No title

PUBLIC LIBRARY of SCIENCE | plosmedicine.org | Volume 1 | Issue 1 | OCTOBER 2004

MEDICINE

Everyone does

because some things just ought to be a universal resource. But with dominant publishing models, researchers and health professionals around the world simply do not have control over or access to the latest medical findings due to copyright restrictions and subscriptions costs.

PUBLIC LIBRARY of SCIENCE | Volume 1 | Issue 1 | OCTOBER 2004

Who holds the copyright to the Universe?

PLoS Medicine is on a mission to achieve a more fair and effective distribution of medical literature. In many ways, PLoS Medicine is like other top-tier journals: it features original, rigorously peer-reviewed research articles and is available online and in print. The powerful difference is that PLoS Medicine is open access, allowing everyone in the world to read, download, copy, forward, print, distribute, and reuse everything we publish, for free, and on the one condition that that you credit the authors and the original work. Go to www.plosmedicine.org to find out more about open access or to submit your own research for publication in PLoS Medicine. Share your science with the Universe.

Expanding access to health research.

www.plosmedicine.org

V olume depletion in malaria S creening for domestic violence T he future of surgical research A ntiretrovirals and lipid changes Published by the Public Library of Science ISSN 1549-1277

Board of Directors Harold E. Varmus, Chairman & Co-founder Patrick O. Brown, Co-founder Michael B. Eisen, Co-founder Nicholas Cozzarelli Brian Druker Paul Ginsparg Allan Golston Calestous Juma Marc Kirschner Lawrence Lessig Richard Smith Beth Weil Editorial Board Jose Acuin, Dasmariñas Hans-Olov Adami, Stockholm Adriano Aguzzi, Zurich Gianni Angelini, Bristol Fred Appelbaum, Seattle Dennis Ausiello, Boston Kumariah Balasubramaniam, Colombo Peter Barnes, London Paolo Beck-Peccoz, Milan Solomon Benatar, Cape Town Ivor Benjamin, Salt Lake City Zvi Bentwich, Rehovot Agnes Binagwaho, Kigali Sally Blower, Los Angeles Andrew Carr, Sydney Timothy Caulfield, Edmonton R. Alta Charo, Madison Mushtaque R. Chowdhury, Dhaka Jonathan Cohen, Brighton Rory Collins, Oxford Charlotte Cunningham-Rundles, New York Adnan Custovic, Manchester John Danesh, Cambridge Sabine Daebritz, Munich Beatriz de Camargo, São Paulo Dorothy Dunlop, Chicago Alain Fischer, Paris Nicholas Fisk, London Ian Ford, Glasgow Eduardo Franco, Montreal Joseph L. Goldstein, Dallas Eduardo Gotuzzo, Lima Deborah Grady, San Francisco Manuel Graeber, London Hank Greely, Stanford Paul Griffiths, London Leif Groop, Lund Simon Hales, Canberra Chris Haslett, Edinburgh Phillipa Hay, Townsville

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David D. Ho, New York Philip Hopewell, San Francisco Richard Hornung, Cincinnati Tom Huizinga, Leiden Steven E. Hyman, Cambridge Patrick Johnston, Belfast Dan Kaseje, Kisumu Martijn Katan, Wageningen Anthony Keech, Sydney Paul Klenerman, Oxford Keith Klugman, Atlanta Davy Koech, Nairobi Clifford Lane, Bethesda Joep Lange, Amsterdam Bruce Lanphear, Cincinnati Kelley Lee, London Cathryn Lewis, London Susan Lightman, London Ed Liu, Singapore Alan Lopez, Brisbane James Lowe, Nottingham David S. Ludwig, Boston William Lynn, London Alberto Malliani, Milan James Marks, Atlanta Malek Massad, Chicago Peter McCluskey, Sydney Anne Merriman, Kampala Lynne Mofenson, Bethesda Gary Nabel, Bethesda Thomas Novotny, San Francisco Malik Peiris, Hong Kong Tom Quertermous, Stanford Jonathan Rees, Edinburgh Andrew Rice, London Philippe Sansonetti, Paris Stefan Schreiber, Kiel Markus Schwaiger, Munich Jaime Sepulveda-Amor, Mexico City Gerald Shulman, New Haven William Sibbald, Toronto Mervyn Singer, London Peter Singer, Toronto Gary Small, Los Angeles Ludvig Sollid, Oslo Awash Teklehaimanot, New York Shoji Tsuji, Tokyo Neil Turner, Edinburgh Patrick Vallance, London Ajit Varki, San Diego Jean-Louis Vincent, Brussels Ralph Weissleder, Boston Nicholas J. White, Bangkok R. Sanders Williams, Durham Clifford J. Woolf, Boston Kim Yancey, Milwaukee

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PLoS Medicine believes that medical research is an international public resource. The journal provides an open-access venue for important, peer-reviewed advances in all disciplines. With the ultimate aim of improving human health, we encourage research and comment that address the global burden of disease.

Executive Director Vivian Siegel Editors Europe Virginia Barbour, [email protected] James Butcher, [email protected] USA Barbara Cohen, [email protected] Gavin Yamey, [email protected] Production Rebecca Kennison, Director Anna Amato, Production Assistant Chelsea E. Scholl, Production Editor Marketing, Sales, & Circulation Cynthia Blair, Director Development & Strategic Alliances Helen J. Doyle, Director Andy Gass, Policy Analyst Staff Isis Choto, Office Administrator Susanne DeRisi, Web Producer Allison Hawxhurst, Executive Assistant Liana Holmberg, Art Director Paul Ocampo, Editorial Assistant Nick Twyman, IT Director Interns Chris Ellis, Dan Engber, Mason Inman, Giovanni Maki, Margaret Shear, Joyce West Volunteers Holly Atkinson, Khloe Sjögren-Cath

October 2004 | Volume 1 | Issue 1

Cover Art Publisher Information PLoS Medicine (ISSN-1549-1277, eISSN-15491676) is published monthly by the Public Library of Science. All works published in PLoS journals are open access, subject to the terms of the Creative Commons Attribution License (http:⁄⁄creativecommons.org/licenses/ by/2.0/). Copyright is retained by the authors. PLoS Medicine is freely available online: http:⁄⁄plosmedicine.org Correspondence Public Library of Science 185 Berry St., Ste. 1300 San Francisco, CA 94107 USA email: [email protected] phone: +1 415.624.1200 fax: +1 415.546.4090 PLoS European Editorial Office European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SD UK phone: +44 (0)1223.494.495 fax: +44 (0)1223.494.468

Get PLoS Medicine in Print The annual print subscription rate for 2005 is US$160. Three free issues, Oct–Dec 2004, are included with your Jan–Dec 2005 subscription. Single copies, including back issues, are US$15. PLoS Medicine Subscriptions P.O. Box 1897, Lawrence, KS 66044-8897 USA email: [email protected] toll-free phone (in USA): 1 800.627.0629 x215 phone: +1 785.843.1235 x215 fax: +1 785.843.1274 online: http:⁄⁄plos.allenmm.com

Heejae Suh Memories of My Childhood etching 16 x 20 in DOI: 10.1371/journal.pmed.0010032.g001

I

have been a print maker for over ten years. During that time, I have traveled from Korea to Japan, Canada, and America, and my memories of these other cultures and people are built into my art. Etching and woodcut are my main media for expression. Using etching, I can show delicate feelings and complicated images, especially of the body and nature. In this work, I show the harmony of humans and nature that I remember from childhood.

Copyright © Heejae Suh ([email protected])

Change of Address File a notification of address change six weeks in advance to: PLoS Medicine ℅ Allen Press, Inc. 810 East Tenth, Lawrence, KS 66044 USA PLoS Medicine is not responsible for issues not received due to improper address, unless a change of address is on file.

Display Advertising email: [email protected] Manuscript Submission online: http:⁄⁄medicine.plosjms.org

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October 2004 | Volume 1 | Issue 1

PUBLIC LIBRARY of SCIENCE

Editorial 1

Prescription for a Healthy Journal The PLoS Medicine Editors

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Message from the PLoS Founders PLoS Medicine—A Medical Journal for the Internet Age

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Volume 1 |

Issue 1

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14

Policy Forum

e10

The Global Health Watch

The Future of Surgical Research

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Mike Rowson, David McCoy, Amit Sen Gupta, Armando de Negri Filho

Robert J. Weil

e13

Steps on the Critical Path: Arresting HIV/AIDS in Developing Countries Julie Gerberding

e31

Michael B. Eisen, Patrick O. Brown, Harold E. Varmus

Neglected Diseases

Synopses

Bernard Pécoul

Which Risk Factors Matter to Whom?

www.plosmedicine.org

New Drugs for Neglected Diseases: From Pipeline to Patients

19 e6

October 2004

How Should the Health Community Respond to Violent Political Conflict?

31 e3

33 e14

Anthony B. Zwi

4 e29

Perspectives Obstetric Fistula in Ilorin, Nigeria

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Getting the Fluid Balance Right in Malaria

e24

T Cells Cause Lung Damage in Emphysema

e25

Andrew Browning

22 e2

5 Learning Forum

Different HIV Drugs Cause Different Lipid Profiles

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Fever, Headache, and Visual Blurring in a 17-Year-Old Woman

e30

36 e7

William Lynn, Sue Lightman

Oats Intolerance in Celiac Disease

6 e23

The PLoS Medicine Debate Should Health Professionals Screen All Women for Domestic Violence?

7 e4

Ann Taket, C. Nadine Wathen, Harriet MacMillan

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25 e20

Peter J. Barnes, Manuel G. Cosio

Health in Action

Case Report

Palliative Care in Africa and the Caribbean

Essays The Birth of Reproductive Health: A Difficult Delivery

Characterization of T Lymphocytes in Chronic Obstructive Pulmonary Disease

Dingle Spence, Anne Merriman, Agnes Binagwaho

27 e5

Generalized Seizure in a Mauritian Woman Taking Bupropion

42 e15

Marie France Lan Cheong Wah, Lan Sem Hing Lan Cheong Wah

e9

Shereen El Feki

PLoS Medicine | www.plosmedicine.org

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October 2004 | Volume 1 | Issue 1

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ifty-one years ago, James Watson and Francis Crick made headlines with their discovery of the structure of DNA. It was an achievement that forever altered our understanding of the world and secured their place in the annals of scientific history. One year ago, at the launch of PLoS Biology, Dr. Watson was asked if he would do anything differently today. He replied, yes—today he’d publish with the Public Library of Science. That’s because Dr. Watson understands the importance of open-access publishing and the enormous potential it has to advance scientific and medical knowledge. He’s not the only one. Within its first year of publication, PLoS Biology has attracted authors and readers from among leading scientists the world over. And the kudos are accumulating. PLoS Biology recently won a World Summit Award and was lauded by MPs in Great Britain’s House of Commons. Even more importantly, PLoS Biology continues to publish some of the best-quality research, as recognized and covered in leading journals andnews outlets. To learn more about open access or to submit your own research for publication, go to www.plosbiology.org. Maybe you’ll have the chance to do something James Watson only wishes he could have.

Celebrating our first anniversary of open-access publishing

“If I could do it over again, I’d publish that paper in PLoS Biology.” Dr. James D. Watson, Nobel Laureate and Chancellor, Cold Spring Harbor Laboratory

Miriam Chua James D. Watson Collection CSHL Archives

Research Articles Distribution of Major Health Risks: Findings from the Global Burden of Disease Study

44 e27

Anthony Rodgers, Majid Ezzati, Stephen Vander Hoorn, Alan D. Lopez, Ruey-Bin Lin, Christopher J. L. Murray, Comparative Risk Assessment Collaborating Group

Assessment of Volume Depletion in Children with Malaria

56 e18

Nevirapine and Efavirenz Elicit Different Changes in Lipid Profiles in AntiretroviralTherapy-Naive Patients Infected with HIV-1 Frank van Leth, Prahpan Phanuphak, Erik Stroes, Brian Gazzard, Pedro Cahn, François Raffi, Robin Wood, Mark Bloch, Christine Katlama, John J. P. Kastelein, Mauro Schechter, Robert L. Murphy, Andrzej Horban, David B. Hall, Joep M. A. Lange, Peter Reiss

Timothy Planche, Myriam Onanga, Achim Schwenk, Arnaud Dzeing, Steffen Borrmann, Jean-François Faucher, Antony Wright, Les Bluck, Leigh Ward, Maryvonne Kombila, Peter G. Kremsner, Sanjeev Krishna

64 e19

An Immune Basis for Lung Parenchymal Destruction in Chronic Obstructive Pulmonary Disease and Emphysema

75 e8

Sandra Grumelli, David B. Corry, LiZhen Song, Ling Song, Linda Green, Joseph Huh, Joan Hacken, Rafael Espada, Remzi Bag, Dorothy E. Lewis, Farrah Kheradmand

The Molecular Basis for Oat Intolerance in Patients with Celiac Disease

84 e1

Helene Arentz-Hansen, Burkhard Fleckenstein, Øyvind Molberg, Helge Scott, Frits Koning, Günther Jung, Peter Roepstorff, Knut E. A. Lundin, Ludvig M. Sollid

Research Articles are accompanied by Patient Summaries written by the PLoS Medicine Editors.

Examining the world’s health from an alternative perspective. See the Policy Forum by Mike Rowson, David McCoy, Amit Sen Gupta, Armando de Negri Filho. Page 31

online FEATURES

PLoS Medicine | www.plosmedicine.org

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eLetters Readers’ Poll eTOC

October 2004 | Volume 1 | Issue 1

A New Frontier

Okay. Maybe it’s not

completely uncharted territory. But the way things work now, most research tends to be published in journals that charge you to read them. PLoS Medicine is on a mission to achieve a more fair and effective distribution of medical literature. In many ways, PLoS Medicine is like other top-tier journals: it features original, rigorously peer-reviewed research articles and is available online and in print. The powerful difference is that PLoS Medicine is open access, allowing you to read, download, forward, print, distribute, and reuse all articles—at no charge and subject only to the condition that you credit the authors and original source. Go to www.plosmedicine.org to find out more about open access or to submit your own research for publication. PLoS Medicine represents the journal of the 21st century—soon your work could be pushing the new frontier.

www.plosmedicine.org

Open access, freely available online

Editorial

Prescription for a Healthy Journal Take monthly, at no cost; reaches six billion The PLoS Medicine Editors

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oday the possibilities for a medical journal are almost limitless. The first medical journals reflected the needs of a closed group of doctors. But medicine, its place in the world, and the dissemination of information have changed utterly. So in starting afresh, what should a new medical journal retain, and what should it ditch? Most obviously, we should throw out the old way of disseminating information. In today’s electronic age, it is no more difficult, and it is only minimally more costly, to provide access to one million people than it is to one person. So the revolutionary idea of anyone being able to read any article is possible. This idea—open access—which completely challenges the old subscription-based publishing model, is the driving force behind the launch of PLoS Medicine. You can download and distribute articles without restrictions (feel free to make a thousand copies, translate articles into other languages, put articles into books—just give the author proper credit). We have also changed the way we involve the academic community in our journal. Our large global editorial board reflects the diversity of medicine today and is intimately involved in what we do. In particular, members of the editorial board are a crucial part of our peer review process. As academic editors they, along with a senior editor at the journal, take research papers through the peer review process in a way that we believe provides the most constructive and fair review. We are delighted that members of our editorial board have also shown their support for our journal by submitting papers to us, even before we launched. What will we publish? The research article on malaria in this issue reflects our priority of publishing papers on diseases that take the greatest toll on health globally. But we will also publish papers reporting a substantial advance in any specialty, whether that advance is in public health, such as the PLoS Medicine | www.plosmedicine.org

paper on the global burden of disease; drug effects, such as the paper on the effect of HIV drugs on lipids; or the molecular understanding of disease, such as the paper dissecting out the immune responses in lung disease caused by smoking. A good general medical journal should also be a place where the global medical community can discuss together what matters to them. The magazine section of PLoS Medicine will be devoted to comment, lively debate, and diverse opinions, in particular giving neglected voices and diseases a place in the limelight. In this issue’s magazine section you will see articles from five continents that cover a huge range of topics, from basic sciences

The revolutionary idea of anyone being able to read any article is possible. (such as the pathology of emphysema) to global public health (such as palliative care in developing countries). You will find diverse opinions—for example, on whether President Bush is helping or hindering Africa’s progress towards tackling HIV, and on whether health professionals should routinely screen women for domestic violence (tell us what you think by taking our poll at www.plosmedicine.org). And you’ll find case-based learning materials on meningitis linked to an online video and an online quiz. Interpretation of results is an essential part of a medical journal’s job. Although we expect that many of our readers will be doctors, we hope readers will range from patients wanting to learn about the latest research on their illness, to teachers wanting to use an article in the classroom, to policymakers. Hence, we have several levels of comment on original research. Perspectives, written 001

by an expert, are aimed at readers who are already familiar with the topic. Synopses, written by PLoS Medicine’s professional editors, should provide any health professional with a quick introduction to an article. Patient summaries provide a starting point for patients to assess the relevance to them of a research paper. We have decided not to be part of the cycle of dependency that has formed between journals and the pharmaceutical industry, an industry that focuses overwhelmingly on the most profitable drugs, thus sidelining many of the world’s health problems. Medical journals have allowed their interests to become aligned with those of the pharmaceutical industry by printing advertisements for drugs, publishing trials designed by drug companies’ marketing departments, and making profits on reprints used as marketing tools. PLoS Medicine will not accept advertisements for pharmaceutical products or medical devices. Our openaccess license allows free distribution of articles, so PLoS cannot benefit from exclusive reprint sales. And we consider as the lowest priority for publication papers that are simply aimed at increasing a drug’s market share without obvious benefit to patients. We will aim to have the highest levels of transparency in our published papers. We require authors to tell us of any possible competing interests; we in turn will tell readers about them. But, information flow should not be just one-way. Our editorial doors (or at least our E-mail boxes) are always open. We want your feedback on the journal: send us an E-mail or submit an eLetter about any article in the journal, take part in our polls, contribute ideas for the magazine section and submit original research. PLoS Medicine is a journal for the global medical community; we invite you to join in.
The editors for PLoS Medicine are Virginia Barbour, James Butcher, Barbara Cohen, and Gavin Yamey. E-mail: [email protected] DOI: 10.1371/journal.pmed.0010022

October 2004 | Volume 1 | Issue 1 | e22

Open access, freely available online

Message from the PLoS Founders

PLoS Medicine— A Medical Journal for the Internet Age Michael B. Eisen, Patrick O. Brown, Harold E. Varmus

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he Internet is awash with medical information. Eight hundred million people have direct access to the Internet [1], and in the United States over 60% have searched for health or medical information on the Web [2]. Go to any search engine and type in the name of a disease or drug, and you will be directed to hundreds of sites, ranging from the sound and useful to the quackish and dangerous. Google “medical” and you get 85 million pages, “drug,” 40 million, and “health,” 230 million. But something is conspicuously missing. The most reliable medical information on the Internet—the contents of peer-reviewed medical journals—is hidden from the public and most of the world’s physicians. Although most medical journals are available online, their publishers limit access to those who choose, and can afford, to pay for access. This should not, and need not, be so. In the 19th and early 20th centuries independent physicians and small medical societies, interested in making the best new medical knowledge available to doctors, students, and the public, began to publish general medical journals containing case reports, ideas for new treatments, and the results of medical experiments. These pioneers took advantage of the best available technology for disseminating information, printing titles like The Lancet, The New England Journal of Medicine, and The Journal of the American Medical Association on cheap paper and selling them to subscribers at a few pennies a copy. For more than a century, printed journals like these were the dominant means of conveying medical knowledge around the world. But technology has changed. The Internet is now the most economical and efficient conduit for the delivery of information to most places. Publishers of medical journals realize this—when the Internet took off, they took their journals online. But

PLoS Medicine | www.plosmedicine.org

Everything we publish is immediately, freely available online throughout the world, with no restrictions on distribution, copying, printing, or legitimate use. while they adapted their means of distribution to the 21st century, they left their business model in the 19th century, continuing to charge readers for access just as they had done for their printed journals. This has been good for business—medical publishing has never been more profitable—but it comes at a huge cost. The established medical publishers have turned their back on the opportunity to make the latest and best medical information available to anyone with an Internet connection. With the launch of PLoS Medicine, we are embracing this opportunity. Everything published in PLoS Medicine is immediately freely available online throughout the world, with no restrictions on distribution, copying, printing, or legitimate use. Of course, it costs us money to publish this journal, and we must cover our expenses. But the fee-for-access business model that made perfect sense for the printed journal is no longer consistent with the mission of medical publishing because it needlessly limits the reach of the medical literature. And so we have adopted a new model. Instead of charging readers for access to our journal, we ask the authors of accepted research articles to pay a publication fee to cover the costs of peer review, editorial oversight, and production. This “open access” business model ensures our financial health as a publisher while allowing us to convey everything we publish to the widest possible audience.

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Of course, we do not expect authors to cover publication costs personally— rather, we expect the government agencies, companies, foundations, research institutions, hospitals, or universities that sponsor the research to pay the fee. These organizations have always considered the wide dissemination of the results of the research they support to be an integral part of their mission. Virtually every leading sponsor of medical research has announced its willingness to pay for open-access publication, the costs of which average less than one percent of the cost of the research itself—a small price to pay to ensure that everyone who could benefit from their research can benefit from it. We realize that not everyone with something important to convey in a medical journal has access to such funds. To ensure that we don’t replace a barrier to access with barriers to publication, we’ve raised money to cover the publication costs of articles whose authors are unable to pay them. And, for every PLoS journal, an author’s ability to pay will never be a consideration in our decision to publish an article. Despite its obvious benefits, openaccess publication has met with fierce opposition. Established medical

Citation: Eisen MB, Brown PO, Varmus HE (2004) PLoS Medicine—A medical journal for the Internet age. PLoS Med 1(1): e31. Copyright: © 2004 Eisen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Michael B. Eisen, Patrick O. Brown, and Harold E. Varmus are the co-founders of the Public Library of Science. Michael B. Eisen is at the Lawrence Berkeley National Laboratory and the University of California, Berkeley, California, United States of America; Patrick O. Brown is at the Stanford University School of Medicine and Howard Hughes Medical Institute, Stanford, California, United States of America; Harold E. Varmus is president and chief executive of Memorial SloanKettering Cancer Center, New York, New York, United States of America. E-mail: [email protected] DOI: 10.1371/journal.pmed.0010031

October 2004 | Volume 1 | Issue 1 | e31

publishers—now businesses more than forces for change—see open-access not as an opportunity to fulfill a mission of public service but as a threat to their lucrative businesses. They contend that their journals still serve the community well, and object that open access threatens their very existence. This is nonsense!

It is our responsibility as publishers and members of the medical community not only to give patients access to the medical literature, but to provide them with tools to use it wisely. The Wellcome Trust, the world’s largest charitable sponsor of biomedical research, seeking to ensure that the results of the science it funds are “disseminated widely and freely available to all,” recently commissioned a thorough analysis of the scientific and medical publishing industry [3]. It concluded that the current market “does not operate in the long-term interest of the research community,” and issued a strong statement in support of open access [4]. Responding to concerns about journal finances, the trust commissioned a detailed economic analysis of open-access publishing [5], based on which it concluded that “the open access model of scientific publishing—where the author of a research paper pays for peer reviewed research to be made available on the web free to all who wish to use it—is economically viable, guarantees high quality research and is a sustainable option which could revolutionise the world of traditional scientific publishing” [6]. (This report, freely available online, is an excellent resource for anyone with questions about the economics of open-access publishing).

PLoS Medicine | www.plosmedicine.org

We know firsthand that the Wellcome Trust is right. In October 2003, we launched our first journal, PLoS Biology, and it is thriving—not only as a destination for the best research in all areas of biology, but also as a resource for students, teachers, and members of the public who have never before had direct access to the product of scientific inquiry (see for yourself at www.plosbiology.org). We are now bringing this success and this spirit to medicine. The world of medical journals needs a fresh infusion of idealism. All of today’s leading medical journals are more than 70 years old, and PLoS Medicine is here to challenge the status quo. We are first and foremost an openaccess publisher working to ensure that everyone has access to the latest medical research and expertise. But we aim to be more than just an openaccess alternative to established general medical journals. We are determined to make PLoS Medicine the best medical journal in the world by providing outstanding original research and new ideas; thought-provoking, educational, and imaginative features for readers; and the fastest, fairest, and most rigorous peer review for authors. As an open-access journal, we see our audience differently than do the conventional medical journals: our audience is composed of medical researchers, physicians, and other health-care providers, patients and their advocates, students, and the public around the world. It will be a great challenge to create a journal that will serve such a diverse audience—we welcome this challenge. We will make it possible for the results of advanced research on infectious diseases to guide treatment in remote clinics thousands of miles away. We will make the results of a clinical trial of a new drug accessible and understandable both to doctors who might prescribe it and to people who might start taking it. We will make research on rare diseases accessible to general practitioners and patients so that they can work together to recognize and treat them.

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Whereas some would argue that medical journals should not be accessible to patients because patients are unable to use the information effectively, we believe it is our responsibility as publishers and members of the medical community not only to give patients access, but to provide them with tools to use the medical literature wisely. Medical research is a partnership between medical scientists and millions of voluntary human participants, conducted largely with public funds. What better way to acknowledge the public’s contribution and ensure their willingness to sponsor and participate in future research than to openly share the product of this research with them? We hope that you will enjoy reading PLoS Medicine and find it useful and provocative. Please share the journal with your colleagues, patients, and friends. Tell us what you want to see, what you like, and what we could do better. Give us your ideas for changes that will make PLoS Medicine a better journal for you and the community. Join us in reinventing the medical journal. References

1. Internet World Stats (2004) Internet usage statistics—The big picture: World Internet users and population stats. Available: http:⁄⁄www.internetworldstats.com/stats.htm. Accessed 30 August 2004. 2. Pew Internet and American Life Project (2003) Internet health resources. Available: http:⁄⁄www.pewinternet.org/pdfs/pip_health_ report_july_2003.pdf. Accessed 30 August 2004. 3. The Wellcome Trust (2003) Economic analysis of scientific research publishing: A report commissioned by the Wellcome Trust, revised ed. Available: http:⁄⁄www.wellcome.ac.uk/en/ images/SciResPublishing3_7448.pdf. Accessed 30 August 2004. 4. The Wellcome Trust (2004) Scientific publishing: A position statement by the Wellcome Trust in support of open access publishing. Available: http:⁄⁄www.wellcome. ac.uk/en/1/awtvispolpub.html. Accessed 30 August 2004. 5. The Wellcome Trust (2004) Costs and business models in scientific research publishing: A report commissioned by the Wellcome Trust. Available: http:⁄⁄www.wellcome.ac.uk/en/ images/costs_business_7955.pdf. Accessed 30 August 2004. 6. The Wellcome Trust (2004) New report reveals open access could reduce cost of scientific publishing by up to 30 per cent. Available: http:⁄⁄www.wellcome.ac.uk/en/1/ awtprerel0404n318.html. Accessed 30 August 2004.

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Open access, freely available online

Synopses of Research Articles Which Risk Factors Matter to Whom? DOI: 10.1371/journal.pmed.0010029

There is a much-quoted saying, attributed to the epidemiologist Geoffrey Rose: “A large number of people exposed to a small risk may generate many more cases than a small number exposed to a very high risk.” This is true for many individual risk factors such as salt intake (linked to high blood pressure and cardiovascular disease) and speeding on the highway (linked to injuries and accidents). Does it apply to many other global health risks? The study by Anthony Rodgers and colleagues suggests that it does. To develop effective health policies, one must understand the existing health risks and disease burdens. On a worldwide scale, this is a tough challenge. The Global Burden of Disease Database, maintained by the World Health Organization (WHO), collects data from countries around the world on risk factors such as tobacco, malnutrition, childhood abuse, unsafe sex, childbirth, and cholesterol levels, as well as on disease burdens, for example depression, blindness, and diarrhea. A large group of scientists from all over the world has developed a framework to analyze these data. To compare different risks or burdens, they calculate disabilityadjusted life-years, or DALYs—the number of healthy life years lost because of a particular disease or risk factor. Rodgers and colleagues used data from the WHO database for 26 risk factors and from 14 epidemiological subregions of the world to calculate the proportion of risk-factor-attributable disease burden in different population subgroups defined by age, sex, and exposure level.

DOI: 10.1371/journal.pmed.0010029.g001

Tobacco is a major player in the global burden of disease (Photo: Bill Branson)

PLoS Medicine | www.plosmedicine.org

For being underweight in childhood, for example—the leading risk factor for global loss of healthy life—they found that only 35% of the disease burden occurred in severely underweight children, the rest occurred in those only moderately underweight. The relative risks for the moderately underweight are much lower, but the number of children in that category is so large that the total attributable burden amounted to almost two-thirds of the total global burden of disease for that risk factor. The analysis confirms—and extends to a global level—previous research showing that many major health risks are important across the range of exposure levels, not just among individuals exposed to high levels of risk. It also points to risk factors that are particularly prevalent among specific populations and age groups, and for which highly targeted interventions could be effective. Despite numerous caveats and limitations of studies like this one, such analyses are essential aids in guiding the distribution of limited funds to lower the burden of life years lost to premature death and disability.

DOI: 10.1371/journal.pmed.0010024.g001

Anopheles gambiae, the principal vector of malaria (Photo: Jim Gathany)

Acidosis is a major cause of death in patients with malaria, although what causes acidosis is still unclear. One possibility is that hypovolemia contributes to the problem, and that rehydration therapy could be of benefit. Now, Sanjeev Krishna and colleagues have shown that in children with severe malaria dehydration is not severe and is not correlated with other measures of disease severity. “The optimum resuscitation approach in severe childhood malaria remains to be defined,” says Nick White (Mahidol University, Thailand), the academic editor of the paper. “The relative advantages of blood, colloids, and crystalloids need to be characterized.”

Every year around 200 million people worldwide contract malaria, of whom over a million die. The vast majority of those who die are children under five years, mostly in Africa, since young children have had little chance to acquire any immunity. Fluid resuscitation is generally considered to be a cornerstone of treatment—but how much fluid should be given? Some researchers believe that surrogate signs of fluid depletion—such as tachycardia, reduced capillary refill time, and reduced urine excretion—suggest that there is substantial volume depletion. The reason that the amount of fluid given matters so much is that giving too much, especially of hypotonic solutions, can lead to electrolyte imbalance, especially hyponatremia and hypokalemia. Research efforts have been hampered by not having an easy way to assess in patients the fluid depletion in different compartments of the body, i.e., total body water and extracellular and intracellular water volume. Krishna and colleagues used heavy-water distribution to calculate the total body water and bromide distribution to determine the extracellular volume in 19 children with moderately severe malaria and 16 with severe malaria in Gabon. By subtracting extracellular volume from total body water, they were able to calculate intracellular volume for each child. They also used a less invasive and more rapid method of determining water volumes based on using bioelectrical impedance to calculate the volume. None of the children were severely dehydrated (defined as more than 100 ml/kg depletion), and only three of the children with severe anemia had fluid depletion, which was moderate (60–90 ml/kg depletion). “This challenges the

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Rodgers A, Ezzati M, Vander Hoorn S, Lopez AD, Lin RB, et al. (2004) Distribution of major health risks: Findings from the Global Burden of Disease study. DOI: 10.1371/journal.pmed.0010027

Getting the Fluid Balance Right in Malaria DOI: 10.1371/journal.pmed.0010024

view that dehydration is a major contributor to the pathology of this frequently lethal disease,” says White. So based on these data, obtained from a carefully studied, albeit small group of children, what should people who treat children with malaria do? The authors’ first recommendation is that clinicians should think again about how vigorously they rehydrate children, and if they have access to ways of assessing fluid volume more precisely, they should do so (not a trivial undertaking in many hospitals where these children are treated). And certainly the methods used by Krishna and colleagues should undergo wider testing in larger groups of children to confirm their usefulness. Until the worldwide efforts to prevent malaria come to fruition, refining the management of infected children will remain a cornerstone of the efforts against this devastating disease. Planche T, Onanga M, Schwenk A, Dzeing A, Borrmann S, et al. (2004) Assessment of volume depletion in children with malaria. DOI: 10.1371/journal.pmed.001001

T Cells Cause Lung Damage in Emphysema DOI: 10.1371/journal.pmed.0010025

T lymphocytes may have an important role in the pathogenesis of smoking-related emphysema, according to a new study by researchers from Houston, Texas, United States. “We now know that T cells are not only present in chronic obstructive pulmonary disease [COPD], but are harmful,” comments Steven Shapiro from Brigham and Women’s Hospital, Harvard Medical School, who was not involved in the study. “We also now have a pathway that could be interrupted to prevent lung destruction in COPD.” Farrah Kheradmand and colleagues took lung samples from 28 ex-smokers who had been admitted to hospital for lung resection: 18 patients had moderate to severe COPD as well as evidence of emphysema, and ten patients had none. The researchers isolated lung lymphocytes from the samples and used two-color flow cytometry to phenotypically characterize the cells. They found that

DOI: 10.1371/journal.pbio.0010025.g001

CT image of the lung of subjects with end-stage emphysema next to a photomicrograph of their resected lung stained with H&E

lymphocytes taken from patients with emphysema expressed more CCR5 and CXCR3 receptors, which are associated with a particular type of T cell called T helper 1 (Th1), than did those from control individuals. By contrast, expression of CCR4 receptors, which are found on T helper 2 (Th2) cells, was very low in both control and emphysema groups. In a separate experiment, Kheradmand’s team showed that lung lymphocytes taken from patients with emphysema secreted more of three other proteins—interferon gamma, monokine induced PLoS Medicine | www.plosmedicine.org

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Different HIV Drugs Cause Different Lipid Profiles DOI: 10.1371/journal.pmed.0010030

Nevirapine and efavirenz are the most commonly prescribed of the class of antiretroviral drugs called non-nucleoside reverse transcriptase inhibitors (NNRTIs). Efavirenz has the advantage of once-daily dosing. In a recent study called the 2NN study (Lancet 363: 1253–1263), it appeared to be only marginally superior to nevirapine in terms of clinical success and virological suppression. Van Leth and colleagues have now shown that while nevirapine and efavirenz both raise high-density lipoprotein (HDL) cholesterol (the “good” type of cholesterol), the overall lipid profile is better with nevirapine than with efavirenz. “These data suggest that nevirapine may be preferable to efavirenz in HIV-infected adults with other cardiovascular risk factors,” says the study’s academic editor, Andrew Carr of St. Vincent’s Hospital in Darlinghurst, Australia. “However, perceived cardiovascular risk is only one factor that would affect the choice between these two drugs.” Van Leth and colleagues prospectively analyzed the lipids of patients enrolled in the 2NN study, a randomized, open-label efficacy study that included adults with HIV who had never been on antiretroviral drugs. All patients were given stavudine and lamivudine and were then randomized into three treatment groups: nevirapine, efavirenz, or both. For the lipid analysis, which was preplanned, the researchers included only the nevirapine and efavirenz groups (417 and 289 patients, respectively). This was because the 2NN study showed that simultaneous use of nevirapine and efavirenz should be avoided—the combination is associated with increased toxicity without increased efficacy. The increase in HDL cholesterol was significantly higher with nevirapine than with efavirenz. There was a decrease in the ratio of total cholesterol to HDL cholesterol with nevirapine and an increase with efavirenz. The study does not prove, however, that the rise in HDL cholesterol seen with NNRTIs (especially nevirapine) actually leads to a reduction in coronary heart disease. “There are no vascular functional data,” says Carr, “or clinical vascular endpoint data that confirm that the statistically significant lipid differences observed are clinically significant.” The study was funded by Boehringer Ingelheim, the manufacturer of nevirapine. The authors clearly state that the company had “a nonbinding input on issues of study design and analyses” but it had “no influence on reporting of the data or the decision to publish.” Despite its limitations, van Leth and colleagues’ study “moves clinicians and patients away from ‘one-size-fitsall’ antiretroviral therapy,” says Carr. “It takes us further along the path of choice of antiretroviral therapy being individualized according to other patient co-morbidities and risk factors, as well as therapy simplicity and side effects.” van Leth F, Phanuphak P, Stroes E, Gazzard B, Cahn P, et al. (2004) Nevirapine and efavirenz elicit different changes in lipid profiles in antiretroviral-therapy-naive patients infected with HIV-1. DOI: 10.1371/journal.pmed.0010019

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by interferon (MIG), and interferon-inducible protein 10 (IP-10)—than control patients. MIG and IP-10 are known to be produced by injured epithelial cells and are ligands for CXCR3 receptors, which are expressed by Th1 cells. Importantly, the researchers were also able to show that isolated peripheral lung macrophages secreted matrix metalloproteinase-12 (MMP12), an enzyme that degrades elastin—a protein important for lung elasticity—in the lungs, in response to IP-10 and MIG. Together these findings, say the authors, indicate that Th1 cells, but not Th2 cells, are required for producing the elastin-destroying lung environment of emphysema. The researchers now intend to investigate the antigens that drive the Th1-based inflammation that underlies emphysema. “Ultimately, we seek to understand the biochemistry of tobacco smoke that triggers inflammation in the first place, and whether such insight might explain other environmentally triggered lung diseases,” explains Kheradmand. “To understand such detailed immune mechanisms, we really need an improved experimental model of disease, and this we are currently working on.” Grumelli S, Corry DB, Song LZ, Song L, Green L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. DOI: 10.1371/journal.pmed.0010008

Oats Intolerance in Celiac Disease DOI: 10.1371/journal.pmed.0010023

Most patients with celiac disease can eliminate their symptoms—at a price: life-long adherence to a gluten-free diet. This means no wheat, rye, barley, and, until recently, no oats. Then some recent studies suggested that oats did not cause the intestinal inflammation characteristic of the disease, and thus oats are now often included in the celiac disease diet. This is good news for patients coping with severe restrictions on what they can and must not eat, but a study by Ludvig Sollid and colleagues in this issue of PLoS

DOI: 10.1371/journal.pbio.0010023.g001

The celiac diet excludes many cereal products (Photo: National Cancer Institute)

PLoS Medicine | www.plosmedicine.org

Ludvig Sollid and colleagues applied the current understanding of celiac disease and a range of molecular pathology tools to studying the response to oats of nine patients with celiac disease. The nine patients were not a random sample: all of them had been eating oats, and four of them had shown clinical symptoms after oats ingestion. The goal of the study was to characterize the intestinal T cell response to oats in these patients, and to relate it to clinical symptoms and intestinal biopsy results. All patients were on a glutenfree diet and ate oats that were free of contamination by other cereals. Three of the four patients who had reported problems after eating oats showed intestinal inflammation typical of celiac disease, and Sollid and colleagues studied intestinal T cells from these three patients. Two of the five patients who seemed to tolerate oats also had oatsreactive intestinal T cells. Functional study of these T cells showed that they were restricted to celiac-disease-associated HLA molecules and that they recognized two peptides derived from oat avenin that are very similar to peptides of gluten. Taken together, the findings show that intolerance to oats exists at least in some patients with celiac disease, and that those patients have the same molecular reaction to oats that other patients have to wheat, barley, or rye. However, identical reactions were also seen in two of the patients who were clinically tolerant to oats. The authors suggest that these reactions could develop into symptomatic disease after some time delay, but there is no proof that the presence of oats-reactive T cells is an indicator of future symptoms or even of enhanced susceptibility to clinical oats intolerance. Oats are not safe for all patients with celiac disease, but future studies are needed to determine the frequency of oats intolerance.

Medicine suggests that oats are not safe in all cases. Like other chronic inflammatory diseases, celiac disease is caused by a complex interplay between genetic and environmental factors, but it is better understood than most. Long believed to be a relatively rare disorder, it is now thought to affect about one in 250 people worldwide. Clinical symptoms are present in less than half of patients and vary considerably. Genetically, almost all patients have one of two predisposing HLA molecules, which determine the context in which their immune system encounters foreign antigens, including gluten proteins found in wheat and other cereals. In individuals with celiac disease, the immune system mounts an abnormal response to gluten, which is characterized by glutenreactive intestinal T cells and by inflammation and compromised function of the small intestine.

Arentz-Hansen H, Fleckenstein B, Molberg Ø, Scott H, Koning F, et al. (2004) The molecular basis for oat intolerance in celiac disease patients. DOI: 10.1371/journal. pmed.0010001

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Open access, freely available online

The PLoS Medicine Debate

Should Health Professionals Screen All Women for Domestic Violence? Ann Taket, C. Nadine Wathen, Harriet MacMillan

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ackground to the debate: The US and Canadian task forces on preventive health recently declared that there is not enough evidence to recommend for or against routine universal screening of women for domestic violence. Yet some experts argue that routine enquiry is justified.

Ann Taket’s Viewpoint: Routinely Asking about Domestic Violence Is Worthwhile Domestic violence is a misunderstood topic. The context of a trusted health professional talking to a woman is one that provides an important opportunity for providing information to counter misconceptions. I deliberately talk about this in terms of asking all women about domestic violence and not in terms of screening women for domestic violence. It is not appropriate or helpful to regard enquiry about being abused as a form of screening. Domestic violence is not a disease present in the body of the person who experiences it—rather it is a health-related risk factor. As such, knowledge of abuse puts health professionals in a position to respond better to the needs of women affected by it. Professionals can respond by providing information on specialist services—usually provided outside the health service—that women may access if they wish. By giving information to affected women, health professionals can also help to reduce women’s sense of isolation and stigmatisation. Asking about experience of domestic violence can be seen as a routine part of history taking, just as health professionals regularly and repeatedly ask patients about their smoking behaviour, alcohol use, weight, and exercise. The prevalence of domestic violence among women is such that, even if it is not a personal issue for the woman concerned, it most likely will be for one or more of her relatives, friends, and neighbours [1]. Since many women experiencing abuse feel alone and ashamed, and their abusers often encourage them to believe that the abuse is their fault, presenting information to counter women’s negative feelings is an important preventive strategy. Most women experiencing domestic violence report that the specialised services that exist to respond to their needs were difficult to find out about [2]. The provision of simple information on the existence of specialised services and how to contact them is relevant to all women. Studies have examined women’s views on being asked about domestic violence. These studies have shown that once they have experienced being asked, they are usually in favour of being asked. This is true both for those who have

DOI: 10.1371/journal.pmed.0010004.g001

Women experiencing violence often feel alone and ashamed

(Illustration: Margaret Shear, Public Library of Science)

experienced or are experiencing abuse, and those who have not [3]. It is only a small minority of women who object to being asked, or who find the question uncomfortable. Women who have experienced abuse particularly value being asked directly. Asking about abuse should be done in a flexible fashion—the particular questions used should respond to the circumstances of the consultation. For example, it is appropriate to ask women about domestic violence as part of a health check in a Well Woman Clinic, but it would be completely inappropriate in a consultation where another adult or a child was present. By being flexible, health professionals can integrate their questioning within a variety of different encounters. Integrating questions about abuse into routine encounters provides for the maintenance of confidentiality and safety. In order to do this, health professionals require training on raising the issue and knowledge about local advice and support services.

The PLoS Medicine Debate discusses important but controversial issues in clinical practice, public health policy, or health in general.

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Certainly all women who disclose that they have been exposed to violence should be provided with options regarding seeking help [13]. Good diagnostic assessment requires that clinicians be able to identify and respond to signs and symptoms of abuse, from patterns of physical injury to mental health concerns, including unexplained pain and depression. Not asking women about exposure to violence during certain diagnostic assessments (such as investigation of chronic pain) may lead to misdiagnosis and a path of inappropriate investigations or treatments that will miss the underlying problem [14]. It is also imperative that clinicians know about the hospital- or community-based services that exist and ensure that there is a system in place to provide appropriate referral [15]. However, what about women presenting without obvious signs and symptoms of domestic violence—such as a woman who comes to the clinic for assessment of an upper respiratory tract infection? Should such women be prompted to disclose whether they are being abused? The woman who is not being abused will answer to that effect, and the appointment can carry on. But for the woman who is experiencing violence, who has not volunteered this information, several factors must be considered. An important issue is whether she is ready—both psychologically and in terms of taking specific actions—to confront the issue. A number of excellent qualitative studies have examined the process that women undertake in acknowledging that they are “victims” of “abuse” and embarking on the often long and difficult journey to avoid, reduce, and ultimately stop the violence in their lives [16,17]. Given the enormousness of that task, the key question becomes the extent to which prompting disclosures of abuse through universal screening will actually help women in this process, and help them in a way that they find meaningful. Any potential benefits of screening must then be weighed against its potential harms, including labelling women, prompting potentially premature disclosure, and triggering possible reprisal violence from the abuser if he discovers she has sought help. The last of these might be particularly exacerbated for the woman with the respiratory tract infection who was unprepared to disclose and did not take necessary precautions. Other potential harms include exposure to the ramifications of laws on mandatory child

Committees on both sides of the Atlantic have rejected the notion of screening women for domestic violence, arguing that there is insufficient evidence of the effectiveness of interventions [4,5]. Part of the reason for this lack of evidence is that the systematic reviews on which these committees based their recommendations often excluded the most important types of evidence that do exist [3,6]. For example, these reviews excluded studies done outside the health service setting—they excluded those based in social services, or in the voluntary or community sector. Some excluded studies show the effectiveness of specialised service provision for women experiencing abuse. In one example of an excluded study, researchers used a randomised design to evaluate an advocacy service for women experiencing domestic violence [7,8]. Women were interviewed six times over two years, and women in the intervention group reported a higher quality of life, decreased difficulty in obtaining community resources, and less violence over time than women in the control group. Other studies showing the value of specialised support services provided outside of the health system provide evidence of the potential benefits of asking about abuse [2]. Systematic reviews have also excluded, or devalued, evidence from qualitative studies. For example, a study of 200 women who had used domestic violence outreach services found that about half were living in situations of domestic violence when they first contacted the service. All of these women reported that the outreach services had helped them to leave the abusive relationship—a valued outcome for them [9]. Given the health impacts on women who experience domestic violence (not to mention their children) and the prevalence of the problem, routinely asking women about abuse should be seen as an important form of primary and secondary prevention for a wide range of health problems.

Nadine Wathen and Harriet MacMillan’s Viewpoint: The Decision to Screen Should Be Based on Evidence Screening tools for domestic violence are abundant, and many are effective at identifying women experiencing abuse [3,10]. However, merely identifying a woman as abused has not been shown to actually improve her quality of life or reduce the violence she is experiencing [6,11]. Furthermore, with one exception [7], we do not know whether interventions for women exposed to violence are effective in reducing violence or improving other healthrelated outcomes. Interventions for abusive men have shown little effectiveness [11,12]. Given the morbidity and mortality associated with domestic violence, it is tempting to suggest that universal screening for abuse should be integrated into routine clinical care, such that all women, regardless of their reason for presenting to a clinical setting, should be “asked the question.” Some argue that this approach is justified by the need to increase awareness of domestic violence as a significant problem with serious health and social consequences, and to make abused women aware that they are not alone in their experience. These are important considerations.

PLoS Medicine | www.plosmedicine.org

DOI: 10.1371/journal.pmed.0010004.g002

There are potential harms from “asking the question”

(Illustration: Margaret Shear, Public Library of Science)

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protection reporting, whereby health providers must report such disclosures to child protection authorities. This can lead to an investigation that potentially increases a woman’s risk of exposure to violence, and in some cases of having her children placed in foster care. Research has shown that many of these potential harms are of concern to women when mandatory universal screening and/or reporting protocols are in place [18]. Finally, from a health system perspective, the opportunity cost of not having used this time with the woman to conduct screening or prevention activities for which there is proven benefit, such as counselling about Pap smears or mammograms, should not be discounted. Given the lack of clear data on the benefits of screening and of the interventions to which women are referred, and the lack of data on potential harms, we and others have concluded the following [3, 19, 20]. Until these questions are answered, the most appropriate health care system approach is the more targeted case-finding or diagnostic method, which focuses health care resources on those in immediate need of care. Our hope is that studies currently underway (for example, those supported by the Ontario Women’s Health Council and the US Centers for Disease Control) will provide information about the effectiveness of domestic violence screening. Let’s base the decision about implementation of screening on evaluations of whether such screening does more good than harm in the lives of women.

requirements, such as when child protection issues are involved. Training and protocols also need to emphasise that the role of routine enquiry is to facilitate, and not force, disclosure. It must remain the woman’s choice as to if, when, and to whom, she discloses.

Wathen and MacMillan’s Response to Taket’s Viewpoint We agree with Ann Taket that domestic violence is not a disease, and that the paradigm of “screening for disease” is problematic in this context. At issue, however, is the question of whether domestic violence should be “talked about” with all women or only in situations where asking about it is part of a specific diagnostic assessment. As with screening for a disease, universal screening for domestic violence should not be implemented unless we are sure that interventions are available to help those identified via screening and that screening plus appropriate treatment will do more good than harm. Professor Taket outlines the importance of integrating discussions about abuse in consultations to raise community awareness. Unfortunately, there is no evidence that this type of consciousness-raising occurs, or if it does, what benefit it might have. Given the lack of effectiveness of educational campaigns in general, it is difficult to be optimistic about this approach. We disagree with her conclusion that existing systematic reviews have “excluded studies done outside the health service setting….” Our review included interventions such as the post-shelter advocacy counselling approach to which Professor Taket refers [11]. This intervention has been recommended by the Canadian Task Force on Preventive Health Care as one to which, where available, clinicians might refer women in these circumstances [19]. However, since shelters themselves have not been adequately evaluated, the value of linking screening to a post-shelter intervention is unclear. Finally, we concur that qualitative studies are invaluable in understanding domestic violence. Such research has provided insight into the complex process that women undertake to address the violence in their lives. Until there is evidence that universal screening actually helps with this process, the focus should be on developing evidence-based approaches to assist women when they do disclose abuse and on training health professionals to respond appropriately to such disclosures.

Taket’s Response to Wathen and MacMillan’s Viewpoint I agree entirely with Nadine Wathen and Harriet MacMillan that practice should be based on evidence. There are further areas of agreement. We agree that there is a lack of knowledge on effective interventions for abusers and on harm occurring as a result of enquiry, and that targeted case finding is important. The key difference that exists between my viewpoint and theirs is the conclusion about whether health professionals should aim to ask all women about domestic violence. Underlying this difference is the issue about how much evidence we need, and of what type. My position is that the evidence that already exists is sufficient to justify the promotion of routine enquiry, aiming to ask all women about their experience of abuse. There is evidence of actual benefits to women—and their children—from interventions provided by specialised services for domestic violence and from brief discussions with health professionals [21]. Aiming to ask all women has several advantages over targeted case finding [22]. It contributes to changing social attitudes to domestic abuse, it is less likely to make women experiencing abuse feel stigmatised, and it is less likely to compromise the safety of women experiencing abuse. Furthermore, health professionals report that their perceptions about which women are being abused, and which are not, are often wrong. The twin issues of women’s safety and harm minimisation are extremely important, for both routine enquiry and targeted case finding. These issues are important reasons why training and protocols for enquiry are necessary. Standard principles of confidentiality should be reinforced in training and protocols, which need to be tailored to relevant legal

PLoS Medicine | www.plosmedicine.org

References

1. Krug G, Dahlberg L, Mercy J, Zwi A, Lozano Generve R, editors (2002) World report on violence and health. Geneva: World Health Organization. Available: http:⁄⁄www.who.int/violence_injury_prevention/violence/ world_report/en/. Accessed 23 July 2004. 2. Taket A, Nurse J, Smith K, Watson J, Shakespeare J, et al. (2003) Routinely asking women about domestic violence in health settings. BMJ 327: 673– 676. 3. Ramsay J, Richardson J, Carter YH, Davidson L, Feder G (2002) Should health professionals screen women for domestic violence? Systematic review. BMJ 325: 314. 4. UK National Screening Committee (2004) Domestic Violence National Screening Committee Policy Position—March 2004. Available: http:⁄⁄www. nelh.nhs.uk/screening/adult_pps/domestic_violence.html. Accessed 9 August 2004. 5. US Preventive Services Task Force (2004) Screening for family and intimate partner violence: Summary of recommendations. Available: http:⁄⁄www. ahrq.gov/clinic/uspstf/uspsfamv.htm. Accessed 23 July 2004.

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Open access, freely available online 6. Nelson HD, Nygren P, McInerney Y, Klein J (2004) Screening women and elderly adults for family and intimate partner violence: A review of the evidence for the U.S. Preventive Services Task Force. Ann Intern Med 140: 387–396. 7. Sullivan CM, Bybee DI (1999) Reducing violence using community-based advocacy for women with abusive partners. J Consult Clin Psychol 67: 43–53. 8. Sullivan C (2000) The community advocacy project: A model for effectively advocating for women with abusive partners. In: Vincent JP, Jouriles EN, editors. Domestic violence: Guidelines for research-informed practice. London: Jessica Kingsley Publishers. pp. 126–143. 9. Humphreys C, Thiara R (2002) Routes to safety: Protection issues facing abused women and children and the role of outreach services. Bristol: Women’s Aid Federation of England. 133 p. 10. MacMillan HL, Wathen CN, Canadian Task Force on Preventive Health Care (2001) Prevention and treatment of violence against women: Systematic review and recommendations. London (Ontario): Canadian Task Force on Preventive Health Care. Available: http:⁄⁄www.ctfphc.org/ Full_Text/CTF_DV_TR_final.pdf. Accessed 23 July 2004. 11. Wathen CN, MacMillan HL (2003) Interventions for violence against women: Scientific review. JAMA 289: 589–600. 12. Babcock JC, Green CE, Robie C (2004) Does batterers’ treatment work? A meta-analytic review of domestic violence treatment. Clin Psychol Rev 23: 1023–1053. 13. Dienemann J, Campbell J, Wiederhorn N, Laughon K, Jordan E (2003) A critical pathway for intimate partner violence across the continuum of care. J Obstet Gynecol Neonatal Nurs 32: 594–603. 14. Cole TB (2000) Is domestic violence screening helpful? JAMA 284: 551–553. 15. Leibschutz JM, Frayne SM, Saxe GN (2003) Violence against women: A physician’s guide to identification and management. Philadelphia: American College of Physicians. 342 p. 16. Landenburger KA (1989) Process of entrapment in and recovery from an abusive relationship. Issues Ment Health Nurs 10: 209–227. 17. Campbell J, Soeken K (1999) Women’s responses to battering over time: An analysis of change. J Interpers Viol 14: 21–40. 18. Gielen AC, O’Campo PJ, Campbell JC, Schollenberger J, Woods AB, et al. (2000) Women’s opinions about domestic violence screening and mandatory reporting. Am J Prev Med 19: 279–285. 19. Wathen CN, MacMillan HL (2003) Prevention of violence against women: Recommendation statement from the Canadian Task Force on Preventive Health Care. CMAJ 169: 582–584.

20. U.S. Preventive Services Task Force (2004) Screening for family and intimate partner violence: Recommendation Statement. Ann Intern Med 140: 382–386. 21. Rhodes KV, Levinson W (2003) Interventions for intimate partner violence against women. JAMA 289: 601–605. 22. Taket AR, Beringer A, Irvine A, Garfield S (2003) Evaluation of the CRP Violence Against Women Initiative, Health Projects: Final report— Exploring the health service contribution to tackling domestic violence. End of study report to the Home Office. London: South Bank University. 137 p.

Citation: Taket A, Wathen CN, MacMillan H (2004) Should health professionals screen all women for domestic violence? PLoS Med 1(1): e4. Copyright: © 2004 Taket et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ann Taket is a professor of primary health care at London South Bank University, London, England. E-mail: [email protected] Nadine Wathen is a research fellow in the Department of Psychiatry and Behavioural Neurosciences at McMaster University, Hamilton, Canada. E-mail: [email protected] Harriet MacMillan is a professor in the Department of Psychiatry and Behavioural Neurosciences and Department of Pediatrics at McMaster University, Hamilton, Canada. E-mail: [email protected] Competing Interests: Ann Taket declares that she has no competing interests. Nadine Wathen holds a Canadian Institutes of Health Research (CIHR)–Ontario Women’s Health Council Fellowship. Harriet MacMillan holds research funding from the CIHR Institutes of Gender and Health; Aging; Human Development, Child and Youth Health; Neuroscience, Mental Health, and Addiction; and Population and Public Health, and from the Ontario Women’s Health Council. DOI: 10.1371/journal.pmed.0010004

Essay

The Birth of Reproductive Health: A Difficult Delivery In 1994, the landmark Cairo Conference promised reproductive health and rights for all. Ten years later, what has been achieved? Shereen El Feki

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bout a decade ago, I went wandering around Cairo’s City of the Dead. This might sound like a grim bit of tourism, but my connection to that vast necropolis runs deep—quite literally, as my family is buried there. After visiting their grave, I rambled through the city’s dusty alleyways, past hundreds of years of history. Yet what I remember most about that day was not one of the many magnificent tombs, but a simple brick building with a sign, of all things, for a family planning clinic.

The Essay section contains opinion pieces on topics of broad interest to a general medical audience.

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I was certainly surprised by my discovery; in retrospect, I should not have been. That part of Cairo is home to hundreds of thousands of people for whom looking after the dead is a way of life. Their fertility invigorates the funereal air: the caretaker of my family’s tomb, for example, had a blooming family of his own living near the grave. Where better to offer family planning than in a place so poor that reproduction seemed more a matter of fate than choice?

The Cairo Conference That visit is a fitting metaphor for the field of reproductive health as a whole. Ten years ago, officials, experts, 010

Citation: El Feki (2004) The birth of reproductive health: A difficult delivery. PLoS Med 1(1): e9. Copyright: © 2004 Shereen El Feki. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: ICPD, International Conference on Population and Development; IPPF, International Planned Parenthood Federation; UNFPA, United Nations Population Fund Shereen El Feki is the healthcare correspondent at The Economist magazine, London, United Kingdom. E-mail: [email protected] Competing Interests: The author declares that she has no competing interests. DOI: 10.1371/journal.pmed.0010009

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and activists from 179 countries also came to Cairo for the International Conference on Population and Development (ICPD). The conference produced a 20-year plan of action that focused on universal access to reproductive health services, including family planning and sexual health; reducing infant, child, and maternal mortality; better education, especially for girls; equality between men and women; and sustainable development. The ICPD’s key achievement was to reorient thinking on reproduction away from narrowly defined, government-dictated population control to a broader appreciation of reproductive and sexual well-being within health care systems, a view driven by individual choice and rights, not official priorities. “The Cairo Conference was a peak moment,” says Sally Ethelston, vice president for communications at Population Action International, one member of a consortium of non-governmental organisations launching a report card to mark the anniversary of the Cairo Conference in early September. “There were times when people were excited that they had accomplished something, and you could see it on their faces.” Today, however, the mood is very different. While progress has been made on some of the plan’s targets, effort has faltered on others. And the conference “camaraderie” that Ethelston describes has given way to conflict between faith and science, over abortion and condoms. Like signs of life in the City of the Dead, the Cairo Conference gave birth to great expectations, some of which have already expired.

Baby Steps Towards Cairo’s Goals So, how far has the developing world come towards meeting the ICPD goals? There has certainly been progress on institutional reform in some countries, according to a recent survey of national policies by the United Nations Population Fund (UNFPA) [1]. For example, more than a third of the 151 countries questioned have introduced legislation on reproductive rights, and almost half have expanded their primary health care services to include family planning. But translating policy into action has been difficult. Overall, the picture

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sub-Saharan Africa, where, on average, 920 women die for every 100,000 live births, compared with 24 deaths per 100,000 live births in Europe [7]. This is all the more distressing, says Vivien Tsu, senior programme officer at the Program for Appropriate Technology in Health, because these women’s lives could be saved through straightforward measures and basic technologies, such as access to skilled midwives, simple drugs like magnesium sulphate for eclampsia and oxytocin for post-partum bleeding, cellular phones to call for help, and transportation to emergency obstetric centres.

Obstacles to Reproductive Health

DOI: 10.1371/journal.pmed.0010009.g001

The birth of a baby on August 15, 2000, brought India’s population to one billion

(Photo: Raghu Rai, on behalf of the David and Lucile Packard Foundation)

is one of patchy success, according to Susheela Singh, director of research at The Alan Guttmacher Institute, a nongovernmental research organisation. Official statistics, as limited as they are for many aspects of reproductive and sexual health, show mixed results. On a positive note, global population growth has slowed to roughly 77 million people a year [2]. But while fertility rates have fallen in some developing countries, such as Mexico, they remain stubbornly high in others, such as Ethiopia [3]. Over the past decade, contraceptive use has grown, but so has demand, and there are now an estimated 201 million women in developing countries whose need for modern birth control goes unmet, resulting in 60 million unintended pregnancies a year [4]. Progress on legalising abortion has been slow, and an estimated 19 million abortions a year still occur under unsafe conditions [5]. Despite growing awareness of sexually transmitted disease, the annual number of sexually transmitted infections remains worryingly high at 340 million worldwide [6]. While infant mortality rates have improved somewhat, maternal mortality figures have barely budged. An estimated 529,000 women still die every year from complications of pregnancy and childbirth. The highest rates are in

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So why hasn’t more been achieved? One problem is certainly money. The 1994 Cairo Conference estimated the cost of implementing programmes for family planning, maternal health, and prevention of sexually transmitted diseases, as well as data collection and analysis in developing countries, at $18.5 billion by 2005—or $24.3 billion in today’s dollars. The goal was to mobilise one-third of the money from donor nations, and the rest from developing countries themselves [8]. Last year, global spending on reproductive health and services reached $14.7 billion, according to estimates from UNFPA, the Joint United Nations Programme on HIV/AIDS, and the Netherlands Interdisciplinary Demographic Institute [8]. Encouragingly, investment has increased since 2001, when the momentum of ICPD seemed to falter and international spending fell to $9 billion. But this is still wide of the mark. While developing countries have failed to meet their conference commitments, it is donor countries that are most remiss: rich country contributions reached an estimated $2.3 billion in 2003 [8], a far cry from the conference target of $6.1 billion (or $8.1 billion in today’s dollars) by 2005. Reproductive health is not alone in waiting for donors to give generously. For all the rhetoric at international summits, few rich countries have lived up to their lofty pledges of debt relief and of dedicating 0.7% of their gross domestic product to overseas development assistance. But as Steve Sinding, head of the International Planned Parenthood Federation (IPPF), points out, there are other reasons

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system, it seems odd not to integrate too for the shortfall. In the past donor such services into the wider battle interest was largely stimulated by fears against HIV. Such centres can offer of a population crisis. When the Cairo not only HIV testing and counselling, Conference reframed issues in terms as well as condoms (against the double of women’s health and reproductive whammy of unwanted pregnancy and rights, rather than an impending HIV infection), but also a broad-based population explosion, Sinding argues, message of good sexual health that can the “demographic rationale” was lost, help protect against HIV and other taking funding with it. sexually transmitted diseases. Moreover, Moreover, there are other issues pre- and ante-natal care provide an competing for international funding, opportunity to stop mother-to-child most notably AIDS. At the time of the transmission of HIV in its tracks. Cairo Conference, 20 million people Where once family planning was the were infected with HIV; today the darling of international donors, HIV is number has grown to an estimated 38 now the cause célèbre. “There’s a lot of million [9]. AIDS threatens to derail the Cairo Conference plan of action. Through maternal-tochild transmission, and widescale orphaning, HIV threatens to reverse small successes at reducing infant and child mortality. By killing off teachers and sapping household incomes, AIDS is sabotaging education. By killing off scarce medical workers and overwhelming fragile health care systems, the disease is compromising reproductive health services. Gender equity is undermined, as women and girls DOI: 10.1371/journal.pmed.0010009.g002 bear the brunt of the epidemic, Condom distribution in Soweto, South Africa as caregivers, breadwinners, or (Photo: Arjen van de Merwe, Population Concern) patients themselves. Roughly half of the money resentment about the spotlight moving spent on reproductive health last year on,” says Kevin O’Reilly, a former went towards HIV/AIDS. And billions reproductive health specialist now at more is on the way, from the likes the department of HIV/AIDS at the of the Global Fund to Fight AIDS, World Health Organization. However, Tuberculosis, and Malaria and the there are now attempts to bring the United States President’s Emergency two together. Meetings earlier this Plan for AIDS Relief, which promises year in Switzerland, New York, and $15 billion over five years to HIV/AIDS Bangkok have led to calls to action to programmes [10]. But much of this strengthen links between programmes money is going into AIDS-specific addressing HIV/AIDS and sexual and programmes that do not address reproductive health. While this should reproductive health more broadly. help in the battle against AIDS, the Even as the world is gearing up to scale money which flows to AIDS should also up AIDS prevention and treatment to benefit reproductive health. millions worldwide, few of the agencies involved come from the world of Ideological Battles reproductive and sexual health. Arguably the most formidable obstacle This is a pity because it means to that union, and indeed further that HIV/AIDS programmes are not progress in improving reproductive making use of valuable infrastructure health, is ideology. Since the Cairo and expertise already on the ground Conference, a fierce battle has emerged in places where AIDS hits hardest. between religious conservatives who Given that 57% of HIV infections in eschew abortion and condoms in sub-Saharan Africa are among women favour of abstinence and fidelity, [9], and that, for many of them, and more liberal voices who argue family planning clinics are their sole for a full armamentarium to tackle contact with the formal health care

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these problems. The clash is loudest in the field of HIV/AIDS, where the President’s Emergency Plan for AIDS Relief allocates a third of its funding for disease prevention to programmes focusing on abstinence and fidelity; public health experts argue that such an approach is ineffective at best, and dangerous at worst, without an equal emphasis on the availability of condoms for all. But the clash resounds in the wider arena of reproductive health as well. Four years ago, the ICPD’s central target—access to reproductive services for all by 2015—failed to make it into the Millennium Development Goals, largely because of political nervousness. But as Kofi Annan, United Nations secretary-general, has pointed out, progress on the other key targets, such as eradication of poverty and hunger, will not be achieved without a focus on women’s rights, education, reproductive health, and family planning. The fight between conservatives and liberals is clearest in the case of the US, which is the world’s leading bilateral donor on reproductive health, spending $429 million this year [11]. However, this money comes with strings attached, says Françoise Girard, a reproductive rights lawyer in New York. Some of these are subtle. For example, Girard points to American pressure on several Asian and Latin American governments—during recent regional meetings to mark the anniversary of the Cairo Conference—not to re-affirm their commitment to the ICPD plan of action, with its emphasis on a full suite of reproductive rights and services. Other strings are more obvious. In 2001, George W. Bush reinstated the Mexico City Policy, otherwise known as the “Global Gag Rule”, which denies US family planning assistance— including money and contraceptive supplies—to any non-American group unless it certifies that it neither performs nor endorses abortion. IPPF, Marie Stopes International, and their local affiliates have been hard hit by the Rule, scaling back services in Kenya, Ghana, and elsewhere that offered essential health care to thousands of women and children.

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Then there is the Kemp-Kasten Amendment, a piece of US legislation which prohibits US assistance to any organisation as deemed by the President that “supports or participates in the management of a program of coercive abortion or involuntary sterilization.” At the behest of conservative supporters, President Bush has used the amendment to withhold $34 million in annual congressional appropriations to the UNFPA for the past three years. The UNFPA says that the $34 million could have been used to prevent 2 million unintended pregnancies, 800,000 induced abortions, 4,700 maternal deaths, and 77,000 infant and child deaths. The White House accuses UNFPA of abetting coercive reproductive practices in China—a claim which the UNFPA strenuously denies. Moreover, a number of international delegations, including one from the US State Department in 2002, have investigated the UNFPA’s activities in China and failed to find evidence to support such allegations. Fortunately, other donors are stepping in to fill the breach: earlier this year, for example, the United Kingdom announced it would raise its contribution to the UNFPA to £80 million over the next four years, as well as increase its support to IPPF by a third. But even if the shortfall is made up, the ill will such clashes have engendered cannot be so easily salved.

They are making their sexual debut at ever earlier ages, against a backdrop of rising sexually transmitted diseases and growing social conservatism, which makes clear information, frank discussion, and free choice on abortion, contraception, and sexual health extremely difficult. More than ever, reproductive health needs strong leaders in rich and poor countries alike to mobilise both money and political commitment. Reproduction is a sexy subject; it is time the world again paid it the attention it deserves.


Useful Links The Cairo Conference: http:⁄⁄www.iisd. ca/cairo.html Population Action International: www.popact.org UNFPA: www.unfpa.org Program for Appropriate Technology in Health: www.path.org The Alan Guttmacher Institute: www.guttmacher.org The Joint United Nations Programme on AIDS: www.unaids.org Netherlands Interdisciplinary Demographic Institute: www.nidi.nl IPPF: www.ippf.org Global Fund to Fight AIDS, Tuberculosis, and Malaria: www.theglobalfund.org The World Health Organization HIV/ AIDS Programme: www.who.int/hiv/en

A Call for Strong Leadership Getting it right on reproductive health cannot wait another decade. The largest generation of young people in history—a whopping 1.2 billion aged 10–19 years—is entering adulthood [1].

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References

1. International Conference on Population and Development (2004) Investing in people: National progress in implementing the ICPD programme of action 1994–2004. Available: http:⁄⁄www.unfpa.org/upload/lib_pub_

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file/284_filename_globalsurvey.pdf. Accessed 3 August 2004. 2. United Nations Population Division (2002) World Population Prospects: The 2002 Revision. Available: http:⁄⁄www.un.org/esa/ population/publications/wpp2002/WPP2002HIGHLIGHTSrev1.PDF. Accessed 9 August 2004. 3. United Nations Population Division (2003) World fertility report 2003. Available: http:⁄⁄www.un.org/esa/population/ publications/worldfertility/World_Fertility_ Report.htm. Accessed 3 August 2004. 4. The Alan Guttmacher Institute and UNFPA (2004) Adding it up: The benefits of investing in sexual and reproductive health care. Available: http:⁄⁄www.unfpa.org/upload/ lib_pub_file/240_filename_addingitup.pdf. Accessed 3 August 2004. 5. World Health Organization (2004) Prevention of unsafe abortion. Available: http:⁄⁄www.who. int/reproductive-health/unsafe_abortion/ index.html. Accessed 3 August 2004. 6. United Nations Commission on Population and Development (2004) Review and appraisal of the progress made in achieving the goals and objectives of the Programme of Action of the International Conference on Population and Development, January 2004 (E/CN.9/2004/3). Available:http:⁄⁄ods-dds-ny.un.org/doc/ UNDOC/GEN/N04/206/70/PDF/N0420670. pdf?OpenElement. Accessed 3 August 2004. 7. World Health Organization, United Nations Children’s Fund, and UNFPA (2003) Maternal mortality in 2000: Estimates developed by WHO, UNICEF, and UNFPA. Geneva: World Health Organization. Available: http:⁄⁄www.who.int/reproductivehealth/publications/maternal_mortality_ 2000/index.html. Accessed 3 August 2004. 8. United Nations Commission on Population and Development (2004) The flow of financial resources for assisting in the implementation of the Programme of Action of the International Conference on Population and Development: A 10-year review (E/CN.9/2004/4). Available: http:⁄⁄ods-dds-ny.un.org/doc/UNDOC/ GEN/N04/206/10/PDF/N0420610. pdf?OpenElement. Accessed 3 August 2004. 9. Joint United Nations Programme on HIV/ AIDS (2004) Report on the global AIDS epidemic. Available: http:⁄⁄www.unaids.org/ bangkok2004/report.html. Accessed 3 August 2004. 10. Gerberding G (2004) Steps on the critical path: Arresting HIV/AIDS in developing countries. PLoS Med. In press. 11. Population Action International (2004) Trends in U.S. population assistance. Available: http:⁄⁄www.populationaction.org/resources/ data_and_graphs/USPopulationAssistance. htm. Accessed 3 August 2004.

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Open access, freely available online

Essay

Steps on the Critical Path: Arresting HIV/AIDS in Developing Countries Julie Gerberding

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s an intern, I took care of the first patients with HIV/AIDS at San Francisco General Hospital, and so I grew up with AIDS in the early days of my medical career. We struggled through the confusion about what was making people so sick, and each new day brought a new discovery about the disease and its consequences. I went through that evolutionary process along with everybody else, and it shaped me in many profound ways. Before long, I recognized that this wasn’t a disease of “those people over there.” This was a disease that could strike anyone, anytime. And as physicians, we had to adjust our thinking about our own vulnerability to occupational risk, and to emphasize prevention, because there wasn’t going to be a cure for a long, long while. And not only physicians had to rethink things—AIDS has reshaped society’s very notions of the most basic human behaviors. I was in Uganda in 1985, early in the AIDS epidemic there. We knew then where that epidemic was going to go, absent an effective vaccine or cure, but few of us could have imagined that it would evolve so quickly without an end in sight. While the people of Africa have achieved a huge amount in tackling HIV/AIDS, particularly in Uganda, the epidemic is far from being under control on that continent and is spreading through other parts of the world with alarming speed.

The Crisis of Human Resources The theme of this year’s International AIDS Conference in Bangkok was “Access for All.” Over the past few years, it has become increasingly apparent that a critical component of assuring access to care and treatment is human capital. Like fiscal capital, human resources are essential to ending the AIDS pandemic. I visited Africa with US Health and Human The Essay section contains opinion pieces on topics of broad interest to a general medical audience.

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DOI: 10.1371/journal.pmed.0010010.g001

Figure 1. Estimated Percentage of Adults in Need of Antiretroviral Treatment Who Are Receiving It, as of March of 2004

(This graphic is based on an image by the World Health Organization, available at http:⁄⁄www.who.int/3by5/en/coverage_march2004.jpg)

Services Secretary Tommy Thompson and many AIDS experts last December, and we saw evidence of this critical need in every country we visited. The miracles of modern science are meaningless without systems and people to deliver them to those in need. The World Health Organization estimates that of the 40 million people worldwide infected with HIV, 6 million need immediate, life-sustaining antiretroviral therapy. Fewer than 400,000 people in developing countries have access to such treatment (Figure 1) [1,2]. There are too few skilled health care workers to provide reliable delivery and administration of these life-saving therapies. According to a recent Institute of Medicine report, and a study sponsored by the US Agency for International Development, the number of health care workers in many African countries is actually shrinking as they are lured to developed countries by better pay and professional

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opportunities (Box 1) [2,3]. Reversing this brain drain is essential over the long-term, as HIV treatment and care will be required for decades. In the short-term, the Institute of Medicine called for expanded efforts “to bring qualified volunteer initiative medical professionals into both urban and rural

Citation: Gerberding J (2004) Steps on the critical path: Arresting HIV/AIDS in developing countries. PLoS Med 1(1): e10. This is an open-access article distributed under the terms of the Creative Commons Public Domain Declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Abbreviations: CDC, Centers for Disease Control and Prevention Julie Gerberding is the director of the Centers for Disease Control and Prevention, Atlanta, Georgia, United States. E-mail: [email protected] Competing Interests: The author declares that she has no competing interests. DOI: 10.1371/journal.pmed.0010010

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Box 1. The Brain Drain: Facts and Figures [2,3] • Only 360 of the 1,200 doctors trained in Zimbabwe during the 1990s continue to practice within the country. • Two-thirds of University of Zimbabwe medical students intend to leave the country after graduating, and one of the country’s major 1,000-bed teaching hospitals lacks a single qualified pharmacist. • In Zambia, only 50 of the 600 doctors trained locally since independence have remained in the country. • In Ghana, 320 nurses are recorded to have been lost in 1999, roughly equivalent to that country’s annual output of nurses; losses for the year 2000 totaled 600. areas to support prevention, care, and training programs” [2]. I could not agree more that addressing the human resource needs will be essential as we move forward—and not just for HIV/ AIDS programs, but for all aspects of public health and health care. It has now been shown, beyond any doubt, that even in resourcepoor countries with the most basic health infrastructure, people get the same benefit from treatment and prevention interventions as those in the rich world [4]. In fact, surveys in Cape Town, Kampala, Khayelitsha, and Senegal found rates of adherence to antiretroviral therapy of 90%–94%, compared with estimates of 70% in developed countries [5,6,7].

We visited a US Centers for Disease Control and Prevention (CDC) program in the very remote areas of Uganda where there are no roads and it is impossible for people to come into population centers to receive HIV testing and other services. Young staff from the CDC are working with Ugandans and community organizations in that area to deliver antiretroviral therapy. Some may think that the difficulties of delivering antiretroviral therapy into such a remote area are overwhelming—and some may question whether this is a sustainable intervention. But once you see firsthand what miracles are possible, your world view changes almost overnight. What we saw was the success of a wonderful home-based treatment and care program for people who don’t have access through other means. And when I say “home-based care,” picture a hut without running water or electricity, where only motorcycles are available to deliver medications. The first step of the program is to provide clean water. Coliforms and other pathogens in the water supply for the household are removed through an inexpensive water vessel fitted with a filter and through a chlorination process. In addition, a cotrimoxazole tablet is given every day, which, in one patient’s words, changed his

life because he began to feel well almost immediately. Not only do the cotrimoxazole prophylaxis and the water treatment improve diarrheal illness, but malarial parasitemia also drops. So that is a very positive, unexpected consequence of just two very simple and inexpensive interventions. Many patients with HIV/ AIDS in Africa also have tuberculosis and are put on tuberculosis therapy in addition to cotrimoxazole. As a result, they begin to feel better even before they begin antiretroviral therapy. We spent time with one of the patients in the home-based care program. As she began to participate in these programs, tests became available to measure her CD4 count. She explained to me what her CD4 count was, what it meant, and how it improved when she started the cotrimoxazole and tuberculosis therapy. She had begun taking three antiretroviral drugs and held up her pill box to explain her regimen in detail. Every week a Ugandan health aide delivered her supply of pills on a CDC motorcycle and monitored her adherence to the treatment. Not only was she extremely reliable in taking her medications, but she also knew more about them and their side effects than most of the patients I treated at San Francisco General. She was also an expert in HIV prevention. I asked her,

When You Have Seen the Faces We hear the numbers—the millions upon millions infected—and we grow numb. That is why we must go to the front lines—the households and communities—and start focusing on each individual living with HIV. I was at the dedication of an AIDS clinic in Kenya. It was raining, and we were waiting outside with our umbrellas. A 12-year-old girl in front of me turned around and leaned her head against my belly and said, “Could you take me to America? I need drugs.” If you take that girl’s face and multiply it a thousand times—that is the memory I bring home from Africa: the faces of the children and their asking, “Why are so many of our parents dying? Why are we dying?”

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DOI: 10.1371/journal.pmed.0010010.g002

Behavior change club at a technical school in Entebbe, Uganda

(Photo: Arjen van de Merwe/World Population Foundation)

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“What do you do to protect your three young sons from this infection?” She replied, “Every day I take them by the hand, and I go out of the house and I say, ‘Do you see that mound of dirt? That is your father’s grave. Your father acquired this fatal infection through sex. Be careful.’” And then she talks to them about the “ABCs” (“A” for abstinence, “B” for being faithful, “C” for condoms). So when you see a story like that unfold in the middle of Africa, it’s impossible not to be hopeful. And yet, it’s also very sobering because we are reminded of our responsibility. The question is not what the international health community is accomplishing in these countries now, but what we could accomplish if we joined together to really fight this war on AIDS. Such a story also inspires hope because you can see the multiplier effect that comes from taking on one problem and can see the way that effort can expand to encompass and address a much greater set of problems.

Beyond ABCs—Diagnosis and Responsibility When we think about successful prevention models in Uganda, “ABC” certainly stands out [8]. However, at this point in the epidemic curve, other letters must also be considered. Most HIV transmission is accounted for by infected people having risky sex with uninfected people. Both in the US and in Africa, studies show that most infected people engaging in risky sex are unaware of their infection status, and that when their infection is diagnosed, they usually take steps to protect the others with whom they are having contact [9,10,11,12]. So let’s add the letter “D” for diagnosis. In fact, improving efforts to help people choose risk avoidance and to diagnose those who are already infected is the cornerstone of the CDC’s new domestic HIV prevention strategy. Diagnosis is extremely important in many African communities, especially where the number of discordant couples—where one individual is infected and the other is not—is high. Sadly, many couples “being faithful” now do not realize that one partner is already infected and are not being reached with diagnostic testing programs. So

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“ABCD” is a concept that I would like to put out on the table as food for thought. Of course, there is another letter that we need to stress: the letter “R,” for responsibility: personal sexual responsibility is a critical component of HIV prevention. Many women and girls become infected after being raped by men or because their social circumstances rob them of the power to refuse sex. Men must be held accountable for greater sexual responsibility and for ending sexual violence and degradation of women and girls. HIV prevention programs need to emphasize responsibility, but not lose sight of the fact that responsibility can be practiced only with personal autonomy, which many women and girls simply do not have.

Expanding the Team to Meet the Needs The innovative programs and ideas emerging in Africa can change the picture of the AIDS epidemic. The purchase of antiretroviral drugs for Africans is not the big challenge. Access to drugs will improve in Africa. The real challenges are delivering drugs in a safe and effective way, monitoring therapy, and sustaining the pipeline of drugs so that ongoing treatment can be guaranteed. In the example of the home-based program in Uganda, we have seen that these challenges can be overcome. Expanding access to prevention, care, and treatment services isn’t going to be easy, but it is certainly possible. It will take unprecedented commitment by people in the public sector, the private sector, faith communities, and community organizations, and perhaps most importantly, individual volunteers who make up their minds to contribute in any way they can. Last fall, the US Peace Corps announced that it was activating programs in some countries that allow volunteers to help communities fight the AIDS epidemic, but this is just one of many steps that are being taken. The US president’s Emergency Plan for AIDS Relief will provide $15 billion, including almost $10 billion in new funds, over five years for international AIDS assistance [13], and I am part of the team that is charged with making this plan

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happen. I look forward to learning from others in the global health community how we can best expand our impact and collectively find a way to support the delivery of prevention messages and life-saving medications to everyone in Africa—and especially to that little girl at the Kenyan clinic who touched my heart.
References

1. World Health Organization. Treating 3 million by 2005: Making it happen. The WHO Strategy. Geneva: World Health Organization. Available: http:⁄⁄www.who.int/3by5/publications/ documents/isbn9241591129/en/. Accessed 4 August 2004. 2. Institute of Medicine (2004) Scaling up treatment for the global AIDS pandemic: Challenges and opportunities. Washington (DC): National Academies Press. In press. Pre-publication uncorrected proofs available: http:⁄⁄books.nap.edu/books/0309092647/ html/index.html. Accessed 22 July 2004. 3. US Agency for International Development (2003) The health sector human resource crisis in Africa: An issues paper. Available: http:⁄⁄www.dec.org/pdf_docs/PNACS527.pdf. Accessed 4 August 2004. 4. Mukherjee J, Farmer PE, Niyizonkiza D, McCorkle L, Vanderwarker C, et al. (2003) Tackling HIV in resource poor countries. BMJ 327: 1104–1106. 5. Orrell C, Bangsberg DR, Badri M, Wood R (2003) Adherence is not a barrier to successful antiretroviral therapy in South Africa. AIDS 17: 1369–1375. 6. Laurent C, Diakhate N, Gueye NF, Toure MA, Sow PS, et al. (2002) The Senegalese government’s highly active antiretroviral therapy initiative: An 18-month follow-up study. AIDS 16: 1363–1370. 7. Oransky I (2003) African patients adhere well to anti-HIV regimens. Lancet 17. Available: http:⁄⁄www.impactaids.org.uk/lancet362.htm. Accessed 28 July 2004. 8. Hogle J, editor (2002) What happened in Uganda? Declining HIV prevalence, behavior change, and the national response. Washington (DC): US Agency for International Development. Available: www.usaid.gov/pop_ health/aids/Countries/africa/uganda_report. pdf. Accessed 22 July 2004. 9. Wenger NS, Kussling FS, Beck K, Shapiro MF (1994) Sexual behavior of individuals infected with the human immunodeficiency virus: The need for intervention. Arch Int Med 154: 1849–1854. 10. Kilmarx PH, Hamers FF, Peterman TA (1998) Living with HIV: Experiences and perspectives of HIV-infected sexually transmitted disease clinic patients after posttest counseling. Sex Transm Dis 25: 28–37. 11. Higgins DL, Galavotti C, O’Reilly KR, Schnell DJ, Moore M, et al. (1991) Evidence for the effects of HIV antibody counseling and testing on risk behaviors. JAMA 266: 2419–2429. 12. Hays RB, Paul J, Ekstrand M, Kegeles SM, Stall R, et al. (1997) Actual versus perceived HIV status, sexual behaviors and predictors of unprotected sex among young gay and bisexual men who identify as HIV-negative, HIV-positive and untested. AIDS 11: 1495–1502. 13. The White House (2003) Fact sheet: The president’s emergency plan for AIDS relief. Available: http:⁄⁄www.whitehouse.gov/news/ releases/2003/01/20030129-1.html. Accessed 22 July 2004.

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Open access, freely available online

Essay

The Future of Surgical Research Robert J. Weil

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n 1996, Richard Horton, editor of the Lancet, chastised much of current surgical research and, in particular, questioned the usefulness of the case series as a predominant form of communication among surgeons [1]. He asked a poignant question: “Does surgical research have a future?” Nearly a decade later, it is important for surgeons and non-surgeons alike to revisit Horton’s challenge.

Why Surgeons Favor Case Series Randomized controlled trials (RCTs) have become the pillar of clinical research. Such trials attempt to obtain an unbiased randomization of patients with respect to known and unknown baseline conditions and to assess the effects of an intervention. However, only a minority of surgical studies involve a valid randomization scheme. The case series remains a favored method of clinical investigation in surgery. Case series are easy to perform, require less resources in terms of personnel and funds, can be performed at a single center, and, for many surgeons, represent a means to illustrate their surgical method and skills. In many instances, case series also serve as valuable intellectual background for future clinical or scientific work. For example, consider Dennis Burkitt’s report on jaw tumors in African children, Alfred Blalock’s initial efforts in cardiac surgery, or, more recently, Starzl and colleagues’ observations, in a small collection of patients, of donor leukocyte chimerism, whereby recipients acquire tolerance to foreign donor cells. In all three cases, the authors’ work led to powerful shifts in our understanding of the biology and treatment of disease [2,3,4]. All were case reports or case series—but under the current paradigm adopted by most journals and evidence-based databases, they would not be valued [5,6,7].

The Essay section contains opinion pieces on topics of broad interest to a general medical audience.

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DOI: 10.1371/journal.pmed.0010013.g001

Surgical research needs to move from case series to RCTs

(Photo by Linda Bartlett, National Cancer Institute)

Barriers to Surgical RCTs There are many reasons why RCTs in surgical patients may be more difficult to perform than those in non-surgical patients. One of the most important— though least understood—is that the complexities of human disease in surgical patients makes them a more difficult group to study. Surgical patients are often heterogeneous in many more ways than non-surgical patients. So it would be inherently easier, for example, to study a new medication for generally healthy young adults with essential hypertension than a surgical technique for older patients with hepatic failure needing transplantation. In addition, while there may be value in studying patients from multiple centers, there may be important differences in the skill levels of different surgeons, either between centers or across the country. For example, the skill levels of surgeons in trials of carotid endarterectomy may be

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greater than those across the surgical community as a whole. This makes the applicability of some surgical RCTs to the wider community less certain than trials of medical therapies. So when it comes to surgical research, for both researchers and Citation: Weil RJ (2004) The future of surgical research. PLoS Med 1(1): e13. Copyright: © 2004 Robert J. Weil. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: NIH, National Institutes of Health; RCT, randomized controlled trial Robert J. Weil is the associate director for basic research at the Brain Tumor Institute at the Cleveland Clinic Foundation, Cleveland, Ohio, United States of America. E-mail: [email protected] Competing Interests: The author was formerly a member of the intramural research program of the National Institute of Neurological Disorders and Stroke at the National Institutes of Health. DOI: 10.1371/journal.pmed.0010013

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funding agencies, it is easier to grapple with a difficult, but ultimately soluble, basic science question than to face the uncertainty of clinical research. Investigators understand these implicit issues and trim their sails accordingly.

Improving the Rigor of Research Nonetheless, too much surgical work is conducted in the less rigorous format of the case series. What can and should be done to improve the rigor of surgical investigation? It would seem that improvements are required from within and beyond the surgical world. First, as Horner observed, and several eminent surgeons have since agreed, reforms must begin within the field itself [1,5,6,7]. Both during surgical training and in the early years of faculty development, surgeons must obtain a thorough grounding in the principles of basic research and proper clinical investigation. Second, surgeons must establish firm and friendly relations with biostatisticians so that the latter may play a strong role in helping to develop adequately powered studies that can answer critical questions raised by new therapies and techniques. This is an especially acute need in an accelerating age of targeted therapies and disease biomarkers. Third, surgeons must re-engage in the clinical research enterprise and resume leadership roles in local and national clinical trials that involve surgical patients. In the United States, for example, an important step in this regard has been the establishment of the American College of Surgeons Oncology Group, which invites surgeons from all sectors, including private practice, to become active participants in well-designed, multiinstitutional trials [5]. Similar efforts are needed on a global level. Finally, similar to the pressures faced by their colleagues elsewhere in academia, surgeon clinicianinvestigators must be nurtured, protected, and valued by their colleagues and medical administrators. The financial health of academic medical centers relies heavily on the generation of clinical revenue, which in many centers falls disproportionately on the shoulders of surgeons. New paradigms for revenue generation and funding of clinical research are needed.

PLoS Medicine | www.plosmedicine.org

Funding for Surgical Research Beyond the walls of the academic medical center, there also needs to be greater recognition of the value of scientifically sound surgical research and clinical investigation. However, the National Institutes of Health (NIH), the major source of biomedical funding in the United States, continues to convey a less welcoming attitude toward surgical research than toward other types of clinical or basic science[8,9]. At the NIH, the principal instrument for performing peer review and making grant funding decisions is the study section, composed of about 10–20 members with expertise in a given field. There are few study sections devoted to

Funding agencies need to recognize the importance of the surgical endeavor to modern medicine. surgically oriented clinical research and only two study sections (from among more than 100) in which surgeons make up even a reasonable minority of the committee members [8]. In comparison to those in other clinical departments, surgical grant proposals are less likely to be funded, and awards, when funded, are smaller [8]. Surgical research is also impeded by processes affecting other types of research as well. The number of researchers under 35 years of age receiving a first RO1 grant, the main NIH mechanism for external funding, in any field, is below 4%. The average age of initial funding for US physicians is about 44 years, and shows a trend toward advancing age that has progressed significantly in the past two decades. Thus, the NIH appears to reward experience and proven results very heavily, which may stifle innovation and likely serves as an innate barrier for younger physician-investigators contemplating research careers [9]. To help correct for this worrisome trend, the NIH created the “K” award system—career development grants designed to help starting researchers gain the experience needed to compete for RO1 grants. However, nearly 40% of the clinicians who receive KO8 awards never apply for RO1 funding

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[10], which suggests that the overall support—both explicit and implicit— for clinical research at the institutional and funding levels is inadequate. Finally, outside the US, surgeons face similar, if not greater problems. This bodes poorly for countries where the cost of evaluating new therapies and technologies may be an unaffordable luxury. These challenges to the surgical research enterprise are therefore global issues and should merit the attention of surgeons, medical institutions, and funding agencies in all countries.

The Future What can be done? On the national and international level, funding agencies need to recognize the importance of the surgical endeavor to modern medicine. Recently, in the US the NIH unveiled a “roadmap” (http://nihroadmap.nih.gov) designed to provide “new pathways to discovery.” Clear, careful, scientific surgical investigation must be part of this roadmap, although it is not specifically mentioned. Outreach efforts to include surgeons in a variety of study sections should be made to ensure that important insights into the pathophysiology and treatment of disease, with which surgeons are concerned on a daily basis, are not overlooked. Additional efforts are needed to improve funding for clinical research, both for individuals at early stages of their careers and for multi-disciplinary clinical research and clinical trials. Locally, and individually, surgeons must join efforts to improve the clinical research enterprise by including training in clinical investigation at an early stage in medical school and during surgical residency training, fostering the careers of young surgeoninvestigators through committed, protected time, participating in local and national clinical research groups, and recognizing that development as a clinical researcher takes time—many years in fact. These efforts may help ensure that surgical research is a vital part of the future of medicine and that it leads to the kind of high-quality work that shapes and remodels the face of medicine. To foster these efforts, surgeons must change and adapt to the currents of modern medical research. If this is successful, the case

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Open access, freely available online series will become the occasional rather than the common form of surgical communication. And surgeons, other clinicians, and, most importantly, basic scientists will be better able to take advantage of the new avenues of biomedical science opening before us. But the case series will always represent one important tool for early studies or uncommon conditions. It remains true that while the method one uses influences the answer one receives, it can be just as important to ask the right questions, which can be

asked even in a series of one patient [11]. And surely that is the place one must begin.
References

1. Horton R (1996) Surgical research or comic opera: Questions but few answers. Lancet 347: 984. 2. Burkitt D (1958) A sarcoma involving the jaws of African children. Br J Surg 46: 218–223. 3. Blalock A, Taussig HB (1945) The surgical treatment of malformations of the heart in which there is pulmonary stenosis or pulmonary atresia. JAMA 128: 189–202. 4. Starzl TF, Demetris AJ, Murase N, Ildstad S, Ricordi C, et al. (1992) Cell migration, chimerism, and graft acceptance. Lancet 339: 1579–1582.

5. Wells SA Jr (2002) Invited commentary: Surgeons and surgical trials—Why we must assume a leadership role. Surgery 132: 519– 520. 6. Barker CF, Kaiser LR (2004) Is surgical science dead? J Am Coll Surg 198: 1–19. 7. Goldstein JL, Brown MS (1997) The clinical investigator: Bewitched, bothered, and bewildered—But still beloved. J Clin Invest 12: 2803–2812. 8. Rangel SJ, Efron B, Moss RL (2002) Recent trends in National Institutes of Health funding of surgical research. Ann Surg 236: 277–287. 9. Kaiser J (2004) Panel weighs starter RO1 grants. Science 304: 1891. 10. Snyderman R (2004) The clinical researcher— An “emerging” species. JAMA 291: 882–883. 11. Collingwood RG (1978) An autobiography. Oxford: Oxford University Press. 172 p.

Neglected Diseases

New Drugs for Neglected Diseases: From Pipeline to Patients Bernard Pécoul

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neglected diseases have no purchasing power, so there is no financial incentive for drug companies to develop the drugs. The basic mechanics of the market-driven system are failing to help these populations. So most scientific research stops at the publication stage or falls through the gaps at different stages of the drug development pipeline (Figure 1) [3].

n wealthy countries, state-funded research has yielded breakthroughs in molecular biology, chemistry, and engineering. These advances have been taken up by the pharmaceutical industry and applied to drug development for a growing range of illnesses and conditions. As a result, patients have access to new drugs that are better tolerated, more specific, and more effective than old ones. In poor countries, however, millions of people have yet to experience the benefits wrought by science. The deadly infectious diseases that plague them, such as sleeping sickness, Chagas disease, and visceral leishmaniasis, fail to arouse the interest of drug developers. The Drugs for Neglected Diseases Initiative (DNDi) is a new, not-for-profit organisation set up to correct this fatal imbalance by developing new drugs for these forgotten patients.

Whose Role Is It, Anyway? It is dangerous to oversimplify the causes of this situation. What share of responsibility for the world’s health is borne by the pharmaceutical industry, which has the know-how and the resources for innovation? Aren’t international organisations also partly responsible? After all, they are the ones who allocate major funding for health programmes and encourage research programmes. And what about public research institutions in rich countries that generate the knowledge used by industry? Governments have the power to influence their research priorities and drug development decisions, either through funding or direct involvement. Unfortunately drugs for neglected diseases are low priority for governments [4]. They tend to prioritise research with potential commercial applications instead.

Dropped off the Radar Screen Most of the drugs still used to treat ‘neglected diseases’ were developed in colonial times. These are often expensive, difficult to administer, and hard to tolerate; several of them are also becoming ineffective because of increasing parasite resistance. Very few new alternatives have been developed in the past decades: between 1975 and 1999, 1,393 new drugs were made available to the public, but only 16 of these were meant for neglected diseases [1]. What makes the lack of drugs more difficult to accept is that scientists know an enormous amount about kinetoplastids, the organisms responsible for sleeping sickness, Chagas disease, and leishmaniasis [2]. The wealth of knowledge generated in this field could easily be used for drug development if the treatment of neglected diseases were perceived as financially attractive. But populations affected by

Citation: Pécoul B (2004) New drugs for neglected diseases—From pipeline to patients. PLoS Med 1(1): e6. Copyright: © 2004 Bernard Pécoul. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: DNDi, Drugs for Neglected Diseases Initiative; R&D, research and development; TDR, UNICEF/UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases; WHO, World Health Organization Bernard Pécoul is the Executive Director of the Drugs for Neglected Diseases Initiative (DNDi), Geneva, Switzerland. E-mail: [email protected]

The Neglected Diseases section focuses attention either on a specific disease or

The author declares that he has no competing interests.

describes a novel strategy for approaching neglected health issues in general. DOI: 10.1371/journal.pmed.0010006

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DOI: 10.1371/journal.pmed.0010006.g001

Figure 1. The Drug Development Pipeline

Because of the gaps in the development pipeline, potential new drugs for neglected diseases often stay stuck at an early stage of development. (Photos: World Health Organization/P.Virot and World Health Organization/Eric Miller)

Global Alliance for TB Drug Development: http://www.tballiance.org

funded by the World Health Organization, the World Bank, and the United Nations Development Programme—can be credited with several important successes in the fight against malaria and leishmaniasis. The Medicines for Malaria Venture and the Global Alliance for TB Drug Development were set up as public–private partnerships to tackle malaria and tuberculosis. These partnerships were made possible by the fact that malaria and tuberculosis are global diseases, affecting patients in the North and South, so there was enough of a market to persuade industry to develop new drugs for these diseases. A different solution, however, was needed for diseases that are limited to tropical countries, are of no military or strategic interest to wealthy countries, and are not supported by markets or patients’ organisations capable of attracting the attention of politicians. This is the kind of solution put forward by the DNDi.

Oswaldo Cruz Foundation: http://www.fiocruz.br

A Collaborative Not-for-Profit

Indian Council for Medical Research: http://icmr.nic.in/home.htm

DNDi is a not-for-profit organisation designed to mobilise resources for R&D of new drugs for neglected diseases. Many people and organisations around the world share an ambition to redress the lack of new treatments for neglected diseases, and bring the benefits of science to forgotten patients. Several of them came together to create DNDi: one humanitarian organisation—Médecins Sans Frontières; five research institutions—the Oswaldo Cruz Foundation from

Responses to the Crisis All is not doom and gloom. In the past few years, there has been some movement on research and development (R&D) for neglected diseases. Despite its broad mandate and limited resources, the Special Programme for Research and Training in Tropical Diseases (TDR)—established and

Box 1. From Pipeline to Patients—Some Key Organizations DNDi: http://www.dndi.org TDR: http://www.who.int/tdr Medicines for Malaria Venture: http://www.mmv.orf

Kenya Medical Research Institute: http://www.kemri.org Ministry of Health Malaysia: http://dph.gov.my/ Pasteur Institute: http://www.pasteur.fr/externe Médecins Sans Frontières: http://www.msf.org

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farnesyltransferase. The initiative is working on validating the kinetoplastid enzyme dihydrofolate reductase as a drug target. Identifying trypanothione inhibitors is also relevant to other trypanosome parasites. These are long term projects. Nifurtimox, a drug for Chagas disease, has been used to treat sleeping sickness since the 1970s in some isolated places. It has never been extended to more people because no one has studied its safety or effectiveness. DNDi will assess its shortterm usefulness by conducting clinical trials on a treatment combination of eflornithine and nifurtimox. DNDi will continue to explore other short- and medium-term projects.

Box 2. New Drugs for Sleeping Sickness Only a few drugs exist to treat sleeping sickness, and they are toxic or difficult to administer. Melarsoprol kills one in 20 patients. Eflornithine requires four daily infusions over 14 days. Given these limited options, DNDi is focusing on identifying new compounds that can cross the blood–brain barrier to treat second stage sleeping sickness. DNDi is using high throughput screening on whole cell trypanosomes to discover novel lead compounds, and is working to identify and optimise inhibitors of the enzyme protein

countries throughout the developing world. The initiative is taking this knowledge of patient needs, matching it with opportunities in R&D, and pushing the most relevant projects through the pipeline. Ultimately, neglected patients will have access to drugs targeting their specific diseases, drugs that were designed with them specifically in mind—such as short-course, low-toxicity treatments that don’t require hospitalisation, or tablets to swallow rather than injections. To identify opportunities in R&D that are both relevant to patient needs and that meet required criteria of scientific merit, DNDi is sending out calls for letters of interest to the scientific community via advertisements in journals and posted on the DNDi website (http:⁄⁄www.dndi.org). These have already pinpointed several promising projects. DNDi is also proactively contacting scientists working on infectious diseases, and surveying published literature for research of interest.

Brazil, the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health Malaysia, and the Pasteur Institute from France; and the TDR (Box 1). The initiative is a virtual organisation with a growing network of academic and R&D expertise at its disposal. The different players involved in DNDi are bringing their knowhow in parasitology and clinical trials, their experience treating neglected patients, and their drug manufacturing capacity. They are pooling these resources to move drugs stuck in the pipeline all the way to the patients themselves. Pharmaceutical companies have a particularly important role to play: they possess vast repositories of molecules, the means to move from development to industrial production, and highly specialised teams of researchers. Their contribution will be crucial to the success of DNDi.

Matching Needs and Opportunities DNDi is a needs-driven initiative—in other words, the needs of patients suffering from neglected diseases are paramount in its search for new drugs to treat them. The organisations that make up DNDi have firsthand knowledge of these needs because they work with patients in disease-endemic

In the Pipeline DNDi’s project portfolio currently holds nine projects at different stages of development to address identified needs for the treatment of visceral leishmaniasis, sleeping

DOI: 10.1371/journal.pmed.0010006.g002

Figure 2. DNDi Projects

DNDi’s project portfolio contains nine projects spread out across the drug development pipeline for the treatment of leishmaniasis, sleeping sickness, Chagas disease, and malaria. HAT, human African trypanosomiasis (sleeping sickness); VL, visceral leishmaniasis.

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Open access, freely available online sickness (Box 2), Chagas disease, and malaria (Figure 2). At discovery stage, DNDi is working on validating the kinetoplastid enzyme dihydrofolate reductase as a potential target for leishmaniasis, trypanosomiasis, and Chagas disease, and on identifying inhibitors of the kinetoplastid enzymes trypanothione reductase and protein farnesyltransferase. It is also conducting high throughput screening on whole cell trypanosomes to discover novel lead compounds. The R&D of new drugs is time-consuming and expensive if the process starts at the early discovery stage, because of the associated risk of project attrition along the way. DNDi is therefore investing resources in several pre-development and development projects as well. These include developing fixed dose combinations of artesunate/amodiaquine and artesunate/mefloquine for use against chloroquineresistant malaria in Africa and Asia, respectively; pushing for registration of paromomycin for use against visceral leishmaniasis in Africa; assessing combinations of existing drugs for visceral leishmaniasis; and evaluating the usefulness of nifurtimox in combination with eflornithine in the treatment of sleeping sickness.

governments in both developed and developing countries should take an active interest in the R&D of new drugs for neglected diseases. In parallel to its own drug development activities, DNDi is working to raise awareness of the neglected disease crisis among key policy- and decision-makers, for instance the European Commission and the National Institutes for Health in the United States.

Conclusion In the poorer countries in the world, over 350 million people are at risk from neglected diseases. Currently available treatments are inadequate or nonexistent, and new solutions are urgently needed. DNDi is working to ensure that the advances of science that have brought health and comfort to wealthy nations also benefit these neglected populations.
References

1. Trouiller P, Olliaro P, Torreele E, Orbinski J, Laing R, et al. (2002) Drug development for neglected diseases: A deficient market and a public health policy failure. Lancet 359: 2188–2194. 2. Torreele E (2003) How the poor die—Getting the research community to address global health needs. The Biochemist 25: 11–14. 3. Médecins Sans Frontières Access to Essential Medicines Campaign and the Drugs for Neglected Diseases Working Group (2001) Fatal imbalance: The crisis in research and development for drugs for neglected diseases. Available: http:⁄⁄www.msf.org/content/page.cfm?articleid=032387D3-7D0949E3-99FC231DBE03F7B7. Accessed 26 July 2004. 4. Global Forum for Health Research (2004) 10/90 report on health research 2003–2004. Available: http:⁄⁄www.globalforumhealth.org/pages/index.asp. Accessed 26 July 2004.

Advocacy for Change Governments can—some might say should—influence drug development choices. DNDi strongly believes that

Perspectives

Obstetric Fistula in Ilorin, Nigeria Andrew Browning

I

n this perspective, Andrew Browning of the Fistula Hospital in Addis Ababa discusses a study on obstetric fistula in Ilorin, Nigeria. The study was originally published in the West African Journal of Medicine [1]. With the journal’s permission, we have made a PDF of the full-text article freely available on our website (see Text S1).

T

he obstetric urogenital fistula has caused women misery ever since they first started delivering children. It was once common worldwide, but with the advent of safe obstetric care during the early part of the last century, the condition has become rare in rich countries. Urogenital fistulae do still occur in developed countries, but unlike in the developing world, they are usually a complication of a difficult pelvic surgery, cancer, or radiation [2].

The Perspectives section is for experts to discuss the clinical practice or public health implications of a published study that is freely available online.

PLoS Medicine | www.plosmedicine.org

Obstetric Fistula: A Disease of Poverty Obstetric fistula—a urogenital fistula from obstructed labour—is now only encountered in countries where health resources are scarce. The shame associated with incontinence drives affected women further into a life of poverty and begging. Many women with fistula either do not know that they can get medical help, or if they do, they are unable to pay. Furthermore, very little scientific research has been published about obstetric fistula and its management, partly because the people treating patients with this condition are working in remote areas, often with very limited resources for research. What has been written consists largely of personal case series and a few epidemiological studies [2]. To date there has been only one randomised trial in the developing world, involving 79 women operated on by a single surgeon in Benin, which found that intra-operative

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intravenous antibiotics did not reduce the risk of failed surgical repair or of objective incontinence [3]. There has been only one study in a developing country comparing different surgical techniques—a retrospective study of 46 patients operated on over a fiveyear period at a hospital in Mumbai, India [4]. This study suggested that a technique called the Martius procedure (which involves grafting of a labial

Citation: Browning A (2004) Obstetric fistula in Ilorin, Nigeria. PLoS Med 1(1): e2. Copyright: © 2004 Andrew Browning. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: UNFPA, United Nations Population Fund Andrew Browning is a Staff Specialist at the Addis Ababa Fistula Hospital, Addis Ababa, Ethiopia. E-mail: [email protected] Competing Interests: The author declares that he has no competing interests. DOI: 10.1371/journal.pmed.0010002

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pad of fat) may be better than simple anatomic repair. What we do know about the obstetric urogenital fistula is that the women who have these injuries are young, usually illiterate, and of a lower socioeconomic background. They are more often primiparous and short in stature, and they have an average length of labour of some 3.9 days. The labour is usually unattended, or if attended, it is by someone unskilled. The women inevitably deliver a stillborn child. About half of the women with fistula are divorced as a direct result of their incontinence [2,5,6,7].

The Injury and its Consequences The initial injury that leads to a fistula results from ischaemic necrosis of the soft tissues of the pelvis due to an impacted presenting part during the long labour. The ischaemia then affects the bladder and vagina (and sometimes the rectum and vagina), resulting in a fistula. The process also affects other pelvic structures. These include the nerves of the sacral plexus, resulting in foot drop and hamstring compartment weakness (foot drop may also be a result of prolonged squatting in labour, injuring the common peroneal nerve as it traverses the head of the fibula). Bony

abnormalities are common, separating or obliterating the symphysis pubis. Up to half of patients develop upper renal tract abnormalities: scarring of the ureter can cause obstructive uropathies [8]. Up to two thirds of women are rendered amenorrhoeic (their periods stop), either from disorders of the hypothalamicpituitary axis or from Asherman syndrome (adhesions in the uterus due to scarring). The vagina may be completely destroyed, as may the cervix, causing an obstructive outflow tract resulting in cryptomenorrhoea (women menstruate, but the sloughed blood and tissue don’t leave the body). The continual leakage of urine over the perineal skin can cause local and painful irritation, termed ‘urine dermatitis’. Bladder stones can occur, as women affected by fistula often drink less to try and pass less urine and the concentrated urine can form calculi. The obstetric injury has been termed a ‘field injury’, as the pathology is broad rather than isolated [9]. The resulting range of injuries can be daunting for health professionals who are working with limited resources.

The Ilorin Experience A recently published retrospective case note review provides new data

DOI: 10.1371/journal.pmed.0010002.g001

Incontinent women face a life of shame and isolation

(Photograph: © 2004 Shaleece Haas. This is an open-access image distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

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What Is Obstetric Fistula? ‘[Obstetric fistula] usually occurs when a young, poor woman has an obstructed labour and cannot get a Caesarean section when needed. The obstruction can occur because the woman’s pelvis is too small, the baby’s head is too big, or the baby is badly positioned. The woman can be in labour for five days or more without medical help. The baby usually dies. If the mother survives, she is left with extensive tissue damage to her birth canal that renders her incontinent.’ Source: UNFPA Campaign to End Fistula: “What is Fistula?” (www.unfpa.org/ fistula/about.htm).

on obstetric urogenital fistula in northern Nigeria. Ijaiya and Aboyeji reviewed 34 cases of fistula managed at the University of Ilorin Teaching Hospital over a two-year period [1]. During this period, there were 32,188 deliveries—thus, the incidence of fistula was 1.1 per 1000 live births. The mean age of the women with fistula was 23.9 years, and 32 of the 34 women were illiterate. Half were primiparous. The most common cause of the fistula was obstructed, prolonged labour—the cause in 28 out of the 34 cases. The most common complications of the fistulae were divorce or separation (eight women) and amenorrhoea (seven women). How does this study compare with other literature on obstetric fistula in Nigeria? First, the incidence reported in the study is lower than that of another hospital study of 22,774 deliveries in Zaria, also in northern Nigeria, which gave an incidence of 3.5 per 1000 deliveries [10]. However, both of these incidence figures are from hospital-based studies, and it is thought that most women do not get to a health facility to deliver their child. So the true incidence of obstetric fistula may well be much higher. In terms of prevalence, it has been estimated that there are up to 800,000 women in Nigeria who have a urogenital fistula from obstructed labour [11]. Second, although the figures given in Ijaiya and Aboyeji’s study differ slightly from those of other publications, their study does reconfirm the trends in aetiology and epidemiology of obstetric fistula in the developing world.

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Box 1. The UNFPA’s Key Strategies to Address Fistula • ‘Postpone marriage and pregnancy for young girls • ‘Increase access to education and family planning services for women and men • ‘Provide access to adequate medical care for all pregnant women and emergency obstetric care for all who develop complications • ‘Repair physical damage through medical intervention and emotional damage through counselling’ Source: UNFPA Campaign to End Fistula: “Fast Facts” (www.unfpa.org/fistula/facts. htm).

References

DOI: 10.1371/journal.pmed.0010002.g002

Surgery at the Fistula Hospital, Addis Ababa

(Photograph: © 2004 Shaleece Haas. This is an open-access image distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

Treatment and Prevention As in other resource-poor countries, many women with obstetric fistula in Nigeria do not get to a surgeon with expertise in fistula repair. There are, however, a few dedicated professionals in Nigeria helping women with fistula and operating on up to 1,600 women a year [11]. Resource-rich countries were able to eradicate the obstetric fistula almost 100 years ago, but the challenge to resource-poor countries is enormous. There are an estimated 2 million women with fistula in the world, with anywhere between 100,000 and 500,000 new cases developing each year [11]. At the world’s current capacity for dealing with the problem, it would take up to 400 years to treat the backlog of patients. Clearly we need many more centres equipped to care for women with fistula. The United Nations Population Fund (UNFPA; www.unfpa.org) and the International Federation of Gynecology and Obstetrics (www. figo.com) are endeavouring to help. UNFPA has already sponsored training workshops on fistula surgery for surgeons and fledgling fistula units in Bangladesh and some parts of Africa.

PLoS Medicine | www.plosmedicine.org

The obstetric fistula is an entirely preventable condition. Several strategies have been proposed to eradicate this condition in developing countries (Box 1), just as it has been eradicated in the developed world. However, to prevent any new cases of obstetric fistula from occurring, there would need to be 75,000 new emergency obstetric centres built in Africa alone [12]. This would require not only funds, but an appropriate number of trained doctors, nurses, midwives, and support personnel. Even if such centres are established, women will need to be convinced of the importance of seeking help without delay for a difficult labour. And then, to be able to receive that help, roads need to be built, transport systems need to be put in place, and communications need to be improved. The obstacles are clearly huge, and with currently very little money and very few professionals available, women with obstetric fistula will sadly be with us for many more years to come.


Supporting Information Text S1. Full Text of Ijaiya and Aboyeji’s Study [1] Found at DOI: 10.1371/journal. pmed.0010002.sd001 (234 KB PDF).

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1. Ijaiya MA, Aboyeji PA (2004) Obstetric urogenital fistula: The Ilorin experience, Nigeria. West Afr J Med 23: 7–9. 2. Hilton P, Ward A (1998) Epidemiological and surgical aspects of urogenital fistula: A review of 25 years experience in south-west Nigeria. Int Urogynecol J Pelvic Floor Dysfunct 9: 189– 194. 3. Tomlinson AJ, Thornton JG (1998) A randomized controlled trial of antibiotic prophlyaxis for vesico-vaginal fistula repair. Br J Obstet Gynaecol 105: 397–399. 4. Rangekar NP, Imdad Ail N, Kaul SA, Pathak HR (2000) Role of Martius procedure in the management of urinary-vaginal fistulas. J Amer Col Surg 191: 259–263. 5. Kelly J, Kwast B (1993) Epidemiological study of vesicovaginal fistulas in Ethiopia. Int Urogyn J 4: 271–273. 6. Tahzib F (1983) Epidemiological determinants of vesico-vaginal fistulas. Brit J Obstet Gynecol 90: 387–391. 7. Ampofo K, Out T, Uchebo G (1990) Epidemiology of vesico-vaginal fistulas in northern Nigeria. W Afric J Med 9: 98–102. 8. Lanundoye SB, Bell D, Gill G, Ogunbode O (1976) Urinary tract changes in obstetric vesico-vaginal fistulae: A report of 216 cases studied by intravenous urography. Clin Radiol 27: 531–539. 9. Arrowsmith S, Hamlin EC, Wall LL (1996) Obstructed labor injury complex: Obstetric fistula formation and the multifaceted morbidity of maternal birth trauma in the developing world. Obstet Gynecol Surv 51: 568–574. 10. Harrison KA (1985) Child-bearing, health, and social priorities: A survey of 22,774 consecutive deliveries in Zaria, northern Nigeria. Brit J Obstet Gynecol 92 (Suppl 5):1–119. 11. UNFPA (2002) The second meeting of the working group for the prevention and treatment of obstetric fistula, Addis Ababa, 30 October–1 November, 2002. Available: http:⁄⁄www.unfpa.org/upload/lib_pub_ file/146_filename_fistula_kgroup02.pdf. Accessed 9 August 2004. 12. Waaldjik K (1998) Evaluation report XIV on VVF projects in northern Nigeria and Niger. Katsina (Nigeria): Babbar Ruga Fistula Hospital. 27 p.

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Open access, freely available online

Perspectives

Characterization of T Lymphocytes in Chronic Obstructive Pulmonary Disease Peter J. Barnes*, Manuel G. Cosio

C

hronic obstructive pulmonary disease (COPD) is a global epidemic of major proportions that is predicted to become the third most common cause of death and fifth most frequent cause of chronic disability by 2020. In developed countries it is mainly caused by cigarette smoking, but the reasons why only a proportion (10%–20%) of smokers develop progressive airflow limitation is currently unknown. The disease is characterized by a chronic inflammatory process predominantly in the small airways and lung parenchyma, with increased numbers of macrophages, neutrophils, and T lymphocytes [1]. The difference between smokers without COPD and smokers with COPD appears to be the intensity rather than the nature of the inflammatory process. This inflammation in the small airways is associated with fibrosis and increases with the severity of airflow limitation [2], which has led to the view that COPD represents an amplification of the normal inflammatory response to inhaled irritants such as cigarette smoke.

T Lymphocytes in COPD T lymphocytes were first reported to be increased in patients with COPD by Finkelstein and colleagues, who showed a correlation between the number of T lymphocytes/mm3 of lung and the extent of emphysema [3]. It was later shown that both CD4+ (T helper) and CD8+ (suppressor/cytotoxic) T cells were increased in the airways and lung parenchyma of patients with COPD, with a predominance of CD8+ cells [4,5]. This is in contrast to the findings in asthma, in which there is a predominance of CD4+ cells, which are predominantly of the T helper 2 (Th2) pattern, with increased expression of

The Perspectives section is for experts to discuss the clinical practice or public health implications of a published study that is freely available online.

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interleukin (IL)-4, IL-5, and IL-13 (see Glossary), and which are associated with an increased number of eosinophils. In smokers who develop COPD there appears to be activation of adaptive immunity, with the infiltration of CD8+ and CD4+ cells in the alveolar walls and small airways and—in patients with the most severe disease—the presence of lymphoid follicles that contain a DOI: 10.1371/journal.pmed.0010020.g001 core of B lymphocytes surrounded by T cells Figure 1. In Emphysema, a Self-Perpetuating Loop May Lead to [2]. This activation Accumulation of Activated Th1/Tc1T Cells in the Peripheral Lung presumably follows on from the initial and then this receptor, IFN-γ inducible protein sustained innate immune response 10 (IP-10, CXCL10), monokine characterized by increased numbers of induced by IFN-γ (CXCL9), and IFNmacrophages and neutrophils; it may inducible T cell α chemoattractant involve the migration of dendritic cells (CXCL11). There is an increase in the from the epithelium to the local lymph expression of IP-10 in the airways of nodes and presentation of antigenic patients with COPD and an increase substances to T cells, resulting in clonal in the number of CXCR3+ cells [8]. expansion of CD4+ and, to an even + greater extent, CD8 cells. The study by Grumelli et al. (2004) Citation: Barnes PJ, Cosio MG (2004) Characterization of T lymphocytes in chronic obstructive pulmonary published in this issue of PLoS disease. PLoS Med 1(1): e20. Medicine takes the story forward [6]. + + The CD4 and CD8 cells appear to Copyright: © 2004 Peter J. Barnes and Manuel G. Cosio. This is an open-access article distributed under be fully activated, as they would be the terms of the Creative Commons Attribution after being presented with antigens, License, which permits unrestricted use, distribution, and reproduction in any medium, provided the and they show predominantly a T original work is properly cited. helper 1 (Th1)/cytotoxic T 1 (Tc1) pattern, with increased expression Abbreviations: COPD, chronic obstructive pulmonary disease; IFN-γ, interferon-γ; IL, interleukin; IP-10, of interferon-γ (IFN-γ) and Th1 interferon-γ inducible protein 10; MMP, matrix metalchemokines. This is consistent with loproteinase; Tc1, cytotoxic T 1; Th1, T helper 1; Th2, the recent demonstration of increased T helper 2 expression of IL-12 in bronchial Peter J. Barnes is professor and head of thoracic biopsies of patients with COPD and medicine at the National Heart and Lung Institute, Imperial College, London, United Kingdom. Manuel activation of the transcription factor Cosio is professor in the Department of Medicine and STAT-4 in T cells, subsequent STAT-4 research director at the Meakins Christie Laboratories, nuclear translocation, and IFN-γ gene McGill University, Montreal, Canada. induction, and thus a Th1 commitment *To whom correspondence should be addressed. in the T cells [7]. E-mail: [email protected] As well as producing the cytokines Competing Interests: The authors declare that they IL-2 and IFN-γ, Th1 and Tc1 cells have no competing interests. also express the chemokine receptor DOI: 10.1371/journal.pmed.0010020 CXCR3 and the ligands that activate

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CXCR3 is expressed on Th1/Tc1 cells, macrophages, and epithelial cells. Release of CXCR3-activating chemokines would attract Th1 and Tc1 cells into the lungs, and these cells then release IFN-γ, which releases more CXCR3 chemoattractants. This results in a self-perpetuating loop that may lead to accumulation of activated Th1 and Tc1 cells in the peripheral lung (Figure 1).

Role of Cytotoxic T Cells It is likely that Th1 cells are the major source of IFN-γ in the lungs of patients with COPD and therefore drive and maintain the T cell response and promote an “immune inflammation” with neutrophils and macrophages. However, it is the role of Tc1 cells that is of particular interest, as these cells are cytotoxic to epithelial cells through the release of granzymes and perforins, which induce apoptosis. Increased concentrations of perforins have recently been reported in the sputum of patients with COPD [9]. In support of this idea there is an increase in the apoptosis of alveolar cells in the lungs of patients with COPD, and this is correlated with the number of CD8+ cells and the severity of emphysema [10].

T Cell Perpetuation The T cell inflammatory response appears in mild COPD but increases markedly with disease severity. It is possible that the initial immune response becomes self-perpetuating because of endogenous autoantigens resulting from inflammatory and oxidative lung injury. There are also antigens in tobacco, but the inflammatory response appears to become independent of smoking status, and there is intense inflammation even in patients who stopped smoking many years previously [2], as seen in the present study by Grumelli et al. [6]. Another possibility is that this chronic immune response is driven, or at least maintained, by chronic infection of the respiratory tract often seen in patients with severe disease, in which there is increased colonization of the lower airways. These infections could act as co-stimulators, or by antigenic mimicry or as polyclonal activators they could provide a persisting antigenic stimulus and maintain the inflammatory process. Further studies on T cell

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receptor usage and expression of surface markers may give further clues as to the driving mechanisms for the increased Th1 and Tc1 cells in COPD.

Proteases COPD is characterized by destruction of the lung parenchyma and loss of elastin due to elastolytic enzymes, such as neutrophil elastase and certain matrix metalloproteinases (MMPs). The predominant MMP in COPD appears to be MMP9, which is released in much larger amounts from alveolar macrophages of patients with COPD than from those of smokers without the disease [11]. The study by Grumelli et al. showed that CXCR3 ligands led to the expression of the elastolytic enzyme MMP12 in alveolar macrophages and that this process was increased in the lungs of patients with COPD. This finding provides a neat link between T cells and alveolar destruction, but is discrepant with other data that have failed to show significant MMP12 release from macrophages of patients with COPD [11].

Therapeutic Implications There are currently no treatments that reduce the relentless progression of COPD, and none that have significant anti-inflammatory effects. However the recognition that an adaptive immune T cell response, most likely driven by antigens, may play an important pathophysiological role in the pathogenesis of COPD has important therapeutic implications. It is possible that T cell inhibitory strategies, such as the use of immunosuppressants, might be effective, although side effects may be a problem, and there is particular concern about increasing the risk of bacterial infection. Another approach might be to block the trafficking of Th1 and Tc1 cells to the lungs by blocking CXCR3, and there is now a search for small-molecule inhibitors of these receptors. Inhibition of IFN-γ signaling might be another approach. The mounting evidence implicating T cells, and thus an adaptive immune response, as an important component of the inflammation in COPD is overwhelming. A better understanding of the immune mechanisms involved in COPD is important, since it might lead us to new and more effective therapeutic approaches to this important disease.


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Glossary CD4+ (helper) T cell: T lymphocyte that enhances the inflammatory response CD8+ (cytotoxic/suppressor) T cell: T lymphocyte that suppresses the inflammatory response CXCR3: Chemokine receptor that is selectively activated by IP-10, monokine induced by IFN-γ, and IFN-inducible T cell chemoattractant Cytotoxic (Tc1) cell: T cell that is characterized by secretion of INF-γ Granzyme: Enzyme released by cytotoxic T cells Interferon-γ inducible protein 10 (IP10, CXCL10): Chemokine of 10 kDa that selectively activates CXCR3 Interferon-inducible T cell γ chemoattractant (I-TAC, CXCL11): Chemokine that selectively activates CXCR3 Interferon-γ (IFN-γ): Protein secreted by Th1 and Tc1 cells Interleukin-4 (IL-4): Protein secreted by Th2 cells that is important in increasing IgE secretion Interleukin-5 (IL-5): Protein secreted by Th2 cells that is important for eosinophilia Interleukin-12 (IL-12): Protein secreted by antigen-presenting cells that promotes differentiation of Th1 cells Interleukin-13 (IL-13): Protein secreted by Th2 cells that is important for IgE secretion Matrix metalloproteinase (MMP): Proteolytic enzyme that degrades connective tissue MMP9, MMP12: MMPs that destroy elastin fibers Monokine induced by interferonγ (MIG, CXCL9): Chemokine that selectively activates CXCR3 Neutrophil elastase: Enzyme released from neutrophils that destroys elastin fibers Perforin: Protein released by cytotoxic T cells that induces apoptosis STAT-4: Transcription factor specifically activated by IL-1 T helper 1 (Th1) cell: T lymphocyte that is characterized by secretion of INF-γ T helper (Th2) cell: T lymphocyte that is characterized by increased secretion of the cytokines IL-4, IL-5, and IL-13; characteristically increased in allergic inflammation

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Open access, freely available online References

1. Barnes PJ, Shapiro SD, Pauwels RA (2003) Chronic obstructive pulmonary disease: Molecular and cellular mechanisms. Eur Respir J 22: 672–688. 2. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, et al. (2004) The nature of smallairway obstruction in chronic obstructive pulmonary disease. New Engl J Med 350: 2645–2653. 3. Finkelstein R, Fraser RS, Ghezzo H, Cosio MG (1995) Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med 152: 1666–1672. 4. Saetta M, Baraldo S, Corbino L, Turato G, Braccioni F, et al. (1999) CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 160: 711–717.

5. O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK (1997) Inflammation in bronchial biopsies of subjects with chronic bronchitis: Inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 155: 852–857. 6. Grumelli S, Corry DB, Song L-Z, Song L, Green L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease. PLoS Med 1: e8. 7. Di Stefano A, Caramori G, Capelli A, Gnemmi I, Ricciardolo F, et al. (2004) STAT4 activation in smokers and patients with chronic obstructive pulmonary disease. Eur Resp J 24: 78–85. 8. Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, et al. (2002) Increased expression of the chemokine receptor CXCR3

and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 165: 1404– 1409. 9. Chrysofakis G, Tzanakis N, Kyriakoy D, Tsoumakidou M, Tsiligianni I, et al. (2004) Perforin expression and cytotoxic activity of sputum CD8+ lymphocytes in patients with COPD. Chest 125: 71–76. 10. Majo J, Ghezzo H, Cosio MG (2001) Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J 17: 946–953. 11. Russell RE, Thorley A, Culpitt SV, Dodd S, Donnelly LE, et al. Alveolar macrophagemediated elastolysis: Roles of matrix metalloproteinases, cysteine, and serine proteases. Am J Physiol Lung Cell Mol Physiol 283: L867–L873.

Health in Action

Palliative Care in Africa and the Caribbean It must be made a public health priority Dingle Spence*, Anne Merriman, Agnes Binagwaho

“If someone is condemned to a premature death because of the injustice of global health inequality, it is doubly unjust for that person to be condemned to an agonising death racked by preventable pain.” [1]

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n many resource-poor countries, death is accompanied by avoidable pain and other distressing symptoms. Unfortunately, governments in these countries usually give care at the end of life a low priority compared with preventive and curative services [2]. This prioritization makes little sense, especially when applied to treating patients with cancer and HIV/ AIDS, since prevention efforts are often failing to reduce the disease burden, while treatments aimed at cure or prolonging life are still too expensive to be made widely available. As three physicians in Jamaica, Uganda, and Rwanda, we believe that providing quality care at the end of life should be seen as a global public health priority. By using relatively lowcost palliative care approaches and community-based strategies, thousands of terminally ill patients in Africa and the Caribbean could be relieved of their pain and suffering.

The Health in Action section is a forum for individuals or organizations to highlight their innovative approaches to a particular health problem.

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Box 1. The WHO Definition of Palliative Care The WHO has defined palliative care as an approach that improves the quality of life for patients and their families facing the problems associated with life-threatening illness, through the prevention and relief of suffering. This

The Burden of Cancer and HIV/AIDS In the countries where we work, the burden of cancer and HIV/AIDS is overwhelming. In Africa about 2.5 million people die annually from HIV/AIDS, and more than 0.5 million die from cancer [3,4]. Sepulveda and colleagues have estimated that each year, at least one in 200 people in the five African countries that they studied (Botswana, Ethiopia, Tanzania, Uganda, and Zimbabwe) need palliative care at the terminal stages of HIV/AIDS or cancer [2]. This figure does not include those needing palliative care for other diseases or those suffering from a serious illness in the pre-terminal stages. Thus, perhaps one in 100 people in these countries needs some level of palliative care each year [2]. In Rwanda, as in most other African countries, infectious diseases are still rife. Health professionals are often

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is done through early identification, careful assessment, and treatment of pain and other problems—physical, psychological, and spiritual. Dying is regarded as a normal process, and death is neither hastened nor postponed [2]. The philosophy of hospice and palliative care acknowledges death, dying, and bereavement as a reality of life. faced with the terrible dilemma of having to choose between saving lives and easing the suffering of the dying.

Citation: Spence D, Merriman A, Binagwaho A (2004) Palliative care in Africa and the Caribbean. PLoS Med 1(1): e5. Copyright: © 2004 Spence et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviation: WHO, World Health Organization Dingle Spence is an associate lecturer at the University Hospital of the West Indies and the director of the Hope Institute, Kingston, Jamaica. Anne Merriman is the medical director of Hospice Africa Uganda, Kampala, Uganda. Agnes Binagwaho is the executive secretary of the National AIDS Control Commission, Kigali, Rwanda. Competing Interests: DS declares that she has no competing interests. AM and AB are on the editorial board of PLoS Medicine. *To whom correspondence should be addressed. E-mail: [email protected] DOI: 10.1371/journal.pmed.0010005

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Indeed the authorities usually believe that any investment in palliative care would be at the expense of providing life-saving treatments for those suffering from curable, often infectious illness. In many Caribbean countries, while the scourge of water- and insect-borne infectious diseases is largely under control, the prevalence rates of HIV in the adult population are some of the highest in the world [5]. In Jamaica, the largest English-speaking country in the Caribbean (population 2.5 million), in 2001, there were an estimated 20,000 people living with HIV and 980 deaths from AIDS [6]. Further, Jamaica’s proximity to the United States means that many people aspire to a lifestyle more representative of a wealthy, industrialized nation, and are thus susceptible to diseases such as cancer, coronary artery disease, and diabetes. Unfortunately, the island’s struggling public health system is often unable to provide adequately for patients with these diseases.

DOI: 10.1371/journal.pmed.0010005.g001

Better distribution of analgesics would improve palliative care provision

The Arguments for Palliative Care Prevention efforts—including health promotion, education, and screening—and treatments aimed at cure or prolonging life are key strategies needed to reduce the burden of HIV/AIDS and cancer in resource-

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Box 2. Dying in Jamaica— A Typical Case Scenario This fictional case scenario gives an impression of the sorts of problems that patients face at the Hope Institute, Kingston—Jamaica’s first public hospice. A 50-year-old woman is diagnosed with inoperable lung cancer. Because of brachial plexus involvement, she experiences severe pain and weakness of her arm. She is treated at Kingston Public Hospital with palliative radiotherapy, which helps the pain for a few months. But then the pain returns, and she requires a high dose of slow-release morphine for pain control. She lives in the mountains, and her house is a two-and-a-half-hour bus ride from Kingston, the capital city. Unfortunately, the public pharmacy in Kingston is unwilling to dispense more

poor countries [7]. However, when it comes to prevention, in many countries the effects of health education, health promotion, and screening programs have yet to make an impact on rates of HIV infection or cancer. When it comes to treatment, the provision of high-quality, affordable treatments for patients with HIV/AIDS and cancer requires the development of appropriate and accessible infrastructure and technology with sustainable funding. At present, access to treatment where we are working is essentially controlled by the ability of the patient to pay. Thus, only about one in 200 people with HIV in Uganda are able to obtain antiretroviral medicines [8]. Furthermore, patients in developing countries often present with far advanced malignant disease, and as many as 80% of people with cancer may be incurable at diagnosis [9]. Given that prevention isn’t taking effect in many places, and curative services are poorly available or inappropriate, we believe that the provision of palliative care (Box 1) in the Caribbean and Africa should be viewed as an urgent public health problem. About 80% of cancer patients will have pain in the terminal phase of their disease [1], and we estimate that at least 25% of HIV/AIDS patients have substantial pain during their illness. Effective and relatively cheap methods exist for controlling pain and

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than a week’s supply of morphine at any one time, because they have limited supplies (there is a shortage of the drug in Jamaica) and because they think the patient’s dose is unacceptably high. So she has to make the exhausting five-hour round trip every week. Her husband’s health has also recently declined, and the woman’s sister now has to care for the patient and her husband. The family now has the financial means to afford only one small meal a day, and they rely on donations from their church community in order to survive. Because the family’s savings dwindle, and the public pharmacy faces further shortages of morphine, the woman with cancer requires multiple admissions to the hospice in Kingston over the last six months of her life in order to get suitable analgesia.

other symptoms. For example, the World Health Organization (WHO) has outlined a relatively cheap way of relieving cancer pain in about 90% of patients, which could be extended to patients with HIV/AIDS [2]. Sadly, most people in Africa and the Caribbean who need pain relief aren’t receiving it [10].

Assessing Patients’ Needs Several studies in East Africa have looked at the experience of dying, the quality of care at the end of life, and patients’ unmet needs [2,11,12]. Recurring themes are (1) unmet physical needs, including the need for relief of pain and other symptoms, (2) the need for food, (3) the high cost or unavailability of appropriate analgesic drugs, (4) the severe financial constraints on the family and caregivers, (5) the need for training of family caregivers, (6) lack of psychosocial support, and (7) social isolation due to the stigma attached to a diagnosis of HIV/AIDS. In the Caribbean, patients’ needs at the end of life appear to be similar to those of patients in many East African countries. A qualitative study in Grenada, in the Eastern Caribbean, showed that people preferred to die at home rather than in hospital and—in the absence of pain relief and muchneeded counseling, information, and financial support—they took solace in spiritual comfort [13]. In Jamaica (Box

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2), although data are scarce, it seems that patients’ needs are very similar to those in Grenada. Christianity is the principal religion of Jamaica, and faith in God and family support are critical factors in patient care at the end of life. Outside of the hospital setting, appropriate analgesics are difficult to access and are often unaffordable. Patients and caregivers are not provided with enough information to help them understand disease processes, and what to expect as the ill person nears death. There is little or no palliative care provision for patients with HIV/AIDS.

Uganda’s Public Health Approach Uganda has made palliative care for patients with AIDS and cancer a priority in its National Health Plan [10]. In 1993, after conducting a feasibility study, Hospice Uganda was established in Kampala, making palliative care available to a population of about 2 million people (Uganda’s population is 22 million people). There are now two other hospices, one in Mbarara serving 1 million people, and one in Hoima serving 350,000 [8]. The hospice care provided by these units is all home-based care. This type of care provision is designed to meet

the cultural and practical needs of the people in Uganda, where most people prefer to die in their own homes, and where people are often buried in their household gardens. Hospice Uganda provides community-based care principally to patients suffering from HIV/AIDS and cancer. Almost all patients coming to the hospice have pain, and a great deal of attention is focused on good pain management. Uganda is only the third African country to have made morphine available and affordable to its patient population. Because of the dearth of legal prescribers (doctors, dentists, and vets only), in May 2004, Uganda changed the statute. This allowed midwives to prescribe pethidine, and allowed clinical palliative care nurses and clinical officers who are specially trained and registered to prescribe morphine. How was Uganda—an African country with a relatively under-funded health service—able to provide a palliative care service? A national program using a public health approach to reach those in need was established following principles outlined in the WHO’s National Cancer Control Guidelines [4]. These guidelines outline the importance

of assessing the magnitude of the problem, setting measurable objectives, evaluating possible strategies, and choosing priorities for initial activities. A series of workshops were held in Uganda between 1998 and 2000, where the WHO’s “little cost, big effect”

Palliative Care Resources for the Developing World African Palliative Care Association Representing Kenya, South Africa, Tanzania, Uganda and Zimbabwe E-mail: [email protected] Hospice Africa (Uganda) Resource and Training Centre PO Box 7757, Kampala, Uganda Tel: +256 41 266 867 / 510089; Fax: +256 41 510087—residence E-mail: [email protected]; [email protected] Centre for Palliative Learning Hospice Association of the Witwatersrand PO Box 87600, Houghton, Johannesburg 2041, South Africa Hospice Information At http://www.hospiceinformation.info. Click on “Training” to search for courses and conferences in palliative care and bereavement. Requires member’s password to access this part of the website but membership is free to people in developing countries—contact hospice information at + 44 (0)870 903 3 903 (telephone), + 44 (0)20 8776 9345 (fax), or info@hospiceinformation. Information is also circulated quarterly by E-mail to members under the title of e-Choices. Palliative Care in Resource-Poor Settings A freely available overview of HIV/AIDS palliative care, written by Kathleen Foley, Felicity Aulino, and Jan Stjernswärd. At http://hab.hrsa.gov/tools/palliative/ chap19.html. Living Well with HIV/AIDS A freely available manual on nutritional care and support for people with HIV/AIDS, by the Food and Agriculture Organization of the United Nations. At http://www.fao.org/DOCREP/005/ Y4168E/Y4168E00.HTM.

DOI: 10.1371/journal.pmed.0010005.g002

Figure 1. The WHO’s Triangle of Foundation Measures

(Adapted with permission from “A Clinical Guide on Supportive and Palliative Care for People with HIV/AIDS” [http:⁄⁄hab.hrsa.gov/tools/palliative/])

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Cancer Pain Relief: A Guide to Opioid Availability A section of this guide, by the WHO, is freely available at http://www.medsch.wisc.edu/painpolicy/ publicat/cprguid.htm. October 2004 | Volume 1 | Issue 1 | e5

measures began to be addressed. The three key measures involve education, increased drug availability, and changes in government policy (Figure 1).

Other African Initiatives Four other African countries— Botswana, Ethiopia, Tanzania, and Zimbabwe—have made the development of home-based care a priority in dealing with the HIV/ AIDS epidemic [2]. Botswana has an operational home-based care program integrated into its national health system, while in the other three countries, care is largely provided through private organizations. But few of the home-based care services in these countries include the capacity for providing effective pain relief [2].

The Next Steps By using strategies such as providing access to an essential short list of relatively cheap generic medications, and other methods recommended by WHO, it has now been proven that palliative care in the African context is affordable and achievable [2, 7,14]. We believe that, following the Ugandan and Botswanan models, palliative care should be integrated into national government strategies. In order to begin to show governments the importance and economic justification for developing a palliative care health policy, it is clear that needs assessments are an essential first step.

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It is likely to be much less expensive to provide community-based care with family and community support at the end of life than to burden already overcrowded hospital wards with patients suffering end-stage disease. There is a long tradition, both in Africa and in the Caribbean, of caring for the disabled, the mentally ill, and the young and elderly sick at home. Both start-up and sustainable funding are enormous issues that will need to be addressed by local governments, international funding agencies, and charitable bodies. Advocating palliative care to decision makers, providing training programs for health professionals, and making medications available and affordable are important challenges. Research in individual countries is needed to assess whether the above recommendations are suitable locally. Hospice Africa Uganda is advocating to other African governments and assessing other African countries where local laws and customs may dictate the most suitable way to provide palliative care together with government support. Partnerships and a public health approach to palliative care must be the way forward.
References

1. Singer PA, Bowman KW (2002) Quality of care at the end of life. BMJ 324: 1291–1292. 2. Sepulveda C, Habiyambere V, Amandua J, Borok M, Kikule E, et al. (2003) Quality care at the end of life in Africa. BMJ 327: 209–213. 3. World Health Organization (2001) World health report 2001. Mental health: New

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understanding, new hope. Geneva: World Health Organization. 178 p. 4. World Health Organization (2002) National cancer control programmes: Policies and managerial guidelines, 2nd ed. Geneva: World Health Organization. 152 p. 5. Joint United Nations Programme on HIV/ AIDS and World Health Organization (2002) AIDS epidemic update: December 2002. Geneva: World Health Organization. Available: http:⁄⁄www.who.int/hiv/pub/epidemiology/ epi2002/en/. Accessed 27 July 2004. 6. Avert.org (2004) Caribbean statistics summary. Available: http:⁄⁄www.avert.org/caribbean. htm. Accessed 20 July 2004. 7. World Health Organization (2003) Project description: A community health approach to palliative care for HIV and cancer patients in Africa. Geneva: World Health Organization. Available: http:⁄⁄www.who.int/cancer/ palliative/projectproposal/en/. Accessed 27 July 2004. 8. Merriman A, Heller KS (2002) Hospice Uganda—A model palliative care initiative in Africa. An interview with Anne Merriman. Innov End-of-Life Care. Available: http:⁄⁄www2. edc.org/lastacts/archives/archivesMay02/ intlpersp.asp. Accessed 26 July 2004. 9. World Health Organization (1996) Cancer pain relief with a guide to opioid availability, 2nd ed. Geneva: World Health Organization. 63 p. 10. Stjernsward J (2002) Uganda: Initiating a government public health approach to pain relief and palliative care. J Pain Symptom Manage 24: 257–264. 11. Murray SA, Grant E, Grant A, Kendall M (2003) Dying from cancer in developed and developing countries: Lessons from two qualitative interview studies of patients and their carers. BMJ 326: 368. 12. Kikule E (2003) A good death in Uganda: Survey of needs for palliative care for terminally ill people in urban areas. BMJ 327: 192–194. 13. Kreitzschitz S, Cox Macpherson C (2003) End of life care. Perspectives from families and caregivers. West Indian Med J 52: 269–274. 14. Merriman A (2002) Palliative medicine: Management of pain and symptoms for cancer and/or AIDS patients in Uganda and other African Countries, 3rd edition. Kampala (Uganda): Hospice Africa Uganda. 129 p.

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Open access, freely available online

Policy Forum

The Global Health Watch Mobilising civil society around an alternative World Health Report Mike Rowson*, David McCoy, Amit Sen Gupta, Armando de Negri Filho

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t the World Health Assembly in May 2003, three civil society groups—the People’s Health Movement, the Global Equity Gauge Alliance, and Medact—discussed the need for civil society to produce its own alternative to the World Health Organisation’s World Health Report. We felt strongly that we needed to produce a global health report that had equity and the right to health at its heart. We also needed a way to monitor the performance of global health institutions themselves. The idea of an alternative to the World Health Report has developed into an initiative called the Global Health Watch, which we are launching next year.

The Three Key Players

DOI: 10.1371/journal.pmed.0010003.g001

Medact (http:⁄⁄www.medact.org) is a United Kingdom–based global health charity, undertaking education, research, and advocacy on conflict, poverty, and the environment. The Global Equity Gauge Alliance (http:⁄⁄www.gega.org.za) was created to participate in and support an active approach to monitoring health inequalities and promoting equity within and between societies. The Alliance currently includes 11 memberteams, called Equity Gauges, located in ten countries in the Americas, Africa, and Asia. The People’s Health Movement (http:⁄⁄www.phmovement.org) is a global network of activists, organisations, and social movements. Its goal is to re-establish health and equitable development as top priorities in local, national, and international policy-making, with comprehensive primary health care as the strategy to achieve these priorities.

The Watch: Examining the world’s health from an alternative perspective

Why an Alternative Is Needed Concerted action by civil society has had tremendous success in the field of

The Policy Forum allows health policy makers around the world to discuss challenges and opportunities for improving health care in their societies.

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(Illustration: Giovanni Maki, Public Library of Science)

international health—global grassroots campaigns on infant feeding, smoking, and drug prices have changed policies and people’s lives. But over the last two decades—at the same time as these campaigns have scored victories—there has, in some parts of the world, been a stagnation and even reversal of the dramatic gains in life expectancy witnessed by many others for much of the 20th century. These reversals, unprecedented outside times of war and famine since the early 1800s and a scandal in a world of enormous wealth and technological prowess, have once more thrown the spotlight on how underlying social and economic problems affect health and health services. The setbacks have also underlined appalling failures of health development policy. Ambitious targets to achieve ‘Health for All’ agreed to at the end of the 1970s by health ministers from around the world have failed miserably; a similar fate seems likely for the targets set out in the Millennium Development Goals for 2015. As a result, there are large question marks hanging over the effectiveness of international health policy. 031

These are the reasons why we have decided to produce Global Health Watch, which we hope will become a regular report on international health issues (Box 1). We believe that civil society campaigners need to look at the broader health agenda— beyond single-issue advocacy. Major concerns about health systems such

Citation: Rowson M, McCoy D, Gupta AS, de Negri Filho A (2004) The Global Health Watch. PLoS Med 1(1): e3. Copyright: © 2004 Rowson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mike Rowson is the executive director of Medact, United Kingdom. David McCoy works for the Secretariat of Global Equity Gauge Alliance, South Africa. Amit Sen Gupta is the co-convener of the People’s Health Movement, India. Armando de Negri Filho is the general coordinator of the Latin American Association of Social Medicine, Brazil, and is president of the International Society for Equity in Health. *To whom correspondence should be addressed. E-mail: [email protected] Competing Interests: The authors declare that they have no competing interests. DOI: 10.1371/journal.pmed.0010003

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Box 1. Global Health Watch—2005 Report Contents Section A: The Politics and Economics of Health in the 21st Century Section B: The Health Care Sector • Health systems that promote social justice • Responding to the commercialisation of health care • The pharmaceutical industry, access to medicines, and intellectual property rights • Human resources: the lifeblood of health systems • Responding to HIV/AIDS • Gene technology and the attainment of health for all • Sexual and reproductive health Section C: Beyond Health Care • Environmental challenges • Militarism and conflict • Water • The right to food: land, agriculture, and household food security • Education Section D: Marginalised Groups • Indigenous peoples • People with disabilities Section E: Monitoring of Institutions and Resource Flows • World Health Organisation • World Bank • World Trade Organisation and trade agreements • Global Fund and Pepfar (United States President’s Emergency Plan for AIDS Relief ) • Monitoring of international promises on aid and debt relief

as poor pay and working conditions for health professionals, creeping commercialisation, and plummeting public investment have not had the attention they deserve. Likewise, broader determinants of health—such as education, water, food, and the environment—are often insufficiently regarded when health policies are formulated. The Watch attempts to focus minds on the need for more integrated planning across sectors and on the creation of health systems that promote social justice rather undermine it.

and from academia and civil society— to help us achieve these objectives. The Watch will also be ‘alternative’ in another sense—it will act as a regular monitor of the policies, governance, and funding of the institutions affecting global health, including the World Health Organisation and World Bank, something no other health report undertakes. We hope to offer proposals for reform, as well as to stimulate further action by civil society to make these institutions more accountable and responsive to the needs of the poor and vulnerable.

How Will the Watch Be Different?

Linking Civil Society Groups

This is how the Watch will be alternative: it will present options for health policy-makers that question the dominant reform agenda that emphasises market-driven and diseased-based approaches to health care. A policy bias against government action and a lack of creative thinking about how governments can shape health care markets to work in favour of equity and social inclusion are unfortunate features of global health debates. More recently, the emphasis has been placed once again on campaigns against specific diseases such as HIV/AIDS and tuberculosis, despite the universally acknowledged importance of building and maintaining health systems that can respond to the broader needs of patients. We hope the Watch will present some alternative and imaginative thinking about how health services can respond creatively to the many challenges they face, with a strong focus on basic principles of equity and universality and avoiding top-down disease-focussed programmes that neglect the broader determinants of health. We have invited some of the most interesting and innovative thinkers in health policy—from both developing and developed countries

It is important to say that the three networks and organisations that have convened the Watch are really just its initiators. In the end we hope the Watch will be backed by as many individuals, organisations, and social movements as possible, strengthening the links between civil society organisations across countries and across healthrelated sectors, and increasing the power and influence of the report itself. Already, many have expressed their interest in the project, and their willingness to contribute: through writing chapters, contributing case studies, and launching the Watch and promoting it in their country when it is finished. Groups from India and Brazil are planning parallel national Watches. We plan to launch the Watch at the second People’s Health Assembly, which will be held in Ecuador in July 2005. We don’t want this report to be addressed just to health activists or health policy-makers or academics. If we are going to create change we need to capture the imagination of the broader health professional community and the public at large. That is why we encourage readers to get involved and tell others about the Watch and to use it to throw down a challenge to those who call the shots at national and international levels.


Section F: Summary and Strategies for Action

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Open access, freely available online

Policy Forum

How Should the Health Community Respond to Violent Political Conflict? We must define better practice and promote organisational learning Anthony B. Zwi

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iolent political conflict, and its impact, is again on the front pages—in Iraq, Afghanistan, and Sudan. While the situation in Darfur is now particularly urgent (see sidebar) [1,2,3,4,5], there are many other settings in which complex political emergencies are undermining health service provision and threatening human rights. Such emergencies have a direct impact on health (see Table 1). They also impair the functioning of health systems through, for example, destruction of infrastructure (such as clinics and vehicles), reduced access to medicines, death of health workers, and weakened national capacity for health policy-making [6]. Such violent political conflicts stir us—the global health community—to discover our own humanity amidst the bloodshed. How best should we respond? Despite unique features in each setting, we must learn lessons from previous conflicts to help guide our response to current and future ones. There are six key lessons that emerge from studying health in conflict settings.

Lessons from Conflict Settings Violent conflict is driven by politics and economics [7]. Complex political emergencies (1) occur within and across state boundaries, (2) have political antecedents typically relating to competition for power and resources, (3) are protracted in duration, (4) express existing social, political, economic, and cultural structures and cleavages, and (5) are often characterised by one sector preying on other parts of the community [8]. Damage to health is not just a side effect but may be the objective for violent groups. In The Policy Forum allows health policy makers around the world to discuss challenges and opportunities for improving health care in their societies.

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Sudan—Conflict and Health The current crisis in Darfur reflects a devastatingly acute episode in the chronic internal conflict that has plagued Sudan since 1983. The cost of this conflict has been enormous: over 2 million lives lost, over 628,000 refugees from Sudan in neighbouring countries, and over 4 million people internally displaced [1]. In southern Sudan, the conflict has led to widespread ill health and has severely compromised the well-being of women and children. Indicators of immunisation, nutrition, primary school completion, and antenatal care are among the worst in the world. About 95,000 children under five years old died last year, most from preventable disease [2]. Statistics from UNICEF are chilling: ‘A girl born in southern Sudan has a better chance of dying in pregnancy or childbirth than of completing primary school….One in nine women dies in pregnancy or childbirth but only one in a hundred girls completes primary school’ [2]. Communities in Darfur face ongoing violence from militia supported by the government of Sudan. The fighting has resulted in large-scale destruction of villages, rape, and kidnapping. About 15,000–30,000 lives are estimated to have been lost from January 2003 to June 2004 [3]. Surveys by Médecins Sans Frontières found death rates of three to five per 10,000 people/day in Mornay and Zalinge villages (the emergency threshold level is set at one death per 10,000/day) [4]. Over 300,000 people are at risk if humanitarian access remains restricted. Of displaced Darfurians, 90% need shelter and latrines, and over half lack access to primary health care [3]. Food insecurity is widespread and is being used as a ‘weapon of war’ [5] resulting in widespread nutritional problems. Despite widespread concern, information gaps abound, and humanitarian agencies report having access to only a fraction of those most affected. Yet, genocide is taking place in real time.

complex political emergencies, we can typically identify three groups: the winners, the ‘conflict entrepreneurs’ (who seek the perpetuation of conflict because they profit economically or politically), and the losers, whose lives and livelihoods are imperilled. Humanitarian and relief agencies increasingly recognise that belligerents may seek to control or manipulate the inflow of humanitarian and relief resources [9]. A political economy perspective helps identify those interests, which may impede the transition to peace [7]. Appreciating context is crucial. The nature of the conflict—its background, history, and the different forms of violence involved—will greatly influence health outcomes. Most conflicts are today intra-national rather than international [10]. Internal conflicts affect 033

populations through forced migration, violence, and human rights abuses including torture, disappearances, and Citation: Zwi AB (2004) How should the health community respond to violent political conflict? PLoS Med 1(1): e14. Copyright: © 2004 Anthony B. Zwi. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Anthony B. Zwi is professor and head of the School of Public Health and Community Medicine at The University of New South Wales, Sydney, Australia. E-mail: [email protected] Competing Interests: The author is chief investigator of a project seeking to explore the links between health and preventing violence, and health and building peace, in five countries in the Asia-Pacific region. This project is funded by AusAID, the Australian development assistance agency. DOI: 10.1371/journal.pmed.0010014

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Table 1. Examples of the Direct Impact of Conflict on Health Health Impact

Causes

Increased mortality

Deaths from external causes, mainly related to weapons Deaths from infectious diseases (e.g., measles, malaria) Deaths from noncommunicable diseases/deaths otherwise avoidable through medical care (e.g., asthma, unavailability of emergency surgery)

Increased morbidity

Injury and illness from external causes (e.g., weapons, landmines, sexual violence) Infectious diseases: water-related (e.g., cholera, typhoid), vector-borne (e.g., onchocerciasis), and others (e.g., tuberculosis, HIV) Reproductive ill health: increased number of stillbirths and premature births, more cases of low birth weight, more delivery complications, longer-term genetic impact of exposure to defoliants or radioactive materials Acute and chronic malnutrition and deficiency disorders Mental illness (e.g., depression, post-traumatic stress disorder)

Increased disability

Physical Psychological Social

Adapted from [7], with permission from the World Health Organization. DOI: 10.1371/journal.pmed.0010014.t001

rape. The forms of violence and types of health damage relate to the phase of the conflict, the sophistication of weapons used, the degree of involvement of regular military forces, the extent of terrorism employed, and the extent to which genocide is intended. Ongoing insecurity and instability may be present even after the ostensible end to the conflict, as in latter-day Afghanistan and Iraq. Challenges to governance, to service delivery, and to the reestablishment of livelihoods may persist for years. A 2003 survey in Iraq found that despite the brief duration of the war and the intent to spare hospitals and clinics from direct attack, many people suffered in the post-war period, primarily as a result of disruption to civil order [11]. Recent reports highlight the difficulties of re-establishing the health system in Iraq—partly because of a failure to appreciate the cultural and health services context [12]. Better care can save lives. Emergency relief efforts are increasingly based upon empirical evidence, and priority

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health issues are much more effectively addressed than previously. Emphasis is typically placed upon disease surveillance, immunisation, control of infectious diseases, reproductive health, water and sanitation, shelter, and nutrition [13]. Mental health, sexually transmitted infections, and HIV have recently attracted additional attention. Standards have improved, can be further improved, and warrant widespread dissemination and application. The more-established humanitarian agencies have accepted that their relief efforts must be as evidence-based as possible. This principle should also apply to the post-conflict period, during which the health of affected communities continues to suffer [14]. We need enhanced accountability for humanitarian action. Despite a developing evidence base for health-related humanitarian action, evaluations of humanitarian activities have found ongoing problems. These include poor standards of delivery,

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About 70% of structures were destroyed in Dili, East Timor, in the violence wrought by Indonesian militia after the referendum in 1999

(Photo: Anthony Zwi.)

duplication of efforts by different agencies, lack of coordination, and failing to learn from prior experience. The Sphere Project has advocated minimum standards for the delivery of humanitarian assistance, and has established a “Humanitarian Charter” (http:⁄⁄www.sphereproject.org). The project’s objectives and achievements have been to improve the quality of humanitarian action and promote a movement concerned with the rights and dignity of those caught up in war and disaster [15,16]. The Active Learning Network for Accountability and Performance in Humanitarian Action (http:⁄⁄www.alnap.org ) seeks to ensure that lessons are learned, distilled, and disseminated. At a meeting in Stockholm in June 2003, key international donors committed themselves to ‘good humanitarian donorship’, which recognises the importance of promoting standards in humanitarian action [17]. However, recent sober reflection suggests that donors and humanitarian agencies could do better: ‘An ailing humanitarian enterprise is labouring under pressures from the external environment over which it has little control, while struggling with issues internal to its own function for which it should take greater responsibility’ [18]. Militarization of humanitarian efforts is problematic. Multinational military forces have played a major part in recent conflicts in Kosovo, East Timor, Sierra Leone, Iraq, and Afghanistan. The military has become increasingly involved not only in

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waging war but also in seeking to win the peace; it is increasingly active in delivering emergency relief. It not only provides services—sometimes necessary to deliver needed relief—but also seeks to ‘win hearts and minds’ while operating within structures responsive to military and foreign policy directives. The result has seen a blurring of the separation between military and humanitarian efforts [19]. This can make humanitarian agencies a target— recent examples include the bombing of United Nations headquarters and the International Committee of the Red Cross in Iraq and the recent, reluctant withdrawal of Médecins Sans Frontières from Afghanistan following the murder of five aid workers [20]. Emerging evidence and good practice in civil-military cooperation highlights the importance of (1) promoting needs-based assistance free of discrimination, (2) civilian-military distinction in humanitarian action, (3) independence of humanitarian organisations from political pressures and interference, and (4) the security of humanitarian personnel [19]. The transition from emergency relief to development is poorly managed. The objectives of humanitarian relief activity (saving lives and livelihoods) differ from those of development (building sustainable systems, promoting equity, building systems of governance, and eradicating poverty). In each phase there are different actors, strategies, and approaches. The increasing politicisation of humanitarian intervention [21,22] brings threats and dangers, undermining key humanitarian principles. The balance between relief and development will vary over time and place; getting the balance right and adequately resourcing the transition warrants careful research, documentation, reflection, and the commitment of appropriate longer-term funding.

What Gaps Remain in Our Knowledge? Despite the knowledge we have gained on responding to violent political conflict, many important gaps remain. We still do not hear the voices of those most affected or of the service providers seeking to assist. The reality of people’s experiences is inadequately appreciated [23]; whatever we learn of their fears, challenges, and suffering

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DOI: 10.1371/journal.pmed.0010014.g002

In southern Sudan, the conflict has affected the well-being of women and girls

(Illustration: Margaret Shear, Public Library of Science.)

is typically represented and reported through sanitised language and media. The language used dehumanises ‘the enemy’ and blunts our senses to the reality of atrocity and to the negative effects of our own countries’ interventions. Within the health sector, ensuring that we hear the voices of service providers and carers will help bring home the reality of system disruption, destruction, and damage and will simultaneously document the mechanisms and potential for effective responses. The new communication technologies provide immense opportunity to ensure that experience is placed in the public domain from where lessons can be drawn and better practice promoted. We know little about how communities and systems survive adversity. In most settings, the inherent ability and ingenuity of people and systems allows them to withstand instability and insecurity. Health personnel and health systems could play a valuable role in these fragile settings—assisting individuals, communities, and systems to further develop their coping strategies,

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adaptations, and responses. But whether health systems do so and how is unclear. Failing to support and maintain these systems may result in much greater challenges when we seek at a later stage to resuscitate them. We also know relatively little about whether the health sector can indeed make a special contribution to building the peace. While it has been forcefully argued that the health sector is uniquely placed to play a role in peace building [24], the evidence for this remains limited [25]. We know little about how health workers see and respond to these challenging roles. The health sector could play a role in demonstrating the values and priorities of government, reflecting the relationship between those with and without resources, and the relationship between those who do and do not have protection. In the aftermath of major periods of violence, the health sector could also help to ensure that the structural inequities that preceded the violence and may have contributed to it, are not reinforced and the same injustices not recreated. But, engagement around health is not always positive: the health system is open to abuse and has been abused by repressive systems.

From Learning Lessons to Sound Policy Perhaps the most important gap of all is between observing lessons and putting them into practice. We urgently need to transform evidence and experience into sound policy. We need more sophisticated policy analyses, more sensitive policy-making, and more relevant research. Policy in these difficult areas will never be entirely evidence-based—often it will at best be ‘evidence-informed’. Our objective must be to promote organisations and systems that are able to reflect on experience, work with partners to critically analyse and learn, and thereby formulate better responses. Violent political conflict will continue to challenge the global health community. International policy-makers and funders must support more extensive documentation and reflection: the building blocks of better practice.


Acknowledgments Natalie Grove assisted in identifying and summarising key source documents cited in this contribution.

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Open access, freely available online References

1. US Agency for International Development, Bureau for Democracy, Conflict and Humanitarian assistance, Office of US Foreign Disaster Assistance (2004) Sudan—Complex emergency. Available: http:⁄⁄www.usaid. gov/our_work/humanitarian_assistance/ disaster_assistance/countries/sudan/fy2004/ Sudan_CE_SR04_06-21-2004.pdf. Accessed 24 July 2004. 2. ReliefWeb (2004) After 21 years of war, the children of Southern Sudan need more than a lifeline. Available: http:⁄⁄www.reliefweb.int/w/rwb.nsf/0/ 1f1c63d6d23057fec1256eb7003a4be4? OpenDocument. Accessed 24 July 2004. 3. Winter R (2004) Humanitarian Crisis in Sudan: Testimony before the committee on foreign relations committee, subcommittee on Africa, United States Senate, June 15, 2004. Available: http:⁄⁄www.usaid.gov/press/speeches/2004/ ty040615_1.html. Accessed 24 July 2004. 4. Medicins Sans Frontieres (2004) Humanitarian situation in Darfur, Sudan—MSF statement to the United Nations Security Council. Available: http:⁄⁄www.msf.org/countries/ page.cfm?articleid=DB8843B3-F57D-4054 82D6530AA6D15E6C. Accessed 25 July 2004. 5. Macrae J, Zwi A (1994) Famine, complex emergencies and international policy in Africa: An overview. In: Macrae J, Zwi A, editors. War and hunger. London: Zed Books. pp. 6–36. 6. Zwi AB, Garfield R, Loretti A (2002) Collective violence. In: Krug EG, Dahlberg LL, Mercy JA, Zwi AB, Lozano R, editors. World report on violence and health. Geneva: World Health Organization. pp. 215–239. 7. Le Billon P (2000) The political economy of war: What relief agencies need to know.

Humanitarian Practice Network. Available: http:⁄⁄www.odihpn.org/documents/ networkpaper033.pdf. Accessed 30 August 2004. 8. Goodhand J, Hulme D (1999) From wars to complex political emergencies: Understanding conflict and peace-building in the new world disorder. Third World Q 20: 13–26. 9. Anderson MB (1999) Do no harm: How aid can support peace—or war. London: Lynne Rienner Publishers. 161 p. 10. Zwi A, Fustukian S, Sethi D (2002) Globalisation, conflict and the humanitarian response. In: Buse K, Fustukian S, Lee K, editors. Health policy in a globalising world. Cambridge: Cambridge University Press. pp. 229–250. 11. Centers for Disease Control and Prevention (2003) Vaccination services in postwar Iraq, May 2003. MMWR Morb Mortal Wkly Rep 8: 734–735. Available: http:⁄⁄www.cdc.gov/ mmwr/PDF/wk/mm5231.pdf. Accessed 19 August 2004. 12. Brown H (2004) An opportunity lost. Lancet 364: 15–18. 13. Toole MJ, Waldman RJ, Zwi AB (2001) Complex humanitarian emergencies. In: Merson MH, Black RE, Mills AJ, editors. Textbook of international public health: Diseases, programs, systems and policies. Gaithersburg (Maryland): Aspen Publishers. pp. 439–513. 14. Ghobarah HA, Huth P, Russett B (2004) The post-war public health effects of civil conflict. Soc Sci Med 59: 869–884. 15. Walker P, Purdin S (2004) Birthing sphere. Disasters 28: 100–111. 16. The Sphere Project (2004) Humanitarian

charter and minimum standards in disaster response, 2nd ed. Oxford: Oxfam. 350 p. 17. Anonymous (2003) International meeting on good humanitarian donorship, Stockholm, 16– 17 June 2003: Meeting conclusions. Available: http:⁄⁄www.reliefweb.int/ghd/imgd.pdf. Accessed 19 August 2004. 18. Donini A, Minnear L, Walker P (2004) The future of humanitarian action: Mapping the implications of Iraq and other recent crises. Disasters 28: 190–204. 19. United Nations Office for the Coordination of Humanitarian Affairs (2004) Civil–military relationships in complex emergencies: An IASC reference paper. Available: http:⁄⁄ochaonline. un.org/DocView.asp?DocID=1219. Accessed 19 August 2004. 20. van Halsema D (2004) Six days surrounding MSF’s decision to withdraw from Afghanistan. Médecins Sans Frontières. Available: http:⁄⁄www.msf.org/countries/page. cfm?articleid=AA5AE5CF-05EA-4D438DB12C6450CBEA7C. Accessed 19 August 2004. 21. Duffield M (2001) Global governance and the new wars: The emerging of development and security. London: Zed Books. 293 p. 22. Macrae J (2001) Aiding recovery? The crisis of aid in chronic political emergencies. London: Zed Books, 2001. 191 p. 23. Pedersen D (2002) Political violence, ethnic conflict, and contemporary wars: Broad implications for health and social well-being. Soc Sci Med 55: 175–190. 24. Santa Barbara J, MacQueen S (2004) Peace through health: Key concepts. Lancet 364: 384–386. 25. Vass A (2001) Peace through health. BMJ 323: 1020.

Learning Forum

Fever, Headache, and Visual Blurring in a 17-Year-Old Woman William Lynn, Sue Lightman a raised white blood cell (WBC) count of 11.1 × 109 per millilitre (normal range, 4.3-10.8 × 109, per millilitre), with 84% neutrophils. Her erythrocyte sedimentation rate was 15 mm in the first hour (normal range, 1–12 mm), and her Creactive protein was less than 5 mg/l (normal range, less than 10 mg/l). A malaria screen was negative, and renal and liver function tests were normal.

DESCRIPTION of CASE

A

17-year-old woman, who was born in Bangladesh, presented to an accident and emergency department in the United Kingdom with a history of being unwell for 24 hours. She had a headache and fever, and was vomiting. On questioning, there was no photophobia or neck stiffness. She lives in the United Kingdom and had been for a holiday to Bangladesh 4 months previously. The family members were all well, and none had similar symptoms. The woman had no previous medical history and was on no medication. On examination, she looked unwell, with a temperature of 38.5 °C, pulse 96 beats per minute, blood pressure 112/52 mm Hg, and a respiratory rate of 16 breaths per minute. She had a Glasgow Coma Score of 14, there was no neck stiffness, and her ocular fundi were said to be normal. There were no other significant findings on examination. Immediate investigations showed a normal blood count apart from

Citation: Lynn W, Lightman S (2004) Fever, headache, and visual blurring in a 17year-old woman. PLoS Med 1(1): e7. Copyright: © 2004 Lynn and Lightman. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: CSF, cerebrospinal fluid; PCR, polymerase; RPE, retinal pigment epithelium; TB, tuberculous; VKH, Vogt-Koyanagi-Harada; WBC, white blood cell William Lynn is a consultant in infectious diseases and the medical director at Ealing Hospital, London, United Kingdom. E-mail: [email protected]. Susan Lightman is professor of Clinical Opthalmology and is head of the Department of Ophthalmology at Moorfields Eye Hospital, London, United Kingdom. Email: [email protected] Competing Interests: The authors are on the editorial board of PLoS Medicine.

The Learning Forum discusses an important clinical problem of relevance to a general medical audience.

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DOI: 10.1371/journal.pmed.0010007

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patient was given 2 g of ceftriaxone intravenously immediately, and ceftriaxone treatment was continued following the CSF results. Aciclovir IV, at a dose of 10 mg/kg every 8 hours, was added to cover the possibility of herpes encephalitis.

What Clinical Diagnoses Were Being Considered? A clinical diagnosis of probable viral meningitis was made pending the results of further investigations. Chest X ray was normal, as was an unenhanced CT brain scan. Lumbar puncture showed a normal cerebrospinal fluid (CSF) opening pressure, and the CSF was not blood stained. Laboratory analysis showed 1,606 WBC/ml (normally there are less than 5 cells/ml), of which 60% were lymphocytes with protein of 1.08 g/l (normally less than 0.6 g/l) and glucose of 2.9 mmol/l (normally greater than 50% of plasma glucose), with a corresponding plasma glucose of 5.7 mmol/l (normal range, 4–9 mmol/ l). No organisms were seen on Gram stain. The CSF was negative for pneumococcal, meningococcal, and Haemophilus antigens, and DOI: 10.1371/journal.pmed.0010007.v001 bacterial culture was subsequently Video 1. Spontaneous Venous Pulsation of the Veins at the Optic Nerve Head negative. The

DOI: 10.1371/journal.pmed.0010007.g001

What Was the Subsequent Differential Diagnosis?

Figure 1. Fundal Appearance of the Patient’s Eye

The large arrow indicates the pink optic nerves; the star shows localised retinal detachment; and the small arrow pointing down shows small, white choroidal granulomas.

The differential diagnosis was now viral, bacterial, tuberculous (TB), fungal, or malignant meningitis, or sarcoidosis. Additional investigations were requested, including polymerase chain reaction (PCR) on CSF for viruses and tuberculosis. There were no risk factors for HIV

Table 1. CSF Changes in the Most Commonly Encountered Types of Meningitis Type of Meningitis

Cells

Glucose

Protein

Diagnostic Tests

Comments

None (normal)

0–5 cells/µl, usually monocytes

>50% of plasma level

<0.5 g/l

Not applicable.

Viral

Mainly lymphocytes

Usually normal

Moderately raised

Viral culture now largely replaced by PCR.

May be CSF neutrophilia early. CSF glucose reduced in mumps meningitis.

Bacterial

Mainly neutrophils

Reduced

Elevated

Gram stain and culture. Bacterial antigen detection for pneumococcus, Haemophilus, and meningococcus. Increasing use of PCR.

Neutrophils may be replaced by lymphocytes in chronic or partially treated infection. Haemorrhagic CSF in anthrax.

Tuberculous

Moderately elevated, usually lymphocytes

Reduced

Markedly elevated, 1–3 g/l

Auramine stain insensitive. Culture takes up to 8 weeks. Sensitivity of PCR is 56% (95% CI 46%–66%) [2].

Occasionally CSF neutrophilia. CSF may be normal or have only minor changes in cerebral abscesses due to tuberculosis (tuberculoma).

Fungal

Moderately elevated lymphocyte count

Reduced

Elevated

Gram stain and culture. India ink stain and antigen detection for Cryptococcus.

CSF changes may be minimal in patients with advanced immunodeficiency (particularly AIDS).

DOI: 10.1371/journal.pmed.0010007.t001

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Papilloedema was thought likely, and no other neurological signs were detected. The original CSF sample was negative on PCR for tuberculosis and herpes simplex virus, C-reactive protein remained at less than 5 mg/l, and a further head CT scan with contrast was normal. The diagnosis was revised to meningoencephalitis. Other agents—such as listeria, tuberculosis (a systematic review found that PCR has a sensitivity of only 56% [95% CI, 46%– 66%] for detecting TB meningitis [2]), and viruses such as others in the herpes group, mumps, and West Nile virus— were considered. The aciclovir was stopped, and quadruple tuberculosis therapy was started.

infection (HIV seroconversion can present with meningitis). Mollaret’s meningitis (recurrent aseptic meningitis associated with herpes simplex virus) was a possibility [1], though this condition DOI: 10.1371/journal.pmed.0010007.g002 characteristically presents as recurrent Figure 2. White Choroidal Infiltrates (Arrow) Seen in VKH Syndrome with Very Pink Optic episodes of Nerve Head apparent aseptic meningitis. The following morning the patient’s temperature returned to normal at 37 °C, and she felt better. Referral was made to the Infectious Disease Service. Twenty-four hours after admission, she was afebrile but clinically worse, with marked headache and vomiting. A day later she spiked a fever of 39 °C. No organisms were cultured from the CSF, urine, or blood. CSF bacterial antigens, cryptococcal antigen, and CSF auramine stain (for mycobacteria) were also all negative. The following day the patient complained of further headache, nausea, blurred vision, and photophobia. In addition, she was noted to have bilateral large pupils, which did not react to light, and very pink optic nerves.

What Did the Eye Signs Mean? The fixed, dilated pupils were of major concern, and urgent ophthalmic review was requested. Examination by the ophthalmologist showed reduced vision at 6/60 right eye and 6/36 left eye. On testing with Ishihara charts, the patient had severely reduced colour vision. Her pupils were large and nonreactive to light or DOI: 10.1371/journal.pmed.0010007.g003 accommodation Figure 3. Localised Retinal Detachment in both eyes.

Table 2: What to Do When the Patient Is Not Getting Better Cause

Explanation

Wrong organism.

The patient is infected but we have chosen incorrect antibiotics. For example, the patient has TB or fungal meningitis or an unusual organism associated with travel.

Right organism but wrong antibiotics.

Antibiotics either chosen incorrectly or the organism is antibiotic-resistant.

Right organism, right antibiotics but pharmacologic failure.

Failure of the antibiotic to penetrate blood-brain barrier, non-compliance (particularly in tuberculosis [8]), or drug interactions (for example, rifampicin enhances the metabolism of fluconazole).

Sequestered site of infection adjacent to the meninges.

Need to consider both intracranial and spinal infection, such as cranial osteomyelitis or a spinal epidural abscess.

Infection treated properly, but a second problem is present or there is another focus of infection.

There may be evidence of a focal source of infection, such as pneumonia, endocarditis, sinusitis, or otitis media. Or the patient may have an unsuspected underlying systemic illness such as malignancy or HIV. One catch here is the development of a drug fever, which may occur, for example, with high-dose beta-lactam antibiotics and rifampicin.

Non-infectious cause of meningoencephalitis.

See Table 3

DOI: 10.1371/journal.pmed.0010007.t002

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The eyes were inflamed, with cells present in the aqueous and vitreous humours. The optic nerves were swollen and very pink (Figure 1), but there was normal spontaneous venous pulsation present—demonstrating that this was not papilloedema (see Video 1). Bilateral choroidal infiltrates with overlying serous retinal detachments were also present.

Harada (VKH) syndrome [3]. All antibiotics were stopped, and high-dose corticosteroids were started at 100 mg prednisolone daily. At one week there was no significant ocular improvement, although the patient’s headache and vomiting were now gone, and she felt much better. Additional immunosuppressive therapy was initiated with cyclosporin and mycophenolate, and within a further week the patient’s What Was the Final Diagnosis and Treatment? vision started to improve, with settling of the ocular signs. Oral corticosteroids were tapered, as was the cyclosporin, and The combination of the clinical symptoms, signs, and by one month the patient’s vision had returned to normal, ocular features was characteristic of Vogt-Koyanagiand the ocular signs continued to Table 3. Non-Infectious Causes of Abnormal CSF settle. By three months the cyclosporin was discontinued, the steroid dose Category Examples CSF Changes was reduced to 5 mg daily, and the mycophenolate dose was tapered. By six Auto-immune/ VKH Lymphocytic with markedly raised protein months all therapy was discontinued. vasculitic and usually normal glucose. At review six months later, the patient remained well, with normal vision and normal optic nerves. Systemic lupus CSF may be normal or show a slightly erythematosus raised cell count and protein. Glucose often low.

DISCUSSION

Vasculitis

Normal or lymphocytic, with raised protein. Glucose may be normal or reduced.

Uncertain aetiology

Sarcoidosis

Lymphocytic with markedly raised protein and usually normal glucose.

Malignant

Leukaemia

Cytology may detect blasts. Raised protein and low glucose.

Lymphoma

Central nervous system involvement mainly in non-Hodgkin’s disease and much commoner with underlying AIDS. Lymphoma cells on cytology. PCR often positive for Epstein-Barr virus.

Solid tumours

Spread to subarachnoid space is rare and can be detected with cytology.

Non-steroidal anti-inflammatory drugs

Lymphocytic with raised protein and normal glucose.

Anti-lymphocyte antibodies, e.g., OKT3

Lymphocytic with raised protein and normal glucose.

Post-spinal anaesthesia

Lymphocytic with raised protein and normal glucose.

Infarction

Raised lymphocytes and protein, normal glucose.

Haemorrhage

Red blood cells more than 400/mm3. Xanthochromia. Usually no increase in WBCs and normal glucose

Drug induced

Cerebrovascular

DOI: 10.1371/journal.pmed.0010007.g004

Figure 4. Total Retinal Detachment, Where Whole Retina is Grey in Colour

DOI: 10.1371/journal.pmed.0010007.t003

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This young patient presented acutely with a fever and some signs suggestive of meningitis. She was initially treated as having viral meningitis, but the CSF findings indicated that other aetiologies needed to be considered. In particular, in a woman who previously lived in and recently visited Bangladesh, with a lymphocytic meningitis and borderline CSF glucose, tuberculosis had to be considered. Initially the ocular symptoms and signs were not a prominent feature, but the signs were likely to have been present when the patient was first seen. The typical CSF changes associated with meningitis of different aetiologies are shown in Table 1. In this case the mixed lymphocytes and neutrophil leucocytosis with a borderline CSF glucose on the patient’s initial CSF sample were consistent with bacterial or TB meningitis. Viral infection was far less likely, as only mumps is consistently

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meningoencephalitis, visual blurring, and deafness. The eye signs are very characteristic and can help to make the diagnosis. The most prominent ocular finding is intensely pink optic nerves (see Figure 1), with severe visual reduction and loss of function, which accounts for the absent pupillary responses. VKH syndrome is usually bilateral, but occasionally the eyes can be affected asymmetrically so that one is very mildly involved. The syndrome is accompanied by marked intraocular inflammation, and there are choroidal infiltrates (Figure 2) associated with serous retinal detachments, which may be localised (Figure 3) or affect the whole retina (Figure 4). It is likely that these detachments are due to the retinal pigment epithelium (RPE) being affected by the underlying inflammatory choroidal granulomas (which heal leaving scars; see Figure 5), and fluid accumulates underneath the retina because of reduced function of the RPE when it becomes inflamed. The disorder DOI: 10.1371/journal.pmed.0010007.g007 is caused by an immune response Figure 7. Poliosis Note white eyelashes on child. to melanin and affects parts of the body where melanin is found. The initiating stimulus for this response is unknown, but T-cells sensitised to melanin-associated antigens are found in the peripheral blood. In the ear, the melanocytes of the inner ear are the target, and the inflammatory response here results in hearing loss and balance problems. In longstanding untreated cases, depigmentation may occur in other sites such as skin (vitiligo; Figure 6) and eyelashes (poliosis; Figure 7), but these are uncommon when corticosteroids and other immunosuppressive agents are used in treatment. Depigmentation of the RPE can also occur, giving a ‘sunset’ appearance to the dark fundus. Treatment with high-dose corticosteroids is essential [3] and should be initially 1–2 mg/kg/day. This treatment can be given orally or intravenously, depending on how unwell the patient is. However, patients commonly need other immunosuppressive agents as well, so as to allow the dose of steroids to be reduced more quickly. Both cyclosporin and mycophenolate are very useful as steroid-sparing agents, with cyclosporin having the advantage of a variable-dose regimen for more rapid onset of action. On the down side, cyclosporin can cause hirsutism, especially in combination with corticosteroids (which can also cause this side effect). Unfortunately, the costs of cyclosporin and mycophenolate may preclude their use in resource-poor settings, with the result that patients may require high-dose corticosteroids for

associated with reduced CSF glucose.

Infectious Causes of Meningitis There is a wide range of infectious causes of meningitis worldwide. The likely infecting organism will be determined by the age and immune status of the DOI: 10.1371/journal.pmed.0010007.g005 patient plus the Figure 5. Scarring When Choroidal situation in which Granulomas Subside the infection was contracted. Thus, in an immunocompetent adult in the UK, enteroviruses are the commonest cause of viral meningitis, with meningococcus and pneumococcus the commonest bacterial agents. Tuberculosis is more common in people who have lived in a highly endemic area. In other parts of the world, the differential diagnosis may include viral infections such as West Nile virus in the continental United States and Japanese B encephalitis in Asia, or other pathogens such as rickettsiae, borrelia (Lyme disease), and protozoa. In the immunocompromised host, listeria must be considered, and there is an increased risk of fungal infection and tuberculosis. Finally, it is important to consider sexual exposure, as both secondary syphilis and HIV seroconversion may present with meningitis. It is important, therefore, in the evaluation and management of patients presenting with a meningoencephalitis that the differential diagnosis be continually reviewed if the patient is not responding to therapy (Table 2). When appropriate investigations have been performed and are negative and symptoms persist, noninfective causes of CSF inflammation must be considered—as turned out to be the case here (Table 3).

VKH Syndrome VKH syndrome [4,5,6] is a systemic disease involving various melanocyte-containing organs. It is rare in white Northern Europeans and white Americans but much more common in people with darker, pigmented skin. For example, among patients presenting with uveitis, about one in ten in Japan and one in 50 in India have VKH syndrome [6,7]. It presents acutely with varying DOI: 10.1371/journal.pmed.0010007.g006 symptoms and signs, which include Figure 6. Vitiligo on Skin of Forearm

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online Take the Quiz online!

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15: 1047–1071. This paper discusses conventional tests, such as culture, and others such as antigen testing and PCR

Key Learning Points • Consider meningitis in the differential diagnosis of a patient presenting with fever and headache.

Rotbart HA (2000) Viral meningitis. Semin Neurol 20: 277–292. The virology, pathogenesis, epidemiology, clinical manifestations, diagnostic studies, and established and potential antiviral therapies for viral meningitis are discussed. A differential diagnosis of the aseptic meningitis syndrome is provided.

• CSF analysis is essential to confirm meningitis and as part of establishing the cause. • Consider non-infectious causes when a patient does not respond rapidly to therapy. • Blurring of vision must be investigated and may help in determining the underlying diagnosis or the presence of papilloedema.

Negrini B, Kelleher KJ, Wald ER (2000) Cerebrospinal fluid findings in aseptic versus bacterial meningitis. Pediatrics 105: 316–319. Polymorphonuclear cell predominance in the CSF does not discriminate between aseptic and bacterial meningitis.

Suggested Reading

Kamondi A, Szegedi A, Papp A, Seres A, Szirmai I (2000) VogtKoyanagi-Harada disease presenting initially as aseptic meningoencephalitis. Eur J Neurol 7: 719–722. This paper describes the neurological and eye signs in VKH syndrome.

Sutlas PN, Unal A, Forta H, Senol S, Kirbas D (2003) Tuberculous meningitis in adults: Review of 61 cases. Infection 31: 387–391. Early suspicion and appropriate longterm anti-tuberculosis therapy together with corticosteroids may reduce mortality and morbidity in patients with TB meningitis.

Seehusen DA, Reeves MM, Fomin DA (2003) Cerebrospinal fluid analysis. Am Fam Physician 68: 1103–1108. Lumbar puncture is frequently performed in primary care, and this review outlines the interpretation of the clinical and laboratory findings.

Redington JJ, Tyler KL (2002) Viral infections of the nervous system. Arch Neurol 59: 712–718. This review is an update on diagnosis and treatment.

Shah KH, Edlow JA (2002) Distinguishing traumatic lumbar puncture from true subarachnoid hemorrhage. J Emerg Med 23: 67–74. The purpose of this article is to assist emergency physicians in distinguishing traumatic lumbar punctures from subarachnoid hemorrhage.

Thomson RB Jr, Bertram H (2001) Laboratory diagnosis of central nervous system infections. Infect Dis Clin North Am

vessels growing into the retina through the damaged RPE (choroidal neovascular membrane).


much longer, with all the concomitant side effects. Inadequate initial treatment may increase the risk of recurrence and longterm complications. Treatment is required until the disease goes into remission. The meningoence-phalitic signs and retinal and choroidal signs settle quickly, often within a week or so, whereas the optic nerve inflammation may take longer to settle. The visual prognosis depends on the degree of permanent damage to the optic nerve and the macula area, which often shows considerable pigment clumping as a result of the damage to the RPE. Relapse affecting the optic nerve, choroids, and retina is uncommon, provided that treatment has been given for long enough. However, recurrent anterior uveitis requiring steroid drops is common. This is not a threat to sight if adequately controlled. As with any other cause of intraocular inflammation particularly associated with choroidal involvement, VKH syndrome can lead to reduced vision via cataracts, glaucoma damaging the optic nerve, and new

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References

1. Tang YW, Cleavinger PJ, Haijing L, Mitchell PS, Smith TF, et al. (2000) Analysis of candidate–host immunogenetic determinants in herpes simplex virus–associated Mollaret’s meningitis. Clin Infect Dis 30: 176–178. 2. Pai M, Flores LL, Pai N, Hubbard A, Riley LW, et al. (2003) Diagnostic accuracy of nucleic acid amplification tests for tuberculous meningitis: A systematic review and meta-analysis. Lancet Infect Dis 3: 633–643. 3. Kamondi A, Szegedi A, Papp A, Seres A, Szirmai I (2000) Vogt-KoyanagiHarada disease presenting initially as aseptic meningoencephalitis. Eur J Neurol 7: 719–722. 4. Read RW (2002) Vogt-Koyanagi-Harada disease. Ophthalmol Clin North Am 15: 333–341. 5. Read RW, Holland GN, Rao NA, Tabbara KF, Ohno S, et al. (2001) Revised diagnostic criteria for Vogt-Koyanagi-Harada disease: Report of an international committee on nomenclature. Am J Ophthalmol 131: 647–652. 6. Mondkar SV, Biswas J, Ganesh SK (2000) Analysis of 87 cases with VogtKoyanagi-Harada disease. Jpn J Ophthalmol 44: 296–301. 7. Wakabayashi T, Morimura Y, Miyamoto Y, Okada AA (2003) Changing patterns of intraocular inflammatory disease in Japan. Ocul Immunol Inflamm 11: 277–286. 8. Wares DF, Singh S, Acharya AK, Dangi R (2003) Non-adherence to tuberculosis treatment in the eastern Tarai of Nepal. Int J Tuberc Lung Dis 7: 327–335.

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Open access, freely available online

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Case Report

Generalized Seizure in a Mauritian Woman Taking Bupropion Marie France Lan Cheong Wah1*, Lan Sem Hing Lan Cheong Wah2 Case report from 1 Faculty of Science, University of Mauritius, Reduit, Mauritius, 2 Port-Louis, Mauritius

Competing Interests: The authors have declared that no competing interests exist. Author Contributions: Both authors contributed equally to preparing the case report. Citation: Lan Cheong Wah MF, Lan Cheong Wah LSH (2004) Generalized seizure in a Mauritian woman taking bupropion. PLoS Med 1(1): e15. Received: July 1, 2004 Accepted: August 13, 2004 Published: October 19, 2004

DOI: 10.1371/journal.pmed.0010015 Copyright: Ó 2004 Marie France Lan Cheong Wah and Lan Sem Hing Lan Cheong Wah. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * To whom correspondence should be addressed. E-mail: lan.sun@ uom.ac.mu

PRESENTATION of CASE 24-y-old woman was admitted to the emergency department having had a generalized seizure (acute loss of consciousness, convulsive movements of her arms and legs, and confusion on regaining consciousness). She was on the sixth day of treatment with 300 mg daily of slowrelease bupropion (Zyban SR) as an aid to smoking cessation. She had a past medical history of tonsillectomy and hay fever, for which she was taking budesonide nasal drops (two drops daily, each drop 200 mcg). She was on no other medication. There was no history of head trauma, liver disease, or alcohol withdrawal. Clinical examination, including neurological examination, was normal. The patient’s weight was 48 kg. Her blood pressure was 130/80 mm Hg. Electrocardiogram showed a sinus tachycardia at 102 beats per minute. Radiography of the skull and a computed tomography scan of the brain without contrast were both normal. The patient’s blood glucose, urea, electrolytes, and liver function tests were all normal. Her serum calcium was 2.01 mmol/l (normal range, 2.0–2.6 mmol/l) and her hemoglobin was 116 g/l (normal range, 120–140 g/l). The bupropion was discontinued, and the patient recovered without any further seizures or other neurological sequelae.

A

Case Discussion Bupropion for Smoking Cessation Originally developed as an antidepressant, bupropion has more recently been licensed in many countries as an aid to smoking cessation. It came onto the Mauritian market as a smoking cessation aid in November 2003. The British National Formulary (www. bnf.org) recommends starting the drug one to two weeks before the target smoking stop date, initially at a dose of 150 mg daily for 6 d and then 150 mg twice daily. The maximum period of treatment is 7–9 wk; treatment PLoS Medicine | http://www.plosmedicine.org

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should be discontinued if abstinence is not achieved by 7 wk. The efficacy of bupropion as an aid to smoking cessation has been shown in randomized, double-blind, placebo-controlled trials [1,2]. But there have also been reports of death, seizure, serum sickness, generalized acute urticaria, myocardial infarction, and psychosis in people taking the drug [3,4,5,6,7]. Our report is of a woman who had a generalized seizure on the sixth day of treatment with bupropion; she had no other risk factors for seizures.

Contraindications to Bupropion To reduce the risk of seizures, the drug should not be given to patients with a current seizure disorder or any history of seizures, with a current or previous diagnosis of bulimia or anorexia nervosa, with a known central nervous system tumour, or to those experiencing abrupt withdrawal from alcohol or benzodiazepines [8,9]. The United Kingdom Medicines Control Agency states that bupropion must not be prescribed in patients with other risk factors for seizures, unless there is a compelling clinical justification for which the potential medical benefit of smoking cessation outweighs the potential increased risk of seizure [8]. Predisposing risk factors for seizure include the following: concomitant use of medications known to lower seizure threshold (including antipsychotics, antidepressants, antimalarials, tramadol, theophylline, systemic steroids, quinolones, and sedating antihistamines), history of head trauma, diabetes treated with hypoglycemics or insulin, history of alcohol abuse, and use of stimulants or anorectic products [8].

The Seizure Risk Bupropion is associated with a dose-related risk of seizure. The Medicines Control Agency states that the incidence of seizures is one in 1,000 based on doses up to the maximum recommended daily dose of 300 mg per day [8]. The seizure risk may be reduced by taking no more than 150 mg (1 October 2004 | Volume 1 | Issue 1 | e15

3. Johnston JA, Lineberry CG, Ascher JA, Davidson J, Khayrallah MA, et al. (1991) A 102-center prospective study of seizure in association with bupropion. J Clin Psychiatry 52: 450–456. 4. Wooltorton E (2002) Bupropion (Zyban, Wellbutrin SR): Reports of deaths, seizures, serum sickness. Can Med Assoc J 166: 68. 5. Loo WJ, Alexandroff A, Flanagan N (2003) Bupropion and generalized acute urticaria: A further case. Br J Dermatol 149: 655–680. 6. Patterson RN, Herity NA (2002) Acute myocardial infarction following bupropion (Zyban). Quar J Med 95: 58–59. 7. Neumann M, Livak V, Paul HW, Laux G (2002) Acute psychosis after administration of bupropion hydrochloride (Zyban). Pharmacopsychiatry 35: 247–248. 8. UK Medicines and Healthcare Products Regulatory Agency(2003) Zyban (bupropion hydrochloride)—Safety update 240702. Available: hppt:// w w w . m c a .g o v .u k / ou r w o rk /m on it o r sa fe qu a l m e d/ sa f et y m es sa ge s/ zyban26702.pdf. Accessed 24 August 2004. 9. Committee on Safety of Medicines and Medicines Control Agency(2001) Reminder: New guidance for bupropion (Zyban). Curr Prob Pharmacovigilance 27: 12. Available: http://www.mca.gov.uk/ourwork/ monitorsafequalmed/currentproblems/cpaug2001.pdf. Accessed 24 August 2004.

pill) at a time and, if taking two daily doses of 150 mg each (two pills per day), ensuring that doses are taken at least 8 h apart (see www.bnf.org). Up to 24 July 2002, in the UK there were 184 reports of seizures suspected as being associated with the use of bupropion [8]. In about half of the reports, patients had a past history of seizures and/or risk factors for their occurrence.

The Presenting Case In the case we have presented, the patient had no current or previous history of seizure disorders, bulimia, or anorexia nervosa. She was on the sixth day of treatment with bupropion, taking 300 mg per day in two separate doses. She had no other predisposing risk factors for seizure. In particular she was taking no prescription medications, or over-the-counter medications (such as those containing ephedrine products) that are known to lower the seizure threshold. While systemic steroids can lower the seizure threshold, systemic absorption of the nasal steroid drops the patient was taking is an extremely unlikely cause for her seizure (she was not taking a high dose of high-strength drops). Her weight (48 kg) may have been an important factor, since bupropion reaches higher plasma levels in smaller individuals—indeed clinical trials of the drug have generally excluded patients weighing under 100 lb (about 45 kg) [2].

Learning Points  This case report is a useful reminder to clinicians that bupropion is associated with a dose-dependent risk of seizures.  In about half of the reports of seizure associated with bupropion, there was a past history of seizures and/or risk factors for their occurrence.

It is extremely important to adequately assess seizure risk before prescribing bupropion. 

References 1. Hurt RD, Sachs DPL, Glover ED, Offord KP, Johnston JA, et al. (1997) A controlled trial of sustained release bupropion and placebo for smoking cessation. N Engl J Med 337: 1195–1202. 2. Jorenby DE, Leischow SJ, Nides MA (1999) A controlled trial of sustained release bupropion, a nicotine patch or both for smoking cessation. N Engl J Med 340: 685–691.

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 Patients should be made aware of the possible adverse effects of the drug.

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Distribution of Major Health Risks: Findings from the Global Burden of Disease Study Anthony Rodgers1*, Majid Ezzati2, Stephen Vander Hoorn1, Alan D. Lopez3, Ruey-Bin Lin1, Christopher J. L. Murray2, Comparative Risk Assessment Collaborating Group 1 Clinical Trials Research Unit, School of Population Health, University of Auckland, New Zealand, 2 Harvard School of Public Health, Boston, Massachusetts, United States of America, 3 School of Population Health, University of Queensland, Brisbane, Australia

Competing Interests: The authors have declared that no competing interests exist. ADL is a member of the editorial board of PLoS Medicine. Author Contributions: AR, ME, ADL and CJLM designed the study. SVH and RBL analyzed the data. AR, ME, SVH, and ADL contributed to writing the paper. Academic Editor: Thomas Novotny, University of California at San Francisco, United States of America Citation: Rodgers A, Ezzati M, Vander Hoorn S, Lopez AD, Lin RB, et al. (2004) Distribution of major health risks: Findings from the Global Burden of Disease study. PLoS Med 1(1): e27. Received: June 2, 2004 Accepted: August 13, 2004 Published: October 19, 2004

DOI: 10.1371/journal.pmed.0010027 Copyright: Ó 2004 Rodgers et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: BMI, body mass index; DALY, disability-adjusted life-year; PAF, population attributable fraction; SBP, systolic blood pressure; SD, standard deviation; WHO, World Health Organization *To whom correspondence should be addressed. E-mail: a.rodgers@ ctru.auckland.ac.nz

ABSTRACT Background Most analyses of risks to health focus on the total burden of their aggregate effects. The distribution of risk-factor-attributable disease burden, for example by age or exposure level, can inform the selection and targeting of specific interventions and programs, and increase costeffectiveness.

Methods and Findings For 26 selected risk factors, expert working groups conducted comprehensive reviews of data on risk-factor exposure and hazard for 14 epidemiological subregions of the world, by age and sex. Age-sex-subregion-population attributable fractions were estimated and applied to the mortality and burden of disease estimates from the World Health Organization Global Burden of Disease database. Where possible, exposure levels were assessed as continuous measures, or as multiple categories. The proportion of risk-factor-attributable burden in different population subgroups, defined by age, sex, and exposure level, was estimated. For major cardiovascular risk factors (blood pressure, cholesterol, tobacco use, fruit and vegetable intake, body mass index, and physical inactivity) 43%–61% of attributable disease burden occurred between the ages of 15 and 59 y, and 87% of alcohol-attributable burden occurred in this age group. Most of the disease burden for continuous risks occurred in those with only moderately raised levels, not among those with levels above commonly used cut-points, such as those with hypertension or obesity. Of all disease burden attributable to being underweight during childhood, 55% occurred among children 1–3 standard deviations below the reference population median, and the remainder occurred among severely malnourished children, who were three or more standard deviations below median.

Conclusions Many major global risks are widely spread in a population, rather than restricted to a minority. Population-based strategies that seek to shift the whole distribution of risk factors often have the potential to produce substantial reductions in disease burden.

Introduction Reliable and comparable analysis of risks to health is an important component of evidencebased policies and programs for disease prevention [1,2]. An important feature of risk assessment, with implications for specific interventions as well as broad prevention policies, is the distribution of disease burden among population subgroups. These subgroups may be defined by demographic factors, such as age and sex, or by socioeconomic status. Subgroups can also be defined by the level of exposure to a risk factor, if exposures are defined in multiple categories or continuously. Understanding the distribution of risk-factor burden is particularly important for targeting interventions. For example, the large number of road traffic accident injuries and deaths among young adult males may be largely associated with binge alcohol consumption by this group. Interventions that focus on this population subgroup and their specific drinking behaviors may be more effective or cost-effective than, for example, raising alcohol taxes, which would have a more diffuse impact on alcohol consumption. On the other hand, the majority of effects from

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Disease Burden of Major Health Risks

minimum exposure distribution as the counterfactual has the advantage of providing a vision of potential gains in population health by risk reduction from all levels of suboptimal exposure in a consistent way across risk factors. Estimates were made for eight age groups (0–4, 5–14, 15–24, 25–44, 45–59, 60–69, 70–79, and 80þ y), both sexes, and 14 Global Burden of Disease subregions (Table 2). PAFs were estimated for mortality and incidence and were applied to regional cause-specific mortality and disease burden from the World Health Organization (WHO) Global Burden of Disease database (Table 1). Burden of disease, reported annually in the annexes of the World Health Report, was expressed in disability-adjusted life-years (DALYs) with methods and assumptions described elsewhere [11]. Aggregate results (both mortality and disease burden) for all ages, sexes, and exposure levels have been reported elsewhere [1,2]. Many risks act simultaneously to cause disease, and joint effects have also been estimated [12], though the separate effects are presented in this paper. The aim of the analyses reported here was to obtain estimates of the distribution of disease burden by age, sex, and exposure level. To make separate estimates of disease burden by exposure level, the relationship in equation 1 was re-estimated with the entire exposure distribution divided into ‘‘narrow bands,’’ with each band corresponding to one level of exposure, and the estimation repeated for each such exposure band.

risk factors such as blood pressure have been found to be among those at moderately elevated levels, motivating interventions beyond those intended for clinical hypertension [3,4,5]. The distributions of the health effects of risk-factor exposure by age and sex or by exposure level have been studied in specific cohorts and for specific risk factors [6,7,8]. Most notably Rose’s seminal work stated that ‘‘a large number of people exposed to a small risk may generate many more cases than a small number exposed to high risk’’ [9]. Using the data from a global and regional assessment of multiple major risk factors, this paper reports the distribution by exposure levels, age, and sex of disease burden attributable to several major risk factors. The findings of this analysis confirm that Rose’s observations have global relevance and also illustrate important new patterns on specific distributions of disease burden for multiple risks, in different age groups, and in populations at various stages of economic development.

Methods The methods and data sources for individual risk factors and for estimating population attributable fractions (PAFs) and disease burden attributable to them have been fully described elsewhere [1,2] and are summarized below. The contribution of a risk factor to disease or mortality relative to some alternative exposure scenario (i.e., PAF, defined as the proportional reduction in population disease or mortality that would occur if exposure to the risk factor were reduced to an alternative exposure scenario [10]) is given by the generalized ‘‘potential impact fraction’’: Zm PIF ¼

RRðxÞPðxÞdx


x¼0

Results The distribution of deaths and DALYs attributable to the risk factors by age and sex is shown in Table 3. Disease burden attributable to being underweight and to micronutrient deficiencies in children was equally distributed among males and females. The total all-age disease burden from iron and vitamin A deficiencies was greater in females because of effects on maternal mortality and morbidity conditions. The disease burden of other diet-related risks, physical inactivity, environmental risks, and unsafe sex (sex with an infected partner without any measures to prevent infection) occurred almost equally in males and females. However, approximately 80% of disease burden from addictive substances and 60%– 90% from occupational risks occured among men (bearing in mind that the assessment considered only formal employment). Women experienced an estimated two-thirds of the disease burden from childhood sexual abuse and all of the burden caused by a lack of contraception. The estimated disease burden from childhood and maternal undernutrition; unsafe water, sanitation, and hygiene; and global climate change (much of whose estimated effects are mediated through nutritional and water variables) was almost exclusively among children under 5 y of age. For these risks, more than 85% of total attributable burden occurred in this age group, with the exception of iron deficiency, for which 30% of burden was borne by women of childbearing age. Only a small fraction of disease burden from the risk factors considered was among the 5–14 y olds. This was because some of the leading diseases of this age group (e.g., injuries and depression) have complex causes that could not easily be included in the current risk-based framework [12]. For other leading diseases of this group (e.g., diarrhea and lower respiratory infections), most epidemiological studies have

Zm RRðxÞP9ðxÞdx x¼0

Zm

ð1Þ

RRðxÞPðxÞdx x¼0

RR(x) is relative risk at exposure level x, P(x) is the population distribution of exposure, P9(x) is the alternative or counterfactual distribution of exposure, and m is the maximum exposure level. The alternative (counterfactual) scenario used in this work is the exposure distribution that would result in the lowest population risk, referred to as the theoretical minimum-risk exposure distribution (Table 1) [1,2]. The theoretical minimum exposure distribution was zero in most cases since zero exposure reflected minimum risk (e.g., no smoking). For some risk factors, zero exposure was an inappropriate choice as the theoretical minimum, either because it is physiologically impossible (e.g., body mass index [BMI] and cholesterol) or because there are physical lower limits to exposure reduction (e.g., ambient particulate matter concentration and occupational noise). For these risk factors, the lowest levels observed in specific populations and epidemiological studies were used as the theoretical minimum. For factors with protective effects (i.e., fruit and vegetable intake and physical activity), a counterfactual exposure distribution was chosen based on levels in high-intake populations (e.g., fruit and vegetable intake in Greece) and the level to which the benefits may continue given current scientific evidence. Using theoretical PLoS Medicine | http://www.plosmedicine.org

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Underweight

Childhood and maternal undernutrition

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Sexual and reproductive health

Non-use and use of ineffective methods of contraception

Unsafe sex

Other nutrition-related High blood pressure risk factors and physical activity High cholesterol High BMI Low fruit and vegetable consumption Physical inactivity

Zinc deficiency

Vitamin A deficiency

Iron deficiency anemia

Risk Factor

Category

6.3%

No unsafe sex

Use of modern contraceptives for all women who want to space or limit future pregnancies

1.3%

All having at least 2.5 h/wk of moderate-inten- 1,922 sity activity or equivalent (4,000 KJ/wk)

Three categories: inactive, insufficiently active (,2.5 h/wk of moderate-intensity activity, or less than 4,000 KJ/wk), and sufficiently active; activity in discretionary-time, work, and transport considered Sex with an infected partner without any measures to prevent infection (represented as parameters of an HIV model) Prevalence of traditional methods or non-use of contraception

149

2,886

2.8% 2.3% 1.8%

4,415 2,591 2,726

3.8 6 0.6 mmol/l (147 6 23 mg/dl) 21 6 1 kg/m2 600 6 50 g intake per day for adults

Level of total blood cholesterol BMI (height over weight squared) Fruit and vegetable intake per day

0.6%

1.9%

1.8%

2.4%

9.5%

Deaths DALYs (Thousands) (Percent of Total Global Burden of Disease)

4.4%

Theoretical Minimum

Children ,1 SD weight-for-age compared to Same fraction of children ,1 SD weight-for- 3,748 the international reference group in 1-SD incre- age compared to the international reference ments; maternal BMI ,20 kg/m2 group; all women of childbearing age with body mass index .20 kg/m2 841 Hemoglobin level (g/dl) estimated from preva- Hemoglobin distributions that halve anemia lence of anemia prevalence, estimated to occur if all iron deficiency were eliminateda 778 Prevalence of vitamin A deficiency, estimated No vitamin A deficiency as low serum retinol concentrations (,0.70 lmol/L) among children aged 0–4 y and among pregnant women (aged 15–44 y) 789 Less than the United States recommended The entire population consuming sufficient dietary allowances for zinc dietary zinc to meet physiological needs, taking into account routine and illness-related losses and bioavailability Level of systolic blood pressure 115 6 6 mm Hg 7,141

Exposure Variable

Table 1. Leading Global Risk Factors, Exposure Variables, Theoretical Minima, and Attributable Deaths and Disease Burden (measured in DALYs) in 2000

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Occupational risks

Environmental risks

Tobacco

Addictive substances

047

Noise

Ergonomic stressors

Airborne particulates

Carcinogens

Risk factors for injuries

Lead Global climate change

Indoor smoke from household use of solid fuels Urban air pollution

Unsafe water, sanitation, and hygiene

Illicit drugs

Alcohol

Risk Factor

Category

Table 1. Continued

Proportions of workers exposed to background, low, and high levels of workplace carcinogens Proportions of workers with background, low, and high levels of exposure High, moderate, and low exposure based on occupational categories High (.95 dBA) and moderate (85–90 dBA) exposure categories

Estimated annual average particulate matter concentration for particles with aerodynamic diameters less than 2.5 lm (PM2.5) or 10 lm (PM10) Current blood lead levels Climate scenarios based on various carbon emissions and concentrations Current proportions of workers exposed to injury risk factors

Current levels of smoking impact ratio (indirect indicator of accumulated smoking risk based on excess lung cancer mortality); oral tobacco use prevalence Current alcohol consumption volumes and patterns Use of amphetamine, cocaine, heroin, or other opioids and intravenous drugs Six scenarios, ranging from regulated water and sanitation with hygiene to no improved water supply and no improved sanitation Household use of solid fuels and ventilation

Exposure Variable

1,804

No alcohol useb

Physical workload at the level of managers and professionals (low) Less than 85 dBA on average over eight working hours

0

0

356

109

0.3%

0.1%

0.4%

0.1%

0.7%

310 Exposure corresponding to lowest rate of work-related fatalities observed: one per million per year for 16–17-y-olds employed as service workers in the United States No work-related exposure above background to chemical or physical agents that cause cancer No work-related exposure above background

0.9% 0.4%

234 154

0.016 lg/dl blood lead levelsc 1961–1990 atmospheric carbon concentrations

0.4%

2.6%

799

1619

No household solid fuel use

3.7%

0.8%

4.0%

4.1%

DALYs (Percent of Total Global Burden of Disease)

7.5 lg/m3 for PM2.5; 15 lg/m3 for PM10

1,730

Absence of transmission of diarrheal disease through water, sanitation, and hygiene

204

4,907

No tobacco use

No illicit drug use

Deaths (Thousands)

Theoretical Minimum

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See Table 1 in Ezzati et al. [2] for disease outcomes and data sources. a The resulting hemoglobin levels vary across regions and age-sex groups (from 11.66 g/dl in children under five in a region of Southeast Asia to greater than 14.5 g/dl in adult males in developed countries) because the other risks for anemia (e.g., malaria) vary. b Theoretical minimum for alcohol is zero, the global theoretical minimum. Specific subgroups may have a non-zero theoretical minimum. c Theoretical minimum for lead is the blood lead levels expected at background exposure levels. Health effects were quantified for blood lead levels above 5 lg/dl where epidemiological studies have quantified hazards. DOI: 10.1371/journal.pmed.0010027.t001

0.6% No abuse

Contaminated injections in health-care settings Child sexual abuse Other selected risk factors

abuse,

contact Prevalence of noncontact abuse, and intercourse

79

0.7% 501 No contaminated injections Exposure to least one contaminated injection

Risk Factor Category

Table 1. Continued

Exposure Variable

Theoretical Minimum

Deaths (Thousands)

DALYs (Percent of Total Global Burden of Disease)

Disease Burden of Major Health Risks

focused on children under 5 y of age and do not provide hazard estimates for older children. The disease burden from other diet-related risks, tobacco, and occupational risks (except injuries and back pain) was almost equally distributed among adults above and below the age of 60 y. For example, 43% of disease burden due to blood pressure and 61% of burden due to tobacco occurred in the 15–59-y age group. More than 90% of disease burden attributable to lack of contraception, illicit drugs, occupational ergonomic stressors, risk factors for injury, and childhood sexual abuse occurred in adults below the age of 60 y. Similarly, 67%–87% of the disease burden attributable to alcohol, unsafe sex, and unsafe health-care injections arose from events occurring between 15 and 59 y of age. Most of the risks whose hazards are concentrated in the younger adults are causes of injuries, neuropsychiatric diseases, maternal conditions, and HIV/AIDS. Assessment by the level of economic and demographic development illustrated that, with the exception of alcohol, which has global presence, the majority of disease burden from risks for mortality and disease among young adults is concentrated in developing countries (see Figure 1 in [2]). Stratification of economic and demographic development was also a determinant of the age distribution patterns for risk factors for chronic diseases, which occurred in younger age groups in developing countries than in developed regions. For example, in highmortality developing regions, 69% of the tobacco burden occurred in people aged 15–59 y, whereas this share was 63% for lower-mortality developing countries and 55% for developed countries. The distributions of attributable risk-factor burden by exposure levels are shown in Table 4 for those risks quantified using categorical variables and in Figure 1 for those with continuous variables. For most of these risks a substantial proportion of attributable disease burden occurred among those with modest elevations of risk. For example, while 35% of the large disease burden from being underweight in childhood, the leading risk factor for global loss of healthy life, occurred in severely underweight children who would be subject to clinical interventions (i.e., more than three standard deviations [SDs] from referent group median), the rest occurred in children only 1–3 SDs below the median. Even though the relative risks for the latter group are much lower, the number of children exposed to risk at this level is so great that the total attributable disease burden amounted to much more than that occurring in the severe category. Similarly, 52% of the attributable burden from physical inactivity occurred among those engaged in some but less than the recommended level of 2.5 h per week of moderate-intensity activity. For unsafe water, sanitation, and hygiene, almost all of the attributable disease burden was distributed among three of the five at-risk exposure categories, with approximately equal levels. This reflects the fact that the exposure categories were defined as the presence or absence of technology-based water and sanitation interventions. During decades of water and sanitation projects, many countries have ‘‘clustered’’ in a limited number of technology groups. There is likely to be large heterogeneity of exposure within each exposure category, however, because of factors such as quantity of water consumed and hygiene behavior [13]. Figure 1 shows that at the aggregated level a substantial proportion of the attributable disease burden for high blood 048

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Table 2. Global Burden of Disease 2000 Subregions WHO Region

Mortality Stratuma

Countries

AFR

D

Algeria, Angola, Benin, Burkina Faso, Cameroon, Cape Verde, Chad, Comoros, Equatorial Guinea, Gabon, Gambia, Ghana, Guinea, Guinea-Bissau, Liberia, Madagascar, Mali, Mauritania, Mauritius, Niger, Nigeria, Sao Tome and Principe, Senegal, Seychelles, Sierra Leone, Togo Botswana, Burundi, Central African Republic, Congo, Coˆte d’Ivoire, Democratic Republic of the Congo, Eritrea, Ethiopia, Kenya, Lesotho, Malawi, Mozambique, Namibia, Rwanda, South Africa, Swaziland, Uganda, United Republic of Tanzania, Zambia, Zimbabwe Canada, Cuba, United States of America Antigua and Barbuda, Argentina, Bahamas, Barbados, Belize, Brazil, Chile, Colombia, Costa Rica, Dominica, Dominican Republic, El Salvador, Grenada, Guyana, Honduras, Jamaica, Mexico, Panama, Paraguay, Saint Kitts and Nevis, Saint Lucia, Saint Vincent and the Grenadines, Suriname, Trinidad and Tobago, Uruguay, Venezuela Bolivia, Ecuador, Guatemala, Haiti, Nicaragua, Peru Bahrain, Cyprus, Iran (Islamic Republic of), Jordan, Kuwait, Lebanon, Libyan Arab Jamahiriya, Oman, Qatar, Saudi Arabia, Syrian Arab Republic, Tunisia, United Arab Emirates Afghanistan, Djibouti, Egypt, Iraq, Morocco, Pakistan, Somalia, Sudan, Yemen Andorra, Austria, Belgium, Croatia, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Israel, Italy, Luxembourg, Malta, Monaco, Netherlands, Norway, Portugal, San Marino, Slovenia, Spain, Sweden, Switzerland, United Kingdom Albania, Armenia, Azerbaijan, Bosnia and Herzegovina, Bulgaria, Georgia, Kyrgyzstan, Poland, Romania, Slovakia, Tajikistan, The Former Yugoslav Republic of Macedonia, Turkey, Turkmenistan, Uzbekistan, Yugoslavia Belarus, Estonia, Hungary, Kazakhstan, Latvia, Lithuania, Republic of Moldova, Russian Federation, Ukraine Indonesia, Sri Lanka, Thailand Bangladesh, Bhutan, Democratic People’s Republic of Korea, India, Maldives, Myanmar, Nepal Australia, Brunei Darussalam, Japan, New Zealand, Singapore Cambodia, China, Cook Islands, Fiji, Kiribati, Lao People’s Democratic Republic, Malaysia, Marshall Islands, Micronesia (Federated States of), Mongolia, Nauru, Niue, Palau, Papua New Guinea, Philippines, Republic of Korea, Samoa, Solomon Islands, Tonga, Tuvalu, Vanuatu, Viet Nam

E

AMR

A B

EMR

D B

EUR

D A

B

C SEAR

B D

WPR

A B

Population (Thousands) 294,078

345,515

325,183 430,932

71,230 139,059 342,576 411,889

218,458

243,184 293,819 1,241,806 154,354 1,532,933

High-mortality developing regions: AFR-D, AFR-E, AMR-D, EMR-D, and SEAR-D. Lower-mortality developing regions: AMR-B, EMR-B, SEAR-B, and WPR-B. Developed regions: AMR-A, EUR-A, EUR-B, EUR-C, and WPR-A. a A, very low child mortality and very low adult mortality; B, low child mortality and low adult mortality; C, low child mortality and high adult mortality; D, high child mortality and high adult mortality; E, high child mortality and very high adult mortality. DOI: 10.1371/journal.pmed.0010027.t002

able to elevated blood pressure, cholesterol, and BMI occurred at lower levels in developing regions compared to developed regions, mainly because of lower age-specific exposure levels in those populations. Figure 2 also shows that the skewness of the distribution of disease burden was not substantially different across different age groups for BMI. This is because the comparatively larger relative risk per unit BMI at younger ages (which leads to more right-hand skew) is counterbalanced by the comparatively lower BMI at younger ages (which leads to left-hand skew). Therefore, the different distributions of BMI-attributable disease burden by region resulted not from the different age structures of populations across major world regions, but rather from the lower age-specific BMI levels in those countries [14]. This is in contrast to blood pressure, for which disease burden in younger age groups occurred at lower exposures because the

pressure, cholesterol, and BMI and low fruit and vegetable intake occurred in the ‘‘mid-range’’ exposures. For example, the second and third quartiles (i.e., half of attributable disease burden) occurred at the following levels: systolic blood pressure (SBP) of 130–150 mm Hg, cholesterol of 5.0–6.1 mmol/l, BMI of 25–32 kg/m2, and fruit and vegetable intake of 150–300 g/d (2–4 servings/d). This was similar to or greater than the amount of disease burden occuring among individuals with risk-factor levels above the commonly used, but arbitrary, thresholds for hypertension, hypercholesterolemia, and obesity indicated in Figure 1. Despite the above finding on the important role of moderately elevated levels in total disease burden, the actual patterns of how disease burden is distributed among exposure levels varied across different regions and risk factors (Figure 2). For example, Figure 2 shows that a comparatively larger fraction of the disease burden attributPLoS Medicine | http://www.plosmedicine.org

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Table 3. Distribution of Risk-Factor-Attributable Deaths and Disease Burden (DALYs) by Age and Sex Category

Risk Factor

Mortality (Percent)

Disease Burden (Percent)

0–4 5–14 15–59 60 Males Females 0–4 5–14 15–59 60 Males Females Childhood and Childhood and maternal 100 0 maternal undernutrition underweight Iron deficiency anemia 72 1 Vitamin A deficiency 85 1 Zinc deficiency 100 0 Other nutrition-related High blood pressure 0 0 risk factors and physical inactivity High cholesterol 0 0 Overweight and obesity 0 0 (high BMI) Low fruit and vegetable 0 0 consumption Physical inactivity 0 0 Addictive substances Tobacco 0 0 Alcohol 1 1 Illicit drugs 0 0 Sexual and reproductive Unsafe sex 16 1 health Non-use and use of inef0 0 fective methods of contraception Environmental risks Unsafe water, sanitation, 68 5 and hygiene Urban air pollution 3 0 Indoor air pollution from 56 0 household use of solid fuels Lead 0 0 Global climate change 86 3 Occupational risks Risk factors for injuries 0 0 Carcinogens 0 0 Airborne particulates 0 0 Ergonomic stressors 0 0 Noise 0 0 Other selected Contaminated injections 10 2 risk factors in health-care settings Child sexual abuse 0 0

0

0

51

49

100 0

0

0

51

49

22 14 0 19

4 0 0 81

45 43 51 49

55 57 49 51

62 86 100 0

6 1 0 0

30 12 0 43

2 0 0 57

45 44 51 54

55 56 49 46

22 26

78 74

48 45

52 55

0 0 0 0

50 57

50 43

55 47

45 53

23

77

53

47

0 0

49

51

57

43

21 30 65 100 77

79 70 33 0 6

50 79 91 80 47

50 21 9 20 53

0 0 3 2 1

48 61 87 98 79

52 39 9 0 2

53 82 85 77 46

47 18 15 23 54

100

0

0

100

0 0

100

0

0

100

13

14

52

48

77 8

13

3

51

49

16 5

81 38

51 41

49 59

12 0 83 0

40 8

49 9

56 49

44 51

41 6 85 28 17 0 0 53

57 5 14 72 83 0 0 35

66 49 94 85 74 0 0 63

34 51 6 15 26 0 0 37

75 88 0 0 0 0 0 16

0 5 0 0 0 0 0 3

16 6 95 51 65 95 89 67

8 1 5 49 35 5 11 13

55 49 93 83 77 59 67 61

45 51 7 17 23 41 33 39

81

22

48

52

0 0

96

4

36

64

0 0 1 0 18

See Table 1, and Figure 1 in [2], for definition of each risk factor, data sources and methods, and total magnitude of mortality and DALYs. DOI: 10.1371/journal.pmed.0010027.t003

age patterns of exposure and relative risk do not entirely compensate.

dominated by absence or limitations of direct studies on exposure, hazard, and background disease burden, rather than parameter uncertainty, such as sampling error. This has motivated innovative assumptions and extrapolations even in the case of the best-studied risk factors in a limited number of countries [6]. While estimates of hazard size in individual studies were as much as possible adjusted for confounding effects, it is likely that residual confounding effects remain to some extent, hence leading to errors in estimation. Extrapolation of hazard from a limited number of studies to other populations is another source of potential error. While the robustness of relative measures of risk has been confirmed for more proximal factors in studies across populations

Discussion The findings reported here should be considered within the context of limited available data and are subject to uncertainty, which varies across risk factors and is generally most marked in developing countries. Full discussion of uncertainty in the basic data sources and parameters is provided elsewhere and includes the uncertainties in estimates of disease incidence, duration, severity, and disability weighting [1,2]. Uncertainty in this risk assessment is by far PLoS Medicine | http://www.plosmedicine.org

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Figure 1. Distribution by Exposure Level of Attributable Disease Burden Due to Selected Continuous Risk Factors Figure 1 shows the distribution of the estimated cardiovascular disease (CVD) burden of disease (in DALYs) attributable to four major continuous risk factors, by exposure levels. Half the attributable burden occurs to the left of the solid vertical line and half occurs to the right. The dashed vertical lines indicate commonly used thresholds—150 mm Hg for hypertension, 6.0 mmol/l for hypercholesterolemia, and 30 kg/m2 for obesity. The blood pressure and cholesterol levels plotted are the estimated usual levels [22], which tend to have a smaller SD than levels based on one-off measurements commonly used in population surveys, because of normal day-to-day and week-to-week fluctuations. For example, the distribution of usual blood pressure is about half as wide as the distribution of one-off blood pressure measures, and so many fewer people would be classified as hypertensive (or hypotensive) if classifications were based on usual rather than one-off blood pressure. Thus, if a population mean SBP was 134 mm Hg, the SD of once-only measures might be 17 mm Hg (with about 18% of the population having one-off SBP over 150 mm Hg), and the SD of usual SBP based on many measures would be about 9 mm Hg (hence about 5% of the population would have usual SBP over 150 mm Hg). DOI: 10.1371/journal.pmed.0010027.g001

exposure distribution, not modeled here, would also lead to an underestimation of events occurring in those who were hypertensive, hypercholesterolemic, or obese. The importance of skewness is emphasized by the observation that the recent increase in the proportion of people who are overweight and obese (e.g., in the United States) has involved not only a shift in the distribution, but also increasing skewness, which has extended the high-exposure tail. Coupled with risk-factor correlation, this should motivate more systematic data collection and reporting to determine mean exposure as well as the complete shape of distribution. The temporal nature of risk-factor exposure also has implications for the current cross-sectional estimates. There is an expectation that the size and rank order of risk-factor burden will alter in coming decades as a result of changes in exposure levels and delays between exposure and hazard. For example, it is predicted that by 2020 the leading risks to health will be (1) unsafe sex, principally because of HIV/AIDS and driven by increasing exposure, and (2) tobacco, because of market expansion of tobacco products in developing countries and delayed temporal effects on major health outcomes such as lung cancer [1]. This analysis in multiple age and exposure categories, or along a continuum of exposures, showed that, globally, a considerable proportion of the disease burden attributable to major risk factors occurred among those with only moderately raised levels, not the extremes such as those that define hypertension, obesity, or severe malnutrition. Further for

[15,16,17], their extrapolation is an important source of uncertainty for more distal risks (e.g., childhood sexual abuse) or those whose effects are heterogeneous (e.g., alcohol and injuries versus alcohol and cancer). Direct exposure data for many risk factors are limited because of both difficulties in their measurement and underinvestment in risk-factor surveillance, especially in developing countries. Of particular relevance to the current analysis is the fact that, due to data limitations, some risks were measured using categorical variables (e.g., indoor smoke from solid fuels, underweight, and physical inactivity) even though the health effects occur along a continuum. Other risk factors were represented using indirect or aggregate indicators (e.g., smoking impact ratio for accumulated hazards of smoking, and motor vehicle accident registries for alcohol-caused accidents) that do not allow quantification of risks along continua of exposure. Two important sources of uncertainty with implications for interventions are correlations among multiple risk factors and the skewness of risk-factor distribution. Because risks are often correlated (e.g., undernutrition, poor water and sanitation, and the use of solid fuels are more common among poor households in developing countries, and smokers are more likely to have poor diets), the contributions of high-exposure groups are likely to be underestimated. In addition to being a source of underestimation at higher exposure levels, risk-factor correlation implies that the same individuals and groups are at the high end of multiple risk factors. Positive (rightward) skewness of PLoS Medicine | http://www.plosmedicine.org

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Table 4. Distribution by Exposure Level of Attributable Burden Due to Selected Categorical Risk Factors Category Type

Risk Factor Physical Inactivity

Underweight

Category Description

Proportion of Total Attributable Burden

Category Description

Proportion of Total Attributable Burden

Water, Sanitation, and Hygiene Category Description

Proportion of Total Attributable Burden

Referent category

At least 2.5 h/wk of moderateintensity activity or equivalent (400 KJ/wk)

–

Same fraction of children below 1 SD weight-for-age as the international reference group

–

Absence of transmission of diarrheal disease through water, sanitation, and hygiene

–

Exposure categories

Some activity, but less than 2.5 h/wk of moderateintensity activity

49%

1–2 SD below standard

20%

Regulated water supply and full sanitation coverage, with partial treatment for sewage

0%

Little or no physical activity

51%

2–3 SD below standard

46%

Improved water supply, basic sanitation, improved access to drinking water, improved personal hygiene, and water disinfected at point of use

39%

3þ SD below standard

35%

Improved water supply and basic sanitation

3%

Basic sanitation but no improved water supply

28%

No improved water supply and no basic sanitation

30%

DOI: 10.1371/journal.pmed.0010027.t004

many chronic-disease risk factors, such as tobacco and high blood pressure, as well as risk factors for injuries and sexual and reproductive health, significant proportions of disease burden occurred from events in middle ages, especially in developing countries. The concentration of disease burden from such a large number of risk factors in those below 60 PLoS Medicine | http://www.plosmedicine.org

years of age illustrates the large, and at times neglected, disease burden from risks that affect young adults in developing nations, with important consequences for economic development [18]. The distribution of risks and their determinants in a population have major implications for strategies of pre052

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Figure 2. Distribution of Attributable Cardiovascular Disease Burden Due to BMI, Blood Pressure, and Cholesterol by Exposure Level, Age, and Level of Development Conventions as for Figure 1. DOI: 10.1371/journal.pmed.0010027.g002

vention. As stated by Rose, risk typically increases across the spectrum of a risk factor [8]. Rose’s work led to one of the most fundamental axioms in disease prevention across risk factors: ‘‘A large number of people exposed to a small risk may generate many more cases than a small number exposed to high risk.’’ The analysis in this work showed that the risk factors for many of the major global diseases, such as lower respiratory infections, diarrhea, ischemic heart disease, and stroke exhibit such behavior, because they are caused by risks that occur along a continuum. Therefore, managing individual, high-risk crises, while appropriate for individuals, can only have a limited preventive effect at the population level and over long time periods because it relies on having adequate discriminative ability to predict future disease, and requires continued and expensive screening for new high-risk individuals. In contrast, population-based strategies that seek to shift the whole distribution of risk factors have the PLoS Medicine | http://www.plosmedicine.org

potential to substantially reduce total disease burden, and possibly over long time periods, if the interventions alter the underlying risk behaviors or their socioeconomic causes. This is particularly relevant in the context of risk factors such as those related to under- or overnutrition that affect societies in all stages of development. For example, a policy to reduce salt content in manufactured foods would result in a leftward shift in the population distribution of blood pressures and a surprisingly large reduction in cardiovascular disease [5]. Another example would be population-level measures that affected energy intake (such as the availability and price of energy-dense foods) and/or expenditure (such as the level of motorization and mechanization)—these can be expected to determine the distribution of a population’s BMI levels, and hence largely determine its level of type 2 diabetes [1]. There were distinct patterns for the distribution of disease burden across risk factors, and across regions at various 053

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Disease Burden of Major Health Risks (International Obesity Task Force), Neville J. Rigby (International Obesity Task Force), Chizuru Nishida (WHO), Anthony Rodgers (University of Auckland). Low fruit and vegetable consumption: Karen Lock (London School of Hygiene and Tropical Medicine), Joceline Pomerleau (London School of Hygiene and Tropical Medicine), Louise Causer (London School of Hygiene and Tropical Medicine), Martin McKee (London School of Hygiene and Tropical Medicine). Physical inactivity: Fiona C. Bull (Loughborough University), Tim Armstrong (WHO), Tracy Dixon (Australian Institute of Health and Welfare), Sandra Ham (United States Centers for Disease Control and Prevention), Andrea Neiman (United States Centers for Disease Control and Prevention), Michael Pratt (United States Centers for Disease Control and Prevention). Tobacco: Majid Ezzati (Harvard University), Alan D. Lopez (University of Queensland). Alcohol: Ju¨rgen Rehm (Addiction Research Institute and University of Toronto), Robin Room (Stockholm University), Maristela Monteiro (WHO), Gerhard Gmel (Swiss Institute for the Prevention of Alcohol and Other Drug Problems), Kathryn Graham (University of Western Ontario), Nina Rehn (WHO), Christopher T. Sempos (University of Buffalo), Ulrich Frick (Psychiatric University Hospital Zurich), David Jernigan (Georgetown University). Illicit drugs: Louisa Degenhardt (University of New South Wales), Wayne Hall (University of Queensland), Matthew Warner-Smith (University of New South Wales), Michael Lynskey (University of New South Wales). Unsafe sex: Emma Slaymaker (London School of Hygiene and Tropical Medicine), Neff Walker (Joint United Nations Programme on HIV/AIDS), Basia Zaba (London School of Hygiene and Tropical Medicine), Martine Collumbien (London School of Hygiene and Tropical Medicine). Non-use and use of ineffective methods of contraception: Martine Collumbien (London School of Hygiene and Tropical Medicine), Makeda Gerressu (London School of Hygiene and Tropical Medicine), John Cleland (London School of Hygiene and Tropical Medicine). Unsafe water, sanitation, and hygiene: Annette Pru¨ss-Ustun (WHO), David Kay (University of Wales), Lorna Fewtrell (University of Wales), Jamie Bartram (WHO). Urban air pollution: Aaron Cohen (Health Effects Institute), Ross Anderson (St George’s Hospital Medical School and University of London), Bart Ostro (California Environmental Protection Agency), Kiran Dev Pandey (World Bank), Michal Krzyzanowski (WHO), Nino Ku¨nzli (University of Southern California), Kersten Gutschmidt (WHO), Arden Pope (Brigham Young University), Isabelle Romieu (Instituto Nacional de Salud Publica de Mexico), Jonathan Samet (Johns Hopkins University), Kirk Smith (University of California at Berkeley). Indoor smoke from household use of solid fuels: Kirk R. Smith (University of California at Berkeley), Sumi Mehta (Health Effects Institute), Mirjam Feuz (Federal Office of Public Health of Switzerland). Lead: Annette Pru¨ss-Ustun (WHO), Lorna Fewtrell (University of Wales), Philip Landrigan (Mount Sinai School of Medicine), Jose´ Luis Ayuso (Universidad Autonoma de Madrid). Global climate change: Anthony McMichael (Australian National University), Diarmid Campbell-Lendrum (WHO), Sari Kovats (London School of Hygiene and Tropical Medicine), Sally Edwards (London School of Hygiene and Tropical Medicine), Paul Wilkinson (London School of Hygiene and Tropical Medicine), Frank Tanser (Medical Research Council of South Africa), David Le Sueur (deceased) (Medical Research Council of South Africa), Michael Schlesinger (University of Illinois at Urbana-Champaign), Natasha Andronova (University of Illinois at Urbana-Champaign), Robert Nicholls (University of Middlesex), Theresa Wilson (University of Middlesex), Simon Hales (University of Otago). Occupational risk factors for injuries: Marisol Concha (Associacion Chilena de Seguridad of Chile), Deborah Imel Nelson (WHO), Marilyn Fingerhut (WHO), Annette Pru¨ss-Ustun (WHO), James Leigh (University of Sydney), Carlos Corvalan (WHO). Occupational carcinogens: Tim Driscoll (University of Sydney), Deborah Imel Nelson (WHO), N. Kyle Steenland (United States National Institute for Occupational Safety and Health), James Leigh (University of Sydney), Marisol Concha (Associacion Chilena de Seguridad), Annette Pru¨ss-Ustun (WHO), Marilyn Fingerhut (WHO), Carlos Corvalan (WHO). Occupational airborne particulates: Tim Driscoll (University of

stages of development. At the extreme of these diverse patterns, for risk factors with acute exposure and acute outcomes, the underlying relationship is considerably more complex. For example while in many societies most alcoholattributable injury (e.g., traffic accidents) involves people who on average drink moderate amounts [19], these people would be on the more extreme, high-risk spectrum in a different dimension: volume of drinking right before the injury. Therefore, if exposure to risk factors is clustered or the risk relationship does not follow a linear pattern, high-exposure groups may indeed play a disproportionately important role [20,21]. In summary, the analysis presented in this paper confirms and extends to a global level previous work indicating that the impact of many major risks is important across their exposure levels, not just among people with particularly high levels [8]. This analysis also illustrates that, beyond the general principle of population-wide shifts, there are important population-specific and risk-factor-specific patterns in how the burden of disease attributable to risk factors is distributed. Systematic assessment of multiple risks within any given population can provide the basis for selecting packages of interventions that include population-wide measures as well as highly targeted interventions provided to much smaller subsections of the population with constellations of major risks [1,5,18].

Acknowledgments This work was sponsored by the National Institute of Aging grant PO1-AG17625. The sponsor had no influence on analysis and results. Anthony Rodgers holds a National Heart Foundation Senior Fellowship. We thank Clarissa Gould-Thorpe for manuscript preparation. We thank Mie Inoue, Doris Ma Fat, Susan Piccolo, Chalapati Rao, Kenji Shibuya, Niels Tomijima, and Marie-Claude von Rulach for assistance with global burden of disease databases and project management. Comparative Risk Assessment Collaborating Group Core, methodology, statistical analysis, editorial and peer review, and writing: Majid Ezzati (Harvard University), Anthony Rodgers (University of Auckland), Alan D. Lopez (University of Queensland), Stephen Vander Hoorn (University of Auckland), Christopher J. L.Murray (Harvard University). Childhood and maternal underweight: Steven Fishman (Johns Hopkins University), Laura E.Caulfield (Johns Hopkins University), Mercedes de Onis (WHO), Monika Blo¨ssner (WHO), Adnan A. Hyder (Johns Hopkins University), Luke Mullany (Johns Hopkins University), Robert E. Black (Johns Hopkins University). Iron deficiency anemia: Rebecca J. Stoltzfus (Cornell University), Luke Mullany (Johns Hopkins University), Robert E. Black (Johns Hopkins University). Vitamin A deficiency: Amy J. Rice (Johns Hopkins University), Keith P, West, Jr. (Johns Hopkins University), Robert E. Black (Johns Hopkins University). Zinc deficiency: Laura E. Caulfield (Johns Hopkins University), Robert E. Black (Johns Hopkins University). High blood pressure: Carlene Lawes (University of Auckland), Stephen Vander Hoorn (University of Auckland), Malcolm Law (St Bartholomew’s and Royal London School of Medicine), Paul Elliott (Imperial College School of Medicine), Stephen MacMahon (University of Sydney), Anthony Rodgers (University of Auckland). High cholesterol: Carlene Lawes (University of Auckland), Stephen Vander Hoorn (University of Auckland), Malcolm Law (St Bartholomew’s and Royal London School of Medicine), Stephen MacMahon (University of Sydney), Anthony Rodgers (University of Auckland). Overweight and obesity (high BMI): W. Philip T. James (International Obesity Task Force), Rachel Jackson-Leach (International Obesity Task Force), Cliona Ni Mhurchu (University of Auckland), Eleni Kalamara (International Obesity Task Force), Maryam Shayeghi PLoS Medicine | http://www.plosmedicine.org

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Disease Burden of Major Health Risks Sydney), N. Kyle Steenland (United States National Institute for Occupational Safety and Health), Deborah Imel Nelson (WHO), James Leigh (University of Sydney), Marisol Concha (Associacion Chilena de Seguridad), Marilyn Fingerhut (WHO). Occupational ergonomic stressors: Annette Pru¨ss-Ustun (WHO), Laura Punnett (University of Massachusetts at Lowell), SangWoo Tak (University of Massachusetts at Lowell), Deborah Imel Nelson (WHO), Marilyn Fingerhut (WHO), James Leigh (University of Sydney), Sharonne Phillips (Occupational Ergonomics). Occupational noise: Deborah Imel Nelson (WHO), Marisol Concha (Associacion Chilena de Seguridad), Marilyn Fingerhut (WHO). Contaminated injections in health-care settings: Anja M. Hauri (Staatliches Untersuchungsamt Hessen), Gregory L. Armstrong (United States Centers for Disease Control and Prevention), Yvan J. F. Hutin (WHO). Child sexual abuse: Gavin Andrews (University of New South Wales), Justine Corry (University of New South Wales), Cathy Issakidis (University of New South Wales), Tim Slade (University of New South Wales), Heather Swanston (University of New South Wales). Poverty and risk: Tony Blakely (University of Otago), Simon Hales (University of Otago), Charlotte Kieft (University of Otago), Nick Wilson (University of Otago), Alistair Woodward (University of & Auckland).

15.

16.

17. 18.

19. 20.

21. 22.

References 1. World Health Organization(2002) The world health report 2002: Reducing risks, promoting healthy life. Geneva: World Health Organization. 250 p. 2. Ezzati M, Lopez AD, Rodgers A, Vander Hoorn S, Murray CJ, Comparative Risk Assessment Collaborating Group. (2002) Selected major risk factors and global and regional burden of disease. Lancet 360: 1347–1360. 3. Rodgers A, Lawes C, MacMahon S (2000) The global burden of cardiovascular disease conferred by raised blood pressure. Benefits of reversal of blood pressure-related cardiovascular risk in Eastern Asia. J Hypertens 18: S3–S5. 4. Cook NR, Cohen J, Hebert P, Taylor JO, Hennekens CH (1995) Implications of small reductions in diastolic blood pressure for primary prevention. Arch Intern Med 155: 701–709. 5. Murray CJL, Lauer JA, Hutubessy RCW, Niessen L, Tomijima N, et al. (2003) Effectiveness and costs of interventions to reduce systolic blood pressure and cholesterol: A global and regional analysis on reduction of cardiovascular-disease risk. Lancet 361: 717–725. 6. Peto R, Lopez AD, Boreham J, Thun M, Heath CW (1992) Mortality from tobacco in developed countries: Indirect estimates from national vital statistics. Lancet 339: 1268–1278. 7. Rodgers A, MacMahon S (1999) Blood pressure and the global burden of cardiovascular disease. Clin Exp Hypertens 21: 543–552. 8. Rose G (1992) The strategy of preventive medicine. Oxford: Oxford University Press. 138 p. 9. Rose G (1985) Sick individuals and sick populations. Int J Epidemiol 14: 32– 38. 10. Miettinen OS (1974) Proportion of disease caused or prevented by a given exposure, trait or intervention. Am J Epidemiol 99: 325–332. 11. Murray CJL, Lopez AD, editors (1996) The global burden of disease. Cambridge (Massachusetts): Harvard University Press. 990 p. 12. Ezzati M, Vander Hoorn S, Rodgers A, Lopez AD, Mathers CD, et al. (2003) Estimates of global and regional potential health gains from reducing multiple selected major risk factors. Lancet 362: 271–280. 13. Curtis V, Cairncross S, Yonli R (2000) Domestic hygiene and diarrhoea— Pinpointing the problem. Trop Med Int Health 5: 22–32. 14. James WPT, Jackson-Leach R, Ni Mhurchu C, Kalamara E, Shayeghi M, et al. (2004) Body mass index and the global burden of disease. In: Ezzati M,

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Lopez A, Rodgers A, Vander Hoorn S, Murray C, editors. Comparative quantification of health risks: Global and regional burden of disease due to selected major risk factors. Geneva: World Health Organization. pp. 497– 596. Eastern Stroke and Coronary Heart Disease Collaborative Group(1998) Blood pressure, cholesterol and stroke in eastern Asia. Lancet 352: 1801– 1807. Law MR, Wald NJ, Thompson SG (1994) By how much and how quickly does reduction in serum cholesterol concentration lower risk of ischaemic heart disease? BMJ 308: 367–373. Horton R (2000) Common sense and figures: The rhetoric of validity in medicine (Bradford Hill Memorial Lecture 1999). Stat Med 19: 3149–3164. Commission on Macroeconomics and Health(2001) Macroeconomics and health: Investing in health for economic development. World Health Organization. Available: http://www.cid.harvard.edu/cidcmh/CMHReport. pdf. Accessed 25 August 2004. Kreitman N (1986) Alcohol consumption and the preventive paradox. Br J Addict 81: 353–363. Lemmens P (2001) Relationship of alcohol consumption and alcohol problems at the population level. In: Heather N, Peters TJ, Stockwell T, editors. International handbook of alcohol dependence and problems. Chichester (United Kingdom): John Wiley and Sons. Skog OJ (1999) Prevention paradox revisited. Addiction 94: 751–757. MacMahon S, Peto R, Cutler J, Collins R, Sorlie P, et al. Blood pressure, stroke, and coronary heart disease. Part I, prolonged differences in blood pressure: Prospective observational studies corrected for the regression dilution bias. Lancet 335: 765–774.

Patient Summary Background. Health policy makers must understand existing health risks and which diseases cause the most health problems. The Global Burden of Disease database, maintained by the World Health Organization, collects information from around the world on risk factors such as malnutrition, childbirth, tobacco use, and cholesterol levels, as well as on diseases such as depression, blindness, and diarrhea. This information can be used to target health interventions. What Did the Researchers Do and Find? These researchers determined for 26 major risk factors the distribution of disease burden by age, sex, and exposure level. They found that many risk factors (such as high blood pressure and high cholesterol levels) occur across populations, and are not confined to one sex or age group. And for most risk factors, exposure to moderate risks (which is usually more common than exposure to severe risk) is responsible for causing most disease. What Do the Results Mean, and Who Will Use Them? Measures that reduce exposure to risk factors across whole populations, if only by a modest extent, can achieve large reductions in disease burden. This information is important for people who set global health policies and priorities. What Are the Problems with the Study? There are many challenges in estimating the impact of different major risks, each of which has distinct effects on a number of diseases. In addition, exposure to some risk factors today will cause disease only many years from now, so the picture will change over time. The major finding of the global distribution of many major risks is secure, but the exact extent of this remains uncertain due to the paucity of data in developing countries.

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Open access, freely available online

PLoS MEDICINE

Assessment of Volume Depletion in Children with Malaria Timothy Planche1,2, Myriam Onanga3, Achim Schwenk1,4, Arnaud Dzeing3, Steffen Borrmann2,5, Jean-Franc¸ois Faucher2, Antony Wright7, Les Bluck7, Leigh Ward6, Maryvonne Kombila3, Peter G. Kremsner2,5, Sanjeev Krishna1,2* 1 Department of Cellular and Molecular Medicine, Infectious Diseases, St. George’s Hospital Medical School, London, United Kingdom, 2 Medical Research Unit, Albert Schweitzer Hospital, Lambare´ne´, Gabon, 3 De´partement de Parasitologie, Mycologie, et Me´decine Tropicale, Faculte´ de Me´decine, Universite´ des Sciences de la Sante´, Libreville, Gabon, 4 Coleridge Unit, North Middlesex University Hospital, London, United Kingdom, 5 Department of Parasitology, Institute of Tropical Medicine, University of Tu¨bingen, Tu¨bingen, Germany, 6 Department of Biochemistry, University of Queensland, St Lucia, Australia, 7 Elsie Widdowson Laboratory, Medical Research Council Human Nutrition Research, Cambridge, United Kingdom Competing Interests: The authors have declared that no competing interests exist. Author Contributions: TP, MK, JFF, MO, PGK, and SK designed the study. TP, SB, and AD analysed patient data; AS analysed the BIA data and constructed the models; AW and LB conducted the analysis of heavy water; and LW analysed the bromide data. TP and SK wrote the first draft of the paper; all authors commented extensively on subsequent drafts. Academic Editor: Nicholas J. White, Mahidol University, Thailand Citation: Planche T, Onanga M, Schwenk A, Dzeing A, Borrmann S, et al. (2004) Assessment of volume depletion in children with malaria. PLoS Med 1(1): e18. Received: May 7, 2004 Accepted: August 16, 2004 Published: October 19, 2004 DOI: 10.1371/journal.pmed.0010018

ABSTRACT Background The degree of volume depletion in severe malaria is currently unknown, although knowledge of fluid compartment volumes can guide therapy. To assist management of severely ill children, and to test the hypothesis that volume changes in fluid compartments reflect disease severity, we measured body compartment volumes in Gabonese children with malaria.

Methods and Findings Total body water volume (TBW) and extracellular water volume (ECW) were estimated in children with severe or moderate malaria and in convalescence by tracer dilution with heavy water and bromide, respectively. Intracellular water volume (ICW) was derived from these parameters. Bioelectrical impedance analysis estimates of TBW and ECW were calibrated against dilution methods, and bioelectrical impedance analysis measurements were taken daily until discharge. Sixteen children had severe and 19 moderate malaria. Severe childhood malaria was associated with depletion of TBW (mean [SD] of 37 [33] ml/kg, or 6.7% [6.0%]) relative to measurement at discharge. This is defined as mild dehydration in other conditions. ECW measurements were normal on admission in children with severe malaria and did not rise in the first few days of admission. Volumes in different compartments (TBW, ECW, and ICW) were not related to hyperlactataemia or other clinical and laboratory markers of disease severity. Moderate malaria was not associated with a depletion of TBW.

Copyright: Ó 2004 Planche et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Conclusions

Abbreviations: 2H2O, heavy water; adj r2, adjusted r2; BIA, bioelectrical impedance analysis; CVP, central venous pressure; ECW, extracellular water volume; ICW, intracellular water volume; IQR, interquartile range; SEE, standard error of the estimate; TBW, total body water volume

Malaria claims one million lives annually, with more than 90% of these being those of children in sub-Saharan Africa [1]. Most deaths of hospitalised children occur in the first 24 h after admission. Even modest improvements in management during this time may improve survival [2]. There is considerable disagreement about the degree to which children with severe malaria become hypovolemic. In east African studies, clinical signs of severe malaria (such as tachycardia, prolonged capillary refill times, and decreased urine volume) have been interpreted as evidence for volume depletion [2,3,4,5,6]. However, determining fluid compartment volumes is the first and most critical step in optimising fluid replacement therapy for children with malaria because clinical assessment of fluid status is difficult and imprecise [7]. Our study was designed to measure total body water volume (TBW) and extracellular water volume (ECW) using nonradioactive tracer dilution techniques and to derive intracellular water volume (ICW). Bromide distributes in the extracellular space so that concentrations measured 2–4 h after administration safely and reliably estimate ECW. Heavy water (2H2O) space represents TBW. ICW is calculated by subtraction of the ECW from the TBW. Tracer dilution methods are expensive and invasive and cannot be repeated at short intervals. Therefore, we simultaneously calibrated a noninvasive technique of bioelectrical impedance

*To whom correspondence should be addressed. E-mail: s.krishna@ sghms.ac.uk

Significant hypovolaemia does not exacerbate complications of severe or moderate malaria. As rapid rehydration of children with malaria may have risks, we suggest that fluid replacement regimens should aim to correct fluid losses over 12–24 h.

Introduction

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analysis (BIA) [8] to estimate the fluid volumes. BIA measures the opposition (impedance) of the body to the flow of a small alternating current between electrodes placed on the hand and the foot, and then estimates TBW and ECW using regression equations derived by calibration against ‘gold standard’ tracer measurements of fluid volumes. We hypothesised that volume changes in fluid compartments would reflect disease severity in malaria and that these changes would be related to established markers of disease severity [9]. We also calibrated BIA assessments in children with moderate and severe malaria with direct measurements of volume of TBW and ECW.

kg/min, i.e., 1.6 or 3.2 ml/kg/h), physicians were free to give any fluid replacement regimen as clinically indicated, including boluses of saline or blood. A strict fluid input/ output chart was kept for each child. A blood transfusion of 20 ml/kg of cross-matched whole blood tested for bloodborne pathogens was given over 4 h if the hematocrit fell below 15%.

Measurement of TBW and ECW A sterile standard dosing solution was prepared by adding 119 ml of 2H2O per litre of 2.315% sodium bromide solution. A baseline sample of blood (3 ml) was drawn, and the plasma frozen at 70 8C for bromide and 2H2O assays. At the start of the study, 2.8 ml/kg of the dosing solution was administered intravenously over 20 min. Four hours after injection a second blood sample was drawn (1.5 ml) for the determination of blood bromide and 2H2O concentrations. Parents were asked to return with their children 28 d after admission; children were examined, and measurement of TBW and ECW repeated. 2 H enrichment was measured in duplicate by isotope ratio mass spectrometry, using a Sira 10 instrument (Micromass, Cheshire, United Kingdom) as described [13]. The precision of the TBW determination was estimated at 0.3% of the value obtained. Batch analysis of bromide enrichment in plasma was performed by high-performance anion-exchange liquid chromatography as described [14]. The intra-assay coefficient of variation for bromide was better than 1.5%. BIA was performed using a SEAC SFB3 multifrequency bioimpedance meter (Impedimed, Brisbane, Australia). An alternating electrical current of 200 lAmp was applied between 2 Ag/AgCl electrodes at the right hand and right foot. Whole body and segmental impedance were measured by rotating the sensing Ag/AgCl electrodes between four sites on the ankles or wrists as described [13]. Each set of measurements was taken at 496 frequencies between 4 kHz and 1012 kHz. Measurements were taken at 0 h, 4 h, 12 h, 24 h, and discharge. The process took about 2 min to perform and was simpler than obtaining an electrocardiogram. Data were analysed with software (Bioimp, version 1.1.0, Impedimed) that uses nonlinear regression to fit measured data to semicircular Cole-Cole plots [15] of reactance against resistance. Resistance and reactance values, obtained from the Cole-Cole plot for specific frequencies (0, 4, 50, and 100; characteristic and infinite kHz) were used in further analysis [13]. Plasma electrolytes were measured (Beckman Coulter, Allendale, New Jersey, United States), and osmolality was calculated using freezing point depression on a Micro Osmometer (Vitech Scientific, West Sussex, United Kingdom). Osmol gap was calculated as described [16]:

Methods The study was conducted at the Albert Schweitzer Hospital, Lambare´ne´, Gabon, and Centre Hospitalier de Libreville, Gabon. It was approved by the ethics committees of the International Foundation of the Albert Schweitzer Hospital, the Gabonese Ministry of Health, and the University of Tu¨bingen. Children (aged 1 to 10 y, inclusive) admitted with suspected severe or moderate malaria were referred to the study team, who assessed them within 15 min, and the children were admitted to the study once informed consent had been obtained from the parents. Malaria was defined as the presence of asexual forms of Plasmodium falciparum in thick or thin blood films. Severe malaria was malaria with one or more of the following features: blood lactate
5 mmol/l, blood glucose  2.2 mmol/l, Blantyre coma score  2, or repeated, observed seizures [2]. Moderate malaria was malaria without any of the features of severe malaria but with a requirement for parenteral treatment because of one or more of the following: a history of frequent (. 2) and recent vomiting (within 12 h), drowsiness, obtundation, or prostration [2]. Alternative diagnoses were excluded clinically.

Assessment and Management On admission children were weighed (undressed) with pediatric scales accurate to within 100 g (Seca, Birmingham, United Kingdom). A history was taken from parents, and the child was examined with particular attention to signs of dehydration, including: capillary refill time, skin turgor, sunken eyes, dry mucous membranes, and absence of tears. Vital signs, blood glucose and lactate concentrations, and hematocrit and parasitaemia were measured every 4 h for the first 24 h and then every 6 h until recovery. If peripheral venous access was impracticable, then a femoral central venous catheter was inserted, and the central venous pressure was measured every 4 h with a manometer zeroed at the midaxillary line. Children were managed in a standard manner as previously described [10,11,12]. All received intravenous quinine (20 mg/ kg salt intravenously as a loading dose given over 4 h, then 10 mg/kg intravenously every 12 h until able to take oral medication) (Quinimax, Sanofi Synthelabo, Paris, France). Hypoglycaemia (blood glucose  2.2 mmol/l) was treated with 25% glucose (2 ml/kg). Convulsions were treated with diazepam (0.3 mg/kg intravenously or 0.5 mg/kg intrarectally) (Roche, Basel, Switzerland), and repeated convulsions were treated with phenobarbital (7.5 mg/kg intramuscularly). In addition to a 5% or 10% dextrose infusion (at least 3 mg/ PLoS Medicine | http://www.plosmedicine.org

OG ¼ MO 

ð1:86 3 ½Naþ Þ þ ½glucose þ ½urea 0:93

ð1Þ

where OG is osmol gap, MO is measured osmolality, [Naþ] is plasma sodium concentration (millimoles/litre), [glucose] is plasma glucose concentration (millimoles/litre), [urea] is plasma urea concentration (millimoles/litre), 1.86 is a correction factor as sodium chloride is only 93% dissociated, and 0.93 is the assumed proportion of water in plasma. 057

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Table 1. Baseline Characteristics of Children with Malaria Characteristic

Severe Malaria

Moderate Malaria

Number Age (mo) Height (cm) Temperature (8C) Number with Blantyre coma score  2 Pulse (/min) Mean arterial pressure (mm Hg) Respiration Rate (/min) Number with capillary refill
2 s Number with lactate
5 mmol/L Number with glucose  2.2 mmol/L Hematocrit (%) Parasitaemia (/lL) (3 103) a Weight (kg)

16 45 (33) / 34 (21–63) 97 (20) / 91 (81–113) 39.3 (1.2) / 39.4 (38.8–40.1) 10/16 156 (22) / 160 (151–166) 73 (10) / 74 (71–77) 46 (19) / 42 (34–51) 10/16 9/16 1/16 23 (8) / 23 (18–29) 78 (0.48–1,341) 14.1 (5.6) / 12.8 (9.7–16.4)

19 40 (26) / 32 (21–46) 95 (19) / 88 (83–89) 38.0 (1.2) / 37.9 (37.4–38.7) 0/19 135 (19) / 132 (120–160) 65 (10) / 63 (60–75) 38 (9) / 38 (32–44) 1/17 0/19 0/19 26 (7) / 27 (19–33) 55 (3,450–555) 13.0 (4.6) / 11.1 (10.1–14.7)

Mean (SD) / median (IQR) shown. a Parasitaemia values are geometric mean (range). DOI: 10.1371/journal.pmed.0010018.t001

Statistical Methods

severe malaria admitted to this study. The median (interquartile range [IQR]) time from admission to administration of tracers was 54 (37–84) min, during which complications such as convulsions or hypoglycaemia were treated. The baseline characteristics of children are given in Table 1. Those with severe malaria had significantly higher pulse rates, mean arterial pressure and blood lactate concentrations, and a longer capillary refill time (p , 0.001), compared to children with moderate malaria. Capillary refill time and blood lactate concentrations were correlated with each other (adjusted r2 [adj r2] = 0.25, p = 0.031). The volume of fluid (including blood) given in the first 4 h of the study was similar in children with severe (median [IQR, range] of 3.7 [2.2–5.8, 2.1–11.4] ml/kg/h) and moderate (median [IQR, range] of 3.3 [2.2–4.2, 1.2–15.1] ml/kg/h) malaria (p . 0.5). There was rapid correction of vital signs in all children (Figure 1), and vital signs were similar in both study groups by 8 h. There were two deaths (4.5 and 6 h after admission), and two children with severe malaria had major persistent neurological deficits (4/16 [25%] with adverse outcomes). Central venous pressure (CVP) measurements were obtained in six children with severe malaria. The median (IQR) CVP on admission was þ6.5 (3–7.5) cm H2O with no significant rise in the first 24 h.

Statistical analyses were carried out using Stata Statistical Software (Releases 6.0–8.0, College Station, Texas, United States). After checking distributions with the Shapiro-Wilks W test, and transforming data logarithmically if necessary, we analysed normally distributed data by two-tailed Student’s t test and nonparametric data with the Wilcoxon sign-rank test. Proportions were compared with Fisher’s exact test, and correlations were assessed by linear regression analysis of Pearson or Spearman. Predictive values for TBW and ECW from impedance measurements were obtained by multivariate analysis in a backward elimination process with p , 0.05 for entry and p , 0.10 for exit, confirmed by forward selection. This analysis was based on the first pair of BIA and isotope dilution measurements after admission. In a second step, whole body BIA models were replaced with segmental BIA models as previously described [13] and compared with whole body estimates. Errors in the BIA estimate and isotope dilution methods were compared. TBW estimates were corrected for fluid input and output during the period of measurement. Sample size was calculated from previously published values [17,18] assuming a mean (SD) for convalescent TBW of 586 (30) ml/kg; we wished to detect a 5% difference in TBW between severe and moderate cases with a power of 90%.

Fluid Volumes Volume determinations using isotope dilution were available at baseline in all but one of the children with severe malaria. The values for the TBW, ECW, and ICW are given in Table 2. The TBW was significantly lower at admission compared with day 28 for the severe cases (p = 0.028) but not for moderate cases (p = 0.109). The mean (SD) TBW was lower in severe than moderate malaria at admission: 524 (44) ml/kg versus 555 (50) ml/kg (p = 0.052). The mean (SD) change in TBW between admission and follow up was 48 (42) ml/kg and 12 (37) ml/kg for severe and moderate cases, respectively. Individual data from the children with severe malaria are shown in Table 3.

Results Between October 1999 and March 2000, 205 children who were judged ill enough to be hospitalised were referred to the study team. One hundred and thirty-two children had malaria, 20 with severe and 35 with moderate disease. Ten children with moderate and two with severe malaria were ineligible for study because of their age, and for seven children (one with severe malaria) consent could not be obtained. One child died before inclusion into the study, leaving nineteen children with moderate and sixteen with PLoS Medicine | http://www.plosmedicine.org

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Bioelectrical Impedance Analysis As predicted by theory [8], there was a strong correlation between height2 divided by impedance at 50 kHz (H2/Z50) and measured TBW. The ‘best fit’ regression equation to predict TBW from BIA was derived using the variables age, weight, and H2/Z50 (standard error of the estimate [SEE] = 0.435, adj r2 = 0.975): TBW ¼ 0:7891 þ ð0:3454 3 W Þ

  H2 þ ð 0:0283 3 AÞ þ 0:0851 3 Z50

ð2Þ

where A is age (months), H is height (centimetres), W is weight (kilograms), and Z50 is impedance at 50 KHz frequency. Disease severity and gender did not contribute significantly to this model, allowing data from admission to be pooled in this prediction equation. Repeating the analysis using impedances measured at other frequencies (4–1012 kHz) did not show a clear advantage, so all data for TBW prediction are given for measurement at 50 kHz. By contrast, ECW was not significantly associated with age or weight, and in agreement with previous studies in babies [19] H2/R0 emerged as the strongest predictive term (SEE = 0.584, adj r2 = 0.753):   H2 ECW ¼ 0:8735 þ 0:2285 3 ð3Þ R0

Figure 1. Vital Signs of Children during the First 24 h after Admission Mean and 95% confidence interval shown. Red circles, severe malaria; blue triangles, moderate malaria. (A) Pulse (per minute), (B) mean arterial pressure (millimetres Hg), (C) respiratory rate (per minute), and (D) blood lactate concentration (millimoles/litre). DOI: 10.1371/journal.pmed.0010018.g001

Bromide space (ECW) measurements were available in all but two children with severe malaria and in all but one of the children with moderate malaria. At baseline, the mean (SD) ECW was significantly lower in children with moderate than in those with severe malaria (p = 0.045). Admission ICW measurements were significantly lower in children with severe malaria than in those with moderate malaria (p , 0.001).

where H is height (centimetres), and R0 is resistance at zero frequency. Again, disease severity and gender did not contribute significantly to this model. Prediction equations

Table 2. Volumes Determined by Tracer Dilution and Electrolyte Measurements Variable

Number TBW (mL/kg) ECW (mL/kg) ICW (mL/kg) Change TBWa (mL/kg) Change ECWa (mL/kg) Change ICWa (mL/kg) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L) Bicarbonate (mmol/L) Lactate (mmol/L) Glucose (mmol/L) Urea (mmol/L) Creatinine (lmol/L) Osmolality mOsm Anion Gap (mmol/L) Omsol Gap (mOsm)

Admission

Follow Up (Day 28)

Moderate Malaria

Severe Malaria

19 555 (50) / 556 (513–589) 237 (49) / 241 (195–268) 318 (41) / 312 (290–350) –30 (32) / –23 (–50, –14) (n = 12)b 16 (33) / 11 (–7, 26) (n = 10)b –10 (26) / –13 (–25 to 10) (n = 10)b 133 (5.0) / 134 (130–136) 3.5 (0.5) / 3.4 (3.1–3.8) 102 (5.1) / 103 (98–105) 16.6 (3.7) / 16 (14–19) 2.4 (1.1) / 2.3 (1.8–3.1) 5.4 (1.5) / 4.8 (4.5, 6.7) 4.5 (3.2) / 3.8 (3.0–4.7) 39 (15) / 38 (28–44) 282 (12) / 282 (278–287) –18 (4) / –19 (–15, –21) 6.3 (4.0) / 4.9 (4.0–8.4)

16 524 (44) / 522 (496–550) 266 (36) / 270 (251–285) 258 (34) / 241 (232–288) –58 (38) / –74 (–78 ,–22) (n = 5)b –30 (56) / –62 (–63, 39) (n = 3)b –69 (22) / –76 (–86 to 44) (n = 3)b 129 (4.5) / 131 (126–132) 3.9 (0.6) / 4.0 (3.2–4.3) 97 (3.4) / 98 (94–100) 16.8 (4.0) / 17 (15–20) 5.7 (3.5) / 5.5 (2.8–6.6) 8.1 (4.4) / 6.5 (5.6 , 10.6) 6.6 (3.4) / 5.9 (4.0–9.2) 65 (34) / 60 (35–88) 292 (12) / 295 (286–298) –20 (5) / –19 (–16, –22) 17.1 (11.8) / 12.9 (10.2–22)

17 561 (30) / 562 (549–581) 247 (30) / 240 (220–260) 314 (33) / 314 (292–343)

136 (2.1) / 137 (135–137) 4.0 (0.3) / 4.0 (3.8–4.1) 104 (2.7) / 104 (102–106) 18.5 (2.8) / 19 (16–21) 1.9 (1.0) / 1.5 (1.3–2.6) 4.6 (0.6) / 4.7 (4.1, 4.9) 3.2 (0.9) / 3.3 (2.8–3.5) 36 (9) / 34 (30–43) 293 (8) / 294 (288–298) –18 (3) / –17 (–16, –21) 11.5 (5.4) / 10.9 (7.9–16.6)

Mean (SD) / median (IQR) shown. a Change in volume as measured by isotope dilution between admission and day 28. b Measurements of ECW and ICW were available at follow up in three cases with severe and ten with moderate malaria (discrepancy due to difficulties obtaining adequate sample volumes). DOI: 10.1371/journal.pmed.0010018.t002

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Table 3. Individual Details of Severely Ill Children Code Age Sex Weight Outcome (mo) (kg) 1 2 3 4 5 6 7

36 34 122 34 98 13 82

F F F F F F F

14.2 12.4 27.7 14.5 22.3 7.5 17.1

8 9 10 11 12

15 16 46 27 77

M F F F F

9.3 10 15.6 10.6 21.3

13 14 15 16

24 29 48 17

M F F M

9.2 11.7 13.1 8.7

Parasitaemia Respiratory Lactate Glucose Haematocrit BCS TBW ECW (/lL) Distress (mmol/L) (mmol/L) (%) (mL/kg) (mL/kg)

Survived 620464 Survived 22106 Survived 248437 Died 30772 Died 240398 Survived 4522 Neurological 480 sequelae Survived 2763 Survived 157502 Survived 315005 Survived 52752 Neurological 139416 sequelae Survived 379814 Survived 1341408 Survived 259992 Survived 279209

No Yes Yes Yes Yes Yes Yes

5.5 4.8 6.5 7.4 13.7 5.5 6.8

17.9 7.0 6.1 14.1 2.3 5.7 14.3

26 18 23 35 22 9 31

5 2 4 0 4 3 0

609 528 –a –a –a 556 –a

307 208 –a –a –a 324 –a

Yes Yes No Yes Yes

13.1 2.8 2.2 1.2 5.8

2.5 7.3 10.8 5.4 10.3

11 19 33 28 30

5 2 0 0 1

479 505 519 446 572

260 270 287 198 277

No No Yes Yes

2.8 5.8 5.0 2.7

4.6 5.7 9.7 5.7

24 15 18 19

2 2 3 2

524 486 544 519

243 263 269 284

a

Missing values because discharge weights unobtainable DOI: 10.1371/journal.pmed.0010018.t003

based on segmental BIA data for ECW and TBW were inferior to whole body BIA models and are therefore not shown here. Errors in BIA estimates of TBW and ECW compared to values measured by isotope dilution are displayed with 95% limits of agreement in Figure 2A and 2B, respectively.

differences in the fluid volume measurements of the four children with adverse and those with good outcomes. There were no relationships found between TBW, ECW, or ICW, or changes in TBW, ECW, and ICW between baseline and discharge, and any of the following markers of severity in malaria: blood lactate concentration, coma score, plasma creatinine concentration, peripheral parasitaemia, blood glucose, coma recovery time, time to walk, time to eat, time to drink, length of hospital stay, or having a history of diarrhoea or vomiting. Admission weight relative to the weight at discharge showed a mean (SD)/median (IQR, range) percentage deficit of 4.3% (4.4%)/4.0% (1.3% to 6.1%, 0.1% to 11.0%) for the children with severe malaria on admission (p = 0.002). For those with moderate malaria the deficit was not significant: 0.3% (4.6%) /0.9% (1.6% to 1.5%, 7.0% to 8.1%) (p = 0.41). As predicted, the deficits in weight and TBW were correlated (adj r2 = 0.67, p , 0.001).

Fluid Volumes from BIA Fluid volume estimates were available from BIA in 14 children with moderate and 11 with severe malaria on admission and 16 with moderate and 12 with severe malaria at discharge. Values for the TBW, ECW, and ICW are shown in Figure 3. BIA-determined TBW was significantly lower in those with severe compared with moderate malaria (mean [SD] of 539 [32] versus 562 [30] ml/kg, p = 0.034). TBW increased from admission to discharge in children with severe malaria by 37 (33) ml/kg or 6.7% (6.0%) (paired t test, p = 0.012), but there were no significant changes in TBW for children with moderate malaria between admission and discharge. ECW did not differ between the severe and moderate groups on admission and did not change significantly between admission and discharge. Serial measurements of ECW did not show any rise during the first 4 d after admission, at discharge, or at day 28 in children with severe or moderate malaria (data not shown). ICW at admission was significantly lower in the severe malaria group than in the moderate malaria group, with a mean (SD) of 293 (17) ml/kg versus 325 (28) ml/kg (p = 0.002). ICW remained unchanged between admission and discharge in children with moderate malaria. In the severe malaria group mean (SD) of ICW at admission was 40 (22) ml/kg, or 11.7% (11.0%) lower at admission than at discharge (p = 0.002). There were no PLoS Medicine | http://www.plosmedicine.org

Electrolyte Measurements The concentrations of plasma electrolytes are shown in Table 2. The mean (SD) concentrations of sodium were significantly lower for the children with severe malaria than for those with moderate malaria (p = 0.036). Plasma potassium concentration and osmolality were higher in children with severe than in those with moderate malaria (p = 0.039 and p = 0.021, respectively). Plasma urea and creatinine were significantly higher in those with severe malaria. Osmol gap was significantly higher in children with severe than in those with moderate malaria (p , 0.001), with 15/16 children in the severe group having an osmol gap 060

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Figure 3. Body Fluid Compartment Volumes Derived from BIA on Admission and Discharge Red circles, severe malaria; blue triangles, moderate malaria. (A) TBW (litres/kilogram), (B) ECW (litres/kilogram), and (C) ICW (litres/ kilogram). DOI: 10.1371/journal.pmed.0010018.g003

Figure 2. Plots of TBW and ECW Estimates from Isotope Dilution and BIA Calculation with Measured Values Filled circles, admission value; open circles, day 28 follow up value; dotted lines, 95% confidence intervals for values. (A) TBW and (B) ECW. D2O, heavy water. DOI: 10.1371/journal.pmed.0010018.g002

pathological process in the evolution of the disease. These findings are also consistent with our previous suggestion that hyperlactataemia arises from tissue hypoxia resulting from microvascular obstruction by infected erythrocytes [12,22] rather than gross hypovolaemia. ECW did not change significantly during hospitalisation. Furthermore, our fluid replacement regimen (median [IQR] 3.7 [2.2–5.8] ml/kg/h) normalised vital signs and blood lactate within 8 to 12 h. Our estimates of fluid compartment volumes in children after recovery from malaria are entirely consistent with previous work in children (range: TBW, 540–640 ml/kg; ECW, 250–320 ml/kg; ICW, 260–340 ml/kg) [17], although our study is the first that we know of to examine fluid status in childhood malaria. Studies in adults with uncomplicated or moderate malaria have given conflicting results [23,24,25,26,27]. In sepsis, ECW increases by up to 50% of TBW because capillary permeability increases by up to 300% of normal. There are no significant increases in ECW in children with severe malaria, confirming earlier studies that indicate that sepsis and malaria syndromes result from different pathophysiological processes [9,28]. Studies on fluorescein angiography in children and adults also confirm there is no increased capillary permeability in severe malaria [23,29]. Furthermore, CVP measurements in a subgroup (6/16) of children were not low, and did not change significantly after

greater than 8.2 mOsm (considered high in United States children [20]).

Discussion We have shown that severe childhood malaria is associated with mild dehydration in most cases, with a mean (SD) depletion of TBW of 37 (33) ml/kg, or 6.7% (6.0%). Only 3/16 children (19%) in our study had moderate volume depletion (. 60–90 ml/kg), and none were severely dehydrated (. 100 ml/kg) [21]. Moderate malaria was not associated with any significant changes in TBW. Consistent with a lower TBW in severe disease, ICW was depleted in children with severe malaria by a mean (SD) of 40 (22) ml/kg, an 11.7% (11.0%) difference. However, we found no relationship between TBW, ECW, and ICW and clinical and laboratory markers of disease severity, in particular the two most important prognostic indicators of fatal outcome: hyperlactataemia and Blantyre coma score [9]. Our findings suggest that the degree of dehydration in children with severe malaria is unlikely to be a primary PLoS Medicine | http://www.plosmedicine.org

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replacement rate of about 3 ml/kg/h [7,36]. In addition, fluid regimens should aim to replace mild fluid deficit within the first 12 to 24 h of admission. The tracer dilution techniques that we have used are expensive and time-consuming and consequently not amenable to large-scale deployment. We took this opportunity to calibrate a much simpler methodology (BIA) to derive TBW and ECW and validated BIA estimations in this population. We are now using BIA measurements to assess much larger numbers of patients (J. Jarvis and S. K., unpublished data). BIA is an excellent noninvasive screening tool that should detect subgroups of children with severe malaria who may be severely volume depleted.

24 h of intravenous fluid replacement. Taken together, these findings do not suggest that volume depletion or increased capillary permeability are important to the pathophysiology of malaria in our population. Hyponatraemia [30] has been attributed to high and possibly inappropriate arginine vasopressin secretion [31] in severe malaria. Our findings (high osmol gap, low ICW, and normal ECW) are more in keeping with sick cell syndrome than with inappropriate arginine vasopressin secretion [32,33]. To conclude that arginine vasopressin is inappropriately elevated, renal function must be normal and volume depletion excluded. No severely ill child in this study fulfilled these criteria. What are the implications of our findings for optimal fluid replacement therapy in malaria? We cannot answer precisely on the basis of measuring fluid compartment volumes because regimens are sometimes designed not only to correct existing fluid deficits and to provide maintenance requirements, but also to rehydrate more vigorously to maintain circulating volume. Such approaches are advocated by others for different populations of children with severe malaria, for example, in a series of studies published from Kilifi, Kenya [4,5,34]. However, estimates of fluid requirements for children with severe malaria have been based upon indirect measurements (such as monitoring vital signs and degree of acidosis) that are potentially misleading because they do not relate to the degree of fluid loss that we measured. Furthermore, adequately powered controlled studies aimed at defining appropriate fluid regimens for severe malaria are lacking, but should take into account our findings as well as the BIA methodology that we have now calibrated to measure fluid compartment volumes in malaria. Indeed, a fluid (0.9% saline) replacement rate of up to 20 ml/ kg in 1 h is a considerably faster rate than we can advocate on the basis of our findings. In any case, BIA can now be used (equations 2 and 3) to measure ECW and TBW noninvasively to guide treatment in patients with severe malaria. There are risks to over-vigorous fluid administration just as there are with inadequate fluid replacement, particularly in hospitals where measuring plasma electrolyte concentrations and providing assisted ventilation are difficult. These risks include pulmonary [3] and cerebral oedema [6] (which occur in adults and children, respectively) and dangerously rapid changes in plasma electrolyte concentrations. Children with severe malaria have a low ICW and are at risk of hypokalaemia if ICW is restored rapidly (, 4 h), particularly when there is relative hyperinsulinaemia due to quinine administration [12]. A correlation between capillary refill time and blood lactate concentrations but none with fluid volume status suggests that prolongation of capillary refill time may be due to the common underlying process of microvascular obstruction. Our findings do not support the widespread use of aggressive fluid volume replacement in children with severe malaria. Clearly, volume depletion indicated by hypotension or by CVP measurements requires more aggressive therapy, but wherever possible plasma electrolyte concentrations should be closely monitored. Because of elevated requirements for glucose in childhood severe malaria (3–6 mg/kg/ min) [35], there is a need to provide a maintenance fluid PLoS Medicine | http://www.plosmedicine.org

Acknowledgments We thank the medical, nursing, and laboratory staff of the Albert Schweitzer Hospital and of the Centre Hospitalier de Libreville, especially Ms. K. Engel, Dr. A. Josseaume, Dr. R. Tchoua, Prof. D. Ngaka, and Prof. R. Tchoua for advice and aid in conducting this study. We also thank M. Nestor Obiang, Batchelili Batchelili, Emmanuel Mozogho, and Frankie Mbandinga for their assistance and Martin Hurl of YSI for loaning the YSI2300 analyser. We also thank Prof. E. Ngou-Milama for his aid in this study. Tim Planche was funded by the Special Trustees of St. Georges Hospital, and this work forms a part of his MD thesis. The funders had no role in study design, data collection and analysis, decision to publish, or & preparation of the manuscript. References 1. Breman JG (2001) The ears of the hippopotamus: Manifestations, determinants, and estimates of the malaria burden. Am J Trop Med Hyg 64(1–2 Suppl): 1–11. 2. Newton CR, Krishna S (1998) Severe falciparum malaria in children: Current understanding of pathophysiology and supportive treatment. Pharmacol Ther 79: 1–53. 3. Hall AP (1976) The treatment of malaria. BMJ 1: 323–328. 4. Marsh K (2003) Management of severe malaria: Implications for research. Br J Clin Pharmacol 55: 460–463. 5. Maitland K, Levin M, English M, Mithwani S, Peshu N, et al. (2003) Severe P. falciparum malaria in Kenyan children: Evidence for hypovolaemia. QJM 96: 427–434. 6. Newton CR, Crawley J, Sowumni A, Waruiru C, Mwangi I, et al. (1997) Intracranial hypertension in Africans with cerebral malaria. Arch Dis Child 76: 219–226. 7. Elzouki AY, Harfi HA, Nazer H (2001) Textbook of clinical pediatrics. Philadelphia: Lippincott Williams and Wilkins. 1,800 p. 8. Anonymous(1996) NIH Consensus statement. Bioelectrical impedance analysis in body composition measurement. National Institutes of Health Technology Assessment Conference Statement. December 12–14, 1994. Nutrition 12: 749–762. 9. Planche T, Agbenyega T, Bedu-Addo G, Ansong D, Owousu-Ofori A, et al. (2003) A prospective comparison of malaria with other severe diseases in African children: Prognosis and optimization of management. Clin Infect Dis 37: 890–897. 10. Krishna S, Nagaraja NV, Planche T, Agbenyega T, Bedo-Addo G, et al. (2001) Population pharmacokinetics of intramuscular quinine in children with severe malaria. Antimicrob Agents Chemother 45: 1803–1809. 11. Nealon C, Dzeing A, Muller-Romer U, Planche T, Sinou V, et al. (2002) Intramuscular bioavailability and clinical efficacy of artesunate in Gabonese children with severe malaria. Antimicrob Agents Chemother 46: 3933–3939. 12. Agbenyega T, Angus B, Bedu-Addo G, Baffoe-Bonnie B, Guyton T, et al. Glucose and lactate kinetics in children with severe malaria. J Clin Endocrinol Metab 85: 1569–1576. 13. Schwenk A, Hodgson L, Wright A, Ward LC, Rayner CF, et al. (2004) Nutrient partitioning during treatment of tuberculosis: Gain in body fat mass but not in protein mass. Am J Clin Nutr 79: 1006–1012. 14. Miller ME, Cosgriff JM, Forbes GB (1989) Bromide space determination using anion-exchange chromatography for measurement of bromide. Am J Clin Nutr 50: 168–171. 15. Cole KS, Cole RH (1941) Dispersion and absorption in dielectrics. I. Alternating current characteristics. J Chem Phys 9: 341–351. 16. Gennari F (1984) Serum osmolality: Uses and limitations. N Engl J Med 310: 102–105. 17. Flynn MA, Hanna FM, Lutz RN (1967) Estimation of body water compartments of preschool children I. Normal children. Am J Clin Nutr 20: 1125– 1128.

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Fluids and Malaria resuscitation in children with severe malaria. Pediatr Crit Care Med 4: 426– 431. 35. Agbenyega T, Planche T, Bedu-Addo G, Ansong D, Owusu-Ofori A, et al. (2003) Population kinetics, efficacy, and safety of dichloroacetate for lactic acidosis due to severe malaria in children. J Clin Pharmacol 43: 386–396. 36. Logan RW (1998) Forfar and Arneil’s textbook of pediatrics, 5th ed. Edinburgh: Churchill Livingston. 2,059 p.

18. Ellis KJ, Wong WW (1998) Human hydrometry: Comparison of multifrequency bioelectrical impedance with 2H2O and bromine dilution. J Appl Physiol 85: 1056–1062. 19. Lingwood BE, Coghlan JP, Ward LC, Charles BG, Colditz PB (2000) Measurement of extracellular fluid volume in the neonate using multiple frequency bio-impedance analysis. Physiol Meas 21: 251–262. 20. McQuillen KK, Anderson AC (1999) Osmol gaps in the pediatric population. Acad Emerg Med 6: 27–30. 21. Adelman RD, Solhaug JS (2000) Pathophysiology of fluids and fluid therapy. In: Behrman RE, Kliegman R, Jenson HB. Nelson textbook of pediatrics, 16th ed. Philadelphia: W. B. Saunders. pp. 189–217. 22. Krishna S, Waller DW, ter Kuile F, Kwiatkowski D, Crawley J, et al. (1994) Lactic acidosis and hypoglycaemia in children with severe malaria: Pathophysiological and prognostic significance. Trans R Soc Trop Med Hyg 88: 67–73. 23. Davis TM, Suputtamongkol Y, Spencer JL, Ford S, Chienkul N, et al. (1992) Measures of capillary permeability in acute falciparum malaria: Relation to severity of infection and treatment. Clin Infect Dis 15: 256–266. 24. Malloy JP, Brooks MH, Barry KG, Wilt S, McNeil JS (1967) Pathophysiology of acute falciparum malaria. II. Fluid compartmentalization. Am J Med 43: 745–750. 25. Sitprija V, Indraprasit S, Pochanugool C, Benyajati C, Piyaratn P (1967) Renal failure in malaria. Lancet 1: 185–188. 26. Feldman HA, Murphy FD (1945) The effect of alterations in blood volume on the anemia and hypoproteinemia. J Clin Invest 24: 780–792. 27. Overman RR, Hill TS, Wong YT (1949) Physiological studies in the human malarial host. I. Blood, plasma, ‘‘extracellular’’ fluid volumes and ionis balance in therapeutic P. vivax and P. falciparum infections. J Nat Mal Soc 8: 14–31. 28. Day NP, Phu NH, Bethell DP, Mai NT, Chau TT, et al. (1996) The effects of dopamine and adrenaline infusions on acid-base balance and systemic haemodynamics in severe infection. Lancet 348: 219–223. 29. Hero M, Harding SP, Riva CE, Winstanley PA, Peshu N, et al. (1997) Photographic and angiographic characterization of the retina of Kenyan children with severe malaria. Arch Ophthalmol 115: 997–1003. 30. Brooks MH, Malloy JP, Bartelloni PJ, Tigertt WD, Sheehy TW, et al. (1967) Pathophysiology of acute falciparum malaria. I. Correlation of clinical and biochemical abnormalities. Am J Med 43: 735–744. 31. Sowunmi A, Newton CR, Waruiru C, Lightman S, Dunger DB (2000) Arginine vasopressin secretion in Kenyan children with severe malaria. J Trop Pediatr 46: 195–199. 32. Guglielminotti J, Pernet P, Maury E, Alzieu M, Vaubourdolle M, et al. (2002) Osmolar gap hyponatremia in critically ill patients: Evidence for the sick cell syndrome? Crit Care Med 30: 1051–1055. 33. Flear CTG, Singh CM (1973) Hyponatraemia and sick cells. Brit J Anaesth 45: 976–994. 34. Maitland K, Pamba A, Newton CR, Levin M (2003) Response to volume

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Patient Summary Background. Although we have known for many years what causes malaria, how it is passed from person to person by mosquito, and how to treat the infection, more than a million people still die of malaria every year, mostly children under five years living in Africa. Children are affected most because they have not had the chance to develop the resistance to malaria that normally builds up over a lifetime when living in places with malaria. As well as being given specific drug treatments against the malaria, children are also often given fluids into their veins, as they appear dehydrated. What Did the Researchers Find? The researchers studied children who were sick with malaria and measured how dehydrated the children were. To do the measurements, they used an accepted technique that required injections into the vein, and also a newer, simpler method that used electrodes. Neither technique suggested that any of the children were severely dehydrated. What Does This Mean for Patients? The study suggests that severe dehydration isn’t a big problem in children with severe malaria. So it may not be necessary to give lots of fluids into the vein. (While treatment of malaria is still being worked out, malaria can, of course, often be prevented by using insecticide-treated bednets.) Are There Any Problems with the Study? The new technique for measuring dehydration will need to be assessed in larger studies: this study is small, so the results may not be entirely accurate. Where Can I Get More Information? The World Health Organization is coordinating many of the initiatives to combat malaria (http:// www.who.int/topics/malaria/en/). Medicines for Malaria Venture is trying to develop new affordable antimalarial drugs (http://www.mmv.org/pages/page_main.htm).

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Open access, freely available online

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Nevirapine and Efavirenz Elicit Different Changes in Lipid Profiles in AntiretroviralTherapy-Naive Patients Infected with HIV-1 Frank van Leth1*, Prahpan Phanuphak2, Erik Stroes3, Brian Gazzard4, Pedro Cahn5, Franc¸ois Raffi6, Robin Wood7,8, Mark Bloch9, Christine Katlama10, John J. P. Kastelein3, Mauro Schechter11,12, Robert L. Murphy13, Andrzej Horban14, David B. Hall15, Joep M. A. Lange1,16, Peter Reiss1 1 International Antiviral Therapy Evaluation Center, Division of Infectious Diseases, Tropical Medicine, and AIDS, Department of Internal Medicine, Academic Medical Center, University of Amsterdam, The Netherlands, 2 Thai Red Cross AIDS Research Center, Bangkok, Thailand, 3 Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, The Netherlands, 4 Chelsea and Westminster Hospital, London, United Kingdom, 5 Fundacion Huesped, Buenos Aires, Argentina, 6 University Hospital, Nantes, France, 7 Sommerset Hospital, Capetown, South Africa, 8 University of Capetown, South Africa, 9 Holdsworth House General Practice, Darlinghurst, Australia, 10 Hospital Pitie-Salpetriere, Paris, France, 11 Hospital Sao Francisco de Assis, Rio de Janeiro, Brazil, 12 Hospital University Clementino Fraga Filoho, Rio de Janeiro, Brazil, 13 Northwestern University, Chicago, Illinois, United States of America, 14 Wojewodzki Szpital Zakazny, Warsawa, Poland, 15 Boehringer Ingelheim, Ridgefield, Connecticut, United States of America

Competing Interests: See section at end of manuscript. Author Contributions: FvL, ES, DBH, JMAL, and PR designed the study. FvL and DBH analysed the data. PP, BG, PC, FR, RW, MB, CK, MS, RLM, and AH enrolled patients. FvL wrote the first draft of the paper. All authors contributed to the writing of the final version of the manuscript. Academic Editor: Andrew Carr, St. Vincent’s Hospital, Australia Citation: van Leth F, Phanuphak P, Stroes E, Gazzard B, Cahn P, et al. (2004) Nevirapine and efavirenz elicit different changes in lipid profiles in antiretroviral-therapynaive patients infected with HIV-1. PLoS Med 1(1): e19. Received: May 18, 2004 Accepted: August 17, 2004 Published: October 19, 2004 DOI: 10.1371/journal.pmed.0010019 Copyright: Ó 2004 van Leth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: 3TC, lamivudine; ART, antiretroviral therapy; bd, twice daily; BMI, body mass index; CHD, coronary heart disease; CI, confidence interval; d4T, stavudine; EFV, efavirenz; HDL-c, highdensity lipoprotein cholesterol; LDL-c, low-density lipoprotein cholesterol; NCEP, National Cholesterol Education Program; NNRTI, non-nucleoside reverse transcriptase inhibitor; NVP, nevirapine; od, once daily; OT, on treatment; PI, protease inhibitor; pVL, plasma HIV-1 RNA concentration; TC, total cholesterol; TG, triglyceride *To whom correspondence should be addressed. E-mail: f.vanleth@ iatec.com

ABSTRACT Background Patients infected with HIV-1 initiating antiretroviral therapy (ART) containing a nonnucleoside reverse transcriptase inhibitor (NNRTI) show presumably fewer atherogenic lipid changes than those initiating most ARTs containing a protease inhibitor. We analysed whether lipid changes differed between the two most commonly used NNRTIs, nevirapine (NVP) and efavirenz (EFV).

Methods and Findings Prospective analysis of lipids and lipoproteins was performed in patients enrolled in the NVP and EFV treatment groups of the 2NN study who remained on allocated treatment during 48 wk of follow-up. Patients were allocated to NVP (n = 417), or EFV (n = 289) in combination with stavudine and lamivudine. The primary endpoint was percentage change over 48 wk in highdensity lipoprotein cholesterol (HDL-c), total cholesterol (TC), TC:HDL-c ratio, non-HDL-c, lowdensity lipoprotein cholesterol, and triglycerides. The increase of HDL-c was significantly larger for patients receiving NVP (42.5%) than for patients receiving EFV (33.7%; p = 0.036), while the increase in TC was lower (26.9% and 31.1%, respectively; p = 0.073), resulting in a decrease of the TC:HDL-c ratio for patients receiving NVP (
4.1%) and an increase for patients receiving EFV (þ5.9%; p , 0.001). The increase of non-HDL-c was smaller for patients receiving NVP (24.7%) than for patients receiving EFV (33.6%; p = 0.007), as were the increases of triglycerides (20.1% and 49.0%, respectively; p , 0.001) and low-density lipoprotein cholesterol (35.0% and 40.0%, respectively; p = 0.378). These differences remained, or even increased, after adjusting for changes in HIV-1 RNA and CD4þ cell levels, indicating an effect of the drugs on lipids over and above that which may be explained by suppression of HIV-1 infection. The increases in HDL-c were of the same order of magnitude as those seen with the use of the investigational HDL-cincreasing drugs.

Conclusion NVP-containing ART shows larger increases in HDL-c and decreases in TC:HDL-c ratio than an EFV-containing regimen. Based on these findings, protease-inhibitor-sparing regimens based on non-nucleoside reverse transcriptase inhibitor, particularly those containing NVP, may be expected to result in a reduced risk of coronary heart disease.

Introduction Numerous large epidemiological studies have unambiguously demonstrated a strong inverse relationship between the plasma concentration of high-density lipoprotein cholesterol (HDL-c) and the incidence of coronary heart disease (CHD) [1,2]. Recent attempts to develop therapies

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treatment for at least 95% of the time during the 48 wk of follow-up (self-reported). Change of d4T and/or 3TC was allowed for reasons of toxicity. Employing such ‘on treatment’ (OT) analysis, allows the best possible assessment of lipid changes that actually result from differences in regimens. Patients in the NVP-od and NVP-bd groups were combined, given that the virologic efficacy of these treatments was comparable and no differences in risk of virologic failure were observed.

aimed at increasing HDL-c as innovative CHD-risk-reducing strategies illustrate the potential of HDL-c as a potent antiatherogenic mediator [3,4,5,6]. Combination antiretroviral therapy (ART) for the treatment of HIV-1 infection has been associated with fat redistribution, insulin resistance, and changes in plasma concentrations of lipids and lipoproteins [7,8,9]. Each of these phenomena is associated with increased CHD risk in the general population. It is not surprising, therefore, that in the setting of HIV-1 infection, increasing exposure to potent combination ART has been demonstrated to be associated with an incremental risk of CHD in a recent prospective study [10]. Interestingly, however, the changes in lipids and lipoproteins differ between patients using an ART regimen containing either a protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor (NNRTI). Whereas many of the PI-based regimens are often associated with increased levels of triglycerides (TGs), total cholesterol (TC), and lowdensity lipoprotein cholesterol (LDL-c) [8,9,11], NNRTI-based regimens importantly differ from PI-based regimens by being associated with marked increases of HDL-c and lesser increases of LDL-c and TGs [12,13]. Notably, the increases in HDL-c demonstrated with NNRTI-containing ART markedly exceed those that may be induced with any of the currently licensed statins or fibrates [14]. Although as yet no clinical data have been generated to support this, these differences between ART regimens raise the expectation that NNRTI-based regimens, particularly in view of their effects on HDL-c, may favourably modify the CHD risk compared with many of the PI-containing regimens. With respect to the two currently commonly used NNRTIs, nevirapine (NVP) and efavirenz (EFV), no detailed comparative data have been reported concerning their effect on plasma lipids and HDL-c in particular. We prospectively analysed lipid and lipoprotein changes in a preplanned substudy of the 2NN trial in which ART-naive patients received stavudine (d4T) and lamivudine (3TC) with the randomly assigned addition of NVP, EFV, or both drugs combined.

Follow-Up and Assessments Plasma samples for prospective determination of lipids and lipoproteins were collected at baseline (before start of treatment) and at weeks 2, 4, 8, 12, 24, 36, and 48. Blood was drawn after a mandatory fast of at least 3 h. The samples were analysed in local laboratories according to predefined protocols. These laboratories were selected by the Virtual Central Laboratory (Zeist, The Netherlands), which selected the laboratories, assured the quality of the analyses and data, and standardised all results. Plasma concentrations of HDL-c, TC, and TGs were assessed by standard enzymatic assays. The concentration of LDL-c was calculated using the Friedewald equation, but only when the concentration of TGs was below 4.5 mmol/l [16]. Because the calculation of LDL-c depends on the measured TG concentrations and these TG levels might be biased because of the relatively short mandatory fasting period, we also calculated the non-HDL-c levels. These are considered to be much less influenced by TG levels. The pVL was measured at a central laboratory (LabCorp, Research Triangle Park, North Carolina, United States) using Ultra Sensitive Roche Amplicor 1.5 (Roche Diagnostics, Basel, Switzerland) with a lower limit of quantification of 50 copies/ml.

Outcome Measurements The primary study outcome was the mean percentage change of HDL-c, TC, TC:HDL-c ratio, non-HDL-c, LDL-c, and TGs between start of allocated treatment and week 48. For each patient at each specific study week we calculated this estimate as concentration at week X minus concentration at baseline divided by concentration at baseline, times 100. Study-week-specific estimates were used for the subsequent analyses. Factors assessed for a possible association with the primary outcome were sex, study region (Asia/Australia, South Africa, South America, Europe/North America), body mass index (BMI) (continuous), increase between start of therapy and week 48 in CD4þ cells (,100, 100–250, or .250 cells/mm3) or decrease in pVL (,2.5, 2.5–3.5, or .3.5 log10), and virologic failure during follow-up. Virologic failure was defined as (1) never having obtained a pVL of less than 50 copies/ml or (2) a rebound to two consecutive pVLs of 50 copies/ml. A single pVL of 50 copies/ml at week 48 was also considered a virologic failure.

Methods Participants and Treatment Allocation The 2NN trial was an open-label study, the main results of which have been published elsewhere [15]. Patients enrolled were 16 y of age or older, ART-naive, and had a plasma HIV-1 RNA concentration (pVL) of at least 5,000 copies/ml. Main exclusion criteria were pregnancy or breastfeeding, abnormal laboratory results at screening, the use of immuno-modulating therapy, or anticipated nonadherence. All patients used d4T (40 mg twice daily [bd] or 30 mg bd when less than 60 kg) and 3TC (150 mg bd). In addition, patients were randomly allocated to NVP at 400 mg once daily (od), NVP at 200 mg bd, EFV at 600 mg od, or NVP and EFV at 400 mg od and 800 mg od, respectively. Patients were included from 65 different study sites in 17 countries in Asia, Australia, North America, South America, South Africa, and Europe. The 2NN study had been approved by the ethics committees of all participating institutions, and all patients had given written informed consent. The current analyses were preplanned. Only those patients were included who used all components of their allocated PLoS Medicine | http://www.plosmedicine.org

Statistical Analyses The analyses included the NVP and EFV treatment groups only. This choice was made since the results of the main 2NN study clearly showed that the simultaneous use of NVP and EFV shouldn’t be recommended in clinical practice in view of 065

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Table 1. Baseline Characteristics of Patients Included in the 2NN Lipid Substudy and the 2NN Main Study

Male, n (%) Age, median (IQR) BMI, median (IQR) Region, n (%) Asia/Australia South Africa South America Europe/North America CD4þ cells, cells/mm3 median (IQR) HIV-1 RNA, log10 median (IQR) CDC-class C, n (%) Risk behaviour, n (%) Heterosexual Homosexual Intravenous drugs Other/unknown

Patients Taking NVP (n = 417)

Patients Taking EFV (n = 289)

Total Patients (n = 706)

Patients in 2NN Main (n = 1,216)

261 (63) 35 (29–42) 20 (18–22)

185 (64) 35 (30–40) 19 (17–22)

446 (63) 35 (30–40) 20 (17–22)

772 (63) 34 (29–40) 19 (17–22)

74 (18) 145 (35) 87 (21) 107 (26) 180 (70–310) 4.6 (4.4–5.4) 89 (21)

62 (22) 108 (37) 61 (21) 58 (20) 190 (70–350) 4.7 (4.4–5.5) 58 (20)

136 (19) 253 (36) 148 (21) 165 (23) 180 (70–330) 4.7 (4.5–5.5) 147 (21)

223 (18) 430 (35) 249 (21) 314 (26) 190 (70–330) 4.7 (4.4–5.5) 253 (21)

244 115 15 43

161 93 8 27

405 208 23 70

695 350 55 116

(59) (27) (4) (10)

(56) (32) (3) (9)

(57) (29) (3) (10)

(57) (29) (5) (10)

IQR, interquartile range. DOI: 10.1371/journal.pmed.0010019.t001

treatment group. Of these, 42 (6.9%) patients in the NVP group and 25 (6.3%) patients in the EFV group did not start their treatment or were considered a ‘study entry violator’ by a (blinded-to-treatment) independent endpoint committee. These patients were excluded from the analyses. From the remaining patients (565 using NVP and 375 using EFV), only those who remained on their assigned treatment during the follow-up were included in the analyses. This resulted in a final sample size of 417 (68.7%) patients in the NVP group and 289 (72.3%) in the EFV group. All of the included patients had at least one measurement of each lipid parameter and could therefore be used in the statistical models. The baseline characteristics of the subset of patients included in the current analyses are summarised in Table 1. These baseline characteristics were comparable with those of all patients enrolled in the main 2NN study. In the NVP group, 148 of the 565 eligible patients (26.2%) were not included in the OT analyses. Of these 96 (65%) were nonadherent (including patients lost to follow-up while on randomised treatment), 30 (20%) changed their NNRTI to EFV, and 22 (15%) changed their regimen by adding a PI. In the EFV group, 76 of the 375 eligible patients (20.3%) were not included in the OT analyses. Of these 54 (71%) were nonadherent, 17 (22%) changed their NNRTI to NVP, 3 (4%) added a PI, and 2 (3%) added a third nucleoside reverse transcriptase inhibitor.

increased toxicity of this combination in the absence of increased virologic efficacy. The mean percentage changes in lipid concentrations were modelled using a mixed model incorporating repeated measurements. This model handles missing data adequately by estimating the outcome of a specific variable based on the available data given the specified covariate structure. The variables (fixed effects) in the model were tested for significance using the Type III F-statistic. The estimates of a specific level of the fixed effect were modelled using the ‘least squared means’ approach. Differences in these estimates between different levels of the variable were tested for significance using the t-statistic. Since the analyses might be biased because of the OT approach or the modelling of data, we performed two sensitivity analyses. The first was an analysis using the same modelling strategy but for an intention-to-treat population including all patients who started their randomised treatment. The second was an analysis using only available data for the OT population, without modelling of data points. Independent risk factors were assessed by multivariable regression analyses. The multivariable analysis included the variable ‘treatment group’ and all predefined variables. Interaction between treatment group and a specific variable was assumed at a p-value less than 0.15. A two-sided p-value less than 0.05 was considered statistically significant in the final analyses. The SAS statistical package was used for analyses (version 8.02, SAS Institute, Cary, North Carolina, United States).

Changes in Lipids and Lipoproteins All changes within the treatment groups in lipid and lipoprotein concentration, as well as in TC:HDL-c ratio were statistically significant. The increase of HDL-c was 8.9% (95% confidence interval [CI], 0.6–17.1) larger in the NVP treatment group (42.5%) than in the EFV treatment group (33.7%). This was statistically significant (p = 0.036) (Table 2). In contrast, the increase

Results Disposition of Patients Of the 1,216 patients included in the 2NN study, 607 were allocated to the NVP treatment group and 400 to the EFV PLoS Medicine | http://www.plosmedicine.org

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Table 2. Lipid Concentrations at Baseline and Week 48 and Mean Percentage Change Lipid

Patients Taking NVP (n = 417) Week 0

TC HDL-c Non-HDL-c LDL-c TGs TC:HDL-c ratio

3.9 1.0 3.0 2.2 1.2 4.2

a

(3.3–4.6) (0.8–1.2) (2.3–3.6) (1.7–2.9) (0.8–1.8) (3.3–5.0)

Week 48

4.9 1.3 3.5 2.8 1.2 3.7

a

(4.1–5.7) (1.1–1.6) (2.7–4.3) (2.2–3.5) (0.8–2.2) (2.9–4.7)

Patients Taking EFV (n = 289)

Percent Week 0 Increaseb

Week 48

Difference (NVP
EFV) Percent Increase (95% CI)

26.9 42.5 24.7 35.4 20.1
4.1

5.0 1.2 3.6 2.9 1.4 4.2

31.1 33.7 33.6 40.0 49.0 5.9

(1.5) (2.7) (2.1) (3.4) (4.4) (1.6)

3.9 1.0 3.0 2.3 1.2 4.2

(3.4–5.9) (0.8–1.2) (2.4–3.5) (1.9–2.9) (0.8–1.7) (3.3–5.0)

(4.3–5.9) (1.0–1.5) (3.0–4.7) (2.4–3.8) (0.9–2.3) (3.3–5.2)

(1.8) (3.2) (2.5) (4.0) (5.3) (1.9)

p-Value for Difference


4.2 (
8.7 to 0.4) 0.073 8.9 (0.6 to 17.1) 0.036
8.9 (
15.4 to
2.5) 0.007
4.6 (
14.9 to 5.7) 0.378
28.9 (
42.3 to
15.5) ,0.001
10.0 (
14.9 to
5.0) ,0.001

All percentage changes within a treatment group were statistically significant. a Units: mmol/l, median (interquartile range). b Mean percentage change (standard error), modeled by repeated measurements. Mean percentage change was calculated at each specific time point for each individual patient as ((concentration[week X] – concentration[baseline]) / concentration[baseline]) 3 100. DOI: 10.1371/journal.pmed.0010019.t002

ratio (
9.8%; 95% CI,
14.7 to
4.9), non-HDL-c (
8.8%; 95% CI,
14.6 to
3.0), and TGs (
24.9%; 95% CI,
37.2 to –12.6). Additionally, the difference between NVP and EFV treatment groups became statistically significant for TC (
4.2%; 95% CI,
8.5 to 0.0) and LDL-c (
14.2%; 95% CI,
28.4 to 0.0) compared to the original OT analysis. The second sensitivity analysis (using only available data for the OT population) also showed comparable estimates (data not shown). The increase in HDL-c for patients who started their ART when their HDL-c levels were, according to the National Cholesterol Education Program (NCEP) guidelines, low (,1.03 mmol/l), normal (1.03–1.55 mmol/l), or high (.1.55 mmol/l) is reported in Table 3. In both treatment groups, the majority of patients had a low HDL-c at the start of therapy. These patients showed the largest increase in HDL-c over 48 wk. Even patients with a normal baseline HDL-c level showed statistically significant, marked increases of HDL-c. The effect of baseline HDL-c level on percentage increase was comparable in both treatment groups (interaction, p = 0.409).

in TC was smaller in the NVP group (26.9%) than in the EFV group (31.1%), but this difference (
4.2%; 95% CI,
8.7 to 0.4) was not statistically significant (p = 0.073). These changes resulted in a decrease of the TC:HDL-c ratio in the NVP group (
4.1%) compared to an increase in the EFV group (þ5.9%; p , 0.001), and a significantly smaller increase of non-HDL-c in the NVP group (difference,
8.9%; 95% CI,
15.4 to
2.5; p = 0.007). The increase of TGs was 28.9% (95% CI,
42.3 to
5.0) smaller in the NVP group (20.1%) than in the EFV group (49.0%; p , 0.001). The difference in LDL-c increase was not statistically significant (35.4% for NVP group; 40.0% for EFV group; p = 0.378). In the first sensitivity analysis (intention-to-treat population), the increases of HDL-c were slightly lower (41.2% for NVP group; 32.4% for EFV group), just as for TC (26.1% for NVP group, 30.4% for EFV group) and non-HDL-c (24.3% for NVP group, 33.1% for EFV group). The TC:HDL-c ratio showed a smaller decrease for patients taking NVP (
2.6%) but a larger increase for patients taking EFV (þ7.2%). The increase in TGs was larger for both patients taking NVP (24.3%) and patients taking EFV (49.3%). The LDL-c increase was somewhat smaller for patients taking NVP (33.1%) but larger for patients taking EFV (47.3%). The difference between patients taking NVP and those taking EFV for HDL-c (8.8%; 95% CI, 1.3
16.3) remained statistically significant, just as the difference in the TC:HDL-c

Multivariable Analysis Factors independently associated with changes in the lipid concentrations were analysed by a multivariable regression analysis (Table 4). Men had a significantly smaller increase of HDL-c, compared to women, but a larger increase of TC. This resulted in an increased TC:HDL-c ratio for men and a

Table 3. Increase in HDL-c Stratified by Baseline HDL-c (NCEP Categories) NCEP Category

Low (,1.03 mmol/l) Normal (1.03–1.55) High (.1.55 mmol/l)

Nevirapine (n = 417)

EFV (n = 289)

n (%)

Percent Increase

251 (60.2) 140 (33.6) 26 (6.2)

53.7 (3.0) 30.8 (4.0)
0.9 (9.5)

a

p-Value

n (%)

Percent Increase

173 (59.9) 95 (32.9) 21 (7.3)

51.1 (4.3) 11.1 (5.7)
5.7 (12.1)

0.698 ,0.001 0.614

a

Mean percentage change (standard error), modeled by repeated measurements. Mean percentage change was calculated at each specific time point for each individual patient as ((concentration[week X] – concentration[baseline]) / concentration[baseline]) 3 100. DOI: 10.1371/journal.pmed.0010019.t003

PLoS Medicine | http://www.plosmedicine.org

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Table 4. Factors Associated with Percentage Change in Lipid Parameters (Multivariable Analyses) TC

HDL-c

TC:HDL-c Ratio

a

Sex Male Female p-Valuec Regiond A B C D p-Value BMI (per unit) p-Value pVL decrease (log10) ,2.5 2.5–3.5 .3.5 p-Value CD4þ increase (cells/mm3) ,100 100–250 .250 p-Value Virologic failure No Yes p-Value

Non-HDL-c

LDL-c

TGs

Base Percent Base Percent Base Increaseb Increase

Percent Increase

Base Percent Base Percent Base Percent Increase Increase Increase

3.9 4.0

24.4 (1.0) 0.9 22.2 (1.3) 1.0 0.103

29.4 (1.9) 4.3 34.3 (2.3) 3.9 0.045

1.4 (1.1)
4.6 (1.4) ,0.001

2.9 3.0

24.5 (1.5) 2.2 19.8 (1.9) 2.4 0.021

34.1 (2.8) 1.3 30.5 (3.5) 1.0 0.326

27.6 (2.7) 12.0 (3.4) ,0.001

4.1 4.0 3.7 3.9

24.2 (1.6) 20.7 (1.2) 25.0 (1.5) 23.3 (1.6) 0.052 0.2 (0.2) 0.179

33.7 (3.0) 33.5 (2.2) 25.5 (2.7) 34.6 (2.9) 0.029
0.7 (0.3) 0.028

4.6 4.4 3.7 4.0


4.2 (1.8)
6.2 (1.3) 4.5 (1.6)
0.5 (1.7) ,0.001 0.7 (0.2) ,0.001

3.1 3.0 2.6 2.8

21.2 (2.5) 16.7 (1.8) 26.8 (2.2) 23.9 (2.4) ,0.001 0.6 (0.3) 0.023

34.9 (4.5) 26.2 (3.2) 31.0 (4.1) 37.2 (4.3) 0.092 1.0 (0.5) 0.037

1.4 1.0 1.1 1.3

21.0 (4.3) 13.5 (3.2) 32.5 (4.0) 12.3 (4.2) ,0.001 1.1 (0.5) 0.022

4.1 4.0 3.8

18.6 (1.5) 1.0 22.5 (1.2) 1.0 28.9 (1.4) 0.9 ,0.001

23.5 (2.6) 3.9 31.4 (2.2) 4.1 40.7 (2.6) 4.4 ,0.001


0.4 (1.6)
0.9 (1.3)
3.5 (1.6) 0.221

3.1 3.0 2.8

18.3 (2.2) 2.5 21.9 (1.8) 2.3 26.2 (2.1) 2.1 0.018

22.5 (3.9) 1.0 29.7 (3.3) 1.1 44.7 (3.9) 1.5 ,0.001

23.4 (3.9) 26.9 (3.2) 9.2 (3.8) ,0.001

4.0 3.9 3.9

21.6 (1.4) 1.0 25.1 (1.2) 0.9 23.3 (1.4) 1.0 0.066

28.3 (2.5) 4.0 37.9 (2.2) 4.4 29.3 (2.6) 4.1 ,0.001


0.7 (1.5)
3.7 (1.3)
0.4 (1.6) 0.073

3.0 3.0 2.9

20.4 (2.0) 2.3 23.6 (1.8) 2.3 22.3 (2.1) 2.3 0.378

28.3 (3.7) 1.1 34.3 (3.2)) 1.2 34.3 (3.9) 1.2 0.312

20.0 (3.6) 18.3 (3.2) 21.2 (3.8) 0.755

3.9 3.9

22.7 (0.8) 1.0 24.0 (1.7) 0.9 0.471

32.5 (1.5) 4.2 31.1 (3.0) 4.2 0.663


2.0 (0.9)
1.2 (1.8) 0.657

3.0 2.8

21.5 (1.2) 2.3 22.7 (2.5) 2.3 0.650

31.2 (2.2) 1.2 33.4 (4.5) 1.2 0.642

14.1 (2.2) 25.5 (4.4) 0.017

0.9 0.9 1.0 1.0

2.4 2.5 2.1 2.3

a

Units: mmol/l. Percentage increase (standard error) between baseline and week 48, modelled by repeated measurements. Mean percentage change was calculated at each specific time point for each individual patient as ((concentration[week X] – concentration[baseline]) / concentration[baseline]) 3 100. c For percentage increase. d A, Asia/Australia; B, South Africa; C, South America; D, Europe/North America. DOI: 10.1371/journal.pmed.0010019.t004 b

increase in LDL-c (p , 0.001), and a 3.6% increase in nonHDL-c (p = 0.002), while the TC:HDL-c ratio declined with 1.6% (p = 0.051). In this analysis, there was also a clear association between pVL decline and change in TGs (6.3% decline per log10; p = 0.002). In general, a smaller CD4þ-cell increase was associated with a smaller increase in lipid concentration, while increases of more than 250 cells/mm3 did not show markedly different effects compared to increases of 100–250 cells/mm3. When analysed as a continuous variable, there was no statistically significant association between CD4þ-cell increase and change in any of the lipid parameters. BMI was independently associated with increases in all lipid parameters, except TC. Although the increases per unit increase in BMI were statistically significant, the magnitude of increases was rather low. All these factors exhibited a similar effect in both the NVP and the EFV treatment group (no significant interactions). There were, however two exceptions. For changes in TGs, the effect of sex and pVL decrease differed between the treatment groups (interaction, p = 0.005 and p = 0.075,

decreased ratio for women, while the increase of non-HDL-c was significantly larger in men. The changes in lipid concentrations varied markedly by region. Patients from Asia/Australia and South Africa had the largest decrease in the TC:HDL-c ratio because of an increase of HDL-c that outweighed the increase of TC. Patients from South America, compared to those from other regions, had a significantly smaller HDL-c increase with a comparable TC increase, resulting in an increased TC:HDL-c ratio. Although patients from Europe showed the largest change in HDL-c, the increases in TC and TC:HDL-c ratio were intermediate. A striking finding is the much larger increase of TGs in patients from South America compared to those in patients from other regions, which to a lesser extent was also seen for nonHDL-c. For all lipid concentrations, except TGs, there was a clear pattern of larger increases of lipid levels with larger decreases of pVL over 48 wk. This was also seen when the pVL increase over 48 wk was analysed as a continuous variable. For each log10 larger decrease in pVL there was a 4.6% increase in TC (p , 0.001), a 7.8% increase in HDL-c (p , 0.001), a 10.2% PLoS Medicine | http://www.plosmedicine.org

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Nevirapine, Efavirenz, and Lipid Changes

Figure 1. Change in Plasma Concentrations of Lipids and Lipoproteins Adjusted for sex, region, pVL decrease, and CD4þ-cell increase. DOI: 10.1371/journal.pmed.0010019.g001

respectively). Men had a significant increase of TGs in both the NVP group (14.1%) and the EFV group (43.3%); women using NVP had no significant TG increase (6.5%), while those using EFV had (15.9%). The effect of pVL decrease on TG increase was quite different for patients taking NVP versus those taking EFV. In the NVP group, the increase in TG concentration was 17.2% for a pVL decrease less than 2.5 log10, 16.6% for a decrease between 2.5 and 3.5 log10, and
6.1% (denoting a decrease) for a pVL decrease more than 3.5 log10. In the EFV group, these estimates were 27.4%, 37.2%, and 26.0%, respectively. Adjusting for the variables included in the multivariable model, the difference between patients taking NVP and those taking EFV in HDL-c increase (9.8%; 95% CI, 3.4
16.3) and decrease of the TC:HDL-c ratio (
11%; 95% CI,
15.1 to
6.8) remained statistically significant. Also, the difference in non-HDL-c increase (
9.5%; 95% CI,
14.6 to
4.4) and TG increase (
27.2%; 95% CI,
38.0 to
16.4) remained statistically significant. The difference in TC increase (
4.4%; 95% CI,
8.0 to
0.8) became statistically significant. The difference between NVP and EFV groups for the increase in LDL-c remained statistically nonsignificant (
6.1%; 95% CI,
14.7 to 2.6). PLoS Medicine | http://www.plosmedicine.org

The adjusted increase of HDL-c was 42.3% and 32.4% for patients taking NVP and EFV, respectively (p = 0.003). The adjusted change in TC:HDL-c ratio was
4.3% for patients taking NVP and þ6.6% for patients taking EFV (p , 0.001). These values were 26.6% and 31.0% for TC (p = 0.020), 24.4% and 33.9% for non-HDL-c (p , 0.001), 17.9% and 45.1% for TG (p , 0.001), and 35.5% and 41.5% for LDL-c (p = 0.168). These estimates were very similar in the two sensitivity analyses (data not shown). The proportional changes of the different plasma lipid concentrations over 48 wk are graphically depicted in Figure 1.

Discussion Initiation of an ART regimen containing NVP or EFV is accompanied by a significant increase of HDL-c, with concomitant increases of TC, non-HDL-c, TGs, and LDL-c. The proportional increase of HDL-c was significantly larger in the NVP treatment group compared to the EFV treatment group, while the proportional increase of TC, non-HDL-c, and TGs was significantly smaller. In the NVP group, the TC:HDL-c ratio decreased, compared to an increase in the EFV group. These observations are different from what is 069

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were of a comparable magnitude to that found for patients taking NVP in this present study (0.97 and 0.55 mmol/l, respectively). However, the HDL-c increase was more than ten times smaller (0.03 mmol/l) in the Riddler et al. study than the 0.36 mmol/l observed in patients taking NVP in the present study, while the mean HDL-c values at which ART was started were comparable in the two studies (1.04 and 1.0 mmol/l, respectively). This indicates that although at least part of the change in TC and LDL-c may reflect a ‘return towards normal’, the magnitude of the HDL-c increase observed in our study must have occurred through additional mechanisms. Since we have no information on the antiretroviral efficacy of the regimens used in the Riddler et al. study, we have to consider that the reported differences between the Riddler et al. study and the present study might be partly due to differences in HIV-1 suppression. However, the type of PI-based regimens used in the Riddler et al. study and the long-term adequate adherence by the patients make large differences in antiretroviral efficacy unlikely. We are currently conducting studies to unravel whether NVP possibly stimulates synthesis of the most important apolipoprotein of HDL-c, apoAI, or alternatively, for instance, decreases the clearance of HDL-c particles. Several studies have convincingly shown that an HDL-c increase is associated with a significant decrease in CHD mortality independent of changes in LDL-c [1,2]. Overall, extrapolation of these studies indicates that a 0.025-mmol/l increase in HDL-c is expected to be associated with a 2%–3% reduction in CHD risk, while an increase of 1.0 mmol/l in LDL-c will increase the CHD risk by 25%. The mean absolute increases in HDL-c and LDL-c were 0.36 and 0.54 mmol/l, respectively, for patients taking NVP, and 0.24 and 0.65, respectively, for patients taking EFV. It can therefore be estimated that, taking the observed effects on both HDL-c and LDL-c into account, the reduction in CHD risk would be 15% for patients taking NVP and 3% for patients taking EFV compared to ART regimens that do not include NNRTIs. Although the differences in absolute concentrations of HDLc and LDL-c may seem modest when comparing the NVP and EFV treatment groups, the combined effect of these changes on CHD risk seems marked. It should be emphasised that these are theoretical estimates, which do not take into account that increases in TGs would be expected to have an opposite effect on CHD risk. The increase in this last parameter is, however, smaller for patients taking NVP than for patients taking EFV. Furthermore, we do not have information on the presence of conventional risk factors for CHD. The actual effect of the lipid changes associated with particular ART regimens on CHD can only be substantiated by clinical endpoint studies.

seen with most PI-based ART regimens, in which higher concentrations of TC, LDL-c, and TGs are reported but without the concurrent higher levels of HDL-c [17,18]. In contrast to a small randomised study (n = 67) that did not show significant differences between NVP and EFV [19], the present study demonstrates a more favourable lipid profile for treatment including NVP than for treatment including EFV in ART-naive patients.

HDL-c Increase and NNRTI Increases of HDL-c with the use of NVP or EFV have been described in studies for patients switching from a PI-based regimen to a NNRTI-based regimen [20,21]. Data for ARTnaive patients starting therapy with an NNRTI-based regimen are scarce. Van der Valk et al. reported an increase of HDL-c of 0.44 mmol/l for patients initiating treatment with didanosine, d4T, and NVP in the Atlantic trial [12]. Tashima et al. reported an increase of HDL-c of 0.21 mmol/l in patients treated with EFV and either zidovudine plus 3TC, or indinavir [13], while Negredo et al. showed an increased HDLc concentration in therapy-naive patients starting a regimen of didanosine, d4T, and EFV (0.34 mmol/l) [22]. The present study showed a clear effect of baseline HDL-c on the proportional increase. The largest increases were seen for patients who had an increased CHD risk based on their low HDL-c level (,1.03 mmol/l) according to NCEP guidelines. But also patients with a normal HDL-c level, who are not at an increased CHD risk, showed marked increases in HDL-c. This baseline effect can likewise be distilled from the other studies, where those with the lowest baseline value (0.93 mmol/l; Atlantic study [12]) showed the largest HCL-c increase, while the smallest increase was seen in the study with the highest baseline value (1.23 mmol/l; Tashima study [13]). The study by Negredo et al. [21], which included patients with similar baseline HDL-c levels as in the present study, showed an increase of HDL-c comparable to that in the present study (0.34 and 0.36 mmol/l, respectively). The much more modest HDL-c-increasing effect of statins likewise shows such a correlation with baseline level in patients without HIV-1. One may postulate that the HDL-c increase merely reflects an adequate suppression of HIV-1 infection (‘return towards normal’). In support, a larger decrease in pVL was associated with a larger increase of HDL-c in the present study. However, the magnitude of the HDL-c increase was only slightly different for patients experiencing virologic failure during these 48 wk (29.5%) and those with complete suppression (34.0%; p = 0.161), and the increases of HDL-c remained statistically significant even after adjustment for pVL decrease. Riddler et al. compared changes in lipid concentrations before seroconversion for HIV-1, initiation of ART, and during ART in patients using different ART regimens, which all but one included a PI [23]. The period between seroconversion and start of ART was characterised by decreases in TC, LDL-c, and HDL-c. Between initiation of ART and the first follow-up visit (mean, 1.3 years), the concentrations of TC and LDL-c increased again to levels that did not differ significantly from before seroconversion. Such a ‘return to normal’ as a result of ART was not seen for HDL-c. The reported increases by Riddler et al. in TC (0.88 mmol/l) and LDL-c (0.41 mmol/l) PLoS Medicine | http://www.plosmedicine.org

Changes in TGs, TC, and LDL-c The data indicate that EFV might have a more detrimental effect on TG levels than NVP. That EFV indeed can be associated with an increase in TGs was shown in two studies, in which sporadical hypertriglyceridaemia was reported in patients starting an ART regimen with EFV but without d4T [24,25]. A difference between NVP and EFV treatment with respect to the TG effect is also in line with a study by Negredo et al. [20]. In this study patients were randomised to either continue their successful PI-based regimen or to change to an NVP-based or an EFV-based regimen. Only patients switching 070

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Nevirapine, Efavirenz, and Lipid Changes

additional year of ART use was associated with a 26% increased risk of myocardial infarction [10]. The latter resembles the results from other retrospective and prospective studies [44,45,46]. The relatively high prevalence of known CHD risk factors in patients with HIV-1, especially smoking [47,48], complicates interpretation of the relation between ART use and CHD. None of these studies allows definitive conclusions to be made about the potentially different degree of risk associated with particular ART regimens.

to the NVP regimen showed a significant decrease in TG levels. The proportional increase in TG levels with both NVP and EFV treatment seems large, but the median absolute TG level at week 48 was still low in both treatment groups (1.2 mmol/l and 1.4 mmol/l, respectively). In the NCEP guidelines, a TG concentration below 1.69 mmol/l is still considered normal [26]. The increase in TG level is therefore probably not clinically meaningful. The differences between patients taking NVP and those taking EFV in changes in TC as well as TGs are unlikely to be explained by the concurrent use of d4T. In both treatment groups, the percentage of patients who used d4T as part of their regimen throughout follow-up was high (96% for the NVP treatment group and 98% for the EFV treatment group). This high rate of d4T use might, however, be responsible for the impression that the increases in TC and TGs seem to continue or even accelerate towards the end of the study period, as opposed to a somewhat declining effect of treatment on HDL-c after 24 wk. A possible explanation for this may be the gradual, progressive worsening of fat redistribution or lipodystrophy that one would expect to occur in this continuously d4T-exposed patient population. Both the incidence and severity of lipodystrophy are particularly increased with d4T-containing ART regimens, and lipodystrophy has been reported to be associated with increased TC and TG levels [27,28,29,30,31,32]. It is therefore conceivable that the lipid changes in the second part of the study represent a combined effect of the NNRTI used and a superimposed effect resulting from gradually worsening fat redistribution. Due to the relatively short mandatory fasting period (3 h), the measured TG concentration might be biased, possibly even more so given that HIV-1 infection may be associated with reduced TG clearance following food intake [33]. As a consequence, the estimates of calculated LDL-c might be biased. TG levels influence changes in non-HDL-c less. The fact that in the present study the increases of non-HDL-c, LDL-c, and TGs are all smaller for patients taking NVP than for patients taking EFV suggests that the LDL-c and TG results are valid despite the potentially short mandatory fasting periods.

Limitations and Possible Biases The selection of patients remaining on their allocated treatment for the full 48 wk might have introduced sampling bias with inflated treatment effects. The reported estimates from this OT analysis were very similar to the estimates from the intention-to-treat analysis. This is caused by the fact that only a few patients who were not included in the OT analyses changed their drug regimen by adding a PI that could potentially influence the lipid estimates. The majority remained on their randomised treatment but were insufficiently adherent (or lost to follow-up on their original regimen) to meet the criteria for being eligible for the OT analysis. Patients replacing their assigned NNRTI by the NNRTI from the other treatment group of the study would have little effect on the lipid estimates, since both NNRTIs show changes in lipid concentrations, which go in the same direction. Another possible reason for the similarity between these two analyses could be the relatively late timing of treatment changes (mean of 75 d for patients taking NVP and 95 d for patients taking EFV). The fact that the second sensitivity analysis also showed comparable results indicates that modelling of data did not affect the lipid estimates. A limitation of the present study is the lack of data on conventional CHD risk factors like smoking. The 2NN being a randomised study, it may be expected that these and other confounding variables are equally distributed over the treatment groups. Possible residual confounding, however, cannot be excluded, and the results should therefore be interpreted cautiously.

Conclusion While awaiting the results of future studies, the less atherogenic lipid profile of patients taking NVP in comparison to those of patients taking EFV may be among the various factors to consider when selecting the most appropriate initial ART regimen, particularly for those patients with HIV-1 with a significant a priori CHD risk. Including such a consideration seems warranted, since treatment of the ART-induced lipid changes with currently licensed lipidlowering agents is not without problems. Most of the available statins except pravastatin and fluvastatin, are metabolised through cytochrome isoenzyme CYP3A4, just as the PIs and NNRTIs are, providing concern for potential drug–drug interactions. Furthermore, statin therapy in patients using a PI-based regimen is in general not able to reduce the lipid concentrations to normal levels [49,50]. Studies on the effectiveness of gemfibrozil in patients on a PI-based regimen show conflicting results [51,52,53]. Finally, the introduction of yet another type of medication in a patient population that often needs to use not only ART but also a considerable amount of concomitant medication might jeopardise treat-

ART and CHD The relationship between ART and CHD has been the subject of several studies, based on either clinical or validated surrogate endpoints (like arterial intima-media thickness [34] or endothelial wall function [35]). Studies examining intima-media thickness in patients with HIV-1 treated with ART, or more specifically with PI-based ART, remain inconclusive as to whether ART use induces accelerated intima-media thickening [36,37,38,39]. PI use may have a detrimental effect on endothelial function in vivo, or accelerate foam cell formation in vitro [40,41]. In a retrospective clinical endpoint study including almost 37,000 patients, Bozzette et al. reported no relation between ART use and hospital admission for cardiovascular events [42], a finding confirmed in the ‘Kaiser Permanente’ cohort [43]. However, the large prospective ‘data collection on adverse events of anti-HIV drugs’ (D:A:D) study, specifically designed to identify the extent to which ART may be associated with increased CHD risk, did show that every PLoS Medicine | http://www.plosmedicine.org

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Nevirapine, Efavirenz, and Lipid Changes M. Rattley, E. Nettleship, D. J. Martin, V. Keyser, T. M. Moraites, M. A. Moorhouse, J. A. Pitt, C. J. Orrell, C. Bester, R. Parboosing, P. Moodley, V. Gathiram, D. Woolf. Switzerland: E. Bernasconi, L. Magenta. Thailand: P. Cardiello, E. Kroon, C. Ungsedhapand. United Kingdom: M. Fisher, E. G. L. Wilkins, E. Stockwell, J. Day, R. S. Daintith, N. Perry, C. Timaeus, J. McIntosh-Roffet, A. Powell, M. Youle, M. Tyrer, S. Madge, A. Drinkwater, Z. Cuthbertson, A. Carroll. United States: S. Becker, H. Katner, D. Rimland, M. S. Saag, M. Thompson, M. Witt, M. M. Aguilar, A. LaVoy, M. Illeman, M. Guerrero. Data Safety and Monitoring Board: J. Gatell (chair), E. Belsey, B. Hirschel. Project management: L. Dam, A. Potarca, M. Cronenberg, L. Kreekel. Data management: R. Meester, J. Khodabaks, H.-J. Botma, N. Esrhir, I. Farida, M. Feenstra, K. Jansen, A. Klotz, M. Mulder, G. Ruiter. Laboratory: C. B. Bass, E. Pluymers, E. de Vlegelaer (Labcorp), R. Leeneman (Virtual Central Laboratory). Boehringer Ingelheim: H. Carlier, E. van Steenberge, P. Robinson.

ment adherence, which is of crucial importance for the sustained success of ART treatment. Use of the novel PI atazanavir may also be considered an attractive option in patients at high risk of CHD, given that it is associated with markedly smaller increases of TC, LDL-c, and TGs compared to previously available PI-based regimens [54,55], but it lacks the concurrently large increase in HDL-c seen with NNRTI-based regimens. The reported increase of HDL-c concentration with NNRTI use is far greater than that seen with conventional lipid-lowering drugs and is of a similar magnitude as the HDL-c increases reported with the most powerful HDL-cincreasing drugs that are currently in clinical development [6,56]. Asztalos et al. reported the effect on HDL-c for five major statins in patients with CHD [57]. They concluded that the HDL-c increase was between 4% (simvastatin) and 11% (pravastatin and lovastatin). Treatment with fluvastatin or lovastatin proved to be most effective in patients with a low baseline HDL-c level [56,58]. Clinical trials assessing the effects of fibrates reported an HDL-c increase of 6% and 11% for gemfibrozil [2,59], and 18% for bezafibrate [60]. Unravelling the mechanism or mechanisms by which NVP and EFV raise HDL-c could contribute to the development of novel interventions aimed at increasing HDL-c and thereby ultimately to reducing CHD risk in the population at large.

Competing Interests FvL has received travel grants and honoraria for presentations from Boehringer Ingelheim and travel grants from GlaxoSmithKline. PP has received research grants and honoraria for speaking from GlaxoSmithKline, Bristol-Myers Squibb, Roche, Merck Sharp and Dohme, and Boerhinger Ingleheim. BG has received grants and honoraria from Abbott Pharmaceuticals, GlaxoSmithKline, BristolMyers Squibb, Gilead, and Boehringer Ingleheim. FR has received research grants and honoraria from GlaxoSmithKline, Roche, BristolMyers Squibb, Abbott, and Boehringer Ingleheim. RW is conducting clinical research supported by GlaxoSmithKline, Boehringer Ingelheim, Pfizer, and Bristol-Myers Squibb. MB is currently conducting clinical studies supported by Boehringer Ingelheim, Bristol-Myers Squibb, and Merck. CK has received grants and honoraria for speaking from Bristol-Myers Squibb, GlaxoSmithKline, Boehringer Ingelheim, Bayer, and Roche. MS has received research grants, travel grants, and honoraria for speaking from Abbott, Bristol-Myers Squibb, Boehringer Ingelheim, GlaxoSmithKline, Gilead, Merck, and Roche. RLM has received honoraria for consultancies from Boehringer Ingelheim and Bristol-Myers Squibb. AH has received honoraria for presentations from Boehringer Ingleheim. DBH is employed by Boehringer Ingelheim, the manufacturer of one of the trial medications. JMAL has received honoraria as an advisor for GlaxoSmithKline, Boehringer Ingelheim, Bristol-Myers Squibb, Roche, Merck Sharp and Dohme, Schering-Plough, Bayer, Shire Pharmaceuticals, Agouron/Pfizer, and Virco/Tibotec. PR, in the last five years, has received honoraria for speaking engagements from Boehringer Ingelheim. & JMAL is a member of the editorial board of PLoS Medicine.

Acknowledgments The study was investigator initiated and financially supported by Boehringer Ingelheim. This company had a nonbinding input on issues of study design and analyses, which did not lead to any significant influence on the resulting design and analyses. The company was allowed to provide comments on the manuscript in progress, but had no influence on reporting of the data or the decision to publish. All investigators, as well as Boehringer Ingelheim, had full access to the data after official closure of the database. Investigators Argentina: H. Laplume´, M. B. Lasala, M. H. Losso, E. Bogdanowicz, R. Lattes, A. Krolewiecki, C. Zala, C. Orcese, S. Terlizzi, A. Duran, J. Ebensrteijn. Australia: M. Bloch, O. Russell, D. B. Russell, N. R. Roth, B. Eu, D. Austin, A. Gowers, D. Quan. Belgium: J. Demonty, R. Peleman, B. Vandercam, D. Vogelaers, B. van der Gucht, F. van Wanzeele, M. M. Moutschen. Brazil: R. Badaro, B. Grinsztejn, M. Schechter, D. Uip, E. N. Netto, S. S.Coelho, F. Badaro´, J. H. Pilotto, A. Schubach, M. L. Barros, O. H. M. Leite, C. R. V. Kiffer, C. T. Wunsch, D. Nunes, A. Catalani, R. de Cassia Alves Lira, T. J. Dossin, M. T. D’Allo´ de Oliveira, S. Martini. Canada: B. Conway, J. J. de Wet, J. S. G. Montaner, C. Murphy, B. Woodfall, P. Sestak, P. Phillips, V. Montessori, M. Harris, A. Tesiorowski, B. Willoughby, R. Voigt, J. Farley, R. Reynolds, S. Devlaming. France: J. M. Livrozet, W. Rozenbaum, D. Sereni, M. A.Valantin, C. Lascoux, B. Milpied, C. Brunet, E. Billaud, A. Huart, V. Reliquet, M. F. Charonnat, M. Sicot, J. L. Esnault, L. Slama. Germany: S. Staszewski, M. Bickel. Greece: M. K. Lazanas, N. Stavrianeas, N. Mangafas, I. Zagoreos, S. Kourkounti, V. Paparizos, C. Botsi. Ireland: S. Clarke, E. Brannigan, N. Boyle. Italy: A. Chiriani, F. Leoncini, F. Montella, L. Francesco, S. Ambu, A. Farese, M. Gargiulo, F. Di Sora, F. Lavria, F. Folgori. Poland: M. Beniowski, A. Boron-Kaczmarska, W. Halota, D. Prokopowicz, D. B. Bander, M. L. P. Leszuzyszyn-Pynka, A. W. Wnuk, E. Bakowska, P. Pulik, R. Flisiak, A. Wiercinska-Drapalo, E. Mularska, A. Witor. Portugal: F. Antunes, R. S. E. Sarmento, M. Doroana, A. A. Horta, O. Vasconcelos. South Africa: S. M. Andrews, C. B. Huisamen, D. Johnson, O. Martin, L.-G. Bekker, G. Maartens, D. Wilson, C. J. Visagie, N. J. David, PLoS Medicine | http://www.plosmedicine.org

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8. Carr A, Samaras K, Thorisdottir A, Kaufmann GR, Chisholm DJ, et al. (1999) Diagnosis, prediction, and natural course of HIV-1 proteaseinhibitor-associated lipodystrophy, hyperlipidaemia, and diabetes mellitus: A cohort study. Lancet 353: 2093–2099. 9. Behrens G, Dejam A, Schmidt H, Balks HJ, Brabant G, et al. (1999) Impaired glucose tolerance, beta cell function and lipid metabolism in HIV patients under treatment with protease inhibitors. AIDS 13: F63–F70. 10. Friis-Moller N, Sabin CA, Weber R, d’Arminio Monforte A, El-Sadr WM, et al. (2003) Combination antiretroviral therapy and the risk of myocardial infarction. N Engl J Med 349: 1993–2003. 11. Periard D, Telenti A, Sudre P, Cheseaux JJ, Halfon P, et al. (1999) Atherogenic dyslipidemia in HIV-infected individuals treated with protease inhibitors. The Swiss HIV Cohort Study. Circulation 100: 700–705. 12. van der Valk M, Kastelein JJ, Murphy RL, van Leth F, Katlama C, et al. 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Patient Summary Why Did the Researchers Do the Study? Drugs used to treat HIV (antiretroviral drugs) help patients to live longer, but they can also have some serious side effects. For example, the longer people take them, the higher the risk they’ll get heart disease. Why? Part of the reason is that many—though not all—antiretroviral drugs cause changes in cholesterol levels in the bloodstream (an increase in the amount of ‘‘bad’’ cholesterol and a reduction in the amount of ‘‘good’’ cholesterol). Two of the most commonly prescribed antiretroviral drugs are nevirapine and efavirenz. Previous smaller studies showed that treatment with either of the drugs could increase the amount of ‘‘good’’ cholesterol. The researchers now wanted to directly compare these drugs to find out what effect they had on patients’ cholesterol levels in a much larger group of patients. What Did the Researchers Do? The scientists studied adults with HIV who had never previously taken antiretroviral drugs. All of the patients then took ‘‘triple therapy’’—a combination of three antiretroviral drugs. Some of the patients took nevirapine as part of their triple therapy, whereas some took efavirenz. The researchers took blood samples regularly for almost a year and measured patients’ cholesterol levels. What Did the Researchers Find? They confirmed that both nevirapine and efavirenz indeed have a beneficial effect on patients’ cholesterol levels. They both increase the amount of ‘‘good’’ cholesterol in the bloodstream. The increase was higher with nevirapine than with efavirenz. What Does This Study Mean for Patients? If your treatment includes nevirapine or efavirenz (particularly nevirapine), this can raise your level of ‘‘good’’ cholesterol. The results of the study may be especially important if you are already at risk for heart disease. In other words, if you have risk factors for heart disease—high blood pressure, diabetes, heart disease running in your family, being a smoker—it may be beneficial for your HIV medications to include nevirapine. If you smoke, you can lower your risk of heart disease by quitting. If you have high blood pressure or diabetes, treating these conditions can also lower your risk of heart disease. What Are the Problems with the Study? Although the researchers showed that nevirapine and efavirenz can have a beneficial effect on cholesterol levels, they haven’t actually shown that this reduces patients’ risk of getting heart disease. The study was funded by the company that produces nevirapine. In theory this could have affected the results (research has shown that company-sponsored studies are more likely to produce results favorable to the company than studies without sponsorship). The study, however, was carried out by a network of independent investigators who state that the company had no influence on the reporting of the results. Where Can I Get More Information? You can get more information on HIV and its treatment from the Terrence Higgins Trust (www.tht.org.uk), AIDS.ORG (www.aids.org), and The Body (www.thebody.com).

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An Immune Basis for Lung Parenchymal Destruction in Chronic Obstructive Pulmonary Disease and Emphysema Sandra Grumelli1, David B. Corry1,2, Li-Zhen Song1, Ling Song1, Linda Green3, Joseph Huh4, Joan Hacken5, Rafael Espada6, Remzi Bag1, Dorothy E. Lewis2, Farrah Kheradmand1* 1 Department of Medicine, Section of Pulmonary and Critical Care, Baylor College of Medicine, Houston, Texas, United States of America, 2 Department of Immunology, Baylor College of Medicine, Houston, Texas, United States of America, 3 Department of Pathology, Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, United States of America, 4 Department of Surgery, Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, United States of America, 5 Department of Radiology, Michael E. DeBakey Veterans Affairs Medical Center, Houston, Texas, United States of America, 6 Department of Surgery, Baylor College of Medicine, Houston, Texas, United States of America

Competing Interests: The authors have declared that no competing interests exist.

ABSTRACT

Author Contributions: D. B. Corry, D. E. Lewis, and F. Kheradmand designed the study. S. Grumelli, L.Z. Song, L. Green, L. Song, J. Hacken, and F. Kheradmand analyzed the data. J. Huh, R. Espada, R. Bag, and F. Kheradmand enrolled patients. D. B. Corry, D. E. Lewis, and F. Kheradmand contributed to writing the paper.

Background

Academic Editor: Peter J. Barnes, National Heart and Lung Institute, United Kingdom

We studied different subsets of lymphocytes taken from portions of human lungs removed surgically to find out which lymphocytes were the most frequent, which cell-surface markers these lymphocytes expressed, and whether the lymphocytes secreted any specific factors that could be associated with disease. We found that loss of lung function in patients with chronic obstructive pulmonary disease and emphysema was associated with a high percentage of CD4þ and CD8þ T lymphocytes that expressed chemokine receptors CCR5 and CXCR3 (both markers of T helper 1 cells), but not CCR3 or CCR4 (markers of T helper 2 cells). Lung lymphocytes in patients with chronic obstructive pulmonary disease and emphysema secrete more interferon gamma—often associated with T helper 1 cells—and interferon-inducible protein 10 and monokine induced by interferon, both of which bind to CXCR3 and are involved in attracting T helper 1 cells. In response to interferon-inducible protein 10 and monokine induced by interferon, but not interferon gamma, lung macrophages secreted macrophage metalloelastase (matrix metalloproteinase-12), a potent elastin-degrading enzyme that causes tissue destruction and which has been linked to emphysema.

Citation: Grumelli S, Corry DB, Song LZ, Song L, Green L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. PLoS Med 1(1): e8. Received: May 28, 2004 Accepted: August 2, 2004 Published: October 19, 2004 DOI: 10.1371/journal.pmed.0010008 Copyright: Ó 2004 Grumelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: COPD, chronic obstructive pulmonary disease; CT, computed tomography; FEV1, forced expiratory volume in 1 s; IL, interleukin; INF-c, interferon gamma; IP-10, interferon-inducible protein 10; I-TAC, interferoninducible T cell alpha chemoattractant; MIG, monokine induced by interferon; MMP, matrix metalloproteinase; PMA, phorbol myristate acetate; Th1, T helper 1; Th2, T helper 2 * To whom correspondence should be addressed. E-mail: farrahk@ bcm.tmc.edu

Chronic obstructive pulmonary disease and emphysema are a frequent result of long-term smoking, but the exact mechanisms, specifically which types of cells are associated with the lung destruction, are unclear.

Methods and Findings

Conclusions These data suggest that Th1 lymphoctytes in the lungs of people with smoking-related damage drive progression of emphysema through CXCR3 ligands, interferon-inducible protein 10, and monokine induced by interferon.

Introduction Chronic inhalation of tobacco smoke causes progressive lung destruction in susceptible individuals, resulting in chronic obstructive pulmonary disease (COPD) and emphysema, two well-described clinical syndromes with poorly understood pathogenesis [1,2,3]. A role for T helper cells in the pathogenesis of obstructive lung disease has been established with asthma, where T helper 2 (Th2) cells are strongly linked to both human and experimental disease [4,5,6,7]. A potential role for T cells in COPD has also been suggested in several recent studies that show CD8þ T cells are increased in the lungs of people who smoke [8,9,10,11]. T cells cause tissue injury through their secreted products such as cytokines; in mice, overexpression of interleukin (IL)-13, a T cell cytokine that is strongly implicated in the pathogenesis of experimental asthma, resulted in increased production of proteases and enlargement of

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Table 1. Clinical and Demographic Characteristics of Participants Emphysema (n = 18) LVRS (n) Small peripheral cancer (n) Benign lesionb (n) Age (mean 6 SE) Percent FEV1 (mean 6 SE) Pack-years smoking (mean 6 SE) QT (mean 6 SE)

8 10 68 45 54 7

6 6 6 6

2 4 6 2

No Emphysema Control (n = 10)

p-Valuea

5 5 65 91 45 4

0.60 ,0.001 0.45 0.54

6 6 6 6

5 3 5 2

a

p-Values are for the comparison of emphysema with control participants (Student’s T test). Histological diagnoses included lipoma, papiloma, and benign scar. LVRS, lung volume reduction surgery; SE, Standard Error; percent FEV1 l/s, percent predicted FEV1 in liters/second; QT, the number of years since the individuals had stopped smoking. DOI: 10.1371/journal.pmed.0010008.t001 b

airspaces reminiscent of emphysema [12]. Further, airway limitation, another characteristic of human asthma, is clinically linked to an accelerated rate of loss of lung function in smoker individuals [13]. It has been suggested, therefore, that asthma and COPD may involve the same type of recruited inflammatory cells, differing only in their location within the lung [14]. Chemokines, their receptors, and cell adhesion molecules regulate migration of immune cells into inflamed tissue [15,16,17,18]. T helper 1 (Th1) cells have been shown to secrete interleukin 2 and interferon gamma (IFN-c), and express a distinct repertoire of chemokine receptors such as CCR5 and CXCR3 [19,20,21]. In contrast, Th2 cells that are biased to produce IL-4 and IL-5 express mainly CCR4 and CCR3 [22,23,24,25]. Immunofluorescent analysis of airway mucosal biopsies in patients with asthma showed that most T cells co-express IL-4 and CCR4, but, in contrast, T cells in airways of patients with COPD and pulmonary sarcoidosis produce IFN-c and express high levels of CXCR3, while lacking CCR4 expression [26]. In addition to T cells, a wide variety of other inflammatory cells have been shown to express distinct chemokine receptors that are critical for their homing, suggesting a universal mechanism for regulating immune responses. Interferon-inducible protein 10 (IP10), monokine induced by interferon gamma (MIG), and interferon-inducible T cell alpha chemoattractant (I-TAC) are three known ligands for CXCR3 produced by normal and injured epithelial cells and T cells that are required for homing of Th1 cells [27,28,29]. In addition to regulation of chemotaxis and homing, other functions have been ascribed to chemokines, including modulation of T cell fate by direct effects on differentiating T cells, and regulation of proteolysis in blood monocytes [19,30]. In this study we determined the dominant T helper phenotype in lung samples from ex-smoker individuals with moderate to severe COPD and emphysema and control individuals with no evidence of smoking-related lung disease. Analysis of chemokine receptor expression on isolated peripheral lung lymphocytes from ex-smokers with COPD/ emphysema indicated that both CD4 and CD8 T helper cells are strongly polarized to the Th1 phenotype compared to T cells isolated from lung tissue of normal individuals or PLoS Medicine | http://www.plosmedicine.org

individuals with non-smoking-related obstructive lung disease. The same cells spontaneously secreted more IFN-c and CXCR3 receptor ligands MIG and IP-10 in the COPD and emphysema group than in the group without emphysema. Further, IP-10 and MIG, but not IFN-c, upregulated macrophage metalloelastase (matrix metalloproteinase [MMP]-12) from isolated lung macrophages. Together, our findings reveal the strong association between COPD/emphysemaand Th1-driven adaptive immunity, suggesting a link to lung destruction mediated by IFN-c, MIG, and IP-10.

Methods Participants Twenty-eight non-atopic ex-smoker individuals (see Table 1) undergoing medically necessary lung resection were serially entered into the study: ten individuals with no COPD and no evidence of emphysema (control group) and eighteen individuals (diseased group) with moderate to severe COPD and evidence of emphysema as determined by pulmonary function tests, high-resolution computed tomography (CT), or conventional CT scan. All participants were ex-smokers who had quit smoking for a mean (SD) of 7 (2) y and 4 (2) y in COPD/emphysema and control groups, respectively. COPD was diagnosed according to the criteria recommended by the National Institutes of Health/World Health Organization workshop summary [31]. Participants in the control and COPD/emphysema groups had similar (mean [SD] of 54 [6] and 45 [5], respectively) ‘‘pack-year’’ smoking histories, where smoking one pack of cigarettes per day each year is defined as one pack-year. All participants were recruited from the surgical clinic at the Michael E. DeBakey Veterans Affairs Medical Center and the Methodist Hospital, and were undergoing lung resection for diagnostic or therapeutic purposes (Table 1). Study protocols were approved by the institutional review board for human studies, and informed consent was obtained from all participants. Participants had no history of allergy or asthma and had not received oral/systemic corticosteroids during the last 6 mo. At the time of study, all participants had been free of acute symptoms suggestive of upper or lower respiratory tract infection for the 6 wk preceding the study. 076

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CT-Based Evaluation for Emphysema

used to measure supernatant concentrations of IP-10, MIG, IL-4, and IFN-c according to the manufacturer’s instructions (R&D Systems and BD Biosciences Pharmingen).

High-resolution CT (two in emphysema group and two in control group) or conventional CT analysis was used to detect emphysema, characterized by the presence of areas of low attenuation contrasted with surrounding normal lung parenchyma [32,33]. CT scans were used by a radiologist to separate participants on the basis of the presence or the absence of any objective evidence for centrilobular, panacinar, or paraseptal emphysema with a detection limit of greater than 3-mm low attenuation density [34].

Detection of MMP12 by Western Blotting, and Real-Time PCR Peripheral blood mononuclear cells and lung macrophages were isolated by positive selection using immunomagnetic beads conjugated with anti-CD14, and cultured in serum-free medium (RPMI, L-glutamine, and Pen/Strep) prior to overnight stimulation with 0, 50, 250, or 500 ng/ml of IFN-c, IL-4, MIG, I-TAC, and IP-10. Supernatants were collected, and MMP12 was detected using anti-human MMP12 (R&D Systems) by Western blotting according to the manufacturer’s instructions. Total cellular RNA was extracted from CD14þ lung macrophages stimulated overnight with rIP-10 (500 ng/ml) in the presence or absence of blocking anti-CXCR3 antibodies (5 lg/ml, R&D Systems). Two-step real-time reverse transcription PCR was used to determine the relative expression of mRNA using the ABI Perkin Elmer Prism 5700 Sequence Detection System (Applied Biosystems, Foster City, California, United States) as described previously [38].

Isolation of Lung Lymphocytes Lung lymphocytes were isolated by modifying established protocols, using a combination of mechanical fragmentation, enzyme digestion, and centrifugation procedures described previously [35,36,37]. Viable lymphocytes were separated from whole lung inflammatory cells (macrophages, eosinophils, and neutrophils) using an immunomagnetic positive separation technique (autoMACS, Miltenyi Biotec, Auburn, California, United States). Briefly, lung leukocytes were labeled with paramagnetic bead-conjugated anti-CD3, -CD19, and -CD56 to positively select T, B, and NK cells, according to the manufacturer’s instructions. Each of the harvested cell populations was used directly for in vitro assays or was cryopreserved in aliquots of 1 3 107 cells for future analysis.

Immunostaining and Histopathology Paraffin-embedded, and fresh-frozen lung sections (5 lm) were immunostained using monoclonal antibodies against human MMP12 (R&D Systems) or non-immune antisera by an immunoperoxidase protocol (Vectastain Elite, Vector Labs, Burlingame, California, United States) and counterstained with hematoxylin as recommended by the manufacturer.

Antibodies The following monoclonal antibodies were purchased from BD Biosciences Pharmingen (San Diego, California, United States): FITC-, Cy5-, and PE-conjugated anti-CD4, -CD8, -CD3, -CD14, -CD69, -CXCR3, -CCR3, -CCR4, and -CCR5. For enzyme-linked immunosorbent assay studies, anti-human antibodies to IFN-c, IL-4, IP-10, MIG, I-TAC, and the appropriate secondary reagents were purchased from R&D Systems (Minneapolis, Minnesota, United States).

Statistical Analysis The Mann-Whitney test (non-parametric, two-tailed) and Student’s T test (two-tailed) were used to compare differences between the two groups of subjects. p , 0.05 was considered statistically significant.

Quantification of Polarized Peripheral Blood and Lung Lymphocyte Subsets

Results Th1 Immune Bias of Peripheral Lung Lymphocytes in Emphysema

Phenotypic characterization of T cells was done by twocolor flow cytometry (Epic XL FL, Beckman Coulter, Allendale, New Jersey, United States) using combinations of the following monclonal antibodies: FITC-conjugated antiCD4, -CD8, and -CD14; PE- and Cy5-conjugated anti-CCR4, -CCR3, -CCR5, and -CXCR3. Freshly isolated lung lymphocytes were resuspended to 1 3 107 cells/ml, and 50 ll of cells was incubated with antibodies to CD3 and CD4 or CD8.

Inflammatory chemokines, cytokines, and their receptors are upregulated at sites of inflammation and play a key role in the recruitment of leukocytes to peripheral tissues in response to injury [17,39]. To detect Th1 polarization, we assessed lung lymphocytes for expression of CCR5 (a receptor for several Th1 chemokines) and CXCR3 (the receptor for IP10, I-TAC, and MIG). We screened for the presence of Th2 cells by assessing T cell expression of CCR4—a receptor for eotaxin/CCL11, macrophage chemoattractant protein 3 (CCL7), and thymus- and activation-regulated chemokine (CCL17) [40,41]—and CCR3, a receptor for eotaxin and related chemokines. Flow cytometry revealed very low expression of CCR3 and CCR4 (1%–3%) in control (n = 10) and emphysema (n = 18) groups, and did not discriminate between these populations (Figure 1A and 1B; data not shown). These findings were in sharp contrast to the enhanced expression of both CCR5 and CXCR3, as shown in representative histograms (Figure 1A). These Th1-specific chemokine receptors were expressed prominently on lung lymphocytes from all participants, but their expression was significantly enhanced in the setting of emphysema (Figure

Intracytoplasmic Cytokine Staining Lung lymphocytes were cultured in the presence or absence of phorbol myristate acetate (PMA)/ionomycin and brefeldin A for 12 h. Cells were harvested, fixed with formaldehyde, permeabilized with saponin, and intracellularly labeled for IFN-c and IL-4, in addition to staining for surface CD69, CD4, and CD8 according to the manufacturer’s recommendations (Fastimmune, BD Biosciences Pharmingen).

In Vitro T Cell Culture and Cytokine Assay Lung lymphocytes were isolated from surgical tissue and cultured in vitro in triplicate for 4 d. Supernatants were collected and stored at –80 8C for future analysis. Standard antibody-based enzyme-linked immunosorbent assay was PLoS Medicine | http://www.plosmedicine.org

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Figure 1. Chemokine Receptor Expression on Peripheral Lung Lymphocytes (A) Single color histograms showing expression of chemokine receptors CCR4, CCR5, and CXCR3 from representative control and emphysema participants. (B) Pooled data from all participants (control, n = 10; emphysema, n = 18) showing percent (median 6 SD) of total lung lymphocytes expressing CCR4 and CCR5. (C) Pooled data from same participants showing percent (median 6 SD) CCR5 expression on CD4 (top) and CD8 (middle) T cells, and CXCR3 expression on unfractionated T cells (bottom) from the same participant groups. (D) Analysis of total lung lymphocyte chemokine receptor (median 6 SD) profiles among participants with emphysema. Participants had either (1) lung volume reduction surgery for emphysema (non-cancer, n = 8) or (2) lung resection for treatment of small peripheral cancer (n = 10). Participants showed similar inflammatory indices as Rdetermined by CCR5 expression. In (B) and (C), *, p , 0.001;  , p = 0.01; z, p = 0.02; , p = 0.007 (Mann-Whitney test) for the comparison of emphysema and control groups. DOI: 10.1371/journal.pmed.0010008.g001

1A–1C). Further, both CD4 and CD8 T cells expressed CCR5 at the same level (Figure 1C). In contrast, we found highly variable expression (0.5%–30%) of CCR4, CXCR3, and CCR5 on peripheral blood lymphocytes isolated from the same participants, and this variation did not correlate with the presence of disease in either group (data not shown). Furthermore, we compared the lung lymphocyte CCR5 and CXCR3 profiles among the eight participants with emphysema alone (lung volume reduction surgery for emphysema; non-cancer) and ten participants with emphysema and accompanying cancer (lung resection for treatment of small peripheral cancer), and found that these two groups cannot be distinguished based on these indices (Figure 1D; data not shown). PLoS Medicine | http://www.plosmedicine.org

Although human lung macrophages are not known to express CXCR3, we suspected based on the immunohistochemical localization of this chemokine receptor that CD14þ cells in the lungs of ex-smoker individuals with emphysema accounted for much of the total lung CXCR3þ immunoreactivity (Figure 2A; data not shown). To confirm this, we determined the percent of total lung cells expressing CD14 and CD11b—which are both markers of monocytes/macrophages—and CXCR3. We found that over 40% of CD14þ cells from participants with emphysema but not control participants were also positive for CXCR3 (Figure 2). In addition, there was a significant negative association between CXCR3 expression on lung T cells and the percent of predicted forced expiratory volume in 1 s (FEV1), based on an R2 078

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Figure 2. Expression of CXCR3 in Lungs of Control and Emphysematous Smoker Individuals (A) Representative forward and side-scatter characteristics of whole lung cells from a participant with COPD and emphysema. Anti-CD11b PEconjugated and anti-CD14 FITC-conjugated antibodies detect lung macrophages (middle), and histogram of mean fluorescence intensity showing anti-CXCR3-Cy5 and control antibodies (cIg) detects lung macrophages in the patient with emphysema. (B) Pooled data from control individuals without (n = 5) and with (n = 8) emphysema. Columns are median, bars represent SD. *, p = 0.009 (Mann-Whitney test) for the comparison of emphysema and control participants. (C) Negative association between CXCR3 expression on CD3þ T cells and FEV1 percentage predicted based on an R2 goodness-of-fit statistic of 0.27 (p = 0.0089, r =
0.52, n = 24). DOI: 10.1371/journal.pmed.0010008.g002

was approximately equal to that of CD8þ/IFN-cþ cells (median [SD], 19[6] versus 16[4], respectively). Because total numbers of CD4þ and CD8þ T cells were approximately equivalent, this suggests that non-CD8þ/IFN-cþ cells are largely CD4þ, and therefore Th1 cells. Finally, the typical Th2 cytokine, IL-4, was not detected in either group, as determined by enzyme-linked immunosorbent assay or intracytoplasmic cytokine staining (Figure 3E; data not shown), confirming the marked Th1 bias of the immune response that underlies smoking-related lung inflammation and emphysema.

goodness-of-fit statistic of 0.27 (Figure 2C; p = 0.0089, r =
0.52). Together, these data indicate that a strong type 1 bias is characteristic of the T cells isolated from the peripheral lung of participants with COPD and emphysema and that this immune phenotype correlates with the lung destruction that is characteristic of this disease. Further, we have shown for the first time, to our knowledge, that CXCR3 expression, a marker of Th1 inflammation, extends to lung monocytes and macrophages.

IFN-c, IP-10, and MIG But Not IL-4 Are Expressed by Lung Lymphocytes

IP-10 and MIG But Not IFN-c Directly Upregulate MMP12 through CXCR3

We sought additional functional data to confirm the apparent Th1 bias of peripheral lung inflammatory cells isolated from ex-smoker individuals. Freshly isolated lung lymphocytes that were not otherwise manipulated secreted high levels of IFN-c, MIG, and IP-10, with significantly greater secretion of both cytokines from lymphocytes of participants with emphysema (Figure 3A–3C). Interestingly, we could not detect appreciable amounts of I-TAC, another known ligand for CXCR3, in lung lymphocytes of control participants or those with emphysema (data not shown). Similar results were obtained using intracytoplasmic cytokine staining of the same cells (Figure 3D), in which PMA/ionomycin stimulation strongly induced IFN-c production from CD69þ/CD8þ lung lymphocytes. Surface staining for CD4 was not feasible with this protocol; however, the percentage of CD8
/IFN-cþ cells PLoS Medicine | http://www.plosmedicine.org

Emphysema and irreversible airway limitation that is characteristic of chronic tobacco smoking are related to the destruction of elastin and the resulting loss of lung elastic recoil. Therefore, to be relevant to the pathogenesis of airway obstruction, type 1 inflammation must be shown to promote lung elastolysis. Because loss of elastin is regulated by proteinases [42], we next determined if expression of MMPs, in particular the elastases MMP9 and MMP12, was regulated by IP-10, MIG, and IFN-c, the principal cytokines detected in emphysematous lung. Indeed, isolated peripheral lung macrophages, but not isolated blood monocytes, secreted MMP12 in response to IP-10 and MIG, but not IFN-c (Figure 4A; data not shown). These findings reflect a specific receptor–ligand interaction because in the presence of a CXCR3 function079

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Figure 3. IFN-c, MIG, and IP-10 Production by Isolated Lung Lymphocytes (A–C) Lung lymphocytes from control individuals and participants with emphysema were cultured without additional stimulation for 3 or 4 d and assessed for secretion of (A) IFN-c, (B) MIG, and (C) IP-10 (control, n = 8; emphysema, n = 12). Columns are median, bars represent SD. *, p = 0.007;  , p = 0.01; z, p = 0.02 for the comparison of emphysema and control participants. (D) The same cells from a representative ex-smoker individual with emphysema were either left unstimulated (No ST) or treated with PMA/ ionomycin (PMA/I) for 24 h and assessed for surface CD8 and CD69 expression and the intracytoplasmic accumulation of IFN-c by flow cytometry. (E) Production of IL-4 by lung lymphocytes. Lung lymphocytes from a representative ex-smoker individual with emphysema were cultured for 24 h with or without PMA/ionomycin stimulation (PMA/I) and assessed for intracytoplasmic IL-4 and IFN-c accumulation by flow cytometry. DOI: 10.1371/journal.pmed.0010008.g003

COPD and suggest a primary role for Th1 cells in controlling the main smoking-related physiologic and structural changes of the lung. Upregulation of CCR5 and CXCR3 on T cells and accumulation of these cells in the lung periphery suggest that aberrant, unremitting pulmonary recruitment of these activated T cells is unique to people with smoking-related lung disease, despite cessation of exposure to the inciting agent, tobacco smoke. We showed that ex-smoker individuals without obstructive lung disease or emphysema have comparatively little Th1-biased inflammation in their lungs; thus, our findings reflect the inflammatory changes that are unique to the COPD microenvironment. Additionally, lung lymphocytes isolated from four lifelong non-smoker individuals with severe obstructive lung disease due to cystic fibrosis or bronchiolitis obliterans did not show a Th1 inflammatory bias of the lung (S. Grumelli, F. Kheradmand, D. B. Corry, unpublished data). This information confirmed our finding that the predominant Th1 bias in COPD/emphysema reflects the microenvironment unique to the lungs of ex-smoker individuals. The prevalence of asthma among people who smoke is currently not known, but in order to study COPD/ emphysema in a population without other confounding variables, people who might have had asthma were excluded,

blocking antibody, IP-10 failed to induce MMP12 (Figure 4B). Furthermore, immunohistochemical studies revealed that lung macrophages of participants with emphysema, but not control participants, specifically express MMP12 (Figure 4C and 4D). Together, these findings indicate that Th1, but not Th2, cytokines and related chemokines are required for establishing the pro-elastolytic lung environment that underlies human emphysema.

Discussion In this investigation, we characterized T cells and lung macrophages isolated from emphysematous and non-emphysematous human lungs. Three principal findings emerge from our study. First, rather than being functionally diverse, as suggested by the heterogeneous nature of humans, lung T cells of ex-smoker individuals with emphysema are relatively homogeneous and characterized by a marked Th1 bias. Second, the principal Th1 chemokines, MIG and IP-10, are linked to a pro-elastolytic lung environment because these cytokines upregulate the elastase MMP12, which is associated with emphysema. Finally, we found no significant expression of Th2 chemokine receptors, such as CCR3 and CCR4, or IL-4 production in lung lymphocytes. Together, our findings demonstrate the role of the adaptive immune response in PLoS Medicine | http://www.plosmedicine.org

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Figure 4. Regulation of MMP12 by Type 1 Cytokines (A) CD14þ, lymphocyte-depleted lung leukocytes were cultured with and without the indicated amounts of recombinant human IP-10 and IFN-c, and supernatants were assessed for the presence of MMP12 by Western blotting. (B) Fold increase relative to unstimulated of MMP12 mRNA from lung macrophages stimulated without (–) and with (þ) 500 ng/ml of IP-10 in the presence or absence of a function-blocking antibody to CXCR3 as determined by real-time PCR. (C and D) Lung tissue from a participant with emphysema (C) shows strong immune staining for MMP12 localized to macrophages (arrows), and (D) shows lung tissue from a control participant without emphysema and with undetectable MMP12. The insets show a high-power view of lung macrophages staining positive (C) and negative (D) for MMP12 (360) *, p = 0.04. DOI: 10.1371/journal.pmed.0010008.g004

proteinase family [45,46]. Cytokines and chemokines are substrates for MMPs, but they also regulate expression of MMPs under pathological conditions [47,48]. We have shown here that IP-10 and MIG, two chemokines that are secreted from lung lymphocytes of participants with emphysema, upregulate specifically MMP12 and thus favor a proteolytic microenvironment that facilitates lung destruction. Strengthening the association between lung macrophages and IP-10/ MIG-dependent MMP12 secretion is the fact that we have demonstrated that in humans macrophages, like T cells, express CXCR3 and that this receptor is required for MMP12 secretion in response to IP-10/MIG stimulation. In addition to defining the predominant immune phenotype of emphysematous lung, these additional findings implicate the principal cell (macrophage), MMP (MMP12), and effector cytokines (IFN-c, IP-10, and MIG) as likely underlying smoking-induced lung destruction. We have further shown that these enzymes may be regulated by proximal immune events driven by Th1 cells or Th1-associated cytokines. A question of major importance for future study is, therefore, the nature of the antigens and adjuvant factors that ultimately drive this inflammatory response. Although this was an entirely human study, our findings show remarkable parallels with studies performed in mice. MMP12 deficiency has been shown to protect mice against emphysema after chronic exposure to cigarette smoke, implying that MMP12 may be the key proteinase in the development of emphysema in this species [49,50]. Studies from both humans and mice therefore firmly suggest the importance of MMP12 in the pathogenesis of emphysema. Interestingly, in addition to solubilizing elastin, MMP12 is the MMP most efficient at degrading a1-antitrypsin, the primary

and thus our findings are restricted to non-asthmatic individuals with emphysema. Our use of T cell chemokine receptor expression analysis to determine recruitment of lung T cells is not without precedent. Analysis by immunohistochemistry of airway mucosa of people with atopic asthma after antigen challenge revealed that large numbers of CCR4þ and CCR8þ T cells express IL-4, and CCR4 expression was prominent in people with severe atopic dermatitis, which decreased upon abatement of disease activity [26,43]. Immunostaining of T cells in synovial fluid from individuals with rheumatoid arthritis showed that virtually all of the T cells associated with inflamed joints expressed CXCR3 and CCR5, representing significant enrichment compared to blood T cells from the same participants. Furthermore, previous studies of smoker individuals with COPD and normal lung function showed the presence of CD8þ/CXCR3þ T cells in the airway epithelium and submucosa [44]. We extend these findings by showing CXCR3 expression on lung macrophages and CD4þ T cells in emphysema patients and the functional interplay between Th1-related chemokines and elastolytic MMPs. In addition to detailing surface chemokine receptor expression, we have functionally confirmed the marked Th1 bias of peripheral lung T cells, demonstrating that either at rest or following stimulation, these cells secrete IFN-c and not IL-4. Our findings therefore confirm the utility of chemokine receptor expression patterns in the initial assessment of T cell effector phenotype. Destruction of lung parenchyma in emphysema is thought to occur through excessive proteolysis mediated by the elastin-degrading enzymes MMP2, MMP9, and MMP12 from the MMP family, and by neutrophil elastase from the serine PLoS Medicine | http://www.plosmedicine.org

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Th1 Immune Cells in Human Emphysema 19. Luther SA, Cyster JG (2001) Chemokines as regulators of T cell differentiation. Nat Immunol 2: 102–107. 20. Kim CH, Rott L, Kunkel EJ, Genovese MC, Andrew DP, et al. (2001) Rules of chemokine receptor association with T cell polarization in vivo. J Clin Invest 108: 1331–1339. 21. Loetscher P, Uguccioni M, Bordoli L, Baggiolini M, Moser B, et al. (1998) CCR5 is characteristic of Th1 lymphocytes. Nature 391: 344–345. 22. Muller KM, Jaunin F, Masouye I, Saurat JH, Hauser C (1993) Th2 cells mediate IL-4-dependent local tissue inflammation. J Immunol 150: 5576– 5584. 23. Kamogawa Y, Minasi LE, Carding SR, Bottomly K, Flavell RA (1993) The relationship of IL-4- and IFNg-producing T cells studied by lineage ablation of IL-4-producing cells. Cell 75: 985–995. 24. Del Prete G (1992) Human Th1 and Th2 lymphocytes: Their role in the pathophysiology of atopy. Allergy 47: 450–455. 25. 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Immunity 12: 121–127. 30. Klier CM, Nelson EL, Cohen CD, Horuk R, Schlondorff D, et al. (2001) Chemokine-induced secretion of gelatinase B in primary human monocytes. Biol Chem 382: 1405–1410. 31. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS (2001) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 163: 1256–1276. 32. Uppaluri R, Mitsa T, Sonka M, Hoffman EA, McLennan G (1997) Quantification of pulmonary emphysema from lung computed tomography images. Am J Respir Crit Care Med 156: 248–254. 33. Satoh K, Kobayashi T, Misao T, Hitani Y, Yamamoto Y, et al. (2001) CT assessment of subtypes of pulmonary emphysema in smokers. Chest 120: 725–729. 34. Madani A, Keyzer C, Gevenois PA (2001) Quantitative computed tomography assessment of lung structure and function in pulmonary emphysema. Eur Respir J 18: 720–730. 35. Upham JW, McMenamin C, Schon HM, Robinson BW, Holt PG (1994) Functional analysis of human bronchial mucosal T cells extracted with interleukin-2. Am J Respir Crit Care Med 149: 1608–1613. 36. Goodacre R, Davidson R, Singal D, Bienenstock J (1979) Morphologic and functional characteristics of human intestinal lymphoid cells isolated by a mechanical technique. Gastroenterology 76: 300–308. 37. Fiocchi C (1985) Lymphoid cells of the gastrointestinal tract. Isolation procedures. Acta Chir Scand Suppl 525: 11–23. 38. Corry DB, Rishi K, Kanellis J, Kiss A, Song Lz LZ, et al. (2002) Decreased allergic lung inflammatory cell egression and increased susceptibility to asphyxiation in MMP2-deficiency. Nat Immunol 3: 347–353. 39. Baggiolini M (2001) Chemokines in pathology and medicine. J Intern Med 250: 91–104. 40. Renauld JC (2001) New insights into the role of cytokines in asthma. J Clin Pathol 54: 577–589. 41. Padrid PA, Cozzi P, Leff AR (1996) Cyclosporine A inhibits airway reactivity and remodeling after chronic antigen challenge in cats. Am J Respir Crit Care Med 154: 1812–1818. 42. Sternlicht MD, Werb Z (1999) ECM Proteinases. In: Kreis T, Vale RD, editors. Guidebook to the extracellular matrix and adhesion proteins. Oxford: Oxford University Press. pp. 503–562. 43. Wakugawa M, Nakamura K, Kakinuma T, Onai N, Matsushima K, et al. (2001) CC chemokine receptor 4 expression on peripheral blood CD4þ T cells reflects disease activity of atopic dermatitis. J Invest Dermatol 117: 188–196. 44. Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, et al. (2002) Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 165: 1404–1409. 45. Barnes PJ (2000) Chronic obstructive pulmonary disease. N Engl J Med 343: 269–280. 46. Cawston T, Carrere S, Catterall J, Duggleby R, Elliott S, et al. (2001) Matrix metalloproteinases and TIMPs: Properties and implications for the treatment of chronic obstructive pulmonary disease. Novartis Found Symp 234: 205–218. 47. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17: 463–516. 48. Kang T, Yi J, Guo A, Wang X, Overall CM, et al. (2001) Subcellular

physiological inhibitor of human leukocyte elastase [51,52]. Thus, chemokine-induced upregulation of MMP12 may orchestrate lung matrix degradation both directly and indirectly through inactivation of a1-antitrypsin. The therapy of COPD and emphysema is currently limited to pharmacologic bronchodilation to relieve dyspnea, antibiotics for intercurrent respiratory tract infection, and vaccination against prominent respiratory pathogens. Aside from efforts to prevent smoking or encourage cessation, there exist no measures that prevent development of emphysema or treat the specific causes of airway obstruction. By providing insight into the immunopathogenesis of COPD, our findings provide genuine hope that future therapies capable of preventing or halting smoking-related lung disease may be possible.

Acknowledgments We thank Dr. A. Clinton White for critical review of the manuscript, Mr. Jeff Scott for technical assistance with flow cytometry, Ms. Pamela Smithwick, Ms. Rose Baglia, clinical research coordinator Dr Michael Reardon, and all the individuals who generously participated in this study. This work was supported by grants from the National Heart Lung and Blood Institute (64061, 72419, 69585) and Baylor College of Medicine (AI 36211). The funding provided by the National Hearth Lung and Blood Institute made our study possible, but it had no impact on any other matters such as study design, data collection and & analysis, decision to publish, or preparation of the manuscript. References 1. Barnes PJ (2001) Potential novel therapies for chronic obstructive pulmonary disease. Novartis Found Symp 234: 255–267. 2. Senior RM (2000) Mechanisms of COPD: Conference summary. Chest 117: 320S–323S. 3. Hogg JC (2001) Chronic obstructive pulmonary disease: An overview of pathology and pathogenesis. Novartis Found Symp 234: 4–19. 4. Corry DB, Grunig G, Hadeiba H, Kurup VP, Warnock ML, et al. (1998) Requirements for allergen-induced airway hyperreactivity in T and B celldeficient mice. Mol Med 4: 344–355. 5. Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, et al. (1998) Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261–2263. 6. Kay AB (1997) T cells as orchestrators of the asthmatic response. Ciba Found Symp 206: 56–67. 7. Fahy JV, Corry DB, Boushey HA (2000) Airway inflammation and remodeling in asthma. Curr Opin Pulm Med 6: 15–20. 8. Cosio MG, Guerassimov A (1999) Chronic obstructive pulmonary disease. Inflammation of small airways and lung parenchyma. Am J Respir Crit Care Med 160: S21–S25. 9. Di Stefano A, Capelli A, Lusuardi M, Caramori G, Balbo P, et al. 2001. Decreased T lymphocyte infiltration in bronchial biopsies of subjects with severe chronic obstructive pulmonary disease. Clin Exp Allergy 31: 893– 902. 10. Turato G, Zuin R, Saetta M (2001) Pathogenesis and pathology of COPD. Respiration 68: 117–128. 11. Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, et al. (1998) CD8þ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 157: 822–826. 12. Zheng T, Zhu Z, Wang Z, Homer RJ, Ma B, et al. (2000) Inducible targeting of IL-13 to the adult lung causes matrix metalloproteinase- and cathepsindependent emphysema. J Clin Invest 106: 1081–1093. 13. Anthonisen NR, Connett JE, Kiley JP, Altose MD, Bailey WC, et al. (1994) Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV1. The Lung Health Study. JAMA 272: 1497–1505. 14. He JQ, Connett JE, Anthonisen NR, Sandford AJ (2003) Polymorphisms in the IL13, IL13RA1, and IL4RA genes and rate of decline in lung function in smokers. Am J Respir Cell Mol Biol 28: 379–385. 15. Baggiolini M, Loetscher P (2000) Chemokines in inflammation and immunity. Immunol Today 21: 418–420. 16. Sallusto F, Lanzavecchia A (2000) Understanding dendritic cell and Tlymphocyte traffic through the analysis of chemokine receptor expression. Immunol Rev 177: 134–140. 17. Luster AD (2002) The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol 14: 129–135. 18. Xie H, Lim YC, Luscinskas FW, Lichtman AH (1999) Acquisition of selectin binding and peripheral homing properties by CD4(þ) and CD8(þ) T cells. J Exp Med 189: 1765–1776.

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49.

50. 51.

52.

Patient Summary

distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils. J Biol Chem 276: 21960–21968. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD (1997) Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 277: 2002–2004. Churg A, Zay K, Shay S, Xie C, Shapiro SD, et al. (2002) Acute cigarette smoke-induced connective tissue breakdown requires both neutrophils and macrophage metalloelastase in mice. Am J Respir Cell Mol Biol 27: 368–374. Gronski TJ Jr, Martin RL, Kobayashi DK, Walsh BC, Holman MC, et al. (1997) Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J Biol Chem 272: 12189–12194. Liu Z, Zhou X, Shapiro SD, Shipley JM, Twining SS, et al. (2000) The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 102: 647–655.

Background. Many people develop long-term lung problems after smoking, including a condition called emphysema. At the very end of the airways are tiny air sacs. In healthy people, the air sacs stretch and relax easily on breathing in and out. But in people with emphysema, the air sacs fill up with air but can’t empty out properly, so air gets trapped, making breathing difficult. While the symptoms of emphysema can be treated, there are no treatments that can reverse the damage to the lung. What Did the Researchers Find? The researchers studied two groups of patients, all ex-smokers who had been admitted to a hospital to have part of their lung removed—some because of cancer, some for other reasons. The researchers studied the lung samples and looked to see exactly what type of immune cells the patients with emphysema had in their lungs and found that most of the immune cells were of one particular type. The researchers also showed that the immune cells could tell other lung cells to produce chemicals that can damage the lung. What Does This Mean for Patients? Lung damage in emphysema may not be caused directly by toxins in cigarette smoke. Instead, if you have emphysema, your body may react to the toxins and produce a special kind of immune cell that is key in causing the lung damage. So perhaps if doctors can find a way to change how this cell behaves, it might be possible to reduce or limit the lung damage. Obviously, not smoking, or stopping smoking, is the best way to prevent COPD or emphysema. What Are the Problems with the Study? The study is quite small, which means that the results may not be completely accurate; in particular, the study did not include detailed information from patients who had never smoked. So it is too soon to say for sure whether these special immune cells really are the link between smoking and lung damage in emphysema. Researchers will need to study many more patients with emphysema as well as people who have never smoked. Where Can I Find More Information? Two places to start are the patient Web pages of the following professional organizations. American Association for Respiratory Care: http://www.yourlunghealth. org/diseases_conditions/copd/ The British Thoracic Society: http://www.brit-thoracic.org.uk/public_ content.asp?pageid=9&catid=21&subcatid=177

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PLoS MEDICINE

The Molecular Basis for Oat Intolerance in Patients with Celiac Disease Helene Arentz-Hansen1, Burkhard Fleckenstein1,2, Øyvind Molberg1, Helge Scott3, Frits Koning4, Gu¨nther Jung5, Peter Roepstorff2, Knut E. A. Lundin1,6, Ludvig M. Sollid1* 1 Institute of Immunology, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway, 2 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 3 Institute of Pathology, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway, 4 Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, Netherlands, 5 Institute of Organic Chemistry, University of Tu¨bingen, Tu¨bingen, Germany, 6 Department of Medicine, Rikshospitalet University Hospital, Oslo, Norway

Competing Interests: The authors have declared that no competing interests exist. LMS is a member of the editorial board of PLoS Medicine. Author Contributions: ØM, KEAL, and LMS designed the study. HAH, BF, ØM, HS, FK, GJ, PR, KEAL, and LMS analyzed the data. FK and GJ contributed synthetic peptides for the study. HAH, BF, ØM, HS, FK, GJ, KEAL, and LMS contributed to writing the paper. Academic Editor: Marco Londei, University College London, United Kingdom Citation: Arentz-Hansen H, Fleckenstein B, Molberg Ø, Scott H, Koning F, et al. (2004) The molecular basis for oat intolerance in celiac disease patients. PLoS Med 1(1): e1. Received: April 1, 2004 Accepted: June 14, 2004 Published: October 19, 2004

DOI: 10.1371/journal.pmed.0010001 Copyright: Ó 2004 Arentz-Hansen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: IEL, intraepithelial lymphocyte; SI, stimulatory index; TG2, tissue transglutaminase *To whom correspondence should be addressed. E-mail: l.m.sollid@ labmed.uio.no

ABSTRACT Background Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance.

Methods and Findings We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine!glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation.

Conclusions We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.

Introduction Celiac disease is a chronic inflammatory condition caused by an inappropriate immune response of intestinal T-cells reactive to gluten proteins of wheat and similar prolamin proteins of related cereals [1]. The majority of the peptides recognized by intestinal T-cells are more immunogenic following deamidation by tissue transglutaminase (TG2). These peptides are invariably presented by HLA-DQ2 or -DQ8, the same HLA molecules that confer genetic predisposition to celiac disease [1]. Gluten-reactive intestinal T-cells can be isolated from virtually all patients with celiac disease but not from normal individuals. The disease goes into remission when harmful cereals are avoided. A gluten-free diet is thus the standard treatment of this disorder. Oats have traditionally been excluded from the gluten-free diet. Several feeding studies, however, have indicated that patients with celiac disease and dermatitis herpetiformis tolerate oats without signs of intestinal inflammation [2–9]. Of note, some of these studies have high patient-dropout rates that may have masked cases of oat intolerance. An in vitro study found no signs of T-cell activation in small intestinal biopsies of celiac disease patients challenged with avenin (the prolamin fraction of oats) [10], and avenins have been predicted to contain only a

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few glutamines that can be deamidated by TG2, presumably making avenins less immunogenic [11,12]. On this basis, oats have been allowed in the gluten-free diet in several countries [13]. It remains to be proven that all celiac disease patients tolerate oats following long-term exposure. A recent study of 39 Finnish patients randomized to eat a gluten-free diet with 50 g of oats daily or a standard gluten-free diet for 1 y reported more intestinal symptoms and more gut inflammation in the group of patients eating oats, although the mucosal integrity was not disturbed [14]. In an open challenge study of 19 adult celiac disease patients using pure oats, one patient developed villous atrophy [15]. This finding prompted us to investigate the phenomenon of oat intolerance further in a selected series of nine adult celiac disease patients, three of whom had clinical oat intolerance. The goal of the study was to characterize the intestinal T-cell response to oats avenin proteins in these patients in detail and to relate this to clinical symptoms and intestinal biopsy findings.

intake of oats but has no mucosal inflammation. Finally, two patients (CD496 and CD507) were referred by a general practitioner and a referring hospital for investigation of complications arising when eating a gluten-free diet, here termed complicated celiac disease. The latter four patients came for gastroduodenoscopy for clinical reasons, and agreed to have extra biopsies taken for research purposes. We were unable to measure serological parameters in these last four patients because no serum samples were taken from them during their clinical course. The study was approved by the regional ethical committee. The participants gave their informed consent.

Histopathological Assessment We took small intestinal biopsies from the horizontal part of the duodenum by gastroduodenoscopy using an Olympus (Tokyo, Japan) GIF-IT140 scope and scored them according to the modified Marsh criteria [16]. Intraepithelial lymphocytes were counted in hematoxilin-eosin-stained sections and enumerated per 100 enterocytes. Five areas per biopsy were counted, each encompassing 50–100 epithelial cells.

Methods Participants

Grain Antigens and Peptides

We studied nine adults with celiac disease who had a history of exposure to pure oats. The oats were derived from a quality-controlled production line and were shown to be free from contamination of other cereals as described elsewhere [15]. The selection of the study participants was not random. Five of the patients (CD359, CD377, CD422, CD431, and CD482) participated in a clinical challenge study consisting of 19 adults with celiac disease who ate 50 g oats daily for 12 wk [15]. One of these patients (CD422) has symptoms and mucosal inflammation on oats consumption as described [15]. Patient CD431 has slight mucosal inflammation when eating oats but is clinically well. The three remaining individuals eat and tolerate oats. All these five patients agreed to undergo gastroduodenoscopy for research purposes. In addition, two other adults with celiac disease (CD446 and CD504) were recruited from our ordinary outpatient clinic. Patient CD446 eats and tolerates oats, whereas patient CD504 has anaphylactoid symptoms after

Oat grains (Regal, Oslo, Norway) were ground and the flour was washed twice with water-saturated 1-butanol. The pellet was dissolved in 45% ethanol overnight and centrifuged. The avenin fraction was precipitated from the supernatant by adding two volumes of 1.5 M NaCl. The precipitate was dissolved either in 0.01 M acetic acid (pH 1.8) and digested with pepsin and subsequently trypsin (pH 7.8) or in 2 M urea / 0.01 M NH4HCO3 and digested with chymotrypsin. Gluten and gliadin (ethanol-soluble proteins of gluten) were isolated from household wheat flour and digested with chymotrypsin as described [17]. Avenin peptides were synthesized on a robotic system (Syro MultiSynTech, Bochum, Germany) using Fmoc/OtBu chemistry and 2-chlorotrityl resin (Senn Chemicals, Dielsdorf, Switzerland). The identity of the peptides was confirmed by electrospray mass spectrometry, and purity was analyzed by reverse-phase HPLC. Treatment of the peptides with guinea pig (Sigma; St.

Table 1. Characteristics of the Included Patients Patient

CD496 CD422 CD507 CD504 CD431 CD359 CD446 CD377 CD482

Born

1934 1961 1944 1963 1970 1951 1954 1949 1949

Sex

F F F F F F F M F

HLA-Type DR

DQ

3, 3, 3, 7, 3 2, 3 3, 1,

2 2 2 2, 2 1, 2 2, 1,

7 7 7 11 3 5 3

3 2 3 2

Clinical Symptoms to Oats

Histology on Oats-Containing Diet

Gluten-Reactive T-Cell Lines

Avenin-Reactive T-Cell Lines

Yes Yes Yes Yes No No No No No

Marsh Marsh Marsh Marsh Marsh Marsh Marsh Marsh Marsh

Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes No Yes No No No Yes

3A 3B 3A 0 1 1 2 0 0

DOI: 10.1371/journal.pmed.0010005.t001

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Figure 1. Histology of Intestinal Mucosa of Two of the Oat-Intolerant Patients Small intestinal biopsies were obtained at diagnosis, after an ordinary gluten-free diet (remission), after introduction of oats, and after withdrawal of oats (recovery). For patient CD496, a biopsy was not taken after she started with a gluten-free diet. Biopsies were scored according to the modified Marsh criteria. Hematoxilin-eosin staining was used, and IEL counts are given in the corners of the photomicrographs. The remission biopsy from patient CD507 was poorly oriented. We therefore melted and reoriented this biopsy (insert). Original magnification: 1003. DOI: 10.1371/journal.pmed.0010001.g001

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Table 2. T-Cell Responses to Avenin, Gluten, and Avenin Peptides in T-Cell Lines Established from Biopsies Stimulated with Avenin Antigen Avenin-Reactive T-Cell Line

CD496.2.1 CD422.2.4 CD507.2.3 CD431.2.0 CD482.2.1

Avenin

Peptide 1490

Peptide Av-a9B


TG2

þTG2

Gluten
TG2

þTG2


TG2

þTG2


TG2

þTG2

6.9 2.8 2.8 2.2 7.7

7.8 9.9 7.8 4.5 7.8

1.2 1.2 6.4 0.9 1.2

3.6 6.3 8.3 1.1 1.4

5.2 2.8 0.9 1.0 1.1

7.2 4.8 1.0 5.4 1.3

2.2 1.7 1.0 0.9 1.1

7.2 9.8 3.7 0.9 1.4

Both untreated and TG2-treated antigens were tested for recognition. The protein antigens were tested at 100 lg/ml, and the peptide antigens were tested at 10 lM. The responses are given as SIs ([T þ APC þ antigen] divided by [T þ APC]), and positive responses are indicated by bold type. DOI: 10.1371/journal.pmed.0010005.t002

Results Clinical and Histological Characteristics

Louis, Missouri, United States) and human recombinant TG2 was performed in the presence of 1 and 2 mM CaCl2, respectively, at 37 8C for 2 h.

Nine adults with celiac disease who had a history of exposure to oats assessed to be free from contamination of other cereals were studied. In some cases they came for gastroduodenoscopy for clinical reasons, in other cases, they agreed to come for research reasons. The characteristics of the patients are given in Table 1. This case series is thus not a consecutive series of ordinary patients with celiac disease. Three of these patients (CD422, CD496, and CD507) were known to exhibit clinical and histopathological signs of oat intolerance. Patient CD422 developed villous atrophy and dermatitis while eating oats, and details of this patient are described elsewhere [15]. Patient CD496 was a 53-y-old woman who was evaluated for complicated celiac disease. Celiac disease was diagnosed in 1987 after 1 y with diarrhea and weight loss; a biopsy showed a Marsh 3C lesion with an intraepithelial lymphocyte (IEL) count of 58/100 enterocytes (range 53–69) (Figure 1). She responded well to a standard gluten-free diet. A control biopsy was not taken. In 2001, she started eating pure oats, but lost weight, going from 55 kg to 44 kg. While eating oats, a biopsy showed a Marsh 3A lesion with an IEL count of 54/100 enterocytes (range 43–62). The oats were discontinued, and she gradually recovered. Some months later, an intestinal biopsy demonstrated a Marsh 1 lesion with an IEL count of 46 (range 28–52). Clinically she is currently well. Patient CD507 was a 59-y-old woman who was also evaluated for complicated celiac disease. She probably had undiagnosed celiac disease since childhood and was diagnosed in 1990 after osteoporotic fractures. A biopsy showed total villous atrophy (Marsh 3C) with an IEL count of 50/100 enterocytes (range 44–54) (Figure 1). She responded well to a standard gluten-free diet. In 1999, a follow-up biopsy showed complete normalization of her mucosa (Marsh 0) with an IEL count of 26/100 enterocytes (range 24–32). In 2000, the patient started eating oats and developed bloating, abdominal pain and iron deficiency. She lost 2 kg in weight. In 2002, while still eating oats, a biopsy showed a Marsh 3A lesion with an IEL count of 50/100 enterocytes (range 38–58). She discontinued eating oats and improved clinically. A new biopsy later in 2002 showed improvement, with a Marsh 1 lesion with an IEL count 32/100 enterocytes (range 24–46). Surprisingly, in late 2003 she was diagnosed with an

T-Cell Assays The generation of T-cell lines, T-cell cloning, and T-cell proliferation assays were performed as described elsewhere [17]. Single biopsy specimens from the patients were challenged in vitro with either a pepsin-trypsin digest or a chymotrypsin digest of avenin. As control, single biopsy specimens were challenged with a chymotrypsin digest of gluten or gliadin. DR3þDQ2þ B lymphoblastoid cells (irradiated 80 Gy) were used as antigen-presenting cells. Positive Tcell responses were defined as a stimulatory index (SI) ([T þ APC þ antigen] divided by [T þ APC]) above 3. Determination of HLA restriction of the T-cells was done by testing inhibition of T-cell proliferation by the monoclonal antibodies B8.11 (anti-DR), SPV-L3 (anti-DQ), 2.12.E11 (antiDQ2), and W6/32 (anti-HLA class I).

Purification of Avenin Fragments A pepsin-trypsin digest of avenin was separated by gel filtration (FPLC, column Superdex Peptide HR 10/30; Pharmacia Biotech, Uppsala, Sweden), and a fraction containing T-cell stimulatory fragments was further separated by reverse-phase HPLC (A¨kta, Pharmacia Biotech; column Jupiter 5l C18, 250 3 4.6 mm, Phenomenex, Torrance, California, United States) using an acetonitrile gradient from 5% to 40% with 1%/min and from 40% to 64% with 3%/min (flow rate 0.9 ml/min, containing 0.05% trifluoroacetic acid).

Mass Spectrometry and Database Searching Electrospray ionization tandem mass spectrometry was performed on a quadrupole time-of-flight hybrid mass spectrometer (Micromass, Manchester, England). For spraying, needles were typically held at 900 V towards a skimmer cone (40 V). In collision-induced dissociation of selected peptide ions (collision gas argon; collision energy 25–35 eV), the generated characteristic b- and y-type fragment ions [18] were detected by the orthogonal TOF mass analyzer. All tandem mass spectrometry spectra were centroided and searched against in the NCBInr database via the Mascot Search Engine (http://www.matrixscience.com). PLoS Medicine | http://www.plosmedicine.org

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Figure 2. Identification of an Epitope in Avenin Recognized by Intestinal T-Cells of Celiac Disease Patients (A) Reverse-phase HPLC of a pepsin-trypsin digest of avenin. Peptides in fraction 25, obtained from gel filtration, were eluted by an acetonitrile gradient (straight line), and 41 fractions were collected. Fractions recognized by T-cell lines in subsequent experiments are indicated by the numbers above the peaks. (B) T-cell recognition of fractions obtained by reverse-phase HPLC. All 41 fractions obtained in (A) were tested for recognition by the intestinal T-cell line 422.2.4 (derived from an oat-intolerant celiac disease patient). The fractions were incubated with DR3-DQ2 homozygous antigenpresenting cells overnight before the T-cell line was added. Specific T-cell responses were measured by 3H-thymidine incorporation. Pepsintrypsin-digested avenins, both TG2-treated and untreated, were used as control antigens. (C) Sequences of the peptides in the stimulatory fractions from reverse-phase HPLC identified by tandem mass spectrometry and overlapping synthetic peptides used for T-cell assays. For better comparison, the amino acid sequence of the avenin precursor protein JQ1047 (gi82331) is taken as a consensus sequence, and deviating residues are underlined. DOI: 10.1371/journal.pmed.0010002.g002

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Figure 3. HLA Restriction and Avenin Peptide Specificity of the Intestinal T-Cell Line 431.2 from Patient CD431 The avenin antigen (at 0.25 mg/ml) and peptides (at 10 lM), treated with TG2 when indicated, were incubated overnight with DR3-DQ2 homozygous antigen-presenting cells before T-cells were added. In the HLA restriction experiments, anti-DR or anti-DQ monoclonal antibodies were added 30 min prior to the T-cells. T-cell responses were measured by 3H-thymidine incorporation and are represented as SIs. DOI: 10.1371/journal.pmed.0010003.g003

nally elongated derivates of these 22-mers. Five peptides identified from fraction 9 correspond to N-terminally truncated and C-terminally elongated variants.

adenocarcinoma in the small intestine, which was removed surgically.

Avenin-Reactive T-Cell Lines Generated from Intestinal Biopsies Challenged with Avenin

T-Cell Recognition of Synthetic Avenin Peptides Four avenin peptides (1488, 1489, 1490, and 1491; Figure 2C) almost completely covering the sequences identified in the reverse-phase HPLC fractions 3, 4, 8, and 9 were synthesized and tested for T-cell recognition. Only peptide 1490 (SEQYQPYPEQQEPFVQQQQ) was recognized by the Tcell lines CD422.2.4, CD496.2.1, and CD431.2 (see Table 2; Figure 3). The recognition of this peptide by the T-cell lines CD422.2.4 and CD431.2 was dependent on TG2 treatment. Tcell line CD496.2.1 responded to the native peptide, but the response was augmented by treatment with TG2. We identified one deamidation site by tandem mass spectrometry (underlined in the above given sequence). This regioselectivity of deamidation conforms to the previously defined specificity of TG2 [11,19]. Several truncation variants of peptide 1490 were also synthesized. The shortest peptides tested were 12-mers; the T-cell line CD431.2 recognized peptide 1505 (YQPYPEQQEPFV) after TG2 treatment and the already deamidated peptide 1504 (YQPYPEQEEPFV) without TG2 treatment (Figure 3). We predict the 9-mer core region binding to DQ2 as PYPEQEEPF, placing the glutamic acid resulting from the deamidation at the P6 position. This is similar to the DQ2-a-I gliadin epitope (PFPQPELPY), which also binds to DQ2 with a glutamic acid at the P6 position [20,21]. Recently, Vader et al. studied whether T-cells generated from celiac disease biopsies stimulated with wheat gluten would cross-react with predicted epitopes of barley, rye, and oats. From these studies they found two broadly reactive polyclonal T-cell lines that responded to peptides from barley hordeins, rye secalins, and the avenin-derived peptides Av-a9A, which is identical to a length variant of 1490 (QYQPYPEQQEPFVQ), and Av-a9B (QYQPYPEQQQPFVQ) [22]. We tested peptide Av-a9B against our T-cell lines and found that it was recognized by T-cell lines from the patients CD422 (line 2.4), CD496 (lines

Responses to TG2-treated avenin were detected in the polyclonal T-cell lines derived from the avenin-challenged biopsies from all three patients who had clinical and histopathological signs of oat intolerance (Table 1). Intestinal T-cell responses to TG2-treated avenin were also found in two of the other six patients. At least one avenin-reactive Tcell line from each patient was expanded. Inhibition experiments using anti-HLA class I and class II monoclonal antibodies demonstrated that these T-cell lines were all restricted to DQ2 (Figure 3; unpublished data), and with the exception of the T-cell line generated from patient CD482, they all gave an enhanced T-cell response to avenin treated with TG2 compared to avenin not treated with TG2 (Table 2). T-cell lines derived from the biopsies challenged ex vivo with avenin gave higher responses to the TG2-treated avenin than to TG2-treated gluten in four of five patients (CD422, CD431, CD482, and CD496, but not CD507; Table 2). Notably, intestinal T-cells specific for TG2-treated wheat gluten and gliadin were identified in control biopsies challenged with gluten in all nine celiac patients (see Table 1).

Identification of a T-Cell Epitope in Avenin To identify the T-cell stimulatory peptides, we initially studied an avenin-specific T-cell line (TCL CD422.2.4) isolated from the oat-intolerant patient CD422. The T-cells weakly recognized one gel filtration fraction (#25) of a pepsin-trypsin digest of avenin. This fraction was further separated by reverse-phase HPLC, and retested for T-cell recognition (Figure 2A and 2B). Stimulatory fractions were subjected to electrospray ionization tandem mass spectrometry (Figure 2C). For fractions 3 and 4, a single 22-mer peptide was identified differing only by an asparagine (fraction 3) and an aspartic acid residue (fraction 4). The two identified peptides from fraction 8 represent C-termiPLoS Medicine | http://www.plosmedicine.org

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2.1 and 2.3), and CD507 (line 2.3) after TG2 treatment (Table 2; unpublished data). From the T-cell line CD496.2.3, we generated a T-cell clone that was specific for the peptide Ava9B after TG2 treatment (Figure 4A). This clone responded also to TG2-treated avenin, but did not display crossreactivity to TG2-treated gluten nor to the TG2-treated 1490 peptide (Figure 4B). The avenin-reactive T-cell line generated from the patient CD482 (CD482.2.1) did not recognize the 1490 peptide nor the Av-a9B peptide. Thus, there exist at least two distinct peptides of oats that can elicit mucosal T-cell responses in celiac disease patients with clinical intolerance to oats.

Location of Epitopes to a Proline- and Glutamine-Rich Region of Avenin The avenin epitopes we identified are localized to a region of avenin uniquely rich in proline and glutamine residues (Figure 5). The presumed 9-mer core region of the avenin epitopes (PYPEQQEPF and PYPEQQQPF) contains three proline residues. The high number of proline residues and the localization of the epitopes to a region rich in proline and glutamine residues bear strong resemblance to features typical of DQ2-restricted T-cell epitopes of wheat gliadin [23,24].

Discussion A number of previous reports concluded that all celiac disease patients tolerate oats. These reports have formed the basis for approving oats in the gluten-free diet for the treatment of celiac disease. The findings reported here demonstrate that oat intolerance exists in some celiac disease patients, and the study provides a molecular explanation for this intolerance. Oats are less related to wheat than are barley and rye. In oats, the prolamines represent much less of the total seed proteins than in the other cereals [25]. In addition, avenins contain about half the amount of proline residues (10%) as the prolamins of wheat (gliadins and glutenins), barley (hordeins), and rye (secalines). On this basis, it is intriguing that the identified avenin epitopes are located in the regions of avenins with the highest content of proline residues, regions also rich in glutamine. This is analogous to the localization of the T-cell epitopes in a- and c-gliadins [23].

Figure 4. Reactivity of an HLA-DQ2-Restricted T-Cell Clone Derived from a T-Cell Line (CD496.2.3) Established by Avenin Stimulation of an Intestinal Biopsy of Patient CD496 T-cell responses were measured by 3H-thymidine incorporation and are represented as SIs. (A) The clone specifically recognizes the avenin peptide Av-a9B after treatment with TG2. The peptides were tested at 10 lM. (B) The clone recognizes avenin but not gluten antigen after treatment with TG2. DOI: 10.1371/journal.pmed.0010004.g004

Figure 5. Amino Acid Sequence of an Avenin (gi 82331, JQ1047) and an a-Gliadin (a2-Gliadin, AJ133612) The proline and glutamine residues are red and blue, respectively. In the avenin, the presumed 9-mer core region of the characterized T-cell epitope is underlined. In the a-gliadin, a 33-mer natural fragment containing six copies of three partly overlapping epitopes (DQ2-a-I, PFPQPQLPY; DQ2-a-II, PQPQLPYPQ; and DQ2-a-III, PYPQPQLPY) is underlined. Note the localization of all the epitopes to regions of the proteins rich in proline and glutamine residues and the high number of proline residues within the 9-mer core regions of the epitopes. DOI: 10.1371/journal.pmed.0010005.g005 PLoS Medicine | http://www.plosmedicine.org

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clinically tolerant to oats and who have minimal or no mucosal pathology after a limited oats challenge. Possibly some of these patients may have latent oat intolerance that will develop into overt disease after prolonged exposure, but this remains speculative. Our observations demonstrate that even if oats seem to be well tolerated by many celiac disease patients, there are patients who have an intestinal T-cell response to oats. Until the prevalence of oat intolerance in celiac disease patients is established, clinical follow-up of celiac disease patients eating oats is advisable. Clinicians should be aware that oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet.

The immunogenicity of gliadin peptides is influenced both by the glutamine residues, which become specifically deamidated by TG2, and by the proline residues, which protect the peptides from proteolysis in the gastrointestinal tract, determine the specificity of TG2, and are crucial for the selective binding to HLA-DQ2 [1]. This study shows that the same features apply to T-cell epitopes of avenin. In humans it is impossible to directly demonstrate that Tcells induce disease. In celiac disease this relates equally to Tcells reactive to gluten and to T-cells reactive to avenin. The fact that avenin-reactive intestinal T-cells, like gluten-reactive T-cells from celiac disease patients, are uniquely restricted by HLA-DQ2 and are activated by TG2-treated peptides speaks strongly in favor of their involvement in the disease pathogenesis. The finding of avenin-specific intestinal T-cells also in individuals with celiac disease that are clinically tolerant to oats does not, as we see it, contradict this assumption. Some patients with celiac disease stay in remission for extended time periods during gluten challenge even if it is likely that they have gluten-reactive T-cells in their intestinal mucosa. Since avenin is less immunogenic than wheat gluten, one would expect an extended time for relapse to be at least as common during oats consumption. It is highly unlikely that the intolerance and the mucosal inflammation observed in our patients could be explained by contamination of the oat flour by wheat, barley, or rye proteins. All the oats consumed were produced in a qualityassessed production line. Our data indicate that avenin can drive mucosal inflammation in that the incubation of the intestinal biopsies with avenin enriches for activated, aveninreactive T-cells. A substantial proportion of the aveninreactive T-cells appear to be specific to avenin. The T-cell clone we established from an avenin-challenged biopsy was reactive to avenin but did not cross-react to wheat gluten, and the T-cell lines from biopsies challenged with avenin responded more strongly to avenin than to gluten in four of five participants. Cross-reactivity at the T-cell clonal level has been demonstrated between wheat gluten, hordein, and secalin antigens [22,26] and likely also exists between gluten and avenin [22]. Even if some of the avenin-reactive T-cells were originally primed to gluten and responded to avenin because of cross-reactivity, they would still participate in an avenin-driven immune response. T-cell reponses to the avenin epitopes described in this paper have been found in T-cell lines derived from intestinal biopsies of patients with celiac disease that were stimulated with gliadin [22]. It is unknown whether any of the patients from whom these T-cells were isolated had clinical symptoms or mucosal inflammation related to oats ingestion. Thus, to our knowledge, the current study is the first to demonstrate a mechanistic link between clinical symptoms of oat intolerance, mucosal inflammation, and avenin-reactive T-cells. Oat intolerance can cause complications in the large group of celiac disease patients who are now regularly consuming oats. At this stage we do not know how frequently such complications may occur. Presumably such complications will not be very common, but only extended clinical follow-up of oats-consuming celiac disease patients will establish the frequency. Monitoring of T-cell responses to avenin epitopes may potentially identify individuals who are at risk of developing oat intolerance. Based on our data, such monitoring will also identify some individuals who are PLoS Medicine | http://www.plosmedicine.org

Acknowledgments This work is supported by the Research Council of Norway, the European Commission (project QLK1-2000-00657), the Norwegian Foundation for Health and Rehabilitation, the Deutsche Forschungsgemeinschaft (SFB 510, Project D4), ZonMW (grant 912-02-028), and the Stimuleringsfonds Voedingsonderzoek LUMC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Jon Matre and Even Lind for referring two of the oat-intolerant patients to Rikshospitalet University Hospital; Marie Kongshaug Johannesen, Elisabeth Reed Eng, Eva Boretti, and Nicole Sessler for excellent technical assistance; and Don Kasarda and Chaitan Khosla for & comments on the manuscript.

References 1. Sollid LM (2002) Coeliac disease: Dissecting a complex inflammatory disorder. Nat Rev Immunol 2: 647–655. 2. Janatuinen EK, Pikkarainen PH, Kemppainen TA, Kosma VM, Jarvinen RM, et al. (1995) A comparison of diets with and without oats in adults with celiac disease. N Engl J Med 333: 1033–1037. 3. Srinivasan U, Leonard N, Jones E, Kasarda DD, Weir DG, et al. (1996) Absence of oats toxicity in adult coeliac disease. BMJ 313: 1300–1301. 4. Hardman CM, Garioch JJ, Leonard JN, Thomas HJ, Walker MM, et al. (1997) Absence of toxicity of oats in patients with dermatitis herpetiformis. N Engl J Med 337: 1884–1887. 5. Reunala T, Collin P, Holm K, Pikkarainen P, Miettinen A, et al. (1998) Tolerance to oats in dermatitis herpetiformis. Gut 43: 490–493. 6. Hoffenberg EJ, Haas J, Drescher A, Barnhurst R, Osberg I, et al. (2000) A trial of oats in children with newly diagnosed celiac disease. J Pediatr 137: 361–366. 7. Janatuinen EK, Kemppainen TA, Julkunen RJ, Kosma VM, Maki M, et al. (2002) No harm from five year ingestion of oats in coeliac disease. Gut 50: 332–335. 8. Størsrud S, Olsson M, Arvidsson LR, Nilsson LA, Nilsson O, et al. (2003) Adult coeliac patients do tolerate large amounts of oats. Eur J Clin Nutr 57: 163–169. 9. Ho¨gberg L, Laurin P, Fa¨lth-Magnusson K, Grant C, Grodzinsky E, et al. (2004) Oats to children with newly diagnosed coeliac disease: A randomised double blind study. Gut 53: 649–654. 10. Kilmartin C, Lynch S, Abuzakouk M, Wieser H, Feighery C (2003) Avenin fails to induce a Th1 response in coeliac tissue following in vitro culture. Gut 52: 47–52. 11. Vader LW, de Ru A, van Der WY, Kooy YM, Benckhuijsen W, et al. (2002) Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 195: 643–649. 12. Piper JL, Gray GM, Khosla C (2002) High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for celiac sprue. Biochemistry 41: 386–393. 13. Thompson T (2003) Oats and the gluten-free diet. J Am Diet Assoc 103: 376–379. 14. Pera¨aho M, Kaukinen K, Mustalahti K, Vuolteenaho N, Ma¨ki M, et al. (2004) Effect of an oats-containing gluten-free diet on symptoms and quality of life in coeliac disease. A randomized study. Scand J Gastroenterol 39: 27– 31. 15. Lundin KEA, Nilsen EM, Scott HG, Løberg EM, Gjøen A, et al. (2003) Oats induced villous atrophy in coeliac disease. Gut 52: 1149–1152. 16. Oberhuber G, Granditsch G, Vogelsang H (1999) The histopathology of coeliac disease: Time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol 11: 1185–1194. 17. Molberg Ø, Flaete NS, Jensen T, Lundin KEA, Arentz-Hansen H, et al.

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(2003) Intestinal T-cell responses to high-molecular-weight glutenins in celiac disease. Gastroenterology 125: 337–344. Roepstorff P, Fohlman J (1984) Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom 11: 601. Fleckenstein B, Molberg Ø, Qiao SW, Schmid DG, von der Mulbe F, et al. (2002) Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 277: 34109–34116. Arentz-Hansen H, Ko¨rner R, Molberg Ø, Quarsten H, Vader W, et al. (2000) The intestinal T cell response to a-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 191: 603–612. Kim CY, Quarsten H, Bergseng E, Khosla C, Sollid LM (2004) Structural basis for HLA-DQ2 mediated presentation of gluten epitopes in celiac disease. Proc Natl Acad Sci U S A 101: 4175–4179. Vader LW, Stepniak DT, Bunnik EM, Kooy YMC, de Haan W, et al. (2003) Characterization of cereal toxicity for celiac disease patients based on protein homology in grains. Gastroenterology 125: 1105–1113. Arentz-Hansen H, McAdam SN, Molberg Ø, Fleckenstein B, Lundin KEA, et al. (2002) Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology 123: 803–809. Shan L, Molberg Ø, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279. Shewry PR, Tatham AS, Kasarda DD (1992) Cereal proteins and coeliac disease. In: Marsh M, editor. Coeliac disease. Oxford: Blackwell Scientific Publications. pp. 305–348. Lundin KEA, Scott H, Fausa O, Thorsby E, Sollid LM (1994) T cells from the small intestinal mucosa of a DR4, DQ7/DR4, DQ8 celiac disease patient preferentially recognize gliadin when presented by DQ8. Hum Immunol 41: 285–291.

Background. Celiac disease is a digestive disease that damages part of the gut (the small intestine) and interferes with absorption of nutrients from food. Patients with celiac disease do not tolerate a protein called gluten, which is found in wheat, rye, and barley. When people with celiac disease eat foods containing gluten, their immune system responds by damaging the small intestine. The disease is quite serious in some patients, but eating a strictly gluten-free diet can eliminate all of the symptoms. Unfortunately, wheat, barley, and rye products like flour are found in many common foods, and patients have to avoid them for the rest of their lives. Previous studies suggested that oats were safe for patients with celiac disease, and as a result, they often form part of a gluten-free diet. What Did the Researchers Find? Contrary to other studies, this one demonstrates that oats intolerance does exist in some patients with celiac disease. These patients have an immune reaction to oats that is similar to the reaction most celiac disease patients have to wheat, barley, and rye. What Does This Mean for Patients? It appears that oats are not safe for all patients with celiac disease. Patients who eat oats as part of a glutenfree diet should discuss their diet and any symptoms with their doctors; doctors should keep in mind that patients might develop symptoms when they eat oats. What Are the Problems with the Study? The researchers studied only a small number of patients, and this study cannot tell us how common oats intolerance is among celiac disease patients. Where Can I Find More Information? US National Institutes of Diabetes, Digestive, and Kidney Disorders: http://digestive.niddk.nih.gov/ddiseases/ pubs/celiac/ Celiac Disease Foundation: http://www.celiac.org/ The Gluten Intolerance Group: http://www.gluten.net/ The Celiac Disease Foundation: http://www.celiac.com/

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