care, some declaring themselves to be ‘Lyme disease experts’. The latter group may be ‘Lyme literate’, often holding a view of Lyme disease best called an ‘alternative reality’. Their non-scientifically-based views of the organism, the disease and its treatment have formed the justification for a counterculture with new rules of practice. Once a physician views a patient with chronic complaints as having an infectious disease, rather than a central sensitivity syndrome, the process of ‘medicalization’ begins. The former starts with the belief that the physician can cure the condition solely with medication, while the latter requires the patient to be a much more active participant in the remedy; the two paths diverge rapidly. In ‘chronic Lyme disease’, antibiotics are ineffective, but nothing shakes faith in the diagnosis of a chronic, seemingly resistant, infectious disease. Some physicians are merely following a trend, with no personal interest in the factual versus the counter-factual view of Lyme

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disease. Some clearly believe their novel, innovative and daring approaches are in the best interests of their patients; such true believers are motivated by nothing more than service to their patients in what they interpret as being an uninformed, often hostile, environment. Some, perhaps starting as ‘true believers’, become aware of benefits other than the satisfaction derived from loyal service, such as money (sales of nutritional supplements and other critical and necessary remedies only from their offices) and notoriety (a following that may electronically span the globe). These may be powerful motivators and serve to perpetuate practice in the alternative reality of ‘chronic Lyme disease’.

15.9 Demands for the ‘Cure’ – the Activist To some degree, ‘chronic Lyme disease’ has become a cultural movement, led by activists working toward the admirable goal of ‘proper’ care for their followers. Many of these leaders have had family members thought to have a missed diagnosis of Lyme disease, or thought to have suffered because their Lyme disease was undertreated, and so these leaders have devoted themselves to making sure no one else suffers from lack of knowledge and access to ‘Lyme-literate’ physicians. However, some of these leaders have adopted a non-scientific belief system, often with prominent belief in the reality of ‘chronic Lyme disease’ and the need for aggressive therapy, using unsubstantiated excesses in dose and duration. In the past, patients with chronic symptoms but no objective findings of specific disease similarly have promoted ‘popular’ explanations such as ‘chronic candidiasis’, ‘chronic Epstein– Barr virus infection’, ‘myalgic encephalitis’, ‘chronic brucellosis’ and many others (Shorter, 1992), all honest attempts to understand and control suffering. The degree of investment in the diagnosis of ‘chronic Lyme disease’, too often manifested by anger, resentment and suspicion, seems far greater than in any of these antecedent movements; these on

occasion have prevented civil discourse. Ad hominem attacks against the disabusing clinician, including allegations of impropriety and self-interest, interfere with their ability to practice medicine properly; no such accusations of possible self-interest brought against ‘Lyme-literate physicians’ are brooked. Such is the desperation that some patients feel that any answer, even one that is illogical and leads to no improvement, is better than mystery, confusion and fear. Even a false answer promises a pathway out of their own personal hell, and offers a plan and some sort of action the patient can take. Removing this assurance of a brighter future and replacing it with a darker and less welldefined road, can be devastating. Politicians have identified these activist groups as powerful lobbies, capable of delivering votes. For example, insurance companies in New Jersey are now compelled to cover 56 days of intravenous antibiotics as a mandatory minimum duration of therapy, despite the fact that this duration of therapy has never been studied or found to be ‘optimum’. In Connecticut, the Attorney General heard the call of these lobbies and demanded the IDSA submit its evidencebased criteria to outside analysis – this was done and an impartial review board found the criteria and policies to be acceptable as written.

15.10 A Rational Approach to Treatment When patients and physicians incorrectly attribute symptoms to an infectious disease, unnecessary antibiotic treatment is often given. Almost 68% of the patients in the Living with Lyme Disease Study who had no evidence of Lyme disease received antibiotic treatment – almost 30% received multiple antimicrobials for months or even years (Hassett et al., 2011). Baker has observed that despite evidence to the contrary, some patients believe that ‘chronic Lyme disease’ results from ongoing infection with B. burgdorferi that requires more than a few months of antibiotic treatment, which is an unprecedented approach for a non-

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life-threatening disease (Baker, 2008). Furthermore, the judicious use of antibiotics is important from an ecological perspective, as their use affects not only the patient to whom they are prescribed but future patients through the creation of new antibioticresistant strains of bacteria (Schiff et al., 2001; Moellering et al., 2007). There is evidence that a multidisciplinary approach combining evidence-based pharmacological and non-pharmacological interventions (e.g. including gentle regular exercise and cognitive-behavioural therapies (CBT)) might best address the symptoms and underlying causes of our patients’ suffering and debility. Thus, medical or drug interventions should focus on symptomatic relief in a manner similar to that recommended for fibromyalgia and other central sensitivity syndromes (Hassett and Gevirtz, 2009; Williams and Clauw, 2009). For a good review of basic pharmacological treatment strategies, see the review by Arnold and Clauw (2010). Furthermore, addressing these same symptoms with adjunctive therapies like CBT plus exercise can serve to boost symptomatic and functional improvement. In using this approach, it is critical to avoid suggesting that the symptoms are psychiatric, because they are not. The treatment approaches for many chronic medical conditions (e.g. diabetes, hypertension, cardiovascular disease) rely on a similar integrative approach combining medication with education, exercise, regaining function and managing stress. Using the principles of comprehensive non-pharmacological pain management represented by the acronym ExPRESS can be quite helpful in organizing the approach to treatment (Hassett and Gevirtz, 2009). Ex is for exercise, which refers to the need for regular, low-impact exercise. P is for psychiatric comorbidity because both depression and anxiety disorders are common in all central sensitivity syndromes and contribute significantly to symptoms and disability. R stands for regaining function, which should emphasize the importance of obtainable goals and activity pacing. Too often chronic pain patients do too much on days that they feel good and too little on days that they feel bad. E is for education, which reminds healthcare

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practitioners that they and patients need to be working from the same illness model. If the patient conceptualizes the symptoms as due to active infection, while the physician views them as due to some other process, treatment adherence will be strongly affected. S is for sleep, which is frequently disturbed and often easily addressed through medication and improving sleep hygiene as many have developed counter-productive habits that exacerbate sleep disturbance. The final S is for stress and the need for stress management, which can include interventions already employed for addressing other ExPRESS factors such as CBT, relaxation techniques, hydrotherapy and gentle exercise to name but a few. However, this approach will probably be greeted with resistance by those with high levels of disease conviction. After all, according to their medical model of the illness, a persistent infection underlies the symptoms; thus, a referral to CBT will seem to come out of the blue. Yet there might be a middle ground – agreeing that the symptoms are real and probably the product of CNS dysregulation could begin to pave the way to a détente.

15.11 In Good Faith Although providing care for these patients may sometimes be frustrating for healthcare professionals, it is imperative that these patients, suffering with symptoms and attendant compromised quality of life, not be dismissed. The illness is not ‘all in their heads’ but often has been implanted in their heads by practitioners of ‘chronic Lyme disease’ medicine. Equally important is that these patients not be ‘medicalized’ into believing that only antibiotics hold the promise of symptomatic improvement. Lessons we have learned from our experiences with patients with fibromyalgia are applicable to patients with ‘chronic Lyme disease.’ We must address their pain, fatigue, anxiety and cognitive symptoms, as well as, when present, the psychological and behavioural processes that contribute to their suffering. Education and reassurance have helped some abandon their belief that they have a chronic incurable

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infectious disease and allowed them to move forward confidently with the rest of their lives. With further study, we may finally understand all the predispositions to this syndrome (neurobiological, genetic and psychological). We believe that these insights will be valuable in understanding and treating this and other forms of central sensitivity syndromes.

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A.C., Kaplan, R.F., Berardi, V.P., Duray, P.H., Larson, M.G., Wright, E.A., Ginsburg, K.S., Katz, J.N. and Liang, M.H. (1994) The long-term clinical outcomes of Lyme disease. A populationbased retrospective cohort study. Annals of Internal Medicine 121, 560–567. Shadick, N.A., Phillips, C.B., Sangha, O., Logigian, E.L., Kaplan, R.F., Wright, E.A., Fossel, A.H., Fossel, K., Berardi, V., Lew, R.A. and Liang, M.H. (1999) Musculoskeletal and neurologic outcomes in patients with previously treated Lyme disease. Annals of Internal Medicine 131, 919–926. Shorter, E. (1992) From Paralysis to Fatigue: a History of Psychosomatic Illness in the Modern Era. New York, The Free Press. Sigal, L.H. (1990) Summary of the first 100 patients seen at a Lyme disease referral center. American Journal of Medicine 88, 577–581. Sigal, L.H. (1994) Persisting complaints attributed to Lyme disease: possible mechanisms and implications for management. American Journal of Medicine 96, 365–374. Sigal, L.H. (1995) Lyme disease: primum non nocere. Journal of Infectious Diseases 171, 423–424. Sigal, L.H. (1996) The Lyme disease controversy. Social and financial costs of misdiagnosis and mismanagement. Archives of Internal Medicine 156, 1493–1500. Sigal, L.H. (1997) The immunology and potential mechanisms of immunopathogenesis of Lyme disease. Annual Review Immunology 15, 63–92. Sigal, L.H. (1999) Antibiotics for the treatment of rheumatologic syndromes. Rheumatic Disease Clinics North America 25, 861–881, viii. Sigal, L.H. (2001) Lyme disease: a clinical update. Hospital Practice (Minneapolis) 36, 31–32, 35–37, 41–42, 47. Sigal, L.H. and Hassett, A.L. (2005) Commentary: ‘What’s in a name? That which we call a rose by any other name would smell as sweet.’ Shakespeare, W. Romeo and Juliet, II, ii(47– 48). International Journal of Epidemiology 34, 1345–1347. Sigal, L.H. and Patella, S.J. (1992) Lyme arthritis as the incorrect diagnosis in pediatric and adolescent fibromyalgia. Pediatrics 90, 523–528. Smith, R.P., Schoen, R.T., Rahn, D.W., Sikand, V.K., Nowakowski, J., Parenti, D.L., Holman, M.S., Persing, D.H. and Steere, A.C. (2002) Clinical characteristics and treatment outcome of early Lyme disease in patients with microbiologically confirmed erythema migrans. Annals of Internal Medicine 136, 421–428. Solomon, S.P., Hilton, E., Weinschel, B.S., Pollack, S. and Grolnick, E. (1998) Psychological factors

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16

Chronic Lyme Disease Adriana Marques

‘The real purpose of the scientific method is to make sure that Nature hasn’t misled you into thinking you know something you actually don’t know.’ Robert Pirsig, Zen and the Art of Motorcycle Maintenance

16.1 Introduction Despite being widely used by both the lay and medical communities; ‘chronic Lyme disease’ is the most confusing term in the Lyme disease field. The term ‘chronic Lyme disease’ is used to describe vastly different patient populations, including patients with objective manifestations of late Lyme disease (for example, arthritis and late neuroborreliosis, addressed in detail in other chapters), as well as patients with post-Lyme disease syndrome and, especially, patients with non-specific signs and symptoms of unclear cause who received this diagnosis based on unproven and non-validated clinical and laboratory criteria.

16.2 ‘Chronic Lyme Disease’ Patients diagnosed with ‘chronic Lyme disease’ have been classified in four categories (Feder et al., 2007) (Table 16.1). Patients in category 1 are diagnosed with ‘chronic Lyme disease’ based on unexplained symptoms without objective or valid laboratory evidence of infection with Borrelia burgdorferi. Patients in category 2 have other recognized diseases and have been misdiagnosed with Lyme

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Table 16.1. Categories of ‘chronic Lyme disease’. Category 1

Symptoms of unknown cause, with no evidence of Borrelia burgdorferi infection

Category 2

A well-defined illness unrelated to B. burgdorferi infection Symptoms of unknown cause, with antibodies against B. burgdorferi but no history of objective clinical findings that are consistent with Lyme disease Post-Lyme disease syndrome

Category 3

Category 4

Reproduced by permission of the Massachusetts Medical Society (from Feder et al., 2007).

disease. Patients in category 3 have symptoms of unknown cause, with antibodies against B. burgdorferi but no history of objective clinical findings that are consistent with Lyme disease. Patients in category 4 fulfil criteria for post-Lyme disease syndrome. Most patients who are labelled as having ‘chronic Lyme disease’ will fall into categories 1 and 2, as evidenced by the difficulty in accruing patients into the placebo-controlled studies of antibiotic treatment in patients with postLyme disease syndrome (category 4), where only 1–10% of the screened individuals were

© CAB International 2011. Lyme Disease: An Evidence-based Approach (ed. J.J. Halperin)

Chronic Lyme Disease

eligible (Marshall, 1999; Kaplan et al., 2003; Krupp et al., 2003; Fallon et al., 2008). There have been a number of studies addressing the issue of overdiagnosis of Lyme disease. In the experience of referral centres, only about one-quarter to one-third of the patients evaluated were thought to have Lyme disease. In comparison, between 50 and 60% of the patients had no present or past evidence of Lyme disease (Sigal, 1990; Steere et al., 1993; Rose et al., 1994; Feder and Hunt, 1995; Reid et al., 1998; Qureshi et al., 2002). Problems contributing to the overdiagnosis of Lyme disease included the use of serological testing in clinical situations with a low pretest probability of Lyme disease, misinterpretation of test results and use of non-validated criteria for interpretation of laboratory results. Symptoms attributed to ‘chronic Lyme disease’ include headaches, neck pain, sleep disturbances, problems with memory, poor concentration, fatigue, irritability and mood swings, depression, anxiety, tremors, lowgrade increase in body temperature, hot flashes, night sweats, sore throat, swollen glands, arthralgias, joint stiffness, back pain, muscle pain, muscle cramps, chest pain and palpitations, abdominal pain, nausea, constipation, heartburn, testicular pain, pelvic pain, menstrual irregularities, blurred vision, floaters, photosensitivity, hyperacusis, tinnitus, lightheadedness, dizziness, decreased libido and weight gain. Even more, ‘chronic Lyme disease’ has evolved into a ‘polymicrobial infectious syndrome’ with patients usually being diagnosed with multiple coinfections (e.g. Babesia, Ehrlichia, Anaplasma, Rickettsia, Bartonella, Mycoplasma), reactivation of herpesviruses and Candida infection, as well as metabolic and hormonal imbalances, immune dysfunction, heavy metal toxicity, allergies, damage by neurotoxins, mitochondrial dysfunction and enzyme deficiencies. The diagnosis is based on ‘clinical judgment’, as ‘tests are not helpful’, and the ‘disease is difficult to treat’ and ‘requires prolonged treatment with multiple antibiotics and supplements’, usually ‘for months to years’, and ‘may not be curable’ (Alexander, 2009; Johnson and Feder, 2010). Evidence is uncontrolled and based on

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the clinical experience of the practitioners. Little formal research has been performed to evaluate this large, heterogeneous group of patients.

16.3 Post-Lyme Disease Syndrome The majority of peer-reviewed studies have addressed patients with post-Lyme disease syndrome. These are patients who have had a documented episode of Lyme disease, who have received treatment with an accepted antibiotic regimen, with resolution or stabilization of the objective manifestation(s) of Lyme disease, who have had persistent or relapsing non-specific symptoms for at least a 6-month period after completion of antibiotic therapy and who have no other condition that could explain their symptoms (Wormser et al., 2006). Studies from patients with erythema migrans (EM) have shown that 0–23% of patients have persistent or intermittent subjective symptoms of mild to moderate intensity 6–24 months after completion of therapy (Nadelman et al., 1992; Strle et al., 1992, 1993; Luger et al., 1995; Gerber et al., 1996; Luft et al., 1996; Dattwyler et al., 1997; Barsic et al., 2000; Arnez et al., 2002; Smith et al., 2002; Nowakowski et al., 2003; Wormser et al., 2003; Cerar et al., 2010) (Fig. 16.1). The most common symptoms are fatigue, arthralgias, myalgias, headache, neck stiffness, paresthesias, sleeplessness, irritability and difficulty with memory, word finding and concentration. Post-Lyme disease symptoms seem to correlate with having had disseminated disease, a greater severity of illness at presentation and delayed antibiotic therapy (Steere et al., 1983; Dattwyler et al., 1990; Weber et al., 1990; Asch et al., 1994; Shadick et al., 1994; Nowakowski et al., 2003; Picha et al., 2006; Ljostad and Mygland, 2010), but not with the duration of initial antibiotic therapy (Wormser et al., 2003; Oksi et al., 2007). Children appear to be less likely to develop post-Lyme disease symptoms (Salazar et al., 1993; Gerber et al., 1996; Wang et al., 1998; Adams et al., 1999; Arnez et al., 1999; Seltzer et al., 2000; Arnez et al., 2002; Eppes and Childs, 2002; Thorstrand et al., 2002).

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A. Marques

Symptomatic

300

Number of individuals

Asymptomatic

200

100

5 be r1 99 6 Lu ft D 19 at 96 tw yl er 19 Ba 97 rs ic 2 Sm 000 ith 20 Ar 02 ne N ow z 20 ak 02 ow sk i2 W C 00 or er m 3 ar se 20 r2 10 00 C er 3 – ar pa 20 tie 10 nt s – co nt ro ls

3

er

r1 99

2

19 9 rle

19 9 St

rle St

Lu ge

G

N ad

el m

an

19

92

0

Fig. 16.1. Symptoms 6–24 months after antibiotic therapy in patients with erythema migrans. Studies: Nadelman et al., 1992; Strle et al., 1992, 1993; Luger et al., 1995; Gerber et al., 1996; Luft et al., 1996; Dattwyler et al., 1997; Barsic et al., 2000; Arnez et al., 2002; Smith et al., 2002; Nowakowski et al., 2003; Wormser et al., 2003; Cerar et al., 2010.

The mechanisms underlying post-Lyme disease symptoms are not known and it is likely that different factors will play a role in an individual case. In many patients, the presence of non-specific symptoms represents the natural evolution of response after therapy. Studies have shown that the percentage of patients reporting symptoms after antibiotic treatment decreases over time. In one study of patients treated for EM, 34% had symptoms at 3 weeks, 24% at 3 months and 17% at 12 months (Wormser et al., 2003). Patients with late manifestations can have a slower response to therapy, may take months to recover and the recovery may be incomplete due to irreversible damage (for example, residual facial weakness after facial nerve palsy) (Dattwyler et al., 1988; Pfister et al., 1991; Steere et al., 1994; Kalish et al., 2001; Berglund et al., 2002; Kindstrand et al., 2002; Borg et al., 2005; Dattwyler et al., 2005; Oksi et al., 2007; Ljostad and Mygland, 2010). In other patients, a post-infective fatigue syndrome may be triggered by Lyme disease, as has been shown to occur with other infections. Prolonged fatigue after infections

is relatively common, and it can be disabling and persistent. In a cohort of patients with acute Epstein–Barr virus infection, Q fever and Ross virus infection, fatigue was predicted by the severity of the acute illness; the incidence was similar after the different infections (Hickie et al., 2006). The case rate for post-infective fatigue syndrome was 35% (87/250) at 6 weeks, 27% (67/250) at 3 months and 9% (22/250) at 12 months. These rates are similar to those reported in patients treated for EM (Wormser et al., 2003). In another study of 301 adolescents diagnosed with acute mononucleosis, 13% fulfilled criteria for chronic fatigue syndrome at 6 months, 7% at 12 months and 4% at 24 months (Katz et al., 2009). The mechanisms that are triggered during the acute illness and that sustain the persistent symptoms in post-infective fatigue syndrome are currently unknown. It is very important to recognize that there is a substantial background prevalence of symptoms attributed to ‘chronic Lyme disease’ in the general population. Musculoskeletal pain is a very common complaint, and chronic pain, fatigue and

Chronic Lyme Disease

sleep disturbances are often reported together (Rohrbeck et al., 2007). Insomnia is also a very common problem, and can be associated with anxiety, depression and pain (Morphy et al., 2007). Surveys have shown that between 5 and 15% of adults report chronic pain, and 8–30% report chronic fatigue (Loge et al., 1998; Picavet and Schouten, 2003; Aggarwal et al., 2006; van’t Leven et al., 2010). A recent study of patients with Lyme disease highlights the caveats of attributing nonspecific symptoms to Lyme disease (Cerar et al., 2010). In this prospective study, patients with EM were assigned to treatment with doxycycline or cefuroxime for 15 days. Each patient was asked to refer a spouse or family member (within 5 years of age) as a control. Patients and controls were followed for 1 year for the presence of fatigue, arthralgias, myalgias, headache, paresthesias, dizziness, irritability and nausea. At 6 months, there was no difference between patients and controls regarding the presence of new or increased symptoms, and at 12 months, controls were more likely to report new or increased symptoms than patients (Fig. 16.1). Similar results were found in a study of children with neuroborreliosis. At 6 months, headache and fatigue were less frequently reported among patients than controls, with no difference in the reporting of loss of appetite, neck pain, nausea and vertigo between the two groups (Skogman et al., 2008). The majority of patients with post-Lyme disease syndrome do not have evidence of coinfection with other tick-borne pathogens (Wang et al., 2000; Klempner et al., 2001; Ramsey et al., 2002; Fallon et al., 2008), but patients with concurrent Babesia (untreated) and B. burgdorferi infection were more likely to be symptomatic for 3 months or longer (Krause et al., 1996). Regarding the role of autoimmunity in post-Lyme disease syndrome, one study showed no association between a class II allele or genotype (Klempner et al., 2005). In a recent paper, the levels of antibodies against neural proteins were found to be higher in patients with postLyme disease syndrome than in patients who recovered from Lyme disease and healthy controls (Chandra et al., 2010). Synovitis persisting for months to years after antibiotic

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therapy in patients with Lyme arthritis (antibiotic-refractory Lyme arthritis) is an unusual complication, most likely due to autoimmunity triggered by the infection in a genetically susceptible individual (Shen et al., 2010). A major concern has been that the symptoms of post-Lyme disease syndrome may be due to a persistent infection with B. burgdorferi. Some of the confusion dates back to the early years following the description of the disease and the discovery of the causative organism. In these early years, non-specific symptoms were classified as part of ‘minor’ late manifestations or complications of Lyme disease, to differentiate from the ‘major’ manifestations, which included arthritis, meningoencephalitis and carditis (Steere et al., 1983; Weber et al., 1988; Dattwyler et al., 1990; Weber et al., 1990). In some cases, nonspecific symptoms were grouped with facial palsy and brief episodes of arthritis (Steere et al., 1983; Dattwyler et al., 1990). Arthritis, meningoencephalitis, carditis and other objective manifestations of Lyme disease are clear evidence of treatment failure and require antibiotic therapy (Wormser et al., 2006). On the other hand, non-specific symptoms frequently resolved without further antibiotic treatment, antibiotic therapy did not hasten their resolution (Nadelman et al., 1992; Asch et al., 1994; Klempner et al., 2001; Krupp et al., 2003; Fallon et al., 2008) and patients did not develop objective manifestations of late Lyme disease (Kalish et al., 2001; Nowakowski et al., 2003). Also, there were no differences in neurocognitive performance between patients and controls (Shadick et al., 1999) and no difference in the frequency of non-specific symptoms between patients with Lyme disease and age-matched controls (Seltzer et al., 2000; Skogman et al., 2008; Cerar et al., 2010). Objective evidence of Borrelia infection in patients with post-Lyme disease syndrome has not been found using PCR (Klempner et al., 2001; Fallon et al., 2008) or culture in Barbour–Stoenner–Kelly (BSK) medium (Klempner et al., 2001; Fallon et al., 2008) or MPM medium (Marques et al., 2000; Klempner et al., 2001; Tilton et al., 2001). It should be noted, however, that B. burgdorferi

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culture and PCR have low sensitivity in most body fluids from patients with Lyme disease (Aguero-Rosenfeld et al., 2005; Wilske et al., 2007). Other tests that have not been helpful to evaluate patients with post-Lyme disease syndrome include changes in C6 antibody levels (Fleming et al., 2004), antibodies in immune complexes (Marques et al., 2005) and the number of CD57+ cells (Marques et al., 2009a). There have been interesting reports of B. burgdorferi being present after antibiotic therapy in dogs and mice as assessed by PCR (discussed in detail by Hovius and Wormser, Chapter 5, this volume), but not by culture (Straubinger et al., 1997; Straubinger, 2000; Barthold et al., 2010; Yrjanainen et al., 2010). A study reported that B. burgdorferi was found by culture in a few mice treated with antitumour necrosis factor antibody either simultaneously or 4 weeks after ceftriaxone therapy (Yrjanainen et al., 2007). A study suggested that these organisms may be attenuated, non-infectious spirochaetes (Bockenstedt et al., 2002), but other studies have shown that the spirochaetes remained present in antibiotic-treated animals and were transmissible to immunocompromised mice, although in significantly lower numbers; moreover, they appeared to be noncultivable (Hodzic et al., 2008; Barthold et al., 2010). There are questions regarding the adequacy of the antibiotic regimens used in these studies (Wormser and Schwartz, 2009). The biological nature of these spirochaetes and their relevance to human disease are currently unknown and further studies are needed. There have been four randomized, placebo-controlled, double-blind studies of antibiotic treatment in post-Lyme disease syndrome (Table 16.2). These studies have shown that prolonged antibiotic therapy offers little benefit and can cause serious adverse effects. The first two studies, one for patients who were IgG seropositive for B. burgdorferi at enrolment and the other for seronegative patients, were published together (Klempner et al., 2001). Patients were randomized to receive intravenous ceftriaxone 2 g daily for 30 days, followed by oral doxycycline 200 mg daily for 60 days, or

matching intravenous and oral placebos. The primary outcome was improvement in the Medical Outcomes Study 36-item Short-form General Health Survey (SF-36) score on day 180 of the study. There were 78 seropositive patients and 51 seronegative patients and all patients had well-documented Lyme disease; most patients complained of pain, fatigue and cognitive changes. The studies were stopped early because a planned interim analysis showed that there was little chance of demonstrating a difference between treatment groups. Intention-to-treat analyses showed no significant differences between patients in the antibiotic groups and those in the placebo groups in the seropositive study, the seronegative study or both studies combined. About one-third of the patients improved, one-third remained unchanged and one-third worsened at the end of the intravenous therapy, at the end of the oral treatment and at 3 months after completion of the treatment. There were two serious adverse events related to treatment. The third study enrolled 55 patients with post-Lyme disease syndrome who had significant fatigue (Krupp et al., 2003). Twenty-eight patients were randomized to ceftriaxone 2 g and 24 patients to placebo intravenously daily for 28 days. The primary clinical end points were improvement in fatigue and mental speed at 6 months. The intent-to-treat analysis showed modest improvement of fatigue with ceftriaxone therapy, with similar results for patients who received therapy and those who completed follow-up. There was no improvement in mental speed or other neurocognitive measures. Three patients in each group discontinued therapy due to side effects and four had to be hospitalized. The fourth study enrolled patients with post-Lyme disease symptoms who were seropositive by IgG Western blot, had objective memory impairment and had received at least 3 weeks of intravenous antibiotic therapy (Fallon et al., 2008). There were only 37 patients enrolled, and they were randomized in a 2:1 ratio to receive 10 weeks of intravenous ceftriaxone (23 patients) or intravenous placebo (14 patients). The authors used as their primary analysis a

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Table 16.2. Placebo-controlled, double-blinded randomized treatment studies in post-Lyme disease symptoms. Regimen and primary end points Results

Serious adverse events

Reference

Patients

Klempner et al. (2001)

Seropositive (78 IV ceftriaxone (2 g/ No significant Two patients had patients) and day) for 30 days, difference was serious adverse seronegative followed by oral observed events associated (51 patients) for doxycycline between patients with treatment that antibodies to B. (200 mg/day) for who received required burgdorferi at 60 days (64 antibiotic or hospitalization (IV the time of patients), or placebo; catheter-related enrolment matching IV and oral approximately pulmonary placebos (65 one-third of the embolism and a patients). The patients gastrointestinal primary outcome improved, onebleed) was improvement third worsened on SF-36 score at and one-third day 180 of the study were unchanged at 30, 90 and 180 days 55 patients with 28 patients received IV There was Four patients had persistent ceftriaxone 2 g/day improvement in serious adverse severe fatigue and 24 patients fatigue but no events associated post-Lyme received IV placebo benefit in with treatment that disease for 28 days. Primary cognitive function required clinical outcomes with ceftriaxone hospitalization were improvement treatment in fatigue score and cognitive function at 6 months 37 IgG Ceftriaxone 2g/day IV Using a complex Nine patients immunoblot (23 patients) or IV data-driven model discontinued positive placebo (14 for analysis, there therapy due to patients with patients) for was borderline adverse events. objective 10 weeks. Follow-up improvement at One patient on memory was completed in 20 12 weeks in the ceftriaxone impairment patients in the ceftriaxone group. underwent and at least ceftriaxone group By 24 weeks, cholecystectomy at 3 weeks of and 12 patients in both groups had week 16 previous IV the placebo group improved similarly antibiotic from baseline therapy

Krupp et al. (2003)

Fallon et al. (2008)

IV, intravenous; SF-36, Medical Outcomes Study 36-item Short-form General Health Survey.

complicated model chosen by a data-driven selection process, which made it difficult to interpret the results (Marques et al., 2009b). Using this complex model, they reported a borderline greater improvement at 12 weeks in the ceftriaxone group. By 24 weeks, both groups had improved similarly from baseline. There were nine patients who discontinued therapy due to side effects and in seven of these patients these side effects were related to the treatment.

These randomized trials have been criticized as offering ‘too little, too late’ and for not ‘addressing the range of treatment options in an actual practice’ (Cameron, 2006; Donta, 2007; Stricker, 2007; Cameron, 2009), based on personal opinion and retrospective, open-label case series and case reports that suggested a possible role of prolonged antibiotic therapy in patients diagnosed with ‘chronic Lyme disease’ (Donta, 1997, 2003). Case series and case reports are classified at

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the lowest level of strength in the hierarchy of evidence-based medicine (Schunemann et al., 2006). Case series studies have many limitations, the primary one being the lack of a control group. Without a comparison group, it is not possible to know whether an outcome is related to an intervention or to a placebo effect, time or chance. Case series studies have a large risk for systematic errors (bias). For example, both patients and physicians’ choices will affect the decision to prescribe a drug to a particular patient. The lack of blinding can affect outcomes, especially for subjective measures. Due to its limitations, no causal inferences about treatment effect can be made from results of case series studies. Case series studies are best used to generate hypotheses that can be tested by stronger study designs.

16.4 Conclusion At this point, the evidence shows that antibiotic therapy, as tested in four randomized placebo-controlled studies, does not offer durable or significant benefit in treating patients with post-Lyme disease syndrome and has considerable risk of treatment-related adverse events. There is a pressing need to investigate other approaches that may help these patients. Because of the significant placebo effect and the variation in symptom intensity over time seen in these patients, studies evaluating therapeutic options should have a randomized controlled design. Studies should also have clearly defined target patient populations. Additionally, research to evaluate patients diagnosed with ‘chronic Lyme disease’ who do not fulfil the criteria for post-Lyme disease syndrome, focusing on definitional issues and heterogeneity among these individuals, is needed in order to better understand and manage the care of these patients.

Acknowledgement This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of

Allergy and Infectious Diseases. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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Shadick, N.A., Phillips, C.B., Sangha, O., Logigian, E.L., Kaplan, R.F., Wright, E.A., Fossel, A.H., Fossel, K., Berardi, V., Lew, R.A. and Liang, M.H. (1999) Musculoskeletal and neurologic outcomes in patients with previously treated Lyme disease. Annals of Internal Medicine 131, 919–926. Shen, S., Shin, J.J., Strle, K., Mchugh, G., Li, X., Glickstein, L.J., Drouin, E.E. and Steere, A.C. (2010) Treg cell numbers and function in patients with antibiotic-refractory or antibioticresponsive Lyme arthritis. Arthritis and Rheumatism 62, 2127–2137. Sigal, L.H. (1990) Summary of the first 100 patients seen at a Lyme disease referral center. American Journal of Medicine 88, 577–581. Skogman, B.H., Croner, S., Nordwall, M., Eknefelt, M., Ernerudh, J. and Forsberg, P. (2008) Lyme neuroborreliosis in children: a prospective study of clinical features, prognosis, and outcome. Pediatric Infectious Diseases Journal 27, 1089– 1094. Smith, R.P., Schoen, R.T., Rahn, D.W., Sikand, V.K., Nowakowski, J., Parenti, D.L., Holman, M.S., Persing, D.H. and Steere, A.C. (2002) Clinical characteristics and treatment outcome of early Lyme disease in patients with microbiologically confirmed erythema migrans. Annals Internal Medicine 136, 421–428. Steere, A.C., Hutchinson, G.J., Rahn, D.W., Sigal, L.H., Craft, J.E., Desanna, E.T. and Malawista, S.E. (1983) Treatment of the early manifestations of Lyme disease. Annals of Internal Medicine 99, 22–26. Steere, A.C., Taylor, E., McHugh, G.L. and Logigian, E.L. (1993) The overdiagnosis of Lyme disease. Journal of the American Medical Association 269, 1812–1816. Steere, A.C., Levin, R.E., Molloy, P.J., Kalish, R.A., Abraham, J.R., Liu, N.Y. and Schmid, C.H. (1994) Treatment of Lyme arthritis. Arthritis and Rheumatism 37, 878–888. Straubinger, R.K. (2000) PCR-based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-day postinfection period. Journal of Clinical Microbiology 38, 2191–2199. Straubinger, R.K., Summers, B.A., Chang, Y.F. and Appel, M.J. (1997) Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. Journal of Clinical Microbiology 35, 111–116. Stricker, R.B. (2007) Counterpoint: long-term antibiotic therapy improves persistent symptoms associated with Lyme disease. Clinical Infectious Diseases 45, 149–157. Strle, F., Ruzic, E. and Cimperman, J. (1992) Erythema migrans: comparison of treatment

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randomized trial of ceftriaxone versus oral penicillin for the treatment of early European Lyme borreliosis. Infection 18, 91–96. Wilske, B., Fingerle, V. and Schulte-Spechtel, U. (2007) Microbiological and serological diagnosis of Lyme borreliosis. FEMS Immunology and Medical Microbiology 49, 13–21. Wormser, G.P. and Schwartz, I. (2009) Antibiotic treatment of animals infected with Borrelia burgdorferi. Clinical Microbiology Reviews 22, 387–395. Wormser, G.P., Ramanathan, R., Nowakowski, J., Mckenna, D., Holmgren, D., Visintainer, P., Dornbush, R., Singh, B. and Nadelman, R.B. (2003) Duration of antibiotic therapy for early Lyme disease. A randomized, double-blind, placebo-controlled trial. Annals of Internal Medicine 138, 697–704. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, J.S. and Nadelman, R.B. (2006) The clinical assessment, treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43, 1089–1134. Yrjanainen, H., Hytonen, J., Song, X.Y., Oksi, J., Hartiala, K. and Viljanen, M.K. (2007) Anti-tumor necrosis factor- treatment activates Borrelia burgdorferi spirochetes 4 weeks after ceftriaxone treatment in C3H/He mice. Journal of Infectious Diseases 195, 1489–1496. Yrjanainen, H., Hytonen, J., Hartiala, P., Oksi, J. and Viljanen, M.K. (2010) Persistence of borrelial DNA in the joints of Borrelia burgdorferiinfected mice after ceftriaxone treatment. Acta Pathologica, Microbiologica et Immunologica Scandinavica 118, 665–673.

17

Lyme Disease: the Great Controversy John J. Halperin, Phillip Baker and Gary P. Wormser

17.1 Background We live in interesting times. As a result of vigorous efforts by well-intentioned but misinformed patient advocates and by a small cadre of their physician supporters, Lyme disease – with fewer annual confirmed cases in the USA than varicella (Hall-Baker et al., 2010) – is repeatedly characterized as epidemic, controversial and difficult to diagnose or treat. Pseudo-documentary movies (Halperin, 2009) have been produced vilifying experts in the field and purporting to demonstrate a medical conspiracy – driven supposedly by unsupported and unsupportable allegations of conflicts of interest – to hide the suffering of the victims of this disorder. The press, politicians and advocates repeatedly portray this as a subject of substantive and legitimate scientific controversy. Yet the scientific evidence is remarkably consistent, providing no real basis for controversy (Sigal, 2007; Weissmann, 2007; Baker, 2010). That fact notwithstanding, the states of Connecticut, Maryland, Minnesota, Massachusetts, New York, Pennsylvania, Rhode Island and others have passed or considered legislation or regulations to assure the provision of demonstrably ineffective prolonged antibiotic treatment (Klempner et al., 2001; Krupp et al., 2003; Fallon et al., 2008) for patients diagnosed with an undefined

disorder termed ‘chronic Lyme disease’. In 2006, the Attorney General of the state of Connecticut opened an investigation of the Infectious Diseases Society of America (IDSA) for issuing evidence-based guidelines for the diagnosis and treatment of Lyme disease, on the legally questionable theory that this clinical guideline represented an anti-trust violation. Although this remarkable action yielded no finding of any anti-trust violation (but ultimately cost the IDSA over half a million dollars in legal and other costs (IDSA, October 2010, personal communication), it did result in a detailed review of the guidelines by an independent panel that endorsed all of the guidelines’ original recommendations (Lantos et al., 2010). What, then, is the basis for this controversy? This strange story begins with the disease’s original characterization in the USA in the early 1970s, when a surprising number of children in Lyme and Old Lyme, Connecticut, were diagnosed as having juvenile rheumatoid arthritis. Efforts by several mothers of affected children led to a more detailed investigation. This ultimately resulted in the pioneering work of Allen Steere and others (Steere et al., 1977), who identified both the tick vector and the responsible bacterial pathogen, Borrelia burgdorferi (Burgdorfer et al., 1982; Benach et al., 1983; Steere et al., 1983). It also resulted in the early creation of multiple vocal patient

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support and advocacy groups, whose members have advocated strongly for the perceived needs and concerns of patients afflicted – or thought to be afflicted – with this disease (www.lymenet.org/Support Groups/). Remarkably, active groups even formed in areas of the USA where Lyme disease is not endemic. Aided by the Internet, these groups have shared information, viewpoints and strategies to lobby for their cause, reinforcing each other’s perspectives and misinformed opinions, thereby setting the stage for the current chaos. The path from there epitomizes the law of unintended consequences. With significant support from both the National Institutes of Health (NIH) and the Centers for Disease Control and Prevention (CDC), several academic groups – primarily those at Yale and Stony Brook – became actively involved in efforts to understand better the full scope of Lyme disease. Development of early serological tests in the 1980s led to the observation that some patients who appeared to have active, disseminated Lyme disease, in whom serological tests would be expected to be positive, did not have measurable antibody responses as evidenced by the ELISAs then in use (Dattwyler et al., 1988). Although this observation was probably the result of limitations in the then-available assays (see Johnson, Chapter 4, in this volume), the notion of seronegative late Lyme disease became firmly implanted in the consciousness of patients and some healthcare providers. In assessing patients in the early 1980s, all with typical signs and symptoms of active Lyme disease, many were noted to have objectively demonstrable cognitive slowing and memory difficulty (Halperin et al., 1988, 1990; Logigian et al., 1990; Krupp et al., 1991), just like many patients with other active infectious or inflammatory disorders. This gave rise to the notion – in some circles– that such symptoms are an essential part of the Lyme disease symptom complex, rather than the non-specific toxic-metabolic encephalopathy seen in patients with many inflammatory diseases. From there, it was a short if illogical step to conclude that these symptoms were sufficiently typical of Lyme disease that their presence – in the absence of

more specific abnormalities or even positive serological tests – justified a diagnosis of B. burgdorferi infection and treatment with antibiotics. As these same symptoms also occur in approximately 2% of otherwise healthy individuals at any given time (Luo et al., 2005), this logic has indeed been problematic. Even worse, these symptoms were misinterpreted as evidence of central nervous system (CNS) infection by B. burgdorferi – a terrifying prospect for symptomatic individuals – despite the fact that early work clearly showed that the vast majority of these patients did not have active nervous system infection (Halperin et al., 1992) (see Halperin, Chapter 13, this volume). All of these false assumptions set the stage for a medical ‘perfect storm’. Reinforced by information from support groups and the Internet, patients with a common but non-specific symptom complex became convinced that their difficulties were caused by an infection for which the diagnostic tools were deeply flawed. Even more frightening, they believed that, if left untreated, this infection would result in irreversible brain damage. Not surprisingly, some physicians – who came to be known as ‘Lyme literate physicians’ or LLMDs – began treating such patients with aggressive courses of antibiotics. When treatment responses were less than satisfactory – and despite the fact that this microorganism has never been shown to develop antibiotic resistance – many patients and LLMDs were reluctant to acknowledge that the underlying premises and logic were deeply flawed. Instead, they invoked a series of ever more creative conjectures – substantiated only by inaccurate or misinterpreted snippets of information – as to why this infection was apparently so difficult to treat. These included assertions that B. burgdorferi cells adopt a cell-wall-free or cyst form (Brorson and Brorson, 1998, 1999; MacDonald, 2006) and/or that they hide intracellularly. Such conclusions were based primarily on extrapolations from in vitro studies, without supporting evidence that this was of any clinical relevance (Wormser et al., 2006). As many of these LLMDs became increasingly invested in this deeply flawed

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disease model, the response to treatment became the final element in this selfreinforcing logic. Early work indicated that patients with acute, active early Lyme disease, as indicated by the presence of an erythema migrans (EM) skin lesion (when large numbers of spirochaetes are presumably present), sometimes exhibited a Jarisch– Herxheimer-like reaction within 24 h after initiation of treatment (Weber et al., 1988; Maloy et al., 1998). This led to the notion that any worsening of symptoms during treatment constituted ‘Herxing’, regardless of the duration of symptoms or treatment at the time of the worsening. The logical inconsistency of postulating that treatmentresistant disease was due to a small number of undetectable bacteria, while at the same time concluding that symptoms arising or worsening during antibiotic therapy were due to the release of large amounts of pharmacologically active bacterial products, was either discounted or never considered. However, this then completed the very tidy but circular conceptual model. If patients improved, even transiently, after treatment, this validated the diagnosis and justified further treatment; the possibility of a placebo effect or natural fluctuation in symptom severity was either never considered or completely rejected. If patients worsened, this was considered to be due to a Jarisch– Herxheimer reaction, similarly validating the diagnosis. If there were no response to therapy, this validated the assumption that this infection is highly resistant to standard antimicrobial therapy. At this point, it is informative to examine in more detail some of the key myths that together resulted in this irrational construct.

17.2 Laboratory Myths – Seronegative Lyme Disease 17.2.1 Serology In 1988, using early whole-cell-sonicate ELISA assays for the serodiagnosis of Lyme disease, a group of scientists at the State University of New York at Stony Brook (SUNY-SB) identified 17 patients who had

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been treated early in the course of Lyme disease but still had symptoms interpreted as evidence of active infection (Dattwyler et al., 1988). Although none of these patients had significant elevations of antibody by ELISA, all had evidence of T-cell immunoreactivity against B. burgdorferi, using a T-cell proliferation assay subsequently found to be non-specific and therefore now felt not to be useful diagnostically. To appreciate how infected individuals could be ‘seronegative’, it is important to understand the factors involved in developing an ELISA for the serodiagnosis of any infection, including infection caused by B. burgdorferi. As many bacterial antigens are shared among B. burgdorferi and other related and unrelated groups of microorganisms, there is considerable immunological crossreactivity, often leading to false-positive serological results (Magnarelli et al., 1987). Consequently, assays must be designed to balance sensitivity and specificity. The greater the sensitivity, the more likely an assay will detect low levels of antibodies specific for Borrelia antigens (as detailed by Johnson, Chapter 4, this volume). However, the assay will then also detect weak cross-reactivities that are not diagnostically significant or meaningful. Before the widespread adoption of the two-tier testing approach in 1995 following a CDC-sponsored conference on Lyme serodiagnosis (Dressler et al., 1993; Anon., 1995), laboratories tried to design single assays to optimize accuracy. This included limiting false-positive results attributable to cross-reactivity by adopting stringent end points for ELISAs (see Johnson, Chapter 4, this volume). However, adoption of the Western blot as an essential component of a two-tiered sequential test represented a major advance, as it enabled the use of validated criteria to confirm the specificity of weakly positive or borderline ELISA results, i.e. results that, in earlier ELISAs, probably would have been considered negative. It is likely that the 17 patients included in the above-described SUNY-SB study included individuals with false-negative ELISAs who would be positive with current assays, patients with post-treatment persistent

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symptoms and perhaps patients who did not really have Lyme disease. Using the currently recommended two-tier approach, most laboratory experts now feel that, except in the first 3–6 weeks of infection before an antibody response has developed sufficiently to be detectable, seronegative Lyme disease is extraordinarily rare. Although the preceding history explains the origins of the myth of seronegativity, there is another, often repeated variation of this argument that is more difficult to understand, namely that serological results become temporarily falsely negative during and because of antibiotic treatment, i.e. the presence of antibiotics in the patient’s system in some way interferes with either the production of antibodies at the time, or the assays for them. Patients often relate that they were told ‘the test was negative because I was on antibiotics’. Not only is there no evidence – or even theoretical rationale – to support such an assertion, there is no precedent for this with reference to any other infectious disease. These two untenable explanations for false-negative serologies are very different from the situation in which a patient is cured very early in infection, in which circumstance the rapid removal of all antigens certainly can lead to an aborted antibody response, because none of the infecting organisms is present long enough to elicit the response. Observations on rabbits infected experimentally with Treponema pallidum (syphilis) provide a useful perspective on this point. Rabbits that received penicillin while incubating infection were ‘either cured or subsequently developed clinically recognizable lesions’ (Hollander et al., 1952). Single subcurative doses of penicillin prolonged the ‘incubation period of experimental syphilis… up to a limit of 30–40 days’, but when lesions developed, all of the animals became seropositive. 17.2.2 Other diagnostic tests Because of the technical difficulty of culturing B. burgdorferi using conventional laboratory methods, and because of the presumed small

number of organisms present in readily obtainable samples, microbiological diagnosis of Lyme disease is generally impractical. Even the extremely technically sensitive and specific PCR, adopted as an alternative to culture, is of remarkably low diagnostic sensitivity with many types of clinical specimens (Lebech et al., 2000; Avery et al., 2005; Roux et al., 2007), again presumably because of the low number of microorganisms present. On the other hand, PCR can detect fragments of DNA from long-dead organisms; DNA has been detected in tissues as long as 7 years after an infection has been microbiologically cured (Rovery et al., 2005). Thus, although such fragments may be specific for B. burgdorferi, their presence does not prove active infection. Consequently, diagnosis has relied almost exclusively on demonstrating a specific host antibody response to the microorganism. However, this too has important pitfalls. The presence of specific antibody – which commonly persists for long periods of time after infection – indicates past or present exposure to relevant borrelial antigens, and does not prove active infection (Hammers Berggren et al., 1993). For most other infections, serological testing typically relies on the demonstration of a fourfold or greater change in antibody titre. In contrast, for Lyme disease the convention has been to rely on a single serological determination. Although adoption of the two-tier testing strategy has provided a reasonable compromise between sensitivity and specificity, test interpretation requires an appreciation of three key elements. Firstly, the criteria used for the Western blot are only to be applied in patients with positive or borderline ELISAs. Without this much measurable antibody, blots should not even be performed. Secondly, the IgM criteria are intended for use only in individuals with early infection (Anon., 1995). By 4–8 weeks after exposure to B. burgdorferi, the much more specific IgG antibody response should be developing and is of much greater diagnostic value (Wormser et al., 2006). In patients with disease of 1–2 months duration or longer, isolated IgM responses are far more likely to be cross-reactive and not specific for

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B. burgdorferi. Thirdly, the bands selected for use in the Western blot were chosen not because they are unique to B. burgdorferi but rather on the basis of statistical considerations that included an analysis of those combinations of bands that provided the best predictive values for well-characterized specimens known to have been obtained from individuals with and without Lyme disease (Dressler et al., 1993). Obviously, laboratories using criteria other than these must establish the validity of their own criteria based on equally rigorous scientific assessments. Efforts continue to develop simpler and more sensitive and specific diagnostic tests for Lyme disease. Although the C6 ELISA assay shows some promise (Philipp et al., 2003; Vermeersch et al., 2009), with accuracy that appears comparable to the two-tier approach, there have not yet been sufficient comparative studies to judge which methodology is preferable.

17.3 Clinical Myths: Lyme Disease is a Clinical Diagnosis Based Entirely on Symptomatology 17.3.1 Background Infectious diseases are associated with a wide array of symptoms. Some symptoms are sufficiently unusual outside the context of that particular disease to have a meaningful positive predictive value supporting that diagnosis. Others are common to a broad range of inflammatory disorders and thus have no diagnostic specificity. As is the case with laboratory diagnostic tests, the extent to which particular clinical signs or symptoms support a diagnosis depends on their sensitivity and specificity. For the diagnosis of Lyme disease, some findings (e.g. EM, bilateral facial nerve palsies or childhood facial nerve palsies) are quite unusual, occurring in very few circumstances apart from Lyme disease. If these occur in an individual living in an endemic area where there is a risk of recent exposure to infected ticks, the probability of the patient having Lyme disease is quite high.

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Other signs or symptoms are of intermediate specificity. Unilateral facial nerve palsy in an adult, radicular pain without a mechanical cause, relapsing large joint oligoarthritis and heart block in an otherwise healthy young individual are less diagnostic. However, such symptoms are suggestive of the diagnosis and, if there has been potential exposure, it is appropriate to consider Lyme disease in the differential diagnosis. Further along the continuum would be lymphocytic meningitis. This can be caused by Lyme disease, but there is heavy epidemiological and symptomatic overlap between this and enteroviral meningitis. Although it would be reasonable to consider Lyme disease in the appropriate context, one must also realize that in many of these patients there will actually be a viral aetiology. At the other end of the spectrum are many symptoms (e.g. fatigue, malaise, headaches, diffuse aches and pains, cognitive slowing) that are common to virtually all inflammatory disorders. If these symptoms are present in the absence of more distinctive and definitive features, they are of no predictive value for the diagnosis of Lyme disease. In this context, even obtaining a serological test is ill-advised, as it is more likely to be misleading than helpful. One historic footnote bears mentioning. In the early 1980s, the academic group at SUNY-SB developed a collaboration with several primary care physicians in eastern Long Island, who were seeing many patients with Lyme disease. In an effort to cast a broad net to identify other symptoms that might be related to the infection, they created a database that included a standard review of systems. This completely generic review of systems subsequently became the questionnaire used by many LLMDs as a Lymespecific symptom inventory. 17.3.2 The assertion that ‘Lyme disease is a clinical diagnosis’ Every diagnosis in medicine relies ultimately on an ill-defined process termed ‘clinical judgement’. ‘Clinical judgement’ assumes that an appropriately knowledgeable

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physician will carefully and correctly gather all relevant clinical, laboratory and epidemiological data and reach a logical conclusion that is congruous with usual and acceptable medical practice. While King Louis XIV of France famously asserted that he was the law (‘Le loi, c’est moi’), diagnoses advanced by physicians are not inherently correct simply because they are asserted by a physician, however sincere the intentions may be. Diagnosis in any given patient requires the appropriate balancing of all the different data elements. A 3-year-old, living in Lyme, Connecticut, with summertime facial nerve palsy probably has Lyme disease. The child might have a negative serology as this disorder may occur before a measurable antibody response has developed. In this case, the initiation of presumptive treatment might well be reasonable. If that child’s father developed acute radicular pain in January after lifting heavy furniture, the probability of the radiculitis being related to Lyme disease is extremely low, even if his serology were positive. If the child’s mother has been completely healthy but feeling exhausted and absented-minded ever since the child and her twin sibling were born, it is unlikely that the fatigue and forgetfulness are due to Lyme disease. In this sense, Lyme disease is a clinical diagnosis in which a capable physician will synthesize all available data specific to that patient. Then, informed by the broader set of evidence-based medical knowledge, that physician will adopt a diagnostic and treatment strategy consistent with current, reasonable medical thinking and practice.

17.4 The Symptom Described as ‘Brain Fog’ The CNS can be affected in one of three ways in patients with Lyme disease (Halperin, 2010; Halperin, Chapter 13, this volume). The most common has nothing to do with brain infection. Individuals with systemic inflammatory or infectious disorders commonly feel tired and cognitively slowed in the absence of any brain infection or direct

involvement of any sort. Actual CNS infection by B. burgdorferi almost always manifests as meningitis. By definition, this disorder consists of inflammation of the lining of the brain, a rather uncomfortable process that is uniformly benign. Very rarely, patients develop parenchymal brain or spinal cord involvement, a disorder most accurately termed encephalomyelitis. Affected patients generally have focally abnormal neurological examinations, abnormal brain or spinal cord magnetic resonance imaging (MRI) scans and inflammatory cerebrospinal fluid (CSF). Many will have demonstrable local production of anti-B. burgdorferi antibodies in the CSF. Although this encephalitis typically causes focal neurological abnormalities, very rarely it may present just as cognitive difficulty. When cognitive difficulties are substantial, or the brain MRI demonstrates significant and potentially related parenchymal abnormalities, CSF should be examined. Inflammatory changes in the CSF would then lead to the diagnosis of encephalitis, with corresponding treatment. Unfortunately, it has become commonplace for patients and their treating physicians to assume that the first, common disorder (a toxic-metabolic encephalopathy) – often referred to by patients as ‘brain fog’ – represents evidence of what, in fact, is the very, very rare phenomenon of direct brain infection (encephalomyelitis) by B. burgdorferi, and that this will progress to severe irreversible brain damage. This assumption has been reinforced by the indiscriminate use of brain single photon emission computed tomography (SPECT) imaging, which, with remarkable frequency, is interpreted as showing cerebral vasculitis in patients with normal neurological examinations, CSF, brain MRI imaging and even brain vascular imaging. Most neurologists find this juxtaposition to be conceptually perplexing, if not frankly irrational. Given this misconception, it should not be surprising that affected patients are so terrified by the misguided fear of a brain-damaging infection that they are willing to undergo extensive and expensive testing and treatment, often at their own expense. Fortunately, the very rare cases of Lyme encephalomyelitis can be

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diagnosed quite easily (Ljostad and Mygland, 2009). The key to correct diagnosis is, as always, sound clinical judgement.

17.5 The Assertion that Lyme Disease is a Potentially Lethal infection One of the more curious aspects of B. burgdorferi infection is how generally benign it actually is. Although heart block and encephalomyelitis could conceivably be lethal, there are only extraordinarily rare cases suggesting Lyme disease was a factor in a patient’s death. Although advocacy groups occasionally cite examples of patients dying from this infection, the objective data contain remarkably little to support this notion. A few case reports suggest that Lyme carditis might have contributed to patients’ demise (Marcus et al., 1985; Lamaison, 2007; Tavora et al., 2008). There are probably as many case reports of deaths due to inappropriate treatment (Patel et al., 2000; Holzbauer et al., 2010). A group at the CDC recently reviewed US death certificate data (Kugeler et al., 2011) from 1999 to 2003. The diagnosis of Lyme disease was listed on 119 of the reviewed death certificates from this period. However, among these, only one patient had symptoms consistent with Lyme disease. It is important to understand that diagnoses listed on death certificates include previously made diagnoses, often with no independent review or substantiation, and in reviewing these data the authors did not have access to medical records or any information other than the terminal events. If this one patient actually did die for reasons related to Lyme disease, a comparison with Lyme disease incidence data during the same period would suggest a mortality rate of approximately 1 per 100,000 of the population. Certainly, in any disease with such extraordinarily low suspected mortality, a causal relationship must be highly suspect.

17.6 Treatment – The Myth that More (and more and more…) is Better Numerous studies have now shown that Lyme disease – even in the presence of

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nervous system infection – can readily be treated with fairly short courses of conventional antibiotics. Well performed studies have repeatedly demonstrated no meaningful or lasting benefit (Klempner et al., 2001; Krupp et al., 2003; Oksi et al., 2007; Fallon et al., 2008) of prolonged courses of treatment. These findings are completely consistent with the known biology of B. burgdorferi, as well as the cumulative knowledge of treatment effects with innumerable other bacterial infections. Despite this, the notion of a need for longer-duration treatment continues in some circles. At least three considerations should be kept in mind. Firstly, as already discussed, many patients being treated for ‘chronic Lyme disease’ do not have an infection with B. burgdorferi, or any other identifiable bacterium. Hence, no amount of antibiotic will cure them. Secondly, as is the case for many infections, some or all of a patient’s symptoms may continue even after the infection has been cured. If a patient has facial nerve palsy, the nerve must still recover from whatever damage it has incurred, even after the precipitating infection has disappeared. An inflamed knee may continue to be painful and swollen, even after the infection has been eradicated. Many patients with significant infections (e.g. bacterial pneumonia) will continue to feel tired and ill for weeks or months after microbiological cure. As symptoms attributed to Lyme disease often do not resolve immediately with treatment and presumed microbiological cure, one can readily understand why patients might feel that the recommended treatment duration is arbitrary and that antibiotic therapy should continue until all symptoms resolve. However, the data for Lyme disease, as in a myriad of other infections, demonstrate that this seemingly logical conclusion is incorrect. Finally, one very real limitation of our diagnostic technology is that there is no definitive laboratory test that confirms cure of the infection. As the immune response typically remains demonstrable for an extended period of time after successful treatment, there is an understandable desire for another laboratory test to which the

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patient can turn as confirmation that the disease is cured. In the absence of such a test, the patient’s uncertainty merges with widely perpetuated misinformation leading to a desire for ever more antibiotic treatment.

17.7 Continuing non-specific Symptoms – the Myth that Bacteria Must be Lurking Somewhere… Several theories have been advanced to explain the persistence of subjective symptoms in patients who have resolved their objective evidence of infection (e.g. EM skin lesion) following antibiotic treatment. One is that spirochaetes persist in unidentified tissue sites and thereby cause fatigue and other non-specific symptoms. Theories on the mechanism of persistence include the persistence of B. burgdorferi intracellularly. Those who invoke this theory apparently do not appreciate that this microbial strategy would not be protective against the antimicrobial effects of tetracyclines, a class of antibiotics that penetrate well into cells. Thus, a persuasive argument against this theory should be the observation that 8 weeks of doxycycline treatment was no more effective than placebo in two studies of patients with post-Lyme disease syndrome (Klempner et al., 2001). In addition, if this theory were valid, refractory disease and/or persistent symptoms would be anticipated to occur significantly more commonly in -lactamtreated patients with Lyme disease compared with tetracycline-treated individuals, which has never been demonstrated (Ljostad et al., 2008). Another theory is that Borrelia and other spirochaetes form cysts that insulate them both from the host’s immune defenses and from the effects of antibiotic therapy. Interestingly, those who have supported this notion have never defined what exactly is meant by a ‘cyst’. What is clear, however, is that under unfavorable in vitro growth conditions, spirochaetes may undergo morphological changes and develop a rounded appearance. These rounded forms could be a survival strategy, as they may remain viable for a period of time. In one

experiment, B. burgdorferi that had been cultured in the absence of serum, a necessary ingredient in growth media, survived for 8 days, although they were no longer viable at 2 weeks (Alban et al., 2000). Even those who reported previously on ‘cyst’ formation by Borrelia have now revised their nomenclature and instead refer to this morphological appearance as ‘round bodies’, a term apparently intended to encompass and replace prior descriptions such as coccoid bodies, globular bodies, spherical bodies, granules, cysts, L-forms, sphaeroplasts and vesicles (Brorson et al., 2009). There are at least three fundamental concerns with these theories that persistence of symptoms is due to persistence of borrelial cells. One is that carefully performed microbiological evaluations have failed to find evidence of B. burgdorferi infection in treated patients with persistent subjective symptoms, including studies that have focused on occult CNS infection (Klempner, 2002; Kaplan et al., 2003; Krupp et al., 2003; Fallon et al., 2008). The second is that four NIH-sponsored, randomized, placebocontrolled trials of intensive antibiotic retreatment of patients in the USA with persistent symptoms found that additional antibiotic therapy either provided no measurable benefit or a benefit so modest or ambiguous that it was outweighed by the risks associated with the treatment (Klempner et al., 2001; Krupp et al., 2003; Fallon et al., 2008). The third is the absence of a plausible mechanism by which spirochaetal persistence, in the absence of a focus of inflammation or elaboration of a toxin, could cause fatigue and other non-specific symptoms. There is clearly ample precedent for latent infections to be asymptomatic, as illustrated by the persistence of Mycobacterium tuberculosis in one-third of the world’s population.

17.8 The State of the Medical Literature – the Assertion of the Controversy The group that calls itself ILADS – the International Lyme and Associated Diseases

Lyme Disease: the Great Controversy

Society – has published a document it titled ‘Evidence-based guidelines for the management of Lyme disease’ (Cameron et al., 2004) and repeatedly asserts that there is a wealth of information that is being ignored by the medical establishment. However, a detailed review (Buerden et al., 2010) of the ILADS document demonstrates that it references no Class I, Class II or even Class III evidence that rebuts the conclusions of the IDSA (Wormser et al., 2006) or American Academy of Neurology (Halperin et al., 2007) guidelines. Moreover, the IDSA guideline has now been reviewed in detail by an independent panel, formed by a process and with membership approved in advance by the Connecticut Attorney General. After more than a year spent reviewing all the available data, the panel found that the conclusions of the original guideline were completely appropriate and that all relevant information had been considered (Lantos et al., 2010). Unable to fight facts with facts, advocacy groups have chosen to accuse the guidelines’ authors of conflicts of interest, a contention sadly supported by statements by the Connecticut Attorney General in a press conference at the termination of his investigation. What is never mentioned, however, is that the legal document that ended the investigation had no allegations, conclusions or reference to there being any conflicts of interest among the panelists (or of there being any anti-trust violation) (Poretz, 2008) – a conclusion further supported by the findings of the independent guideline review panel. The concept that the recommendations could be influenced by conflicts of interest is a curious one. Firstly, the conclusions are in agreement with all other guidelines published by respected medical organizations (Halperin et al., 2007; Ljostad and Mygland, 2009; O’Connell, 2009; Mygland et al., 2010; British Infection Association, 2011). Moreover, the guidelines recommend short courses of inexpensive generic antimicrobials and testing approaches that are widely available from multiple commercial sources. The guideline contained no mention of vaccines. Consequently, following the guidelines’ recommendations could in no way enrich any

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of the authors. (In contrast, the authors of the ILADS guideline included a principal in a company that markets Lyme disease diagnostic testing favored by LLMDs, as well as practitioners who derive substantial clinical practice revenue from providing the care recommended in their guideline – none of which was mentioned in that document.) Some have suggested that the IDSA guideline might serve to advance the authors’ academic careers, but most of the authors have already achieved senior academic rank. For them, working on this guideline constituted a tremendous amount of work with the only reward being the anticipated reaction from patient advocacy groups and LLMDs. In summary, there was nothing in the guideline that could lead to personal profit for any of the authors. It is clear that, despite focusing their rage and indignation on the authors of the various guidelines, the advocacy groups’ real fight is with the notion of evidence-based medicine. The ILADS guideline demonstrates a remarkable lack of understanding of this process. Included statements consistently refer primarily to the authors’ personal anecdotal observations. Many outside ILADS would welcome a rigorous, scientific study of the issues they raise. If a fraction of the time, money and energy that has been spent on inappropriate care and advocacy had instead been invested in scientific studies to understand better the pathophysiology of the disorder they refer to as ‘chronic Lyme disease’, we would probably all be in a much better position to help the unfortunate individuals whose lives have been severely disrupted by this symptom complex.

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Index

Page numbers in bold refer to illustrations and tables acrodermatitis chronica atrophicans (ACA) 55, 107, 130, 133, 142–143, 145 see also lesions; skin activists 228, 242, 260 see also LLMDs adhesins 36–37 aetiology/etiology 55–56, 143, 145, 221, 238, 263 Afzelius, A. 29, 54, 141, 190 age-specific incidence 105–108, 130, 131, 143, 144, 148, 155 see also children agents 1, 14, 89, 101–102 see also ticks; vectors Amblyomma americanum 10, 161 American Academy of Neurology 225, 267 American College of Physicians (ACP) 83 amino acid sequences 55, 180 5-aminoimidazole-4-carboxamidoribonucleotide (AICAR) 203 amoxicillin administration method 135 adverse effects 167 culture positivity after treatment 93 dosage 118, 133, 167, 168, 185, 201, 202, 226 recommended use 117 treatment duration 135, 166, 202 Anaplasma spp. 4, 135, 167 see also coinfection anaplasmosis 9, 14, 135, 225 animal models 89–95 anti-trust violation 259 antibiotic-refractory Lyme arthritis 198, 202–203 antibiotics adverse effects 136, 252

assessment 91, 252 disease persistence after treatment 89–95 dosage/duration sufficiency 92 efficacy 95, 116–117, 118, 146 failure 93 incorrect choice 233 ineffectiveness 259 minimum concentrations 115–116 oral courses 119 pain persistence after treatment 194–195 recommendations 202 resistance 116, 196, 233 response failure 59 retreatment 121 studies 252, 253 therapy 115–122, 183, 185, 200, 234–235, 259 treatment outcomes 171 unnecessary treatment 242, 259 see also amoxicillin; ceftriaxone; doxycycline antibodies anti-C6 serum 94 bactericidal action 39 cerebrospinal fluid 225, 264 detected 105 detection 74 development 13 immune responses 80 levels 73, 192, 251 measurement 74, 213 production 40 response development 132 role 38–39 tests 132–133, 134, 224 271

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antibiotics continued titres rise 93 see also antigens; immunoglobulin antigens epitope containing 62 exposed 17 expression variation 132 heterogeneity 55 human leukocyte 61 identification 224 immune response role 62, 73, 195–196 prominent 36–38 protective immune response 40 recognition 78 retained 60 see also antibodies; bacteria; toxins; VlsE antimicrobials administration methods 119 dosage 226 Jarisch-Herxheimer-like reaction 117, 165, 185, 225, 261 prolonged treatment 242 role 95 unrealistic expectations 122 anxiety 232, 236, 238 aporia 204 arthralgias 191 see also symptomatology, systemic symptoms arthritis antibiotic-refractory 119–120 causes 60, 107, 115 chronic 42 described 60 development time after tick bite 93 diagnosis rarity, UK and Ireland 131–132 epidemic 221 European manifestations 145 immune responses 80 joints 35 juvenile rheumatoid 29, 259 late manifestation 59–61, 79, 223 mouse strains development 91 non-human primates development 94 persistent, antibiotic-resistant 60–61 rarity 57, 131–132 reactive 201 recurrences 164–165 reported 130 severity 41, 63 treatment recommendations 119–120 see also joints arthrocentesis 225 Asia 104–105 Aspects of Lyme Borreliosis 140 assays 61–62, 74, 75, 82–83, 133, 164, 263 see also ELISA; serology

Index

assumptions false 260 see also misdiagnosis AtheNA Multi-Lyte test system 82 attractants, heat; CO2 62 autoimmunity 42, 60–61, 198, 251 azithromycin 133, 166, 167, 168 B-cells 39–40, 180, 197 Babesia spp. 4, 11, 30, 135 babesiosis 9, 14, 30, 135, 225 bacteria lurking somewhere myth 266 resistance 39 shape 36, 37 see also Borrelia spp. Bannwarth’s syndrome 58, 132, 223 Barbour-Stoenner-Kelly (BSK) medium, culture 34, 161 biopsies 64, 82, 83, 161, 184, 186 birds 2, 7, 10–11, 128 bites anatomical locations 147 awareness 147 detection 155 hypersensitivity reaction 160 local reaction size 160 rash onset time after bite 163, 222 recall 155, 156, 163 repeat 168, 170 risk reduction strategy 227–228 seasonality 147, 161 spider 162 BL (borrelial lymphocytoma) see lymphocytoma blots 134 see also immunoblots; Western blots Borrelia spp. B. burgdorferi sensu lato complex B. afzelii 30, 89, 107, 108, 128, 130 B. burgdorferi American subtypes 180 enzootic cycle 29 establishment as Lyme disease agent 54 geographical evolutionary patterns 4 global epidemiology 100–109 haematogenous dissemination 159 infections 8, 100–109 introduction new sites 11 isolation 127 life cycle 31–33 persistence 89–95 protein profile 36 B. garinii 30, 89, 107, 128, 144 biological classification 31 genospecies 30, 32, 74, 101, 101, 115 less common species 3, 14, 33, 101, 128, 161 B. burgdorferi sensu stricto 101, 102, 107, 159 biology 29–42, 89, 102, 105, 107

Index

cell-wall-free form assertion 260 cyst formation assertion 260, 266 endemic areas 55, 104, 105, 107, 128 evolution, asexual lineages differentiation 55 frequency 149 geographical origin 30, 32, 74, 101, 101, 115 identified 55 immune system interactions 54–65 infections 115, 116–117, 118, 146–147 intracellular hiding assertion 260 isolates 31, 55 species list 55, 141 UK and Ireland 128 borreliosis (Lyme disease) defined 140 described 100 original characterization 259 bovine borreliosis 14 brain 210, 239–240, 264 Burgdorfer, Willy 54 C6 antigen peptide 62, 82, 93, 164, 263 canine model 92–93 cardiac see heart cardiomegaly, prognosis 186 see also heart cardiomyopathy 183–184, 185 see also carditis; heart carditis diagnosis 146 erythema migrans connection 81, 159 European rarity 144–145 manifestations 59, 165, 181, 182–183, 184, 186, 223 mortality 265 prognosis 186 seasonality 179 symptoms 181 untreated, outcomes 164 see also heart CDC (Centers for Disease Control and Prevention) 164 see also guidelines; recommendations; reporting cefotaxime 119 ceftriaxone adverse effects 119, 167, 168 C6 antibody levels effects 93 dosage 185, 252, 253 efficacy 91 parenteral 165 preferred parenteral agent, reasons 119 serum half-life 91–92 treatment 183, 217 treatment duration 92, 118, 166, 217, 252, 253 cefuroxime axetil adverse effects 165, 167

dosage 133, 165, 167, 168, 185, 201, 202 recommended use 117 subjective complaints after treatment 169 treatment duration 133, 201, 202, 251 cellulitis 161, 162 Centers for Disease Control and Prevention (CDC) 164 central nervous system (CNS) diagnosis challenge 213 disorders 217 inflammation 225 Lyme disease effect 264–265 Lyme disease involvement 212–215 neuroborreliosis 94 pain processing augmentation 240 treatment 133, 202 central pain syndromes 240 central sensitivity syndromes 239–240, 241 cephalosporins 116, 167, 201 cerebrospinal fluid (CSF) abnormalities 58 examination need debate 217–218 index 225 inflammatory 264 invasion by B. burgdorferi 94 isolates 55 pleocytosis 145–146 test 213 cerebrovascular disease 211 chemokines 64, 159, 197 chemoprophylaxis 116–117 children common presentations 131 dosage adjustment 166 doxycycline contraindication 167 electrocardiographic abnormalities 179 epidemiology 221–229 juvenile rheumatoid arthritis incidences 259 manifestations 29, 181, 202, 212, 223, 225 treatment 133, 135, 166, 168, 225, 226 see also age-specific incidence cholestyramine 200 chondromalacia patella (osteoarthritis) 194 chronic Lyme disease attributed symptoms 249 concept 228–229, 240, 248–254 cultural movement 242 diagnosis 235–236 evidence lack 227 myths 228, 241 treatment failure concept 251 undefined disorder 259 clarithomycin 167 classification 30–31, 248 climate 8, 9–12, 105 coexistence 57 cognition 211–212, 215, 236, 237, 243, 260 see also brain; memory

273

274

coinfection 13, 14, 30, 120, 163, 223, 249 comorbidity 236–237 complaints, subjective 169, 201, 232, 250–251 complement 38, 39 complications objective extracutaneous 165 see also arthritis; carditis; meningitis conduction system disease 181–182 conflicts of interest 267 congenital Lyme disease 227 controversy 136, 259–267 see also myths corticosteroids 91, 93, 185, 202, 218 countries affected 9, 103, 104–105, 147 cross-reactivity 61, 198, 214, 261, 262–263 cultural movement 242 cultures confirmation 168 impracticality 262 limited value 163 mediums 33–34, 161 methods 33–34 not recommended 192 routine diagnosis inappropriateness 61 sensitivity 224 see also tests cure debate 121–122, 142 cytokines 41–42, 64, 159, 197, 201 data, animal, availability 95 debris 196, 197, 200–201, 234 decompensation, acute cardiovascular 185 deer 3–4, 6, 9, 10, 16, 33 DEET (N, N-diethyl-meta-toluamide) 227 defence mechanism, immune-mediated 40 depression 232, 236, 238 dermatitis 108, 162, 223 see also acrodermatitis chronica atrophicans; erythema migrans; skin detection 74, 82, 83, 105, 146–147 see also serology; tests diabetes 210–211 diagnosis clinical 154, 263–264 commitment, chronic Lyme disease 235–236 differential 159–161, 162, 191, 193–194, 263 history taking 159, 191 laboratory 61–62, 145–146, 163–164, 261–263 methods 141, 184–185, 193, 224–225 microbiological, impracticality 262 physical examination value 159 reconsideration 202 see also misdiagnosis specific host antibody response reliance 262 technical, limitation 265–266 validation 261 see also ELISA; immunoblots; PCR; serology; tests

Index

Diagnostic and Statistical Manual for Mental Disorders IV 239 diapause (dormancy) 8 disease conviction 235–236 increase 9, 148 phases 89 stages 57 dissemination 82, 159, 222–223 distribution 2, 9, 57, 101–102 DNA (deoxyribonucleic acid) detection 30, 82, 83, 262 dogs 92–93 doxycycline administration methods 117–118, 133, 165 advantages 167 adverse effects 167 contraindication, children 167 culture positivity after treatment 93 dosage 94, 167, 185, 201, 202, 252 efficacy 117, 165, 166, 217, 266 pharmacodynamic properties 92 prevention role 228 single dose 17, 228 subjective complaints after treatment 169 treatment duration 93, 118, 120, 166, 202, 251, 252 uses 116, 168, 251 see also antibiotics drugs 116, 185, 202, 225 see also antibiotics; treatment echocardiography 184 ecology 127–128 education programmes 17 electrocardiographic abnormalities, children 179 ELISA (enzyme-linked immunosorbent assay) C6 assay 164, 263 diagnosis confirmation 186 immunoblotting replacement proposal 164 index calculation 214 negativity 79–80 results improvement 263 sensitivity 77, 82 skipping 78–79 encephalitis 12, 13, 14, 120, 212–213, 223, 264 encephalomyelitis 59, 131, 135, 264–265 encephalopathy 210, 211–212, 213, 214–215, 223 see also neuropathy endemicity countries affected 9, 103, 104–105, 107, 128, 147 tourist destinations 129–130 vectors 31, 146, 161 see also epidemiology endocardium, inflammation 181 enzyme immunoassays (EIA) 74, 75, 133 enzyme-linked immunosorbent assay see ELISA

Index

epicardium 181 epidemiology children 221 enzootic disease 1–2, 3, 4, 14, 29, 38 Europe 146–149, 179 global 100–109 seasonality 146, 155, 179 UK and Ireland 127, 128–130 USA 155, 179 see also endemicity epitopes 62, 198–199 erythema chronicum migrans now erythema migrans EM 154 erythema migrans (EM) anatomical location 58 characteristics 155–157 defined 141–142 described 54, 57–58 diagnosis 145, 154–164 differentiation from bite hypersensitivity 160 duration factors 156–157 epidemiology 155 first UK reported case 127 increase 130 incubation period 148 manifestation 57, 107, 130 neurological disease connection 81 outcomes 171 patients characteristics 156 physical examination findings 158 previously erythema chronicum migrans 154 recognition 221 reinfection 170–171 repeat episodes 168 symptoms 157, 249, 250 tick association 29–30, 161–163 treatment 117, 120, 164–170, 167, 190 see also lesions; palsies; rash erythromycin 165, 167 etiology/aetiology 55–56, 143, 145, 221, 238, 263 entomological risk index 14 Eurasia 1 Europe 31, 55, 101, 104, 107, 140–149 European Federation of Neurological Societies (EFNS) 130, 132, 133 European Lyme radiculitis 212 European Union Concerted Action on Lyme Borreliosis (EUCALB) 130, 132, 140 evaluation, laboratory 192 evidence 30, 227, 251–252, 261 Evidence-based guidelines for the management of Lyme Disease 267 evidence-based medicine 241, 243, 267 examination neurological 211 physical 158, 159, 191, 192 see also diagnosis; tests

275

experts (so-called) 241 see also LLMDs exposure 15, 16, 136, 194 see also prevention; risk ExPRESS 243 extracellular matrix (ECM) 38 eyes 145 fatigue chronic 203–204, 250, 251 population rates 215 search for an explanation 240–241 syndromes 239, 250, 252 fear 228–229 feeding 4, 12–13, 56, 63 females 105–106, 179 see also gender-specific incidence fibromyalgia 203–204, 232, 234, 238–239, 240 fibromyalgia-like syndromes 195 fibrosis, endocardial 180–181 flagella 36, 37, 62 see also pathogens flavivirus 13 flu-like symptoms 197, 232 fluid removal 201, 225 fluoroquinolones 116, 167 Garin-Bujadoux neurological triad 208, 212 Garin-Bujadoux-Barnworth syndrome (meningoradiculoneuritis) 130–131, 145 gender-specific incidence 105–108, 130, 143, 148, 171, 179 genetics 3–4, 31, 35–36, 38 genome 35–36, 56, 141, 149 Germany 106 gout 193 guidelines antibiotics use 201 diagnosis 74, 121, 130 evidence-based 267 prevention 121 review 199–200, 259, 267 treatment 120, 121, 130, 133, 167 see also recommendations Guillain-Barré syndrome 131 Haiku 84 Health Protection Agency (HPA) 128 heart block 167, 181–182, 186, 223, 234, 263 conduction system disease 181–182 disease 120 dysmetabolic syndrome 193 enlargement, prognosis 186 failure, chronic 183 involvement in Lyme disease 179–186 manifestations, clinical 59, 181–182

276

Index

heart continued pacing 182, 185–186, 223 tissue, spirochaete effect 35 see also carditis Herxing 261 history, Lyme disease 29–30 hosts availability 10 diversity 1, 6–7 immunocompromised 40, 64, 91, 92, 169–170, 180 interactions 63 reservoirs 7, 30, 127, 128, 146, 195 response 38–42 seeking 7, 146 tick dispersal role 11 types 5, 31 vertebrate 4–7, 32 human, studies 180–181 human granulocytic anaplasmosis (HGA) 120, 163 human granulocytic ehrlichiosis now human granulocytic anaplasmosis hydroxychloriquine 200, 203 identification, gold standard 163 IgG 75, 78–79, 134, 214, 262–263 see also immunoglobulin IgM 75, 78–79, 80, 134, 262 see also immunoglobulin ILADS (International Lyme and Associated Diseases Society) 266–267 illness model 241 imaging 184–185, 211, 264 immune complex formation 192 immune response adaptive 64, 196 antigen-specific 73, 195–196 development 62 dysregulation 60 evolution 80 human 170 induction 63 innate 40–41, 63, 64 stimulation 132 tolerance breakdown 198 immune system 39, 54, 195, 196–198, 216 immune/inflammatory response 196 Immunetics 62, 76, 82 immuno-pathogenesis 197, 199 immunoassays 74, 75, 133 immunoblots bands 82 clinical usefulness 80 faint bands 79 recombinant 132–133 scoring 76, 79–80 second tier serology 75

standardizing difficulty 81 striped 82 techniques 78 see also Western blot immunodeficiency 38–39, 40, 64, 91, 92, 180 immunoflorescence assays (IFAs) 74, 75 immunogen 198 immunoglobulin (IG) anti-borrelial 39 assays 62 binding fragments 39 criteria 262 cross-reactivity 180 production 40 profile determination 75, 78–79, 134 response 62, 80, 262–263 secretion 40 seroprevalence 129 synthesis increase 214 see also antibodies immunoglobulins, profile determination 75, 78–79, 134 immunoprophylaxis 146 immunosuppression, transient 93, 94 in good faith 243–244 incidence areas 105, 259 assessment difficulty 147–148 by country 9, 103, 104–105, 147 by region 102–104 determination 12 local variation 129 mapping 83 ranges, reported 104 rates 103, 104 see also age-specific incidence; gender-specific incidence infection active 60, 261 concurrent 251 current 233 multiple 13 see also coinfection rate 146–147 risk 17, 57 secondary 199 sources 13–14, 56, 101, 102 stages 57 transfusion-associated 108 types 120 see also transmission Infectious Diseases Society of America (IDSA) 120, 167, 225, 259, 267 infectivity 34, 36, 37, 38 infiltrates 180, 181, 186, 211 inflammation causes 194, 196, 234

Index

continuing symptoms 265 drivers 197 induction 41, 79 initiation and modification mechanism 40–41 manifestations 181, 192 mediators 197 reaction 197–198 see also debris inflammatory cardiac infiltrate, prognosis 186 infliximab 91 insomnia 251 intention-to-treat analysis 252 interfaces 12–17 interferon (IFN) 64, 180, 197 interleukin (IL) 41–42, 197, 217 International Lyme and Associated Diseases Society (ILADS) 266–267 intracranial pressure management 212, 225–226 invariant NKT cells (iNKT) cells 41, 180 Ireland 127–136 isolates 30, 31, 55, 107, 127, 149 Ixodes spp. see ticks Jarisch-Herxheimer-like reaction 117, 165, 185, 225, 261 joints clinical signs 196 dysfunction 194 infection 145, 164, 196 inflammation 165, 191, 192 manifestations 208 see also arthritis; knee; rheumatology judgement, clinical 263–264 juvenile rheumatoid arthritis incidences 29, 259 killing complement-mediated 39 see also natural killer T (NKT) cells knee 190, 192, 193, 194, 201, 265 see also joints; rheumatology landscape, exposure risk factor 15–16 late Lyme disease 59, 81, 250, 260 law, unintended consequences 260 legislation 259 leptospirosis 116 lesions anatomical location 156, 158, 222 central clearing 156 cutaneous 29 described 89, 157 diameter 156 growth rate 156 heat 157 homogeneously erythematous 57–58 multiple 115, 156, 158–159, 165, 222 secondary 58, 158, 222

277

size 154, 156 see also acrodermatitis chronica atrophicans; erythema migrans; rash; skin lethality 265 life cycles 4–9, 31–33, 56 lipopolysaccharide (LPS) 55–56 lipoproteins 35, 55–56, 63 see also proteins liver, sinusoids 41 Living with Lyme Disease Study 242 LLMDs (Lyme Literate Physicians) 241, 242, 260–261, 267 LNB (Lyme neuroborreliosis) see neuroborreliosis localization 222 locations, anatomical, Borrelia 34–35 louping ill virus 135 Lyme borreliosis: Biology, Epidemiology and Control 147 Lyme disease, described 232 Lyme Literate Physicians (LLMDs) 241 lymphadenopathy 158 lymphocytes 181, 198 lymphocytoma 58, 107, 142, 145, 223 lymphoma, cutaneous 143 Macaca mulatta, rhesus macaques 94 macrolides 117, 133, 166, 167 macrophage 180 magnetic resonance imaging (MRI) 184–185, 211, 264 males 106, 179 see also gender-specific incidence manifestations clinical European 140–145 first descriptions 141 frequency, Europe 148 immune system 57–61 stages 222–223 strain differences influence 159 cutaneous 34–35 early 225, 226 infection 118, 120 late 79, 80, 223, 226, 250 major 251 objective 115, 154 rare 145 systemic 222 mapping 11, 83 Masters’ disease 161 matrix metalloproteinase (MMP) 60 medicalization 241, 243 see also antibiotics; drugs; treatment medication 185 see also antibiotics; drugs; treatment membrane 37, 39

278

memory 252, 260 see also cognition meningitis diagnosis 213 effects 22, 264 lymphocytic 131, 167, 212, 263 presentation 212 seasonality 212 symptoms 144 treatment 225 treatment duration recommendation 120 meningoencephalitis 131, 223 meningoradiculoneuritis (Garin-BujadouxBarnworth syndrome) 130–131, 145 methotrexate 203 metronidazole, non-use advocation 167 mice 40, 41, 56, 64, 90–92, 180 see also murine model microscopy, polarizing 193 Millon Clinical Multiaxial Inventory (MCMI) 237 mimicry, molecular 180, 198–199, 216–217 minocycline 217 misdiagnosis antimicrobial response lack 225 chronic Lyme disease 248 diagnosis reconsideration 202 lupus 241 ongoing infection 200 radiculopathy 131 rashes 161 warning issued 136 see also chronic Lyme disease molecules, adhesion 180 monoarthritis 190, 192–194, 201, 202 monocyte, infiltration 181 mononeuropathy 94, 216 morphology 36, 37 mortality 6–7, 8–9, 265 motility 35, 36 murine model 30, 41, 63–64, 90–92, 179–180 muscles, arthritis effect 203 musculoskeletal complaints 201 musculoskeletal consequences 190–195, 199 myalgias 191, 195, 238–239 myocardium, inflammation 181 myths 228, 261–263, 266 see also chronic Lyme disease natural killer T (NKT) cells 41, 180 necrosis 91, 181, 217, 252 nerves 216 nervous system 35, 117–118, 208–218 see also neuroborreliosis neuritis 144, 212 neuroblastoma cells 199 neuroborreliosis causes 211

Index

chronic Lyme disease investigations 251 classification 35 data value 128 defined 143–144 early diagnosis 145–146 European perspective 143–144 nervous system disorders 94, 211–212 presentations 131, 132 protean manifestations 215–216 seasonality 130 species causing 107 treatment 130, 133, 135, 225 see also nervous system; palsies neurological, definition 209 neurology abnormalities 164 Bbsl infection 121 diseases 81, 168–169, 209–210 disorders, mechanisms 217 function, acute focal changes 210 illness complex 208 involvement 223 learned responses 209–210 manifestations 115, 130 nervous system diseases 210–211 see also meningitis; nervous system neuropathy 59, 60, 80, 210–211, 216 neurotransmitters 239 non-steroidal inflammatory drugs (NSAIDS) 202, 225 North America 57, 102–104 not Lyme 232–244 notifiable diseases 128 see also reporting notions, misguided 228 observations 57 Ockham’s razor, dulling 199 oesophagitis 167 oligoarthritis 192–194, 201, 263 organism presence 196 organisms, long-dead DNA detection 262 organs 145, 196, 234 see also heart osteoarthritis (chondromalacia patella) 193, 194 outcomes 135, 164–165, 166–167, 168–170, 202, 225–227 outdoor activities studies 14–15 outer-surface proteins (Osps) A (OspA) 12–13, 34, 55, 61, 79–80, 180, 217 B (OspB) 36–37, 79–80 C (OspC) 37, 62, 133, 159 changes 56 overdiagnosis 183, 249 see also misdiagnosis pacemakers 182, 185–186, 223

Index

pain characterization 240 chronic 239–240, 251 complaints 250–251 explanation search 240–241 management, non-pharmacological 243 mechanisms 240 pathophysiology 239–240 persistence 194–195 palpitation 181 palsies cranial nerve 158, 168, 223 facial nerve continuing symptoms 265 CSF examination debate 217–218 occurrence before measurable antibody response 216 occurrence percentage USA 212, 223 treatment 225 treatment failure 166–167 treatment trial percentage 165 unresolving 234 see also nervous system paralysis facial 80, 81 see also palsies parenchyma 210, 212 parenteral regimens 119, 165, 217 pathoanatomy 210–211 pathogen-associated molecular patterns (PAMPs) 64 pathogenesis 32, 62–64, 179–181, 195–196, 197, 199 pathogens bacterial identification 259–260 encephalitis virus 120 enzootic transmission 7–8 evolutionary history 4 interface, ticks 12–17 species 141 pathophysiology, nervous system 216–217 pathways 41, 63 patients asymptomatic 168 behaviours 209–210, 237 characteristics 156 untreated, outcomes 164–165 PCR (polymerase chain reaction) analysis detection yield 192–193 diagnosis inappropriateness 61, 82 DNA fragments detection 262 limitations 73 low diagnostic sensitivity 262 positivity 94 sensitivity 83 penicillin 116, 119, 165, 185, 217, 262 peptide binding 61

279

peripheral nervous system (PNS) 210–211, 215– 216, 217 permethrin 227 Peromycus leucopus, reservoir host 195 persistence complaints 201 infection 90–95, 233, 251 mechanism theories 266 pain 194–195 post-treatment 89–95, 120–121, 194–195 psychological aspects 232–244 symptoms 226–227 see also chronic Lyme disease; post-Lyme disease syndrome personality disorders 237 pesticides 227 phagocytes 39 pharmacology 243 phenology (seasonal cycle development) 7–8 see also seasonality phenomenology 209–210, 216 phenoxymethylpenicillin 117, 166 photosensitivity 167 physicians 241–242, 260–261, 267 plasmid 35–36, 37 pleocytosis 94, 145–146 poly-microbial infectious syndrome 249 polyarthralgias 191 polyarthritis 194 polymerase chain reaction see PCR polymorphonuclear leucocytes (PMNs) 180 positive effect 237, 238 post-infectious immune dysregulation 60 post-Lyme borreliosis symptoms 89 post-Lyme disease, see also chronic Lyme disease post-Lyme disease syndrome (PLDS) 121, 122, 135, 215, 232–244, 248, 249–254 post-treatment, long-term outcome 168–169 PR intervals 181–182, 185, 186 prednisone 93 pregnancy 169–170 presentations clinical 55, 130–132, 212 see also manifestations; symptoms prevalence 13, 84 prevention antimicrobial prophylaxis 228 insect repellents 227 personal measures 16–17, 116, 135–136 rodent host vaccination 17 showering 108 strategies 15 tick bites risk reduction methods 227–228 tick checks 17, 108, 116, 135–136, 227 tick removal 146, 227–228 vaccines 13, 17, 79, 168 primate model, non-human 94

280

pro-inflammatory molecule release 196–197 probability, pre-test 83, 84 prognosis 168–169, 186, 225 see also outcomes prophylaxis 146, 228 see also prevention proteins 12–13, 36, 38, 62, 197, 198 see also lipoproteins; outer-surface proteins pseudo-tumour cerebri-like syndrome 212 psoriasis 194 see also rash; skin psychiatry 210, 236–237 psychology 232–244 psychopathogenesis 204, 239 psychosocial risk factor 237 pumps, multidrug efflux 116 QTc interval prolongation 182 quinolinic acid release 217 radiculoneuritis 58, 135, 144, 212, 216, 263 see also neurology radiculopathy 131, 212, 216, 223 rash anatomical location 161, 222 annular/bull’s eye appearance 156 cause 222 description 3–4, 162 duration 156 misdiagnosis 161 onset time after bite 163, 222 resolution 135 STARI 161–163, 166 see also erythema migrans; lesions receptors 40, 41, 63–64, 180 recommendations 74, 118, 133–135, 167, 225 see also guidelines records, kept Russia 104 redwater fever 135 regulations 259 see also guidelines; law reinfection 170–171 relapse 171 relapsing fever (RF Borrelia) 14, 31, 116 repellents, insect 227 repolarization abnormalities 182 reporting 102, 104, 128, 129, 147, 155 responses RF Borrelia 14, 31, 116 rhesus macaques, Macaca mulatta 94 rheumatology 29, 190–204, 208, 259 Rickettsia spp. 14, 135 risk areas 15, 83, 102, 194 entomological risk index 14 exposure 194 factors 14, 15, 108, 238

Index

insect repellent adverse effect 227 Ixodes nymph density link 57 levels 228 management 136 occupational 15, 129 recreational 129 reduction 16 residential 129 see also prevention rodent populations 4–5 see also mice; murine model roxithromycin, trial treatment failure 166 Salp15 protein 12 Schereschewsky’s method, culture 33 scintigraphy 184–185 screening 128, 133 Scrimenti, R. J. 54 seasonality epidemiology 146, 155, 179 infection 105, 148, 154, 179 Ixodes ricinus activity 147 meningitis 212 neuroborreliosis 130 onset-peaks 105 reinfection 170–171 synchrony 11–12 tick exposure 136 transmission 7–8, 32–33, 56, 105 seeding 57 sequence analysis 30–31, 55, 180 sera, protective capacity 39 seroconversion 131, 132 serodiagnosis 61, 74–81 serology diagnostic testing USA 73 early development 260 essential diagnosis tool 145–146 false-negative 261–262 methods 186 newer tests 81–82 not recommended 228 positivity 192 proper use understanding 190–191 results predictive value 83, 224, 228 seronegative Lyme disease myth 261–262 two-tiered 61–62, 74–81, 163–164, 183–184, 261, 262 see also diagnosis; ELISA seronegativity 260, 261–262 seropositivity 76, 79–80, 81, 128–129, 133, 183 seroprevalence 129 serosurvey 30 serum immune complexes presence 192 sex-specific incidence 105–108 see also gender-specific incidence shape, bacterial 36, 37

Index

shrubs, tick density factor 15 sigma factors (Ó) 38 signalling, TLRs 41, 63–64 single photon emission computed tomography (SPECT) 264 sinusoids, liver 41 skin biopsies 64, 82, 83, 161 disorders, confusion with EM 161, 162, 165 isolates 55 manifestations 34–35, 130, 141–143 scaling 157 see also acrodermatitis chronica atrophicans; erythema migrans; lesions; lymphocytoma; rash sleep disturbance 195, 251 somatization disorders 239 see also anxiety; depression somatoform 239 southern hemisphere, incidence 105 southern tick-associated rash illness (STARI) 161– 163, 166 spirochaetaemia 58, 158–159 spirochaetes cultivation 33–34 cyst formation 260, 266 infection types 120 isolation, white mice blood 30 morphological changes 266 persistence in unidentified tissue sites 266 presence after anti-biotic therapy 252 primary vectors distribution 2 spondyloarthopathies 201 standards 74–82, 163 see also guidelines Steere’s classic description, musculoskeletal features 201 steroids 157, 202, 218 Streptoccus pyogenes M 180 stress-response systems 237–238 studies 168, 180–181, 242, 252, 253, 261–262, 263 subolesin protein 13 sulfonamides, non-use advocation 167 SUNY-SB study 261–262, 263 surveillance 100 survival factors 11, 266 symptomatology brain fog 264–265 clinical diagnosis role 263–264 common symptoms 260 constitutional symptoms 58 continuing symptoms 249, 250, 265, 266 misinterpretation 260 multiple symptoms 235 non-specific symptoms 228, 249, 250, 251, 266 relief 243 subjective symptoms 117, 120–121, 165, 233–234, 266

symptoms misinterpretation 260 systemic symptoms 115, 157–158, 159, 211 see also fatigue; manifestations; persistence; presentation synchrony, seasonal 11–12 synovectomy 202 synovial fluid 83, 192, 193, 201 synovitis 119–120, 193, 197, 200, 251 synovium 60 syphilis (Treponema pallidium) 33, 116, 117, 200, 214, 262 T regulatory (Treg) cells 42, 60, 198 T-cells 41–42, 61, 64, 180, 197 taxonomy 30–31 techniques, molecular 30 tests 73–84, 132–133, 164, 241, 252, 262–263 see also assays; culture; diagnosis; serology tetracyclines 116, 165, 166 see also doxycycline Th17 cells 197 therapies creative 200 intravenous 202 molecular biology 203 oral 202 physical 203 prolonged 200, 234–235, 259 recommended 118 success standard 121–122 unapproved 202–203 see also antibiotics; treatment ticks (Ixodes spp.) behaviours 7, 105, 146–147 biogeography 2–4 density 10, 15 dispersal 10, 11 distribution 83, 101, 104 evolutionary history 2–4 feeding 4, 12–13, 56, 63 genetic bottlenecks 3–4 guilds 3, 12, 13 habitats 3–4, 14–15, 108, 127–128 hard 31 importation estimates 11 integrated management 16 interface role 12–17 life cycles 4–9, 56 nymphs 14, 57, 83, 105, 146, 228 populations 3–4, 8–9, 33, 129 range expansion 3, 9, 11–12 role in Lyme disease evidence 30 seasonality 7–8, 136 species I. dammini 3 I. hexagonus 3 I. ricinus species complex 1

281

282

Index

ticks (Ixodes spp.) continued species continued I. pacificus (Western black-legged tick) 7, 8, 30, 102, 104 I. persulcatus (taiga tick) 6, 89, 102, 146 I. ricinus (castor bean tick; sheep tick) 2–3, 8, 11, 101, 127, 147 I. scapularis (black-legged tick: deer tick) 3, 4, 6–7, 8, 9, 30 I. uriae (seabird tick) 2 transmission role 89, 101, 102, 105, 135, 146–149, 221 as vectors 1–17 see also bites; hosts; vectors tigecycline, dosage regimens 92 tinea (ringworm) infection 161, 162 tissue predilection 63 toll-like receptors (TLRs) 41, 63–64 toxins 63, 200 transformylase 203 transmission alternative modes 108 competence 12 cycle 32 enzootic 1–2, 4, 7–8, 14 intensity, Europe 104 interfaces 12–17 method 100, 221 seasonality 7–8, 32–33, 56, 105 time requirement 17, 36, 56, 108, 116, 155–156 transstadial 31 see also bites; ticks; vectors traps, ecological 9 treatment adverse effect 253 attempts, early 141 chemoprophylaxis 116–117 duration 119, 133, 146, 168, 226 failure 166, 170, 233, 251 inappropriate 136, 265 issues, animals 90 localized 117 long-duration 265 methods 199–204, 217–218, 226 post-antibiotic 122, 232 rational approach 242–243 recommendations 119, 133–135, 167, 225, 259

resistance, postulating 261 success standard judging 121 trials 165–167, 168 unnecessary 241, 242–243 see also antibiotics Treg (T regulatory) cells 42, 60, 198 Treponema pallidium (syphilis) 33, 116, 117, 200, 214, 262 triad neurological 208, 212 see also meningitis, lymphocytic; cranial neuritis; radiculoneuritis trials 165–167, 168 tropics, incidence 105 TROSP-A proteins 13 UK and Ireland 127, 128–130, 131–132 United Kingdom 127–136 urticaria 162 vaccine 13, 17, 79, 168 vectors bridge 6, 12, 14 control strategies 14–17 endemicity 31, 146, 161 geographical distribution 2 global distribution 31, 101–102 identification 259 silent enzootic cycles 3 ticks 1–17 transmission competence 12 see also agents; ticks (Ixodes spp.) Virablots 82 Viramed 82 virulence 36–38, 39, 158, 197 viruses 13, 162 VlsE 38, 62, 75–76, 82, 93, 133 see also antigens website, tick activity 146 Western blot 61–62, 183–184, 190–191, 192, 261, 262, 263 see also immunoblots whole DNA–DNA hybridization (WDDH) 30 zoonosis 56–57 zymosan 79