Pediatric Infectious Diseases Revisited (Birkhauser Advances in Infectious Diseases)

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Birkhäuser Advances in Infectious Diseases BAID

Series Editors Axel Schmidt University Witten/Herdecke Faculty of Medicine Alfred-Herrhausen-Str. 50 58448 Witten Germany Stefan H.E. Kaufmann Max-Planck-Institut für Infektionsbiologie Department of Immunology Schumannstrasse 21/22 10117 Berlin Germany

Manfred H. Wolff University Witten/Herdecke Faculty of Biosciences Stockumer Str. 10 58448 Witten Germany

Pediatric Infectious Diseases Revisited Edited by Horst Schroten and Stefan Wirth

Birkhäuser Verlag Basel Boston Berlin

·

·

Editors Horst Schroten Pediatric Infectious Diseases Department of General Pediatrics University Children’s Hospital Moorenstr. 5 40225 Düsseldorf Germany

Stefan Wirth Children’s Hospital HELIOS Klinikum Witten-Herdecke University Heusnerstr. 40 42283 Wuppertal Germany

Library of Congress Control Number: 2007920789 Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de

ISBN 978-3-7643-7997-1 Birkhäuser Verlag, Basel - Boston - Berlin The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2007 Birkhäuser Verlag, P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TFC ' Cover design: Micha Lotrovsky, CH-4106 Therwil, Switzerland Cover illustration: Art work by Martina Ziegler Printed in Germany ISBN-10: 3-7643-7997-9 e-ISBN-10: 3-7643-8099-3 ISBN-13: 978-3-7643-7997-1 e-ISBN-13: 978-3-7643-8099-1 987654321 www. birkhauser.ch

Contents List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Preface

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ix

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

Rudolf H. Tangermann, Hanna Nohynek and Rudolf Eggers Global control of infectious diseases by vaccination programs . . . . . . . . .

1

Duncan Steele Potential impact of rotavirus vaccination on the mortality of children in developing countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43

Sieghart Dittmann Controversially discussed indications for immunization . . . . . . . . . . . . . . . .

71

Axel Schmidt Gonorrheal ophthalmia neonatorum: historic impact of Credé’s eye prophylaxis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95

Susanna Cunningham-Rundles and Deborah Ho Lin Malnutrition and infection in industrialized countries . . . . . . . . . . . . . . . . . . 117 Matthew Jukes Better education through improved health and nutrition: Implications for early childhood development programs in developing countries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Shigenobu Kimura and Yuko Ohara-Nemoto Early childhood caries [ECC] and childhood periodontal diseases . . . . . 177 Rüdiger Adam, Kwang Sik Kim and Horst Schroten Role of the blood-brain barrier and blood-CSF barrier in the pathogenesis of bacterial meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

vi

Contents

Ian A. Clark and Michael J. Grifﬁths The molecular basis of paediatric malarial disease . . . . . . . . . . . . . . . . . . . . . . 239 Wilbert Mason Epidemiology and etiology of Kawasaki disease . . . . . . . . . . . . . . . . . . . . . . . . 273 Hien Q. Huynh Helicobacter pylori infection in children

................................

297

Adilia Warris and Ronald de Groot Human metapneumovirus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317 John V. Williams Avian inﬂuenza viruses: a severe threat of a pandemic in children? . . . . 345 Nanette B. Silverberg Human papillomavirus infections in children . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Patrick Gerner New treatments for hepatitis B and C in children and adolescents . . . . . 391 Andreas H. Groll, Julia Koehler and Thomas J. Walsh Invasive fungal infections in children: advances and perspectives . . . . . . 405 Kwang Sik Kim Pediatric aspects of bioterrorism

........................................

473

David Nadal Pediatric infectious diseases – Quo vadis 2015? . . . . . . . . . . . . . . . . . . . . . . . . . 485 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

List of contributors Rüdiger Adam, Pediatric Infectious Diseases, Department of General Pediatrics, University Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf, Germany; e-mail: [email protected] Ian A. Clark, School of Biochemistry and Molecular Biology, Australian National University, Canberra ACT 0200, Australia; e-mail: [email protected] Susanna Cunningham-Rundles, Host Defenses Program, Department of Pediatrics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA; e-mail: [email protected] Sieghart Dittmann, Hatzenporter Weg 19, 12681 Berlin, Germany; e-mail: [email protected] Rudolf Eggers, Expanded Programme on Immunization Plus, World Health Organization, Geneva, Switzerland; e-mail: [email protected] Patrick Gerner, Zentrum für Kinder- und Jugendmedizin, HELIOS Klinikum Wuppertal, Heusnerstr. 40, 42283 Wuppertal, Germany; e-mail: [email protected] Michael J. Grifﬁths, Department of Paediatrics, Newcastle General Hospital, Newcastle upon Tyne, U.K.; e-mail: [email protected] Andreas H. Groll, Infectious Disease Research Program, Center for Bone Marrow Transplantation and Department of Pediatric Hematology/Oncology, Children’s University Hospital, Albert-Schweitzer-Str. 33, 48129 Münster, Germany; e-mail: [email protected] Ronald de Groot, Department of Pediatrics, Radboud University Nijmegen Medical Centre, and the Nijmegen University Center for Infectious Disease, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands; email: [email protected] Deborah Ho Lin, Department of Pediatrics Host Defenses Program, Weill Medical College of Cornell University, New York, NY 10021, USA; e-mail: [email protected] Hien Q. Huynh, Department of Pediatrics, Stollery Children’s Hospital, Aberhart Centre #1, Room 9222, 11402 University Avenue, Edmonton, AB, Canada T6G 2J3; e-mail: [email protected] Matthew Jukes, Harvard Graduate School of Education, Appian Way, Cambridge, MA 02138, USA; Partnership for Child Development, Department of Infectious Disease Epidemiology, Imperial College School of Medicine, Norfolk Place, London W2 1PG, UK; e-mail: [email protected]

viii

List of contributors

Kwang Sik Kim, Johns Hopkins University School of Medicine, 200 North Wolfe Street/Room 3157, Baltimore, MD 21287, USA; e-mail: [email protected] Shigenobu Kimura, Department of Oral Microbiology, Iwate Medical University School of Dentistry, 1-3-27 Chuodori, Morioka, Iwate 020-8505, Japan; e-mail: [email protected] Julia Koehler, Children’s Hospital Boston, Harvard Medical School, Division of Infectious Diseases, 300 Longwood Avenue, Boston, MA 02115, USA; e-mail: [email protected] Wilbert Mason, Los Angeles Children’s Hospital, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA; e-mail: [email protected] David Nadal, Abteilung für Infektiologie und Spitalhygiene, Kinderspital Zürich, Universitäts-Kinderkliniken, Steinwiesstrasse 75, 8032 Zürich, Switzerland; e-mail: [email protected] Hanna Nohynek, National Public Health Institute, Department of Vaccines, Helsinki, Finland; e-mail: hanna.nohynek@ktl.ﬁ Yuko Ohara-Nemoto, Division of Oral Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Japan Axel Schmidt, Institute of Microbiology and Virology, Faculty of Medicine, University Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany; e-mail: [email protected] Horst Schroten, Pediatric Infectious Diseases, Department of General Pediatrics, University Children’s Hospital, Moorenstr. 5, 40225 Düsseldorf, Germany; e-mail: [email protected] Nanette B. Silverberg, Department of Dermatology, St. Luke’s-Roosevelt Hospital Center, 1090 Amsterdam Avenue, Suite 11D, New York, NY 10025, USA; e-mail: [email protected] Duncan Steele, Initiative for Vaccine Research, Department of Immunisation, Vaccines and Biologicals, World Health Organisation, Geneva, Switzerland; e-mail: [email protected] Rudolf H. Tangermann, Polio Eradication Initiative, World Health Organization, 20, Avenue Appia, 1211 Geneva 27, Switzerland; e-mail: [email protected] Thomas J. Walsh, Immunocompromised Host Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA; e-mail: [email protected] Adilia Warris, Department of Pediatrics, Radboud University Nijmegen Medical Centre, and the Nijmegen University Center for Infectious Disease, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands; e-mail: [email protected] John V. Williams, Division of Pediatric Infectious Diseases, Vanderbilt University Medical Center, D-7235 Medical Center North, 1161, 21st Avenue South, Nashville TN 37232-2581, USA; e-mail: [email protected]

Preface The fifth volume of Birkhäuser Advances in Infectious Diseases is focused on pediatric infectious diseases. In modern medicine, the discipline pediatric infectious diseases is an important medical specialty. The successful prevention of childhood diseases like diphtheria, tetanus and pertussis has made a major contribution to the improvement of public health. Understanding the biology of causative agents and the pathogenesis is an essential step in achieving control and elimination of disease. Today pediatric infectious diseases research is closely interconnected with other disciplines. This volume addresses vaccination, historical, epidemiological and sociocultural issues as well as clinical and molecular biological aspects of pediatric infectious diseases. New insights into the pathogenesis of infection are presented and an update on diagnostics, prevention and treatment of pediatric bacterial, viral, fungal and parasitic diseases is provided. The role of emerging new pathogens is also pointed out. Finally, the future perspectives of pediatric infectious diseases are highlighted. Therefore, this book aims at an interdisciplinary audience of clinicians and non-clinicians: pediatricians, infectious disease researchers, virologists, microbiologists as well as public health scientists and politicians. We would like to sincerely thank the staff of Birkhäuser publishers, and notably Dr. Beatrice Menz, for editing this volume of the Advances in Infectious Diseases series. Most of all we would like to thank all our colleagues who are international experts and scientists in their respective field and who generously shared their knowledge in the broad interdisciplinary area of pediatric infectious diseases with us. Düsseldorf/Wuppertal, Germany, December 2006

Horst Schroten Stefan Wirth

Glossary ABCD ABLC ACIP AD ADIP AEFI AGA ANCA ARDS ARF ARI ART AZT

amphotericin B colloidal dispersion amphotericin B lipid complex Advisory Committee for Immunization Practices auto-disable (syringe-needle unit) accelerated development and introduction plan adverse events following immunization appropriate for gestational age antibodies to neutrophil cytoplasmic antigens adult respiratory distress syndrome acute renal failure acute respiratory infection Anti-retroviral therapy zidovudine

BBB BMEC BMI

blood-brain barrier brain microvascular endothelial cell body mass index

CAA CAG CD CF CFR CGD CIS CLEAR CM CMA CNS COPD CP CSF CVI CVO

coronary artery abnormalities cyctotoxin-associated gene Crohn’s disease cystic fibrosis case fatality rate chronic granulomatous disease Commonwealth of Independent States Collaborative Exchange of Antifungal Research (registry) cerebral malaria cow’s milk allergy central nervous system chronic obstructive pulmonary disease choroid plexus cerebrospinal fluid Children’s Vaccine Initiative circumventricular organ

DAMB DQ DTP

amphotericin B deoxycholate Development quotient diphtheria/tetanus/pertussis (vaccine)

xii

Glossary

EAE ECC ECD EDV EM EMEA EMS EPI

experimental autoimmune encephalomyelitis early childhood caries early childhood development epidermodysplasia verruciformis electron microscopy European Medicines Evaluation Agency emergency medical service Expanded Program on Immunization

FC FDA FTT

fluorocytosine Food and Drug Administration (USA) failure to thrive

GAVI GCF GERD GIVS GMT GTF GVHD

Global Alliance for Vaccine and Immunization gingival crevicular fluid gastroesophageal reflux disease Global Immunization Vision and Strategy geometric mean titer glucosyltransferase graft-vs.-host disease

HA HAART HBIG HBMEC HbS HBsAg HBV HCV Hib HMGB1 hMPV HPAI hPIV HPV HPV HRCT HSCT HUVEC

hemagglutinin highly active antiretroviral treatment hepatitis B immunoglobulin human brain microvascular endothelial cell line sickle cell haemoglobin hepatitis B surface antigen hepatitis B virus hepatitis C virus Haemophilus influenzae type b high mobility group box1 (protein) human metapneumovirus highly pathogenic avian influenza human parainfluenza virus human papilloma virus human papillomavirus high-resolution computed tomography hematopoietic stem cell transplant/transplantation human umbilical vein endothelial cell

IBD ICC ICP IDO IE IFFIm IMCI IPN IPV

inflammatory bowel disease Interagency Coordinating Committee intracranial pressure indoleamine 2,3-dioxygenase infected erythrocyte International Finance Facility for Immunization Integrated Management of Childhood Illnesses infantile periarteritis nodosa inactivated poliovirus vaccine

Glossary

xiii

ITP IUGR IVIG

idiopathic thrombocytopenia intrauterine growth retardation intravenous immunoglobulin

JORRP

juvenile onset-recurrent respiratory papillomatosis

KD

Kawasaki disease

LAMB LPS

liposomal amphotericin B lipopolysaccharide

MALT mucosa-associated lymphoid tissue MDP muramyl dipeptide MMP matrix metalloproteinase MMR measles, mumps and rubella MNT maternal and neonatal tetanus MPO myeloperoxidase MRI magnetic resonance imaging MS multiple sclerosis MSCRAMM microbial surface component recognizing adhesive matrix molecules NA NICU NID NIS NT

neuraminidase neonatal intensive care unit National Immunization Day Newly Independent States neonatal tetanus

OME OPC OPV OR ORT

otitis media with effusion oropharyngeal candidiasis oral polio vaccine odds ratio oral rehydration therapy

PAF PAMP PATH PBMEC PCM PCV PCZ PEM pIgR PMNL Pnc PPI PPV PRGP

platelet-activation factor pathogen-associated molecular pattern Program for Applied Technology in Health porcine brain microvascular endothelial cell line protein-calorie malnutrition Pnc conjugate vaccine posaconazole protein-energy malnutrition polymeric immunoglobulin receptor polymorphonuclear leukocyte pneumococcus proton pump inhibitor Pnc polysaccharide vaccine proline-rich glycoprotein

RBC

red blood cell

xiv

Glossary

RED RES RRV RSV RTI

Reach Every District (vaccination strategy) reticuloendothelial system rhesus rotavirus respiratoy syncytial virus respiratory tract infection

SA SAE SAGE SARS SCID SGA SIA SIDS SIGN SNP SSPE SUV SVCC

superantigen sepsis-associated encephalopathy Strategic Advisory Group of Experts severe acute respiratory syndrome severe combined immunodeficiency small for gestational age supplementary immunization activities sudden infant death syndrome Safe Injection Global Network single-nucleotide polymorphism subacute sclerosing panencephalitis small unilamellar vesicle shell vial centrifugation culture

TNF TSST TT

tumor necrosis factor toxic shock syndrome toxin tetanus toxoid

UC UCI UNICEF

ulcerative colitis Universal Child Immunization United Nations Children’s Fund

VEGF VLP VVM

vascular endothelial growth factor virus-like particle vaccine vial monitor

WHIM

warts/hypogammaglobulinemia/recurrent bacterial infections/ myelokathexis (syndrome)

Pediatric Infectious Diseases Revisited ed. by Horst Schroten and Stefan Wirth © 2007 Birkhäuser Verlag Basel/Switzerland

1

Global control of infectious diseases by vaccination programs Rudolf H. Tangermann1, Hanna Nohynek2 and Rudolf Eggers1 1World

Health Organization, Geneva, Switzerland; 2National Public Health Institute, Department of Vaccines, Helsinki, Finland R. Tangermann and R. Eggers are staff members of the World Health Organization. The authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions, policy or views of the World Health Organization.

Abstract In both industrialized and developing countries, childhood immunization has become one of the most important and cost-effective public health interventions. National immunization programs have prevented millions of deaths since WHO initiated the ‘Expanded Program on Immunization’ in 1974. Smallpox was eradicated in 1979, poliomyelitis is on the verge of eradication, and two thirds of developing countries have eliminated neonatal tetanus. Global immunization coverage was at 78% in 2005. Through their impact on childhood morbidity and mortality, immunization programs are contributing to reaching the ‘Millennium Development Goal 4’ – a two-thirds reduction of under-five mortality by 2015. However, the failure to reach more than 20% of the world’s children with existing vaccines was responsible for at least 2.5 million of an estimated 10.5 million deaths of children under 5 years, mainly in developing countries. Of these deaths, 1.4 million could have been prevented by vaccines currently recommended by WHO. Rapid progress in our understanding of the pathogenesis of infectious diseases, immunology, and biotechnology has increased the number of candidate vaccine antigens available. Pressures are growing on public health decision makers to establish evidence-based ways to decide which new vaccines should be introduced on a large scale into national immunization programs. The gap in access to new vaccines between the developing and industrialized worlds is still wide, and wealthy countries are still the first to introduce and use new vaccines. Interest from countries and partner agencies in vaccination, as one of the most cost-effective public health interventions, continues to be strong, also due to rapid progress in biotechnology and vaccine development and the emergence of global infectious disease threats, including HIV/AIDS, SARS, and influenza. The establishment of the Global Alliance for Vaccines and Immunization has focused global activities to support vaccination programs through raising considerable funds, and to assist especially poorer countries in improving and expanding their vaccination programs. Global efforts concentrate on further reducing the gap in the access to all existing vaccines between industrialized and developing countries.

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Introduction In both industrialized and developing countries, child immunization has become one of the most important and cost-effective public health interventions [1, 2]. National immunization programs have prevented millions of deaths since WHO initiated the ‘Expanded Program on Immunization (EPI)’ in 1974 [3]. Smallpox was eradicated in 1979 [4], poliomyelitis is on the verge of eradication [5], and two thirds of developing countries have eliminated neonatal tetanus (NT).1 Global immunization coverage, as measured by the reported infant coverage with the third dose of diphtheria–tetanus–pertussis (DTP) vaccine (DTP3), was at 78% worldwide in 2005 [6] (Fig. 1), as compared to 20% in 1980. By the end of 2004, 153 of 192 WHO Member States had introduced hepatitis B (HepB) vaccine and 92 countries had introduced Haemophilus influenzae type b vaccine (Hib) into routine infant vaccination programs [7, 8], even though both vaccines are still underused in developing countries. The estimated number of deaths (from measles, pertussis and NT) prevented through childhood immunization in 2003 was more than 2 million. Infant HepB vaccination in 2003 was estimated to prevent a future 600 000 adult deaths, which would have occurred without vaccination, due to chronic liver disease and liver cancer. However, the failure to reach > 20% of the world’s children with existing vaccines was responsible for at least 2.5 million of an estimated 10.5 million deaths of children < 5 years in 2002 (Fig. 2), mainly in developing countries. Of these deaths, 1.4 million could have been prevented by vaccines currently recommended by WHO: > 500 000 due to measles, nearly 400 000 due to Hib, nearly 300 000 due to pertussis, and 180 000 NT deaths [9, 10]. An additional 1.1 million children < 5 years are estimated to have died worldwide in 2003 from rotavirus and pneumococcal disease, against which effective vaccines exist,2 but are not yet used in developing countries [10]. Through their impact on childhood morbidity and mortality, immunization programs are already contributing considerably to reaching the ‘Millennium Development Goal 4’ – a two-third reduction of < 5 mortality by 2015 [11]. It was estimated that improving coverage with the basic six EPI vaccines could potentially reduce < 5 mortality by 13%, with another 10% mortality reduction possible following the introduction and more widespread use of Hib, pneumococcal, rotavirus and meningococcal vaccines. In industrialized countries, mortality reduction is not the main driving force of national vaccine programs. Programs in wealthy countries recognize and mostly adhere to global vaccination goals set by WHO, and address

1 WHO Geneva: Maternal and neonatal tetanus (MNT) elimination web site at http://www. who.int/immunization_monitoring/diseases/MNTE_initiative/en/index2.html 2 See the chapter by Dr. Steele of this volume on rotavirus and section on pneumococcal vaccines later in this chapter

Global control of infectious diseases by vaccination programs

3

Figure 1. Annual third dose of diphtheria-tetanus-pertussis vaccine (DTP3) coverage globally and by Region, 1980–2005. Source: WHO/UNICEF estimates, 2006

Figure 2. Percentage of deaths from vaccine-preventable diseases (VPDs) globally among children < 5 years, by disease, 2002. An estimated 2.5 million deaths of children < 5 years worldwide (of a total of 10.5 million deaths in this age group) are caused by diseases for which vaccines are currently available. (†) Diphtheria, hepatitis B (HepB), Japanese encephalitis, meningococcal disease, poliomyelitis, and yellow fever. In older age groups, approximately 600 000 HepB deaths are preventable by routine immunization.

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Rudolf H. Tangermann et al.

potential life years saved through vaccination in cost-effectiveness analyses. Main motivators for vaccination programs in industrialized countries are morbidity reduction and improvements in quality of life, indirect societal savings and also moral causes [12]. As for vaccination programs anywhere in the world, the access to the best and most effective vaccines available is seen as a right of every child. Rapid progress in understanding of infectious disease pathogenesis, immunology, and biotechnology has increased the number of candidate vaccine antigens available, many of which have entered clinical phases of testing for safety, immunogenicity and eventually efficacy. Pressures are growing on public health decision makers, advisers and implementers to establish transparent and evidence-based ways to decide which new vaccines can and should be introduced on a large scale into national immunization programs. While the gap in access to new vaccines between the developing and industrialized world remains wide (see below), rich countries are still the first to introduce and use new vaccines. This is illustrated by the recent licensing of the first human papilloma virus (HPV) vaccine (see later in this chapter), the second possibly cancer-preventive vaccine since HepB. HPV vaccine is now being recommended by the Advisory Committee of Immunization Practices to be included into the U.S. immunization program. Interest in vaccination programs from countries and partner agencies continues to be strong, due to the cost effectiveness and measurable public health impact of vaccination, particularly on recent progress towards global polio eradication [5] and measles mortality reduction. Other reasons for which vaccination remains a high priority in public health are the rapid progress in biotechnology and vaccine development, and the emergence of global infectious disease threats, including HIV/AIDS, SARS, and influenza. The establishment of the Global Alliance for Vaccines and Immunization (GAVI) in 2000 [13] has focused global activities to support vaccination programs through raising considerable funds, and assisting especially poorer countries in improving and expanding their vaccination programs. WHO and UNICEF, together with other immunization partners, have recently elaborated a long-term strategic plan for 2006–2015, the Global Immunization Vision and Strategy (GIVS) [8], to guide country programs and coordinate efforts of the international immunization partnership. This chapter describes the main currently used global immunization policies and strategies, discusses progress towards improving access of all children to vaccines worldwide, including remaining gaps between developing and industrialized countries, and provides short updates on the current status of priority and new vaccines.

Global control of infectious diseases by vaccination programs

5

Immunization policies and strategies The ‘Expanded Program on Immunization’ Established in 1974 [3], the EPI targeted to achieve 80% immunization coverage of children under the age of 12 months by the year 1990. The immunization goal was further reinforced by the Alma-Ata Declaration in 1978 [14], which identified primary health care, including immunization, as the key strategy for achieving “Health for All by the Year 2000”. Interest in immunization was greatly boosted by the global eradication of smallpox in 1977 [4]. While progress towards improving overall coverage was slow in the first half of the 80s, the UN Secretary-General, in 1985, called for all countries to reach at least 80% infant coverage (Universal Child Immunization, UCI). Following renewed efforts in developing countries and by immunization partner agencies, the UCI goal was achieved in 1990. Up to the early 1990s, the EPI concentrated on establishing the necessary infrastructure (vaccine cold chain, transportation, training of staff) to deliver vaccines to children, and on monitoring coverage. The program then added specific disease control goals during the 1990s: polio eradication, and accelerated control of measles and of maternal and NT (MNT) elimination. The Children’s Vaccine Initiative (CVI), which operated between 1990 and 1999, was a first and innovate attempt to create a global public-private partnership to support global vaccination and make new vaccines available to all children. However, impact of the CVI was not as strong as expected, mainly because critically important partners, such as the major vaccine manufacturers, were not yet sufficiently represented in the initiative. Since 2000, the GAVI3 has been very successful at re-focusing immunization activities globally. Many strategies outlined by the GIVS document support the GAVI objectives [8]: the introduction of new vaccines, the increasing integration of immunization with other health interventions, and strengthening national immunization programs within the health system context. In GIVS, new goals for the global and national EPI programs were set and supported by a wide collaboration of partners. Among others, the goals called for were: – by 2010, achieve 90% coverage of children under 1 year of age nationally in each country, with at least 80% coverage in every district; – by 2010, reduce measles mortality by 90% compared to the 2000 levels, and – by 2015, reduce overall morbidity and mortality from vaccine-preventable diseases by two-thirds compared to the 2000 level. 3 GAVI partners include governments in industrialized and developing countries, UNICEF, WHO, the Bill and Melinda Gates Foundation, the World Bank (WB), NGOs, foundations, vaccine manufacturers, and technical agencies such as the US Centers for Disease Control and Prevention (CDC)

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Table 1. Routine immunization schedule for infants recommended by the EPI Vaccine Birth BCG Oral polio DTP Hepatitis B* Scheme A Hepatitis B Scheme B Haemophilus infl. type B Yellow fever Measles

X X† X

6 weeks X X X X X

Age 10 weeks X X X X

14 weeks

9 months

X X X X X X** X***

†In

polio-endemic countries. is recommended in countries where perinatal transmission of HBV is frequent (e.g., in South-East Asia). Scheme B may be used in countries where perinatal transmission is less frequent (e.g., in sub-Saharan Africa). **In countries where yellow fever poses a risk. ***A second opportunity to receive a dose of measles vaccine should be provided for all children. This may be done either as part of the routine schedule or in a campaign. *Scheme A

Routine infant immunization Table 1 shows the ‘basic’ immunization schedule recommended by the EPI/ WHO [15], which is followed in low-income and most lower middle-income4 developing countries. Schedules in most upper middle- and high-income countries start later (e.g., 2 months), with longer intervals between doses [16, 17]. While the basic EPI schedule, with some variation, is still followed by many developing countries, vaccination schedules in middle-income and industrialized countries vary considerably, for historical, epidemiological, and economical reasons (compare the 2006 U.S. Child and Adolescent Immunization Schedule, Table 2). WHO keeps track of and publishes national immunization schedules [18]. To protect mothers and neonates against tetanus, WHO recommends implementing a five-dose tetanus toxoid (TT) schedule [19] for women of childbearing age, especially where most women in this age group have not previously received TT when they were young [20]. The different EPI contacts during the first year of life present opportunities for health education of mothers and caretakers and to deliver other basic health care interventions. For example, the measles contact at 9 months of age is used in many developing countries to administer vitamin A to children. In developing countries, routine immunization services are delivered most commonly by midwives or nurses in a health center, offering vaccination either daily or on specific days of the week, depending on the number of children attending each day. Where health centers have large catchment 4 Based on the classification of the WB by gross national income; of 208 economies with populations of > 30 000, including 184 WB member countries, 54 are ‘low’, 58 are ‘lower middle’, 40 are ‘upper middle’ and 56 are ‘high’ income.

Table 2. US recommended childhood and adolescent immunization schedule, as published by the Centers for Disease Control and Prevention at www.cdc.gov/nip/acip

Global control of infectious diseases by vaccination programs 7

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areas, regular additional ‘outreach services’ through staff based at the health center may be organized to reach children who live too far away from the center, and to trace children who did not come back for follow-up doses. In other areas it may be necessary to set up mobile services, which are more costly, because vaccination teams need vehicles and spend 2 or more days to reach hard-to-access population groups [21]. Any contact with a child in a facility offering EPI services, whether health center or hospital, should be used to screen the vaccination status of both the child and its mother (TT) and to offer vaccines and a basic package of non-vaccine preventative child health services. Missing opportunities to vaccinate, such as during a visit to the health facility for other reasons, still constitute a major factor contributing to low coverage.

Booster doses and ‘second opportunity’ for measles vaccination Few vaccines give life-long protection after the primary series. To maintain immunity beyond childhood, booster doses are needed. To maximize returns of scarce resources, however, WHO recommends considering adding booster doses to immunization programs once they have reached routine coverage levels of 80% or higher. Boosting with BCG is not recommended, as there is no evidence of its efficacy [22]. Since many developing countries have now reached 80% coverage, they have begun to include booster doses in their schedules, based on epidemiological patterns of diseases, available resources, and health infrastructure. Events like the diphtheria epidemic in Eastern Europe in the early 1990s, or the recognition that pertussis-infected adults contribute to community spread [23] triggered renewed interest in, and importance attached to, booster doses. While high coverage with one dose of measles vaccine will reduce measles morbidity and mortality, a second vaccine dose is needed for more efficient measles reduction, or to achieve measles elimination [24]. This ‘second opportunity’ for measles vaccination is not intended as a true ‘booster dose’ but to give a second chance to seroconvert for children who did not respond to the first dose, and also to reach children who missed the first dose. Increasingly, additional measles vaccine doses in developing countries, intended to reduce measles mortality or to move towards measles elimination, are delivered through campaigns. As the EPI programs mature, WHO encourages adopting routine two-dose measles schedules, to sustain gains in measles mortality reduction [25].

Supplementary immunization activities Immunization campaigns to supplement routine programs to increase coverage – now often referred to as supplementary immunization activities

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(SIAs) – were used first during the early phase of EPI to rapidly increase coverage to reach the 1990 ‘universal child immunization’ (UCI) goal, at that time often with poor results. More recently, SIAs are no longer used mainly to boost overall coverage, but have become the main tools for disease eradication and elimination initiatives – to achieve global polio eradication, reduce measles mortality, mainly in Africa, for measles elimination (in WHO Regions with a measles elimination goal [25]), and for TT campaigns to eliminate MNT, targeting child-bearing age women. SIAs typically target all children in a particular age group, according to disease epidemiology (5 years for polio campaigns, from 9 months to < 15 years for initial measles campaigns), and regardless of previous immunization status. SIAs are used in many countries to provide other interventions, most commonly vitamin A supplementation [26], but also, for example, insecticide-treated bed nets for malaria prevention [27], or de-worming medication. With appropriate support from donors and partners, and with adequate planning, implementation and monitoring/evaluation, recent experience with SIAs to reduce measles mortality and for polio eradication has been good overall. However, there has also been considerable discussion and controversy about the effects of vaccination campaigns on routine immunization programs and primary health care, particularly about the impact, whether positive or negative, of the polio eradication initiative. Some observers believe that polio eradication has detracted from health service delivery and has been detrimental to an integrated approach to health systems development [28]. Several large field studies on the impact of the polio eradication initiative on health systems concluded that, while SIA planning and implementation may have been detrimental in the short term to general health services, positive longterm synergies exist between polio eradication and health systems [29] (building vaccine-preventable disease surveillance, strengthening cold chain and management and planning for routine immunization, distribution of Vitamin A), but that these synergies must be more systematically exploited [30].

The vaccine cold chain EPI programs established a system of vaccine transport and storage at appropriate temperature – the cold chain – to assure that vaccine potency is maintained. This vaccination strategy component is particularly critical in tropical developing countries, where logistics and lack of reliable power supply and refrigeration equipment are frequent problems. The WHO recommends that the storage temperature for vaccines used in the EPI at health facilities be between 2 °C and 8 °C, a temperature range determined by the heat sensitivity of oral poliovaccine (OPV) and sensitivity to freezing of other vaccines (DTP, TT, HepB). Live vaccines (OPV, measles, BCG, yellow fever) can be stored in freezers at –20 °C. UNICEF and WHO, in collaboration with manufacturers, have set standards [31] for technologically

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appropriate cold-chain equipment and helped to develop such equipment, such as ice-lined refrigerators, which can maintain appropriate storage temperature for up to 16 h during power cuts, or refrigerators run on kerosene, gas and solar power in areas without grid electricity. Vaccine temperature is monitored over time during international and domestic vaccine transport using temperature-sensitive cards. More recently, vaccine vial monitors (VVMs) [32], attached to each vial of vaccine procured through UNICEF, have greatly facilitated vaccine use in the field, particularly to extend the ‘cold chain’ into remote areas during polio eradication campaigns. VVMs measure and indicate ‘cumulative’ heat exposure by changing color once vaccine potency is threatened. It was realized more recently that inappropriate freezing of freeze-sensitive vaccines is also a problem in many countries, potentially affecting the potency of vaccines with adjuvants (HepB, combination vaccines) [33].

Immunization safety and adverse events following immunization The goal of immunization is to protect the individual and the community from vaccine-preventable diseases. While modern vaccines are safe and effective, no vaccine is entirely without risk. Effective vaccines may produce some undesirable side effects, which are mostly mild and self limited. Many of the adverse events attributed to the administration of a vaccine are actually not caused by the vaccine, but are either due to programmatic or human error (particularly in developing countries), or are simply coincidental events, which are not causally related to vaccine administration [34]. Surveillance for adverse events following immunization (AEFIs) in many developing countries has confirmed that most adverse events temporally associated with vaccination were not causally but only incidentally associated with vaccination. In cases where the vaccine of the vaccination program is the cause of an AEFI, events resulting from inappropriate handling of vaccines (‘program error’) are much more common than severe events related to properties of the vaccine itself [35]. Examples for reported serious adverse events related to program error are vaccine reconstitution with the wrong diluent, administration of dangerous drugs for vaccines, contamination of multi-dose vials leading to abscesses or sepsis, or transmission of blood-borne diseases (HIV, hepatitis B or C) through contaminated needles or syringes. If allegations regarding vaccine-related AEFIs are not rapidly and effectively investigated and clarified, confidence in a vaccine or the immunization program can quickly be undermined, even if the vaccine or the vaccination program is not at fault, with possible dramatic consequences for acceptance of vaccination and disease incidence. As successful immunization programs continue to reduce the incidence of vaccine-preventable diseases, there is increasing public concern, particularly in industrialized countries, about possible risks attributed to vaccines.

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During the past decade, different vaccine antigens have been accused of contributing to increases of non-infectious diseases. Recent examples of these are false allegations linking measles-mumps-rubella vaccine to autism (United Kingdom), attributing multiple sclerosis to administration of HepB vaccine (France), and linking Hib vaccine to diabetes mellitus (Finland). Also, when a disease has been eradicated, even extremely rare adverse events may no longer be acceptable. Following the interruption of wild poliovirus transmission in three WHO Regions, the only polio cases that still occur in OPV-using countries are vaccine associated, which has caused many countries to switch to inactivated poliovirus vaccine. In Finland, the increase in BCG-related osteitis cases, while the incidence of tuberculosis (TB) remains very low, led to switching from universal to risk-group BCG vaccination. The programmatic importance of vaccine and immunization safety issues, including the need for monitoring and rapid investigation of AEFIs, has been increasingly highlighted by WHO. A Global Advisory Committee on Vaccine Safety was established [36], which has issued position papers on vaccine safety issues, such as the use of thiomersal as preservative in vaccines, or the safety of HepB vaccines.5 All countries are advised to establish a system of monitoring and investigating AEFIs, and to train key health staff on AEFI surveillance, and on how to communicate effectively with the media on vaccine safety issues. High-income countries are starting to utilize new information technology and vaccine registers to monitor AEFIs in a timelier manner. Through linking of vaccine registry information to diseasespecific registry information, different advanced epidemiological methods can be utilized to try to understand potential cause–effect relationships. The safe administration of vaccines is an essential component of immunization safety, the importance of which was not fully recognized during the initial phase of the global EPI. Because of the large-scale improper use of both re-sterilizable and single-use injection equipment (inadequate sterilization, re-use of disposable needles and syringes) [37] in developing countries, WHO and UNICEF have promoted universal use of auto-disable (AD) syringe-needle units. AD syringes can only be used once because of an internal locking mechanism, and have now been widely introduced into immunization programs in developing countries [38]. UNICEF now ‚bundles’ vaccine shipments with AD syringes and disposal boxes to ensure that safe injection practices are maintained. It is estimated that < 10% of all injections given worldwide are related to immunizations, and activities to promote the safety of injections in the immunization context are handled in the broader context of overall injection safety. The Safe Injection Global Network (SIGN)6, a global partnership of interested parties, aims to prevent transmission of blood-borne 5 Position papers on immunization safety can be found at http://www.who.int/vaccine_safety/en/ 6 Information on the SIGN project can be found at http://www.who.int/injection_safety/sign/en/

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disease by reducing the number of unnecessary injections, and ensuring the safety of all injections, including those who apply vaccines, as well as by ensuring safe injection-waste disposal. Another emphasis has been on proper disposal of injection equipment, such as the use of ‘sharps’ boxes, appropriate disposal pits, and incinerators to prevent infection of health workers through accidental needle stick injuries and reduce risk to communities [39]. There is also progress in developing needle-free injection technologies, particularly focusing on jet injectors with exchangeable nozzles.

Program monitoring and surveillance for vaccine-preventable diseases The main aim of an immunization program is to reduce the incidence of, and in some cases to eradicate a disease. Disease-specific morbidity and mortality can best be monitored through disease surveillance systems. In poor countries, surveillance data are often not very reliable: case detection and confirmation is erratic, and laboratory equipment and reagents may not exist. Other means to help maintain and improve the quality of immunization programs are monitoring immunization coverage, measuring antibody and cellular immunity responses, and testing vaccine efficacy using different observational epidemiological methods, as well as monitoring the quality of disease surveillance (completeness and timeliness of reporting) [40]. Program monitoring and surveillance data should be available at national, sub-national and particularly at the district level. For immunization programs, main quality indicators include immunization coverage for the vaccines used, the ‘drop-out rate’, which measures the proportion of children who start but do not return to finish the vaccine schedule (mainly measured between the BCG and DPT3 contact), and the extent of missed opportunities for immunization. Other program components monitored include injection and immunization safety, cold-chain maintenance and social mobilization and information activities [41]. In developing countries, coverage is monitored by the ‘administrative method’ – a comparison of routine reports of the number of doses given to children to the estimated population in that age group, or through surveys [42]. Coverage data from different sources and at different levels has often shown considerable discrepancies. WHO and UNICEF have reviewed and compared reported ‘administrative’ and survey coverage data for all countries since 1980, and then developed ‘best coverage estimates’ for each country [10]. Best estimates are updated annually, and are often lower than results obtained by the administrative method. However, the iterative processes now used to derive coverage estimates have much improved the accuracy of available coverage data, with continuously declining discrepancies. Many middle- and high-income countries have better demographic data available for more precise estimation of coverage: total or sample popula-

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Figure 3. Global vaccine-preventable disease laboratory network. The designation employed and the presentation of material on this map do not imply the expression of any opinion whatsoever on the part of the secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.

tion method is used in these countries. Increasingly, individualized ‘numerator data’ are available, which allow evaluatation of timeliness of vaccinations in addition to coverage. Surveillance for vaccine-preventable diseases is an essential program component to measure the impact of vaccines used in routine immunization programs. Surveillance should provide ‘data for decision-making’ through the ongoing systematic collection, analysis and interpretation of surveillance data, which enables program managers to take decisions on planning, implementation and evaluation of immunization programs. High-quality surveillance remains particularly critical for polio eradication [43] and regional measles elimination [44] efforts, to detect remaining chains of virus circulation and reliably monitor progress towards interruption of transmission. Reliable surveillance data are also critical to establish baseline ‘disease burden’7 [45] in countries considering introducing a new vaccine into their immunization program. Laboratory confirmation is important for some vaccine-preventable diseases, particularly those with eradication or elimination goals. A global poliovirus lab network (Fig. 3) consisting of 145 laboratories all around the world [46] provides critical information to the polio eradication effort, 7 WHO’s immunization programme maintains a web site on vaccine-preventable disease burden estimation at http://www.who.int/immunization_monitoring/burden/estimates_ burden/en/index.html

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including primary virus isolation from stool specimens, intratypic differentiation to distinguish wild- from vaccine-type polioviruses, and genetic sequencing of isolated viruses to track transmission paths of virus strains around the world. A global measles laboratory network has been established, which has utilized much of the polio laboratory infrastructure; often housed at the same institutions as polio labs, measles labs use similar systems for specimen transport, data management, communication and reporting of results. The measles network’s primary roles are confirmation of suspected measles cases using IgM testing and genetic characterization of measles viruses. Measles laboratories also perform serological diagnosis of yellow fever in countries in Africa and Latin America where yellow fever is prevalent. Regional rotavirus laboratory networks are also emerging in some regions [47]. Together with the planned expansion of the African ‘Paediatric Bacterial Meningitis Laboratory Surveillance Network’, a global vaccine-preventable disease laboratory (both virological and bacteriological) network is evolving [48], which will be a crucial component of the future of vaccination described in GIVS.

Current status, remaining problems, and progress achieved The global immunization program has been supported by a degree of commitment and cooperation by the health sector and many other partners, within and outside of government, and from both the public and private sector, which has not been seen before for other health programs. However, the wider benefits of immunization are not reaching all children. Children in lesser developed countries still have less access to immunization services than those in wealthier countries, often because political commitment to, and funding available for, health is low, and health service delivery systems are weak and badly managed. Typically, the range of vaccines accessible to poorer children is smaller, and they are at greater risk from unsafe immunization practices. While some low-income countries have made substantial progress in increasing coverage, coverage remains low in others. While global aggregate coverage was relatively stagnant at 70–75% throughout the 1990s (Fig. 1), coverage increased during the 2000s and reached 78% (DTP3 coverage) in 2005 (UNICEF/WHO best estimate8), with relatively greatest increases in Africa. Such aggregate global coverage masks wide variations both between and within sub-regions [49] (Fig. 1). In 2004, DTP3 coverage was over 90% in industrialized countries, countries of Central Europe, the former Soviet Union (Commonwealth of Independent States, CIS) and Latin America and the Caribbean, while coverage was 88% in countries of the Middle East and 8 Available at http://www.who.int/immunization_monitoring/en/globalsummary/ wucoveragecountrylist.cfm

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Figure 4. Estimated 28 million infants were not fully immunized (DTP3), 2005, representing a 78% global immunization coverage.

North Africa, 86% in East Asia and the Pacific, 67% in South Asia and 65% in sub-Saharan Africa. At these coverage levels globally, 28.2 million of the estimated 125 million newborns in 2005 were not fully immunized, including 12.1 million in South Asia (8 million in India alone), 8.7 million in subSaharan Africa, and 4.6 million in East Asia and the Pacific (Fig. 4). Also, regional averages conceal variations in coverage between countries. Some developing countries – notably Bangladesh, and Latin American countries, increased coverage substantially, while coverage rates actually fell in other low-income countries, particularly in parts of sub-Saharan Africa. In 2005, coverage in Somalia was 35%, and in Nigeria 25%, down from coverage rates which were twice as high one decade earlier. In Europe, the economic and social changes following the break-up of the Soviet Union triggered a considerable decline in investment in immunization services and in immunization rates in countries in east and central Europe and countries of the former Soviet Union (CIS), which led to the re-emergence of diseases such as diphtheria. A major diphtheria epidemic occurred in the early 1990s in Eastern Europe, in which more than 30 000 people died [50]. There continues to be great disparity between vaccines available in the high-income countries of Europe and those with economies in transition. In many developing countries, children are not reached by immunization because they either live in remote areas beyond the reach of health services, or because they are not accessible in conflict zones [51]. Children may also be excluded because their parents fail to register their birth, or do not make use of existing immunization services. Great inequalities exist between the

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poorest and wealthiest population groups [52] within some countries: the wealthiest 20% of children in India, Nigeria and Cote d’Ivoire, for example, are four times more likely to be immunized than the poorest children in the same country. Immunization ‘drop-out’, i.e., failure to complete the full immunization schedule, is also highest among the poorest population groups. To identify and target children who remain unvaccinated, countries have increasingly introduced and strengthened district-level monitoring and surveillance activities, which reflects crucial differences in coverage and disease incidence, often concealed by province- or national-level averages, and allows taking corrective action. Many of the children who are not reached by vaccination either live in remote, hard-to-access areas, or belong to hard-to-reach groups, like nomads and seasonal migrants. Reaching and vaccinating these children involves the use of outreach and mobile teams, and is much more costly than immunizing children in an urban area. Low coverage in many densely populated urban or peri-urban slums and lowincome areas, due to lack of health services, presents another challenge. The “Reach Every District” (RED) strategy9, launched jointly by WHO and UNICEF in 2003, is a new approach aimed at assisting developing countries to strengthen immunization services at district level. Fifty-three countries have implemented the RED strategy, which encourages supportive supervision, strengthening of district immunization management, regular outreach services, community links with service delivery, improved data management, and improved planning based upon data, also using lessons learned through polio eradication. GIVS [8] recommends that, to reach everybody targeted for immunization, national programs should use a combination of approaches, including both routine services and SIAs (immunization campaigns), attempting to reach every child at least four times per year. National commitment to immunization services should be strengthened by assuring that human resources and financial planning for immunization is included in national budget allocations, in the wider health sector context. GIVS proposes that comprehensive multi-year national immunization plans (cMYPs), including detailed budgets and yearly workplans, should become a main tool to develop and maintain sustainable, well-performing immunization services. CMYPs for immunization provide countries with a method for identifying critical areas and resource needs, and with opportunities to track progress. At least 40 countries are now developing these cMYPs [49], which include cost estimates for all immunization activities and outline future initiatives to improve vaccine coverage and extend vaccination to unreached populations. GIVS stresses the importance of the district level in planning, implementing, and evaluating immunization services, and 9 RED strategy WHO website at http://www.who.int/immunization_delivery/systems_policy/ red/en/index.html

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endorses the continued use of the RED approach (see above) to accomplish this objective. GIVS also highlights the importance of communication and social mobilization activities to inform communities and ensure there is community demand for immunization and confidence in its benefits and safety. Communities and non-governmental organizations (NGOs) and other interest groups should be directly engaged in immunization activities.

Immunization service delivery In many developing countries, a main contributing factor to ill-functioning immunization services is the fact that overall health services, often due to years of neglect and under-investment, are poorly managed and unable to meet the basic health needs of the population. In these settings, the immunization infrastructure – buildings, vehicles and vaccine cold-chain equipment – is in a poor, often non-functional state. Storage in badly maintained cold-chain equipment may compromise vaccine potency, and non-functional sterilizing equipment may leave injection equipment contaminated. Weak managerial skills, poor staff pay and motivation, failure to plan and budget effectively, and the lack of effective disease surveillance and reporting systems undermines the effectiveness of disease control and immunization systems, which are left unable to provide services to those in greatest needs. There is an alarming mismatch in some countries between the health needs of the population and the size of the health workforce, the mix of skills available, and the geographical location of health workers, with a severe shortfall of health personnel in rural areas in most developing countries (e.g., 85% of the population in Cambodia live in rural areas, but only 13% of health workers are based there) [53]. In some countries (Somalia, Afghanistan, South Sudan), conflict has destroyed or severely compromised the health infrastructure. Public health systems in sub-Saharan Africa are overwhelmed by the increasing burden of HIV/AIDS, exacerbated by HIV-related illnesses, absenteeism and deaths among health workers. Since overall ‘system-wide barriers’ such as human resource capacity, logistics and overall financial resources seriously affect immunization services, these barriers will need to be addressed in joint action with all other parts of the health sector. However, efforts to strengthen immunization services can also help to reduce overall barriers to the equitable delivery of health services, for example by capitalizing on the well-established access of immunization services to children and women. Linkage of immunization contacts with routine health checks, or with the delivery of other essential health interventions, such as vitamin A, de-worming treatments and insecticide-treated bed nets to prevent malaria, has considerable impact on child health and reducing child mortality.

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Immunization services can also assist through establishing ‘best practices’, which offer opportunities to strengthen overall health services. For example, polio eradication has in most developing countries led the way in strengthening national disease surveillance systems, including the establishment of a global virological laboratory network (see Fig. 3), and in strengthening cold-chain systems. Polio eradication activities have also shown that it is possible to reach each and every child in a country, including those in hard-to-reach or conflict-affected areas [51] or which are hard to access for other reasons. In many countries, the district-level micro-planning approach (‘bottom-up’) [54] used to prepare for polio campaigns has been very helpful to better define and map populations for routine immunization; microplanning lessons learned during polio eradication now form a core component of the RED strategy.

Improving access to under-used and new vaccines Even though the market for vaccines in developing countries is potentially huge, with < 130 million children born each year, vaccines for developing countries currently account for only 18% of the global US$ 6 billion vaccine market. While a number of new vaccines have become available over the last two decades, most poorer countries have not been able to pay for them in the public health services. This has widened the divide in access to new vaccines between wealthy and poorer countries. Even in wealthy countries it is no longer self-evident that a new vaccine gets introduced universally: the inclusion of pneumococcal conjugate vaccine has more than doubled the vaccine budget in those countries where introduced. Cost-effectiveness calculations have gained an important role in the decision-making about the introduction of new vaccines in many countries. In addition to lack of funding, the inadequate disease surveillance and reporting systems in developing countries made it difficult to establish the disease burden and potential benefits and cost effectiveness of new vaccines. Lack of demand for a newly introduced vaccine can have a long-term impact on both supply and price. A vicious circle ensues, which keeps the vaccine out of reach of developing countries: manufacturers will limit the scale of production if demand in developing countries is low or uncertain, and the low production volume ensures that prices remain high. Unequal access to Hib vaccine is an example. While the widespread use of Hib vaccines since the early 1990s has almost eliminated Hib-related disease in developed countries, many developing countries have had neither the capacity to establish the burden of Hib disease, nor the resources to afford the vaccine. As a result, an estimated 4.5 million unvaccinated children died in developing countries from Hib-related diseases, mainly pneumonia and meningitis, in the same period.

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Vaccine-manufacturing research agendas still neglect needs of children in developing countries. There are three main underlying problems: the low demand for new vaccines in developing countries, the neglect by manufacturers of vaccines for mainly developing country markets, which are considered ‚low profit’, and differences in the prevalence of causative organisms between developed and developing countries (e.g., different spectrum of pneumococcal serotypes between industrialized countries and the developing world, see above). New vaccines go through a lengthy and very costly research and development phase, with investments of more than US$ 500 million or more per vaccine, and periods of 12–15 years until licensing. Initially high prices are set for new vaccines, so that development costs are recouped and a profit can be made. The manufacturer’s exclusive rights to the vaccine are patentprotected for an initial 20-year period. Only then can other manufacturers start to produce the vaccine without paying royalties, which will lead to price reductions. Through the support of the GAVI and the GAVI Fund, major progress was achieved in making under-used and new vaccines available in developing countries. Within Phase 1 of GAVI support for new vaccines, countries were eligible to apply for vaccines and funding to introduce HepB vaccine, Hib vaccine and yellow fever vaccine as required. Breakthroughs in the development of new vaccines are occurring, which revolutionize the way vaccines are conceptualized, produced, and administered. It will be critical that the needs of both developed and developing countries are taken into account when setting vaccine research and development agendas. Combination vaccines that include DTP with other antigens (e.g., HepB and Hib) simplify vaccine delivery and will be increasingly available during the next decade. Wider use of combination vaccines in developing countries will depend on making them ‘affordable’ for developing country immunization programs. Decisions on the introduction of under-used or new vaccines into national immunization programs must be based on evidence showing the target disease burden, the safety of the vaccine on individual and population level, and on economic analyses defining the extent to which a new vaccine is ‚affordable’ and cost effective, and to assure that its use is sustainable in the long run, within the country’s budgeting and planning context. Countries should be empowered to evaluate their own needs and priorities, particularly to enable them to determine which of a number of several new vaccines will be easiest to integrate into the immunization program and represents the best opportunity for the investment of limited resources. GAVI has established innovative mechanisms to support the development and introduction of new vaccines, such as the ‘accelerated development and introduction plans’ (ADIPs) for two new priority vaccines – rotavirus and pneumococcal conjugate vaccine. ADIPs include efforts to assist countries to establish credible forecasts of vaccine demand (based

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mainly on disease burden and vaccine efficacy) early in the vaccine cycle, i.e., before manufacturers begin the lengthy development and scale-up process. Early demand forecasts will also allow countries to secure sustainable financing from national and external sources. It is hoped that the ‘ADIP’ strategy could advance the introduction of rotavirus and pneumococcal vaccines by 6 or more years in developing countries. Similarly, the introduction and wider use of Hib vaccine is supported by an international partnership of interested parties through the ‘Hib Initiative’ (see below).

Funding of immunization programs National governments in all countries have primary responsibility to assure the sustainable financing of their national immunization program. However, as routine immunization coverage has not improved or fallen in many lowincome countries, and newer vaccines remain out of reach for those children in greatest need, consensus has grown that equal access to vaccines should be considered as a ‘global public good’, and that financing of immunization, particularly for the poorest countries, should be a joint global responsibility. While self-sufficiency remains the ultimate goal, the GAVI works with countries towards increasing the financial sustainability of immunization programs, as measured by a country’s ability to mobilize both domestic and external funding on a reliable basis, and to use funds efficiently to achieve immunization targets. This is accomplished by strengthening national capacity for financial planning within the immunization program and the Ministry of Health, by committing increased national budget allocations for vaccines, and by using the existing Interagency Coordinating Committees (ICCs) for immunization to ensure adequate and appropriate donor support to the government. The GAVI channels resources to a country’s immunization programs through the GAVI Fund (formerly The Vaccine Fund). While the GAVI Board sets the policies for selecting which countries and programs may access GAVI Fund resources, the GAVI Fund manages existing funds and raises new financial resources, and channels them to developing countries’ health systems. The support provided by the GAVI to date – in the form of multi-year grants to countries to support immunization services, new and under-used vaccines and injection safety – has been critical in many developing countries. Grants are made based on a strict application process in which country proposals are reviewed by a panel of independent experts drawn from a wide geographic and technical base. As of April 2006, a total of almost US$ 3.3 billion has been raised in traditional funding from government and private sources, including US$ 1.7 billion actually received. Of this amount, US$ 1.5 billion has been committed to directly support countries, with US$ 603 million disbursed.

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In addition, France, Italy, Spain, the United Kingdom, Sweden, Norway, Brazil, South Africa and other countries have recently committed nearly US$ 4 billion to immunization over the next decade, using an innovative new mechanism called the ‘International Finance Facility for Immunization (IFFIm)’. By borrowing against commitments made by the donors, the IFFIm will raise funds, which will be disbursed through the GAVI Fund. Within Phase 1 of GAVI support, 75 low-income countries (with a per capita gross national income of less than US$ 1000 per year) have received support. The resources that have been received have been used to help to (a) strengthen healthcare delivery systems, (b) boost coverage with established vaccines (against diphtheria, tetanus, pertussis, TB, measles and polio), (c) introduce under-used vaccines where needed (hepB, Hib and yellow fever); (d) ensure immunization safety, and (e) accelerate the development of, and affordable access to, priority new vaccines for developing countries (e.g., against rotavirus, pneumococcal disease and meningitis types A and C). Approximately two thirds of the resources received by GAVI-eligible countries are used to purchase vaccines and supplies, while one third supports capacity strengthening and infrastructure. In Phase 2 of GAVI support starting in 2006, 72 countries are eligible to receive help, and further areas of country support are initiated. Countries will be able to apply for funding support to reduce health system barriers to improved primary health care and vaccination programs, thereby addressing fundamental barriers to improved vaccination coverage and program efficiency. In addition to HepB, Hib and yellow fever vaccines, it is anticipated that the GAVI will provide support to the introduction of further new vaccines, after having considered their investment potential through an investment case process. Both conjugate pneumococcal and rotavirus vaccines are expected to gain support from GAVI, followed in future by other, newer vaccines as they become available for general use. In addition to the support directly to countries, the GAVI provides funding for specific research projects or areas of agency support through its workplan. Thus, areas such as vaccine management, healthcare waste management and coverage reporting quality improvement are supported by GAVI. It will be critical for the international immunization partners to continue to secure and sustain financing for immunization, including through longterm commitments by existing public and private funding entities and new long-term financial mechanisms, to support research, development, production and use of new vaccines.

Brief updates on priority current, under-used and new vaccines While several existing vaccines, such as those against Hib, yellow fever, influenza, pneumococcus, Japanese encephalitis and rubella, are readily

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Table 3. Current and future vaccines and supportive technologies.

from [8].

available but under-used, new vaccines against rotavirus, certain pneumococcal serotypes targeted with conjugate vaccines, meningococcus and HPV have recently been licensed and are gradually being introduced in high-income countries. At the same time, research on vaccines against major infectious diseases such as malaria, HIV/AIDS, TB and pandemic influenza is underway, as well as against some ‘orphan’ infectious disease, including leishmaniasis and hookworm infestation (see Tab. 3). The following short summaries provide updates on the most important current, under-used and new priority vaccines.10

10 Please note that rotavirus disease and rotavirus vaccines are described in the chapter by Dr. Steele of this volume.

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Poliomyelitis: progress towards eradication Following significant progress towards interrupting wild poliovirus transmission in the Americas, all Member States of WHO passed a resolution in 1988 to eradicate polio globally by the year 2000 [55]. The global eradication initiative is based on implementing the following main strategies: (a) to maintain the highest possible routine infant immunization coverage against polio, (b) to conduct large-scale SIAs11 with OPV over a few days, using house-to-house vaccine delivery, and targeting children aged < 5 years, regardless of previous immunization status, and (c) to detect circulating wild poliovirus through maintaining high-quality surveillance for all cases of acute onset flaccid paralysis in children < 15 years in all countries, with stool specimen collection and laboratory testing for wild poliovirus [56]. While the initial goal of global eradication by the year 2000 was not met, progress has been extraordinary nevertheless. Supported by an international polio eradication partnership spearheaded by Rotary International, WHO, UNICEF and the U.S. CDC, and involving millions of health workers and volunteers, the number of polio-endemic countries12 was reduced from > 125 in 1988 to only 4 during 2005: Nigeria, India, Pakistan and Afghanistan. Three WHO Regions have already been certified free of indigenous wild poliovirus: the Americas (Western Hemisphere), Western Pacific and European Region, which together encompass 134 countries and territories, with more than 3 billion total population. The transmission of type 2 wild poliovirus, which was last found in 1999, has been interrupted globally [57]. Type 3 wild poliovirus transmission is now restricted to small foci in northern Nigeria and northern India, and a joint virus reservoir between southern Afghanistan and central Pakistan. Monovalent OPVs (types 1 and 3), which result in significantly higher type-specific seroconversion rates compared to trivalent vaccine, were re-licensed in 2005. Monovalent OPV1 has been extensively used in both endemic countries and those affected by outbreaks, and was critical in stopping indigenous transmission in Egypt. Use of monovalent OPV3 has begun in high-risk areas of northern India, and monovalent OPV3 will be used in the other remaining type 3 wild virus foci. Since 2003, virus exported from the remaining endemic areas, mainly from Nigeria, re-infected 25 previously polio-free African and Asian countries and resulted in several major outbreaks. However, transmission and outbreaks following importation dating back to 2003–2004 have stopped, and outbreaks beginning in 2005 are resolving. New importations and outbreaks in 2006 – Bangladesh, Democratic Republic of Congo, Namibia – were detected

11 SIAs are conducted either at the national, ‘National Immunization Days (NIDs)’, or subnational level, ‘sub-NIDs’. 12 Countries where circulation of indigenous wild poliovirus has never been interrupted

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early and are likely to be contained rapidly, because response activities were initiated more timely than for the 2003–2005 series of outbreaks. It has now been recognized that SABIN-strain polioviruses have the potential to both revert to neurovirulence and start to circulate, particularly in areas with low population immunity [58]. Since 1999, six polio outbreaks caused by circulating vaccine-derived polioviruses have been recorded, which were all rapidly controlled with SIAs using OPV. The highest priority for the global polio initiative in 2006 is to urgently interrupt virus transmission in the remaining endemic countries, where intensified eradication activities continue, including large-scale SIAs with monovalent OPV1 or trivalent OPV, depending on the epidemiological situation, every 6–8 weeks throughout the year. With continued high frequency of SIAs in the polio-affected countries, active or ‘silent’ refusals have become an issue negatively impacting on the quality of campaigns in some population groups. To ensure community acceptance and compliance, social mobilization and communication activities have become critical to the success of SIAs, and will be a key priority in 2006. Community awareness of the risks of wild poliovirus transmission needs to improve, including the public’s understanding of the need for repeated campaigns and of the benefits of multiple doses of OPV for children. Continuing and worsening conflict situations in parts of Afghanistan and Somalia have become a serious impediment to interrupting transmission in these areas, since very limited or no access to the affected areas makes it very difficult or even impossible to vaccinate children. While progress in Asia, particularly in Pakistan and India, continues, Nigeria, particularly in ten states in northern Nigeria where SIAs continue to miss > 40% of target children, remains the single greatest threat to global polio eradication through possible renewed international spread of wild polioviruses.

Measles: progress towards mortality reduction and elimination Despite the availability of measles vaccination for over 40 years, an estimated > 30 million cases of measles, with > 500 000 deaths from measles, occurred among children aged < 5 years in 2002. In many communities in measles-endemic areas, the protective effects of the vaccine are well-known and the vaccine is in high demand. However, throughout the 1990s, reported global routine immunization coverage with measles vaccine was only about 70%. In developing countries with the goal of measles mortality reduction, measles vaccine should be given at 9 months of age. In these settings, the measles dose is given more than 6 months after the last EPI contact, and drop-out rates may be high. Based on criteria for the feasibility of global disease eradication, after polio, measles was the next disease singled out for regional elimination and possible eradication within the next 10–15 years [59, 60]. Four WHO

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Regions have established regional measles elimination goals: the Americas (by 2000), European Region (by 2010), Eastern Mediterranean Region (by 2010), and Western Pacific Region (by 2012) [61]. The regional measles elimination initiatives are part of a global initiative to achieve measles elimination in the four Regions as planned, and to reduce measles mortality by 50% by 2005, compared with the 1999 level. This latter target has been achieved [61a] as a result of efforts in high-measles-burden countries, with the support of the ‘Measles Partnership’. Global measles control is based on four main strategies [62]: – achieving high routine immunization coverage with measles vaccine given at 9 months of age – providing a ‘second opportunity’ for measles vaccination either through the routine immunization program or measles SIAs targeting the age group in which most susceptibles have accumulated, both to increase the chance that children not vaccinated before now get a dose of measles vaccine, and to allow children who did not sero-convert to the first dose to gain immunity – establishing an effective system to monitor coverage and conduct measles surveillance with integration of epidemiological and laboratory information – improving clinical management of every measles case, e.g., administering Vitamin A. Following the initial large catch-up campaign, follow-up measles SIAs are conducted at regular intervals (e.g., every 3–5 years), targeting children born since the initial catch-up campaign. On the basis of well-planned and intense implementation of these strategies in all countries, the last measles case from endemic transmission in the Americas, which was also the first WHO Region to interrupt transmission of indigenous wild poliovirus, occurred in November 2002 [63].

Maternal and neonatal tetanus: progress towards elimination Since WHO in 1989 called for global elimination of MNT,13 the estimated number of deaths from NT, a disease almost exclusively linked to poverty, was reduced from an estimated 800 000 worldwide in the 1980s to 180 000 in 2002. Despite this impressive progress, the goal of eliminating MNT by 2005 has not yet been achieved. While MNT has been essentially eliminated in the Americas and northern Africa [64] as of end-2005, 49 countries remained that were considered as not having eliminated MNT, including large countries like China, India and Nigeria [65]. Main reasons for missing the global elimination goal are continued relatively low TT coverage of 13 < 1 NT case per 1000 live births at district level.

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Figure 5. Countries using Hib vaccine in routine immunization program, by Hib vaccine coverage, 2004. Source: WHO/UNICEF estimates, 2005

pregnant and child-bearing age women: opportunities to vaccinate pregnant women visiting antenatal clinics or other health centers offering immunization are frequently missed. Also, in many developing countries, mothers continue to deliver under unhygienic circumstances. To overcome the rather slow progress towards MNT elimination, a “high-risk approach” has been introduced, which targets all women of childbearing age in high-risk areas using campaign-style immunization (SIAs) with three doses of TT (or Td) with an interval of at least 4 weeks between doses 1 and 2, and of at least 6 months between doses 2 and 3. Promotion of clean deliveries is also part of this approach. Between 1999 and 2005, approximately 64 million women worldwide received at least two doses of TT through this strategy.

Haemophilus influenzae type B vaccine Wherever thorough studies have been performed, Hib has been shown to be an important cause of childhood meningitis and a major cause of bacterial pneumonia in children. Although little population-based incidence data are available from most of Asia and the newly independent States of the former Soviet Union, Hib is estimated to cause at least 3 million cases of serious disease and hundreds of thousands of deaths globally, each year. The most

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important manifestations of Hib disease, pneumonia and meningitis, are seen mainly in children < 5 years of age, particularly infants. Several different Hib conjugate vaccines are available, which are all highly effective. Their use has virtually eliminated invasive Hib disease from much of the industrialized world, and from The Gambia [66, 67]. In vaccine efficacy trials and case-control studies in Africa and Latin America, Hib vaccine reduced the incidence of overall pneumonia [68]; in Indonesia, the vaccine protected against invasive Hib disease but not against pneumonia [69]. In 1998, WHO recommended that Hib vaccine should be included in routine infant immunization, as appropriate to national capacities and priorities. More recently, the WHO Immunization Strategic Advisory Group of Experts (SAGE) recommended global implementation of Hib vaccination unless robust evidence exists of low disease burden or overwhelming impediments to implementation [70]. Hib conjugate vaccines have now been introduced in 92 countries worldwide (Fig. 5); however, most of these countries are high- or middle-income countries of Western Europe, the Americas, and the Middle East. In Asia and Africa, lack of disease burden data, lower disease burden (Asia) and relatively high vaccine cost ($ 2.50 per dose) has so far impeded the introduction of Hib vaccine into routine immunization programs. With a grant from the GAVI, the Hib Initiative14, a global consortium of academic and public health experts, works on evidence-based decision making regarding the use of the Hib vaccine at the country level. The Hib Initiative provides a focus on national-level decisions about vaccination through strategic coordination among partners and donors, support for studies to measure disease burden, and advocacy for Hib vaccine introduction. GAVI funds Hib vaccine introduction in several African countries in Africa, and will expand this support to additional eligible countries. Where the disease burden is unclear, the Hib Initiative is collaborating with governments and researchers to further define the scope of the disease. One example of such a project is a collaborative research among the Indian government and local researchers in three sites to define the burden of Hib disease in India. It is expected that this project will help support decisions on Hib vaccination programs throughout South Asia. To support local surveillance capacity for bacterial vaccine-preventable diseases, WHO has established a network of laboratories to assist in diagnosing and confirming bacterial meningitis in children. In many areas, these regional bacteriological laboratory networks for meningitis are now expanding their capacity to perform blood cultures in anticipation of the surveillance needs associated with newer vaccines such as pneumococcal vaccines.

14 http://www.hibaction.org/about.html

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Hepatitis B vaccine Even though safe and efficacious vaccines have been available for more than 20 years, HepB infection remains a significant public health problem globally, and is second only to tobacco as a recognized cause of a major cancer in humans. The majority of infections and chronic HBV surface antigen (HBsAg) carriers are caused by vertical (mother-to-child) and horizontal (child-to-child) transmission. While rarely causing acute hepatitis in young children, 90% of those infected perinatally and 30% infected in early childhood will become long-term HBsAg carriers, at high risk for chronic liver disease and liver cancer. An estimated 600 000 deaths every year are attributed to chronic HBV infection and its serious consequences, including liver cirrhosis and hepatocellular cancer [71]. HepB vaccine is considered to be very cost effective in endemic countries [71]. The vaccine was found to be highly effective in reducing carrier rates from > 8% to < 2% in immunized groups of children in a number of countries, including The Gambia, Hong Kong (SAR), Singapore, Taiwan (China), and Alaska [72]. The incidence of hepatocellular carcinoma in children of 10–14 years of age in Taiwan fell significantly 10 years after a universal infant HepB vaccine program was initiated [73]. The World Health Assembly recommended in 1992 that all countries should integrate HepB vaccine into their routine infant immunization programs by 1997. High coverage with the primary vaccine series among infants has the greatest overall impact on the prevalence of chronic HBV infection in children and should be the highest HBV-related priority. Lack of awareness of the link between early infection and delayed serious morbidity and mortality in adults [74] has been one of the reasons for the delayed introduction of the vaccine into infant immunization programs around the world. Different schedules are used for HepB immunization in national programs, depending on the local epidemiological situation and programmatic considerations (see Tab. 1). In countries where a high proportion of HBV infections are acquired perinatally, the first dose of HepB vaccine should be given as soon as possible (< 24 h) after birth. In countries where a lower proportion of HBV infections are acquired perinatally, the relative contribution of perinatal HBV infection to the overall disease burden, and the feasibility and cost effectiveness of providing vaccination at birth, should be carefully considered before a decision is made on the optimal vaccination schedule. Catch-up strategies targeted at older age groups or groups with risk factors for acquiring HBV infection should be considered as a supplement to routine infant vaccination in countries of intermediate or low HepB endemicity. In such settings, a substantial proportion of the disease burden may be attributable to infections acquired by older children, adolescents and adults. In all countries, large-scale routine vaccination of infants rapidly reduces the transmission of HBV.

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As of 2005, 158 of 192 WHO Member States have introduced HepB vaccination in their routine infant immunization schedules. This is a sevenfold increase compared to the number of countries using this vaccine in 1990, resulting from continued global advocacy for universal infant HepB vaccination, for which disease burden data is now well established [75], and a sharp drop in the price of the vaccine, now about $0.27 per dose of single antigen vaccine, and the assistance for the purchase and delivery of HepB vaccine from the GAVI. The target of the GAVI is for all its focus countries with adequate immunization systems to introduce this vaccine into routine immunization programs by 2007. The availability of this first ‚vaccine against cancer’ to the majority of the world’s children will have a significant impact on long-term morbidity and mortality from chronic liver disease and hepatic cancer.

Yellow fever vaccine Yellow fever is endemic in tropical regions of Africa and South America where 44 countries (33 in Africa and 11 in South America) are considered to be at risk. In francophone Africa, intensive preventive mass vaccination campaigns nearly eliminated yellow fever during the 1950s, but subsequently vaccine coverage waned and epidemics occurred in the 1980s. Currently, 500 million people are considered at risk for the disease in Africa. Although WHO Member States are required to report yellow fever cases under the International Health Regulations, reported data underestimate the true incidence of the disease. Studies indicate that yellow fever morbidity and mortality are underestimated by a factor of 10–500; every year, an estimated number of 200 000 cases and 30 000 deaths are estimated to occur. Since the late 1980s, there has been a reemergence of yellow fever epidemics [76]; more than 80% of all yellow fever cases reported to the WHO were from Africa. Of the 33 “at-risk” countries in Africa, 16 reported at least one outbreak from 1980 to 1999. During the period 2000–2004 alone, 16 countries reported one or more outbreaks, with a total of 1927 cases and 425 deaths reported. Yellow fever control strategies include preventive vaccination (routine and supplementary mass campaigns), case-based surveillance with laboratory confirmation and rapid vaccination response in the event of an outbreak. The most cost-effective approach is to incorporate yellow fever vaccine in the routine national immunization program. This will prevent more yellow fever cases and deaths than emergency vaccination responding to outbreaks. The World Bank’s 1993 Development Report [77] strongly endorsed adding yellow fever vaccine to national immunization programs of at-risk countries. A study in Nigeria [78] estimated that the cost of routinely providing yellow fever vaccine through the national program would be about US$0.65 per

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fully immunized child. The cost of emergency vaccination would be much higher, about US$7.84 per person. All countries at risk in the Americas, and 22 of the 33 African countries have included the vaccine in their routine immunization program. However, coverage is generally poor in Africa, lagging behind measles vaccine coverage, even though both vaccines are supposed to be given at the same visit. Routine coverage improved in many countries once GAVI began in 2000 to support routine yellow fever vaccination in GAVI-eligible countries at risk for yellow fever. In 2002, the GAVI Board accepted to fund a 6 million-dose vaccine stockpile for outbreak response and preventive campaigns (SIAs) to reduce the number of susceptibles in wide age groups; these SIAs began in some countries in 2004. Yellow fever case-based surveillance was set up in 15 of 33 African countries at risk, and a laboratory network consisting of 22 laboratories was established. Most of these laboratories currently test samples and report to WHO. Although much progress has been achieved in yellow fever control in Africa, a large proportion of the population remains susceptible in countries at-risk, creating the potential for future outbreaks, which could be particularly explosive if they occur in urban areas. Advocacy and further resource mobilization are urgently needed to accelerate the progress made thus far in achieving yellow fever control.

Pneumococcal vaccines Streptococcus pneumonia or pneumococcus (Pnc), is considered as one of the major bacterial pathogens causing a multitude of childhood infections [79]. The spread of HIV infection has increased the incidence of Pnc disease, especially in many resource-poor countries where anti-HIV treatment is not readily available. Children infected with HIV/AIDS are 20–40 times more likely to contract Pnc disease than those without HIV/AIDS [80]. According to WHO more than 1.6 million people die every year from Pnc infections – primarily pneumonia and meningitis – including more than 800 000 children < 5 years old; 40% of all acute lower respiratory tract infection, and 35% of all meningitis in children is caused by Pnc. For each invasive, potentially deadly Pnc infection, there are from 10- to over 100-fold milder clinical infections caused by Pnc. Pnc disease can be prevented by (a) direct protective effect of the vaccine on vaccinated individuals (both Pnc polysaccharide vaccine, PPV, and Pnc conjugate vaccine, PCV) and/or (b) indirect protective effect via reduced transmission of the pathogen to susceptible, nonvaccinated individuals (PCV only, since the mucosal protection provided by PPV is insignificant). The 23-valent PPV is recommended and used mostly in high-risk group children > 2 years of age since the vaccine is poorly immunogenic in younger

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children [81]. To date, four different types of PCVs have been developed for large-scale clinical trials. They consist of different selection of Pnc serotypes ranging from 7 to 11, and different carrier proteins. All are immunogenic and safe on individual level. So far only the 7-valent PCV with mutant diphtheria toxoid as carrier protein has been licensed (in 76 countries by early 2007), but formally introduced into immunization programs in only 15 countries. The public health impact of the vaccine has been unexpectedly high: in the U.S., where the 7-valent conjugate vaccine has been used in the national program for children since 2000, over two thirds of the impact of the vaccine is obtained via the indirect herd effect, and is seen as a significant reduction in invasive Pnc disease in adults [82, 83]. Recent cost-effectiveness estimations have shown that life years across ages can now be gained at much lower cost [84], compared to earlier estimates [85]. The public health benefit arising from both the direct and indirect effects is further enforced by the reduction of the incidence of vaccine-preventable Pnc strains resistant to antimicrobials [86]. A Phase III trial of a 9-valent Pnc conjugate vaccine in the Gambia unexpectedly showed that overall, allcause mortality in study children was decreased by 16% [87], indicating that Pnc vaccines may eventually become powerful tools with impact on overall global childhood morbidity and mortality. The limiting factor turning countries away from introducing PCV into national childhood programs both in rich and resource-poor countries has been the inhibitive cost of the vaccine. This, coupled with the underestimation of both overall Pnc disease burden and lack of understanding of the potential of the herd impact, has meant that so far (by early 2007) only 16 countries have included Pnc vaccine into routine immunization programs. GAVI currently supports efforts towards the early introduction of Pnc conjugate vaccine in three developing countries: Bangladesh, the Gambia and Kenya [88].

Meningococcal vaccines Neisseria meningitidis, or meningococcus, causes serious bacteremic disease globally. In the so-called meningitis belt of sub-Saharan Africa, large epidemics occur every 5–10 years. Asymptomatic carriage of meningococcus is very common during times when outbreaks occur, while symptomatic disease caused by meningococcus mostly manifests as rapidly advancing meningitis and sepsis with high case fatality rate and approximately 20% of surviving cases developing neurological sequelae. Serotypes and groups (A, B, C, W, Y) causing meningococcal disease vary by geographic location and time. While responsible for most meningococcal disease in sub-Saharan Africa, group A meningococcus has been almost non-existent in Europe and the U.S. for over 50 years. In Europe overall, approximately two thirds of the reported cases have been caused by serogroup B, about one third by serogroup C,

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with a small number of cases caused by serogroups Y, W-135, or A [89]. In several European countries (United Kingdom, Ireland, Spain, Netherlands, Germany) where serogroup C has reached relatively high levels, a new monovalent meningococcal C conjugate vaccine was introduced on a nation-wide scale, targeting young children and teenagers (catch-up vaccination), which rapidly changed the epidemiology of the disease during this decade. Since the licensure of the new 4-valent meningococcal conjugate vaccine in the U.S. in 2005, this vaccine is now recommended for prevention of meningococcal infection in pre-teens, adolescents and high-risk adults. This recommendation is largely based on newer epidemiological data showing a considerable risk of meningococcal disease in late adolescence, most of which is preventable with vaccine [90, 91]. In other high-income countries, the older meningococcal polysaccharide vaccine, composed of capsular polysaccharide, is still recommended to children from 2 to 10 years of age, and to travelers to endemic or epidemic areas. In developing countries struggling with outbreaks and the changing serogroup profile of meningococcus, the polysaccharide vaccine has remained the cheapest alternative, although it does not protect the very young. It is bought in significant amounts annually. An important Meningitis Vaccine Project was launched in 2001 under the auspices of GAVI, WHO, the Gates Foundation and Program for Applied Technology in Health (PATH) to develop a bivalent A and C group conjugate vaccine for the endemic countries with direct African country involvement in the development work. A two-pronged vaccine introduction strategy is envisioned: (1) one-dose mass vaccination campaigns with a group A containing meningococcal conjugate vaccine for 1–30 year olds, and (2) routine infant immunization with one, two or three doses of meningococcal conjugate vaccines integrated with routine EPI schedules. The project includes clinical evaluation (sites, protocols) of meningococcal conjugate vaccines (“MenAfriVac”) as well as licensing strategies, which need to be adapted to both routine and mass vaccination strategies. The Phase I study was carried out in India, i.e., the country of production, and the Phase II studies will start in latter part of year 2006 in Mali and the Gambia. Following licensure, two or more countries will be chosen for initial introduction of conjugate vaccine. Discussions held with the WHO AFRO, African health ministries and other African representatives have highlighted the need to select countries based on specific criteria, for example, burden of meningococcal disease, epidemiological and laboratory capacity, capacity for vaccine delivery, and status of other vaccination efforts (i.e., polio eradication, measles elimination).

Human papillomavirus vaccine Cervical cancer is the leading cause of cancer mortality among women in developing countries. Approximately 500 000 new cervical cancer cases are

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estimated to occur annually, leading to about 250 000 deaths each year [92]. Over 99% of cervical cancer cases are linked to genital infection with HPV, which is the most common viral infection of the reproductive tract worldwide [93] and infects an estimated 660 million people annually. The most prevalent oncogenic HPV strains associated with cervical cancer is HPV type 16, but types 18, 45, 33 and 31 have also been identified. HPV types 16 and 18 account for 65–70% of cervical cancers globally, although the proportion varies in different regions. The burden of disease attributable to HPV infection is, however, not limited to cervical cancer, but includes an even greater proportion of pre-malignant cervical lesions, as well as anal, penile and other reproductive system cancers. Additionally, low-risk HPV types, such as 6 and 11 are responsible for 90% of genital warts or condylomas. While HPV infection resolves spontaneously in the majority of people, it can develop into chronic infection which, in some women and if not treated, may progress to cervical cancer. The peak incidence of HPV infection occurs in adolescents and young women, while cervical cancer typically follows 20–30 years later. The disease represents a major health inequity, as 80% of cervical cancer deaths occur in developing countries [94], where pelvic examination and treatment of pre-cancerous lesions is often not available. Industrialized countries have greatly reduced deaths from cervical cancer through screening programs that allow early detection and treatment. Secondary prevention programs for cervical cancer exist in developing countries, but are mostly under-funded and sub-optimally managed; they have not resulted in the profound reductions in cervical cancer morbidity and mortality observed in the industrialized countries of Europe and North America. The definitive identification of certain types of HPVs as the etiological agents in cervical carcinogenesis led to the rapid development of HPV vaccines [95], and their subsequent testing in human populations with excellent results. To date, sub-unit bivalent (types 16 and 18) and quadrivalent (types 6, 11, 16, and 18) HPV vaccines have been developed and found to be highly immunogenic. They elicit significant humoral and robust cell-mediated immune responses at levels higher than those observed in naturally acquired infections. These vaccines are also highly efficacious in preventing persistent type-specific infections as well as associated cervical cytological abnormalities and pre-cancerous lesions. Because HPV is spread by sexual contact, and the high-risk years for infection are roughly from ages 18 to 25, the best subjects for vaccination are thought to be pre-adolescents or adolescents. The first HPV vaccine licensed in the USA in mid-2006 was a quadrivalent vaccine,15 which has already been recommended for routine use for girls and women aged 11–26 years of age by the U.S. Advisory Committee on Immunization Practices (ACIP). 15 Gardasil® by Merck

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The introduction of HPV vaccine constitutes an effective new strategy to reduce morbidity and mortality from cervical cancer, but will not replace screening and early treatment. Also, while there has been considerable recent progress in vaccine development, the natural history of HPV and cervical cancer and geographic variations in the type-specific prevalence of HPV present unique challenges related to the introduction and acceptance of HPV vaccines. It will not be easy to communicate the public health benefit of preventing a very common, albeit usually harmless, sexually transmitted infection that has only a remote possibility many years in the future of progressing to cervical cancer. The impact of a vaccine, particularly if administered to young adolescents, will not be measurable for decades to come – the amount of time it would take for girls to reach an age when they might otherwise have developed cancer. Socio-cultural issues regarding the vaccination of pre-adolescent and adolescent with HPV vaccine will need to be addressed with great sensitivity. Studies are under way to prepare for HPV vaccine use in developing countries, particularly to find out which sociocultural factors will determine vaccine acceptance and reaching sufficient coverage. Guidelines on HPV vaccine use need to be developed through an integrated approach with adolescent health, reproductive health and cancer control programs at national and international levels.

Other new vaccines under development There are several other new vaccine antigens in different preclinical and clinical phases of development, which, if successful and eventually implemented in national programs, will have a major impact on public health globally. These include vaccines against malaria, HIV, and TB, as well as against dengue fever, schistosoma, different enteric pathogens, Streptococcus A and others. The three most urgently needed vaccines today are vaccines to prevent HIV/AIDS, TB and malaria. Together, these three diseases account for over 5 million deaths worldwide each year, about half of all deaths from infectious diseases. There is no effective vaccine against HIV/AIDS or malaria. The existing widely used TB vaccine (BCG) offers only limited protection against childhood forms of the disease. Safe and cost-effective vaccines against each of these diseases would prevent millions of deaths every year and help countries in their social and economic recovery. They would also help lower the increasing threat of antimicrobial resistance to existing treatments in the worst-affected countries. However, current levels of investment in vaccine research and development do not reflect the magnitude of the threat that these diseases pose to this and future generations. Although HIV/AIDS and TB also occur in the developed countries (albeit at a much lower level) and a malaria vaccine would be useful for the expanding travelers’ market, most of the vaccine sales would be in the developing world. The uncertain demand for new

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vaccines in developing countries has deterred vaccine manufacturers from long-term investment in the development of vaccines against HIV/AIDS, malaria and TB, which remain three of the most scientifically challenging vaccines ever investigated. Several formidable scientific obstacles have so far have prevented these much-needed vaccines reaching licensure and large-scale production. The pathogen may be so variable that it has the potential to escape vaccineinduced protection within a short period of time (malaria, HIV). For other diseases, such as dengue virus, the pathogenic mechanism of the disease or the protective antigenic epitope may not be known to the level of detail needed. The status of development of new vaccines against TB can illustrate the hurdles of new vaccine development in general. The existing BCG vaccine is the most frequently used vaccine worldwide, is low in cost, and protects infants against severe forms of disease, such as TB meningitis and miliary TB. However, the efficacy of BCG against pulmonary forms of disease is variable [96]. Genomic sequencing of Mycobacterium tuberculosis has opened the way towards a more rational approach to screening for antigens with protective capacity against TB. Promising approaches to TB vaccine development include protein subunit vaccines, DNA vaccines expressing protective M. tuberculosis genes, rationally attenuated live M. tuberculosis vaccines and modifications to BCG to boost its immunogenic properties. New live mycobacterial vaccines will benefit from the experience with BCG and BCG production; candidate vaccines are likely to have both good priming and initial protection. Like BCG, they also are expected to provide an adjuvant effect for other vaccines given at the same time. The main issues with new live mycobacterial vaccines relate to quality control and mutant stability. These new vaccines will have to be as safe as BCG, but at the same time significantly more efficacious, which will make it difficult to assess them clinically. The new subunit vaccine candidates, on the other hand, have better stability, are likely to be good for boosting rather than priming, and could be combined with other vaccines. Main concerns for sub-unit vaccines are that repeated use of the same vectors (such as MVA-antigen 85A) may decrease their efficacy, and adjuvants may be needed to obtain the protective effect, which will most likely increase cost. There is also some concern about risk of enhancement of pathology. The most effective future TB vaccination strategy may be to combine different vaccine candidates, using a prime-boost approach, as described in a recent comprehensive review [97] of ‘state of the art’ and future perspectives of TB vaccine development.

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Technical consultation on imbalances in the health workforce (2002) WHO/ EIP/OSD/02.3. World Health Organization, Geneva 54 World Health Organization (2000) Key elements for improving supplementary immunization activities for polio eradication. World Health Organization, Geneva WHO/V&B/00.22 55 World Health Assembly (1988) Global eradication of poliomyelitis by the year 2000. World Health Organization, Geneva (Resolution WHA41.28) 56 Dowdle WR, Cochi SL (2002) Global eradication of poliovirus: history and rationale. In: BL Semler, E Wimmer (eds): Molecular biology of picornaviruses. ASM Press, Washington, DC 57 World Health Organization (2001) Transmission of wild poliovirus type 2: apparent global interruption. Wkly Epidemiol Rec 76: 95–97 58 Kew OM, Sutter RW, de Gourville EM, Dowdle WR, Pallansch MA (2005) Vaccine-derived polioviruses and the endgame strategy for global polio eradication. Annu Rev Microbiol 59: 587–635 59 Dowdle WR, Hopkins DR (1998) The Eradication of Infectious Diseases: Dahlem Workshop Report. John Wiley & Sons, Chichester 60 De Quadros CA, Olive JM, Hersh BS (1996) Measles elimination in the Americas: evolving strategies. JAMA 275: 224–229 61 Expanded Programme on Immunisation (2006) Progress in reducing global measles deaths, 1999–2004. Wkly Epidemiol Rec 81: 90–94 61a Wolfson LJ, Strebel PM, Gacic-Dobo M, Hoekstra EJ, McFarland JW, Hersh BS (2007) Has the 2005 measles mortality reduction goal been achieved? A natural history modelling study. Lancet 369: 165–166 62 WHO and UNICEF (2005) WHO/UNICEF Joint Statement: Global plan for reducing measles mortality 2006–2010. Geneva (WHO/IVB/05.11) 63 PAHO (2004) XVI Meeting of the Technical Advisory Group on Vaccine Preventable Diseases, Mexico City. 3–5 November 2004. Final Report. Accessed June 30 at http://www.paho.org/English/AD/FCH/IM/TAG16_FinalReport_ 2004.pdf 64 World Health Organization (1999) Progress towards the global elimination of neonatal tetanus, 1990–1998. Wkly Epidemiol Rec 74: 73–80 65 Vandelaer J, Birmingham M, Gasse F, Kurian M, Shaw C, Garnier S (2003) Tetanus in developing countries: an update on the maternal and neonatal tetanus elimination initiative. Vaccine 21: 3442–3445 66 Adegbola RA, Secka O, Lahai G (2005) Elimination of Haemophilus influenzae type b (Hib) disease from the Gambia after the introduction of routine immunization with a Hib conjugate vaccine: a prospective study. Lancet 366: 144–150 67 Steinhoff MC (1997) Haemophilus influenzae type b infections are preventable everywhere. Lancet 349: 1186–1187 68 de Andrade AL, de Andrade JG, Martelli CM, e Silva SA, de Oliveira RM, Costa MS, Laval CB, Ribeiro LH, Di Fabio JL (2004) Effectiveness of Haemophilus influenzae b conjugate vaccine on childhood pneumonia: a casecontrol study in Brazil. Int J Epidemiol 33: 173–178 69 Gessner BD, Sutanto A, Linehan M (2005). Incidences of vaccine-preventable

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Dagan R (2004) The potential of pneumococcal conjugate vaccines to reduce antibiotic resistance. Adv Expe Med Biol 549: 211–219 Cutts FT, Zaman SM, Enwere G (2005) Efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in The Gambia: randomised, double-blind, placebo-controlled trial. Lancet 365: 1139–1146 Ray GT, Whitney CG, Fireman BH, Ciuryla V, Black SB (2006) Pneumococcal vaccination in developing countries. Lancet 367: 1880–1882 Schmitt HJ, Booy R, Weil-Olivier C, Van Damme P, Cohen R, Peltola H (2003) Child vaccination policies in Europe: a report from the Summits of Independent European Vaccination Experts. Lancet Infect Dis 3: 103–108 Baltimore RS (2006) Recent trends in meningococcal epidemiology and current vaccine recommendations. Curr Opin Pediatr 18: 58–63 Centers for Disease Control and Prevention (CDC) (2005) Prevention and control of meningococcal disease. Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 54 (RR-7): 1–21 World Health Organization (2005) World Health Report 2004. WHO, Geneva Baseman JG, Koutsky LA (2005) The epidemiology of human papillomavirus infections. J Clin Virol 32S: S16–S24 Parikh S, Brennan P, Boffetta P (2003) Meta-analysis of social inequality and the risk of cervical cancer. Int J Cancer 105: 687–691 Jansen KU, Shaw AR (2004) Human papillomavirus vaccines and prevention of cervical cancer. Annu Rev Med 55: 19–31 Fine PE (1995) Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346: 1339–1345 Kaufmann SH (2006) Envisioning future strategies for vaccination against tuberculosis. Nat Rev Immunol 6: 699–704

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Potential impact of rotavirus vaccination on the mortality of children in developing countries Duncan Steele Initiative for Vaccine Research, Department of Immunization, Vaccines and Biologicals, World Health Organization, Geneva, Switzerland

Abstract The global burden of rotavirus infection and associated mortality in infants and young children has led to the international prioritization of the development of a rotavirus vaccine. In recent months, two new rotavirus vaccines have been licensed by the multinational pharmaceutical industry and are currently being introduced into routine childhood immunization schedules in the Americas and Europe. However, for the full impact of these rotavirus vaccines to be felt they need to be introduced into Africa and Asia where the bulk of rotavirus associated mortality occurs. Several questions regarding the efficacy of the vaccines in these settings remain, as well as questions of supply and pricing of the vaccines.

Introduction Diarrheal diseases cause approximately two million deaths in infants and young children in developing countries every year, constituting ~18% of all childhood deaths [1, 2]. The most recent review of diarrheal mortality indicates that, although global mortality due to diarrheal disease has declined dramatically over the two past decades, the annual incidence of diarrheal episodes per child in developing countries has remained high [2]. Of the numerous microbial causes of diarrheal illness in young children, one agent is associated to a disproportionate degree with the observed morbidity and mortality in this vulnerable population. Rotavirus is recognized as the major enteric pathogen associated with this high burden of disease and mortality in infants and young children in developing countries. The development of a safe and effective rotavirus vaccine is considered a priority by the international community such as the World Health Organization (WHO) and the Global Alliance for Vaccines and Immunization (GAVI). Rotavirus vaccines have been identified as potentially having a significant impact on reducing childhood mortality and contributing to achieving Goal 4 (i.e., reducing childhood mortality) of the Millennium

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Development Goals [3]. The review presented here focuses specifically on the potential of rotavirus vaccines for reducing childhood mortality.

Burden of rotavirus disease Rotavirus is the single most important enteric pathogen associated with high mortality in infants and young children in developing countries, and is associated with more than an estimated 600 000 deaths annually [4, 5]. Thus, new estimates indicate that rotavirus is responsible for ~5% of total childhood mortality [5, 6]. The reported incidence rates of rotavirus infection do not vary significantly between industrialized countries and the developing countries of Africa and Asia, indicating that socio-economic improvements in water and sanitation may not reduce rotavirus diarrhea [7]. Nevertheless, the inequity in healthcare means that the vast majority of both diarrheal and rotavirus deaths are in children in the poorest countries of the world [1, 8]. In these poor countries, about 1 child in 200 will die of rotavirus disease [6]. This has prompted the international prioritization of rotavirus vaccines as a primary strategy for the reduction of the mortality associated with this infection. General improvements in the overall severity, management and outcome of diarrheal diseases, due to such global interventions as oral rehydration therapy (ORT) and the Integrated Management of Childhood Illnesses (IMCI), have been observed. However, the effect of these improvements on rotavirus infection, per se, has not been significant, indicating that the successful interventions against the bacterial and parasitic microbes causing diarrheal illness, may have a much less dramatic effect on rotavirus infection. In fact, as estimates of the mortality due to diarrheal diseases decline globally, the proportion of hospitalizations due to severe rotavirus infection has increased, and this is taken as a likely marker for the severity of the diarrheal episode and the potential risk of death due to the infection or complications of the infection. The latest reviews of rotavirus infection from various regions, including Africa, Asia and Latin America as well as Europe indicate that rotavirus is associated with 25–60% of all hospital admissions for diarrheal diseases [9–13]. Rotavirus-associated illness has been estimated to result in approximately 25 million clinic or emergency room visits and about 2 million hospitalizations in children less than 5 years of age annually [8]. This tremendous burden of disease and associated mortality is concentrated in the developing countries of Africa and Asia, where over 82% of the rotavirus-associated mortality occurs [8]. Thus, for rotavirus vaccines to truly impact on childhood mortality, the vaccines need to be introduced into these regions and countries. The current status of rotavirus vaccine development globally has never been more promising. In early 2006, two live oral attenuated rotavirus vac-

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cines completed large-scale safety and efficacy evaluation in Latin America, Europe and the USA [14, 15]. These vaccines have been licensed by the multinational pharmaceutical industry in the USA (RotaTeq®, Merck & Co., Inc., Pennsylvania) and in Europe (Rotarix®, GSK Biologicals, Belgium), which are important as these represent the countries of manufacture, and in many other individual countries. In addition, these licensed vaccines are already being utilized in routine childhood immunization in some countries in the Americas. Finally, several other candidate rotavirus vaccines are under clinical development in partnership with various emerging vaccine producers in developing countries. The impact of introducing these two rotavirus vaccines in Europe and the USA and in countries in Latin America should contribute significantly to help reduce both the high numbers of hospitalizations and the costs associated with this, as well as reduce the limited rotavirus-associated deaths in these regions [16, 17]. Nevertheless, the true effectiveness of rotavirus vaccines to impact on diarrheal disease mortality in infants and young children in developing countries still needs to be ascertained.

Epidemiology of rotavirus in young children Rotaviruses are ubiquitous in nature, infecting virtually all young children by the second or third year of life resulting in the high burden of disease and morbidity in both developed and developing countries [7, 18]. However, it is clear that differences exist in the epidemiology and the distribution of rotavirus strains between developing countries and industrialized countries. Following a WHO recommendation for specific standardized studies in Africa and Asia on the epidemiology of rotavirus and strain surveillance [19], a more systematic investigation was utilized to examine rotavirus infection in developing countries. Regional rotavirus networks have been established and have contributed significantly to our current knowledge [10, 20]. Differences in the epidemiology of rotavirus infection between developing countries and industrialized countries do exist, and may have some consequences for vaccine strategies (Tab. 1). First, the primary rotavirus infection, which is usually the most serious episode, tends to occur earlier in infants in these regions. For instance, in many countries in Africa and Asia, almost three-quarters of infants will acquire their primary rotavirus infection before their first birthday [9, 10, 20–24]. Typically, symptomatic rotavirus infection occurs most frequently in children between 3 and about 18 months of age, resulting in mild to severe acute watery diarrhea with a subsequent loss of fluids and electrolytes [18, 25]. Neonatal infections are generally asymptomatic, perhaps due to protection conferred by passively acquired maternal antibodies or an immature intestinal epithelial system [26, 27], or by viral characteristics [28]. Finally, re-infections in older children and adults are common but tend to be sub-

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Table 1. Implications for rotavirus vaccine defined by the differences in the epidemiology of rotavirus infection between developing countries and developed countries (adapted from [7]) Epidemiology Age of first infection - Percent infected by 12 months - Median age of infection

Developed country

Developing country

Implications for vaccine trials

40%

75%

Vaccine must be given earlier.

12–18 month 6–12 months Potential interference of maternal antibody with vaccine take.

Seasonality

Winter peak All year round

Year round exposure to infants, earlier age of acquisition. Need for earlier vaccination.

Case fatality

Low

High

Mixed infection with other enteropathogens

Uncommon

Common

Outcomes and measurements in vaccine trial design.

Multiple virus serotypes

One major type circulating

More than one type circulating

May limit vaccine take, and affect diarrhea outcomes of trial design. Necessitate additional vaccine doses. Vaccine efficacy may be challenged in trial design. Vaccine candidates may need different formulations

clinical and probably reflect the natural immunity offered by the primary infection. Secondly, rotavirus infections exhibit a seasonal pattern in temperate countries, where most rotavirus infections occur during the winter [18]. However, in tropical countries and in most developing countries, rotavirus tends to occur year round, although with some increased activity during the cooler months of the year [9, 10, 20–24, 29]. Thirdly, many of the children in developing countries have additional factors, such as malnutrition, concomitant infections and co-morbidity, and potentially multiple enteric pathogens, which may all exacerbate the subsequent disease consequences with rotavirus infection.

What have natural history studies shown? Natural rotavirus infection has been shown to be highly protective against subsequent infection associated with disease, although not against reinfection. Early studies showed that neonatal rotavirus infection, although asymptomatic, conferred protection against subsequent severe rotavirus diarrhea, although re-infection was common [26, 30]. Furthermore, longitudinal natural history studies following infants from birth to approximately 2 years of age in Mexico and Guinea Bissau, have

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confirmed that a primary rotavirus infection, whether associated with symptomatic or asymptomatic infection, confers significant protection against disease associated with subsequent re-infection [22, 31]. In Mexican infants, the primary rotavirus infection conferred 77% protection against all symptomatic rotavirus infection and 87% against serious rotavirus diarrhea [31]. This phenomenon has been seen in several other studies and stimulated the concept that a live, orally administered rotavirus vaccine would confer protective efficacy against severe rotavirus disease.

Characteristics of rotavirus important for vaccine development The two outer capsid structural proteins of the rotavirus virion, VP7 and VP4, elicit the production of distinct neutralizing antibodies in the host and thus are considered important in vaccine development. The VP7 and VP4 also determine the serotype of the virus strain by the specificity of this antigen to elicit neutralizing antibody response in the host. The genes encoding these two proteins segregate separately, and this has led to a binomial system of classification for the VP7 glycoprotein (G types) and the protease-sensitive VP4 (P types) [32]. However, the importance of the neutralizing antibody response in vaccine development is less clear and will be discussed below. It is assumed that the neutralizing antibody response in the serum reflects the “vaccine take” and the magnitude of the response and the specificity of the immune response to the virus.

What is the role of the VP7 G types? The outer capsid layer of the virion is a glycoprotein and constitutes the major neutralization antigen of the viral particle. Early studies showed that VP7 elicited an immune response in the host [33] and studies with hyperimmune sera could distinguish rotavirus serotypes [34]. However, genotyping methods, using a multiplex, nested PCR assay to type the VP7 gene have become convenient and popular for typing of the VP7 characteristics of the strain [35–37]. The VP7-based genotype and the neutralizing antibody-based serotype systems of analysis and characterization have been compared and correlate completely [32]. Although at least ten VP7 G types are recognized among the human rotaviruses, five are identified globally to be common (G1–G4, and G9) [35–38]. Some of the other G types are found to be important regionally, e.g., G5 strains were detected in Brazil [37, 39, 40], G8 strains are prevalent in Africa [41–43] and G10 strains in India [44]. As the serotype distribution is believed to be important epidemiologically, and to have potentially vaccine-related efficacy, strain diversity and surveillance studies are a prime research tool for ongoing studies [36, 38].

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What is the role of the VP4 P types? The VP4 is a non-glycosylated protein of the outer capsid and has been identified to have several important functions, which include being the viral receptor, and having hemagglutinin, neutralization and virulence characteristics [32]. Both VP4 and VP7 proteins act as antigens in neutralizing immune responses and contribute to the diverse antigenic complexities of rotaviruses [33, 45]. The diversity and distribution of VP4 have also been investigated widely in molecular epidemiology studies, but these have centered on genotyping of the VP4 gene due to difficulties of generating immunological reagents for VP4 serotyping. Identification of the VP4 types thus has both a serotype (indicated by a number when known) and the genotype is indicated in square brackets, e.g., P1A[8] [32]. To date, studies have identified more than 20 types in nature [18, 32, 46], although only three occur commonly in human rotaviruses globally [36, 38]. The most common types are designated as P1A[8], P1B[4] and P2[6], although some strains occur sporadically, e.g. P[9] and P[14] [36, 37]. Once again, regionally some other VP4 types have been found to occur more commonly. For instance, in India, P[11] strains have been identified commonly [44], while in Africa, the P[6] strains occur commonly [10].

Will genetic diversity influence vaccine efficacy? Potentially, the number of reassortant strains, with variations of the VP7 and VP4 genes that could occur in nature is enormous, but fortunately this seems to be generally biologically restricted and there is a relationship between the VP7 G type and the VP4 P types that co-segregate. Therefore, five human rotavirus strains occur commonly: P[8]G1, P[4]G2, P[8]G3, P[8]G4 and P[8]G9, although some variations are observed. Comprehensive analyses of the numerous rotavirus molecular epidemiology and genetic diversity studies have revealed several important observations. First, when the global distribution of rotavirus strains is examined, there is a definite difference in the P-G combinations of strains in different geographic regions. For instance, the four most common rotavirus strains (P[8]G1, P[8]G3, P[8]G4 and P[4]G2) represented more than 90% of the strains in North America and Europe, but only 68% of the strains in Latin America and Asia and only 50% of the strains in Africa [10, 37]. Secondly, the distribution of strains with unusual P-G combinations was highest in Africa, followed by Asia and Latin America, highlighting the complexity of the molecular epidemiology of rotavirus strains in developing countries [37, 38]. Finally, mixed rotavirus infections with different strains occur relatively commonly in developing countries strains (10–15%) and this can result in naturally occurring reassortant strains with multiple unusual P-G configurations [35, 37, 38, 47].

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Thus, there is a complex array and diversity of rotavirus strains circulating in developing countries. The current vaccine strains – licensed or in development – have not been effectively evaluated in this context, despite the recommendation from WHO to do so [19]. Clinical trial data indicate that the licensed vaccines may protect against rotavirus strains that are not included in the vaccine (discussed below), although this question remains to be addressed.

Pathogenesis and clinical presentation Rotavirus particles were first visualized in humans by thin-section electron microscopy (EM) of the duodenal mucosa of an infant with acute watery diarrheal illness [48]. The distinctive viral particles were soon identified in the feces of infants and young children with gastroenteritis worldwide [49]. Rotaviruses were found to be an important etiological agent of acute infantile diarrhea, and were soon recognized as the most important of the known etiological agents of severe diarrheal illness in infants and young children [18]. Rotavirus particles are shed in large numbers in the feces during the acute infection and are transmitted by the fecal-oral route. The viral particles are relatively stable in the environment [50], which exacerbates the rapid and efficient transmission of the infection. Speculation on the respiratory transmission of rotaviruses, due to the seasonality and rapid transmission of the infection [29], has not been substantiated by clinical or laboratory studies, although rotavirus has occasionally been recovered from the respiratory tract [51, 52]. Nevertheless, aerosol droplet and person-to-person spread does seem to be a primary mode of transmission. Rotavirus infection has a short incubation period of between 1 and 3 days. The disease is characterized by the sudden onset of acute watery diarrhea, often accompanied by fever and vomiting [53, 54]. Although most rotavirus infections are relatively mild, approximately 1 in every 5 children will develop symptoms and dehydration severe enough to warrant a visit to a medical facility, and as many as 1 in 65 will be admitted to hospital and approximately 1 in every 293 children will die of rotavirus infection [8]. Rotavirus infection is often accompanied by serious fluid and electrolyte loss with dehydration, especially in small infants, which is related to severe damage to the intestinal epithelial cells [55]. Typically, the acute infection lasts for 3–5 days with diarrhea and fever, and vomiting is a predominant early symptom of rotavirus infection, which may undermine the effectiveness of ORT.

Immunity against rotavirus infection The actual immunological mechanism by which protection against rotavirus disease occurs is unknown, whether after natural infection [31] or

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after immunization [56]. Rotavirus infection does result in both serum and intestinal antibody and in general does protect against severe diarrheal illness upon subsequent infection [22, 26, 30, 31]. Although the role of intestinal neutralizing antibody is generally accepted to play an important role in protection against disease, consistent results have been difficult to obtain, possibly due to difficulties in experimental design and the use of different animal models. However, questions do remain on what the actual mechanism(s) of protective immunity are and whether serum antibody, which is measured in all the vaccine trials, is indicative of clinical protection.

What is the role of serum antibody in protection? Confounding results of the role of serum antibody have been identified in studies examining the natural history of rotavirus infection [31, 57, 58], vaccine trials [56, 59] and adult challenge studies (summarized in [60]). In brief, most studies have indicated that the presence of serum antibodies serves as a good surrogate marker for protection [60], although it is believed that other effector mechanisms of the immune response are also important [57, 60]. However, whether the serum antibodies are an active component of the protective immune response or just a correlate of protection is difficult to assess from these studies. For instance, natural rotavirus infection in young children shows differences in different settings. In Danish children, IgA correlated with protection against rotavirus illness, but IgG did not [61], whereas in Bangladeshi infants, IgG was reported to correlate with protection against subsequent symptomatic infection [62], and in some other studies both immunoglobulins were shown to correlate with protection. Furthermore, in vaccine trials in young children, a significant degree of protection was usually observed when serum antibodies were present, although this correlated better with overall immune response and not serotype-specific neutralizing antibody [59, 60, 63, 64]. Clinical trials utilizing monovalent rotavirus vaccine strains indicated that serotype-specific neutralizing antibody responses against the circulating rotavirus strains might be important in protection (reviewed in [56]). Thus, evidence that neutralizing antibody is important in protection is based on empirical data from early vaccine clinical trials. Nevertheless, in other vaccine trials in children, protection did not always correlate with neutralizing antibody [59, 63, 64]. However, several studies in human populations have indicated that neutralizing antibodies are not the major mechanism in protection. For instance, in separate neonatal studies, the serotype of the endemic neonatal strain that infected the newborn babies was able to confer protection against other

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circulating serotypes in those same infants [26]. It is also clearly recognized that in older infants there is usually a severe primary rotavirus infection that in most cases confers protection against subsequent severe rotavirus disease upon re-infection even with different serotypes [22, 31]. Occasionally, when re-infection is associated with symptomatic disease, the infecting strain is usually of a different VP7 serotype, which may indicate that serotype-specific neutralizing antibody does have a role to play. Studies investigating the T cell responses in humans have been limited. However, lymphoproliferative assays have indicated that children did develop measurable levels of circulating rotavirus-specific T cells after a primary infection [65]. Furthermore, recent studies have shown that virusspecific CD4+ and CD8+ T cells secreting interferon-a, were elevated after rotavirus infection [66, 67].

Different approaches to vaccine development Two philosophical approaches have been utilized for the development of live oral rotavirus vaccines, based essentially on the putative role of serum neutralizing antibody in the role of protection. On the one hand, the concept of the need for serotype-specific neutralizing antibody for protection has resulted in the approach of the multivalent reassortant rotavirus vaccines, such as the rhesus quadrivalent [64] or the bovine pentavalent strains [68, 69]. Thus, although monovalent rhesus (G3) and monovalent bovine strains (RIT, WC3 and UK, which are all G6) have been tested in clinical settings, with different results, reassortant vaccines covering the four common human rotavirus VP7 serotypes have been developed as discussed below. An alternative approach is based on the premise that natural rotavirus infection generates a broad protective response against re-infection and that protective efficacy is generated by alternative immune responses in addition to the neutralizing antibody response [67].

Rotavirus vaccine development Rotavirus vaccine development was initiated relatively soon after the discovery of the virus due to the early recognition of the burden of disease and mortality in infants and young children universally. Within 10 years, rotavirus vaccine trials were being prepared utilizing a live attenuated oral vaccine approach based on several observations, including (i) that primary natural infection led to protection against severe disease upon re-infection [26, 70], (ii) the antigenic relatedness of animal and human rotaviruses [34, 71], and (iii) early animal studies that indicated that protection against rotavirus disease was mediated primarily by intestinal immunity [72].

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Table 2. Live oral rotavirus vaccine candidates which are currently licensed or in clinical development Vaccine strain

Type of vaccine

Company/ developer

Status

Inventor

Licensed vaccines RotaShield®

Quadrivalent reassortant rhesus rotavirus strain with human rotavirus VP7 genes for G1-G4

Wyeth Ayerst (USA)

Licensed in USA (1998)

AZ Kapikian, NIH

LLR

Monovalent lamb rotavirus

Lanzhou Institute (China)

Licensed in China (2000)

ZS Bai, Lanzhou Institute

RotaTeq®

Pentavalent reassortant bovine strain with human rotavirus VP7 and VP4 genes to G1–G4 and P[8]

Merck & Co., Inc. (USA)

Licensed in USA (2006)

HF Clark, Wistar Institute

Rotarix®

Monovalent human rotavirus strain G1P[8]

GlaxoSmith Kline (Belgium)

Licensed in Mexico and in Europe (2006)

R Ward, Gamble Institute

Clinical development UK

Multivalent reassortant bovine strain with human rotavirus genes

NIH with vaccine producers in Brazil, China and India

Phase 2 data available

AZ Kapikian, NIH

RV3

Monovalent human neonatal rotavirus strain

University of Melbourne with BioFarma, Indonesia

Phase 2 data available. High titer strains

RF Bishop, University of Melbourne

Indian/USA consortium

Phase 1 data available

MK Bhan, RI Glass, HB Greenberg, CD Rao et al.

116E and I321 Monovalent humanbovine reassortant strains Adapted from [6]

Although there are several licensed rotavirus vaccines and several under development (Tab. 2), the discussion in this review focuses on the two vaccines that have been developed by the multinational pharmaceutical industry and which are closest to be able to impact the global mortality due to rotavirus infection. These two vaccines have been evaluated for efficacy and safety in large clinical trials [14, 15], and licensed internationally by the FDA and/or the European Agency (EMEA).

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Reassortant rotavirus vaccines using animal rotavirus strains The protection observed by the primary rotavirus infection and the antigenic relatedness of animal and human rotaviruses, stimulated the “Jennerian” approach to rotavirus vaccination, which relied on immunization with animal rotavirus or animal-human reassortant rotavirus strains [56, 73]. Thus, attenuated animal rotavirus strains produced the first rotavirus vaccine candidates (Tab. 2) and continue to be a major source of the rotavirus vaccine development currently. The early rotavirus vaccine candidates included bovine strains (RIT4237 and WC3) and the monovalent parent rhesus rotavirus (MMU18006). After variable results with the monovalent rhesus G3 strain [74–76] and with the monovalent bovine strains, which carried a G6 serotype specificity that is not found in human strains [77–79], the approach shifted to developing reassortant vaccine strains [56, 59]. The concept of the Jennerian approach – that animal rotaviruses were attenuated for human disease – was modified to generate reassortant vaccine strains carrying the VP7 gene of one of the four most common human rotavirus VP7 strains (G1–G4) on the genetic background of the animal rotavirus strains. These reassortant vaccine candidates were developed to yield multiple strains that would offer a multivalent serotype exposure upon immunization, but which kept the attenuated nature of the parent strain [56, 59]. This approach yielded the tetravalent reassortant rhesus rotavirus vaccine candidate, which was licensed as RotaShield®.

Rhesus-human reassortant rotavirus vaccine The quadrivalent rhesus-human reassortant rotavirus vaccine is based on the rhesus rotavirus (RRV) strain, which shares G3 specificity with human rotaviruses. Three reassortant rhesus strains with the VP7 gene from human rotaviruses for G1 (human strain D), G2 (strain DS-1) and G4 (strain ST3), respectively, were created [80]. The vaccine candidate consists of a pool of these reassortant strains with the parent strain RRV, but early studies showed each reassortant strain to be similar to the parent RRV strain in infants with regard to safety, reactogenicity, shedding and immunogenicity [56]. These studies also showed that protective efficacy was associated with the serological response as measured by the serum IgA response [56, 81]. A series of efficacy trials were conducted in different populations and at different vaccine concentrations (104 pfu and 105 pfu per dose) and in general showed consistent protective efficacy against all rotavirus diarrhea (50–60%) and against severe rotavirus disease (70–100%) (reviewed in [56]). The pivotal phase III efficacy trials, using three doses of the vaccine at 4 × 105 pfu per dose, showed protection against any rotavirus diarrhea of between 50–60% and against severe rotavirus diarrhea requiring hospitalizations or rehydration of 70–100% [82–84]. The protective efficacy was

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exhibited against different circulating VP7 serotypes and was evident over two to three rotavirus seasons. On the basis of these results, RotaShield®, was licensed in the United States by Wyeth Ayerst in 1998 and quickly implemented into the routine immunization schedule for USA infants [85]. However, within 9 months and after over half a million US infants had received the vaccine, there was a reported association of the vaccine with intussusception [86]. Although RotaShield® was licensed in the USA, the vaccine is no longer produced and has not been evaluated in clinical trials in children in developing countries. Questions remain whether the vaccine should have finished clinical evaluation in the developing world due to the high risk-benefit of a rotavirus vaccine where mortality is high due to rotavirus disease [87, 88]. The debate about the actual risk of the RotaShield® vaccine with intussusception continues [88, 89]; however, the vaccine was withdrawn by the manufacturer in October 1999, and the recommendation for its use was withdrawn by the Advisory Committee for Immunization Practices (ACIP). It remains unavailable today. The major safety concern currently is whether the new rotavirus vaccines will have the same association with intussusception, and it is likely that this can only be addressed in large post-marketing surveillance studies once the vaccines are introduced. This has been specifically requested by the WHO and will be specifically pertinent to all future rotavirus vaccines [90].

Reassortant WC3 bovine-human rotavirus vaccine WC3 is a bovine rotavirus, bearing a G6P7[5] serotype, which is not found among human rotaviruses. The vaccine development is well described in earlier reviews [59, 69] and shows the safety and immunogenicity of the quadrivalent vaccine candidate [91, 92] and the final pentavalent reassortant vaccine with the reassortant strains containing the G1–G4 and P1A[8] human rotavirus genes [68, 69]. The parent strain, WC3, was consistently found to be safe and immunogenic in early studies, with neutralizing antibody responses in 71–97%, although the immune response was specific to bovine rotavirus [59, 93]. Various reassortant combinations with human rotavirus genes for serotypes G1–G4 and/or P1A[8] on the bovine WC3 background have been generated [59, 68]. A series of clinical trials utilizing the monovalent WC3 reassortant strains with human rotavirus G1 or G2 specificity illustrated the safety and immunogenicity of the vaccine components, and also illustrated that the immune response to the bovine rotavirus VP4 was significant and should be included in future vaccine candidates [94]. The pentavalent WC3 reassortant rotavirus vaccine candidate, which consists of reassortant strains with each of the human rotavirus genes G1–G4 and P[8], was recently licensed as RotaTeq® by Merck & Co., Inc.,

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based a series of clinical trials that are described elsewhere [68, 69]. The pivotal safety and efficacy study was also recently reported [15]. The vaccine showed 74% protection against any rotavirus-associated diarrhea and 98% efficacy against severe rotavirus disease and was protective against all four human rotavirus strains (G1–G4) included in the vaccine and G9 strains which are not in the vaccine, but which share the VP4 P[8] genotype [15]. This study was also designed to examine any potential risk of association with intussusception and so enrolled over 70 000 infants in 11 countries in the US, Europe and Latin America. The infants were 6–12 weeks of age and received three doses of either the pentavalent vaccine or placebo in a blinded, randomized fashion. Active surveillance for cases of intussusception was conducted with adjudication by an independent safety monitoring board. Overall, 27 cases of intussusception were identified during a full year’s follow-up of each subject, although these were evenly distributed between vaccine (12) and placebo groups (15). Only two cases were identified in the 14-day window after any dose and these were evenly split [15, 68]. Thus, this vaccine is licensed in the USA and has been recommend for use in universal immunization of American infants by the ACIP.

Monovalent lamb rotavirus (LLR) A monovalent lamb rotavirus strain (G10P[12]) was isolated in primary calf kidney cells in China in 1985 and has been developed as a vaccine after multiple passaging [19]. The vaccine strain was developed at the Lanzhou Institute for Biological Products, and has been evaluated in clinical trials in China, showing a serum neutralizing response in 61% of vaccinees [19]. The trials were conducted in slightly older children and the immune responses resemble a “booster” response in these children, as it exhibits a similar elevation in titer of neutralizing antibody to all G1–G4 strains. This vaccine was licensed for use in China in 2000 and has been utilized in the private market since then [Zhi Sheng Bai (inventor), personal communication].

Reassortant UK bovine-human vaccine A second bovine-human reassortant vaccine is based on the bovine strain UK (also G6P7), and contains the human rotavirus genes for serotype G1– G4 and P1A[8] reassorted onto the bovine rotavirus UK background [95]. The individual components of the vaccine were shown to be safe and immunogenic following two doses, as indicated by the presence of serum IgA [96]. Subsequently, the quadrivalent VP7-specific vaccine was administered in three doses at 105 plaque-forming units (pfu) to infants with concomitant childhood immunizations [97]. There was no adverse reaction with the other

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concomitant vaccines and 95% of the infants developed neutralizing antibody responses to the vaccine strain. Several vaccine producers in Brazil, China and India are intending to license-in the UK vaccine strains and produce them locally on site. A full clinical development program will be required and efficacy trials with the vaccine candidate are planned in developing countries where the vaccines are to be produced. Although this development will take a number of years, the eventual capacity to produce supplies of vaccine and the likely prices of these vaccines should benefit the global market for rotavirus vaccines and particularly their introduction into other developing countries.

Monovalent human rotavirus vaccine strains The concept of a monovalent human vaccine strain is predicated on the premises that (i) natural infection confers protection against subsequent disease [22, 26, 30, 31], and (ii) that neutralizing antibody is not the only immune effector of protection and that other immune factors do play a role in clinical protection [67].

Attenuated human rotavirus strain (89-12) A naturally circulating human rotavirus strain associated with diarrheal disease was identified to confer natural immunity to subsequent rotavirus infection in infants and young children [58]. The strain (89-12), which was recovered from the stools of a 15-month-old toddler with rotavirus diarrhea, was shown to be protective against rotavirus disease in the following season. [58]. The rotavirus strain is G1P1A[8], which is the most predominant human rotavirus strain circulating globally and constitutes about 55% of all human rotaviruses [38]. The strain 89-12 was adapted to tissue culture and serially passaged to attenuate the strain as a vaccine candidate [98]. A clinical trial of the attenuated 89-12 vaccine strain was seen to offer 89% protective efficacy against any rotavirus disease and 100% against severe rotavirus infection in the subsequent season [99], and this protection was shown to extend over at least 2 years [100]. Initial trials demonstrated that the vaccine strain was safe and immunogenic and that after two doses, nearly every child (94%) developed an immune response. The parent strain has been further developed by GlaxoSmithKline Biologicals who further attenuated the strain by passage in tissue culture, before cloning and purifying the end product (now designated strain RIX4414) [101]. This vaccine strain has been evaluated in several immunogenicity and efficacy studies globally including in Finland [102], Latin America [103] and Singapore [104]. The vaccine was first licensed in Mexico in 2004, based on clinical efficacy data generated in a phase III efficacy trial

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in Brazil, Mexico and Venezuela, where 1986 infants were vaccinated at 2 and 4 months with different vaccine concentrations at approximately 104, 105 and 106 ffu [103]. Immunogenicity was detected in 60–65% of the infants and the vaccine conferred protection of 68–87% against severe rotavirus infection and 61–92% against rotavirus hospitalizations [103]. The safety and efficacy study with this vaccine recruited over 60 000 infants in 11 Latin American countries and in Finland, who received two doses of the vaccine at 2 and 4 months of age in a randomized, double-blind, placebo-controlled study [14, 101]. The efficacy of the vaccine was shown to be 85% clinical protection against both rotavirus-associated hospitalization and against severe rotavirus gastroenteritis. The VP7-type specific efficacy was 91% against wildtype G1P[8] strains (homologous to the vaccine), and was 87% against strains bearing only the P[8] antigen (strains G3P[8], G4P[8] and G9P[8]) [14]. Only 14 wild-type strains with a G2P[4] specificity were detected; these strains are of special interest as neither antigen is included in the vaccine. In the safety cohort, 25 cases of intussusception were reported by active surveillance and hospital record capture methods – 13 cases occurred within 31 days post-administration of any dose with 6 cases in the vaccine group and 7 in the placebo group. The remaining 12 cases occurred after 31 days from administration and up to a year’s follow-up, and were detected in the vaccine group (3) and the placebo group (9), indicating that there was no increased risk of intussusception [14]. Following the safety and efficacy clinical data that was generated in a large phase III study in Latin America and Finland [14], the vaccine was licensed by the EMEA in 2006. This licensure is significant because it represents the licensure within the “country of manufacture“ and is significant for the international community for possible future procurement. In essence, the vaccine dossier has now been submitted to the WHO for the process of pre-qualification, which would enable developing countries to apply for procurement of the vaccine by the GAVI. This is a crucial step towards introducing the vaccine in some of the poorest countries of the world and where rotavirus mortality is high [5, 8]. Following the recommendation by WHO for the parallel evaluation of new rotavirus vaccines in developing countries in Africa and Asia [19], clinical trials examining specific issues for infants in developing countries (such as the potential interaction of the vaccine with oral poliovirus vaccine (OPV), dose-ranging studies and immunogenicity trials) have been completed in South Africa and Bangladesh [105–107]. Efficacy studies with this vaccine are ongoing in Africa and the results should be available in 2008.

Neonatal human rotavirus strain (RV3) Neonatal rotavirus infection in Melbourne, Australia was reported to confer clinical protection against subsequent rotavirus disease in infants [26]. This

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study and other longitudinal surveillance studies [22, 31, 58, 70] indicated that natural infection with a single wild-type rotavirus invariably conferred protection against moderate to severe rotavirus disease upon re-infection. The naturally attenuated neonatal rotavirus strain identified in this study (RV3) was developed as a vaccine candidate due to these observations. The vaccine candidate was shown to be safe and well tolerated in phase I trials in adults, children and infants [108]. A phase II trial administering three doses of vaccine at 105 ffu at 3, 5 and 7 months of age showed an immune response in only 46% of vaccines [109]. However, those infants with an immune response were partially protected against rotavirus disease in the 2nd year, supporting the observation that this strain offered protection after natural infection. The vaccine strain has been adapted to a WHO-approved Vero cell line and produced at a higher titer, and further clinical trials and development with BioFarma, Indonesia are planned (Graeme Barnes, personal communication).

Naturally occurring neonatal bovine-human reassortant strains Neonatal rotavirus strains identified in India also conferred protection against subsequent rotavirus disease [30]. Strain 116E was identified to be a naturally occurring reassortant strain (G9P8[11]), between bovine and human rotaviruses with only the VP4 gene derived from a bovine rotavirus. A second naturally occurring bovine–human rotavirus strain in neonates was detected in Bangalore. Strain I321 carries G10P8[11] specificity and is predominantly a bovine strain, with only two human rotavirus non-structural proteins present [110]. These strains are being developed further as vaccine candidates by an international consortium consisting of Indian and US collaborators [110].

Challenges to rotavirus vaccine development What challenges can ostensibly remain for rotavirus vaccines, at the time that two safe and efficacious rotavirus vaccines are licensed internationally and on the verge of being introduced in multiple countries in the Americas and in Europe? Certainly, these vaccines will reduce the tremendous costs associated with rotavirus-associated illness and hospitalizations. Nevertheless, for rotavirus vaccines to reach their full potential and impact significantly on reducing childhood mortality, the vaccines need to be introduced in the developing countries of Africa and Asia, where the bulk of global rotavirus mortality lies [5, 7, 8]. There are several challenges to the successful introduction and implementation of rotavirus vaccines in these regions [111].

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Efficacy in developing countries in Africa and Asia The WHO has recommended consistently that the efficacy of the new generation rotavirus vaccines needs to be evaluated in the developing countries of Africa and Asia where the burden of disease and the mortality due to rotavirus disease is highest [4, 19, 112]. The rational reasons for this have been described previously [73, 111], but include issues such as: (i) differences in the immunogenicity and/or efficacy of other oral enteric vaccines, such as OPV and cholera vaccines in populations living in developing countries; and (ii) differences in the epidemiology and strain diversity of rotavirus strains circulating in developing countries, which seem to be different. Some of the potential questions why a rotavirus vaccine may not be as effective in a developing country population have been investigated. For instance, the immune response to one of the new rotavirus vaccines was demonstrated to be lower in an African infant population [106, 107], and is under evaluation in an Asian infant population. The other vaccine has not been evaluated in this group. Both commercial licensed rotavirus vaccines will be evaluated for clinical efficacy in developing country populations in Africa and Asia, as was recommended by the WHO [19, 112]. Secondly, both the RRV (RotaShield®) and monovalent human vaccine (Rotarix™), have been evaluated in malnourished infant populations. Although the study numbers were relatively small, both studies indicate that there was not a reduced efficacy associated with malnourished status of the infants [113, 114]. Thirdly, the co-administration of a live attenuated OPV has been examined and the immune responses and geometric mean titers (GMT) of the response to the three polio virus serotypes was not shown to be detrimentally affected by the Rotarix™ vaccine [106, 107], as was also seen with the RRV vaccine [115] and is being evaluated with the pentavalent rotavirus vaccine by Merck. The definitive clinical efficacy studies in infant populations in Africa and Asia, where infants will get the vaccine under the most testing situations (e.g., age of immunization, high maternal antibody, high co-morbidity of other enteric infections, co-administration of OPV, etc.) are ongoing and will only yield results in late 2008 or 2009.

Safety of the vaccines with regards to intussusception The reported association of the RotaShield® vaccine with intussusception, a rare but serious type of bowel obstruction found in infants worldwide, has had a lasting effect on rotavirus vaccine development [86, 116]. A clear temporal relationship between receipt of the vaccine and the development of intussusception was demonstrated with cases of intussusception clustering between 3 and 14 days following immunization with the first dose of the vaccine [117]. The age at time of receipt of the first dose of RotaShield®

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appears to have influenced the risk of intussusception post immunization. No cases of intussusception occurred in infants vaccinated at < 60 days of age despite 16% of all first doses received at that age [89]. Therefore, the risk of intussusception following RotaShield® was highest in infants who received their first dose of vaccine after 3 months of age, perhaps coinciding with the “natural” high-risk period for intussusception. The reasons for the association between the RRV vaccine and the development of intussusception are not known. In addition, it is not known if non-US populations would have the same risk of intussusception following receipt of this vaccine. The two licensed commercial rotavirus vaccines (Rotarix®, GSK Biologicals and RotaTeq®, Merck & Co., Inc.) completed Phase III clinical trials in 2005, with one or other of the vaccines being given to over 130 000 infants in Latin America, Europe and the U.S. in placebocontrolled studies with no association identified between receipt of the vaccine and intussusception [14, 15]. However, the safety and efficacy of these vaccines outside a clinical trial setting have not yet been demonstrated and need to be evaluated in the real world setting in developing countries [90]. Any risks associated with the newly developed rotavirus vaccines will only be identified after further trials or postlicensure surveillance studies. Therefore, it is likely that post-licensure surveillance in countries that are introducing the rotavirus vaccines will be the final harbinger of the long-term success of the new generation rotavirus vaccines.

Costs and cost effectiveness of vaccines The future utilization of the new rotavirus vaccines in the populations that need them most, will depend on costs of the vaccine and mechanisms for funding the vaccine procurement for these countries. In turn, this will depend on the cost effectiveness of the rotavirus vaccines. During the last few years, several studies to define the economic burden of rotavirus disease and the impact and cost effectiveness of rotavirus immunization have been conducted and have been reviewed, including studies in Asia [118, 119]. The main drivers of costs and cost effectiveness vary by setting, and were identified as the burden of disease, vaccine effectiveness, timing of vaccination, vaccine cost, additional immunization program costs, model structure, and study perspective. Nevertheless, by various measurements described by the World Bank as indicative of the “cost effectiveness“ of vaccines, the new rotavirus vaccines are definitely seen as cost effective [118–120].

Supplies of vaccine and financing GAVI will soon be reviewing an investment case for the potential future procurement of rotavirus vaccines for infants in some of the poorest coun-

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tries of the world. The investment case has evaluated a demand forecasting model of the numbers of doses of vaccines required during the next decade, and has examined a range of pricing options for the costs of rotavirus vaccines for developing countries. Both industrial partners have committed to a tiered pricing of their vaccines. The decision by GAVI whether to procure these vaccines for countries with the highest rotavirus mortality should impact directly on the Millennium Development Goals and help to reduce childhood mortality associated with rotavirus infection.

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with acute diarrhea in Blantyre, Malawi from 1997 to 1998. Predominance of novel P[6]G8 strains. J Med Virol 57: 308–312 Matthijnssens J, Rahman M, Yang X, Delbeke T, Arijs I, Kabue JP, Muyemba JT, van Ranst M (2006) G8 rotavirus strains isolated in the Democratic Republic of Congo belong to the DS-1 like genogroup. J Clin Microbiol 44: 1801–1809 Das S, Gentsch JR, Cicirello HG, Woods PA, Gupta A, Ramachandran M, Kumar R, Bhan MK, Glass RI (1994) Characterization of rotavirus strains from newborns in New Delhi, India. J Clin Microbiol 32: 1820–1822 Offit PA, Blavat G (1986) Identification of the two rotavirus genes determining neutralization specificities. J Virol 57: 376–378 Rao CD, Gowda K, Reddy BSY (2001) Sequence analysis of theVP4 and VP7 genes of non-typeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses. Virology 276: 104–113 Iturriza-Gomara M, Isherwood B, Desselberger U, Gray JJ (2001) Reassortment in vivo. A driving force for diversity of human rotavirus strains isolated in the UK between 1995 and 1999. J Virol 75: 3696–3705 Bishop RF, Davidson GP, Holmes IH, Ruck BJ (1973) Virus particles in epithelial cells of duodenal mucosa of children with gastroenteritis. Lancet 2: 1281–1283 Flewett TH, Davies HA, Bryden AS, Robertson MJ (1974) Diagnostic electron microscopy of faeces II. Acute gastroenteritis associated with reovirus-like particles. J Clin Pathol 27: 608–614 Ansari SA, Springthorpe VS, Sattar SA (1991) Survival and vehicular spread of human rotaviruses. Possible relation to seasonality of outbreaks. Rev Infect Dis 13: 488–491 Santosham M, Yolken RH, Quiroz E, Dillman L, Oro G, Reeves WC, Sack RB (1983) Detection of rotavirus in respiratory secretions of children with pneumonia. J Pediatr 103: 583–585 Zheng BJ, Chang RX, Ma GZ, Xie JM, Liu Q, Liu XR, Ng MH (1991) Rotavirus infection of the oropharynx and respiratory in young children. J Med Virol 34: 29–37 Rodriguez W, Kim H, Arrobio J, Brandt CD, Chanock RM, Kapikian AZ, Wyatt RG, Parrott RH (1977) Clinical features of acute gastroenteritis associated with human reovirus-like agent in infants and children. J Pediatr 91: 188–193 Staat MA, Azimi PH, Berke T, Roberts N, Bernstein DI, Ward RL, Pickering LK, Matson DO (2002) Clinical presentations of rotavirus infection among hospitalized children. Pediatr Infect Dis J 21: 221–227 Holmes IH, Ruck BJ, Bishop RF, Davidson GP (1975) Infantile enteritis viruses: Morphogenesis and morphology. J Virol 16: 937–943 Midthun K, Kapikian AZ (1996) Rotavirus vaccines: An overview. Clin Microbiol Rev 9: 423–434 Gorrell RJ, Bishop RF (1999) Homotypic and heterotypic serum neutralising antibody response to rotavirus proteins following natural primary infection and re-infection in children. J Med Virol 59: 204–211 Ward RL, Bernstein DI (1994) Protection against rotavirus disease after natural infection. J Infect Dis 169: 900–904

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Clark HF, Offit PA, Ellis RW, Eiden JJ, Krah D, Shaw AR, Pichichero M, Treanor JJ, Borian FE, Bell LM, Plotkin SA (1996) The development of multivalent bovine rotavirus (strain WC3) reassortant vaccine for infants. J Infect Dis 174S: s73–80 Jiang B, Gentsch JR, Glass RI (2002) The role of serum antibodies in the protection against rotavirus disease: An overview. Clin Infect Dis 34: 1351–1361 Hjelt K, Grauballe PC, Paerregaard A, Nielsen OH, Krasilnikoff PA (1987) Protective efficacy of pre-existing rotavirus-specific immunoglobulin A against naturally acquired rotavirus infection in children. J Med Virol 21: 39–47 Clemens JD, Ward RL, Rao MR, Sack DA, Knowlton DR, van Loon FP, Huda S, McNeal M, Ahmed F, Schiff G (1992) Sero-epidemiologic evaluation of antibodies to rotavirus as correlates of the risk of clinically significant rotavirus diarrhea in rural Bangladesh. J Infect Dis 165: 161–165 Bernstein DI, Smith VE, Sander DS, Pax KA, Schiff GM, Ward RL (1990) Evaluation of WC3 rotavirus vaccine and correlates of protection in healthy infants. J Infect Dis 162: 1055–1062 Kapikian AZ, Hoshino Y, Chanock RM, Perez-Schael I (1996) Efficacy of a quadrivalent rhesus rotavirus-based vaccine aimed at preventing severe rotavirus diarrhea in infants and young children. J Infect Dis 174S: s65–s72 Offit PA, Hoffenberg EJ, Santos N, Gouvea V (1993) Rotavirus-specific humoral and cellular immune response after primary symptomatic infection. J Infect Dis 167: 1436–1440 Jaimes MC, Rojas OL, Gonzalez AM, Cajiao I, Charpilienne A, Pothier P, Kohli E, Greenberg HB, Franco MA, Angel J (2002) Frequencies of virus-specific CD4 and CD8 T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults. J Virol 76: 4741–4749 Ward RL (2003) Possible mechanisms of protection elicited by candidate rotavirus vaccines as determined with the adult mouse model. Viral Immunol 2003; 6: 17–24 Heaton PM, Gouveia MG, Miller JM, Offit P, Clark HF (2005) Development of a pentavalent rotavirus vaccine against prevalent serotypes of rotavirus gastroenteritis. J Infect Dis 192: S17–S21 Clark HF, Offit PA, Plotkin SA, Heaton PM (2006) The new pentavalent rotavirus vaccine composed of bovine (strain WC3) human rotavirus reassortants. Pediatr Infect Dis J 25: 577–583 Bernstein DI, Sander DS, Smith VE, Schiff GM, Ward RL (1991) Protection from rotavirus re-infection. Two year prospective study. J Infect Dis 164: 277– 283 Estes MK, Graham DY (1985) Rotavirus antigens. Adv Exp Med Biol 185: 201–214 Woode GN, Crouch CF (1978) Naturally occurring and experimentally induced rotaviral infections of domestic and laboratory animals. J Am Vet Med Assoc 173: 522–526 Bresee JS, Parashar U, Widdowson MA, Gentsch JR, Steele AD, Glass RI (2005) Update on rotavirus vaccines. Pediatr Infect Dis J 24: 947–952 Christy C, Madore HP, Treanor JJ, Pray K, Kapikian AZ, Chanock RM, Dolin

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R (1986) Safety and reactogenicity of the live attenuated rhesus monkey rotavirus vaccine. J Infect Dis 154: 1045–1047 Flores J, Perez-Schael I, Gonzalez M, Garcia D, Perez M, Daoud N, Cunot W, Kapikian AZ (1987) Protection against severe rotavirus diarrhea by rhesus rotavirus vaccine in Venezuelan infants. Lancet 1: 882–884 Santosham M, Letson GW, Wolff M, Reid R, Gahagan S, Adams R, Callahan C, Sack RB, Kapikian AZ (1991) A field study of the safety and efficacy of two candidate rotavirus vaccines in a Native American population. J Infect Dis 163: 483–487 Hanlon P, Hanlon L, Marsh V, Byass P, Shenton F, Hassan-King M, Jobe O, Sillah H, Hayes R, M’Boge BH et al (1987) Trial of an attenuated bovine rotavirus vaccine (RIT 4237) in Gambian infants. Lancet 1: 1342–1345 Lanata C, Black RE, del Aguila R, Gil A, Verastegui H, Gerna G, Flores J, Kapikian AZ, Andre FE (1989) Protection of Peruvian children against rotavirus diarrhea of specific serotypes by one, two or three doses of the RIT4237 attenuated bovine rotavirus vaccine. J Infect Dis 159: 452–459 Georges-Courbot MC, Monges J, Siopathis MR, Roungou JB, Gresenguet G, Bellec L, Bouquety JC, Lanckriet C, Cadoz M, Hessel L et al (1991) Evaluation of the efficacy of a low passage bovine rotavirus (strain WC3) vaccine in children in Central Africa. Res Virol 142: 405–411 Midthun K, Greenberg HB, Hoshino Y, Kapikian AZ, Wyatt RG, Chanock RM (1985) Reassortant rotaviruses as potential live rotavirus vaccine candidates. J Virol 53: 949–954 Vesikari T, Green KY, Flores J, Kapikian AZ (1992) Protective efficacy against serotype 1 rotavirus diarrhea by live oral rhesus reassortant rotavirus vaccines with human rotavirus VP7 serotype specificity. Pediatr Infect Dis J 11: 535–542 Rennels MB, Glass RI, Dennehy PH, Bernstein DI, Pichichero ME, Zito ET, Mack ME, Davidson BL, Kapikian AZ (1996) Safety and efficacy of the high dose rhesus-human reassortant rotavirus vaccines: Report of the national multi-centre trial. Pediatrics 97: 7–13 Perez-Schael I, Guntinas MJ, Perez M, Pagone V, Rojas AM, Gonzalez R, Cunto W, Kapikian AZ (1997) Efficacy of the rhesus rotavirus based quadrivalent vaccine in infants and young children in Venezuela. N Engl J Med 337: 1181–1187 Joensuu J, Koskenniemi E, Pang XL, Vesikari T (1997) Randomised placebo controlled trial of the rhesus-human reassortant rotavirus vaccine for prevention of severe rotavirus gastroenteritis. Lancet 350: 1205–1209 Centers for Disease Control and Prevention (1999) Rotavirus vaccines for the prevention of rotavirus gastroenteritis among children – Recommendations of the Advisory Committee on Immunization Practices. MMWR 48: 1–23 Centers for Disease Control and Prevention (1999) Intussusception among recipients of rotavirus vaccine – United States 1998–1999. MMWR 48: 577– 581 Bresee JS, El Arifeen S, Azim T, Chakraborty J, Mounts AW, Podde G, Gentsch JR, Ward RL, Black R, Glass RI, Yunus M (2001) Safety and immunogenicity of tetravalent rhesus based rotavirus vaccine in Bangladesh. Pediatric Infect Dis J 20: 1136–1143

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Simonsen L, Morens DM, Elixhauser A, Gerber M, van Raden M, Blackwelder WC (2001) Effect of rotavirus vaccination programme on trends in admission of infants to hospital for intussusception. Lancet 358: 1224–1227 89 Simonsen L, Viboud C, Elixhauser A, Taylor RJ, Kapikian AZ (2005) More on RotaShield and intussusception: The role of age at the time of vaccination. J Infect Dis 192: S36–S43 90 World Health Organization (2006) Report of the WHO Global Advisory Committee on Vaccine Safety. Wkly Epidemiol Rec 81: 15–17 91 Clark HF, White CJ, Offit PA, Ellis RW, Krah D, Shaw AR, Eiden JJ, Pichich M, Treanor JJ (1995) Preliminary evaluation of safety and efficacy of quadrivalent human-bovine reassortant rotavirus vaccine. Pediatr Res 37: 172 92 Clark HF, Bernstein DI, Dennehy PH, Offit P, Pichichero M, Treanor J, Ward RL, Krah DL, Shaw A, Dallas MJ et al (2004) Safety, efficacy and immunogenicity of a live quadrivalent human-bovine reassortant rotavirus vaccine in healthy infants. J Pediatr 144: 184–190 93 Clark HF, Furukawa F, Bell LM, Offit PA, Perella PA, Plotkin SA (1986) Immune response of infants and children to low passage bovine rotavirus (strain WC3). Am J Dis Child 140: 350–355 94 Clark HF, Borian FE, Plotkin SA (1990) Immune protection of infants against rotavirus gastroenteritis by a serotype 1 reassortant of bovine rotavirus WC3. J Infect Dis 161: 1099–1104 95 Hoshino Y, Jones RW, Chanock RM, Kapikian AZ (1997) Construction of four double gene substitution human x bovine rotavirus reassortant vaccine candidates. J Med Virol 51: 319–325 96 Clements-Mann ML, Makhene MK, Mrukowicz J, Wright PF, Hoshino Y, Midthun K, Sperber E, Karron R, Kapikian AZ (1999) Safety and immunogenicity of live attenuated human-bovine (UK) reassortant rotavirus vaccines with VP7-specificity for serotypes 1, 2, 3, or 4 in adults, children and infants. Vaccine 17: 2715–2725 97 Clements-Mann ML, Dudas R, Hoshino Y, Nehring P, Sperber E, Wagner M, Stephens I, Karron R, Deforest A, Kapikian AZ (2001) Safety and immunogenicity of live attenuated quadrivalent human-bovine (UK) reassortant rotavirus vaccine administered with childhood vaccines to infants. Vaccine 19: 4676–4684 98 Bernstein DI, Smith VE, Sherwood JR, Schiff GM, Sander DS, deFeudis D, Spriggs DR, Ward RL (1998) Safety and immunogenicity of live, attenuated human rotavirus vaccine 89-12. Vaccine 16: 381–387 99 Bernstein DI, Sack DA, Rothstein E, Reisinger K, Smith VE, O’Sullivan D, Spriggs DR, Ward RL (1999) Efficacy of live, attenuated human rotavirus vaccine 89-12 in infants: A randomised placebo-controlled trial. Lancet 354: 287–290 100 Bernstein DI, Sack DA, Reisinger K, Rothstien E, Ward RL (2002) Second year follow-up evaluation of live, attenuated human rotavirus 89-12 vaccine in healthy infants. J Infect Dis 186: 1487–1489 101 De Vos B, Vesikari T, Linhares A, Salinas B, Perez-Schael I, Ruiz-Palacois G, Guerro MdL, Phua KB, Delem A, Hardt K (2004) A rotavirus vaccine for

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prophylaxis of infants against rotavirus gastroenteritis. Pediatr Infect Dis J 23: S179–S182 Vesikari T, Karvonen A, Korhonen T, Espo M, LeBacq E, Forster J, Zepp F, Delem A, De Vos B (2004) Safety and immunogenicity of RIX4414 live attenuated human rotavirus vaccine in adults, toddlers and previously uninfected infants. Vaccine 22: 2836–2842 Salinas B, Perez-Schael I, Linhares A, Ruiz-Palacios GM, Guerrero MdL, Yarzabal JP, Cervantes Y, Costa Clemens SA, Damaso S, Hardt K, DE Vos B (2005) Evaluation of the safety, immunogenicity and efficacy of an attenuated rotavirus vaccine, RIX4414. Pediatr Infect Dis J 24: 807–816 Phua KB, Quak SH, Lee BW, Emmanual SC, Goh P, Han HH, De Vos B, Bock HL (2005) Evaluation of RIX4414, a live attenuated rotavirus vaccine in a randomized double-blind, placebo-controlled, phase II trial involving 2464 Singaporean infants. J Infect Dis 192: S6–S16 Zaman K, Sack DA, Yunus M, Arifeen SE, Azim T, Podder G, Faruque ASG, Karim S, Luby S, Breiman RF. Rotavirus vaccine trials in Bangladesh. 8th Commonwealth Congress on Diarrhea and Malnutrition, 6–8 February 2006. Dhaka, Bangladesh Steele AD, Tumbo JM, Armah GE, Reynders J, Scholtz F, Bos P, de Beer MC, van der Merwe CF, Delem A, De Vos B (2004) Immunogenicity and reactogenicity of a new live attenuated oral rotavirus vaccine (RIX4414) when administered concurrently with poliovirus vaccines in African infants. International Congress of Paediatrics, 15–20 August, 2004. Cancun, Mexico Steele AD, Tumbo JM, Reynders J, Scholtz F, Bos P, de Beer MC, van der Merwe CF, Delem A, De Vos B (2006) Comparison of two different regimens (two doses versus three doses) in terms of reactogenicity and immunogenicity of the live attenuated human rotavirus vaccine RIX4414 in South African infants. 11th Asian Conference on Diarrhea Disease and Nutrition. 8–10 March, 2006, Bangkok, Thailand Barnes GL, Lund JS, Adams L, Mora A, Mitchell SV, Caples A, Bishop RF (1997) Phase I trial of a candidate rotavirus vaccine (RV3) derived from a human neonate. J Paediatr Child Health 33: 300–304 Barnes GL, Lund JS, Mitchell SV, de Bruyn L, Piggford L, Smith AL, Furmedge J, Masendycz PJ, Bugg HC, Bogdanovic-Sakran N et al (2002) Early phase II trial of human rotavirus vaccine candidate RV3. Vaccine 20: 2950–2956 Glass RI, Bhan MK, Ray P, Bahl R, Parashar U, Greenberg HB, Rao CD, Bhandari N, Maldonado Y, Ward RL et al (2005) Development of candidate rotavirus vaccines derived from neonatal strains in India. J Infect Dis 192: S30–S35 Glass RI, Bresee JS, Turcois R, Fischer TK, Parashar U, Steele AD (2005) Rotavirus vaccines: Targeting the developing world. J Infect Dis 192: S160– S166 World Health Organization (2006) Report of the WHO Strategic Advisory Group of Experts (SAGE). Wkly Epidemiol Rec 81: 8–9 Linhares AC, Carmoi KB, Oliveira KK, Freitas RB, Bellesi N, Monteiro TAF, Gabbay YB, Mascarehas JD (2002) Nutritional status in relation to the efficacy

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of the rhesus human reassortant tetravalent rotavirus vaccine (RRV-TV) in infants in Belem, Para State, Brazil. Rev Inst Med trop S Paulo 44: 13–16 Perez-Schael I, Salinas B, Tomat M, Linhares AC, Ruiz-Palacios GM, Guerrero ML, Costa-Clemens SA, Bouckenooghe A, Yarzabal JP. Human rotavirus vaccine RIX4414 is efficacious in malnourished children. 9th International Symposium on Double-stranded RNA viruses, 21–26 October, 2006. Cape Town, South Africa Migasena S, Simasathien S, Samakoses R, Pitisuttitham, Sangaroon P, van Steenis G, Beuvery EC, Bugg H, Bishop RF, Davidson BL, Vesikari T (1995) Simultaneous administration of oral rhesus human reassortant tetravalent human rotavirus (RRV-TV) and oral poliovirus vaccine (OPV) in Thai infants. Vaccine 13: 168–174 Murphy BR, Morens DM, Simonsen L, Chanock RM, La Montagne JR, Kapikian AZ (2003) Reappraisal of the association of intussusception with the licensed live rotavirus vaccine challenges initial conclusions. J Infect Dis 187: 1301–1308 Murphy TV, Gargiullo PM, Massoudi MS, Nelson DB, Jumaan AO, Okoro CA, Zanardi LR, Setia S, Fair E, LeBaron CW et al (2001) Intussusception among infants given an oral rotavirus vaccine. N Engl J Med 344: 564–572 Podewils LJ, Antil L, Hummelman E, Bresee JS, Parashar U, Rheingans R (2005) Projected cost effectiveness of rotavirus vaccination in Asia. J Infect Dis 192: S133–S145 Walker DG and Rheingans R (2005) Cost-effectiveness of rotavirus vaccines. Expert Rev Pharmaco-economics Outcomes Res 5: 593–601 World Health Organization (2006) Report of a WHO Meeting on the cost estimation and cost effectiveness of rotavirus vaccines. Wkly Epidemiol Rec 81: 350–353

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Controversially discussed indications for immunization Sieghart Dittmann Hatzenporter Weg 19, 12681 Berlin, Germany

Abstract The indication for immunization in general or indications for selected vaccines are sometimes controversially discussed by parents, the media and even by some parts of the medical community. This controversial discussion can cause confusion for people who want to make decisions about immunization for their children or themselves. There is clearly a need for accurate and evidence-based information about indications and effectiveness of vaccines, as well as about the risks from natural diseases compared with potential risks of adverse events following immunization. This chapter deals with (i) immunization as a safe and very effective disease prevention measure, (ii) indications for immunization of selected risk groups, and (iii) contraindications and false contraindications. The first part raises the most controversial questions (many diseases already disappeared due to improved socioeconomic conditions before vaccines were introduced; when a disease is gone there is no need to continue with immunization; natural immunity is better than vaccine-induced immunity; many vaccines are useless and not able to prevent disease; multiple immunizations overload the immune system; some vaccines are not safe and cause more complications than the natural disease) and tries to provide evidence-based answers. The second part deals with controversially discussed indications/contraindications for selected risk groups such as pregnant and breast-feeding women, pre-term babies, individuals with chronic diseases or immunodeficiency, patients with bleeding disorders and patients receiving anticoagulant medication. In the last part, genuine contraindications against distinct vaccines are discussed as well as health conditions falsely believed by the physician or the health worker to constitute a contraindication.

Introduction The impact of immunization on the health of the world’s people is hard to exaggerate. However, the continued success of immunization programs depends on a high level of public confidence in their effectiveness and safety. Immunization in general or indications for selected vaccines are increasingly often controversially discussed by parents, the media and some parts of the medical community. This controversial discussion can cause

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confusion for people who want to make responsible, informed decisions about immunization for their children and themselves. There is a need for accurate information about indications and effectiveness of vaccines as well as about the risks from natural diseases compared with those from adverse events following immunization. This chapter tries to respond to some controversial discussions on indications. Three topics will be discussed: – Immunization is safe and one of the most effective disease prevention measures – Indications for the immunization of selected risk groups – Contraindications and false contraindications

Immunization is safe and one of the most effective disease prevention measures Seven most often used controversial arguments are discussed:

Controversial argument 1 Most vaccines are not really indicated, because many of the so-called vaccine-preventable diseases already disappeared due to the improved socioeconomic conditions before vaccines were introduced.

Counter-argument Improved socioeconomic conditions as well as the development of antibiotics have undoubtedly had a great impact on disease incidence, disease-related complications and death. However, the immediate and direct impact of vaccines is absolutely significant. A few examples may underline the effectiveness of selected immunization programs, many more examples could be given. – The implementation of oral poliovirus vaccine (OPV) mass immunization in Germany eliminated poliomyelitis within a few months (East Germany)/few years (West Germany) (Fig. 1) [1]. – Due to decreasing OPV coverage in Albania, a large re-appearance of poliomyelitis occurred in 1996. OPV mass immunization stopped the outbreak within few weeks (Fig. 2) [2]. _ In November 1999, a newly developed conjugated meningococcal group C vaccine was recommended for all children and adolescents in England and Wales. By 2003, cases and deaths due to meningococcal group C disease were reduced by more than 90% (Fig. 3) [3]. – The success of immunization programs implemented in the US Pink Book, 8th edition 2004: Appendices) is another convincing example (Fig. 4) [4].

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Figure 1. Implementation of polio immunization (OPV) in Germany. Start of massive campaigns in East (1960) and West (1962).

Figure 2. Polio outbreak in Albania – Outbreak control with OPV, April–December 1996.

Controversial argument 2 In a country where a disease no longer exists, there is no longer an indication to continue with immunization against this disease.

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Figure 3. Success of immunization program with conjugated meningococcal serogroup C vaccine, England 1999-2003.

Figure 4. Impact of immunization programs, USA 1945–2002.

Counter-argument It is true that following the implementation of nationwide immunization programs in industrially developed countries in the second half of the last

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century, morbidity and mortality of childhood diseases, such as diphtheria, poliomyelitis, measles, and pertussis in children, decreased to very low levels or even levels close to elimination. These diseases are much less common now, but the bacteria and viruses that cause them are still present. Travelers can carry diseases from country to country, and if an individual is not immunized he/she could be at serious risk. It is also important to realize that some people cannot have vaccines because of certain medical conditions or severe allergies. These people are susceptible to disease, and their only hope of protection is that people around them are immune and cannot pass disease along to them. A successful immunization program depends on the cooperation of every individual to ensure the good of all. Many examples can be provided where unjustified counter-propaganda, religious barriers, or program management failures negatively influenced the acceptance of immunization and caused the re-emergence of vaccine-preventable diseases. – Pertussis: In the 1970s in Japan, a near boycott of the vaccine due to suspected vaccine complications caused the recurrence of epidemic pertussis with hundreds of deaths. Similarly, because of aggressive publicity concerning central nervous system damage following immunization using wholecell pertussis vaccine in UK, coverage rates fell from 75% to about 25% during the mid-1970s, and major epidemics re-emerged. The re-implementation of pertussis immunization programs (whole-cell vaccine in UK and acellular vaccine in Japan) brought pertussis back under control. – Poliomyelitis: Two outbreaks of poliomyelitis occurred in particular religious communities of 200 000 individuals dispersed throughout the Netherlands who refused immunization. One outbreak caused 110 cases in 1978, and the second outbreak caused 71 cases in 1992. There was only a single case of poliomyelitis among other Dutch people as the Dutch population in general is well protected through inactivated poliovirus vaccine (IPV) immunization programs achieving high coverage rates. However, contact cases from the 1978 outbreak occurred in religious groups in North America. – Diphtheria: In 1958–1959, a near-universal childhood diphtheria immunization program began throughout the Soviet Union, and by 1963, the incidence of diphtheria had decreased by > 90%. Epidemic diphtheria re-emerged in the Russian Federation in 1990, spread to all Newly Independent States (NIS) of the former USSR by the end of 1994, and developed into the largest diphtheria epidemic in the world since the implementation of diphtheria immunization programs. In 1995 and 1996, more than 90% of all diphtheria cases and deaths reported worldwide occurred in the NIS. As a result of the political and socioeconomic changes in the former USSR, various factors contributed to the epidemic, including decreasing immunization coverage in children, immunity gaps in adults, altered public perception of the benefits and risks of immunization, population movement, deteriorating health infrastructure, initial shortages of

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Table 1. Comparison of severity of diseases and complications following immunization Disease

Severity of disease

Complications following immunization

Diphtheria

Case fatality rate (CFR) 1–7%, nerve paralysis and myocarditis often occur

Rarely, allergic reactions, anaphylaxis and peripheral neuritis following DTP may occur

Hepatitis B

1% (Western Europe. North America) to 10% (Asia, Pacific, sub-Saharan Africa) of population are chronically infected; about 1 in 4 chronic carriers develop cirrhosis or liver cancer

In rare cases, allergic reactions or anaphylaxis may occur

H. influenzae disease

CFR: meningitis, 5%; epiglottiditis, 1%; about 1 in 4 survivors has permanent brain or nerve damage

In case of fever young children may develop febrile seizures, allergic reactions are very rare

Influenza

Causes increased hospitalization rates and excess mortality in high risk groups, particularly the elderly and the chronically ill

In rare cases allergic reactions, vasculitis or thrombocytopenia may occur; Guillain-Barré syndrome is reported in about 1 in 1 million vaccinees

Measles

4% of patients develop pneumonia, and 1 in 1000 encephalitis; CFR of measles encephalitis, 10%; and 40% have permanent brain damage; rarely subacute sclerosing panencephalitis (SSPE) occurs

In rare cases, allergic reactions or anaphylaxis may occur; in case of fever young children may develop febrile seizures

Meningococcal disease, invasive

Meningitis, septicemia; CFR ~10%; ~10–20% permanent damage (CNS, physical sequelae range from necrosis to amputation following extensive gangrene, bone lesions and skeletal growth disturbances

Following conjugated MenC vaccine only local and systemic reactions reported

Mumps

4% of patients develop meningitis, occasionally mumps causes deafness; 1 in 5 males past puberty develop inflammation of testes

Allergic reactions are rare; in case of fever young children may develop febrile seizures; meningitis may occur but has not reported following mumps vaccines based on the ‘Jeryl Lynn’ and ‘Jeryl Lynn’-derived strains

Pertussis

CFR (due to pneumonia or encephalopathy) in infants about 1%

In rare cases, allergic reactions, anaphylaxis, hypotonic-hyporesponsive following DTP may occur; following whole-cell pertussis vaccine, rare cases of encephalopathy occurred

Pneumococcal Meningitis, septicemia, bacteremia; disease in CFR ~5%; ~ 20% permanent children, invasive damage (CNS, hearing loss, learning disabilities)

Following conjugated pneumococcal vaccine only local and systemic reactions reported, allergic reactions are very rare

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Table 1 (continued) Disease

Severity of disease

Complications following immunization

Poliomyelitis

CFR 5%; 1 in 2 survivors is permanently paralyzed

Vaccine-associated paralytic poliomyelitis may occur following OPV; IPV has an excellent safety profile

Rubella

50% of adolescents and adults develop arthritis/arthralgia; very rarely thrombocytopenia or encephalitis; 9 of 10 babies infected during the first 10 weeks of pregnancy will develop major congenital abnormalities (CRS)

In case of fever young children may develop febrile seizures, allergic reactions are very rare; arthritis/ arthralgia may rarely occur in (preferably female) adolescents/adults

Tetanus

CFR: 10%, much higher in older individuals

In rare cases allergic reactions, anaphylaxis and peripheral neuritis may occur

vaccine, and delays in implementing control measures. Since 1995, aggressive control measures, including mass immunizations as the core element of the strategy, were implemented in close collaboration between the NIS and international donors and stopped the epidemics [2].

Controversial argument 3 Natural immunity is better than vaccine-induced immunity.

Counter-argument While vaccine-induced immunity may diminish with time, ‘natural’ immunity, acquired through natural disease persists usually longer and often lifelong. However, for most vaccines, individuals can receive booster immunization(s) if the vaccine-induced immunity falls to a low level. Therefore, vaccine-induced immunity can also protect lifelong. The problem is that ‘natural’ diseases have a high risk of serious illness and occasionally death. Natural disease is far more risky than immunization (see Tab. 1).

Controversial argument 4 Many vaccines are not indicated because they are often useless and many people get the disease despite being immunized.

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Counter-argument First, no vaccine is 100% effective. For reasons related to the individual, not all immunized individuals develop immunity. Most vaccines are effective for 85–95% of recipients. Second, in countries with high immunization coverage, the people who have been immunized vastly outnumber those who have not. How these two factors work together to result in outbreaks although the majority of cases have been immunized can be easily understood by looking at the following example: In a dormitory of 300 students, the entire student body is exposed to measles, none has ever had measles. Of these, 295 have had two doses of measles vaccine, the efficacy rate for two doses of measles is at least 95%; and the 5 non-immunized students will get measles, of course. However, of the 295 students, who have been immunized, we would expect approximately 5% (15 students) not to respond to the vaccine and they, too, become infected. Therefore, 15 of 20, or about 70%, of the cases occur in students who have been fully immunized. Under circumstances of high coverage those individuals who were immunized and did not respond outnumbered those who had not been immunized. This does not prove the vaccine did not work: 100% of the students who had not been immunized got measles, compared with approximately 5% of those who had been immunized [5]. We should also note that illness in immunized individuals is usually much less severe than in those who were not immunized.

Controversial argument 5 Multiple immunizations or combination vaccines overload the immune system.

Counter-argument The increase in the number of vaccines given to children, and preferably administered as combination vaccines, has led to concerns about the possible adverse effects of the aggregate vaccine exposure, especially on the developing immune system. However, in day-to-day life, all children and adults confront enormous numbers of substances that provoke a reaction from the immune system, and the immune system responds to each of these in various ways to protect the body. Studies of the diversity of antigen receptors indicate that the immune system can respond to an extremely large number of antigens. Scientists estimate that the immune system can recognize and respond to hundreds of thousands, if not millions, of different organisms. In the face of these normal events, it seems unlikely that the number of separate antigens contained in childhood vaccines would represent an appreciable added burden on the immune system that would be

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Table 2. Content of immunogenic proteins and polysaccharides in vaccines – 1900 vs 2000 1960s

1900 Vaccine Smallpox

Total

Proteins ~200

~200

Vaccine Smallpox

2000

Proteins ~200

Vaccine

Proteins/ polysaccharides

Measles-mumpsrubella

24

Diphtheriatetanus

2

Diphtheria-tetanus

2

Pertussis (whole-cell)

~3000

Pertussis (acellular)

2–5

Poliomyelitis

15

H. influenzae type b

2

Hepatitis B

1

Total

Plus additional vaccines included in the US vaccination schedule

~3200

Total Western European countries

46-49

Varicella

69

Pneumococcal conjugate vaccine

8

Total US

123–126

immunosuppressive. We should also consider that the number of antigens received by children during routine childhood immunization has actually decreased compared with immunization programs used during the 20th century, in particular the 1960s. The replacement of whole-cell pertussis vaccine by acellular pertussis vaccine (introduced in most European and North American countries as well as in Australia, Japan and many other regions) decreased the content of immunogenic proteins and polysaccharides from approximately 3000 to 50–125 (Tab. 2, adapted from [6]). The authors of carefully designed studies concluded that there is no evidence that adding vaccines to combination products increases the burden on the immune system. Young infants have a great capacity to respond to multiple vaccines. Increased reactogenicity following the receipt of combination vaccines has also not been a major issue. Combining antigens usually does not increase adverse effects, but it can lead to an overall reduction in adverse events. Neither the licensing agencies nor the national advisory boards on immunization would recommend the simultaneous administration of any vaccines or the use of combination vaccines until studies have confirmed the safety and efficacy. What is the practical justification for the use of a combination vaccine or several vaccines during the same visit? First, we want to immunize children as early as possible to give them protection during the vulnerable early months of their lives. Second, it means fewer

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office visits, which saves parents both time and money and may be less traumatic for the child [5–8].

Controversial argument 6 Vaccines are not indicated because they are not safe and cause much more complications than natural disease.

Counter-argument Vaccines are among the safest tools of modern medicine. Following immunization, local and/or systemic reactions may develop such as redness, swelling or tenderness at the injection site, or a mild fever, but these reactions are most often minor and temporary. Serious side effects can happen, but are extremely rare. On the other hand, the dangers of vaccine-preventable diseases are many times greater than the risk of a serious adverse reaction to the vaccine. Examples for both the severity of diseases and complications following immunization have been provided in Table 1. All vaccines are manufactured according to strict manufacturing guidelines. Before vaccines are licensed they are tested for safety and efficacy in carefully designed clinical trials. All vaccine manufacturing facilities and vaccine products are licensed by the national or supranational licensing authorities such as the European Medicines Evaluation Agency (EMEA) or the (US) Food and Drug Administration (FDA). In addition, every vaccine lot is safety-tested by the manufacturer. The results of these tests are reviewed by the licensing authority, which may repeat some of these tests as an additional protective measure. The licensing authorities also inspect vaccine-manufacturing facilities regularly to ensure adherence to manufacturing procedures and product-testing regulations, and review in most countries the adverse event reports searching for unusual patterns of licensed vaccines [5, 7, 8].

Controversial argument 7 Instead of preventing diseases vaccines cause diseases.

Counter-argument Vaccines have been spuriously linked by various researchers to asthma, autism, Crohn’s disease, diabetes, multiple sclerosis (MS), permanent brain damage, and sudden infant death syndrome (SIDS). Is there any evidence for the causal relationship between immunization and the diseases mentioned?

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Asthma There is no evidence that vaccination causes or worsens asthma. It is especially important that children with asthma be vaccinated like other children, as catching a disease like whooping cough can make an asthma attack worse [7, 8].

Autism At the end of the 1990s, concerns about the safety of the measles, mumps and rubella (MMR) and thimerosal-containing vaccines as possible causes of autism and other neuro-developmental disorders were raised. Various careful designed studies have been undertaken (particularly in Denmark, Finland, Sweden, the United Kingdom and the United States) to evaluate if there is any evidence for an association between MMR and thimerosal-containing vaccines and neuro-developmental disorders, particularly autism. Recently, two major independent vaccine safety committees (the Immunization Safety Review Committee of the Institute of Medicine, US National Academy of Sciences; and the Global Advisory Committee on Vaccine Safety) examined the hypotheses. The main conclusions of the committees are as follows: the evidence favors rejection of a causal relationship between MMR vaccine and autism as well as a causal relationship between thimerosal-containing vaccines and neuro-developmental disorders including autism. However, in response to the controversy over the safety of thimerosal, various manufacturers developed thimerosal-free versions of vaccines, particularly childhood vaccines; they are now licensed in many countries worldwide [5, 7–12].

Crohn’s disease Although the risk of Crohn’s disease (inflammatory bowel disease, IBD) is higher for those who have relatives with IBD, there are no data to suggest that measles vaccine will increase or decrease this risk. Measles vaccine is recommended for children with a family history of IBD unless there is another specific reason not to immunize [5, 7, 8, 13, 14].

Diabetes In 1997, a study from Finland suggested a link between Haemophilus influenzae type b (Hib) vaccination and type 1 diabetes. However, subsequent reanalysis of the data did not support such a link. The conclusion that there is no causal link between any of the childhood vaccines and diabetes has also

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been supported by a subsequent review of the literature, and the conclusions of two workshops held in the USA in 1998. The Institute for Vaccine Safety at the Johns Hopkins School of Public Health held a workshop in Baltimore, Maryland: Analyzing all available data on the pathogenesis of diabetes, autoimmunity, epidemiology, biostatistics, and adverse events following immunization, the workshop found no evidence that changing the routine childhood immunization would increase or decrease the risk of developing type 1 diabetes. A further meeting discussing the same problem of diabetes and immunization has been held in Bethesda, Maryland. The consensus was that existing studies in humans do not indicate an increase in type 1 diabetes attributable either to any vaccine or to the timing of the vaccine [8].

Influenza vaccine may cause influenza disease Although some believe that the vaccine causes influenza, this is not possible as it is not a live virus vaccine. As some people experience adverse events such as a mild fever after the vaccine, it is understandable that they may confuse these symptoms with actually having the ‘flu’ [8].

Multiple sclerosis The precise cause of MS, a presumed autoimmune disease, is unknown. There is no evidence that hepatitis B vaccine causes MS. Concerns about hepatitis B vaccination arose in France, after a few reports of a possible link between hepatitis B vaccine and MS. However, when the French data were examined closely, the rate of MS in immunized people was not significantly different from the expected population rate. Subsequent studies have found no increase in incidence of MS, or even relapse of MS, after hepatitis B vaccination. Worldwide use of over a billion doses of hepatitis B vaccine has not resulted in increased incidence of MS, as would be expected if there were a causal connection. The Medical Advisory Board of the (US) National Multiple Sclerosis Society has concluded that there is no evidence of a link between hepatitis B vaccination and MS. The Immunization Safety Committee of the Institute of Medicine reviewed the available data on immunization and hepatitis B and concluded that the evidence favors rejection of a causal relationship between hepatitis B vaccine administered to adults and incident multiple sclerosis or multiple sclerosis relapse [5, 7, 8, 15].

Sudden infant death syndrome Deaths do occasionally occur shortly after vaccination but the relationship is simply an incidental association, as SIDS tends to occur in babies of

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2–6 months of age whether they are vaccinated or not. Extensive studies have conclusively shown that SIDS is not caused by immunization. When a number of well-controlled studies were conducted during the 1980s, the investigators found, nearly unanimously, that the number of SIDS deaths temporally associated with diphtheria, tetanus toxoid and pertussis (DTP) immunization was within the range expected to occur by chance. In addition, some studies have found a lower rate of SIDS in immunized children. The Institute of Medicine reported that all controlled studies that have compared immunized versus non-immunized children have found ‘either no association … or a decreased risk … of SIDS among immunized children’ and concluded that the evidence does not indicate a causal relation between vaccines and SIDS [5, 7, 8, 16].

Indications for the immunization of selected risk groups Immunization during pregnancy Many authors take the conservative position that the use of vaccines during pregnancy should generally be avoided at any stage of the pregnancy, since definitive studies on the level of risk have not been carried out. Other authors take a more balanced position: They consider that there is no convincing evidence that pregnancy should be an absolute contraindication to the use of standard vaccines. With regard to live vaccines, only smallpox vaccine has been shown to cause fetal malformation. Despite concerns that attenuated rubella vaccine virus might cause congenital abnormalities, rubella vaccine (either monovalent or as MMR) has been given to pregnant women (usually inadvertently) without harm to the fetus. Even though the rubella vaccine virus can infect the fetus if given in early pregnancy, there is no evidence that it causes congenital rubella syndrome in infants born to susceptible mothers immunized during pregnancy, and rubella immunization during pregnancy is not an indication for termination. To date, congenital varicella syndrome has not been identified in women who have been accidentally immunized in early pregnancy. Furthermore, no evidence exists of risk from immunizing pregnant women with inactivated virus or bacterial vaccines or toxoids. Resulting from these considerations the following is concluded: – Although only of theoretical concern, pregnancy is a contraindication for measles, mumps, rubella, and varicella vaccines. Women of child-bearing age should avoid becoming pregnant for 1 month after immunization – Persons who receive MMR vaccine do not transmit the vaccine viruses to contacts; transmission of varicella vaccine virus to contacts has been reported, but is rare. MMR and varicella vaccines could be administered, when indicated, to the children and other household contacts of pregnant women

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– If a pregnant woman is likely to be at significant risk of an infection that can be prevented by other live (other than MMR or varicella vaccines), inactivated or toxoid vaccines, then the vaccine should be used: In case of a significant risk of poliomyelitis, IPV can be given if the series of injections can be completed before the anticipated exposure; pregnant women who must travel to areas where the risk for yellow fever is high should receive yellow fever vaccine, because the limited theoretical risk from immunization is substantially outweighed by the risk for yellow fever. Conversely, if the risk of infection from a particular disease is not immediate and significant, then the relevant vaccine should not be used, or its use should be postponed until after the pregnancy. In some cases, changing travel plans can eliminate the risk of exposure and therefore the need for immunization. – Women in the second and third trimesters of pregnancy have been demonstrated to be at increased risk for hospitalization from influenza. Therefore, routine influenza immunization is recommended for healthy women who will be beyond the first trimester of pregnancy (i.e., * 14 weeks of gestation) during influenza season. Women who have medical conditions that increase their risk for complications of influenza should be immunized before the influenza season, regardless of the stage of pregnancy [8, 17].

Immunization of pre-term babies Despite their immunological immaturity, pre-term babies should be immunized according to the recommended schedule and precautions at the usual chronological age, provided that they are doing well and that there are no contraindications to vaccination. Birth weight and size are not factors in deciding whether to postpone routine vaccination of a clinically stable premature infant, except for hepatitis B vaccine (see below). OPV, which might spread the live vaccine virus to other babies in the hospital, should not be given until the time of discharge. Alternatively, IPV can be used. Studies have demonstrated that decreased seroconversion rates might occur among certain premature infants with low birth weights (i.e., < 2000 g) after administration of hepatitis B vaccine at birth. However, by chronological age 1 month, all premature infants, regardless of initial birth weight or gestational age are as likely to respond as adequately as older and larger infants. A premature infant born to HBsAg-positive mothers and mothers with unknown HBsAg-status must receive immunoprophylaxis with hepatitis B vaccine and hepatitis B immunoglobulin (HBIG) ) 12 h after birth. If these infants weigh < 2000 g at birth, the initial vaccine dose should not be counted towards completion of the hepatitis B vaccine series, and three additional doses of hepatitis B vaccine should be administered, beginning when the infant is age 1 month. The optimal timing of the first dose

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of hepatitis B vaccine for premature infants of HBsAg-negative mothers with a birth weight of < 2000 g has not been determined. However, these infants can receive the first dose of the hepatitis B vaccine series at chronological age 1 month. Premature infants discharged from the hospital before chronological age 1 month can also be administered hepatitis B vaccine at discharge, if they are medically stable and have gained weight consistently. All pre-term babies born at less than 28 weeks of gestation or with chronic lung disease should be offered the 7-valent pneumococcal conjugate vaccine at 2, 4 and 6 months of age with a fourth dose at 12–18 months of age, and a 23-valent pneumococcal polysaccharide vaccine booster during the 3rd year of life [8, 17].

Immunization of individuals with chronic diseases Chronic diseases (such as asthma, chronic lung and heart diseases, congenital heart diseases, cystic fibrosis; celiac disease; diabetes and other metabolic diseases; renal dysfunction, nephrotic syndrome and other chronic organ failures; stable neurological conditions and Down’s syndrome) in children and adults increase the risk from infectious diseases and are known to predispose to complications of infectious diseases. In general, children and adults belonging to these groups at risk should be immunized according to the schedules recommended in a given country and as a matter of priority. The small potential risk from immunization outweighs by far the much greater risk from complications of vaccine-preventable disease. Although the considerations are valid for the majority of immunizations in children and adults with chronic diseases, the risks from influenza and pneumococcal disease and their prevention through immunization should be considered as a matter of priority. This includes the use of influenza vaccine in severe asthma, chronic lung disease, congenital heart disease and Down’s syndrome; pneumococcal conjugate vaccine in children with renal failure, persistent nephrotic syndrome and certain anatomical abnormalities; and pneumococcal polysaccharide vaccine in adults with certain chronic medical conditions mentioned above. Note: Recommendations for use of influenza and pneumococcal polysaccharide vaccine are somewhat similar; the two vaccines can be co-administered at the same visit [7, 8].

Immunization of individuals with impaired immunity Immunodeficiency conditions are grouped into primary and secondary disorders. Primary disorders are inherited and include humoral (B lymphocyte) immunodeficiencies, cell-mediated (T lymphocyte) immunodeficiencies, disorders of the complement and phagocytic function. Secondary disorders are acquired and occur in individuals with HIV infection, asplenia,

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malignant neoplasms, transplantation(s) or immunosuppressive or radiation therapy [18]. The immunization of individuals with impaired immune systems presents several problems. Firstly, the immune response to vaccines may be inadequate and, secondly, there is a risk that some live vaccines may themselves cause progressive infection. Degrees of immunodeficiency vary from insignificant to profound, and this should be taken into account when considering a schedule of vaccination, as should the risk of acquisition of the infection one is trying to prevent. Although it may be logical to give higher or more frequent doses of vaccines to these patients, in many cases there are insufficient data to advocate such measures. Because of the uncertainty of the immune response in some immunodeficient patients, it may be useful to measure post-vaccination antibody titers in groups such as children who have received hemopoietic stem cell transplants. Concerning vaccine response and immunodeficiency, considerable data on immunization in HIV-infected individuals, particularly children, are available, and provide valuable reassurance about immunogenicity, effectiveness and safety of vaccines administrated to the immunocompromised, whereas experience with immunization in persons with other specific disorders is lacking and mainly based on theoretical considerations. Moss and colleagues [19] have recently provided an overview on the most important studies of immunization in HIV-infected children. Table 3 summarizes the data on immunogenicity and effectiveness. The studies under review show wide variations in the age of immunization, the number of vaccine doses received, the interval between immunization and assay, the type of antibody assay used and the degree of immunosuppression. In general, seroconversion rates and geometric mean titers are lower in HIV-infected children than in uninfected children and infected children are more likely to lose antibody within few years after immunization. Placental transfer of maternal antibodies may be impaired in HIV-infected women. This correlates with an improved response to measles vaccine administered at 6 months of age. Studies in progress evaluate the immunogenicity of measles immunization at 6 and 9 months of age in HIV-infected children. Experience in southern Africa suggests that the measles incidence can be reduced in regions of high HIV prevalence by maintaining high immunization coverage coupled with periodic supplemental campaigns.

Current general recommendations for vaccine use in immunodeficient individuals For immunodeficient individuals, the general recommendations are: – BCG and smallpox vaccines are always contraindicated - OPV should not be given to the patient or to the patient’s parents or siblings; IPV should be used instead.

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Table 3. Immunogenicity and effectiveness of immunization in HIV-infected children (adapted from [19]) Vaccine

Seroconversion rate

Geometric mean titer (GMT)

Diphtheriatetanus toxoid

40–100%

Lower than More rapid uninfected decline than children in uninfected children

Pertussis (wP/aP)

Lower than in uninfected children

Hepatitis B

25–50%

More rapid decline than in uninfected children

Hib conjugated

37–86%

Lower than More rapid uninfected decline than children in uninfected children

Meningococcal

Antibody per- Booster sistence response

No effect in children after extra or higher doses

Effectiveness Studie Field

no longterm follow-up studies

Rapid antibody increase due to immunological memory

No data available

Pneumococcal

Better antibody response to conjugated vaccine than that of PS vaccine

In Ugandan adults 23-valent PS vaccine did not prevent invasive disease

BCG

Tuberculin test not a good predictor of protection; no data to permit definite conclusions re effectiveness of BCG in HIV-infected children

Polio vaccine

> 90% after 3 doses

Measles

17–100%, median value 60%

No studies; polio eliminated from several high HIV prevalence countries More rapid decline than in uninfected children

Generally poor

Yellow fever Much lower than in uninfected children

– Immunodeficient travelers should not be given live oral cholera or typhoid vaccines; Vi polysaccharide typhoid vaccine should be used instead. – Yellow fever vaccine is only indicated if the patient must travel to an area where there is a high risk of yellow fever. Most immunodeficient patients

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should obtain exemption certificates of immunization ratified by health authorities and immigration departments where international immunization requirements are the only reason for yellow fever immunization. MMR and varicella-zoster vaccines may be given to children with HIV infection who do not have evidence of severe immunosuppression. – Contacts of immunodeficient patients: healthy siblings and close contacts of immunodeficient children should be immunized with MMR and varicella-zoster vaccines to prevent them from infecting their immunodeficient sibling; there is no risk of transmission of the MMR vaccine viruses and there is an almost negligible risk of transmission of varicella-zoster vaccine virus; these close contacts should be given IPV and not OPV when being given routinely scheduled vaccines. – Morbidity and mortality from influenza and invasive pneumococcal disease are increased in all significantly immunodeficient patients. They should receive annual influenza immunization and either 7-valent pneumococcal conjugate vaccine or 23-valent pneumococcal polysaccharide vaccine, depending on their age; although the immune response to pneumococcal polysaccharide vaccine may be suboptimal in those individuals, the vaccine is nevertheless strongly recommended [5, 7, 8, 17].

Immunization and corticosteroid administration In adults, daily doses of oral corticosteroids in excess of 60 mg prednisolone (or equivalent), and in children doses in excess of either 2 mg/kg per day for more than 1 week or 1 mg/kg per day for more than 4 weeks, are associated with significant immunodeficiency. However, even lower doses may be associated with some impairment of immune response. For adults treated with systemic corticosteroids in excess of 60 mg/day, live vaccines (such as MMR, OPV, varicella-zoster and BCG) should be postponed until at least 3 months after treatment has stopped. Children on daily doses of 2 mg/kg per day of prednisolone or equivalent for less than 1 week, and those on lower doses or alternate-day regimens for longer periods, may be given live virus vaccines. The use of inhaled steroids is not a contraindication to the use of live vaccines [8].

Recommendations for immunization of HIV-infected children and women of childbearing age In collaboration with UNICEF, WHO has established guidelines [20] for immunization of HIV-infected children and women of childbearing age with recommended vaccines (Tab. 4). It is recommended that individuals with known or suspected asymptomatic HIV infection receive all recommended vaccines as early in life as possible, according to the nationally

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Table 4. WHO/UNICEF recommendations for immunization of HIV-infected children and women of childbearing age [20] Vaccine

Asymptomatic HIV infection

Symptomatic HIV infection

BCG

Yes

No

Birth

DTP

Yes

Yes

6,10,14 weeks

OPV*

Yes

Yes

0, 6, 10, 14 weeks

Measles

Yes

Yes

6 and 9 months

Hepatitis B

Yes

Yes

As for uninfected children

Yellow fever

Yes

No

Tetanus toxoid

Yes

Yes

Optimal timing of immunization

5 doses

*IPV can be used as an alternative for children with symptomatic HIV infection

recommended schedules. Because of the risk of early and severe measles infection, these infants should receive a dose of standard measles vaccine at 6 months of age with a second dose as soon after age 9 months as possible. Individuals with symptomatic HIV infection can receive all recommended vaccines except BCG and yellow fever vaccines. In asymptomatic children, the decision to give BCG should be based on the local risk of tuberculosis (TB): where the risk of TB is high, BCG is recommended at birth or as soon as possible thereafter, in accordance with standard policies for immunization of non-HIV-infected children; in areas where the risk of TB is low, but BCG is recommended for routine immunization, BCG should be withheld from individuals known or suspected to be infected with HIV. Similar recommendations exist in many countries, as an example the recommendations of the (US) Advisory Committee on Immunization Practices (ACIP) for immunization of immunocompromised children [21] are provided in Table 5. There are minor differences between recommendations for HIV-infected and other immunodeficient children. Currently, IPV is used as the vaccine of choice without any contraindication due to immunodeficiency.

Immunization of patients with MS Adults with MS should be given influenza and pneumococcal polysaccharide vaccines. There is clear evidence that these patients have an increased risk of complications following natural influenza and pneumococcal disease, whereas the administration of these vaccines is not associated with an increased risk of exacerbations of MS [8, 17].

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Table 5. Contraindications for childhood vaccines – ACIP [6, 7] Vaccines

Immunize?

Family history Note 1: Do not give OPV to a member of a household with a family history of immunodeficiency until the immune status of the recipient and other children in the family is documented Note 2: Varicella vaccine should not be administered to a person with a family history of congenital or hereditary immunodeficiency in parents or siblings unless that person’s immune competence has been clinically substantiated or verified by a laboratory

OPV Varicella All others

See Note 1 See Note 2 Yes

In household contact

OPV All others

No Yes

In recipient (hematological and solid tumors, congenital immunodeficiency, long-term immunosuppressive therapy, including steroids) Note 3: Varicella vaccine should not be administered to persons who have cellular immunodeficiencies, but persons with impaired humoral immunity may be vaccinated. A protocol exists for use of varicella vaccine in patients with acute lymphoblastic leukemia (ALL).

OPV MMR Varicella All others

No No See Note 3 Yes

In recipient (asymptomatic) Note 4: Varicella vaccination should be considered for asymptomatic or mildly symptomatic HIV infected children with age-specific T cell percentages of 25% or higher Note 5: MMR vaccination is recommended for all asymptomatic HIV-infected persons who do not have evidence of severe immunosuppression and for whom measles vaccination would otherwise be indicated

OPV Varicella MMR All others

No See Note 4 See Note 5 Yes

In recipient (symptomatic) Note 6: MMR vaccination should be considered for all symptomatic HIV-infected persons who do not have evidence of severe immunosuppression or of measles immunity

OPV Varicella MMR All others

No See Note 4 See Note 6 Yes

In household contact

OPV All others

No Yes

Immunodeficiency

HIV infection

Immunization of patients with bleeding disorders and patients receiving anticoagulant therapy Intramuscular injection may lead to hematoma formation in patients with bleeding disorders and to pressure necrosis, muscle contractures or nerve compression in patients with severe coagulopathies. On the other hand, these patients have an increased risk for acquiring hepatitis B and at least the same risk as the general population of acquiring other vaccine-preventable diseases. When hepatitis B or any other vaccine is indicated for a patient with a bleeding disorder or a person receiving anticoagulant therapy,

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the vaccine could be administered intramuscularly if, in the opinion of a physician familiar with the patient’s bleeding risk, the vaccine can be administered with reasonable safety by this route. A fine needle () 23 gauge) should be used for the immunization and firm pressure applied to the site, without rubbing, for * 2 min. The patient or family should be instructed concerning the risk for hematoma from the injection. Patients with platelet counts of less than 50 × 109/L should not receive intramuscular injections. The subcutaneous or intracutaneous route should be considered as an alternative to the intramuscular route in patients with bleeding disorders. Children with inherited coagulopathies should receive factor replacement prior to intramuscular injection [8, 17].

Immunization of recent recipients of human immunoglobulin With the exception of yellow fever vaccine, the immune response to live viral vaccines may be inhibited by normal human immunoglobulin. Therefore, live virus vaccines should be given 3 weeks before or 3 months after a dose of immunoglobulin. If an individual is under medical treatment with high-dose or intravenous immunoglobulin, the physician who initiated this treatment should be consulted [8].

Immunization and breast-feeding Breast-fed infants should be immunized according to routinely recommended schedules. Although live vaccines multiply within the mother’s body, the majority has not been demonstrated to be excreted in human milk. Rubella vaccine virus might be excreted in human milk. However, the virus usually does not infect the infant. Where infection has occurred in an infant, it has been mild because the virus is attenuated. Inactivated, recombinant, subunit, polysaccharide, conjugate vaccines and toxoids pose no risk for mothers who are breast-feeding or for their infants [8, 17]. Special recommendations for the immunization of hematopoietic stem cell transplant (HSCT) recipients and for solid organ recipients before transplantation exist [22–25].

Contraindications and false contraindications Contraindications Contraindications to immunization dictate circumstances when vaccines should not be given because the condition in an individual increases the risk for a serious adverse reaction following immunization. The majority of con-

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traindications are temporary, and the vaccine can be given later. However, in many cases immunization is delayed or denied because of conditions falsely believed by the physician or the health worker to constitute a contraindication. The World Health Organization and the majority of countries have established and periodically updated lists of contraindications (and often also false contraindications) to offer expert advice for physicians and health workers involved in immunization for individual cases where doubt occurs. Genuine contraindications are few and the numbers of individuals to whom they apply are fewer still. The various lists of contraindications include mainly: – acute illness – altered immunity – pregnancy – severe adverse events after a previous dose – children with neurological disorders – anaphylaxis and allergy to vaccines and vaccine constituents. Depending on the individual vaccines, contraindications are provided specifically.

False contraindications Conditions that are NOT contraindications to immunization are called ‘false contraindications’. Examples are the following conditions: – minor illness, such as upper respiratory infection or diarrhea, with temperature < 38.5 °C – asthma or other atopic manifestations – family history of convulsions – treatment with antibiotics, low-dose or locally acting corticosteroids – dermatoses, localized skin infection – chronic diseases of heart, lung, kidney and liver – stable neurological conditions, such as Down’s syndrome – history of jaundice after birth – prematurity – malnutrition – mother pregnant – in incubation period of illness. Some of these conditions increase the risk from infectious diseases and such individuals should be immunized as a matter of priority [17, 26].

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References 1 2 3

4

5 6

7

8

9 10 11

12

13

14

15 16 17

Dittmann S (2006) Elimination der Poliomyelitis. Polio-Nachrichten 2: 11–12 Dittmann S (2001) Vaccine safety: risk communication – a global perspective. Vaccine 19: 2446–2456 Campbell H, Ramsay M, Gungabissoon U, Miller E, Andrews N, Mistry A, Mallard R, Borrow R (2004) Impact of the meningococcal C conjugate vaccination programme in England. Summary Surveillance Report from the Health Protection Agency, Centre for Infections Immunisation Department to end December 2004. Centers for Disease Control and Prevention (2002) Epidemiology and prevention of vaccine-preventable diseases. In: Atkinson W, Hamborsky J, McIntyre L, Wolfe S (eds): The Pink Book, 9th edn, Appendix G: Reported cases and deaths for vaccine-preventable diseases. Public Health Foundation, Washington, D.C. Six common misconceptions about vaccination and how to respond to them. htpp://www.cdc/nip/publications/6mishome.htm (accessed August 14, 2006) Offit PA, Quarles J, Gerber MA, Hackett CJ, Marcuse EK, Kollman TR, Gellin BG, Landry S (2002) Addressing parents’ concerns: do multiple vaccines overwhelm or weaken the infant’s immune system? Pediatrics 109:124–129 Public Health Agency of Canada (2002) Talking with patients about immunization. In : Canadian Immunization Guide 2002, Public Health Agency of Canada, Ottawa, 42–54. Responding to questions and concerns about immunization. In: Australian Immunization Handbook, 8th edn 2003, online. htpp://www9.health.gov.au/ immhandbook (accessed August 14, 2006) Global Advisory Committee on Vaccine Safety (2003) MMR and autism. Weekly Epidemiol Rec 78: 18 Global Advisory Committee on Vaccine Safety (2005) Thiomersal: neurobehavioural studies in animal models. Wkly Epidemiol Rec 80: 3–4 Institute of Medicine Immunization Safety Reviews: Measles-mumps-rubella vaccine and autism. National Academy Press, Washington DC 2001. http://www. cdc.gov/nip/news/iom-04–24.htm (accessed August 14, 2006) US Centers for Disease Control. Vaccines and autism – references. http:// www.cdc.gov/nip/vacsafe/concerns/autism/autism-ref.htm (accessed August 14, 2006) Davis RL, Kramarz P, Bohlke K, Benson P, Thompson RS, Mullooly J, Black S, Shinefield H, Lewis E, Ward J et al (2001) Measles-mumps-rubella and other measles-containing vaccines do not increase the risk for inflammatory bowel disease. Arch Pediatr Adolesc Med 155: 354–359 US Centers for Disease Control. Measles vaccine and inflammatory bowel disease – references. http://www.cdc.gov/nip/vacsafe/concerns/autism/ibd. htm#references (accessed August 14, 2006) (2002) Immunization Safety Review: Hepatitis B Vaccine and Demyelinating Neurological Disorders. National Academy Press, Washington, D.C. (2003) Immunization Safety Review: Vaccinations and Sudden Unexpected Death in Infancy. National Academy Press, Washington, D.C. (2002) Recommendations of the Advisory Committee on Immunization

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Practices (ACIP). General recommendations on immunization. Morb Mortal Wkly Rep 51: No RR-2. American Academy of Pediatrics (2003) Immunocompromised children. In: LK Pickering (ed): 2003 Red Book: Report of the Committee on Infectious Diseases. 26th ed. Elk Grove Village, IL, 69 Moss WJ, Clements CJ, Halsey NA (2003) Immunization of children at risk of infection with human immunodeficiency virus. Bull World Health Organ 81: 61–70 EPI Vaccines in HIV-infected Individuals. htpp://www.who.int/vaccines-diseases/diseases/HIV.shtml (accessed August 14, 2006) Contraindications for childhood vaccinations. htpp://www.cdc.gov/nip/recs/contraindications.htm (accessed August 14, 2006) (2000) Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. Recommendations of CDC, the Infectious Disease Society of America, and the American Society of Blood and Marrow Transplantation. MMWR 49: RR-10 Avery RK, Ljungman P (2001) Prophylactic measures in the solid-organ recipient before transplantation. Clin Infect Dis 33 (Suppl 1): 15–21 Stark K, Günther M, Schönfeld C, Tullius SG, Bienzle U (2002) Immunisations in solid-organ transplant recipients. Lancet 359: 957–965 Ljungman P (2004) Immunization in the immunocompromised host. In: SA Plotkin, WA Orenstein (eds): Vaccines, 4th edn. Saunders, Philadelphia, 155– 168 (1998) Contraindications for vaccines used in EPI. Wkly Epidemiol Rec 63: 279–281

Pediatric Infectious Diseases Revisited ed. by Horst Schroten and Stefan Wirth © 2007 Birkhäuser Verlag Basel/Switzerland

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Gonorrheal ophthalmia neonatorum: Historic impact of Credé’s eye prophylaxis Axel Schmidt Axel Schmidt, Institute of Microbiology and Virology, Faculty of Medicine, University Witten/ Herdecke, Stockumer Str. 10, 58448 Witten, Germany

Abstract In the pre-antibiotic era gonorrhea showed a high prevalence also in industrialized countries. In Germany, more than 10% of all newborns developed gonorrheal ophthalmia neonatorum. Clinical courses of gonorrheal ophthalmia neonatorum were quite different in their severity but often caused significant impairment of eyesight up to total blindness in more than 5%. This accounted for 25–40% of cases of blindness in Germany. It was Carl Siegmund Franz Credé (1819–1892), a German obstetrician, who introduced the eye prophylaxis of eye drops containing 2% silver nitrate solution to every newborn child in his clinic in Leipzig on June 1st 1880. The incidence of gonorrheal ophthalmia neonatorum immediately decreased from 10% to 0%. Credé actively communicated these results and immediately published them in four publications within a time period of 3 years. These publications, which are discussed here, are written in a very pragmatic and strictly clinical style, ignoring new basic scientific insights into the microbiology of gonorrhea and the discovery of the corresponding pathogen, the “Micrococcus” by Albert Neisser, which Credé considered unimportant for his purposes. Against a high degree of opposition by many physicians, Credé put all enthusiasm into the call for education of midwives in this technique. Credé knew that this was the central way to ensure that all newborns could obtain this prophylaxis, including outpatients and home deliveries. Credé’s eloquence led to the rapid spreading of “his” eye prophylaxis over the rest of the world. The concentration of silver nitrate was often reduced from 2% to 1% thereafter and in most countries the performance of this prophylaxis was rapidly enforced by law. By introducing this method, Credé saved or improved the eyesight of millions of people – a significant contribution to obstetrics, neonatology and pediatrics, ophthalmology and mankind. Still today, in the antibiotic era, other topical regimens for antiseptic prophylaxis against ophthalmia neonatorum are often referred to as “Credé’s prophylaxis”.

“However, the broad use of silver as a powerful clinical tool against infections is still in the future, because its full range of activity remains to be elucidated.” Q.L. Feng et al., 2000 [1]

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The endangered eyesight In the pre-antibiotic era, i.e., until almost the middle of the 20th century, gonorrhea and ophthalmia neonatorum showed a high prevalence also in industrialized countries [2–7]. In the middle of the 19th century more than 10% of all newborns in Germany developed gonorrheal ophthalmia neonatorum. Clinical courses of gonorrheal ophthalmia neonatorum were quite different in their severity but often caused a huge and irreversible damage to the eyes with a significant impairment of eyesight up to total blindness as final outcome of the disease in more than 5% of the infections. This accounted for 25–40% of cases of blindness in Germany [8–11]. What about silver as a broadly acting antiseptic?

Carl Siegmund Franz Credé, introducer of the antiseptic eye prophylaxis with silver nitrate Carl Siegmund Franz Credé (23.12.1819–14.03.1892) (Fig. 1) [8, 12–15] was born in Berlin where he went to school and studied medicine, with the exception of one semester at the university of Heidelberg (Germany). The principle of “nihil nocere” – an attempt to keep necessary treatment approaches as mild and gentle as possible – was his general philosophy in medicine. After several years of postgraduate study in Austria, France, Belgium and Italy, he returned to Berlin in 1847 and was appointed assistant in obstetrics at Berlin’s clinic of obstetrics, where he remained until 1852. In 1850 he became a “Privatdozent” (university teacher) in obstetrics. In 1852 he was appointed Director of the Berlin School of Midwives and Physician in Chief to the inpatient division of obstetrics and gynecology of the Berlin Royal Charité Hospital. In 1856 Credé was appointed Professor of Obstetrics and Director of the inpatient hospital in Leipzig, Germany where he retired in 1887 because of his poor health condition due to prostate cancer. Within the time in Leipzig he became “Nestor of German midwifery” [8]. During his time in Berlin he made a significant contribution to obstetrics by introducing a new and safer method for the delivery of the placenta (“Credé’scher Handgriff”/Credé’s method) [16, 17]. Credé was a consistently modest person and did not claim priority for this method. This method is still used today in emergencies such as hemorrhage after delivery. The affiliation with Leipzig gave him the chance of fully living his talents as a clinician, academic teacher and administrator, and his department became very prestigious. He personally focused on obstetrics being convinced that improvements in obstetrics are a key parameter to reducing the number of gynecological impairments. The famous obstetrician and gynecologist Gerhard Leopold was Credé’s son-in-law [8]. Credé wrote several textbooks and original articles; he took over the editorship of gynecological journals of high reputation and was awarded the

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Figure 1. Carl Siegmund Franz Credé (1819–1892)

“Senckenberg Preis/Senckenberg Award” due to his outstanding achievements in obstetrics and medicine [15]. Further, he received the prestigious post of a “Geheimer Medicinalrath/Aulic Counsellor”. After 1860, Credé began to work on optimizing warming devices for premature and feeble tiny children (“Erwärmungswanne”) [18], which he established at his department thereafter – a significant contribution to obstetrics and a precursor of the incubators for newborns today. Whereas the “Credé’scher Handgriff” and the “Erwärmungswanne” were mostly recognized by the public in the lifetime of Credé, he introduced an eye prophylaxis for ophthalmia neonatorum (“Credé’sche Prophylaxe”), which achieved highest recognition especially amongst physicians [15]. The prophylactic application of “Argentum nitricum/silver nitrate” 1:50 aqueous solution was introduced in all newborns from June 1st 1880 onwards in the Leipzig obstetrics department.

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Credé wrote three consecutive publications with the same title on this topic “Die Verhütung der Augenentzündung der Neugeborenen” [19–21] (Prevention of inflammatory eye disease in the newborn) in the Journal “Archiv für Gynäkologie” between 1881 and 1883. The first [19, 22] focused on methodological aspects of the eye prophylaxis and will be the core issue of this chapter. His second publication presented more cases, and stressed the performance by midwives and by general practitioners. The third summarized his results and comprehensively addressed new aspects of etiology and practicable everyday prevention of ophthalmia neonatorum by his method. The second and third paper are discussed on the background of the “revolutionary” first one later in this chapter. In 1884, Credé summarized central aspects of his three publications in a booklet version in English [23]. An abbreviated English translation, translated by the WHO [22], of the first paper is given below. For systematic purposes, the original of the first paper of Credé in German language [19] is attached to this chapter as an “Addendum”.

“Prevention of Inflammatory eye disease in the newborn. Information from the Maternity Clinic Leipzig by Credé” [22] “I am (…) publishing the following information concerning the prevention of inflammatory eye disease in the newborn (…) in this Archive because the disease is almost invariably caused by infection during delivery and is therefore directly related to a diseased condition of the female genitals. Responsibility for prevention of the disease must also lie solely with obstetricians and midwives. I shall confine my remarks exclusively to the practical question of prophylaxis. (…) My request for further testing of the prophylaxis I am recommending is therefore addressed to those of my colleagues who work in maternity hospitals or obstetric clinics and (…) are frequently confronted with this condition. Most obstetricians would probably share my view that the case of vaginal catarrh and infections that are so frequently encountered are attributable to gonorrheal infection and that the discharge remain infectious long after the specific symptoms of gonorrhea have disappeared; moreover, in some cases where there is virtually no further trace of discharge, the infection may still be considered to have occurred in the mother’s vagina when an inflammatory eye condition develops in the first few days after birth. Transmission of the infectious substance from another child with eye disease is inconceivable (…) inasmuch as every child who is suffering from inflammatory eye disease is moved with its mother to a ward that is entirely separate in all respects from the maternity ward. The possibility of mothers infecting their children, for example through fingers soiled by lochial dis-

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charge, is also remote because the child’s cot is always placed beyond reach of the mother, who only comes into contact with the child when the nurse places it on her breast. I am therefore convinced (…) that all affected children in (…) hospital (…) were infected solely by direct transmission of vaginal discharge to the eye during delivery. The infected eye usually begins to show symptoms of disease 2 or 3 days after birth, but also sooner or later – the sooner, the more serious the condition. (…) I have set myself the doubtless worthwhile task of finding effective ways and means of preventing this disease (…) and of detecting the infectious discharge. I initially focused on ensuring extensive and effective treatment and cleansing of the diseased vaginas of pregnant and delivering women. But the results were poor and unsatisfactory; although there were fewer cases of eye disease (…). I then began to disinfect the children’s eyes themselves and from then on the success recorded was surprisingly encouraging. My experiments proceeded as follows: first, the vaginas of all pregnant and delivering women admitted to the hospital with gonorrhea or chronic vaginal catarrh were cleaned out with lukewarm water or a light solution (2:100) of carbolic or salicylic acid as frequently as possible – every half hour in the case of delivering women. The incidence of eye disease declined but the problem persisted (…). In October 1879, I carried out my first test involving the introduction of prophylactic eye drops into the newborn babies immediately after birth, using a borax solution (1:60) because it seemed to be the mildest and least caustic substance. This was only done, however, in the case of children whose mothers were ill and whose vaginas had been cleansed during the whole delivery process in a manner described above. From December 1879, I replaced the borax by solutions of Argentum nitricum (1:40), which were injected into the eyes shortly after birth. The eyes were carefully washed beforehand with a solution of salicylic acid (2:100). The children of sick mothers who were treated in this way remained healthy, while other children who had not been given preventive treatment (…) still fell ill, in two cases quite seriously. From 1 June 1880, all eyes without exception were disinfected immediately after birth by means of a weaker solution of Argentum nitricum (1:50). (…) a glass stick was used to introduce a single drop of liquid into each eye, which was gently opened by an assistant and which had been cleaned beforehand with ordinary water. Then the eyes were cooled for 24 h with a canvas cloth soaked in salicylic water (2:100). The numerous vaginal douches, on the other hand, were abandoned (…). All children treated in this way remained free from even mild attacks of inflammatory eye disease, although many mothers showed advanced symptoms of vaginal blenorrhea (…). Only one child (…) fell ill on the 6th day with a moderate inflammation of the conjunctiva of the left eye, without swelling of the

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eyelid, which healed within 3 days. It emerged that, quite by chance, owing to pressure of work, the prophylactic eye drops had not been administered to this child. To date, no adverse effect on the treated eyes has been observed. Not infrequently the administration of the eye drops is followed by a slight hyperemia and in some cases by slight increased secretion from the conjunctiva in the first 24 h. Then these symptoms disappear. They could perhaps be avoided if further tests indicate that a weaker solution of Argentum nitricum is sufficient. As has been shown, the procedure is simple, (…) completely without risk and seemingly reliable in terms of its effect. (…) my set of observations is (…) still sufficiently extensive and striking to warrant further urgent application of the procedure. I wish to lay special emphasis on the finding that the desired effects are achieved through disinfection of the eyes themselves rather than the vagina. It is to be hoped that the future will tell whether the eye procedure that I have been using is the best and most reliable one (…). For the time being, I have no reason to deviate from my own method. Needless to say, the successful banishment of inflammatory eye diseases at least from maternity hospitals and clinics would constitute a major achievement in many respects. Lastly, I wish to present some figures for cases of inflammatory eye disease observed in this maternity hospital in recent years. (…). Year

Number of births

Number of cases of inflammatory eye disease

Percentage

1874

323

45

13.6

1875

287

37

12.9

1876

367

29

9.1

1877

360

30

8.3

1878

353

35

9.8

1879

389

36

8.2

1880 (until 31 May)

187

14

7.6

1880 (from 1 June to 8 December)

200

1*

0.6

*This is the case in which the eyes were not disinfected; the figure should therefore read 0.0%

In the first paper (1881; [19]) Credé strictly focused on practical aspects of prophylaxis of ophthalmia neonatorum. It was recognized that the way of transmission was by direct contact with vaginal excretions. He described hygienic procedures of cleaning the vagina, described several interim stages of eye drops applied to the newborn, and ended up with the abandonment of vaginal douches/extensive cleaning of the vagina and introduction of

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the consequent direct eye prophylaxis in every delivered newborn with a single drop of 2% silver nitrate solution per eye applied to the middle of the cornea by a glass rod from June 1st 1880 onwards. This prophylactic method was declared as highly efficacious, easy to handle and without adverse effects apart from a slight hyperemia and some increased secretion from the conjunctiva within the first 24 h in some cases. Already in this paper Credé recommended that this procedure of eye prophylaxis should also be put into the hands of midwives. Etiologically, Credé only mentioned an “Infektionsstoff” (contagious agent) as reason for the disease; further microbiological aspects – including Neisser’s new discovery of 1878/1879 – are not addressed. The second paper (1881; [20]) verified the effectiveness of this procedure by reports of an additional 400 new cases (first paper [19]: 200 cases) including 300 newborns treated with a simplified regimen. In contrast to the method described first, in the simplified regimen, the cord was cut and the newborn was washed. Thereafter the eyes were wiped clean with water, and a 2% silver nitrate solution was applied by the same way as mentioned before. In contrast, no consecutive treatment/manipulations at the eyes were performed. None of the 400 newborns developed ophthalmia neonatorum. In this paper, Credé highlighted that the application of 2% silver nitrate solution directly into the newborn’s eye has to be performed immediately after the first manipulations, as mentioned above, after delivery. Further, for the first time, Credé addressed the aspect of introducing this method of eye prophylaxis to general practitioners active in obstetrics for prophylaxis of corresponding newborn outpatients. In particular, the need for putting the prophylaxis into the hands of midwives was stressed again. In addition, the aspect of treatment for ophthalmia neonatorum by stronger solutions of silver nitrate was addressed for the first time. The most critical/political aspect coming up in this paper was the suggestion – as mentioned before – of giving the prophylaxis into the hands of midwives, which meant breaking with a prestigious medical privilege in obstetrics by apparently by-passing the outstanding authority of the physician/obstetrician. Credé suggests that every midwife should obtain a bottle of 2% silver nitrate solution and a corresponding glass rod. His interest was that hereby ophthalmia neonatorum could be eradicated. Microbiologically, the disease was attributed to a “Contagium” as the causative agent without more detailed discussions and without presentation of Neisser’s actual new insights. The third paper (1883; [21]) gave a synopsis of Credé’s overall experiences on ophthalmia neonatorum and was divided into two parts. The first part focused on the aspect of prevention and the second part on the aspect of etiology of ophthalmia neonatorum. In the relatively short first part of this paper, Credé stated that he considered the issue of prophylaxis for ophthalmia neonatorum as solved. The method suggested by him appeared easy to handle, safe and effective. He gave the advice not to deviate from this proposed method, as in some insti-

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tutions, where modified procedures were performed, poorer results were achieved. The second part of this paper was announced to focus on etiological aspects of ophthalmia neonatorum. Indeed, this was only partly the issue, and this part of the paper was in many perspectives highly political. The transmission of ophthalmia neonatorum via direct vaginal contact was reaffirmed and aspects such as duration of the delivery period, gender of the newborn, etc., were discussed from the etiological perspective. Credé stated that he considered the “Diplococcus Neisser” the most probable causative pathogen (“specifisch gonorrhoeisches Virus Diplococcus Neisser”). This one sentence of his series of publications was Credé’s only hint at Neisser’s tremendous achievements concerning the etiology of gonorrhea (Neisser’s second comprehensive publication on the etiology of gonorrhea had been published 1 year before in 1882). In the following part, Credé stated that it was his achievement, having obtained the insight that vaginal douches were almost ineffective and that the contagious agent had to be destroyed sufficiently, that the prophylactic efforts, which had not been performed before, were put into place. As a method for the sufficient destruction of the contagious agent he stated again the administration of 2% silver nitrate solution directly into the eyes of every newborn child, including consecutive hygienic precautions to prevent a later inoculation of the child’s eye by vaginal discharge from the mother. As dose justification for the 2% silver nitrate solution he cites a study from Hecker [24], who performed the eye prophylaxis with a 1% silver nitrate solution. Of 133 children, 4 developed ophthalmia neonatorum in this study, although even Hecker pointed out that compliance was poor within this study, and it still remained unclear to the reader if the eyes were washed with NaCl solution afterwards, as described in the paper in case of treatment for ophthalmia neonatorum with aqueous silver nitrate solution. Credé ignored all of this argumentation and insisted that the 1% silver nitrate solution was ineffective for the prophylaxis of gonorrheal ophthalmia neonatorum, which he considered as an undisputable justification for his 2% regimen. Afterwards, a long, enthusiastic plea for giving the prophylaxis into the hands of midwives was given again. It was discussed that even potential misuse by midwives could not cause significant disadvantages in contrast to the tremendous advantages of a broad application of this prophylaxis. A lot of concerns against giving the prophylaxis into the hands of midwives, which were brought forward by physicians, were cited, discussed and declared invalid. At the end of this manuscript, Credé highlighted that on January 31st 1883 his prophylactic eye regimen was enforced by law for cases of hospital deliveries in Austria. The procedure should – by law – be performed only by physicians; indeed, Credé did not oppose in this special case. Nevertheless, he encouraged every country to release such a law.

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The complementary booklet on this issue (fourth publication), written in English language (1884; [23]), gave a comparable synopsis on gonorrheal ophthalmia neonatorum and its prophylaxis such as given in the third paper. Fascinating is, how extremely precise and concerned Credé was with issues he was dealing with. In the English publication, for example, he gave a very detailed description of the solution, its storage and the glass rod being applied. It was described that the solution of silver nitrate should be kept in a dark bottle made of glass with a glass stopper. The glass rod to be used should be 15 cm in length, 3 mm thick and rounded at both ends. The little bottle and glass rod had to be stored in a small drawer in the swaddling table. The solution had to be renewed every 6 weeks, but it was pointed out that it was not critical, concerning safety and efficacy, if the solution was accidentally used for a longer period of time. No room for personal freedom was left open concerning this issue. This description was an excellent reflection of Credé’s personality. With him, nothing was left open to accident and/or to spontaneous occurrence.

Ludwig Sigesmund Albert Neisser and insights into etiology and pathophysiology of gonorrhea at Credé’s time Ludwig Sigesmund Albert Neisser (1855–1916) was a German physician and bacteriologist [25–32]. He was a school classmate of Paul Ehrlich (1854–1915) in Breslau – former Germany – and studied medicine mainly in Breslau thereafter. Consecutively, he started specializing in dermatology, although he primarily intended to specialize in internal medicine but could not get an appointment as assistant in Breslau. Apart from working on echinococcosis (PhD thesis), leprosy and syphilis, he was the person who discovered the “Micrococcus” as the causative pathogen of gonorrhea. As a basis for this discovery, the botanist Ferdinand Cohn (1828–1898) taught Neisser Robert Koch’s (1843–1910) smear test for the microscopic examination of bacteria. Julius Friedrich Conheim (1839–1884) and Carl Weigert (1845–1904) taught him bacterial staining techniques, including the methylene blue staining technique. Further, Neisser had access to an excellent innovative Zeiss microscope that was equipped with Ernst Abbe’s (1840–1905) innovative condenser system and an oil-immersion object lens system. This equipment allowed him detailed microscopic examinations, which were not the usual “state of the art” in 1879, the year when Neisser discovered the “Micrococcus” microscopically. Finally, in 1879 Neisser published a paper “Über eine der Gonorrhoe eigenthümliche Micrococcenform” (“A form of Micrococcus typical for gonorrhea”) [33]. In this paper he was the first person to describe that a very typical form of a somewhat peach-like (semmelartig) “Micrococcus/Diplococcus” (“Micrococcus” [33, 34], “Micrococcenhaufen” [33], “Semmelform” [33, 34], “Diplococcus” [34]) was always found as sole bacteria in a large quantity

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in genital smears of patients suffering from symptomatic gonorrhea. He mostly observed this “Micrococcus” topologically associated with inflammatory cells and/or epithelia. Further, he stated that the best diagnostic results were obtained using the methylene blue staining technique, and that the microscopic picture was extremely typical for the disease and for him to be certain of the association. Beside a few doubts and the recommendation for a scientific proof, based on something like the later-discussed, communicated and published Koch-Henle postulates (1875–1885; ideas arising and postulated by Robert Koch in the late 1870s, “Wollsteiner Zeit”) [35], Neisser was at that time rather convinced that the “Micrococcus” was the causative agent of gonorrhea. This statement was the milestone of Neisser’s discovery of the “Micrococcus” as causative pathogen of gonorrhea. He further stated that he found this “Micrococcus” in eye smears of gonorrheal eye infections in adults and children. He already started with cultivation approaches in 1879, which were at that time not successful, probably due to the fact that Neisser’s poor health condition restricted the time he could spend on his scientific activities beside his clinical duties. In 1882 he published a second paper “Die Micrococcen der Gonorrhoe” (“Micrococci and gonorrhea”) [34]. This paper was a very comprehensive, but from the author’s view, somewhat unconventionally structured review paper in which Neisser – in the beginning – points out in a disappointed manner that it took over a year after his first publication for other scientists to pick up the topic of the “Micrococcus” and gonorrhea, and publish new insights on this issue. In this paper Neisser (a) extensively repeated his observations as stated in [33], and additionally (b) gave a drawn picture of the “Micrococcus” and its different division stages, (c) pointed out that other colleagues had also verified his observations (e.g., Aufrecht, Bókai, Brieger, Ehrlich, Gaffky, Haab, Hirschberger, Leber, Sattler, and Weiss), (d) reported that he successfully treated a case of gonorrheal eye infection with silver nitrate solution, (e) reported that he – beside other colleagues – had been successful in cultivating the “Micrococcus” in 1881/1882, (f) gave information on Bókai’s successful inoculation experiments with cultured “Micrococcus” material in volunteer male students achieving an acute and typical genital gonorrhea, (g) gave information that the “Micrococcus” was apathogenic on inoculating the conjunctiva of dogs and rabbits, and (h) gave information on current treatment options and the pathophysiology of gonorrhea, highlighting genital and ocular gonorrheal infections. Indeed, the name of Carl Credé was never mentioned. As Bókai’s insights appeared not to have convinced Neisser, he repeatedly stated in this paper [34] that the strict proof for the “Micrococcus” as causative agent of gonorrhea still had to be produced. This seems an exaggerated skeptic statement considering this broad data basis and the aspect that Neisser repeatedly stated in this paper that the “Micrococcus” could always, and only, be found in case of symptomatic gonorrhea. In contrast, the Koch-Henle postulates became overemphasized within the scientific

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community at that time, which also put Neisser under an extreme pressure concerning the validity of his insight that the “Micrococcus” was the causative agent of gonorrhea, due to the categorical force of demonstrating that a pathogen unambiguously fulfilled these postulates. Finally, it was Neisser’s friend and school classmate Paul Ehrlich who named Neisser’s “Micrococcus” the “Gonococcus”. Therefore, a lot of Neisser’s students named Neisser the “Father of Gonococcus” [30].

Discussion of Credé’s activities and his “four publications with the same title” In Credé’s case there was an urgent medical need for an effective prophylaxis against gonorrheal ophthalmia neonatorum. Credé realized that vaginal douches were almost ineffective in preventing ophthalmia neonatorum, and that a strong antiseptic agent for prophylactic application at the ocular infection site – the newborn’s eye – was needed. Potential irritative side effects had to be tolerable at this sensitive organ. Further, he recognized the threat of re-contamination of the eye by vaginal discharge especially in the first weeks after delivery so that strict hygienic requirements as well as teaching and education on this aspect became necessary and were introduced. Credé reduced the concentration of the 2.5% silver nitrate solution (eye drops) that he used initially to 2.0% and immediately recognized that he was “on the safe side” with this regimen, reducing the incidence of ophthalmia neonatorum from approximately 10% to 0%. Adverse effects of chemical eye irritations were considered insignificant or even almost ignored most probably due to the high medical benefit obtained by this technique. In contrast, Hecker’s tests and results with 1% silver nitrate solution were not properly analyzed, although, to our knowledge today, a 1% solution of silver nitrate would also have been appropriate with comparable effectivity but less irritative adverse effects at the eye. Credé was convinced of the urgent need of enforcing such an eye prophylaxis as soon as possible and there was obviously no time left for him for a proper dose finding study, e.g., verifying or falsifying Hecker’s observations. Further, Credé solely acted as a clinician and did not join the scientific activities around the Micrococcus/Diplococcus and gonorrhea discussion, which were driven by Neisser and were highly contemporary, within just years 1879 (first paper of Neisser describing the Micrococcus microscopically [33]) to 1882 (Neisser’s second, review publication on the Micrococcus with positive cultivation and inoculation results [34]). To the present author, it still remains an open historic miracle, as to why these two outstanding persons, Credé and Neisser, did not recognize each other appropriately. Credé could have obtained a lot of additional scientific merits by joining these discussions and activities, but despite this he strictly focused on the clinical aspects of prophylaxis of ophthalmia neonatorum, most probably to

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speed up and to enhance the pressure for an establishment of his prophylactic regimen as soon as possible. Credé, therefore, focused strictly on the obstetrician’s/neonatological aspect, ignoring all recent new microbiological discoveries concerning gonorrhea within his papers published in 1881 [19, 20], 1883 (here the “Micrococcus Neisser” is only mentioned in one short sentence as most probable pathogenic agent for gonorrheal ophthalmia neonatorum [21]) and 1884 (in English [23]). The author has never seen such a focused, condensed and straightforward approach without any scientific detour from the streamlined intentions as that performed by Credé. Credé additionally recognized that the midwives were the central persons/institution for rapidly spreading this prophylactic regimen to hospitalized patients/deliveries and outpatients. This led to a high degree of controversies with his colleagues as many of them realized this as a form of undermining the authority of the physician and obstetrician. Credé wrote two outstanding books for midwives, where the aspect of ophthalmia neonatorum was also included [36, 37]. Further, demonstrating his positive attitude towards the responsibilities of midwives, Credé became appointed “Nestor of German midwifery” [8]. This reflects that Credé had no concerns regarding a potential conflict of interests and/or competence between physicians/obstetricians and midwives. By this pragmatic, clinical, non-academic, and consequent way, by 2 years after Credé’s first publication on prophylaxis of ophthalmia neonatorum ([19]; 1881), his prophylaxis became enforced by law for clinical deliveries in Austria in 1883, which is highlighted in his 1883 publication [21]. Here Credé claimed that all countries should introduce his method and should enforce it by law [21]. His four publications on ophthalmia neonatorum all have the same title, are easy to read, clearly structured, and in most parts highly repetitive, and do not allow alterations of the suggested prophylactic regimen. For today’s understanding they appear almost like a guideline or kind of a directive. Even the famous ophthalmologist Lucien Howe (1848–1928) [38] was so impressed by this approach that he established it in the “New World”. Nevertheless, he used a weaker concentration of 1% silver nitrate as did many other physicians and countries by law. In addition, silver acetate was used alternatively instead of silver nitrate in many places [39]. In summary, Credé made a great contribution to mankind, broadly “enforcing” the eye prophylaxis against gonorrheal ophthalmia neonatorum within 2 years without spending any unnecessary time for the final “i-dot” of optimization of this technique.

Credé’s prophylaxis today In recent times, especially after the discovery and development of potent antibiotics, the etiology of ophthalmia neonatorum has changed signifi-

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cantly. Despite cases of ophthalmia neonatorum due to Neisseria gonorrhoeae infections, chlamydial eye infections in the newborn became more the primary focus compared to gonorrheal eye infections [40–45]. Silver nitrate and acetate show no sufficient activity in prophylaxis of chlamydial eye infections and exhibit irritative adverse effects of chemical conjunctivitis including consecutive psychological adverse effects (impairment in eye-to-eye contact in early maternal-infant attachment), which are currently under discussion [46–50]. Many antibiotic and other aseptic kinds of eye prophylaxis have therefore been considered and evaluated for prophylaxis of ophthalmia neonatorum [51–55]. At present, aseptic eye prophylaxis with povidone-iodine at different concentrations (preferably 1–2.5%) is often recommended [56–63]. Nevertheless, one aspect did not change: the name for the procedure itself. Independent of which compound the prophylactic eye drops contain, the procedure of eye prophylaxis against ophthalmia neonatorum is often still today declared as “Credé’s prophylaxis” [64–67]. Further, with the program “VISION 2020”, the WHO states that ophthalmia neonatorum is still an important health issue today [68, 69], which reflects its persistent actuality.

Some confusion about Carl Credé? Some confusion might arise as two other Carl Credé are described within medical history. Some brief information is given here to avoid confusion:

Benno Carl Credé Benno Carl Credé (also: Carl Benno Credé; 1847–1929) was Carl Siegmund Franz Credé’s son [70–72]. Despite his christian name Benno, he also appears under the name Carl Credé in the literature. Credé studied medicine and specialized as a surgeon in Dresden, Germany, thereafter. Scientifically, Credé followed the steps of his father in so far as performing research activities on silver in colloidal form, which he introduced into medical practice in 1897. This was possible by collaboration with the company “Chemische Fabrik von Heyden”, and led to the development of the “Collargolum Credé” (“Collargol”) [73, 74] for systemic, parenteral therapy. Thus, it should be highlighted that the “Collargolum Credé” goes back to Benno Carl Credé and not to his father, Carl Siegmund Franz Credé. Wrong information concerning Credé is deriving from the internet, which is found in the biography of Otto Spiegelberg [75]: “After the closing of the Monatsschrift für Geburtshilfe und Frauenkrankheiten (appeared 1853–1869) Spiegelberg and Carl Benno Credé (1847–1929) in 1870 founded the Archiv für Gynäkologie, of which almost every volume contained a

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contribution of his”. Actually, the editors of the “Archiv für Gynäkologie” were “(Otto) Spiegelberg” and “Credé”, in this case Carl Siegmund Franz Credé and not his son, Benno Carl Credé.

Carl Credé Carl Credé (Carl Credé-Hoerder; 1878–1952) [76, 77], a physician, had an uncle – who was also a physician – with the name Dr. Hoerder. Later on Carl Credé incidentially took over the name Carl Credé-Hoerder. He further used his synonym “Credo”. He was politically extremely active and a co-founder of the “Verein sozialistischer Ärzte” (Association of physicians following socialists’ ideology). In his position as physician he specialized in gynecology and obstetrics. Due to his political activities, Credé spent several years in prison, following the (unjustified?) accusation of violation of the German law concerning abortion (§218 StGB; Germany). In 1913, he published a scientifically less significant paper on “Die Augeneiterung der Neugeborenen, Pathologie, Therapie und Prophylaxe” [77] (Ophthalmia neonatorum, etiology, pathology, therapy and prophylaxis). In the present author’s estimation, the motivation for this publication was to be brought together with the ideas of Carl Sigmund Franz Credé, possibly to cause confusion. If or how Credé was related to Carl Siegmund Franz Credé remains historically unclear.

Acknowledgement I would like to express my thanks for assistance especially to the staff of the “ZB MED/Deutsche Zentralbibliothek für Medizin” (The German National Library of Medicine) in Cologne, Germany.

Addendum “Die Verhütung der Augenentzündung der Neugeborenen. Mittheilungen aus der geburtshülflichen Klinik in Leipzig von Credé [19]“ Die folgenden Mittheilungen über die Verhütung der Augenentzündung der Neugeborenen veröffentliche ich deshalb nicht in einem Fachjournale der Ophthalmologie, sondern in diesem Archiv, weil die Krankheit fast ausschließlich durch eine Infection während des Geburtsactes entsteht, also mit einer Erkrankung der weiblichen Genitalien unmittelbar zusammenhängt. Auch muss die Verhütung der Krankheit allein in die Hände der Geburtshelfer und Hebammen gelegt werden. Ich beschränke mich ausschließlich auf die praktische Frage der Prophylaxe.

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Im Allgemeinen kommen die Augenentzündungen der Neugeborenen seltener in den höheren Ständen vor, häufig schon im Proletariate, aber in den Entbindungsanstalten gehören sie zu einer fortlaufenden, höchst lästigen Plage und Sorge. Deshalb wende ich zunächst meine Aufforderung, die von mir empfohlene Prophylaxis weiter zu erproben, an diejenigen Herren Collegen, welche in Entbindungsanstalten oder in geburtshülflichen Polikliniken thätig sind, und, gleich mir, häufige Erkrankungen zu beobachten haben. Wohl von den meisten Geburtshelfern wird meine Ansicht getheilt werden, dass die so überaus häufig vorkommenden Katarrhe und Entzündungen der Vagina auf gonorrhoischer Infection beruhen und dass die Ansteckungsfähigkeit des Secretes noch fortbesteht, nachdem lange die specifisch gonorrhoischen Erscheinungen verschwunden sind, ja dass in Fällen, wo fast kein Secret mehr gefunden wird, doch noch die erfolgte Ansteckung in der Mutterscheide stattgefunden hat, wenn in den ersten Tagen nach der Geburt eine Augenentzündung sich entwickelt. Eine Übertragung des Infectionsstoffes von einem anderen augenkranken Kinde ist für die Leipziger Entbindungsanstalt völlig auszuschließen, da jedes inficirte augenkranke Kind mit seiner Mutter auf die Krankenstation verlegt wird, welche von der Station der Wöchnerinnen nach allen Richtungen hin vollständig getrennt ist. Auch können die Wöchnerinnen die Kinder mittels ihrer Finger, welche etwa durch Lochialsekret verunreinigt wären, kaum inficiren, weil die Kinder stets von den Müttern so weit entfernt in ihren Bettchen liegen, dass die Mütter sie nicht erreichen können und nur dann mit den Kindern in Berührung kommen, wenn diese ihnen von den Wärterinnen an die Brust gelegt werden. Somit bin ich nach meinen Beobachtungen und Einrichtungen der Ueberzeugung, dass fast ohne Ausnahme die in der hiesigen Anstalt erkrankten Kinder nur durch eine directe Uebertragung des Vaginalsecretes in das Auge während des Geburtsactes inficirt werden. Die Erkrankung des inficirten Auges beginnt in der Regel etwa zwei bis drei Tage nach der Geburt, aber auch früher und später, je früher, desto intensiver. Ich habe mir nun schon seit längerer Zeit die gewiss lohnende Aufgabe gestellt, die Mittel und Wege zu finden, wie man die für so viele Augen verderbliche Krankheit verhüten, wie am besten dem ansteckenden Secrete beikommen könne. Meine ersten Bemühungen erstreckten sich auf eine möglichst ausgedehnte zweckmässige Behandlung und Reinigung der kranken Vagina der Schwangeren und Gebärenden. Die Resultate waren jedoch gering, nicht befriedigend; die Zahl der Erkrankungen der Augen nahm zwar ab, aber sie verschwanden nicht. Darauf begann ich die Desinfection der Kinderaugen selbst und fortan wurden die Erfolge überraschend günstig. Der Gang meiner Versuche war folgender: Zuerst wurden bei allen MIT GONORRHOE ODER CHRONISCHEM VAGINALKATARRH in die Anstalt kommenden Schwangeren und Gebärenden reinigende Ausspülungen der Vagina

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mittels lauwarmen Wassers oder leichter Carbol- oder Salicylsäurelösungen (2:100) möglichst häufig, bei Gebärenden jede halbe Stunde gemacht. Die Erkrankungen der Augen wurden seltener, hörten aber nicht auf, ja verliefen in einigen Fällen noch hartnäckig und bösartig. Im October 1879 machte ich den ersten Versuch mit prophylaktischen Einträufelungen in die Augen der Neugeborenen gleich nach der Geburt und bediente mich einer Lösung von Borax (1:60), weil ich dieses Mittel für das mildeste, wenigst ätzende hielt. Es geschah dies aber zunächst nur bei Kindern von kranken Müttern, bei denen gleichzeitig die oben angeführten Ausspülungen der Scheide während der ganzen Geburt gemacht worden waren. Auch diese Methode führte nicht zum gewünschten Ziele, und ich nahm vom December 1879 statt des Borax Lösungen von Argentum nitricum (1:40), welche bald nach der Geburt in die Augen eingespritzt wurden. Vor der Einspritzung wurden die Augen mit einer Lösung von Salicylsäure (2:100) sorgfältig gewaschen. Die so behandelten Kinder kranker Mütter blieben gesund, indess andere Kinder, welche selbst und ebenso ihre Mütter, weil wir letztere für nicht erkrankt hielten, nicht prophylaktisch behandelt worden waren, erkrankten immer noch, zwei ziemlich heftig. Vom 1. Juni 1880 an wurden nun alle Augen ohne Ausnahme gleich nach der Geburt desinficirt und zwar in der Weise, dass eine schwächere Lösung von Argentum nitricum (1:50) gewählt, auch die Flüssigkeit nicht mehr eingespritzt, sondern nur mittels eines Glasstäbchens in jedes durch einen Gehülfen sanft geöffnete, vorher mit gewöhnlichem Wasser gereinigte Auge ein einziger Tropfen Flüssigkeit eingeträufelt wurde. Dann wurden die Augen 24 Stunden lang mit in Salicylwasser (2:100) getränkten Leinwandläppchen gekühlt. DIE ZAHLREICHEN VAGINALDOUCHEN WURDEN DAGEGEN GÄNZLICH AUFGEGEBEN und kamen nur aus anderen Gründen, die ganz unabhängig von den Vaginalkatharren waren, zur Anwendung. SÄMMTLICHE SO BEHANDELTE KINDER SIND VON AUGENENTZÜNDUNGEN, SELBST LEICHTESTEN GRADES, VERSCHONT GEBLIEBEN, obwohl manche der Mütter hochgradige Scheidenblenorrhöen und trachomatöse Wucherungen zeigten. Nur ein Kind (Jahresnummer 339) erkrankte am SECHSTEN Tage an einer mässigen Entzündung der Conjunctiva des linken Auges, ohne Schwellung des Augenlides, welche nach drei Tagen wieder geheilt war, und stellte sich heraus, dass bei diesem Kinde im Drange der Geschäfte zufällig die prophylaktische Einträufelung nicht gemacht worden war. Irgend ein Nachtheil für die so behandelten Augen haben wir bis jetzt nicht beobachtet. Nicht selten folgt der Einträufelung eine geringe Hyperämie, ab und zu auch eine etwas verstärkte Secretion der Conjunctiva in den ersten 24 Stunden. Dann verschwinden auch diese Erscheinungen. Vielleicht sind sie zu vermeiden, wenn die weiteren Versuche ergeben sollten, dass eine schwächere Lösung des Argentum nitricum genügt. Das Verfahren ist demnach sehr einfach, überall von einigermaassen geschickten Händen leicht auszuführen, ganz gefahrlos und, wie es scheint, zuverlässig in der Wirkung.

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Meine Beobachtungsreihe ist freilich noch zu klein, um ganz sichere Schlüsse zuzulassen, immerhin aber gross und namentlich frappant genug, um zu weiterer Anwendung dringend aufzufordern. DEN HAUPTWERTH MÖCHTE ICH IN DIE ERFAHRUNG LEGEN, DASS NICHT DIE DESINFECTION DER VAGINA, SONDERN NUR DIE DER AUGEN SELBST ZUM GEWÜNSCHTEN ZIELE FÜHRT. Ob nun gerade das von mir geübte Verfahren an den Augen das beste und sicherste sei, oder ob noch bessere gefunden werden können, wird hoffentlich die Zukunft lehren. Zunächst habe ich keinen Grund, von meiner Methode abzuweichen. Sollte es gelingen, die Augenentzündungen auch nur aus den Entbindungsanstalten und den Polikliniken zu verdrängen, so wäre schon dadurch ein Gewinn von grosser Tragweite nach verschiedenen Richtungen hin erreicht, was ich hier wohl nicht näher auseinanderzusetzen brauche. Schließlich theile ich ganz kurz eine Zahlenreihe über die in den letzten Jahren in der hiesigen Entbindungsanstalt beobachteten Augenentzündungen mit. Vielleicht sind anderswo die Erkrankungen der Vagina und demnach die der Kindesaugen weniger häufig, als gerade in Leipzig, dessen eigenthümliche, von vielen, auch grösseren Städten abweichende Verhältnisse besonders in Betracht gezogen werden müssen.

Jahr

Zahl der Geburten

Zahl der Augenerkrankungen

Procentsatz

1874

323

45

13,6

1875

287

37

12,9

1876

367

29

9,1

1877

360

30

8,3

1878

353

35

9,8

1879

389

36

8,2

1880 (bis 31. Mai)

187

14

7,6

1880 (vom 1. Juni bis 8 Decbr.)

200

1*

0,6

*Es ist dies der Fall, bei welchem die Augen nicht desinficirt wurden; also eigentlich sind 0,0% zu verzeichnen.

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Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO (2000) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52: 662–668 Quelmaltz ST (1740) De caecitate infantum floris albi materni ejusque virulanti pedisseque dissertatione. PhD Thesis, Leipzig Gibson B (1807) On the common cause of puriform ophthalmia of new-born children. Edinburgh Med Surg J 3: 159–161 Ammon FA v (1825) Pathology of the eye bulbus and its adnexes during the illness entitled ophthalmitis of the new-born child (“Das pathologische Verhalten des Augapfels und seiner Häute während des Verlaufs der sogenannten Augenentzündung neugeborener Kinder”). In: Hecker’s Annalen, Vol 1, TCF Enslin, Berlin, 129–142 Bednar A (1848) Treatment of ophthalmia neonatorum (“Über die Behandlung der Augenentzündung der Neugeborenen”). Zschr k k Ges Ärzte 5: 139–145 Mackenzie W (1854) Practical Treatise on the Diseases of the Eye. Longmann, London Noeggerath E (1872) Die latente Gonorrhoe im Weib (Latent Female Gonorrhea). Seiler, Bonn Oriel JD (1991) Eminent venerologists 5: Carl Credé. Genitourin Med 67: 67–69 Kibel MA (1981) Silver nitrate and the eyes of the newborn – a centennial. S Afr Med J 60: 979–980 Novak JM (1984) Current status of Credé prophylaxis. Am J Optometry Physiol Optics 61: 340–346 Schaller UC, Klauss V (2001) Is Credé’s prophylaxis for ophthalmia neonatorum still valid? Bull WHO 79: 262–263 Dunn PM (2000) Dr. Carl Credé (1819–1892) and the prevention of ophthalmia neonatorum. Arch Dis Child Fetal Neonatal Ed 83: 158–159 Rosenberg J (1892) In memoriam, Carl Siegmund Franz Credé. Am J Obstet Gynecol 25: 780–783 Leopold G (1891) Talk in remembrance of Carl Siegmund Franz Credé (“Carl Siegmund Franz Credé. Gedächtnisrede”). Arch Gynäkol 42: 193–212 Author unknown (Dr. L) (1892) Karl Credé. Illustrierte Zeitung, press release, no 2543 Credé CSF (1854) Manual expression of the placenta (“Handgriff zur Entfernung der Plazenta”). Klinische Vorträge über Geburtshilfe, Hirschwald, Berlin, 599– 600 Speer H (1957) Carl Siegmund Credé, placental expression, and the prevention of ophthalmia neonatorum. Obstet Gynecol 10: 335–339 Credé CSF (1884) Incubation systems for early for date and weak small children (“Ueber Erwärmungsgeräthe für frühgeborene und schwächliche kleine Kinder”). Arch Gynäkol 24: 128–147 Credé CSF (1881) Die Verhütung der Augenentzündung der Neugeborenen (“Prevention of inflammatory eye disease in the newborn”). Arch Gynäkol 17: 50–53

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Holmes KK, Counts GW, Beatty HN (1971) Disseminated gonococcal infection. Ann Intern Med 74: 979–993 Wilcox RR (1972) A world look at the venereal disease: recrudescence of the venereal diseases. Med Clin North Am 56: 1057–1071 Armstrong JH, Zacarias F, Rein MF (1976) Ophthalmia neonatorum: a chart review. Pediatrics 57: 884–892 Handsfield HH, Hodson KK (1973) Neonatal gonococcal infection: orogastric contamination with Neisseria gonorrhoeae. J Am Med Assoc 225: 697–701 Huber-Spitzy V, Arocker W, Schmidt C (1987) Ophthalmia neonatorum. Klin Mbl Augenheilk 191: 341–343 Lehrfeld L (1935) Limitations of use of silver nitrate in prevention of ophthalmia neonatorum. J Am Med Assoc 104: 1468–1469 Robson KS (1967) The role of the eye-to-eye contact in maternal-infant attachment. J Child Psychol Psychiatry 8: 13–25 Nishida H, Risemberg HM (1975) Silver nitrate ophthalmic solution and chemical conjunctivitis. Pediatrics 56: 368–373 Butterfield PM, Emde RN, Svejda MJ (1981) Does the early application of silver nitrate impair maternal attachment? Pediatrics 67: 737–738 Graf H, Retzke U, Schilling C, Schmidt M (1994) Reaction of the ophthalmic front region to the Credé-prophylaxis (“Die Reaktion des vorderen Augenabschnittes auf die Credé-Prophylaxe”). Zentralbl Gynäkol 116: 639– 642 Greenberg M, Vandow JE (1961) Ophthalmia neonatorum: evaluation of different methods of prophylaxis in New York City. Am J Public Health 51: 836–845 CDC (Centers for Disease Control) (1989) Sexually transmitted diseases treatment guidelines. MMWR 38: 8 Chen JY (1992) Prophylaxis of ophthalmia neonatorum: comparison of silver nitrate, tetracycline, erythromycin and no prophylaxis. Pediatr Infect Dis J 11: 1026–1030 Seiga K, Shoji T (1993) Chemoprophylaxis of ophthalmia neonatorum through vertical infection. Evaluation of Crede’s method using norfloxacin and gentamycin. Jpn J Antibiot 46: 331–336 Kramer A, Behrens-Baumann W (1997) Prophylactic use of topical anti-infectives in ophthalmology. Ophthalmologica 211 (S1): 68–76 Benevento WJ, Murray P, Reed CA, Pepose JS (1990) The sensitivity of Neisseria gonorrhoeae, Chlamydia trachomatis, and herpes simplex type II to disinfection with povidone-iodine. Am J Ophthalmol 109: 329–333 Isenberg SJ, Apt L, Yoshimori R, Leake RD, Rich R (1994) Povidone-iodine for ophthalmia neonatorum prophylaxis. Am J Ophthalmol 118: 701–706 Isenberg SJ, Apt L, Wood M (1995) A controlled trial of povidone-iodine as prophylaxis against ophthalmia neonatorum. N Engl J Med 332: 562–566 Reimer K, Fleischer W, Brogmann B, Schreier H, Burkhard P, Lanzendorfer A, Gumbel H, Hoekstra H, Behrens-Baumann W (1997) Povidone-iodine liposomes – an overview. Dermatology 195 (S2): 93–99 Kramer A, Below H, Behrens-Baumann W, Muller G, Rudolph P, Reimer K

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(2002) New aspects of the tolerance of povidone-iodine in different ex vivo models. Dermatology 204 (S1): 86–91 Isenberg SJ, Apt L, Campeas D (2002) Ocular applications of povidone-iodine. Dermatology 204 (S1): 92–95 Simon JW (2003) Povidone-iodine prophylaxis of ophthalmia neonatorum. Br J Ophthalmol 87: 1437 Isenberg SJ, Apt L, Del Signore M, Gichuhi S, Bermann NG (2003) A double application approach to ophthalmia neonatorum prophylaxis. Br J Ophthalmol 87: 1449–1452 The Editorial Board (1980) Centenary of Credé prophylaxis. Am J Ophthalmol 90: 874–875 Tietze KW (1994) Credé’s prophylaxis. Report of a commission of the German Authority of Health (“Die Credé Prophylaxe. Bericht einer Kommission des Bundesgesundheitsamtes”). Ophthalmologe 91: 551–552 Watten RG (1994) Reinvention of visual fatigue: accumulation of scientific knowledge or neglect of scientific history? Ophthalmic Physiol Opt 14: 428– 432 Editorial Board (2002) New aspects in prophylaxis of ophthalmia neonatorum (Credé prophylaxis). Wien Klin Wochenschr 114: 171–172 Laga M, Meheus A, Piot P (1989) Epidemiology and control of gonococcal ophthalmia neonatorum. Bull WHO 67:471–477 [Erratum: Bull WHO (1990) 68: 690] Gilbert C, Foster A (2001) Childhood blindness in the context of VISION 2020 – The Right to Sight. Bull WHO 79: 227–232; http://www.scielosp.org/scielo. php?pid=S004296862000100300011&script=sci_arttext&tlng=en http://www.matrikel.unizh.ch/pages/45.htm http://www.internet-taubenschlag.de/medizin/bericht7 http://www.uni-kiel.de/anorg/lagaly/group/klausSchiver/Lottermoser.pdf Hille M (1897) On Dr. Credé’s Antiseptics: Silver and Silver-Salts and their Usage within Dental Medicine (“Ueber Dr. Credé’s neue Antiseptica: Silber und Silbersalze und deren Anwendung in der Zahnheilkunde”). “Separatabdruck” (Reprint) from Deutsche Monatsschrift für Zahnheilkunde 15 (5). A. Pries, Leipzig, 1–9 Beyer JL (1904) Use of Kolloidal Metalls in Medicine: Silver and Mercury (“Über die Verwendung kolloider Metalle – Silber und Quecksilber – in der Medizin”). L. Simion, Berlin, Germany http://www.whonamedit.com/doctor.cfm/2028.html Albrecht B, Albrecht G (1989) Carl Credé, 1878–1952, an unjustifiedly accused physician (“Carl Credé, 1878–1952, als Arzt unschuldig verurteilt”). In: B Albrecht, G Albrecht (eds): Diagnoses. Remembrances on Physician of the 20th Century (“Diagnosen. Ärzteerinnerungen aus dem 20. Jahrhundert”). Der Morgen, Berlin, 294–307 Albrecht B, Albrecht G (1989) Carl Credé. In: Albrecht B, Albrecht G (eds): Diagnoses. Remembrances on physician of the 20th century (“Diagnosen. Ärzteerinnerungen aus dem 20. Jahrhundert”). Der Morgen, Berlin, 416–417

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Malnutrition and infection in industrialized countries Susanna Cunningham-Rundles and Deborah Ho Lin Department of Pediatrics Host Defenses Program, Weill Medical College of Cornell University, New York, NY 10021, USA

Abstract Malnutrition is a major cause of immune deficiency that directly affects the acute phase response and leads to greater frequency and severity of common infections. Primary malnutrition is not uncommon in wealthy industrialized societies due to poverty, lack of education, food allergies, inappropriate or limited diet, or eating disorders. Inadequate intake of micronutrients including vitamin A, E, calcium, iron and zinc are prevalent among children under 10 years of age and often unrecognized. Although chronic infectious diseases are less prevalent in industrialized countries, infections with HIV, Mycobacterium tuberculosis and hepatitis C virus are significant problems and parasitic infections may appear among immigrant populations. Obesity is becoming increasingly common in children and may enhance risk of serious complications of common infections. Adequate nutrition is critically important for the development of the immune system, immune response to environmental antigens and pathogens, and for the maintenance of host defense. In children with congenital anomalies or medical conditions affecting growth, poor nutrient status will have a disproportionate effect on development, immunity, and susceptibility to infection since nutrients are cofactors in immune response. Defects in T cell immunity lead to increased susceptibility to intracellular pathogens, reactivation of viral infections, and development of opportunistic infections. Zinc deficiency inhibits Th1 cytokine responses, thymic hormone activity, and lymphopoiesis. Vitamin A deficiency is associated with severity of many infections including measles, rotavirus, HIV, and bacterial infections. Selenium deficiency is associated with HIV progression. Nutrient cofactors of innate immune response include 1,25-dihydroxyvitamin D3, which is a direct regulator of antimicrobial responses. The overall impact of chronic subclinical malnutrition in children may determine the quality and duration of immune response to vaccines and may be an important topic for future research.

Introduction Malnutrition is a major cause of immune deficiency that leads to greater frequency of common infections, increasing their severity and impeding clinical resolution. Infection also imposes a metabolic stress through activation of the acute phase response that is more difficult to resolve in malnutri-

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tion. The combination often produces a vicious cycle, which leads to chronic infection. Malnutrition is a leading cause of death in children less than 5 years of age in less-developed countries. Where lack of food availability, poor sanitation, lack of safe water, endemic infections and general poverty are widely prevalent, malnutrition is generally appreciated as a major cause of clinical infection. Although food is apparently available in wealthy industrialized societies, primary malnutrition is not uncommon due to poverty, lack of education, food allergies, inappropriate or limited diet, or eating disorders and should be considered as a possible root cause or cofactor in frequent infection or failure to resolve infection. Furthermore, children comprise a significant part of the increasingly large immigrant populations in industrialized urban settings where they may live in impoverished circumstances and have less access to health care. Such children are especially vulnerable to the effects of nutrient deficiency. For infants and toddlers, adequate nutrition is critically important for the development of the immune system, immune response to environmental antigens and pathogens, and for the maintenance of host defense. In children with congenital anomalies or medical conditions affecting growth, poor nutrient status will have a disproportionate effect on development, immunity, and susceptibility to infection. For children with secondary malnutrition, specific macronutrient and micronutrient supplements are an essential part of disease management, due to the additional metabolic burden associated with chronic illness, as indicated by inflammation, anemia, and altered gastrointestinal (GI) function.

Pathophysiology of malnutrition Malnutrition can be classified as either primary or secondary [1]. Primary malnutrition is caused by inadequate calorie and nutrient intake. In developed societies, calorie intake is usually presumed to be adequate. However, inadequate intake of micronutrients including vitamins A and E, calcium, iron and zinc are prevalent among children of 1–10 years of age and often unrecognized, especially in minority populations [2]. Primary malnutrition in infants can also occur through child neglect or accidental nutrient insufficiency [3, 4]. For example, a genetic defect impairing zinc transport into breast milk from maternal blood can lead to zinc deficiency in infancy [5]. Eating disorders associated with psychosocial disorder are a common cause for primary failure-to-thrive in children [6]. Other causes include inadequate diet due to food intolerance or imposition of special diets unsuited to growing children. Vegetarian, macrobiotic or vegan diets in children may be associated with low vitamin D, reduced cobalamin, and perhaps iron. However, lacto-ovo-vegetarian children may consume diets closer to expert recommendations than omnivores and their pre-pubertal growth is at least as good [7, 8]. The presentation of malnutrition is outlined in Table 1.

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Table 1. Presentation of malnutrition Protein calorie malnutrition: marasmus

Chronic wasting, underweight Low weight for height, stunting, short stature

Protein calorie malnutrition: kwashiorkor

Peripheral edema, depigmenation, hepatomegaly Often develops at weaning

Micronutrients

Iron: (?) anemia, infections, pica Zinc: (?) skin lesions, diarrhea, alopecia, infections Copper: (B) infections, e.g., protozoal infections Zinc and copper: (?) hypoproteinemia, anemia Selenium: (?) muscle aches, pains, cardiomyopathy, infections Vitamin A: (?) keratomalacia, night blindness, infections Vitamin C: (?) leg pain, bleeding gums, petechial hemorrhage

Secondary malnutrition can be caused by reduced intake of food, malabsorption, impaired nutrient utilization, and nutrient losses associated with chronic infection and many other clinical conditions as well. Examples include inflammatory bowel disorders, celiac disease, chronic anemia, renal disorders, and cystic fibrosis (CF). In both primary and secondary malnutrition, understanding of the relevant genetic mechanisms can be helpful in approaching the clinical manifestations. Genetic mechanisms of malnutrition that affect susceptibility to infectious disease include mutations affecting metabolism of the trace elements zinc, iron, and copper, and several vitamins as well as those underlying complex, inherited disorders such as CF and celiac disease. Primary malnutrition impairs immunity impeding host response to infection, but these effects are reversible with nutrient repletion. However, calorie and nutritional supplement alone cannot resolve the secondary malnutrition with organic etiology. Protein-calorie malnutrition (PCM), sometimes termed protein-energy malnutrition (PEM), is the most common cause of secondary immune deficiency in the world because of wide spread chronic and seasonal food shortages, as well as chronic poverty, the deprivations of war, and maternal malnutrition [9]. The deficiencies associated with PCM usually are multiple, involving varying degrees of calorie, protein, vitamin, and mineral deficits. Classically, PCM is divided into two types – marasmus and kwashiorkor. Marasmus occurs in total calorie deficiency, with chronic wasting and gross underweight. Kwashiorkor occurs due to protein deficiency in the diet, which may be high in calories. The growth retardation is moderate, but these

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children often appear apathetic and miserable, with various problems such as characteristic dermatitis, brittle reddish tinged hair, edema, moon faces, hepatosplenomegaly, anemia, and hypoalbuminemia. Both marasmus and kwashiorkor often have concomitant vitamin and mineral deficiencies. In industrialized countries, the edematous presentation of kwashiorkor often delays or prevents recognition of this form of protein malnutrition. The causes of protein deficiency include use of low protein milk substitutes such as rice “milk”, which contains no milk product, and other beverages, which may be provided by caregivers in response to perceived food intolerance or food aversion [10–12]. Selective micronutrient deficiencies can occur when food and calorie intake is adequate. Iron, copper and zinc deficiencies are the most common due to dietary insufficiency. Results from a large double-blind trial of fortified milk in preschool children show that this intervention can reduce morbidity from diarrhea, respiratory infections and other illnesses, as well as improve iron status and growth. [13] Selenium deficiency occurs primarily in parts of the world where selenium levels are low in the soil. As a constituent of selenoproteins, selenium is needed for the functioning of neutrophils, macrophages, NK cells, and T lymphocytes. Mild selenium deficiency is relatively widespread and appears to worsen viral infection [14]. Selenium and vitamin E deficiency in the mouse have been shown to promote the virulence of Coxsackie B3 virus and influenza by inducing genetic changes in the genomes of the viruses [15]. Selective micronutrient deficiency frequently occurs in patients with underlying systemic illnesses, chronic viral infection and in low birth weight infants [16, 17]. In some cases, the adverse effects have long-term effects [18]. Obesity is a specialized form of malnutrition that is becoming increasingly common in children, raising concerns about type 1 diabetes, cardiovascular disease, and risk of cancer. A recent study has reported that low-grade inflammation, as determined by serum levels of high-sensitivity C-reactive protein, while significantly increased in children with type 1 diabetes, a high level was even more pronounced in apparently healthy juveniles with primary obesity [19]. Uncomplicated morbid obesity in adolescents may be accompanied by alterations in the levels of circulating T cells and cytokine response [20]. Other studies show that regulation of natural killer (NK) function and proliferative response to mitogens in vitro are affected [21, 22]. Leptin, the product of the ob gene, is a pleiotropic molecule that regulates food intake through metabolic and neuro-endocrine functions, has cytokine-like activities and is a major regulator of immune function [23]. Leptin is acutely increased during infection and inflammation [24]. Primary leptin deficiency is associated with obesity and altered immune function [25]. Although the relationship between obesity and susceptibility to infections is not well defined, there is consensus that postoperative infections, other nosocomial infections, and risk of serious complications of common infections are enhanced in obesity [26]. A role for fetal programming that links

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early growth compromise to subsequent development of obesity and the metabolic syndrome has been postulated [27].

Immune dysfunction and malnutrition Malnutrition in the neonatal period and early childhood can lead to severe immune deficiency and high mortality. Effects on the immune system are broad, involving all limbs of the immune system, with impaired T cell responses secondary to effects on thymic architecture and function being the most common. The link between malnutrition and infection is readily observable. For children living a rural environment in a developed country, one study reported that bacterial infections were discovered in one third of all patients hospitalized for malnutrition [28]. Malnutrition was also frequently found among adults hospitalized for nosocomial infections in another study [29]. Host response to infection is also altered in malnutrition. Thus, children who were well nourished were found to show a relative increase in B lymphocytes in response to bacterial infection, while B cell response was significantly reduced in malnourished children [30].

Innate immune defects The innate immune system provides the first line of defense against infection. Defense mechanisms include barrier functions, which require both anatomic components such as specialized epithelium, products such as mucus, and soluble mediators such as cytokines, interferons (IFNs), lysozymes, and defensins. Loss of barrier function due to malnutrition promotes infection. Studies in a mouse model of visceral leishmaniasis have shown that malnutrition promoted visceralization through loss of lymph node barrier function after Leishmania donovani infection. This was caused by excessive production of prostaglandin E2, and decreased levels of IL10 and nitric oxide (NO) [31]. The effect of diet on mucosal integrity is a key measure of nutritional rehabilitation in infants [32]. Protein deficiency predisposes both to skin and mucosal atrophy and compromises barrier function. Chemotaxis, phagocytosis, and microbial killing mechanisms are potentially impaired in malnutrition through reduced production of key mediators including complement C3, leukotrienes, cathelicidin antimicrobial peptide and leptin [33–36]. Children who are malnourished mount a partial acute phase response to infection and this defect is more marked in children with the edematous form [37]. The activities of innate immune cells such a neutrophils, monocytes, macrophages, and dendritic and NK cells are affected by altered nutrient levels [38–43]. These effects can be particularly critical in the perinatal period of immune development [44, 45].

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The development of immune response in the neonate occurs in the context of initial microbial antigen exposure when neonates are also vulnerable to bacterial infection due to immature innate and adaptive immune response. Neonates have deficiencies of innate cellular immunity including decreased production of IFNs, IL-12/IL-23, and IL-18, proinflammatory cytokines, and impaired monocyte response to IFN-a and to lipopolysaccharide (LPS) [46]. Response to bacterial antigens involves microbial antigen binding to the Toll-like receptors (TLRs), which recognize conserved molecular products derived from various classes of pathogens, such as gram-positive (TLR-2) and gram-negative (TLR-4) bacteria, leading to production of inflammatory cytokines and chemokines. Nutrient regulators of this process include 1,25dihydroxyvitamin D3, which is an immune system modulator that induces expression of the TLR4 coreceptor CD14. 1,25-Dihydroxyvitamin D3 is a direct regulator of antimicrobial innate immune responses and causes secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa, a well-known major pathogen in CF [35]. As shown by studies in experimental gnotobiotic models, protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, and metabolic processes central to nutrition, and the development of mucosal (oral) tolerance. Nutrient status affects the development of this system [47, 48]. The role of commensals in maturation of the TLR system is currently being studied [49]. Thymic atrophy caused by PCM is associated with hormonal imbalance, loss of leptin, and increase in serum glucocorticoid level. Leptin levels normally increase acutely during infection and inflammation [24], but this does not occur in PCM. The reduction of serum leptin levels and insulinlike growth factor-1 (IGF-1) in marasmus and kwashiorkor [50] may compromise response to infection. Loss of immune function in malnourished children correlates with low leptin levels, and refeeding leads to increase in leptin levels and immunological recovery [51].

Adaptive immune defects Defects in T cell immunity are characteristic of malnutrition and lead to increased susceptibility to intracellular pathogens, reactivation of viral infections, and development of opportunistic infections [1, 52]. Malnutrition activates the metabolic switch that controls T cell activation and apoptosis [53]. The effects of undernutrition in infancy may extend beyond this period due to effects on programming that are now becoming appreciated. A study of antibody response to typhoid vaccine among adolescents has shown that the likelihood of mounting an adequate response was diminished among the group who were small for gestational age compared to those who were appropriate for gestational age at birth [54]. Age-related effects of malnu-

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trition on immune response have also been found in adults. A recent study in healthy volunteers consisting of younger and older adults showed that short-term fasting had a significant effect on total, helper, and cytotoxic T and B lymphocytes and that this response was significantly and negatively affected by older age [55]. T cell deficiencies in malnutrition are directly attributable to profound lymph node germinal center depletion and thymic atrophy, which can appear similar to primary immune deficiency [56]. Lymphopenia is common. The T cell functional defects resemble those of congenital thymic aplasia as in Di George syndrome [50, 56]. The selective effect of malnutrition on the thymus gland is due to apoptosis-induced thymocyte depletion, affecting the immature CD4+ and CD8+ cells, as well as a decrease in cellular proliferation. Hormonal imbalance, involving decrease of leptin and consequent increase in serum glucocorticoid hormone levels can be reversed with nutritional rehabilitation [57]. Morphological changes in thymic epithelial cells are associated with decreased thymic hormone production. Lymphopenia is commonly observed in malnutrition with an incidence of about 25% in children with fatal malnutrition. This effect on hematopoiesis is now understood as the result of a critical regulatory effect on both B and T cell development that is caused by accompanying zinc deficiency [58]. The absolute number of T cells is directly decreased by zinc deficiency. CD4+ T cells are reduced more than CD8+, resulting in a reversed CD4:CD8 ratio. Other effects of zinc deficiency include defective T cell activation, reduced maturation to a memory phenotype, and impaired cytokine synthesis. T cell deficiencies in zinc deficiency may approach the severity seen in children with combined immunodeficiency (SCID) or advanced HIV infection. The humoral immune system is generally relatively preserved in malnutrition. Serum levels of IgA1, IgA2 and C4 tend to be higher than in normal children, while serum level of C3 and the proportion of B cells are significantly lower [43]. IgE levels are lower even among asthmatic children [59]. Response to immunization tends to be normal and therefore vaccines remain effective [60]. Malnourished children at risk for tuberculosis (TB) often do not respond to Bacille Calmette-Guerin (BCG) immunization, shown by negative tuberculin skin test, and have an increased risk for developing disseminated systemic TB compared to well-nourished children who usually have mild localized disease and rarely present with hematogenous spread [61]. Current studies show a low protective effect of BCG vaccination against all forms of TB among vaccinated children as defined by visible scar and somewhat better efficacy against extra-pulmonary TB [62]. The role of nutritional status in children at the time of vaccination has not been fully evaluated. Since disseminated BCG infection in immune deficiency remains a serious concern, studies to examine the interaction with malnutrition would be informative [63].

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Specific micronutrient deficiencies Micronutrients have a major impact on immune response, through antioxidant activities and modulation of cytokine expression. Antioxidant enzymes, such as copper, zinc, and manganese superoxide dismutases, require trace metals for biological activity, and these enzyme reactions protect against oxidative damage caused by free radical formation during immune response and other biological reactions. Intracellular redox balance has a signaling role in immune cell development and function, and the antioxidant effects of micronutrients regulate cytokine production [64]. Iron deficiency anemia in children leads to impaired cell-mediated immunity, such as decrease of T cell number, abnormal delayed cutaneous hypersensitivity, IL-2 production, and reduced bactericidal activity of neutrophils [38, 65]. Vegetarian diet and Helicobacter pylori infection can be causes of iron deficiency anemia [8, 66]. Pangastritis is more common in children whose H. pylori infection is accompanied by anemia [67]. Zinc deficiency is associated with primary immune deficiency disorders such as common variable immune deficiency or hypogammaglobulinemia, Di George syndrome and IgA deficiency, as well as other conditions including fetal alcohol syndrome, sickle cell disease due to hyperzincuria, celiac disease, enteritis and diarrhea. Zinc deficiency due to loss occurs in epidermolysis bulosa and in `-thalassemia due to chelation protocols required to remove excess iron secondary to chronic blood transfusion for anemia. Zinc deficiency inhibits Th1 cytokine responses, thymic hormone activity, and lymphopoiesis [1, 58, 68]. Acrodermatitis enteropathica, a genetic defect in zinc absorption, presents in infancy as skin lesions (acute dermatitis or hyperkeratotic plaques), diarrhea, alopecia, and increased susceptibility to infection, and is resolved with zinc supplementation [68]. Because zinc competes with copper for GI uptake, zinc supplements may induce copper deficiency, and may cause neutropenia [69]. Selenium is an important micronutrient for health [70] and is critical for antioxidant function acting via the selenium-dependent enzyme, glutathione peroxidase, to protect cellular membranes and organelles from peroxidative damage. Neither toxic nor deficient levels in soil are commonly found in Europe [71, 72], yet deficiency is fairly common due to variable bioavailability [72, 73]. Soil deficiencies of selenium and iodine are common in some countries such as New Zealand, Australia, Finland, and in parts of China [74]. Some studies have suggested that risk of cancer is increased in selenium deficiency [75, 76]. Selenoproteins are an important component of the antioxidant host defense system affecting leukocyte and NK cell function [77]. Selenium is emerging as a critical micronutrient in host defense against viral infection since selenium deficiency is associated with progression in HIV disease and in viral shedding [78, 79]. Vitamin A has long been appreciated as a significant factor in the severity of infection such as measles, rotavirus diarrhea, and HIV in the mal-

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nourished host. Pure deficiency is uncommon, but neonates and children less than 5 years of age are at risk. Vitamin A deficiency, which affects 140 million pre-school children worldwide, is associated with severity of many infections including measles, rotavirus, and HIV [80, 81]. Low vitamin A levels are associated with the occurrence of chronic bacterial infections and splenomegaly, as well as high neopterin levels in common variable immune deficiency or hypogammaglobulinemia [82]. In these patients supplementation in vivo led to improved immune function in vitro. Vitamin C is a free radical scavenger that serves as an important antioxidant. Vitamin C concentrations in the plasma and leukocytes decline during infections and stress. Supplementation with antioxidant vitamins including vitamin C has been shown to improve immune response to group A streptococcal infection compared to penicillin alone [83]. Supplementation may enhance phagocytosis and NK cell activity [84], increase levels of the antioxidant plasma glutathione levels, and inhibit Fas-induced apoptosis of monocytes. H. pylori infection is associated with a decrease in gastric juice ascorbic acid concentration, and this effect is greater in children with the CagA-positive strain A [67]. Both vitamin C and astaxanthin, a carotenoid, show antimicrobial activity against H. pylori that may be mediated through immune mechanisms [85]. Vitamin C is used to treat recurrent furunculosis in patients with deficient neutrophil function, and may lower the incidence of colds associated with acute physical stress. This may be related to the finding that vitamin C reduces muscle release of IL-6 [86]. No substantial evidence supports the view that megadoses of vitamin C decrease the severity or frequency of respiratory infection. However, recent studies show that vitamin C selectively influences intracytoplasmic cytokine production in vitro. [87] Current studies suggest that vitamin E deficiency is common in US toddlers [88]. Vitamin E supplementation enhances proliferative response in vitro [89] and improves IL-2 cytokine response [90]. Vitamin E deficiency causes reduced transferrin receptor internalization in the mouse, which suggests restriction of intracellular iron stores that would be needed for cellular function and proliferation [91]. Vitamin E may influence T cell function by downmodulating PGE2. Improvement in eczema and reduction in serum levels of IgE in atopic subjects has recently been reported [92]. A recent study has shown that antioxidant deficiency is common in a very large cohort of CF patients. Carotenoid and vitamin E deficiencies were found to occur early in the course of the disease and antioxidants were observed to decrease with bronchial infection [93].

Malnutrition syndromes of childhood Malnutrition syndromes of childhood are especially important for host defense since long-term effects may occur due to the continuing interaction

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of the immune system with potentially infectious pathogens. In addition, there are long-term implications for response to immunization and duration of protection. A general outline of how nutrients affect immune function is shown in Table 2.

Intrauterine growth retardation Low birth weight infants are classified as SGA (small for gestational age), which can also occur in full-term infants, or as AGA (appropriate for gestational age), which is the more common presentation of prematurity. Intrauterine growth retardation (IUGR) is usually associated with placental insufficiency, congenital infection, maternal smoking, exposure to toxins, or a combination of factors. In developing countries, IUGR can be caused by a prenatal deficiency of calcium, vitamin A, B1, and E; and folate. Clinically, low birth weight infants, including AGA premature infants with no evidence of infection, have impaired cell-mediated immunity, diminished cytokine responses, and reduced phagocyte function [94–96]. SGA infants have smaller thymic glands and deficient cytokine responses relative to the AGA infants. It is not surprising that IUGR is linked to poor future health, postnatal infections, sudden infant death syndrome, hypertension, ischemic heart disease, insulin resistance and diabetes. Zinc supplements given to SGA infants have been shown to reduce infectious mortality [97].

Failure to thrive Failure to thrive (FTT) can be caused by primary malnutrition, malignancy, and toxin exposure, congenital anomalies (i.e., Bloom’s syndrome, Russell Silver syndrome, immune deficiency, GI disorders, and psychosocial/eating disorders. For most cases of FTT, the causes can be found with a comprehensive history/physical, and limited laboratory studies. Some cases of true FTT have an unknown etiology, with simple under-nutrition due to behavioral abnormalities and inadequate parenting the most common cause. Infants require nutrient rich diets to sustain growth and development. Although rare, exclusively breast fed infants can show signs of growth abnormalities. One cause can be a maternal genetic abnormality in zinc transport into milk, resulting in severe zinc deficiency in the infant [98]. It is widely appreciated that many so-called health food diets and beverages that may be harmless for adults are not appropriate for infants due to their need to maintain continued growth and their unique requirement for additional nutrients. Cases of severe nutritional deficiency, even kwashiorkor, can be caused by consumption of “health food beverages” as an alternative nutrient source for children with perceived food allergies [10].

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Table 2. Mechanisms of nutrient action Nutrient

Target

Effect

Zinc

T, B, NK cells, Deficiency increases GALT (gas- infection, impairs lymtrointestinal- phopoiesis associated lymphoid tissue)

Deficiency causes cytokine shifts, activation of the HPA axis, T cell apoptosis

Antioxidant vitamins, C, E, and carotenoids

Monocytes, T cells, neutrophils, NK cells

Anti-inflammatory effects Decreases PGE2 production Increases IFN-a, IL-4 production Enhances phagocytosis

Vitamin A

Monocytes, Deficiency leads to neutrophils, infections, morbidity, T, B, NK cells, mortality GALT

Glutamine

T cells, monocytes, GALT

Increased thymus Increases lymphocyte prolifweight, T cell response, eration, cytokine response, phagoIgA, autoimmunity? cytosis, MHC class II expression

Arginine

T cells. monocytes, GALT

Increased immune response thymic weight, phagocytosis and killing

Increases thymocyte proliferation, modulates TNF-_ production, increases production of ROS. phagocyte function

Saturated/ unsaturated fatty acid ratio

T cells, NK, monocytes

NK, T cells response, phagocytosis

Regulation of adhesion molecules, membrane fluidity

Essential fatty acids n-6, n-3

Monocytes, neutrophils T cells

Cellular immunity

Deficiency or excess: decreases chemotaxis, phagocytosis, NK activity

Eicosapentanoic acid

Monocytes, T, and NK cells

Anti-inflammatory

High levels decrease IL-2, IFN-a, ICAM-1, superoxide production

Decosahexanoic acid

Monocytes, T cells

Anti-inflammatory

Signal transduction MHC class II

Deficiency causes oxidative stress, increases reactive oxygen species, DNA damage

Mechanism

Decreased Th-2 cytokine and IgA response, NK, B cells, Decreased cilia, microvilli, mucin, abnormal keratin

Gastrointestinal disease Short bowel syndrome (congenital atresia or surgical resection), and inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohn’s disease (CD) are commonly associated with chronic malnutrition due to poor GI absorption. Bacterial infections may contribute to intestinal inflammation in genetically susceptible hosts. Malabsorption due to lactose intolerance and gluten-sensitive enteropathy are common causes of GI disease. Prolonged parenteral nutrition, while essential, often correlates with impaired immune responses due to loss of antigenic stimuli, caloric and micronutrient insufficiency. Change in normal flora can result in micro-

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infections of the GI tract, which provokes inflammation, motility abnormalities and worsening of malabsorption. Both UC and CD appear to be multigenic disorders with evidence of familial segregation. Recent studies show that the development of oral tolerance is defective in both UC and CD. In UC patients, clinical investigators have reported that failure to induce tolerance to a neo antigen is associated with disease expression [99]. Inflammatory cytokines have been implicated in the pathogenesis of UC, possibly linked with gene polymorphism of the IL-1 receptor antagonist. Antibodies to neutrophil cytoplasmic antigens (ANCAs) and to mucin are often present in UC, with generalized hyper reactivity to cow’s milk protein, either cellular or antibodies, often present. CD is associated with increased numbers of circulating memory CD4+ T cells and activated mucosal T cells with defective proliferative responses. An abnormal immune response towards endogenous bacteria may be causative. A genetic defect in tolerance induction in CD has been identified [99]. Variants of NOD2, an intracellular sensor of bacteria-derived muramyl dipeptide (MDP), increase susceptibility to CD [100]. Altered taste and anorexia can cause inadequate dietary intake and lead to zinc deficiency. Celiac disease is a genetically determined chronic inflammatory intestinal disease induced by an environmental precipitant, gluten, that often presents without clear GI symptoms. Celiac disease may be characterized by damage to the small intestinal mucosa caused by the gluten fraction of wheat proteins and similar alcohol-soluble proteins (prolamines) of barley and rye in genetically susceptible subjects [101]. Clinical severity varies from silent to severe. FTT is the most frequent presentation in the pediatric age group. Increased frequency of other diseases such as type 1 diabetes or autoimmune thyroiditis, Down’s syndrome, Turner’s syndrome, or IgA deficiency, is found in family members of celiac patients. In developed countries, the prevalence of celiac disease among children and adults with type 1 diabetes exceeds the prevalence in the general population [101]. Reduced levels of vitamin E have also been reported [102]. Exclusively breastfed children with biopsy-proven celiac disease are significantly less likely to present with FTT [103].

Cystic fibrosis Severity of pulmonary infection often correlates with the degree of intestinal involvement and nutritional status. Lung function correlates with nutritional status [104]. One large study has shown that levels of specific antioxidants vitamin A, vitamin E, carotenoids, and glutathione were lower in CF patients than in controls, decreasing during acute exacerbation, and increasing after antibiotic treatment. Antioxidant levels were decreased with bronchial infection [93]. Only vitamin A and carotenoid were linked with body mass index (BMI). Intestinal inflammation may be a fundamen-

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tal feature of CF. Inflammatory markers such as soluble IL-2 receptor and eosinophilic cationic protein are often increased. Infections are worsened by diminished immune responsiveness, possibly related to abnormal zinc turnover, reduced thymulin activity, and reduced IL-2 and NK activity. Both copper and zinc are reduced in CF [105, 106]. Nutritional therapy includes dietary supplements, increased fat and protein absorption with oral pancreatic enzymes, supplemental fat-soluble vitamins (vitamin K), and omega-3 long-chain polyunsaturated fatty acids, such as docosahexaenoic acid. Evaluation of growth requires a specialized approach [107].

Malnutrition and food allergy Development of immune tolerance towards food and environmental antigens is a central requirement for gut homeostasis. The neonate encounters food, environmental antigens and microbes after birth. A functional relationship between the composition of normal commensal microflora and presence or absence of allergies and atopic disease in children has been recently shown by several groups. These findings include a report of reduced colonization with lactobacilli and higher counts of aerobic bacteria in a large study of allergic children [108] and the demonstration that characteristic differences in neonatal gut flora precede development of allergic responses [109]. A Th2-skewed immune response prevails systemically in the neonate, and contact with microbial antigens acts to repolarize this orientation gradually during the first months of life [110]. Studies strongly suggest that absence of exposure to appropriate microbial signals and lack of a Th2 to Th1 switch is associated with allergic disease in high-risk children. The primary mediators now appear to be regulatory T cells and dendritic cells, which down-regulate inflammatory response through production of IL-10 and transforming growth factor (TGF)-`1. Milk intolerance can be associated with failure to develop immune tolerance mediated by T regulatory cells [111].

Food intolerance and food allergy Food intolerance is defined as a reproducible adverse reaction to the ingestion of food or any of its components, i.e., proteins, carbohydrates, fats, and additives. Such adverse reactions include toxic, metabolic, and allergic reactions. Common forms of food intolerance include cow’s milk allergy (CMA), lactose intolerance that causes carbohydrate malabsorption, and gluten-sensitive enteropathy (celiac disease). CMA and other food allergies of childhood are often transient; more than 85% of children lose their sensitivity to most allergenic foods within the first 3–5 years of life. Most common allergenic foods in childhood include egg, cow’s milk, wheat, and soy. In the case of infants and toddlers, milk and egg are important source

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of calcium and protein, and dietary restriction may have long-term effects on growth and development. CMA may cause acute diarrhea [112]. Human milk also contains a number of immune-modifying substances, such as IgA antibodies toward bacteria, fungi, foods, and inhalants, and even inhalant allergens, as well as cytokines and chemokines. The protective effect for infection and prevention of atopy development is promoted both by specific immunologically active elements in milk and prebiotic oligosaccharides [113, 114]. Milk allergy is associated with IgA deficiency [115]. A low IgA content in maternal milk may lead to defective exclusion of food antigens and thus predispose an offspring to develop food allergies [116]. In addition, levels of TGF-`, a regulator of the mucosal immune system, may be important. TGF-` induces IgA production and oral tolerance. Inadequate production of TGF-` has been reported in children with CMA alone, and as part of multiple-food allergy presentations. This was associated with increased systemic pro-allergenic IL-4 responses on intestinal antigen contact [117]. Lactose intolerance is most common cause of carbohydrate malabsorption, with unabsorbed carbohydrate undergoing bacterial fermentation in the colon, producing gas and fluid. Maturational lactose deficiency may occur in premature infants. Both early and late onset congenital defects may occur at any age, with increased incidence in certain populations. Lactose intolerance may be associated with infection or develop in chronic infection such as HIV or parasitic infection [118–121].

Eating disorders Infantile anorexia was first described in a series of case studies, and was initially thought to be a separation disorder [6]. These children exhibit extreme food refusal and frequently fail to take in sufficient calories to sustain growth, and as a result display acute and/or chronic malnutrition. Eating disorders, such as bulimia (BN) and anorexia nervosa (AN), in childhood are characterized by a seriously undernourished state. In contrast, changes in the immune system have been less clear-cut and do not appear to follow the more typical types of malnutrition, such as PEM. In general, adaptive immunity seems to be preserved over long periods and susceptibility to viral infection is not common outside the advanced stages of disease. However, altered cell-mediated immunity in AN and BN is reflected in lymphocyte subset balance and poor response to delayed hypersensitivity tests [122]. A recent study compared healthy women to both underweight AN and normal-weight BN patients and reported that both patient groups had decreased plasma levels of leptin, prolactin, and 17`-estradiol. Plasma levels of cortisol were increased in AN, but not in BN, women. In bulimics, circulating leptin was inversely correlated with the duration of the illness and the frequency of bingeing/vomiting [123].

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Obesity Obesity develops when energy intake exceeds expenditure. Human obesity often becomes a permanent condition, and is thought to involve changes in the neural-endocrine network, which regulates energy intake, expenditure and storage [124]. Plasma leptin and insulin are signals in this system. Obesity-prone individuals may have an inborn reduction in their catabolic responses to glucose, leptin and insulin [125]. Involvement of immune pathways in obesity is also likely, as suggested by the role of leptin signaling in immune regulation. One study has revealed a possible relationship between a common inherited IL-6 promoter single-nucleotide polymorphism (174 G/ C), serum leptin and BMI [126]. Obesity is usually characterized by elevated circulating leptin levels, which may contribute significantly to the reported low-grade systemic inflammation. One hypothesis is that obesity involves altered metabolism secondary to changes in microflora. The gut microbiota as a whole is essential for production of short chain fatty acids from polysaccharides, and has been shown to regulate host metabolism through direct effects on fat storage [127]. Leptin secretion is linked to the functions of the hypothalamic-pituitary-adrenal axis and the immune system in response to infection, as shown by a study of leptin and cortisol response in acute sepsis in which survivors had higher levels of leptin [128]. Congenital leptin deficiency is a rare cause of severe early onset obesity characterized by absence of leptin, and carries a high risk of death due to infection in childhood [25, 129]. Generally, the incidence and severity of specific types of infectious illnesses are higher in obese persons and may also be linked to poor antibody responses to antigens in overweight subjects. A direct role for viral infection in obesity has also been proposed [130]. In vitro studies have shown that weight loss in obesity may be associated with improved immune function [22].

Chronic infection Infection causes metabolic disturbance that leads to short-term shifts in circulating levels of certain nutrients in association with the acute-phase response. In the presence of underlying malnutrition, infection may not be resolved, and a vicious cycle may be established. Although chronic infectious diseases are less prevalent in industrialized countries, infections with HIV, Mycobacterium tuberculosis (MTB), and hepatitis C virus (HCV) are significant problems that interact with nutritional status and immune response. Where there are recent immigrant populations, otherwise highly unusual parasitic infections must be considered in seeking the cause of metabolic disturbances in children living in industrialized countries [131, 132]. The role of malnutrition in pediatric HIV disease was appreciated as a significant cause of stunting and delayed maturation before the availability

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of effective anti-retroviral treatment and continues to be a significant area of investigation. Oral candidiasis and lower respiratory tract infections are more common than in children with non-HIV-associated immune deficiency [133]. Malnutrition, intestinal dysfunction, and immune impairment have been shown to increase the progression of HIV disease in children, and nutritional intervention in the form of total parenteral or enteral feeding can improve both nutritional status and CD4 count [134]. Children with HIV-associated FTT have similar levels of energy expenditure compared to HIV-positive children who have normal growth but show more advanced disease, severe immune suppression, increased viral burden, increased IL-6 activity, decreased total serum protein, and decreased IGF-1 levels [135]. Deficiencies of micronutrients are common in HIV-infected persons. Micronutrient impairment is causally associated with the course of HIV infection and immune dysfunction. This occurs due to malabsorption, altered metabolism, gut infection, and altered gut barrier function. Selenium deficiency increases the virulence of HIV and enhancing disease progression, while supplementation reduces high levels of IL-8 and TNF_ [136]. Vitamin A may increase the risk of HIV-1 transmission through breast milk [137]. In contrast, multivitamin supplementation of breastfeeding mothers with B, C, and E reduces child mortality and HIV-1 transmission through breastfeeding among immunologically and nutritionally compromised women. Supplementation in children with HIV-1 improves overall health. Generally, declining rates of MTB in industrialized countries have lead to less rigorous surveillance. In countries such as Canada, where BCG immunization has been used for selected indigenous population with higher risk of MTB, greater emphasis is placed on adverse reactions to BCG. However, MTB is an important opportunistic pathogen and can lead to significant infection in persons with nutritional insufficiency such as AN [138]. International adoptees are at high risk for acquisition of MTB and progression to active TB infection [139]. Many children have been vaccinated with BCG and, due to the mistaken belief that this always results in a positive Mantoux test and should be ignored, adds to the complexity of evaluation. Current studies indicate that less than 50% of infants given BCG shortly after birth have reactive Mantoux test results at 12 months of age and almost all vaccinated infants have nonreactive skin test results by 5 years of age [140]. The natural history and clinical manifestations are different in children and are associated with the age at infection and the host immune status. Vitamin D deficiency is associated with an increased risk for TB infection. Studies using in vitro systems indicate that 1,25-dihydroxyvitamin, the most active form of the vitamin, enhances mycobacterial killing by increasing NO production. Aerosol-challenge with Mycobacterium bovis in the NO synthase 2 deficient (NOS2–/–) mouse leads to increased mycobacterial colonization and lesion formation compared to wild-type mouse.

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Infected NOS2–/– mice developed severe necrotizing pyogranulomatous inflammation [141]. In these studies, lung colonization and lesion area of vitamin D-deficient mice exceeded that of vitamin D-replete mice, regardless of NOS2 phenotype, demonstrating a fundamental role for vitamin D. However, effects of vitamin D on colonization, but not lesion area, were more pronounced in NOS2+/+ mice than in NOS2–/– mice, suggesting NOindependent effects of vitamin D as well. Primary malnutrition increases the incidence and exacerbates clinical manifestations of MTB infection. Experimental studies in the mouse have shown that PCM reduces production of IFN-a, TNF-_ and NO after MTB infection, leading to a decreased granulomatous reaction, higher lung bacillary load, and a more fatal TB course than in well-nourished control mice, and that this can be reversed by restoring a diet with normal protein content [142, 143]. One study of US immigrants reported that the most common pathogens were Trichuris trichiura, Giardia lamblia, and Ascaris lumbricoides. Giardia lamblia was more prevalent in the younger than 5-year-old age group, and helminths were more prevalent in the 6- to 10-year-old age group. No helminths were found in immigrants who had been in the US for more than 3 years. Infection caused by intestinal parasites irritate the GI tract, cause pain, anorexia, flatulence, tenderness, and affect the host nutrition directly as a result of inflammatory and non-inflammatory diarrhea. Host response mechanisms include accelerated epithelial cell turnover [144]. Trace element deficiencies affect the host pathogen interaction. Examples include the exacerbating effect of selenium deficiency on Trypanosoma cruzi, which is responsible for Chagas disease [145]. Malnutrition can cause an imbalance in T cell subpopulations that may lead to a defective T cell maturation and a decreased specific anti-Ascaris IgE response and worsens infection with A. lumbricoides [146]. Malaria causes the most serious nutritional consequences of any major parasite. It infects the placenta and compromises blood flow to the fetus, causing low birth weight. It also causes PCM in pregnant and lactating women and young children. Anemia, recurrent fever with acute-phase cytokine responses, vomiting and anorexia all produce adverse nutritional consequences in an already fragile child or pregnant women. Recent investigation suggests that micronutrients such as vitamin A, vitamin E, and zinc, may improve the morbidity of malaria through immune modulation and alteration of oxidative stress [147]. The lack of an effective HCV vaccine and the risk of mother-to-child transmission may increase the number of children with vertically acquired HCV that ultimately go on to develop liver fibrosis or cirrhosis [148]. There appear to be no direct effects of HCV on growth in the first 5 years [149]. Chronic HCV infection is usually asymptomatic, although viremia and liver enzyme increases are found in children [150], but significant liver disease may occur [151]. In patients with liver cirrhosis, PEM is a frequent finding and a risk factor influencing survival [152].

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The overall impact of chronic subclinical malnutrition in children may determine the quality and duration of immune response to vaccines and may be an important topic for future research.

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Gordon JI (2004) The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 101: 15718–15723 Bornstein SR, Licinio J, Tauchnitz R, Engelmann L, Negrao AB, Gold P, Chrousos GP (1998) Plasma leptin levels are increased in survivors of acute sepsis: associated loss of diurnal rhythm, in cortisol and leptin secretion. J Clin Endocrinol Metab 83: 280–283 Otero M, Lago R, Gomez R, Dieguez C, Lago F, Gomez-Reino J, Gualillo O (2006) Towards a pro-inflammatory and immunomodulatory emerging role of leptin. Rheumatology (Oxford) 45: 944–950 Dhurandhar NV (2004) Contribution of pathogens in human obesity. Drug News Perspect 17: 307–313 Schwarzwald H (2005) Illnesses among recently immigrated children. Semin Pediatr Infect Dis 16: 78–83 Salas SD, Heifetz R, Barrett-Connor E (1990) Intestinal parasites in Central American immigrants in the United States. Arch Intern Med 150: 1514–1516 Barker D, Younger N, MooSang M, McKenzie CA (2004) HIV serostatus and recovery from severe childhood malnutrition. A retrospective matched casecontrol study. West Indian Med J 53: 89–94 Guarino A, Spagnuolo MI, Giacomet V, Canani RB, Bruzzese E, Giaquinto C, Roggero P, Plebani A, Gattinara GC (2002) Effects of nutritional rehabilitation on intestinal function and on CD4 cell number in children with HIV. J Pediatr Gastroenterol Nutr 34: 366–371 Johann-Liang R, O’Neill L, Cervia J, Haller I, Giunta Y, Licholai T, Noel GJ (2000) Energy balance, viral burden, insulin-like growth factor-1, interleukin-6 and growth impairment in children infected with human immunodeficiency virus. AIDS 14: 683–690 Baum MK, Miguez-Burbano MJ, Campa A, Shor-Posner G (2000) Selenium and interleukins in persons infected with human immunodeficiency virus type 1. J Infect Dis 182 (Suppl 1): S69–73 Fawzi WW, Msamanga GI, Hunter D, Renjifo B, Antelman G, Bang H, Manji K, Kapiga S, Mwakagile D, Essex M et al (2002) Randomized trial of vitamin supplements in relation to transmission of HIV-1 through breastfeeding and early child mortality. AIDS 16: 1935–1944 Hotta M, Nagashima E, Takagi S, Itoda I, Numata T, Kobayashi N, Takano K (2004) Two young female patients with anorexia nervosa complicated by Mycobacterium tuberculosis infection. Intern Med 43: 440–444 Miller LC (2005) International adoption: infectious diseases issues. Clin Infect Dis 40: 286–293 Starke JR, Correa AG (1995) Management of mycobacterial infection and disease in children. Pediatr Infect Dis J 14: 455–469; quiz 469–470 Waters WR, Palmer MV, Nonnecke BJ, Whipple DL, Horst RL (2004) Mycobacterium bovis infection of vitamin D-deficient NOS2–/– mice. Microb Pathog 36: 11–17 Boelaert JR, Gordeuk VR (2002) Protein energy malnutrition and risk of tuberculosis infection. Lancet 360: 1102 Chan J, Tian Y, Tanaka KE, Tsang MS, Yu K, Salgame P, Carroll D, Kress Y,

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Better education through improved health and nutrition: implications for early childhood development programs in developing countries Matthew Jukes Harvard Graduate School of Education, Appian Way, Cambridge, MA 02138, USA Partnership for Child Development, Department of Infectious Disease Epidemiology, Imperial College School of Medicine, Norfolk Place, London W2 1PG, UK

Abstract Before children reach school age they must negotiate threats from a number of diseases. More than 50% of child deaths are caused by pneumonia, diarrhea, malaria, measles, malnutrition and HIV. For those who survive, health and nutrition can affect children’s development. School readiness depends on cognitive, motor and socio-emotional development, which can be affected by, among other things, undernutrition, iron deficiency anemia and malaria. There is clear evidence of the benefits of preschool health and nutrition interventions to tackle these three conditions. For malnourished children, psychosocial stimulation can be as effective as nutritional supplementation in compensating for delayed cognitive development. In general, interventions in this preschool age group have substantial and consistent effects on development and education, which are generally larger than for school-age children. Effects are seen in all dimensions of school readiness – cognitive, motor and socio-emotional development – but are perhaps greatest for motor development. They also have a greater impact on the most disadvantaged children and can help to promote equity in educational outcomes. Overall, evidence suggests that early childhood health and nutrition interventions have the potential to make a major contribution to achieving the goal of Education for All.

Introduction Public health interventions to promote child survival have long been a priority for governments and development agencies. However, beyond issues of mortality, the role of health and nutrition in promoting child development and educational outcomes is increasingly being recognized [1, 2]. This chapter examines how common pediatric conditions affect children’s cognitive, motor and socio-emotional development and consequently their readiness for school. Evidence of the impact of health and nutrition interventions on child development is reviewed and the potential for their inclusion in early childhood development (ECD) programs is considered.

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Health and nutrition problems in preschool children It is becoming apparent that treating health and nutrition problems in preschool children (< 5 years old) is important for two reasons. First, these children account for more than 50% of the global gap in mortality between the poorest and richest quintiles of the world’s population and second, they bear 30% of the total burden of disease in poor countries. There are an estimated 600 million preschool children worldwide [3] and they have severalfold higher case fatality rates for many infections, therefore keeping them healthy gives them a better survival rate in childhood and adulthood. Out of 100 children born in each year, 30 will most likely suffer from malnutrition in their first 5 years of life, 26 will not be immunized against the basic childhood diseases, 19 will lack access to safe drinking water and 40 to adequate sanitation and 17 will never go to school. In developing countries, every fourth child lives in abject poverty, in families with an income of less than $1 a day. As a consequence nearly 11 million children each year – about 30 000 children a day – die before reaching their fifth birthday, mostly from preventable causes. Of these children, 4 million die in their first month of life. Of the 10.5 million children that died in 1999, 99% were from developing countries and of these 36% were in Asia and 33% in Africa. In many of the world’s poorest countries, child mortality rates have either not changed or else they have worsened. In sub-Saharan Africa, child mortality averages 173 deaths per 1000 live births, and in South Asia 98 deaths per 1000 – many times the industrialized country average of 7 deaths per 1000. More than 50% of all child deaths (< 5 years old) are due to five communicable diseases, which are treatable and preventable. These are pneumonia, diarrhea, measles, malaria and HIV/AIDS. For those who survive, poor health and nutrition has an impact on their lives, which is less apparent but which nonetheless has serious implications for their development and their education. This impact is considered in the following section.

Impact of health and nutrition on school readiness Common conditions of poor health and nutrition can affect education in a number of ways. First of all, health and nutrition has an impact on children’s access to education, particularly where disease leads to serious physical or mental disabilities. However, this chapter addresses the impact of health and nutrition on children’s ability to learn once they do enroll in school – their ‘school readiness’. This impact on school readiness may have knock-on effects for children’s educational achievement and attainment, particularly where effects of disease and poor nutrition on brain development persist as cognitive impairments or emotional problems throughout the school-age years.

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School readiness refers to a range of competencies that preschool children should possess to benefit from the school environment. In order to be ready for school, in this sense, children require certain cognitive skills, such as language abilities and numeracy, a level of physical and motor development, and appropriate socioemotional development. Each of these factors will be given individual consideration in reviewing the evidence for an effect of preschool health and nutrition on school readiness.

Undernutrition Effects on cognitive development Undernutrition (also called ‘protein energy malnutrition’) is a general term applied to children with heights and weights below age-referenced criteria. It typically results from a severe or chronic lack of a range of essential nutrients rather than from just a lack of protein. This complicates the discussion of the cognitive consequences of undernutrition because several different causal factors may be involved, each potentially associated with a different means of affecting brain and behavior. Nevertheless, evidence suggests that undernutrition impairs children’s mental development in the early years, through one mechanism or another. A low height or weight for age is associated with impairment in developmental levels of young children (see [4] for a review). For example, in Guatemala the length and weight of 1–2-year olds was related to their scores on a test on infant mental development [5]. Children hospitalized with severe malnutrition show lower developmental levels, but not more so than in children hospitalized for other reasons [6]. Similarly, on recovery the development levels of severely malnourished children remain impaired but this is likely attributable to chronic undernutrition rather than the acute episode itself [7]. Quality evidence of the relationship between nutrition and cognitive development comes from intervention trials that fall into two categories: preventative and therapeutic. We look here at these in turn. In many countries steps have been taken to prevent malnutrition in children by beginning nutritional supplementation in pregnancy and continuing in infancy. This approach has been successful in improving cognitive development. In Guatemala, such a supplementation program found small improvements in cognitive function for children between 3 and 7 years [8]. Supplementation in Mexico from shortly after birth and throughout the first 3 years was found to improve children’s school performance and language skills [9]. In addition, from 8 months of age, supplemented children became increasingly active and by 2 years of age were showing eight times more activity than non-supplemented children. A similar program with high-risk mothers in Bogotá, Colombia was successful in improving the mental development of

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their children at 18 months and also their language skills at 36 months [10]. One group of mothers in this study received education on how to stimulate cognitive development in their children. This program improved children’s language skills assessed at 18 months and 36 months. In addition, the nutritional supplementation and maternal education program worked synergistically: supplementation improved the effectiveness of stimulation (or vice versa) such that the benefit of receiving both interventions was greater than the sum of the independent benefits of the two interventions. A final finding is worthy of note from this study: Overall girls benefited more from the program than boys. This study is fairly unusual in reporting such an effect. However, if gender differences were found to be common in children’s response to nutritional supplementation, this would have important implications for the gender equity goals of Education for All. One study in Kenya [11] found a benefit of a school-feeding program for children’s educational outcomes. Children were given a breakfast meal throughout and an ECD class, and improvement was found in educational achievement but not in tests of cognitive function, and was only evident in schools with an experienced teacher. The improvement in educational achievement was around 0.4 SD. Results from therapeutic trials also provide strong evidence of a link between nutritional supplementation and cognitive development. These studies have typically involved remedial nutritional supplementation of malnourished children. In Bogotá, Colombia children from a poor urban area who underwent four periods of an educational stimulation and nutritional supplementation program between the ages of 42 and 84 months showed a gain in general cognitive ability of 0.80 SD in comparison with a group who received the same treatment for only one period between the ages of 74 and 84 months [12]. In so doing, these children closed the gap in IQ between themselves and a group of richer urban children. In this study, children received both nutritional supplements and education, and it is not possible to decipher which of these two interventions was most influential in improving children’s cognitive abilities. A more recent study in Jamaica helped resolve this issue by giving poor, urban and undernourished children aged 9–24 months a 2-year program of either nutritional supplements, stimulations, both interventions or neither intervention. The gains in overall development quotient (DQ), an IQ equivalent for infants and young children, were impressive. Nutritional supplementation accounted for an increase of 6.1 DQ points (0.66 SD) over 2 years, while stimulation improved DQ by 7.3 points (0.79 SD). The effects of the two interventions were additive (receiving both interventions was better than receiving only one of them) but there was no interaction between them (nutritional supplementation did not improve the effectiveness of the stimulation program, for example). Significantly, the children who did receive both treatments effectively closed the gap in DQ between themselves and adequately nourished children [13].

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Long-term effects on cognition The above studies show that undernutrition leads to impaired school readiness in terms of cognition. The reason for concern about delayed school readiness is that children are likely to perform less well at school as a result. But is there evidence of this? It is certainly possible that differences in school readiness at the age of school entry may lead to poor achievement, which in turn leads to drop out and repetition, and thus deficits become compounded. On the other hand, mental development can be quite robust to early difficulties. For example, large differences in language abilities in the preschool years typically even out in the early years of primary school. The following reviews the evidence that preschool undernutrition has longterm effects. Beginning with the most profound nutritional insults, severe malnutrition in early childhood has a long-term effect on development. Children in Jamaica who were admitted to hospital suffering from severe malnutrition between the ages of 6 and 24 months were found to lag behind adequately nourished children, who had been hospitalized for other reasons at ages 7, 8, 9 and 14 years, on a range of IQ tests. At 14 years they were substantially delayed in overall IQ (1.50 SD below the control group), vocabulary (1.33 SD) and tests of educational achievement, even after accounting for differences in the background of the two groups of children [14]. These are substantial differences that are far from unique. Similar results have been found in more than a dozen other studies [15]. Other results from experimental interventions strengthen the evidence for a long-term effect of nutrition on cognition and also demonstrate the potential for reducing the gap between severely undernourished children and their peers. The study in Jamaica found that a 3-year program to teach mothers how to improve the development of their child (aged 6–24 months at the beginning of the program) conferred significant long-term benefits on undernourished children. At age 14 years, the undernourished children whose mothers had taken part in the education program were only 0.28 SD behind adequately nourished children on overall IQ scores and 0.68 SD ahead of undernourished children who had not taken part in the intervention. It is clear that severe malnutrition has a substantial long-term effect on child development. Of potentially greater concern is the effect that mild and moderate malnutrition has on child development, given the high prevalence of this condition amongst children in developing countries. This issue has again been addressed by researchers in Jamaica who followed 127 undernourished children for 8 years. As discussed above, these children received a 2-year program of nutritional supplementation, psychosocial stimulation, both interventions or neither intervention. Four years after the end of interventions, perceptual/motor skills – but not other cognitive skills – were superior in those children who had received stimulation [16]. The same skills were also superior for children who had originally received a nutritional

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supplement and whose mothers had the highest verbal intelligence. One explanation for this interaction was that the most intelligent mothers were also the ones giving children the most stimulation. There were no effects of the intervention on general cognitive abilities or on memory, although each intervention group had higher scores than the control subjects on more of these cognitive tests than would be expected by chance. Thus, stimulation, and to a lesser extent supplementation, had modest effects on children’s cognitive abilities over 4 years. The study also compared the stunted children taking part in the original intervention with other children from similar backgrounds, but who were known not to be stunted at the time of the interventions. These non-stunted children had higher scores on the general cognitive factor than previously stunted children, although they were no better in perceptual-motor skills or memory. There were similar findings 8 years after the end of the intervention. Children who received stimulation as infants had a higher IQ (by 0.42 SD) at ages 11–12 years, while supplementation had no effect on cognitive abilities of children at this age. Again, children who were stunted before 2 years of age had a lower IQ (by 0.60 SD) and performed less well on eight out of nine cognitive tests (effect size range 0.38 SD to 0.61 SD) at age 11–12 years than children who were not stunted before 2 years of age [17]. A more recent study in Vietnam [18] adds to our understanding of the interaction between educational and nutritional interventions in early childhood. In this study, children aged 0–3 years in five communities were given nutritional supplements. In two of these communities children took part in an ECD project at ages 4–5 years. At ages 6–8 years those who had received both interventions scored 0.25 SD higher on the Raven’s Progressive Matrices Test (a test of non-verbal reasoning) than those who had received only the nutritional intervention. The effect was particularly pronounced for those who were stunted at the time of testing. Amongst stunted children, those who had received both interventions scored significantly better (0.67 SD) than those who had only received the nutrition intervention. Furthermore, the ECD intervention appeared to counteract the impact of stunting on cognitive abilities, whereas those who had received nutritional supplements but no ECD intervention showed a large (~0.5 SD) difference between stunted and non-stunted children (Fig. 1). In another long-term follow-up study in Guatemala, children given nutritional supplements prenatally and in the immediate postnatal period (up to 2 years) were found to perform better as adolescents (aged 13–19 years) on tests of vocabulary, numeracy, knowledge, and reading achievement [19]. Interestingly, these benefits were found only for those children of low socio-economic status. In tests of reading and vocabulary, the effect of supplements was most evident for children with the highest levels of education. Performance in tests of memory and reaction time were better in supplemented children, although the improvement did not depend on

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Figure 1. Impact of two preschool interventions in Vietnam on cognitive abilities of children aged 6–8 years.

socio-economic status or education. A later study of women in this cohort [20] found a positive effect of the nutritional intervention on educational achievement but only for those who had completed primary school. The studies in Jamaica and Guatemala show that a fairly sustained program of nutritional supplementation and/or psychosocial stimulation, lasting for 2 years, can have long-term benefits for children’s development. A study in Indonesia shows that even a 3-month program of supplementation can have long-term effects [21]. Children supplemented before 18 months were found to have improved performance on a test of working memory at age 8 years, although no effect was observed on other measures of information processing, vocabulary, verbal fluency and numeracy.

Undernutrition and motor development Motor development is an important aspect of school readiness and can often be closely associated with cognitive development. Four studies were found that reported the impact of nutritional supplementation on motor development. Three of the studies were reported above and found a greater impact of the intervention on motor development than on cognitive development. A third study found an impact on motor development but not on cognitive development. The first study found improvement in motor development of

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infants in Taiwan by 8 months of age [22] following supplementation during pregnancy and early infancy. The second study is the preventative trial in Columbia [10]. At 18 months this program was successful in improving the motor development of their children to a greater extent than their mental development. In another preventative trial in West Java, Indonesia [23], a short-term intervention – only 90 days of nutritional supplementation beginning after pregnancy – found improvements in the motor development of children at between 6 and 20 months of age. No impact was found on mental development. Finally, in the Jamaican study, giving nutritional supplementation and/or psychosocial stimulation to undernourished children, larger gains were found for the locomotor sub-scale of the assessment battery than for mental development – a 12.4 point (1.04 SD) increase was found due to supplementation (compared with 6.1 points for mental development) and 10.3 points (0.87 SD) due to stimulation (compared with 7.1 points for mental development). A possible interpretation of these results is that nutritional supplementation is more important for motor development than for mental development. Four years after the end of interventions, motor skills were superior in those children who had received stimulation [16].

Socio-emotional development Evidence on social and emotional development is more scarce than evidence on mental and motor development. This is due in part to the difficulty in measuring development in this domain and the time-consuming observation techniques that are typically involved. But some evidence suggests that both chronic and acute malnutrition is associated with changes in social and emotional development in young children. For example, in Kenya, undernourished infants were found to be less sociable than adequately nourished infants [24]. Acute episodes of severe undernutrition can lead to increased apathy, decreased activity and a less frequent and less thorough exploration of the environment [15]. After the acute episode, all behavior returns to normal except for the thoroughness of exploration of the environment. Similar to motor and cognitive development, aspects of social and emotional behavior can be improved by interventions. The program in Mexico [9], which gave nutritional supplements from shortly after birth and throughout the first 3 years, was found to improve adaptive behavior and personal and social behavior in addition to the cognitive improvements reported above. Similarly, the supplementation program with high-risk mothers in Bogotá, Columbia found improvements in personal and social skills as well as the cognitive and motor improvements reported above [10]. Children who enter school with poor socio-emotional developmental levels are a concern because they are less able to adapt to the school and less able to learn. The link between socio-emotional development and cognitive development is clear. For example, in Kenya, children who were

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undernourished at 6 months were also less sociable, and those who were less sociable at 6 months had lower development scores at 30 months and poorer verbal comprehension scores at 5 years [24]. However, poor socio-emotional development is a concern in its own right for the school-age child. In addition, there is good evidence from Jamaica that nutritional deficiencies in early childhood have a long-term impact on socio-emotional outcomes. Children who were stunted before aged 2 years in this study were more likely to have conduct disorders aged 11–12 years [25]. However, those who received psychosocial stimulation during early childhood as part of this program were found in a recent follow-up to be less anxious and depressed with fewer problems of poor attention and low self-esteem [26]. There were no such beneficial effects from children who received nutritional supplementation as part of this program. It is not clear from this study how such long-term effects arose. It is possible that they represent the continuation of social and emotional benefits of the psychosocial intervention, which were already evident in early childhood. Alternatively, they may have resulted from, for example, improved cognitive abilities that resulted from the intervention and led to increased self-esteem and other positive psychosocial outcomes. However, taking findings of short-term and long-term effects together, there is strong evidence that undernutrition can lead to poor socio-emotional outcomes, which will affect school readiness.

Timing It might be expected that nutritional deficits in the first year of life have the greatest impact on development. However, evidence does not bear this out. A study in Colombia found that giving nutritional supplements to children between 6 months and 36 months of age had a greater impact on cognitive development at 36 months than supplements given to the mother in the third trimester of pregnancy and then to the child up to 6 months of age, and the same impact as a continuous supplementation running from the third trimester of pregnancy to 36 months [10]. A longer-term study in the Philippines found that malnutrition in the second year of life actually had a greater impact on the performance of 8-year-old children on a non-verbal test of intelligence than malnutrition in the first year of life [27]. Other studies support early supplementation. In Indonesia, children supplemented before – but not after – 18 months of age were found to have improved performance on a test of working memory at age 8 years [21]. Another study in the Philippines found that children stunted in the first 6 months were more likely than those stunted later on to have impaired cognitive performance at 8 years of age [28]. This however was explained by the fact that the children suffering the earliest bouts of malnutrition also suffered the most severe and persistent malnutrition. A confounding factor

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such as this is a reminder of the difficulty in interpreting findings related to timing effects of nutritional deficiencies on cognitive development. At present, there is no strong evidence that early (first year of life) interventions with children suffering from or at risk of malnutrition are more effective than interventions at a later age.

Maternal behavior A child’s development is shaped by a complex interaction of factors in its environment. Just as a child’s active interaction with its environment is crucial for development so is the active engagement of others in their environment. Nutrition can play a part in this too. In Egypt and in Kenya, maternal behavior towards toddlers was found to be influenced by the nutritional intake of the child more than that of the mother [29], with poorly nourished children more likely to be carried by their mother and in general stay closer to their mother than adequately nourished children [30]. In addition to the effect child malnutrition has on maternal behavior, evidence from Mexico suggests that mothers of malnourished children behave differently towards their children even before the onset of malnutrition [31]. They were less likely than other mothers to reward the successes of their child, were less affectionate and talked less to them. This could be because mothers of children who become malnourished are less well educated than other mothers [14]. In addition, mothers of malnourished children may often be poorly nourished themselves, which in turn affects their behavior. In Kenya, it was found that although toddlers were protected from the effects of temporary food shortages, their mothers were not and maternal nutritional deficiencies led to changes in the quality of motherchild interactions [32]. These findings have clear implications for children’s development. We have seen that psychosocial stimulation is perhaps the most important factor preventing poor cognitive outcomes in malnourished children. If these children typically receive poor levels of stimulation from their parents – for whatever reason – the lack of stimulation is likely to compound the effects of nutrition on their development.

Low birth weight A number of the intervention studies reported above begin nutritional supplementation before birth in recognition of the importance of prenatal nutrition. Children with a low birth weight or more generally, those born small for their gestational age (SGA) have poor developmental outcomes with implications for school readiness. Differences between SGA babies and those of normal birth weight typically do not appear in the first year

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of life [33], although this can depend on environmental factors. In Brazil, developmental delays were observed only in SGA babies who also received little stimulation in the home. Similarly, low birth weight affects infant development to a greater extent in the homes of illiterate mothers as compared to literate mothers. Deficits in developmental levels appear with high-risk infants in the second year with clear significant differences apparent by the third year. Some deficits were also found in the development levels of SGA babies between the ages of 4 and 7.

Breast feeding The percentage of infants who are exclusively breastfed in the first 6 months of life fell from 43% in 1998 to 34% in 2004 [34]. In Western and Central Africa the figure is only 20%. This is of concern because breast feeding is associated with a moderate long-term improvement in cognitive development. A review of 17 studies in developed countries estimated that breast feeding led to an improvement of 3.2 IQ points (~0.21 SD), which was fairly stable across the lifespan from 3 to 50 years of age [35]. Low birth weight babies benefit most from breastfeeding, gaining 5.2 IQ points (0.35 SD) compared with a gain of 2.7 points (0.18 SD) for children of normal birth weight. The effects of breastfeeding also depend on the length of time that infants were breastfed. Scandinavian children breast fed for longer than 6 months were found to have improved cognitive tests outcomes at 5 years compared with children who were breastfed for less than 3 months [36]. However, it is difficult to be certain about such findings since mothers who choose to breastfeed are often more educated or more wealthy and this could explain some of the difference in IQ scores [37], although review studies do attempt to account for such factors in their estimates of IQ differences [38]. In general, the evidence is not conclusive but is strongly suggestive of a link between breast feeding and cognitive ability in later life.

Iron-deficiency anemia Iron deficiency and mental development: Children < 2 years A number of studies have found that infants with iron deficiency have lower developmental levels than iron-replete children. Lower scores on the Mental Development Index and the Psychomotor Development Index of the Bayley Infant Development Scales for iron-deficient children have been found with 12-month-old children in Chile [39], 12- to 23-month-old children in Costa Rica and [40], 6- to 24-month-old children in Guatemala [41], and 12- to 18-month-old children in Indonesia [42].

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Only one rigorous randomized controlled trial has been conducted on the impact of iron supplementation on children less than 2 years of age in a low-income country that has met rigorous criteria for experimental design (a double-blind randomized controlled trial). This study in Indonesia [42] gave iron supplementation (iron sulfate) or placebo to iron-deficient children aged 12–18 months. Those receiving iron supplementation showed impressive gains in the Bayley Scales of Infant Development. Their Mental Development Index rose by 19.3 points (1.3 SD). This represents a substantial improvement by children receiving iron supplementation. At the end of the 4-month trial, these children had similar developmental levels to those who were not iron deficient in the first place. Other studies have conducted supplementation trials over a similar time period (* 12 weeks), although none had the same rigorous experimental design. One other study in Indonesia succeeded in eliminating differences between iron deficient and iron-replete children after supplementation, while in two other studies, in Chile [39] and Costa Rica [40], there was no observed effect of supplementation. However, in the Costa Rica study, children whose iron status recovered completely also showed improvement in their mental and psychomotor development indices. A number of shorter term trials (< 15 days) have also been conducted. There is no evidence of improvement of iron-deficient children in such trials [43]. Taken together, the evidence from all trials suggests that iron supplementation can improve the development of children under 2 years if sustained over a sufficiently long period of time (~12 weeks).

Iron deficiency and mental development: Children aged 2–6 years A number of studies have compared iron-deficient/anemic children with iron-replete children. Working in the preschool age group, Pollitt et al. [44] found that Guatemalan children with iron-deficiency anemia took longer to learn a discrimination task than their iron-replete peers. The difference between the two groups was substantial in this test (> 3 SD), although there were no differences in two other tests. Similarly, Soewondo et al. [45] found that Indonesian children with iron-deficiency anemia were slower than iron-replete children in a categorization task, although the two groups performed similarly on tests of learning and vocabulary. No such differences were found with younger children in one study in India [46]. All five studies in the preschool age group have found improvements in the cognitive function of iron-deficient children following iron supplementation, including improvements in a learning task [44, 45] and in an IQ test [46]. One study in Zanzibar [47] gave 12 months of iron supplementation and deworming treatment to children aged 6–59 months from a population in which iron deficiency was common. They found that iron supplementation improved preschoolers’ language outcomes by 0.14 SD.

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One study has looked at the impact of iron supplementation in a preschool setting. This study [48] was conducted with 2–6-year olds in informal settlements in East Delhi. Children who received 30 days of iron supplementation had improved attention in class, as rated by their teachers. The improvement was around 0.18 SD in comparison with the control group. However, there was no impact on a measure of general cognitive development. All these studies indicate that iron deficiency can lead to substantial impairments in cognitive development, which are likely to impair children’s readiness for school. What is the evidence that such deficiencies have longterm implications for children’s school achievement? The most comprehensive study to address this question followed a group of Costa Rican infants for more than 10 years [49, 50]. At 12–24 months of age, 30 of the group of 191 infants had moderate anemia and received treatment. At age 5 years, formerly anemic infants performed less well on a range of tests of non-verbal intelligence, after accounting for differences between the two groups in a number of variables such as socio-economic status, birth weight, maternal IQ, height and education. Verbal skills were more equally matched between groups. At age 11–12 years, the formerly anemic group performed more poorly in writing and arithmetic, and spatial memory. Older children only were poorer in a selective attention test. A number of other studies have found similar long-term effects of iron deficiency [43]. Anemic infants in Chile [51] were later found to have lower IQs and poorer performance on a range of tests of verbal and visual abilities at 5 years of age. Studies have attempted to quantify the relationship between infant anemia and later cognitive impairment. A study with infants in Israel [52] found that a reduction in hemoglobin levels of 10 g/l at 9 months was associated with a reduction of 1.75 IQ points at 5 years of age (although no effect on developmental levels was found at 2 and 3 years of age). Children in the anemic group were found to be learning less well and to be less task-oriented than control children in second grade [53]. The results from these studies should be interpreted with a degree of caution. None of the studies reported in this section allows causal inferences to be drawn. In each study, the anemic group most likely differed from the control groups on a number of background variables such as socio-economic status. One study [51] found that, in comparison to the control group, the homes of anemic infants were less stimulating and their mothers were more depressed and less affectionate. Thus, we cannot be sure that differences in performance between groups are not attributable to these other background characteristics, even though comprehensive attempts were made to control for them statistically in most studies. Notwithstanding this caveat, the evidence of the effect of anemia and iron deficiency on the brain, on the behaviors of infants, preschoolers and their caregivers, and the suggestion that the effect is a long-term one, combine to make a persuasive case for early intervention to prevent iron deficiency.

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Iron deficiency and motor development Iron supplementation is found to have a substantial impact on the motor development of infants and also a significant effect on older preschool children. One study in Indonesia gave iron supplementation (iron sulfate) or placebo to iron-deficient children aged 12–18 months and scores on the Psychomotor Development Index of the Bayley Scales of Infant Development rose by 23.5 points (1.6 SD). Most studies find cognitive or motor impacts of around 0.2–0.4 SD, but this study in Indonesia shows that iron supplementation can have truly substantial effects on development. A study with older (6–59 months) preschool children in Zanzibar [47] found that 12 months of iron supplementation and deworming treatment improved preschoolers’ motor outcomes by 0.18 SD. Such effects found with children of enrollment age persist into the school-age years. In Costa Rica, formerly anemic infants performed poorly on motor tests at 5 years of age and again aged 11–12 years [50]. Anemic infants in Chile [51] were also later found to perform poorly on a range of tests of motor function.

Socio-emotional development There is clear evidence that iron-deficiency anemia affects social and emotional development. In Costa Rica [40], infants with iron-deficiency anemia were found to maintain closer contact with caregivers; to show less pleasure and delight; to be more wary, hesitant, and easily tired; to make fewer attempts at test items; to be less attentive to instructions and demonstrations; and to be less playful. In addition, adults were found to behave differently towards iron-deficient children, showing less affection and being less active in their interactions with these children. Such findings have serious implications for the amount of stimulation children receive, both from their own exploration of the environment and in the stimulation they receive from their caregivers. When these infants were followed up at age 11–12 years [49], the formerly anemic group was more likely to have a number of behavioral problems. They were more anxious and depressed, had more attention problems, social problems and behavioral problems overall. They were also more likely to repeat grades at school and to be referred for special service.

Iodine deficiency Iodine is required for the synthesis of thyroid hormones. These hormones, in turn, are required for brain development, which occurs during fetal and early postnatal life [54]. Mental development is affected by both maternal

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hypothyroidism (a deficiency in maternal thyroid activity), which affects development of the fetal brain during the third trimester, and hypothyroidism in the newborn, which affects postnatal brain development. In either case, a spectrum of neurological disorder can ensue, from severe mental retardation associated with cretinism to more subtle neurological impairments. Nearly 50 million people suffer from iodine-deficiency disorderrelated brain damage. A relatively small proportion of these (< 10%) are cretins with the remainder suffering more mild impairments. Iodine supplementation in pregnancy reduces cretinism and improves IQ and school achievement between 8 and 15 years of age in one study [55] and between 14 and 16 years of age in another [56]. The clear evidence from these intervention studies is supported by findings of impaired cognitive function in adults and children living in iodinedeficient areas. An estimate based on an analysis of 21 studies suggests that general intelligence is 0.40 SD lower in iodine-deficient areas [57]. However, there is no clear evidence for the cognitive benefits of targeting preschool children with iodine supplementation.

Other micronutrients A few other micronutrients have been studied in relation to their effect on the cognitive development of young children. There is a growing literature on zinc and mental development. In the UK, children with dyslexia were found to be deficient in zinc and have higher concentrations of toxic metals in their sweat and hair [58]. Animal studies show that zinc deficiency in offspring causes impaired learning, which can be corrected by zinc supplementation [59]. One study has been conducted to investigate the impact of maternal zinc supplementation on cognitive development. This study in Bangladesh [60, 61] gave zinc (30 mg daily) or placebo (cellulose) to pregnant women from 4 months’ gestation to delivery. At 6 months, the children whose mothers had been given zinc supplementation had poorer outcomes in both mental development and psychomotor development indices. This is likely due to an imbalance of micronutrients and suggests caution should be exercised when targeting single micronutrient deficiencies for supplementation.

Disease Cognitive impacts of malaria The most significant infectious disease for the mental development of young children is cerebral malaria. In addition to the mortality and severe neurological sequelae associated with cerebral malaria, many children suf-

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fer more subtle cognitive deficits, which may affect their ability to learn later on in life. In Kenya, children aged 6–7 years were studied 3–4 years after hospitalization due to cerebral malaria with impaired consciousness [62] and were found to be 4.5 times more likely than other children from similar backgrounds to suffer cognitive impairment ranging from severe learning difficulties requiring care to mild cognitive impairments. Almost half of such children had had no neurological problems at the time of hospitalization. Similarly, in Senegal children aged 5–12 were found to have impaired cognitive abilities due to a bout of cerebral malaria with coma before the age of 5, possibly due to a primary deficit in attentional abilities [63]. A third study in the Gambia looked at children who suffered from cerebral malaria that was not accompanied by neurological symptoms at the time [64]. These children had poorer balance 3.4 years after recovery implying some impaired motor development. However, no other cognitive deficit was found. In addition to the direct effects on cognitive function, an episode of cerebral malaria can leave an individual with an increased chance of epileptic episodes, which in turn can lead to cognitive impairment [65]. Cerebral malaria is clearly a major cause of cognitive impairment in preschool children. However, the incidence of serious attacks of malaria declines sharply in the school years. Is there evidence that early childhood malaria continues to be a problem for children’s learning? Only one study has investigated the long-term impact of early childhood malaria prevention on subsequent cognitive development. This study in the Gambia [66] found that children who were protected from malaria for three consecutive transmission seasons before the age of 5 years had improved cognitive performance at age 17–21 years. For those who had received the longest protection from malaria, the improvement in cognitive function was around 0.4 SD. There was also clear evidence of the impact of malaria protection on educational attainment. Children who had been protected from malaria in early childhood stayed at school for around 1 additional year (see Fig. 2). Malaria can be prevented. Use of insecticide-treated bed nets is effective [67] and is listed as one of the Millennium Development Goal quick wins [68]. Use of anti-malarial drugs for intermittent preventive treatment or to treat clinical attacks may help reduce the burden of this disease [69].

Socio-emotional impacts of malaria The effects of cerebral malaria extend beyond the cognitive domain. Psychotic episodes have been reported following bouts of cerebral malaria in Nigeria [70, 71]. However, it is not clear to what extent such episodes are common in preschool children or if other socio-emotional sequelae are present in this age group.

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Figure 2. Impact of early childhood malaria prevention on years of schooling in the Gambia.

Cognitive impacts of HIV infection There is little evidence on this issue from developing countries but research in high-income countries has demonstrated that HIV infections are associated with lower IQ and academic achievement and impaired language in the late preschool and early school-age years [72], and with poorer visual-motor functioning in older children [73]. This is likely to be due in part to the effects of HIV on cognitive development before children enroll in school. Studies including children from infancy to school age find that such deficits in cognitive function can be reduced or reversed with antiretroviral therapy (ART) [74–76]. A wide age range of children took part in these studies, spanning preschool and the school-age years. It seems likely that therapy directed specifically at preschool children will be beneficial, although one study [77] found that improvement in cognitive abilities in response to 36 months of ART was greater for children older than 6 years compared with younger children.

HIV infection and socio-emotional development A number of studies have found that the adaptive behavior (skills required for everyday activities) of children living with HIV improves after treatment. In one study [76], after 6 months of zidovudine (AZT) treatment, almost all behavioral domains assessed (communication, daily living, socialization, but not motor skills) showed significant improvement

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overall. In another study infants with HIV-associated encephalopathy (degenerative brain disease) were rated as more apathetic and nonsocial in their behavior than nonencephalopathic infants. Older children (mean age around 8 years) with encephalopathy had significantly higher scores on scales measuring depression, autism, and irritability compared to nonencephalopathic patients from this age group. A subgroup of patients showed a significant decrease in these elevated scores after a 6-month course of AZT.

Orphanhood HIV/AIDS brings with it many other factors that may affect children’s education. Children living with HIV/AIDS are more likely than other children to have lost one or both parents. Evidence suggests that children living with HIV/AIDS suffer from psychosocial problems. One study in Tanzania has found increased rates of depression in AIDS orphans [78]. A more recent study in Zimbabwe [79] found that orphans had a higher rating on a measure of depression than non-orphans by 0.13 SD for boys and 0.20 SD for girls. Female orphans were also more likely to suffer from poor self-esteem. Both of these studies were conducted with older children. Further evidence is required for preschool children.

Worms Evidence on the cognitive impact of worm infections comes mainly from the school-age years. School children in South America, Africa and South-East Asia who are infected with worms perform poorly in tests of cognitive function [80]. When infected children are given deworming treatment, immediate educational and cognitive benefits are apparent only for children with heavy worm burdens or with nutritional deficits in addition to worm infections [81–85]. One study in Jamaica [83] found around a 0.25-SD increase in three memory tests attributable to treatment for moderate to heavy infection with whipworm (Trichuris trichiura). However, for most children, treatment alone cannot eradicate the cumulative effects of lifelong infection nor compensate for years of missed learning opportunities. Deworming does not lead inevitably to improved cognitive development, but it does provide children with the potential to learn. Children in Tanzania who were given deworming treatment did not improve their performance in various cognitive tests, but they did benefit more from a teaching session in which they were shown how to perform the tests [86]. Performance on reasoning tasks at the end of the study was around 0.25 SD higher in treated children than in those who still carried worm infection. The treated children’s performance was similar to children who began the study without infection. This suggests

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that children are more ready to learn after treatment for worm infections and that they may be able to catch up with uninfected peers if this learning potential is exploited effectively in the classroom. It is likely that worm infections have a similar impact for preschool children. Infections are prevalent in this age group, although worm loads typically do not reach peak intensity until the school-age years. A study in Kenya showed that 28% of 460 preschool children (0.5–5 years) harbored hookworm infection, 76% were anemic and that anemia was more severe in those children with hookworm [87]. Evidence of a cognitive impact of worm infections in preschoolers is not clear. Two studies [48, 88] have demonstrated cognitive improvements in preschool children following combined treatment for worm infections and iron-deficiency anemia. However, neither study was able to disentangle the effects of the two treatments.

Other parasitic infections Infection with Giardia lamblia has been associated with mental development. Giardia is a protozoan parasite that is ingested and inhabits the gastrointestinal tract. It contributes significantly to caseloads of diarrhea. One study in Peru [89] followed a cohort of children some of whom had had diarrheal diseases, parasitic infection and severe malnutrition in the first 2 years of life. Severe malnutrition at this age was associated with an IQ 10 points (0.67 SD) lower at age 9 years. Those who had suffered two or more episodes of Giardia lamblia per year scored 4.1 points (0.27 SD) lower than children with one episode or fewer per year. It is likely that this association is due to Giarda infection causing, or acting as an index of, malnutrition.

Otitis media (Glue Ear) Otitis media is an inflammation of the middle ear cavity often resulting from spread of infection from the nose or throat. In acute cases, pus is produced pressurizing the eardrum and causing perforation in chronic cases. Otitis media is common in developed and developing countries [90]. Around 6% of primary school and preschool children were found to have chronic otitis media with effusion (OME) in Vietnam [91] and South India [92]. In Tanzania, 9.4% of rural and 1.4% of urban school children were found to have chronic OME. OME has mild effects on language development [93] and other cognitive skills. The effect depends on the length of infection and caregiver environment [94]. Children from low socio-economic status backgrounds are more likely to suffer effects of OME. Although research has not documented the effect of OME on cognitive development in developing countries, this result suggests that the effect may be greater than in developed countries.

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Meningitis Meningitis has a high prevalence in developing countries, with associated mortality and risk of severe neurological problems for survivors. Other survivors of meningitis do not have obvious neurological problems and yet suffer long-term behavioral problems. In Ghana, survivors of meningitis aged 2–73 were more likely to suffer from feelings of tiredness (odds ratio = 1.47) and were more often reported by relatives to have insomnia (odds ratio = 2.31) [95]. However, meningitis infection did not affect school attendance amongst school-age cases. Studies in developed countries have found that children who appear well after bacterial meningitis have more nonspecific symptoms like headache, and more signs and symptoms indicating inattention, hyperactivity and impulsiveness than their siblings [96]. Survivors of bacterial or viral meningitis go on to perform less well at school, to be more likely to repeat a grade and to be referred to a special needs school. They are also more likely to have behavioral problems in the home [97]. Cognitive abilities are also affected. Survivors of meningitis have lower IQs than their peers (~0.3 SD) at ages 7 and 12 years [98] with no sign of the gap narrowing with age. Conversely, behavioral problems of meningitis survivors are greater than their peers and actually increase with age.

Programmatic responses Addressing the educational consequences of health and nutrition problems outlined in the previous sections requires an integrated life-cycle approach including maternal child health programs and integrated management of childhood illnesses during infancy, ECD and early childhood care and education programs during early childhood, moving to school health programs during the school-age years. However, the focus of this report is on interventions that can be delivered through ECD programs.

Interventions: What works? Table 1 summarizes the impact of health interventions on cognitive development in early childhood. Only studies giving strong evidence from experimental interventions are included. Such evidence is available for four types of intervention: iron supplementation, iron supplementation and deworming, psychosocial stimulation of malnourished children and nutritional supplementation. Most interventions are aimed at a specific target group (iron-deficient or malnourished children), although two interventions are aimed at all children in a community.

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Table 1. Impact of health interventions on development during early childhood Study

Country

Intervention

Age

Sample Effect characteristics size

Outcomes

Jukes et al. [48]

India

Iron (30 d) + 2–6 yrs deworming

ECD pupils

0.18 SD Attention

Seshadri and Golpadas no. 1 [46]

India

Iron (60 d)

5–8 yrs

Anemic vs. non-anemic

+ve

Seshadri and Golpadas no. 2 [46]

India

Iron (60 d)

5–6 yrs

Anemic vs. non-anemic boys

0.33 SD Verbal IQ 0.67 SD Performance IQ

Soewondo et al. [45]

Indonesia

Iron (56 d)

4 yrs

Anemic vs. non-anemic

+ve No effect

Stoltzfus et al. [88]

Zanzibar

12 mo iron + deworming

6–59 mo

Community

0.14 SD Language

McKay et al. [12]

Columbia

Nutrition + education from 42 mo

84 mo

Malnourished 0.80 SD Cognitive ability

GranthamMcGregor et al. [13]

Jamaica

Psychosocial stimulation

9–24 mo

Malnourished 0.67 SD Mental development

GranthamMcGregor et al. [13]

Jamaica

Nutritional supplementation

9–24 mo

Malnourished 0.79 SD Mental development

Vermeersch and Kremer [11]

Kenya

School feeding

4–6 yrs

ECD pupils

IQ

Learning task 3 cognitive tests

0.4 SD Educational (for sub achievement sample)

The first striking thing about Table 1 is that all studies have demonstrated a positive impact. Note that Table 1 is not a selective account of health interventions that have worked. Rather, it is a summary of all experimental interventions found in the literature. In contrast with interventions in other age groups, it is notable that a positive impact on at least one cognitive test was found in every case. The size of the impacts are also worthy of note. Where the size of the impact is quantified, all interventions aimed at nutritionally disadvantaged groups improve cognitive abilities by at least 0.67 SD. In the context of the literature on improving cognitive abilities, these are remarkably large effects, equivalent to an increase of 10 IQ points or lifting a child from the 25th percentile to the 50th percentile of the ability distribution. The two studies that showed modest effects were targeted at a community cohort rather than a nutritionally disadvantaged population. Table 2 shows the studies that have followed-up preschool health interventions and assessed their cognitive impact in the long term. Three of the studies tracked participants to adolescence and found that improvements

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Table 2. Long-term impact of health interventions in early childhood on cognitive and educational outcomes Study

Country

Intervention

Age

Sample characteristics

Effect size

Outcomes

GranthamMcGregor et al. [14]

Jamaica

Maternal education

14

Severely malnourished

0.68

IQ

Walker et al. [17]

Jamaica

Stimulation

11–12

Stunted

0.38

IQ

No effect

Education tests

+ve

Working memory

Chang et al. [25] Pollitt et al. [21]

Indonesia

Nutritional supplements

8

Initially > 18 months

Jukes et al. [66]

Gambia

Malaria prevention

14–19

Community 0.25–0.4 cohort

Cognitive function

in cognitive function persisted. Both studies in Jamaica found sizeable long-term effects of psychosocial stimulation or education, but no effects of nutritional supplementation. The malaria prevention study in the Gambia is of interest because relatively large impacts were found even though the intervention was provided for a community cohort rather than targeted at a sub-population. All four studies support the hypothesis that the cognitive benefits of preschool health interventions in terms of school readiness carry through to benefit children’s education in the long term. Overall, evidence presented in this chapter shows clear benefits for education of tackling three health and nutrition conditions in early childhood: undernutrition, anemia and malaria. Table 3 illustrates the scale of the impact of these three conditions by combining data on prevalence and on the cognitive impact of treating the conditions. Table 3 shows that, for each condition, at least 190 million children under 5 suffer cognitive deficits equivalent to between 3 and 24 IQ points. The shift in ability distribution caused by these diseases creates between 2.5 million and 61 million additional cases of mental retardation (IQ< 70). All three conditions are easily preventable. The following section outlines current best practice for addressing these conditions.

Undernutrition The most obvious way in which ECD programs can address chronic undernutrition is through school-based feeding programs. Evidence discussed shows such programs can be effective in improving child development and school readiness. However, such programs can be costly and often difficult

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Table 3. Estimated global impact of malaria, anemia and stunting on cognitive development Developing country prevalence

Total cases (millions)1

Malaria

~50%

270

0.2

0.4

3

6

3.56

8.65

Stunting

31%

190

0.2

1.6

3

24

2.50

61.15

Anemia

40%

219

0.3

0.7

4.5

10.5

4.78

16.22

1Based 2Using

Effect size

Equivalent loss in Additional cases of IQ points per child mental retardation (millions)2

Lower Upper Lower Upper Lower Upper estimate estimate estimate estimate estimate estimate

on a developing country population of under-5s of 548 million [34]. the definition of mental retardation as IQ < 70.

to sustain. Experience from programs with school-age children [99] suggests that these programs are most effective when significant cost burdens are borne by the community. One example has been presented in this review of a preschool feeding program partly funded by parents, which had a significant impact on preschool attendance and achievement [11]. Beyond school feeding, current recommendations are for counseling mothers and caretakers to improve feeding practices and improved management of malnutrition [100].

Iron-deficiency anemia Supplementation with iron, for example through ingestion of ferrous sulfate or folic acid, is an effective way of combating iron-deficiency anemia. This intervention has been used successfully in India at a cost of around $2 per child. Cost can be further reduced and sustainability improved through teacher delivery of supplements in ECD programs. Iron-deficiency anemia can also be controlled through fortification of food with iron. Where malaria is common, iron supplementation can have adverse consequences for mortality and morbidity [101] and current recommendations are that caution should be exercised when providing supplementation in such areas. Programs in these areas should be targeted at those who are anemic or are at risk of iron deficiency [102].

Malaria Current priorities for malaria control in endemic areas include the use of insecticide-treated bed nets and prompt and effective treatment, includ-

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ing presumptive treatment, with artemisinin-based combination therapy. ECD programs may have a role for promoting both of these strategies. Intermittent preventative treatment [103] has been successful in controlling malaria amongst infants, but further research is required, particularly in the preschool age group.

Health or education interventions: Targeting disease or symptoms? It might be expected that tackling the root cause of a disease is more important than dealing with its consequences for development, as sure as prevention is better than cure. However, the one study to test this hypothesis in the long term found the reverse. The study of malnourished children in Jamaica found a long-term effect of psychosocial stimulation but no long-term effect of nutritional supplementation. Both interventions had an immediate effect on the developmental levels of its preschool participants but the effects of the nutritional supplement waned with time in an interesting way. Eight years after the intervention nutritional supplements had an effect on cognitive ability only for children whose mothers had high verbal intelligence (a proxy for the amount of stimulation they would have received). In the later follow-ups no impact of the nutritional supplements was apparent. It seems that stimulation is a key part of intervention. We saw in the review of literature that nutritional problems have serious consequences for the amount of stimulation children receive. Perhaps the crucial element in combating this effect is to ensure that young children receive sufficient stimulation. There is certainly plenty of evidence in support of an interaction between health and education interventions. Low birth weight has been shown in separate studies to be a risk factor for mental development only for children who also received insufficient stimulation in the home and (in a separate study) only for children of illiterate mothers. A study in Vietnam found that nutritional supplementation alone was insufficient to equalize cognitive performance between stunted and non-stunted children. Only in villages receiving both nutritional supplementation and an ECD intervention did cognitive development improve in both stunted and non-stunted children. In another example from Kenya, the educational achievement of children benefited from a school-feeding program only in schools with experienced teachers. Related to this, the study of the long-term effect of nutritional supplements in three villages in Guatemala found that supplementation only had a long-term effect for participants who subsequently went on to have the most schooling. These findings parallel others from school-age children. For example, a study with school children in Tanzania [86, 104] found that deworming alone was insufficient to improve the cognitive abilities of children infected with these parasites, whereas a teaching intervention combined with the deworming did improve reasoning skills.

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These findings have programmatic implications. First, it is clearly more effective to prevent the onset of health and nutrition problems rather than to cure them. Second, where remediation is necessarily, or where health or nutrition problems commonly reoccur (for example with seasonal variations in nutritional intake or in the transmission of disease, or where communities are constantly exposed to diseases for which there are no simple preventative measures), educational interventions, such as ECD programs should be considered as important as health interventions in the programmatic response to problems of health and nutrition.

Promoting equity through preschool health interventions The burden of disease is borne disproportionately by the poor. In addition, the impact of disease on education is greatest for the poor. In the preceding review we saw examples where lack of breast feeding, or otitis media infection led to cognitive impairments only for children of the least educated mothers. There are also examples where the impact of one condition is greater for children suffering from other problems of health or nutrition [105, 106]. Conversely, preschool health interventions tend to provide the greatest benefit to disadvantaged children. For example, long-term educational benefits of a nutritional supplementation program in Guatemala were found only for those children of low socio-economic status. Many other examples exist in the literature on school-age children. For example, giving breakfast to children in Jamaican schools improved cognitive function on the same day to a greater extent for children with chronic malnutrition [107]. Similarly, gender differences in the effect of interventions favor girls. For example, iron supplementation is found to improve preschool attendance for girls more than boys [108], and malaria prevention increases enrolment for girls but not boys [66]. Health and nutrition interventions therefore offer a way of promoting equity in education and by benefiting vulnerable children to the greatest extent. If ECD health and nutrition projects are explicitly targeted at the poorest in society (or at least ensure that coverage extends to the rural poor and other hard-to-reach group), the impact of equity will be all the greater.

Conclusions Extensive research has been conducted on the educational effects of early childhood health and nutrition interventions. The breadth and depth of this research allows for a number of general conclusions to be drawn. First, early childhood health and nutrition interventions have a consistently large impact on cognitive development. Second, health and nutrition interventions have the largest impacts for the preschool age group, but are also

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effective in older children. Third, early childhood health and nutrition interventions improve all aspects of school readiness, but greatest impacts are seen for motor development. Fourth, early childhood health and nutrition interventions promote equity. Fifth, the best evidence of educational benefits is found for feeding programs, iron supplementation and malaria prevention. Sixth, preschool education programs can be as effective as health and nutrition interventions in mitigating the educational impact of poor health and nutrition. Early childhood health and nutrition interventions clearly have a major role to play in an expanding system of early childhood development programs and their efforts to achieve quality Education for All.

Acknowledgements This article was written with support from the UNESCO Education for All Global Monitoring Report 2007. I would like to thank Don Bundy for valuable comments on earlier drafts of this chapter.

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Nyamukapa CA, Gregson S, Lopman B, Saito S, Watts HJ, Monasch R, Jukes MCH. HIV-associated orphanhood and children’s psychosocial disorders: theoretical framework tested with data from Zimbabwe; submitted Watkins WE, Pollitt E (1997) “Stupidity or worms”: Do intestinal worms impair mental performance? Psychol Bull 121: 171–191 Dickson R, Awasthi S, Williamson P, Demellweek C, Garner P (2000) Effects of treatment for intestinal helminth infection on growth and cognitive performance in children: systematic review of randomised trials. BMJ 320: 1697– 1701 Nokes C, Grantham McGregor SM, Sawyer AW, Cooper ES, Robinson BA, Bundy DA (1992) Moderate to heavy infections of Trichuris trichiura affect cognitive function in Jamaican school children. Parasitology 104: 539–547 Nokes C, McGarvey ST, Shiue L, Wu G, Wu H, Bundy DA, Olds GR (1999) Evidence for an improvement in cognitive function following treatment of Schistosoma japonicum infection in Chinese primary schoolchildren. Am J Trop Med Hyg 60: 556–565 Simeon DT, Grantham McGregor SM, Callender JE, Wong MS (1995) Treatment of Trichuris trichiura infections improves growth, spelling scores and school attendance in some children. J Nutr 125: 1875–1883 Simeon DT, Grantham McGregor SM, Wong MS (1995) Trichuris trichiura infection and cognition in children: results of a randomized clinical trial. Parasitology 110: 457–464 Partnership for Child Development. Effect of antheltmintics on children’s growth and cognitive abilities after 3 months and 16 months; submitted Brooker S, Peshu N, Warn PA, Mosobo M, Guyatt HL, Marsh K, Snow RW (1999) The epidemiology of hookworm infection and its contribution to anaemia among pre-school children on the Kenyan Coast. Trans R Soc Trop Med Hyg 93: 240–246 Stoltzfus RJ, Kvalsvig JD, Chwaya HM, Montresor A, Albonico M, Tielsch JM Savioli L, Pollitt E (2001) Effects of iron supplementation and antihelmintic treatment on motor and language development of preschool children in Zanzibar: double blind, placebo controlled study. BMJ 323: 1389–1393 Berkman DS, Lescano AG, Gilman RH, Lopez S, Black MM (2002) Effects of stunting, diarrhoeal disease, and parasitic infection during infancy on cognition in late childhood: a follow-up study. Lancet 359: 564–571 Berman S (1995) Otitis media in developing dountries. Pediatrics 96: 126–131 Balle VH, Tos M, Dang HS, Nhan TS, Le T, Tran KP, Tran TT, Vu MT (2000) Prevalence of chronic otitis media in a randomly selected population from two communes in southern Vietnam. Acta Oto-Laryngol 543: 51–53 Rupa V, Jacob A, Joseph A (1999) Chronic suppurative otitis media: prevalence and practices among rural South Indian children. Int J Pediatr Otorhinolaryngol 48: 217–221 Casby MW (2001) Otitis media and language development: A meta-analysis. Am J Speech Lang Pathol 10: 65–80 Roberts JE, Burchinal MR, Zeisel SA, Neebe EC, Hooper SR, Roush J, Bryant D, Mundy M, Henderson FW (1998) Otitis media, the caregiving environment, and language and cognitive outcomes at 2 years. Pediatrics 102: 346–354

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Hodgson A, Smith T, Gagneux S, Akumah I, Adjuik M, Pluschke G, Binka F, Genton B (2001) Infectious diseases – Survival and sequelae of meningococcal meningitis in Ghana. Int J Epidemiol 30: 1440–1446 Berg S, Trollfors B, Hugosson S, Fernell E, Svensson E (2002) Long-term follow-up of children with bacterial meningitis with emphasis on behavioural characteristics. Eur J Pediatr 161: 330–336 Koomen I, Grobbee DE, Jennekens-Schinkel A, Roord JJ, van Furth AM (2003) Parental perception of educational, behavioural and general health problems in school-age survivors of bacterial meningitis. Acta Paediatr 92: 177–185 Grimwood K, Anderson P, Anderson V, Tan L, Nolan T (2000) Twelve year outcomes following bacterial meningitis: further evidence for persisting effects. Arch Dis Child 83: 111–116 Del Rosso JM (1999) School feeding programmes: improving effectiveness and increasing benefit to education. Partnership for Child Development, London WHO (2006) Child and adolescent health: Child feeding and nutrition. http:// www.who.int/child-adolescent-health/NUTRITION/child.htm Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, Dhingra U, Kabole I, Deb S, Othman MK, Kabole FM (2006) Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 367(9505): 133–143 WHO (2006) Iron supplementation of young children in regions where malaria transmission is intense and infectious disease highly prevalent. WHO, Geneva Chandramohan D, Owusu-Agyei S, Carneiro I, Awine T, Amponsa-Achiano K, Mensah N, Jaffar S, Baiden R, Hodgson A, Binka F, Greenwood B (2005) Cluster randomised trial of intermittent preventive treatment for malaria in infants in area of high, seasonal transmission in Ghana. BMJ 331: 727–733 Grigorenko E, Sternberg R, Ngorosho D, Nokes C, Jukes M, Alcock KJ, Lambo J, Ngorosho D, Nokes CA, Bundy DAP (2006) Effects of antiparasitic treatment on dynamically-assessed cognitive skills. J Appl Dev Psychol 27: 499–526 Jukes MCH, Drake L, Bundy DA. School health, nutrition and educatin for all: Levelling the playing field. CABI Publishing, Wallingford, UK; in press Jukes MC, Nokes CA, Alcock KJ, Lambo JK, Kihamia C, Ngorosho N, Mbise A, Lorri W, Yona E, Mwanri L et al (2002) Heavy schistosomiasis associated with poor short-term memory and slower reaction times in Tanzanian school children. Trop Med Int Health 7: 104–117 Simeon DT, Grantham McGregor S (1989) Effects of missing breakfast on the cognitive functions of school children of differing nutritional status. Am J Clin Nutr 49: 646–653 Bobonis G, Miguel E, Sharma C (2006) Iron deficiency anemia and school performance. Journal of Human Resources 41: 692–721

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Early childhood caries and childhood periodontal diseases Shigenobu Kimura1 and Yuko Ohara-Nemoto2 1Department of Oral Microbiology, Iwate Medical University School of Dentistry, 1-3-27 Chuodori, Morioka, Japan; 2Division of Oral Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki, Japan

Abstract Dental caries and periodontal diseases are one of the most prevalent diseases affecting adults and children in industrialized countries. The major causative factor in both diseases is the microbial biofilm (dental plaque) formed on teeth and oral epithelial surfaces, and early childhood caries and periodontal diseases are both plaque-induced infectious diseases caused by endogenous bacteria. However, it is also evident that the colonization of the putative pathogenic bacteria in plaque is not sufficient for the initiation and onset of these plaque diseases. In dental caries, it is apparent that the association of dietary fermentable carbohydrates, especially sucrose, is implicated in the etiology. Moreover, recent studies also acknowledge the significant role of the local environmental conditions in plaques. In periodontal diseases, the host response plays a major role in the outcome of the diseases. The present review addresses the pathogenic bacteria and microflora and the etiology of early childhood caries and childhood periodontal diseases.

Introduction The mouth is sterile at birth, and thereafter the successive transmission of microorganisms occurs by passive contamination from foods, water and the saliva of individuals intimate with the baby. However, the mouth is highly selective for microorganisms, since the process of acquisition of resident microflora depends on the interrelationship of the microenvironment in physical, chemical and/or biological characteristics and the infective ability of the organisms encountered during development of oral microflora (“window of infectivity”) [1]. Furthermore, the ecological conditions within the mouth vary on the tooth eruption and the change from primary to the permanent dentition. Thus, the mouth is not a uniform habitat for microbial growth and colonization. Previous studies with regard to the distribution of bacteria in various sites in the human oral cavity demonstrated four distinct habitats within the mouth; the buccal epithelium, dorsum of the tongue, supragingival tooth surface, and gingival crevice (subgingival

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tooth and crevicular epithelial surfaces) [2]. Among these, the latter two habitats are important for etiology of dental caries and periodontal diseases, since cariogenic and periodontopathic bacteria often colonize the supragingival tooth surface and gingival crevice, respectively. The oral microflora formed on some surface (such as a tooth or an epithelial surface) is a microbial biofilm called dental plaque. In dental plaque, the interactions of the component species results in a metabolic efficiency and diversity that is greater than the sum of its constituent species, and form an organized bacterial mass that cannot be readily removed by flushing with water sprays. Although constituent bacteria may differ among subjects, in general, supragingival plaque contains predominantly Gram-positive bacteria often including cariogenic bacteria. In contrast, subgingival plaque is composed predominantly of Gram-negative organisms and often contains anaerobic periodontopathic bacteria. Therefore, dental caries and periodontal diseases are both plaque diseases, and the major therapeutic approach for treatment is the mechanical removal of the plaques. However, it is also evident that the colonization of the putative pathogenic bacteria in plaque is not sufficient for the initiation and onset of these plaque diseases. In dental caries, it is apparent that the association of dietary fermentable carbohydrates, especially sucrose, is implicated in the etiology, suggesting that dental caries is a dietary-conditioned oral infectious disease. In periodontal diseases, the host response plays a major role in the outcome of the diseases. The present review addresses the pathogenic bacteria and microflora and the etiology of early childhood caries (ECC) and childhood periodontal diseases.

Early childhood caries Dental caries is one of the most prevalent diseases affecting people in industrialized countries. Caries of enamel surfaces (enamel caries) is particularly common in children (ECC) and young subjects up to the age of 20 years, while root surface caries is frequently observed in elder individuals with gingival recession exposing the vulnerable cementum to microbial colonization (Fig. 1) [3]. The ECC lesion invariably originates as small demineralized area on the external surface of erupted teeth, i.e., the enamel, which is the most highly calcified tissue, composed of 95% hydroxyapatite. The lesion can progress through dentin and into pulp centripetally increasing in size and depth. Demineralization of hydroxyapatite is caused by acids, particularly lactic and formic acids, which are produced from the microbial fermentation of dietary carbohydrates, resulting in the transport of the calcium and phosphate ions away into the surrounding environment. Thus, the dietary carbohydrates and infection of cariogenic bacteria on the surface of tooth enamel are essential factors in the development of ECC.

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Figure 1. Structure of tooth and periodontal tissues. The structure of tooth and periodontal tissues are indicated in left half. The right half illustrates the host-parasite relationship in gingival crevice.

Mutans streptococci and etiology of ECC The first description regarding the association of Streptococcus mutans with human caries was made by Clarke in 1924 [4], and numerous studies have been performed to elucidate the causative relationship between specific oral bacterial species and dental caries. In animal experiments including monkeys, gerbils, mice, rats and hamsters, most studies indicated the cariogenicity and the transmissibility of mutans streptococci, although organisms other than mutans streptococci occasionally induce variable levels of dental caries in animals [2]. In humans, many epidemiological surveys have also found a strong association between mutans streptococci and dental caries. Thus, mutans streptococci are now considered to play an important role in the development of dental caries in humans as well as animals. Mutans streptococci are Gram-positive, facultatively anaerobic cocci, currently known to be composed of seven species (Tab. 1). Among them, S. mutans and S. sobrinus are the species recovered from human oral microflora. An individual harbors either one or both species of mutans streptococci in the mouth, especially in the supragingival plaque, and the occurrence rate of S. mutans is commonly higher than that of S. sobrinus. The most important virulent factor of mutans streptococci as cariogenic bacteria is attributed to a group of enzymes, glucosyltransferases (GTFs), which catalyze the formation of water-insoluble and -soluble extracellular polysaccharides, glucans. Water-insoluble glucans enable the microorganisms to adhere to the tooth surface. GTFs transfer a glucose moiety derived from sucrose, disaccharide of glucose and fructose, to the end of growing glucan molecule: n · sucrose A (glucose)n + n · fructose

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Table 1. Oral streptococcal phylogenetic groups and species Mutans streptococci Phylogenetic group

Species

Serotypes

Natural habitat

mutans group

S. mutans S. sobrinus S. cricetus S. rattus S. dewnei S. ferus S. macacae

c/e/f d/g a b h c c

human human hamster rat monkey rat monkey

anginosus group

S. anginosus S. intermedius S. constellatus

salivarius group

S. salivarius S. vestbularis S. thermophilus

mitis group

S. sanguinis S. gordonii S. parasanguinis S. oralis S. mitis S. crista

where sucrose is the sole substrate for GTFs. Because the hydrolysis of sucrose is exergonic (6G˚ = –6.6 kcal/mol), the formation of glucan is irreversible. Glucan commonly contains _(1A 6) glycosidic linkages and is soluble, while glucan containing _(1A 3) glycosidic bonds in addition to the _(1A 6) glycosidic linkages becomes insoluble. S. mutans and S. sobrinus produce three and four GTFs, respectively, whose cooperative actions are essential for the adhesive water-insoluble glucan synthesis that leads to cariogenic plaque formation on tooth surfaces. The general features and biological characteristics of GTFs have been extensively reviewed [5–9]. In addition to the production of water-insoluble glucan, the properties of cariogenic bacteria that correlate with their pathogenicity include the ability to rapidly metabolize carbohydrates to acid, and to survive and grow under acidic conditions (acidogenicity and aciduricity). Mutans streptococci, as well as some other streptococci and lactobacilli, are potently acidogenic and aciduric. With the supply of sucrose, by GTFs of mutans streptococci synthesize the adhesive water-insoluble glucan, producing lactate by homolactic fermentation, which accounts for the augmented virulence of these bacteria in hosts that frequently consume high-sucrose diets. Epidemiological observations support the sucrose-caries-mutans streptococci association; an

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increase in the rate of dental caries occurs with increased levels of mutans streptococci in the dental plaques and a decrease in the rates of dental caries among patients who were urged to reduce their frequency and level of sucrose consumption [10]. Thus, mutans streptococci are the primary cariogenic bacteria in ECC. It is most likely that mutans streptococci can adhere to smooth tooth surfaces through the de novo synthesis of the adhesive glucan from dietary sucrose by their GTFs, whereby they can colonize and grow exclusively at these sites and produce lactate causing the demineralization of enamel. The model of ‘sucrose-caries-mutans streptococci association’ explains the impact of sucrose (compared with glucose, fructose, starch, and sorbitol) on caries in studies of humans [11, 12].

An alternative etiology of dental caries Despite a number of findings supporting the sucrose-caries-mutans streptococci association, an alternative hypothesis of the etiology of development/ onset of dental caries has been proposed by Marsh [13]. The hypothesis (the ‘ecological plaque hypothesis’) is based on the in vitro observation regarding relationship between pH and growth of plaque bacteria in a mixed culture mimicking plaque microflora. In this experiment, it was demonstrated that a decrease of pH could induce a marked growth of mutans streptococci as well as Lactobacillus casei, and that a microbial shift with the predominance of these cariogenic bacteria and with the reduction of non-mutans streptococci such as S. sanguinis and S. oralis in the mixed culture could observe under lower (but not neutral) pH conditions. The ecological plaque hypothesis does not exclude the sucrose-caries-mutans streptococci association, but acknowledges also the significant role of the local environmental conditions in plaques. It is proposed that cariogenic mutans streptococci may also be present at sound sites, but at levels too low to be clinically relevant, and that frequent metabolism of fermentable carbohydrates in plaque induces the critical pH condition, which leads to the growth of acid-tolerating cariogenic bacteria. This hypothesis may explain the data of Nyvad and Kilian [14], who indicated that the composition of non-mutans streptococcal microflora in plaque could be a factor that governs the cariogenic potential of mutans streptococci.

Reevaluation of the colonization of mutans and other oral streptococci in childhood and its relationship to ECC According to the ecological plaque hypothesis, the development/onset of ECC could be associated not only with the colonization of cariogenic mutans streptococci but also the local environmental conditions including the colonization of non-mutans streptococci in plaque. Although the

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distribution of non-mutans streptococci has been monitored in some early cross-sectional studies, most of these clinical studies employed conventional identification assays with culture methods, and are still limited in scope. Mitis-salivarius agar containing crystal violet and tellurite is widely used in isolating of oral streptococci [15]. Crystal violet and tellurite inhibit the growth of most Gram-negative bacilli and most Gram-positive bacteria except streptococci. For selective culture for mutans streptococci, mitis-salivarius agar supplemented with bacitracin, an antimicrobial agent for oral streptococci except mutans streptococci, is commonly used, although some researchers reported the inhibition in growth of some species of mutans streptococci [16, 17]. Recently, the polymerase chain reaction (PCR) has been applied for detection of bacterial species including mutans streptococci and other oral streptococci [18–22]. The PCR method offers a rapid and highly sensitive means of specific identification when compared to other identification assays, including culture and immunological methods. How the colonization of mutans and other oral streptococci in plaque of children varies with age, and the relationship between colonization of these bacteria and caries development are still not clear, since the reported studies monitored only mutans streptococci, or focused on the bacteria in saliva. Thus, species-specific PCR assays for mutans and other oral streptococcal species targeting GTF and 16S rRNA genes have been developed, which enable specific detection of 0.5–10 pg genomic DNA, corresponding at least to 100 CFU bacteria. Using the species-specific PCR assays, the colonization of S. mutans, S. sobrinus, S. gordonii, S. sanguinis, S. oralis, S. anginosus and S. salivarius in plaque samples from children was assessed in relation to caries prevalence. In the plaque samples from 320 children (0–15 years old, 20 subjects from each year of age), S. mutans (68.8%) was most frequently detected among the seven streptococci, and S. sobrinus and S. gordonii were more rare (15.0% and 17.2%, respectively). The percentages of S. mutans- and S. anginosus-positive subjects increased with age, while the percentage of S. sanguinis-positive subjects decreased. The proportional changes with age of increase of S. mutans and S. anginosus and decrease of S. sanguinis may be attribute to the gradual increase of caries scores with age. The subject-based analysis noted a significant positive correlation between S. mutans colonization and the caries score. Furthermore, there was a tendency to elevated caries scores in the group of children with mixed colonization of S. mutans and S. sobrinus, in accordance with the data of Seki et al. [23], who examined 20 variables in a univariate analysis to predict caries development in preschool children. Although the etiological involvement of mutans and other streptococci in plaque cannot be fully determined by itself, cross-sectional surveys have the advantage that a large number of subjects can be analyzed, and the dynamism of development of cariogenic and non-cariogenic microflora can be monitored. The cross-sectional survey using species-specific PCR assays indicated that

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many oral streptococcal species including mutans streptococci can colonize quite early in childhood without development of ECC, and thereafter proportional changes of microflora could occur on some tooth surfaces, where the composition of non-mutans streptococcal microflora may affect the local environmental conditions in plaque that governs the cariogenic potential of mutans streptococci.

Preventive strategies of ECC In theory, ECC can be prevented by (1) eliminating cariogenic bacteria, especially mutans streptococci, from plaque microflora, (2) increasing resistance of tooth surfaces against acid attack, and (3) avoiding frequent high-sucrose diets. Regarding the first category, mechanical debridements of supragingival plaque is effective. However, the infection of the tooth surfaces is the primary point of attack and the pioneer bacteria of oral streptococci including mutans streptococci are adsorbed within 2 h after cleaning. To maintain clean conditions at the tooth surface, topical chemotherapy using antimicrobial agents, glucan-hydrolyzing enzymes, and vaccination and passive immunization against mutans streptococci have been developed or investigated. Fluoride has long been known to have anti-caries effects, most of which are ascribable to (a) the interaction with the surface of enamel of erupted teeth to form fluoroapatite that strengthen the resistance of enamel to acid attack [24], and (b) the enhancement of local remineralization of the partially demineralized enamel surfaces [25]. Furthermore, it was demonstrated that fluoride has the ability to suppress the growth of mutans and other cariogenic bacteria [26, 27], and, especially under low but not neutral pH conditions, can slow the acid production at low concentrations (1 mmol/l) [13, 28]. Since dental caries is a dietary-conditioned oral infection, avoiding the frequent intake of fermentable carbohydrates, especially sucrose, in diets is important for the prevention of ECC. The historical association of dietary sucrose with caries is strong and many epidemiological studies revealed the relationship between caries prevalence and sugar consumption [11, 29]. A number of sugar substitutes (non-cariogenic sweetener) that are hardly fermented by plaque bacteria have been developed. Among them, sugar alcohols including xylitol and sorbitol are effective as non-cariogenic sugar substitutes through the inhibition of intracellular metabolisms of carbohydrates. In particular, xylitol has a unique caries-reducing effect, the so-called xylitol futile cycle. The agent is transported into mutans streptococci by the fructose-bacterial phosphotransferase system, and it enters a futile cycle of phosphorylation, dephosphorylation and eventual expulsion. The xylitol futile cycle leads to the reduction of cell growth and results in the elimination cariogenic mutans streptococci in the plaque [30].

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Since early acquisition of mutans streptococci is a major risk factor for ECC [31] and future caries experience [32, 33], preventing the transmission of these organisms to naive infants’ mouths is another potential strategy. Recent studies demonstrate that acquisition of mutans streptococci in infants occurs not only by vertical transmission from mothers but also by horizontal transmission from individuals in intimate proximity [34]. Moreover, primary oral infection of mutans streptococci may occur occasionally in predentate infants [35]. Thus, the reduction of the mutans streptococcal reservoir in the mother as well as sibling(s) and the infant’s caretaker(s) is needed for effective prevention of ECC.

Infective endocarditis caused by oral streptococci The most predominant pathogens of infective endocarditis are the bacterial species in the oral cavity such as mutans and other oral streptococci [36]. The tooth-tissue interface can be a typical portal for bacteria to enter the body, and nearly all physical entries of the organisms into the bloodstream. In the case of the subjects with cardiac valvular abnormalities, the organisms entering the blood stream can potentially attach and grow, and then cause the infective endocarditis. Furthermore, the glucan-synthesizing ability of these streptococci may play an important role in the etiology, since cell-bound glucan could promote the establishment of mutans and other oral streptococci on the heart valves [37, 38]. Thus, the adherence-promoting ability of glucan synthesized by mutans streptococci appears to be the initial step in the pathogenesis of infective endocarditis as well as dental caries.

Childhood periodontal diseases Periodontal diseases can be grouped broadly into gingivitis and periodontitis, and each can be further divided according to the disease characteristics (e.g., chronic, aggressive) and the contributing factors, including related systemic conditions and disorders [39]. On the basis of histopathology, gingivitis is characterized by inflammation confined to the gingiva, and periodontitis denotes destruction of periodontal tissues that involve the gingiva, the periodontal ligament, root cementum and the supporting bone (alveolar bone) (Fig. 1). From the epidemiological aspect, gingivitis is more prevalent in childhood than periodontitis. The periodontal diseases are well recognized to be initiated by some selected microorganisms, so-called periodontopathic microorganisms, in subgingival plaque and/or supragingival plaque adjacent to gingival crevice. It is also evident that the onset and progress of these inflammatory diseases are based on the balance between the periodontopathic microorganisms

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and the host-defense against them (host-parasite relationship) [40]. In other words, the children with related systemic conditions and/or disorders could be easily afflicted with periodontal diseases. Thus, the host-defense as well as periodontopathic bacteria play an important role in the outcome of childhood periodontal diseases. The following sections address: (i) clinical and etiological aspects of childhood periodontal diseases, (ii) pathogenic bacteria, (iii) onset of periodontal diseases – host-parasite relationship, and (iv) transmission of periodontal bacteria.

Clinical and etiological aspects of childhood periodontal diseases Plaque-induced gingivitis without other local contributing factors is a bacterially elicited inflammation of the marginal gingiva, and is the most common gingivitis among children as well as adults. Etiological involvement of supragingival plaque in the gingivitis has been demonstrated by the pioneer experiments by Löe and coworkers [41]. They showed that the increase in severity of gingivitis was directly proportional to the amount of accumulated supragingival plaque, and that gingivitis was eliminated by the removal of the bacterial plaque. The early finding of a strong cause-and-effect relationship between the amount of plaque and the severity of gingivitis has led to a major emphasis on prevention of gingivitis by the reduction in amount of plaque. From the standpoint of the etiological model of ‘host-parasite relationship in periodontal diseases’, however, the plaque-induced gingivitis appears to be a typical periodontal disease in that the increased virulence of periodontopathic bacteria surpasses the host defense. Therefore, the gingivitis cannot be cured only by the reduction in amount of plaque, but by the elimination of pathogens in the plaque. In fact, a recent study demonstrates that there is no correlation between plaque amounts and severity of periodontal inflammation in children with deciduous dentition [42]. In this report, it has been also indicated that clinical manifestations of gingivitis are more severe in adults than in children and adolescents, whereas the accumulation of dental plaque are almost equal between them. Since periodontal bacteria appear to inhabit the child’s oral cavity as described below, these observations suggest that the host defense in childhood is more effective in opposing the periodontal pathogens than that of adults, resulting in prevention of the onset and/or progression of gingivitis. However, it should be remembered that the children with related systemic conditions and/or disorders could be easily afflicted with periodontal diseases. Thus, steroidaddicted subjects, young people during pubertal development, patients with endocrine disorders and juvenile diabetes mellitus patients may suffer from gingivitis (e.g., drug-influenced gingivitis, puberty-associated gingivitis and diabetes mellitus-associated gingivitis) due to a decline or alterations in host defenses.

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Prevalence of periodontitis is extremely low in childhood, compared to that in adults. In a population-based study of 3896 Swedish children (7–9 years old), it was found that only 32 children (0.8%) exhibited alveolar bone loss [43]. Since the sulcular epithelium around temporary (deciduous) tooth is thicker than that of a permanent tooth, a likely explanation of the difference in prevalence may be that the host defense could more efficiently prevent an invasion of periodontal bacteria to the gingival epithelium in children with deciduous dentition than in adults with permanent dentition. However, the fact that periodontitis is also a rare disease even in children with mixed and permanent dentitions suggests other preventive factors. The host defense seems more effective in arresting the developing of periodontitis in children for some reason. The term ‘early-onset periodontitis’ has now been renamed to ‘aggressive periodontitis’. Early-onset periodontitis represents a group of highly destructive periodontitis in young subjects that includes prepubertal, juvenile and rapidly progressive periodontitis. Prepubertal periodontitis develops just after eruption of temporary teeth in such subjects. Juvenile periodontitis starts from puberty in late teens to 20s, and rapidly progressive periodontitis has been characterized as a highly destructive periodontitis that usually has an onset before 35 years of age. However, the age-dependent classification of this type of periodontitis is neither adequate nor practical in either the etiological or the clinical aspect, since the scientific basis for using the patient’s age of disease onset as a classification division of periodontitis is lacking, and similar dysfunction/malfunction in host-defense mechanisms can be observed in most of the early-onset periodontitis patients. Thus, the highly destructive forms of periodontitis have been renamed to aggressive periodontitis. The former ‘prepubertal periodontitis’ and ‘juvenile periodontitis’ are categorized into two forms based on the localization of lesions: one is a localized type in which severe periodontal destructions are limited to first molars and incisors, and the other is generalized (defused) type in which destruction of the periodontal tissue widely progresses in all teeth. Clinical manifestations of the localized type of this periodontitis (localized aggressive periodontitis) are as following: accumulation of the dental plaque is usually limited and gingival inflammation is rarely observed, although resorption of alveolar bone occurs. A likely explanation of the localization to first molars and incisors is that these are the teeth with greater probability for being at risk when the disease started, as other teeth had not yet erupted. On the other hand, extensive alveolar bone resorption and significant gingival inflammation with severe plaque accumulation are commonly observed in generalized type of the aggressive periodontitis (generalized aggressive periodontitis). Etiological factors reported as being associated with aggressive periodontitis include the specific bacterial pathogens, especially Actinobacillus actinomycetemcomitans for localized aggressive periodontitis, and functional abnormalities of peripheral blood polymorphonuclear leukocytes (PMNL) [44]. The PMNL is the principal cell of the gingival crevicular exudate.

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Figure 2. Gingival inflammation and periodontal destruction in a patient with Chediak-Higashi syndrome.

PMNL come into direct contact with plaque bacteria in the gingival crevice and actively phagocytose them. The protective function of PMNL in human periodontal diseases is demonstrated by the fact that patients with PMNL disorders, e.g., Chediak-Higashi syndrome (Fig. 2) [45, 46], lazy leukocyte syndrome [47], cyclic neutropenia [48], chronic granulomatous disease [49] and diabetes mellitus [50, 51], have usually rapid and severe, aggressive periodontitis. Quantitative analyses using a flow cytometer revealed that about 50% of the patients with localized and generalized aggressive periodontitis, but not chronic periodontitis (formerly adult periodontitis), exhibited depression of phagocytic function of peripheral blood PMNL (Tab. 2) [52]. The depressed phagocytic responses could be due to cell-associated

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Table 2. Prevalence of the periodontitis patients exhibiting depressed phagocytic function of peripheral blood PMNLa Aggressive periodontitis Localized type

Generalized type

Chronic periodontitis

% Phagocytosis

53%

(8/15)b

46% (6/13)

6% (3/52)

d-Phagocytosis

67% (10/15)

46% (6/13)

6% (3/52)

aPhagocytic

function was assessed by means of the percentage of phagocytosing cells (% Phagocytosis) and the degree of phagocytosis by one PMNL (d-Phagocytosis). Depressed phagocytic function was defined when 2 SD below the mean of those in healthy subjects. bPatients exhibiting depressed phagocytic function/total patients.

defect(s) of the PMNL, and therefore it remains unchanged after periodontal treatments, suggesting that the depression of PMNL phagocytosis in both types of aggressive periodontitis may not be a transient phenomenon associated with the local periodontal status. In contrast, the capacity of the PMNL to mount intracellular oxidative burst reaction was much higher in both types of aggressive periodontitis and chronic periodontitis than that in the control [53]. On an individual basis, the elevated capacity of oxidative burst showed a significant positive correlation to clinical periodontal parameters, and decreased to normal levels after periodontal treatments. These findings suggest that the PMNL with marked increase in oxidative metabolic capability (‘primed’ PMNL) could be a significant component of the host defense to not only aggressive but also chronic periodontitis, as seen in other systemic bacterial infections [54]. Thus, the host defense, especially by PMNL, plays an important role in the outcome of childhood periodontitis. Nevertheless, the association of the unique and specific pathogens must be taken account in the pathogenesis of childhood periodontitis. The functional abnormalities of PMNL is implicated in the pathogenesis of both forms of aggressive periodontitis (localized and generalized), but the clinical manifestations of the two periodontitis are clearly distinguishable, as described above. Furthermore, the prominence of A. actinomycetemcomitans is cited as a feature in only the localized type.

Pathogenic bacteria Although the pathogens of periodontal diseases were presumed to be microorganisms habiting in the dental plaque, none of periodontopathic bacteria were identified until the late 1970s. The main reason is because these bacteria are obligatory anaerobes and do not, or hardly, proliferate under the conventional aerobic culture conditions. Therefore, a development and prevalence of anaerobic culture methods were required to isolate and identify the periodontopathic bacteria. Clinical and basic

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studies, including animal trials, have recently revealed that some selected microorganisms that colonize in subgingival plaque in gingival crevice, the so-called periodontopathic microorganisms, can cause periodontal diseases. Several groups of periodontopathic bacterial species are considered to be responsible for each form of clinical manifestations of the periodontal diseases. These include Gram-negative obligatory anaerobic rods: P. gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythensis (formerly Bacteroides forsythus); Gram-negative facultative anaerobic rods: A. actinomycetemcomitans, Capnocytophaga spp., Campyrobacter rectus and Eikenella corrodens; and oral spirochetes: Treponema denticola (Tab. 3). P. gingivalis is considered to be a major pathogen of chronic periodontitis in adults and generalized (but not localized) aggressive periodontitis [55]. This microorganism possesses several virulence factors for periodontopathogenicity, including fimbriae, proteolytic enzymes and lipopolysaccharide (LPS) [56]. Fimbriae of P. gingivalis are involved in the attachment with the host cells; they specifically bind to salivary component proteins of proline-rich protein (PRP) and proline-rich glycoprotein (PRGP) [57]. In addition, they significantly interact with extracellular matrix proteins, fibronectin and laminin [58, 59]. Therefore, P. gingivalis cells can bind to tooth surface and upper gingival sulcus, which are covered with saliva. Although a deeper portion of the gingival sulcus is not contaminated with saliva, P. gingivalis can bind sulcular epithelial cells via interaction with extracellular matrix proteins and may invade sulcular epithelial cells (Fig. 1). It has been also reported that the fimbriae exhibit a variety of biological and immunological activities in the infectious process [60]. Large amounts of the proteolytic enzymes of gingipains and collagenase are produced by P. gingivalis, and these proteases have the abilities to destroy periodontal tissue directly or indirectly [61, 62]. Furthermore, since P. gingivalis is asaccharolytic, proteolytic dipeptides are uptaken and used as an energy source. Ammonia, propionate and butyrate produced from amino acids can disrupt the host immune system and be toxic against the gingival epithelium. LPS from the outer membrane of the bacteria also elicits a wide variety of responses that may contribute to inflammation and host defense. At established periodontal disease lesions, infiltration of inflammatory lymphocytes, especially B cells and plasma cells, are significant [63], and this event seems to correlate with the polyclonal or oligoclonal B cell activation by P. gingivalis LPS [56, 64]. A. actinomycetemcomitans was originally isolated from a localized aggressive periodontitis patient [65], and has been recognized to be involved in this type of aggressive periodontitis, since early experiments indicated the positive relationship between the periodontal destruction and the high levels of serum antibodies to the bacteria. The reported virulent factors of the bacteria include LPS and an exotoxin, leukotoxin, which has cellular toxicity against human PMNL and monocytes. However, substantial evidence demonstrates that not all A. actinomycetemcomitans can produce leukotoxin,

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Table 3. Prevalence of periodontopathic microorganisms in plaque and saliva from children (119 children aged 2-15 years) Periodontopathic microorganisms

Detection ratio (%) Plaque only

Saliva only

Both

Porphyromonas gingivalis

1.5

3.0

0.6

Prevotella intermedia

0.6

2.4

0.3

Prevotella nigrescens

26.2*

11.3

5.7

Tannerella forsythensis

6.3

15.5*

2.4

Actinobacillus actinomycetemcomitans

10.1

38.7*

30.4

Capnocytophaga ochracea

7.4

34.8*

40.8

Capnocytophaga sputigena

12.5

33.0*

33.6

Eikenella corrodens

15.5

25.6*

10.7

Campyrobacter rectus

21.4

16.4

11.6

Treponema denticola

0

0.6

0

*Significantly higher detection ratio between plaque and saliva samples by Fisher’s exact probability test (p < 0.01).

and leukotoxin-non-producing strains of the bacteria were also recovered from localized aggressive periodontitis patients [66]. Furthermore, recent studies using a PCR detection method revealed that the prevalence rate of the microorganism was relatively high even in periodontally healthy children; it was greater than 50% in saliva, and 30% in subgingival plaque of Japanese children (2–15 years old) [67, 68]. Thus, A. actinomycetemcomitans appears to be an early colonizer in the human oral cavity. However, the accumulation of the bacteria to a critical amount in plaque may contribute as the predisposing factor for the onset and/or progression of localized aggressive periodontitis especially in children with systemic risk factors such as functional impairments/abnormalities of PMNL. In addition to the two periodontopathic bacteria described above, Capnocytophaga sputigena, C. ochracea, E. corrodens, Campylobacter rectus, P. intermedia, P. nigrescens, T. forsythensis and T. denticola have been proposed as possible periodontophatic pathogens in some types of periodontitis, although most of these bacteria can be often or occasionally detected in the plaque microflora of periodontally healthy children [67].

Onset of periodontal diseases – Host-parasite relationship The onset of periodontal diseases, especially periodontitis, is based on the balance in host-parasite relationship in gingival crevices. The gingival crev-

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ice (sulcus) is a groove between the tooth surface and the sulcular epithelium that extends from the free surface of the junctional epithelium to the level of the free gingival margin. The junctional epithelium forms a collar around the tooth. The gingival crevice is bathed in saliva that contains a lot of antibiotic agents, such as lysozyme, lactoferrin, peroxydase and secretory IgA. In addition, the sulcular epithelium acts as a physical barrier against intruders. Furthermore, serum antimicrobial components consecutively exude to the gingival crevice through the junctional epithelium, termed gingival crevicular fluid (GCF). GCF originates from plasma exudates, and thus contains IgG, IgA, complements and cellular elements. It is noted that 95% of the cellular elements are PMNL and the remainder are lymphocytes and monocytes, even in the GCF from clinically healthy gingival crevice [40]. This suggests that PMNL in plasma emigrate actively to gingival crevices and play an important role in the localized host defense within the gingival crevice. Although the colonization of periodontopathic bacteria in gingival crevice does not necessarily induce infection that causes destruction of the periodontium, the acquisition of the putative pathogens is a prerequisite process for developing periodontal diseases. In adults, periodontopathic bacteria are detected from periodontally healthy sites as well as diseased sites, although the number of the microorganisms is generally lower than that in diseased sites [55, 69]. In children, however, less information is available on periodontopathic bacterial infection in their plaque. Our recent longitudinal investigations by means of PCR method using the periodontopathic bacterial species-specific primers for 16S rRNA genes indicated that seven out of ten bacteria, i.e., C. rectus, E. corrodens, A. actinomycetemcomitans, Capnocytophaga ochracea, C. sputigena, T. forsythensis and P. nigrescens were frequently found in both subgingival plaque and saliva from 119 periodontally healthy children (2–15 years old) [67]. In contrast, P. gingivalis, T. denticola and P. intermedia were rarely detected in plaque and saliva from children. These findings indicate that the colonization of many putative periodontopathic bacteria can occur quite early in childhood without development of periodontal diseases, and may become common members in the microflora of plaque and saliva in children. However, the oral infection/colonization of P. gingivalis, T. denticola and/or P. intermedia could be an occasional and transient phenomenon. The child’s oral cavity is assumed to be possibly colonized by P. gingivalis based on the premise that the bacteria specifically interact with the saliva proteins, PRP and PRGP, and with extracellular matrix proteins of the sulcular epithelium as in the adult’s oral cavity. Therefore, the low prevalence rate of P. gingivalis, T. denticola and P. intermedia observed in the study suggest that the child host-defense of antibiotic components in saliva and GCF efficiently prevent the initial colonization and/or proliferation of these periodontal pathogens, resulting in the arrest of periodontal diseases in healthy children. Regarding other putative periodontopathic bacteria including C. rectus, E. corrodens,

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A. actinomycetemcomitans, C. ochracea, C. sputigena, T. forsythensis and P. nigrescens, the pathogenic role in periodontal diseases is still not clear. Together with the observation in adults that a relatively lower, but significant, number of the periodontopathic bacteria are detected from periodontally healthy gingival crevices, it is likely that the initially colonized bacteria having pathogenic potentials are efficiently controlled by the host-defense mechanisms so that they do not to reach a critical level of accumulation in the healthy gingival crevice. If the host-defense is not efficient, as is the case in children with functional impairments/abnormalities of PMNL, the bacteria with no or little pathogenic potentials could be a factor that governs the periodontopathic potential. In fact, the children with Down’s syndrome often develop severe early-onset of periodontal diseases. These subjects demonstrate an early decline in the host-defense ability including malfunction of PMNL due to premature senescence. Our microbial observation in children with Down’s syndrome indicated that the seven putative periodontopathic bacteria (C. rectus, E. corrodens, A. actinomycetemcomitans, C. ochracea, C. sputigena, T. forsythensis and P. nigrescens) were detected with greater frequency in Down’s syndrome patients than in healthy control children [70]. Furthermore, the cluster group characterized by the additional infections with P. gingivalis, T. denticola and P. intermedia to the seven putative periodontopathic bacteria showed the highest severity in periodontal parameters, suggesting that this particular predisposing condition probably permits the colonization of these periodontopathic bacteria and allows their growth, resulting in the onset of periodontal diseases in these children.

Transmission of periodontal bacteria Periodontal diseases are caused by dental plaque bacteria, and thus can be classified as infectious diseases by indigenous bacteria. It has been demonstrated that children whose parents were colonized by the BANA-positive periodontpathic species including P. gingivalis, T. denticola, and T. forsythensis were 9.8 times more likely to be colonized by these species, and children whose parents had clinical evidence of periodontitis were 12 times more likely to be colonized the species [71]. Concordance in colonization of T. forsythensis, P. intermedia and P. nigrescens within children and their parents was also observed in Japanese families [72]. In addition, vertical transmission of A. actinomycetemcomitans was reported in families from Finland [73], and was estimated between 30% and 60% in the Netherlands [74]. Compared with A. actinomycetemcomitans, the case of P. gingivalis is still controversial; vertical as well as horizontal transmission was speculated in a study of 564 members of American families [75], whereas vertical (parentsto-children) transmission has rarely been observed in the Netherlands [74], in Finland [73], and in the research of 78 American subjects [76]. In the later reports, since horizontal transmission of P. gingivalis between adult family

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members was considerable, it was suggested that P. gingivalis commonly colonizes in an established oral microbiota. According to these observations, vertical and horizontal transmission of periodontal pathogens may be controlled by periodontal treatment involving elimination of the pathogen in diseased individuals and by oral hygiene instructions.

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by peripheral blood polymorphonuclear leukocytes in human periodontal diseases. J Periodont Res 28: 197–203 Bass DA, Olbrantz P, Szejda P, Seeds MC, McCall CE (1986) Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection. J Immunol 136: 860–866 Hamada S, Holt SC, McGhee JR (eds) (1991) Periodontal disease: Pathogens and host immune responses. Quintessence Publishing Co., Tokyo Genco R, Hamada S, Lehner T, McGhee J, Mergenhagen S (eds) (1994) Molecular pathogenesis of periodontal disease. ASM Press, Washington, DC Amano A, Shizukuishi S, Horie H, Kimura S, Morisaki I, Hamada S (1998) Binding of Porphyromonas gingivalis fimbriae to proline-rich glycoproteins in parotid saliva via a domain shared by major salivary components. Infect Immun 66: 2072–2077 Kontani M, Ono H, Shibata H, Okamura Y, Tanaka T, Fujiwara T, Kimura S, Hamada S (1996) Cysteine protease of Porphyromonas gingivalis 381 enhances binding of fimbriae to cultured human fibroblasts and matrix proteins. Infect Immun 64: 756–762 Amano A, Nakamura T, Kimura S, Morisaki I, Nakagawa I, Kawabata S, Hamada S (1999) Molecular interactions of Porphyromonas gingivalis fimbriae with host proteins: kinetic analyses based on surface plasmon resonance. Infect Immun 67: 2399–2405 Hamada S, Amano A, Kimura S, Nakagawa I, Kawabata S, Morisaki I (1998) The importance of fimbriae in the virulence and ecology of some oral bacteria. Oral Microbiol Immunol 13: 129–138 Holt SC, Kesavalu L, Walker S, Genco CA (1999) Virulence factors of Porphyromonas gingivalis. Periodontol 2000 20: 168–238 Potempa J, Banbula A, Travis J (2000) Role of bacterial proteinases in matrix destruction and modulation of host responses. Periodontol 2000 24: 153–192 Okada H, Shimabukuro Y, Kassai Y, Ito H, Matsuo T, Ebisu S, Harada Y (1988) The function of gingival lymphocytes on the establishment of human periodontitis. Adv Dent Res 2: 364–367 Kimura S, Koga T, Fujiwara T, Kontani M, Shintoku K, Kaya H, Hamada S (1995) Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis. FEMS Microbiol Lett 130: 1–6 Newman MG, Socransky SS (1977) Predominant cultivable microbiota in periodontosis. J Periodontal Res 12: 120–128 Kaplan JB, Schreiner HC, Furgang D, Fine DH (2002) Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients. J Clin Microbiol 40: 1181–1187 Kimura S, Ooshima T, Takiguchi M, Sasaki Y, Amano A, Morisaki I, Hamada S (2002) Periodontopathic bacterial infection in childhood. J Periodontol 73: 20–26 Ooshima T, Nishiyama N, Hou B, Tamura K, Amano A, Kusumoto A, Kimura S (2003) Occurrence of periodontal bacteria in healthy children: a 2-year longitudinal study. Community Dent Oral Epidemiol 31: 417–425 Dzink JL, Socransky SS, Haffajee AD (1988) The predominant cultivable

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Role of the blood-brain barrier and blood-CSF barrier in the pathogenesis of bacterial meningitis Rüdiger Adam1, Kwang Sik Kim2 and Horst Schroten1 1Pediatric

Infectious Diseases, Klinik für Allgemeine Pädiatrie, Universitätsklinikum, Düsseldorf, Germany; 2Pediatric Infectious Diseases, Johns Hopkins Hospital, Baltimore, Maryland, USA

Abstract Despite significant progress in prevention, diagnosis and therapy acute bacterial meningitis remains an important cause of high morbidity and mortality in the pediatric population with no significant improvement in the outcome in recent years. Further amelioration in treatment can only result from a better understanding of the pathophysiological events that occur after activation of the host’s inflammatory pathways secondary to initial bacterial invasion. The need for improved management strategies is highlighted by the observed increase in antibiotic resistance of microbial pathogens and recent developments in the pharmacological treatment of meningitis patients with dexamethasone, which might adversely influence delivery of drugs to the central nervous system (CNS). In this respect the cellular and molecular events at the blood-CNS barriers come to the focus of attention. It has become evident that these anatomical and functional barriers with their differentiated functionality and vast surface area centrally contribute to the development of bacterial meningitis. This holds true not only for their role as a port of entry into the CNS but also as key players in the pathophysiological cascade following bacterial invasion into the brain. Important aspects that have to be considered are the unique anatomical and functional features of the blood-brain barrier and the bloodcerebrospinal fluid barrier, and their distinct interactions with the variety of pathogens responsible for the development of bacterial meningitis.

Introduction In spite of marked progress in diagnostic procedures, improvement in intensive care and introduction of new antimicrobials, bacterial meningitis still remains a serious, sometimes life-threatening disease in children. A high number of survivors are left with persistent neurological or neuropsychological sequelae. To improve present strategies and to develop new options in diagnostic, prevention and therapy, knowledge and understanding of pathogenesis and pathophysiology of bacterial meningitis is of utmost importance. It is well established that most cases of bacterial meningitis

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develop through hematogenous spread of bacteria after crossing peripheral mucosal barriers. Even though major insights in pathophysiological events have been derived from experimental animal and in vitro models in recent years, many aspects of the subsequent invasion of the central nervous system (CNS), the role of the blood-brain barrier (BBB) and even more the blood-cerebrospinal fluid (CSF) barrier, remain incompletely understood. It has become clear that these anatomical and functional barriers play a central role as a port of entry into the CNS but also as key players in the pathophysiological cascade following bacterial invasion into the brain. They are involved in the often deleterious events secondary to the host immune response and are also important for therapeutic issues.

Bacterial meningitis Bacterial meningitis as the most common serious infection of the CNS continues to be an important cause of morbidity and mortality in children. The causative organism varies with age, immune function and immunization status. The majority of cases are associated with an infection with Streptococcus pneumoniae and Neisseria meningitidis, whereas Haemophilus influenzae type b (Hib) infections have been virtually eradicated as a result of routine vaccination policies. Streptococcus agalactiae, Escherichia coli and Listeria monocytogenes are the most common meningitis pathogens in neonates [1–3]. Bacterial meningitis typically presents with the triad of headache, fever and meningism in adolescents, but the clinical picture can vary widely in younger children [3]. Despite the development of highly effective antibiotics, improvement of early diagnosis and intensive care management, the disease is fatal in 5–40% of the cases depending on the etiological agent and the patient’s age [2, 4]. Neurological sequelae develop in up to one third of children and adults who survive an episode of bacterial meningitis [5]. These sequelae can be related to direct damage of neuroacoustic structures with following hearing impairment, and to disturbances of CSF dynamics and cerebral blood flow with consequent hydrocephalus, brain edema and intracranial pressure. They can also be caused by direct damage of brain parenchymal tissue leading to focal sensory-motor deficits, neuropsychological impairment, or seizures [6]. Despite all improvements in early detection and antibiotic treatment, the rate of sequelae has proven to be rather unchanged in recent years [2, 7]. One main reason for this unacceptable rate of complications is the incomplete knowledge about the pathogenesis of this disease, even though experimental studies with cell cultures and animal models have substantially contributed to our understanding of the interactions of bacterial pathogens with mammalian cells and their entry into the CNS.

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Figure 1. Pathogenetic cascade of bacterial meningitis [9]. With friendly permission of Springer.

The pathogenetic cascade Apart from external protection by the skull and the leptomeninges, the CNS is protected against blood-borne pathogen invasion by effective cellular barriers. Thus, a meningitis pathogen can gain access to the CNS through a defect within the external barriers, be it a congenital malformation such as a dermal sinus or a myelomeningocele, accidentally acquired or iatrogenic, e.g., after a neurosurgical procedure. An infection per continuitatem from purulent mastoiditis or sinusitis is also possible. In the vast majority of cases, however, a pathogen reaches the CNS by hematogenous seeding, after running “a biological gauntlet of host defenses” [8]. It has become an accepted pathogenetic concept that the disease typically progresses through several interconnected phases of interactions between the pathogen and the host (Fig. 1).

Mucosal colonization and invasion Initially, mucosal surfaces of the host’s upper respiratory and gastrointestinal tract are colonized by bacterial pathogens. The bacteria must attach to the mucosal epithelium and resist clearance by mechanical and immunological mechanisms. All meningeal bacterial pathogens seem to express a

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range of surface proteins that facilitates pathogen-host cell interaction. This event is followed by bacterial penetration of the mucosal epithelium either transcellularly or paracellularly, depending on the organism. Many pathogens niftily use host-specific transport mechanisms to safely transverse this epithelial barrier.

Survival within the bloodstream Once the bacteria gain access to the bloodstream, they must overcome the host defense to survive, disseminate and replicate to a sufficiently high density within the blood. Several studies have suggested that a threshold level of bacteremia is necessary for a successful invasion into the CNS. To remain viable, bacterial phase variable switching of surface elements, such as the polysaccharide capsule, seems to be a prerequisite to counteract opsonophagocytosis and complement-mediated cell lysis [10]. The population of organisms recovered from blood or CSF in the acute phase of bacteremia or meningitis is the believed to be the progeny of a few founder bacteria, often a single clone, mostly suited to survival within the bloodstream [11].

Breaching of blood-CNS barriers and replication in the CSF Reaching the blood-CNS barriers, the bacteria then attach to and transgress them through mechanisms that will be outlined in more detail below. It became evident that the host defense mechanisms within the brain are notably ineffective in eliminating invading bacterial pathogens. Bacterial multiplication within the subarachnoid space is facilitated by the virtual absence of host defensive factors such as complement and immunoglobulins, the limited number of endogenous antigen-presenting cells and the limited exchange of immune cells and mediators due to restrictive barriers [12]. As these bacterial compounds are formidable immunological stimuli, various cells within the CNS [e.g., resident leptomeningeal phagocytes, microglia, choroid plexus (CP) epithelia, endothelial cells, astrocytes] are activated to produce a wide array of proinflammatory cytokines. There is a substantial body of evidence that tumor necrosis factor-_ (TNF-_), interleukin-1` (IL1`) and interleukin-6 (IL-6) play a central role in this setting [13, 14].

Local intraventricular inflammation After reaching a critical bacterial concentration and subsequent stationary growth phases or after treatment with antibiotics, a number of bacterial cell wall products, toxins and DNA are released into the CSF compartment [2, 15]. In gram-positive pneumococci for example, peptidoglycans, lipoteichoic

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Figure 2. Concept of “maximal inflammation” [9]. With friendly permission of Springer.

acid and pneumolysin are liberated after activation of autolytic hydrolases (Lyt A-C) [8]. In gram-negative infections such as meningococci, lipopolysaccharide (LPS) and non-LPS compounds are released during growth and lysis [15, 16].

“Maximal CNS inflammation” In this critical phase of meningitis a sequence of parallel and dependent deleterious events leads to maximal leptomeningeal inflammation. A substantial body of evidence mainly derived from animal and in vitro models shows that cytokines, chemokines, proteolytic enzymes, and oxidants together with an influx of leukocytes are essentially involved in the inflammatory cascade that leads to tissue destruction and brain dysfunction during bacterial meningitis [17] (Fig. 2).

The blood-CNS barriers The homeostasis in the brain is an unconditional prerequisite for correct neuron function. Thus, several barrier systems are present in the brain regulating the distribution of substances between the blood stream and the CNS. Of all these CNS interfaces the BBB is not only dominant with regard to the surface area available for interchange with the CNS compartment but also with regard to coverage by scientific examinations. Neglecting other

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anatomical sites of interchange, it is yet infrequently regarded as the only blood-CNS-barrier. Theoretically, blood-derived substances can gain access to the CNS access at various different anatomical sites [18]: 1. the CP with high perfusion, a wide surface area and tight barrier properties despite fenestrated capillaries due to tight junctions at the epithelial lining 2. the circumventricular organs with fenestrated capillaries but a tight ependymal cell lineage of so-called tanycytes [19] 3. the ependymal lining covering the surface of intracerebral ventricles with a less tight cellular layer (gap junctions) and correspondingly less restrictions to extracellular fluid to communicate with CSF 4. the whole subarachnoid space with a network of tight capillaries in the pia mater and arachnoid mater 5. dural venous sinuses, pial and intracerebral veins or postcapillary venules Whether these barriers function as a port of entry during bacterial meningitis is most likely dependent on the nature of the invading microorgamism. The major barriers are described below.

The blood-brain barrier The BBB is a dynamic membranous interface between the systemic circulation and the brain, protecting it and maintaining its homeostasis. Its anatomical base constitutes a complex system of brain microvascular endothelial cells (BMECs) (Fig. 3A). These cells are ensheathed by astrocytic outgrowths, which are referred to as astrocytic end-feet, necessary to maintain barrier properties, and associated pericytes, important for structural support and vasodynamic capacity [20]. The BMECs are unique insofar as their cellular clefts are sealed by tight junctions that closely join adjacent cells, resulting in a transendothelial electrical resistance of 1000–2000 1·cm2 [21]. Paracellular diffusion of molecules larger than Mr 200–400 and the formation of extracellular fluid is thus inhibited [22]. Transcellular passage of solutes is also impeded, as the endothelial cells have only a limited pinocytotic capacity and lack endothelial fenestrations [12]. The BBB eliminates (toxic) substances from the endothelial compartment and supplies the brain with nutrients and other (endogenous) compounds, while restricting the entrance of potentially harmful substances, e.g., bacteria and circulating toxins. It does so by specific ionic channels, transporters, energy-dependent pumps and limited receptor-mediated endocytosis [20, 23]. However, during infectious diseases of the CNS, the BBB integrity may be lost and permeability may be increased.

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Figure 3. Parenchymal cells of the blood-brain barrier (BBB) and blood-CSF barrier. (A) Schema for the components of the BBB. The endothelial cells of the cerebral capillaries lack fenestrations and are tightly joined by zonulae occludentes (see arrows). Astrocyte foot processes extensively abut the outside surface of the endothelium. The darkened area is the interstitial space surrounding the capillary wall (N, neuron). (B) Cross-section of a choroidal villus. A ring of choroid epithelial cells surround the interstitial fluid and adjacent vascular core. The basolateral surface of the cells has interdigitations, whereas the outer CSF-facing apical membrane has an extensive microvilli system. Arrows point to the tight junctions between cells at their apical ends [24].

The blood-CSF barrier The second system that prevents the free passage of substrates between blood and brain is the blood-CSF barrier, represented by the CP epithelium. The CP is comprised of a vascularized stromal core surrounded by epithelial cells that are aligned in villi. The CP capillaries have a much bigger diameter than cerebral microvasculature (~50 +m vs. 8 +m, respectively) [25], the perfusion of about 5 mL/min/g is about tenfold faster than the average cerebral blood flow [24]. The cell surface is greatly increased due to an array of microvilli on the CSF side and basolateral interdigitations directed towards the basal membrane [26]. The CP surface area calculated from animal experiments is believed to be much bigger than previously appreciated, especially when put into relation to the BBB interface [27, 28]. The epithelial cells are sealed by tight junctions, which become indispensable since the endothelium of CP capillaries is fenestrated, non-continuous and has ‘window’-like openings being highly permeable to hydrophilic substrates. Thus, it is the CP epithelial cells welded by tight junctions that constitute the anatomical basis of the blood-CSF barrier [24] (Fig. 3B). The CPs are located throughout the fourth ventricle near the base of the brain and in the lateral ventricles inside the right and left cerebral hemisphere. They are known to be centrally involved in CSF formation and

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actively regulate the concentration of molecules within the CSF by numerous transport mechanisms [29]. Whereas a number of molecular carriers are responsible for controlling the influx of nutrients into the CSF, a potent efflux apparatus promotes the discarding of noxious substances in the CNSto-blood direction [28].

Circumventricular organs Circumventricular organs (CVOs) are situated at several strategic locations around the ventricles of the brain. They are midline structures within the ependymal lining bordering the 3rd and 4th ventricle. The most outstanding morphological characteristic of CVOs is a dense and intricate network of mostly fenestrated capillaries, making it readily accessible to blood-borne substances, some of which effect the functions of the subfornical organ [30]. With the exception of the subcommisural organ, the fenestration of blood vessels makes the CVOs part of the blood-CSF barrier. CVOs are recognized as important sites for blood-brain communication as neurosecretory products gain access to the bloodstream and blood-borne substances can be detected by neuronal structures. The term CVOs comprises the following organs: pineal gland, median eminence, neurohypophysis, subfornical organ, area postrema, subcommissural organ, organum vasculosum of the lamina terminalis (Fig. 4). Sometimes the CP is also included as well as the intermediate and neural lobes of the pituitary [26]. Collectively, the ependymal and capillary surface areas of the CVOs are relatively small, likely accounting for less than 1% of the ventricles and brain capillary bed, respectively. Despite these diminutive transport interfaces, a possible role in microbial CNS invasion is conjectural, since involvement of these areas during inflammation and leucocyte invasion has been reported [31].

Gateways into the brain It is still unclear why many pathogens principally have the potential to initiate meningitis, but only a relatively small number of them account for the vast majority of cases. The crucial step for all microorganisms after invasion of the host is the attachment and subsequent penetration of the structures that separate the CNS from the periphery. For most pathogens, however, the exact port of entry into the brain remains unclear. Nonetheless, observations on cell culture and animal models as well as histological experiments allow conclusions about the primary site of invasion to be drawn. It has to be kept in mind, though, that multiple routes into the CNS compartment may be used simultaneously.

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Figure 4. Sagittal view of the anatomical relationship among the circumventricular organs (CVOs), which are located on the midline of the brain (AP, Area postrema; SFO, subfornical organ; ME, median eminence; PI, pineal gland; OVLT, organum vasculosum of the lamina terminalis) [24].

Importance of threshold bacteremia Even though the exact sites of entry might not be exactly known, several studies suggest the probability of developing meningitis to be directly related to the concentration of bacteria in the blood and to their exceeding a critical threshold. For example, Dietzman et al. [32] reported a higher incidence of E. coli meningitis in neonates who had bacterial counts in blood > 103 CFU/mL (6 out of 11 cases, 60%) compared to those with bacterial counts less than 103 CFU/mL (1 out of 19 cases, 5%). Such associations between a certain degree of bacteremia and subsequent disease have also been described for all other pathogens relevant for meningeal infections such as Hib [33, 34], S. agalactiae [35], S. pneumoniae [36, 37], E. coli [32, 38] and, with some conflicting data, N. meningitidis [37, 39]. The infection of the CSF compartment possibly appears as a kinetic process with bacteria entering from blood and being cleared into the cerebral venous sinuses within the CSF flow. Bacteria have been shown to exit from the CSF to the venous blood through the arachnoid villi [40]. The balance of bacterial ingress and egress is proposed to be important in the establishment of meningitis and its severity [41].

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The blood-brain barrier Many investigations on meningitis pathogenesis focus on the BBB or its morphological correlate, the endothelium of the cerebral microvasculature. As outlined below, a respectable number of both in vitro and in vivo models are available. One reason for this emphasis on the BBB is its assumed preponderance regarding surface area in comparison with the other bloodCNS barriers. As one researcher puts it, “the cerebral microvasculature was chosen for morphological assessment in this study because it represents the dominant site of the BBB. The surface area of the cerebral microvasculature is 5000-fold greater than the surface area of capillaries supplying the circumventricular organs, rendering the former more pertinent for this investigation” [42]. This view, however, has been seriously questioned by other researchers who believe the ratio to be more in the area of 1:10 taking into account more recent data derived from calculations on neonatal rat CP extrapolated to conditions in humans [27, 28]. Others have put their emphasis on the cerebral vasculature because it plays a dominant role in the pathophysiology of bacterial meningitis after the initial stages of blood-CNS barrier breakdown [43]. In an infant rat model of S. pneumoniae meningitis, brain tissue examinations from animals with positive CSF cultures revealed histopathological signs of inflammation predominantly within the meningeal region [44]. In cryostat sections of infant rat brain cortical slices, S-fimbriated E. coli strains have been shown to bind specifically to the luminal surfaces of cerebral endothelial cells besides binding to CP epithelial cells and ependymal cells [45]. In contrast, gram-negative rods were present in the subarachnoid space predominantly around the perivascular areas not in the CP, pointing towards the BBB as being the major gateway into the CSF [38]. In a mouse model, the animals that developed pneumococcal meningitis after intranasal inoculation and treatment with hyaluronidase, showed a significant inflammatory infiltrate predominantly composed of polymorphonuclear leukocytes preferentially around the leptomeningeal blood vessels, suggesting them to be the area of blood-CNS barrier breaching [46]. Challenge of mice with S. agalactiae by intraperitoneal injection led to bacteremia and subsequent meningitis. Histopathological studies of brain and meninges of animals with positive CSF cultures principally revealed that bacteria and leukocytic infiltrate distributed surrounding the meningeal vessels and the perivascular spaces within the cerebral cortex [35]. Histological examination of brain tissue from a fatal case of meningococcal disease revealed attachment of N. meningitidis on the CP and microvascular endothelium, indicating that both loci may be used by meningococci for invasion of the meninges [47]. Despite decades of investigation on microbial interactions at the bloodCNS barriers, there remains a distinct paucity of studies clearly pointing towards the cerebral vasculature as the primary site of CNS invasion for

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certain pathogens. Probably due to this dilemma, some authors independently cite a reviewing feature in the News of the American Society of Microbiology (ASM News) [48] as the only reference for microbial BBB invasion [49–51]. However, abundant experimental studies have demonstrated that receptors for various meningeal pathogens are present on cells of cerebral capillaries potentially mediating attachment or penetration of the BBB [52, 53].

The blood-CSF barrier For many important meningitis pathogens certain experimental data suggests the CPs to be involved in bacterial entry into the brain. Whether the blood-CSF barriers represent the primary sites of invasion or one of several ports of entry remains to be clarified. Insufficient availability of suitable in vitro models throughout recent decades may be in part responsible for the lack of supportive data. In the fatal case of an infant having succumbed to fulminant infection with N. meningitidis, histopathological investigations of brain sections at autopsy revealed the greatest number of bacteria attaching to CP capillaries (68% in CP vs. 7% in meningeal capillaries). No meningococci were found to be adhered to the plexus epithelial cells. Interestingly the bacteria isolated from the CSF expressed significantly more PilC protein than blood isolates, suggesting this adhesin plays an important role in attachment and invasion of meningococci [47]. In Hib meningitis, early studies on infant rats suggested that invasion from the bloodstream occurred via the dural sinus veins, while other studies favored the cribriform plate or the CPs to be the main site of entry into the brain. The latter notion was supported by infant rat models with serial CSF sampling from infected animals. Here, at least in the early phases of infection before an assumed equilibrium within the CSF compartments has occurred, the highest density of bacteria was found in the CSF of the lateral ventricles in comparison to the lumbar and cortical subarachnoid space or the cisterna magna, respectively, suggesting an entry of bacteria primarily via the CPs [54]. This observation is supported by studies on primates, in which the CP has been found to be the site of earliest histopathological changes during Hib infections [55]. Another line of evidence favoring the CP to be the main site of bacterial entry is derived from the observation that, in experimental meningitis of infant primates, a concordance of bacterial density in the CSF between the lumbar subarachnoid space and the cisterna magna was observed even at low bacterial concentrations (i.e., in early stages of the disease) [56]. Since the CSF flow is unidirectionally circulating from the ventricles down to the lumbar region, the presence of bacteria in the ventricular fluid suggests entry via the CPs.

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Another pathogen, Streptococcus suis, which accounts for both human and porcine meningitis cases, is also suspected of entering the CNS primarily via the blood-CSF barrier. In a porcine animal model, infected pigs were killed at the earliest clinical signs of meningitis. In these cases, in which a low bacterial density within the CSF can be assumed, streptococci were almost exclusively detected in the CP epithelium [57]. The lack of diffuse parenchymal lesions in most S. suis cases of meningitis suggests access to the CNS via the CPs. Experiments with E. coli strains possessing S-fimbriae demonstrated specific binding sites on CP epithelial cells, to a lesser extent also to endothelial cells of the CP core besides vascular endothelial cells and ependymal cells. In this work, which was performed on cryostat brain sections of neonatal rats, pre-treatment of the slices with neuraminidase or a fimbrial analogue abolished attachment of E. coli, demonstrating the specificity of these interactions [45]. In contrast, in infant rats with experimental hematogenous E. coli meningitis, gram-negative rods were demonstrated around the perivascular area, not in the CP [38]. Thus, entry of E. coli into the CNS via the CP may be unlikely and additional studies are needed to clarify this issue. A study on experimental listeriosis in mice showed that after subcutaneous injection the animals developed meningitis displaying a mixed inflammatory infiltration in the ventricular system, especially in the CPs. Inflammatory lesions were associated with the presence of L. monocytogenes within phagocytic cells. It is suggested that choroiditis and meningitis developed as a consequence of hematogenous dissemination of L. monocytogenes within mononuclear phagocytes and penetration of these cells into the ventricular system through the CP [58]. In addition, invasion of the CNS via the blood-CSF barrier may also be facilitated by the high blood flow in the CPs of up to 500 mL/g/min [24], which allows putative delivery of a relatively high number of pathogens to this site via blood stream.

Experimental models for blood-CNS-barrier observations Animal models A number of animal models have been successfully established to study cellular and molecular mechanisms of microbial invasion into the brain. Apart from bacterial species used or animals selected as a host, the information obtained from these models is very much dependent on the mode of inoculation. Intranasal, orogastral, intravenous/intracardial, subcutaneous or intraperitoneal inoculation primarily focus on events on “the blood side”, e.g., bacterial and host factors that determine the pathogen’s fate within the bloodstream and the potential of CNS invasion. In contrast, experimental models using direct inoculation into the CSF rather highlight pathogenetic

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events on “the brain side”. Notwithstanding bypassing the microbial permeation of blood-CNS barriers artificially, these models have the advantage of reliably inducing lethal infections with reproducible bacterial inocula over a predictable time course [59, 60]. These animal models have contributed considerably to the study of pathogen and host factors such as bacterial virulence traits, microbial invasion genes, intracellular signaling cascades and modes of cellular permeation. Furthermore, they have helped in understanding the complications of meningeal inflammation and evaluating potentially useful agents for treatment therapy [61, 62]. An infant rat model has been widely used to mimic human neonatal bacterial meningitis. An important advantage of this model lies in the development of meningitis after bacterial hematogenous spread similar to human newborn meningitis. The pathogenesis of meningitis has been studied essentially with two major pathogens, Hib [63, 64] or E. coli [65–68] using many different routes of inoculation (nasopharyngeal, orogastric, subcutaneous, intraperitoneal or intracardial). For other purposes an infant rat model with intracisternal inoculation of S. pneumoniae has been used [69, 70]. Other important meningitis models are performed with adult animals by direct systemic or intracerebral inoculation mostly in rabbits [71, 72], rats [73] or mice [74]. In recent years, knockout mice with targeted deletion of specific genes have become a powerful tool in investigating the roles of the different adhesins, cytokines, proteases, and oxidants involved in the inflammatory cascade during bacterial meningitis [75].

Cell culture models To identify and study cellular and molecular mechanisms of microbial permeation of the blood-CNS barriers, it has become important to model the blood-CNS barriers in vitro [76, 77]. Both primary and immortalized cell culture systems have been established. One of the major potential benefits of these in vitro systems in comparison to animal models lies in the possibilities to measure cellular responses to a variety of stimuli without the risk of interference by possible contributions of other cell types such as neuroglia or resident macrophages. Furthermore, no experimental bias is risked by changes in functional and structural characteristics of the blood-CNS barriers.

Blood brain barrier As outlined above the BBB principally consists of a tight microvascular endothelium, a basal membrane and the pericytic sheath that have to be crossed by bacteria when entering the CNS. The central component of all

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models is the BMEC. BMECs are usually harvested from brain homogenates, purified on dextran gradients and cultured alone or together with supporting glial cells. Many mammal BMECs have been used: rat, mouse, dog, dogs, cattle and human [78–84]. Models using peripheral endothelial cell such as human umbilical vein endothelial cells (HUVECs) have also been introduced, but these systemic endothelial cells are likely not appropriate targets for meningitic bacteria [52]. Extending the potential of cell monolayers, several coculture systems have been developed. Bilayer systems consisting of endothelial and epithelial cocultures separated by a porous membrane offer added complexity of multiple layers that might more closely resemble the in vivo situation and allow examination of microbial penetration and associated effects [85]. Multiple studies have indicated that coculturing of BMECs with astrocytes or neuroglia on opposing sides of a permeable support has mutual benefits as endothelial cells facilitate astrocyte differentiation but, more importantly, astrocytic metabolism contributes to the formation of BBB properties in BMECs (reviewed in [86]). These culture systems were employed in studies on bacterial interactions with cerebral endothelium, e.g., using S. pneumoniae [83, 87] or E. coli [88, 89]. Primary BMEC isolation is laborious, time consuming and the cells are difficult to maintain in native tissue culture and suffer from contamination. In addition, these cells often lose their typical features such as Factor VIII Rag or a-GTP upon subcultivation. Immortalizations and spontaneous transformations have been reported for mouse, rat, cow and human-derived brain endothelial cells. The best-studied system so far is a human brain microvascular endothelial cell line (HBMEC) that has been derived from a brain biopsy of an adult female with epilepsy. The HBMEC were immortalized by transfection with simian virus 40 large-T antigen [90]. This cell line has proven invaluable in multiple experiments on bacterial interaction with the BBB. Many different bacterial species have been examined, e.g. S. agalactiae [91], S. suis [92], S. pneumoniae [83], N. meningitidis [93], Staphylococcus aureus [94], and H. influenzae [95]. In addition to a bovine cell line [90], a porcine counterpart of HBMEC, an immortalized porcine brain microvascular endothelial cell line (PBMEC/ C1-2) has recently been established by lipofection with simian virus 40 small and large T-antigens [96]. It was shown to maintain its morphological and functional characteristics and was used in several investigations with S. suis [49, 97] and Haemophilus parasuis [98]. BMEC cells in vitro as models of the BBB should exhibit substantial properties of cerebral microvascular endothelium. At best they should express tight junction proteins (such as claudins, occludin and ZO-1 and 2) and adherens junction proteins (such as VE-cadherin and `-catenin) spatially separated to morphologically demonstrate features of a polarized monolayer. Functionally, this should translate to a limited permeability to

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paracellular tracers (e.g., inulin, sucrose, mannitol or dextran) and to ions, resulting in low permeability coefficients and high transendothelial electrical resistance, respectively [99, 100].

Blood-CSF barrier As mentioned earlier, the tight CP epithelial lining constitutes the structural correlate of the blood-CSF barrier. The establishment of in vitro models of CP epithelial cells has been a challenge for many years. Several preparation methods of primary cells have been established, all based on the initial experiments with rat and cow cells [101, 102]. Subsequently, other working groups have been successful in culturing primary CP epithelial cells including other species: rabbit [103, 104], rat [105–107], cow [102] and swine [108]. However, many primary cultures have been problematic regarding contaminating fibroblasts. The CP epithelial cells are principally isolated with enzymatic digestion after mechanical pre-treatment and cultured either on flat bottom culture dishes or in permeable filter inserts, where they are able to maintain a hydrostatic pressure difference between apical and basolateral compartment and, thus, are able to establish an effective hydrodynamic barrier. Just recently our working group has adopted a primary porcine CP cell model [108] for studies of bacterial interactions at the blood-CSF barrier. We were the first to demonstrate a bacteria-CP interaction in vitro using S. suis [109–111] (see also p. 216). Several CP cell lines have been established from rat [112], mouse [113] or sheep [114] with varying quality regarding typical markers, phenotypes and especially barrier function. Therefore, their impact regarding questions on CNS infections has been limited so far.

Microbial translocation across the blood-CNS barrier Recent studies on E. coli have elegantly shown that successful crossing of the BBB by circulating bacteria requires, as mentioned above, a certain degree of bacteremia, a direct attachment of the microbe to and subsequent invasion of the endothelial cells, a rearrangement of the BMEC actin cytoskeleton and the traversal of the cells alive [1, 53, 115]. Once attachment to the tight blood-CNS barriers has occurred, several pathogen-specific strategies can be employed to migrate across and gain access to the CSF space (Fig. 5): – disruption of tight cell-to-cell contacts and passage between the cells (paracellular route) – direct or indirect invasion of the endothelial cells, permeation and release in a vital state on the contralateral side of the barrier (transcellular passage)

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Figure 5. Possible strategies of microbial penetration of blood-CNS barriers [9]. With friendly permission of Springer.

– penetration of the barrier attached to or phagocytosed by leukocytes during their diapedesis (direct or ‘modified’ Trojan horse mechanism) – destruction of the barrier by cellular injury, e.g., due to release of cytotoxic enzymes or bacterial fragments.

Transcellular passage Just like overcoming the nasopharyngeal barriers, pathogens use several host transport systems to breach the blood-CNS barrier. For N. meningitidis, interaction between surface proteins (Opc) with endothelial integrin receptors is important [116]. S. pneumoniae utilizes the internalization of platelet-activation factor (PAF) receptor via binding of phosphorylcholine and is likewise incorporated. While a fraction of the internalized pneumococci dies, others transverse the cells via transcytosis [117]. A similar mode of action is known for S. agalactiae. They are also internalized by “induced transcytosis” after attachment to fibrinogen, even though in higher densities they might also damage the cellular barrier by release of toxins (see also below) [52, 118, 119]. E. coli displays attachment and invasion characteristics specific for cerebral endothelial cells. They adhere to and invade HBMEC using several capsular and fimbrial epitopes and can be found within intracellular vacuoles of HBMEC [67]. Bacterial proteins necessary for bacterial invasion have been identified, i.e., IbeA, IbeB, YijP and CNF1 [52, 65, 67]. Using the

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host cytoskeleton they are able to transverse the BBB and reach the CNS in a vital state. Other pathogens believed to breach the BBB by transcellular passage are L. monocytogenes [120], Mycobacterium tuberculosis [121], and fungal pathogens such as Candida albicans [122] and Cryptococcus neoformans [123]. Figure 5 illustrates possible strategies of microbial penetration of blood-CNS barriers [9].

Paracellular/intercellular passage If cerebral endothelial cells are confronted with high bacterial loads, other factors besides the transcellular passage supposedly become relevant. Both the `-hemolysin production of S. agalactiae and the pneumolysin of S. pneumoniae are capable of damaging the endothelial layer integrity, thus possibly allowing direct paracellular passage of bacteria [52, 124]. In studies on Hib, it has been suspected that the bacteria cross the BBB paracellularly [125]. Borrelia burgdorferi is also suspected of reaching the subarachnoid space after paracellular penetration, although some aspects point at a transcellular route as well [126]. Protozoans such as Trypanosoma brucei at least partly penetrate endothelial linings via a paracellular mechanism, although recently transcellular permeation has been documented [127].

Transmigration via leucocytes (Trojan horse mechanism) Pathogens with the ability to survive within phagocytes can take advantage of being phagocytosed and reach the brain when their “Trojan horses” migrate through blood-CNS barriers. Such mechanisms have been suggested for Brucella spp., M. tuberculosis and L. monocytogenes [128, 129]. It is of interest that, in some events, Listeria are even able to spread by retrograde neuronal transport from the periphery to the CNS [130]. Whether this intraaxonal movement is pathogenetically relevant in humans is not known yet. Intracellular survival in macrophages has also been demonstrated for S. agalactiae [131] and E. coli [132], but it is unclear whether this property has any relevance to transversal of blood-CNS barriers. In S. suis, a “modified Trojan horse” mechanism, in which bacteria transverse blood-CNS barriers by adhering to diapeding macrophages, rather than residing in phagosomes within them, was discussed [133, 134].

Interactions between bacteria and blood-CNS barrier cells As stated earlier, one key factor for microbial entry into the subarachnoid space is the ability to reach a critical and sustained bacterial concentration

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in the bloodstream. Consequently, the ability of a pathogen to escape the host defenses is crucial for meningeal invasion. However, high level of bacteremia per se is not sufficient for the development of meningitis. Bacterial adhesins and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are believed to be centrally involved in binding to blood-CNS barrier cellular receptors or interactions with extracellular matrix proteins [1, 51]. Such interactions can then promote attachment to and invasion of BMEC or CP epithelial cells, a prerequisite for bacterial penetration of the blood-CNS barriers.

E. coli In extensive studies with E. coli K1 and HBMEC, it has been shown that several microbial determinants contribute to a successful traversal of the BBB (Fig. 6). Fimbrial proteins such as FimH or membrane proteins such as OmpA mediate attachment to the cerebral endothelium via ligand-receptor interaction and contribute to subsequent invasion [67, 135]. Other structures such as S-fimbriae, previously shown to facilitate bacterial adhesion, failed to demonstrate a pivotal role for invasion in ensuing experiments [45, 136, 137]. Components of K1 E. coli, identified as Ibe proteins, AslA, TraJ, and cytotoxic necrotizing factor 1 are believed to contribute to HBMEC invasion, even though the exact mechanisms why these E. coli determinants are required for invasion yet remain incompletely understood (summarized in [1]). Several signal transduction pathways, e.g., phosphatidylinositol 3-kinase, focal adhesion kinase, Rho GTPases and others, have been shown to be involved in bacterial invasion of human BMEC, most likely through their effects on actin cytoskeleton rearrangements [53] (Fig. 6).

S. pneumoniae Initial attachment of S. pneumoniae involves the recognition of host cell receptor glycoconjugates [138]. Subsequently, the bacteria invade BMEC in part via interaction between pneumococcal surface component phosphorylcholine and the BMEC PAF receptor [117]. This has been shown by partial inhibition of pneumococcal invasion of BMEC by a PAF receptor antagonist. Phosphorylcholine decoration was found to be up-regulated in pneumococci retrieved from CSF samples of experimentally infected rodents [139]. Choline-binding protein SpsA mediates pneumococcal adherence to and invasion of mucosal epithelial cells by a human-specific interaction with the polymeric immunoglobulin receptor (pIgR) [140] and might be involved in crossing the blood-CSF barrier.

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Figure 6. Microbial and host factors that contribute to successful crossing of E. coli across brain microvascular endothelial cells (BMECs) (O-LPS, O-lipopolysaccharide) [1].

In addition, the PavA protein, which shows a close relationship to fibronectin-binding proteins of other streptococcal species, was identified as a pneumococcal adhesin for fibronectin. In an experimental mouse meningitis model, pneumococcal strains deficient in PavA showed substantially reduced adherence to and internalization of HBMEC [141]. Pneumolysin, a major virulence factor of S. pneumoniae, was shown to damage endothelial cells and to be an important component for compromising the BBB [83]. Ependymal cells were shown to be damaged in a rat meningitis model by pneumolysin and hydrogen peroxide [142]. Infection with S. pneumoniae led to a loss of ciliae, decrease in their beat frequency and damage to their ultrastructure [143].

N. meningitidis Several groups had previously reported that encapsulation of N. meningitidis impedes interaction with epithelial or endothelial cells preventing their invasion or transversal [144]. It was reasoned that relevant binding sites such as the bacterial outer membrane proteins Opa and Opc proteins were masked by the capsule [145]. It has recently been shown in a study with mutants unable to inactivate capsule expression that fully encapsulated meningococci are well capable of adhering to HBMEC. Invasion of N. meningitidis in HBMEC was mediated by Opc binding to fibronectin, thus anchoring the bacteria to the _5`1-integrin receptor on human BMEC surface [116]. Invasion of N. meningitidis into HBMEC has been shown to involve c-Jun kinases 1 and 2 (JNK1 and JNK2) as their inhibition significantly

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reduced meningococcal invasion in HBMEC [93]. Another factor essential for meningeal invasion by N. meningitidis seems to be an adhesin located at the tip of type IV pili, PilC. Meningeal invasion of meningococci was associated with an increase in the expression of this adhesin [146].

S. agalactiae (Group B streptococci) Invasion of HBMEC by S. agalactiae was shown to require active bacterial DNA, RNA, and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. The streptococcal polysaccharide capsule reduced the invasive ability of the organism [119]. The bacteria were found inside membrane-bound vacuoles within the cells, suggesting the bacteria might induce their own uptake. A streptococcal adhesin just recently identified for HBMEC is the fibrinogen-binding protein fbsA, which mediated attachment to the BBB but failed to support invasion of the cells [118]. Using microarray systems and knockout bacteria a recent study determined the `-hemolysin of S. agalactiae to be the principal provocative factor for activation of HBMEC. It was found that streptococcal infection induced a highly specific and coordinate set of genes known to orchestrate neutrophil recruitment, activation and enhanced survival (e.g., CXC family chemokines IL-8, Gro-_ and `, IL6, granulocyte-macrophage colony stimulating factor (GM-CSF), myeloid cell leukemia sequence 1 and intercellular adhesion molecule 1). The bacterial capsule, in contrast, was believed to rather conceal the pathogen’s surface to diminish host recognition. The authors concluded that the innate immune response of the BBB endothelium to S. agalactiae is to activate circulating neutrophils under modulation by specific bacterial virulence determinants [91].

S. suis In studies using a porcine microvascular endothelial cell line, S. suis was shown to adhere to the cells [49, 97]. In addition, intracellular survival and some degree of invasion were observed. A cytolysin was noted to be mainly responsible for endothelial damage [49]. Besides damaging a cellular barrier with the help of suilysin, S. suis was shown to bind to porcine and human plasminogens on its surface; this could then be activated into an endogenous plasminogen activator. As acquisition of plasmin activity is a mechanism by which invasive bacteria can enhance their capabilities to destroy cell integrity this capacity may affect blood-CNS barrier permeability and contribute to the invasive potential of S. suis [147]. Experiments using porcine CP epithelial cells highlighted that S. suis is also able to markedly affect the barrier function and cell integrity of the

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CP epithelium [110]. Further investigations revealed that the infection with S. suis induced cell death both by apoptosis, indicated by strain-dependent DNA fragmentation and caspase activation, and by necrosis, shown by the increase of cell membrane permeability and release of nuclear high mobility group box 1 protein [111].

BBB disruption and pleocytosis After bacteria have accomplished invasion into the CNS, they multiply and induce the release of a multitude of proinflammatory and toxic compounds, leading to the hallmarks of bacterial meningitis, the disintegration of bloodCNS barriers and the infiltration of leukocytes with subsequent pleocytosis. Animal experiments failed to demonstrate a close association between blood-CNS barrier breakdown and CSF pleocytosis [148–150], and clinical observations have shown that either pleocytosis without significant CNS barrier dysfunction [151, 152], apurulent courses of bacterial meningitis [153] or blood-CNS barrier dysfunction in neutropenic patients [154] do occur. This has led to the conclusion that initial bacterial entry into the CNS per se takes place without pleocytosis and blood-CNS barrier breakdown, and that bacteria can then induce inflammation or other alterations such as pleocytosis or increased BBB permeability [1]. Although not in the focus of this review, it is of note that cerebral edema, increased intracranial pressure and altered cerebral blood flow occur in bacterial meningitis, resulting in neuronal injury.

Leukocyte recruitment CSF pleocytosis is a result of leukocyte extravasation from the circulation into the extravascular space after chemotactic attraction [155, 156]. It occurs through a tightly controlled multistep process governed by the sequential activation of adhesion receptors and their ligands on both leukocytes and the endothelium [157, 158]. The multistep paradigm postulates that four sequential steps (capture, activation, adhesion strengthening, transmigration) are involved in this cascade. The initial capture, the ‘tethering’ of leukocytes as well as subsequent rolling are mediated by adhesion molecules such as P-, E-, and L-selectin, and their corresponding carbohydrate ligands. Firm adhesion of leukocytes to the endothelium is subsequently mediated by a family of integrins, which have to be ‘activated’ by a proinflammatory cytokine (e.g., IL-1`), chemokines (e.g., IL-8), complement products, or bacterial cell wall components to reach adequate avidity [159]. Macrophage antigen 1 (MAC-1; CD11b/CD18) from the Ig superfamily of adhesion receptors is the predominant integrin involved in neutrophil binding to their endothelial ligands. Intercellular adhesion

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molecule (ICAM)-1 exhibits low constitutive levels on the cell surface of the resting endothelium but is markedly induced by exposure to inflammatory stimuli and is the most important endothelial ligand for MAC-1. In experiments with HBMEC, challenge with S. agalactiae led to the up-regulation of a number of CXC chemokines for recruitment of neutrophils, GM-CSF for bone marrow stimulation of neutrophils, ICAM-1 for adhesion of neutrophils, and Mcl-1 for prevention of neutrophil apoptosis, demonstrating the interconnection between microbial infection and leukocyte activation [91]. Infection of HBMEC with L. monocytogenes led to a significant expression of ICAM-1 [160] as well as HBMEC challenge with Plasmodium falciparum-infected erythrocytes [161]. Antibodies directed against the adhesion molecules MAC-1 or ICAM-1 profoundly attenuated invasion of neutrophils during experimental meningitis and led to significant reductions in intracranial complications such as brain edema formation [162]. An animal model of experimental autoimmune encephalomyelitis (EAE) demonstrates the involvement of the CP in leukocyte recruitment. Using immunohistochemistry and in situ hybridization, expression of VCAM-1, ICAM-1 and MAdCAM-1 has been observed on the CP epithelial cells in combination with a complete absence of these structures on the fenestrated endothelium [163].

Immunological properties of the blood-CNS barrier Induction of inflammation Cells of the intracerebral microvasculature and the CP epithelium are, among many other cells of the CNS, capable of expressing several cytokines and other proinflammatory molecules [164]. In humans, the classic proinflammatory cytokines such as TNF-_, IL-1`, and IL-6, as well as a great variety of other cytokines, are present in CSF during meningitis. In addition, CXC and CC chemokines have been found in the CSF of these patients [13, 165]. Concentrations of IL-1`, but not IL-6 and TNF-_, are associated with significantly worse disease outcome or disease severity [14].

Chemokine production at the blood-CNS-barrier Numerous observations highlight that the cerebral endothelium is capable of releasing an array of factors for leukocyte attraction. In experimental studies it was shown that HBMEC are capable of secreting IL-8 in response to challenge with S. agalactiae [118]. After infection of HBMEC with N. meningitidis, the endothelial cells were also shown to respond with IL-8 production with the p38 mitogen-activated (MAP) kinase being centrally involved [93].

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S. suis infection of HBMEC led to the production of IL-8 and MCP-1 by the endothelial cells in a time- and dose-dependent manner [50]. The presence of binding sites for MCP-1 and MIP-1_ on human brain microvessels [166] suggests that chemokines produced locally by perivascular astrocytes and microglia either diffuse or are transported to the endothelial cell surface, where they are immobilized for presentation to leukocytes [167], a process that has been demonstrated in peripheral endothelium with the chemokine IL-8 [168]. Following stimulation with LPS, TNF-_, IFN-a, and IL-1` alone or in combination, HBMEC released significant amounts of RANTES and MIP1` [169].

Cytokine release at the blood-CNS-barrier Various studies have demonstrated that BMECs are well capable of producing and secreting proinflammatory cytokines including IL-1_ and `, IL-6, and GM-CSF [167, 170, 171]. In a BBB in vitro model, infection of HBMEC with N. meningitidis resulted in the release of IL-6 by the endothelial cells [93]. Using the same BBB model, challenge of HBMEC with S. suis led, apart from secretion of chemokines, to the production of IL-6 as well [50]. An established example of brain microvascular endothelial activation during an infectious disease is the cerebral manifestation of malaria. IL-1` and TNF-_ are predominant cytokines released during the disease by the cerebral endothelium [172].

Macrophages along the blood-CNS barriers It has been known for some time that several subpopulations of resident macrophages are associated with the CNS. However, defining their role in microbial infection is difficult, as the number of morphological and functional studies is limited and few types of cells in neuroimmunology have prompted so much controversy as have the members of the monocyte lineage in the CNS [173]. In the pathogenesis of bacterial meningitis, these macrophages could act as sentinels at the interface between CNS and the circulation.

Blood-brain barrier Perivascular macrophages are a minor population in the CNS situated adjacent to endothelial cells immediately beyond the basement membrane of medium to small vessels [174]. They constitute a subpopulation of resident macrophages in the CNS that by virtue of their strategic location at

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the BBB potentially form a first line of defense against invading bacteria, and may play a role in the regulation of the inflammatory response during bacterial meningitis [175]. Recent studies on the putative function of these cells have used a rat model of pneumococcal meningitis with depletion of meningeal and perivascular macrophages by intraventricular injection of mannosylated clodronate liposomes [176]. This depletion aggravated clinical symptoms and resulted in higher bacterial titers both in the blood and the CSF. In addition, a decreased CSF pleocytosis despite elevated relevant chemokines (e.g., MIP-2), cytokines (e.g., IL-6) and a higher expression of vascular adhesion molecules (e.g.,VCAM-1) was observed [177].

Blood-CSF barrier Subpopulations of resident macrophages associated with the ventricular space comprise a family of specific histiocytes that constitute the epiplexus (“Kolmer”) cells and supraependymal cells apart from free-floating phagocytes [178]. Immunohistochemical studies on rat brains have revealed extensive populations predominantly on the ventricular side of the CP [179]. Very few functional observations have been made so far. LPS injected intraperitoneally in infant rats led to a vigorous up-regulation of complement receptor 3, leukocyte common antigens and major histocompatibility complex (MHC) classes I and II, suggesting an immunoregulatory role [180]. Upon injection of LPS and following in situ hybridization of rat brains, IL1_ and IL-1` as well as IL-1 receptor antagonist mRNA expression were noted primarily within the CPs and the CVOs. Interestingly, characterization of the cell types expressing IL-1 mRNA identified the cells as belonging to the monocyte/macrophage lineage [181]. In this respect, it is of note that recent observations confirmed the CPs to contain extensive populations of dendritic cells in rat and in humans [182], in the latter even bearing the potential of acting as a reservoir or port of entry for HIV-1 infection [183]. In a study using environmental scanning and confocal electron microscopy MHC class II-positive cells were found in abundance in the CPs of rat brains. The dendriform morphology and large size of these epiplexus/macrophage-like cells led to the assumption that these cells could indeed represent “real” dendritic cells ideally situated to sample CSF-borne antigens functioning as sentinels at the blood-CSF barrier [184].

Innate immunity The CNS orchestrates an organized innate immune response during systemic bacterial/viral infection. This inflammatory response, characterized

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by the expression of Toll-like receptors (TLRs), cytokines, chemokines and proteins of the complement system, is predominantly elicited in the CVOs and the CPs, i.e., structures that are devoid of the BBB and in close contact with the circulation environment. The inflammatory stimulus extends progressively into microglia across the brain parenchyma and may lead to an adaptive immune response [185]. Distinct TLRs have been proposed as key molecules in the selective recognition of main pathogen associated molecular patterns (PAMPs) that are released by either gram-negative (LPS) or gram-positive bacteria (peptidoglycan) [186]. In recent studies, murine challenge with LPS demonstrated a constitutive expression of both TLR4 and CD14, in structures that can be reached by the bloodstream: the CVOs, the CPs and the leptomeninges. These data provided the anatomical evidence that an exogenous ligand (LPS) has an endogenous receptor (CD14) in the brain in regions that can be reached by the systemic circulation. It has been proposed that this might allow intracellular signaling and then rapid transcription of pro-inflammatory cytokines, first within these organs and thereafter throughout the brain parenchyma in response to cell wall components of gram-negative bacteria [187]. In addition, this response could have been modulated by activation of TLR2 by peptidoglycan of gram-positive bacteria [188]. Challenge of human embryonic cell lines selectively overexpressing TLRs with live S. pneumoniae, Hib, and N. meningitidis showed that the bacteria use distinct sets of TLR2, -4 and -9 to trigger inflammatory responses. Heat-inactivated pneumococci or meningococci did not elicit comparable responses [189]. This is in line with observations in a murine model of experimental meningitis where TLR2 participated in sensing and activating the initial immune response to intracisternal challenge with S. pneumoniae. Nonetheless, other TLRs such as TLR4 were believed to be additionally involved [190]. Other observations on the interaction of BMEC and N. meningitidis point to TLR-independent mechanisms [16].

Restriction of microbial growth Besides confining entry of blood-borne pathogens into the CNS by means of tightly sealed cell-to-cell interfaces, HBMECs display distinct antimicrobial properties [191]. Experiments from our laboratory demonstrated that bacteria such as Staphylococcus aureus as well as intracellular parasites such as Toxoplasma gondii were restricted in their growth in HBMECs after stimulation with interferon-a [94, 192]. Activation of indoleamine 2,3dioxygenase (IDO) with subsequent degradation of the essential amino acid L-tryptophan has been found to be the principle antimicrobial mechanism. The in vivo relevance of this mechanism is emphasized by studies on patients suffering from bacterial meningitis [193]. For example, in children with purulent meningitis, concentrations of kynurenine, the primary meta-

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bolic product of tryptophan degradation, were more than 40 times higher than in healthy controls [194]. We have recently shown that IDO activation also accounts for growth restriction of S. suis in experiments with primary porcine CP epithelial cells after activation with proinflammatory cytokines [109]. The CP as a source of tryptophan degradation has been shown in an early study on the rabbit brain, where highest IDO activity could be demonstrated in the CP [195]. Teleologically, inducible tryptophan depletion by the brain endothelium would be particularly advantageous at the strategically important interface between blood and brain parenchyma. In the light of related antimicrobial action and IDO expression in neighboring cells in the CNS such as astrocytes, microglia and neurons [196], the cerebral microvasculature or the CPs could act in concert with them by collectively reducing tryptophan influx into the brain tissue, restricting the amount of tryptophan freely available to the pathogen. The blood-CNS interfaces thus not only seem to play a role as barriers against microbial penetration, but also once invasion has occurred.

Conclusion Many aspects of the pathophysiology of bacterial meningitis have been clarified in recent years. It has become clear that neuronal damage is not caused by the initial bacterial infection but results from host reactions to the invading pathogen. Nevertheless, important issues still need to be addressed and await further exploration to approach pharmacological options that supplement antibiotic treatment. Apart from ameliorations of therapeutic measures, broadening the focus on other blood-CNS barrier interfaces could offer new insights in pathophysiological events. In times where antibiotic resistance of microbial pathogens increases and new modalities in treating meningitis patients such as the application of dexamethasone drastically influence the plethora of cellular and molecular events, penetration of the blood-CNS barriers with suitable drugs might gain more attention.

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The molecular basis of paediatric malarial disease Ian A. Clark1 and Michael J. Griffiths2 1School

of Biochemistry and Molecular Biology, Australian National University, Canberra, Australia; 2Department of Paediatrics, Newcastle General Hospital, Newcastle upon Tyne, UK

Abstract Severe falciparum malaria is an acute systemic disease that can affect multiple organs, including those in which few parasites are found. The acute disease bears many similarities both clinically and, potentially, mechanistically, to the systemic diseases caused by bacteria, rickettsia, and viruses. Traditionally the morbidity and mortality associated with severe malarial disease has been explained in terms of mechanical obstruction to vascular flow by adherence to endothelium (termed sequestration) of erythrocytes containing mature-stage parasites. However, over the past few decades an alternative ‘cytokine theory of disease’ has also evolved, where malarial pathology is explained in terms of a balance between the pro- and anti-inflammatory cytokines. The final common pathway for this pro-inflammatory imbalance is believed to be a limitation in the supply and mitochondrial utilisation of energy to cells. Different patterns of ensuing energy depletion (both temporal and spatial) throughout the cells in the body present as different clinical syndromes. This chapter draws attention to the over-arching position that inflammatory cytokines are beginning to occupy in the pathogenesis of acute malaria and other acute infections. The influence of inflammatory cytokines on cellular function offers a molecular framework to explain the multiple clinical syndromes that are observed during acute malarial illness, and provides a fresh avenue of investigation for adjunct therapies to ameliorate the malarial disease process.

Introduction Although many species of malarial parasite exist, only Plasmodium falciparum, vivax, ovale, and malariae are classically associated with human infection. The former two species are most frequently associated with malarial disease in humans, with severe malarial disease almost exclusively associated with P. falciparum infection. Falciparum malaria is responsible for considerable morbidity (300–500 million annual clinical cases) and death across the globe, with a particular burden of mortality among children in sub-Saharan Africa. Infection with P. vivax is rarely fatal, but is associated with considerable morbidity outside the African continent. It should also be recalled that malaria causes social and economic disruption on a uniquely large scale [1].

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Severe adult malaria is a clinical syndrome originally classified using 10 defining and 5 supportive (often overlapping) clinical features unified by the presence of asexual malarial parasites in the peripheral blood smear [2]. Based on observations of children in coastal Kenya, paediatric severe malaria has similarly been distilled into three main (again often overlapping) clinical syndromes, anaemia, respiratory distress (an indicator of an underlying metabolic acidosis) and impairment of consciousness [3]. These clinical syndromes are discussed below. In the review mentioned above [3], the authors’ judicious use of the term impaired consciousness, rather than cerebral malaria (CM), promoted the useful concept that the neurological features (and in-turn the underlying mechanisms) associated with severe malaria are not necessarily unique to malarial disease. Indeed, over 60 years ago, it was noted that the clinical features of malaria can resemble those exhibited in patients with fulminant bacterial or viral infections [4]. Severe malaria has been intensively studied, and there appears to be a complex interplay between host infection and disease. This is highlighted by the different clinical manifestations of severe malaria exhibited by children and adults. These differences are undoubtedly, in part, a function of patient age. However, age is just one of a series of interacting factors, e.g. geographical region, level of malaria transmission, degree of previous malaria exposure, length of illness prior to treatment and host immunity that may influence the clinical presentation of severe malaria. This variation in clinical presentation has been mirrored by a similar multitude of proposals regarding the functional mechanisms underlying pathogenesis of severe malaria. One concept of pathogenesis consistently articulated has been the ‘mechanical theory’. Historically, this theory was developed from two fundamental differences between P. falciparum and P. vivax infection. Firstly, erythrocytes parasitised with P. vivax do not sequester. Secondly, death following P. vivax infection is rare. Consequently, pathogenesis is believed to be due to obstruction of micro-vascular flow by erythrocytes containing mature-stage falciparum parasites adhering to the endothelium (termed sequestration). More recently the ‘cytokine theory of disease’ has also gained credence. This theory can be applied to disease following both falciparum and vivax infection. The lower mortality associated with P. vivax being explained by a relatively milder degree of pro-inflammatory imbalance during the host’s response to P. vivax infection. The main theme of this chapter is to examine the increased understanding of the functions of inflammatory cytokines gained over the past 15 years, and explore how these insights are changing attitudes in malarial disease research. We also discuss how two theories (mechanical and cytokine) can, as proposed first in a recognisable form at least 65 years ago [5], be complementary.

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Table 1. Comparison of Kenyan children and Papua-New Guinea adults admitted to hospital using the WHO classification Kenyan children [7]

Adults Papua-New Guinea [19a ]

Prevalence

Mortality

Prevalence

Mortality

Coma*

10.0

16.8

17.1

41.7

Severe anemia**

17.6

4.7

10

0

Hypoglycaemia

13.2

21.7

5.7

75

Circulatory collapse

0.4

71.4

0

Renal Failure

0.1

0

22.9

37.5

Spontaneous bleeding

0.1

0

0.1

100

Haemoglobinaemia

0.1

50

0.1

Acidosis

63.6

21.4

Repeated convulsions

18.3

6.8

0.3

0

Defining criteria

Pulmonary oedema

Supporting criteria Impaired consciousness

8.2

6.0

37.1

11.5

Jaundice

4.7

11.9

45.7

25

Prostration

12.2

5.2

Hyperpyrexia

10.6

1.6

20

7.1

Hyperparasitaemia

8.9

4.3

40

28.6

* Childhood coma is defined by a Blantyre coma score * 2. ** The childhood definition for severe anemia does not include a cut off for parasitaemia. Modified from [11].

Severe malaria in children compared to adults The majority of the clinical cases of malaria occur in sub-Saharan Africa. Nevertheless, malaria also accounts for considerable morbidity and mortality in other continents particularly South East Asia [6]. In malaria-endemic regions (e.g. sub-Saharan Africa), where the resident population have continuous exposure to malarial parasites, most of the severe cases are seen in children [7]. In hypoendemic regions (e.g. South East Asia), where parasite exposure is more intermittent, cases of severe malaria are also common in adults (Tab. 1). Clinical features associated with malaria mortality vary between children and adults, but acidosis and coma are associated with malarial mortality in both populations [7, 8]. Acute renal failure (ARF) and pulmonary oedema,

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a marker for adult respiratory distress syndrome (ARDS), are almost exclusively reported among adults [9, 10], whereas mortality associated with hypoglycaemia is frequently reported among children [11]. Why malarial disease displays such age-related differences in pathophysiology is unclear. However, these differences are not exclusive to malaria. ARDS, which is more frequently observed as a complication of trauma in adults compared with children [12], is believed to reflect an exaggerated pro-inflammatory response within the lung [9]. A possible lead for future studies on these age-related differences in malaria is suggested by a report of peritoneal macrophages collected from healthy adults producing much less interleukin (IL)-10 (an anti-inflammatory cytokine), but the same levels of pro-inflammatory cytokine, than those from healthy children, giving adults a much higher pro-inflammatory status [13, 14]. The mechanism of malarial ARF pathogenesis is postulated to be multifactorial, involving mechanical, haemodynamic, and immunological factors [15]. The observation that ARF is more frequently observed as a complication of trauma in adults than children [12] suggests that age-related variations in cytokine response may again influence pathogenesis. Hypoglycaemia is regarded as a more frequent complication of sepsis in paediatric populations compared with adults [16]. Hypoglycaemia in children may, in part, be associated with a higher basal metabolic rate, and lower glycolytic [17] and gluconeogenic substrate reserves compared to adults [18]. However, these substrates are not always limiting during acute paediatric malaria, suggesting functional impairments of glucose metabolism may also occur [19]. Such functional impairments may, in part, be influenced by increases in inflammatory cytokines as the infection progresses.

How might P. falciparum cause this complex disease? Once the malarial parasite was identified as the cause of disease, it quickly became apparent that illness and death were linked with parasite invasion into bloodstream and subsequent parasite growth within (and release from) the erythrocytes. By the start of the 20th century, two major theories, capillary blockage and toxicity of the parasites themselves, had been proposed to explain morbidity and mortality. Thus, the study of malarial disease is not a settled story requiring regular updates, but one containing, from its beginning, an unresolved tension. Vascular occlusion and malarial toxin (nowadays vascular occlusion and inflammatory cytokines) have been alternative approaches to understanding malarial disease as a whole, as well as the coma, for over a century, and the two have often been discussed side by side [5, 20, 21]. The presence of hyperlactataemia, hypoglycaemia, and metabolic acidosis, all three consistent with a patient being forced to rely on anaerobic glycolysis for energy production, have provided a consensus that hypoxia is central to disease pathogenesis in falciparum malaria. As sum-

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marised below, the modern literature offers two main theories for cellular hypoxia during infection; insufficient oxygen delivery to cells and impaired oxygen utilization within the cells. Both mechanisms may be governed by the host inflammatory cytokine response to infection. This chapter focuses on how an increased understanding of the molecular functions of cytokines during disease demonstrates a closer alignment between the pathogenesis of falciparum infection and other systemic infectious diseases.

Inflammatory cytokines and malarial disease One hundred and twenty years ago, Golgi (of the Golgi apparatus [22]), noted onset of malarial fever and illness at a predictable short interval after the regular shower of new parasites were released from bursting red cells. The nature of the putative toxin so released was much discussed in the first decade of the 20th century [23]. It was assumed to be directly toxic, in the manner of tetanus toxin. The proposal that malarial products were not harmful in themselves, but only through causing the infected host to harm itself through generating toxic amounts of molecules (pro-inflammatory cytokines) that, in lower concentrations, inhibit growth of malarial parasites did not arise until 1981 [24]. Indeed, acceptance of the broad applicability of this concept to infectious disease in general is now sufficient for its evolution to be a subject for research [25]. Tumour necrosis factor (TNF) is regarded as a major player, malaria being the first disease in which it was proposed to cause systemic illness and pathology [24]. Multiple TNF promoter polymorphisms have since been independently associated with severe malaria across several geographical populations [26]. A longitudinal study in Burkino Faso has also demonstrated several TNF promoter polymorphisms associated with the regulation of host-parasite density [27]. The TNF concept has since begun to dominate the sepsis literature [28], and the virulence of different strains of influenza, a disease that is a standard clinical misdiagnosis for imported malaria, has recently been expressed in terms of their capacity to induce TNF [29]. The critical role of TNF in both malaria and influenza pathogenesis is consistent with the clinical similarities between the diseases. Indeed, TNF infusions in tumour patients produce side effects mimicking both diseases [30], as discussed below. Although TNF is the prototype pro-inflammatory cytokine linked with severe malaria, other cytokines (and mediators) including interferon (IFN)a [31], its corresponding receptors IFN-a receptor-1 [32] and IFN-_ receptor-1 [33], IL-1 [34], IL-4 [35] and IL-10 [36] have all be identified through genetic association analysis to be linked with their potential regulation of malarial disease severity. All the above cytokines typically act as homeostatic agents, but can cause pathology if produced excessively. When this happens they also induce a late-onset, but long-acting cytokine termed the high mobility

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Table 2. Some changes common to systemic inflammatory states, including falciparum malaria Cytokines

– TNF, IL-1, iNOS, IFN-a raised – MIF, IL-10, and HO-1 raised – a/b T cells increased – S100A8–S100A9 complex raised – Procalcitonin raised – S100A12 raised – HMGB1 raised – ICAM, VCAM and p-selectin raised

Consequences

– Insulin resistance – Hyperlactataemia – Hypertriglyceridaemia – Hypoglycaemia – Metabolic acidosis – Hyponatraemia – Coagulopathy – Thrombocytopaenia – Decreased red cell deformability

group box 1 (HMGB1) protein, which prolongs and amplifies inflammation [37, 38]. This molecule, normally in the cellular nucleus and previously known only for several physiological functions, now shows great promise as a therapeutic target in sepsis, in that countering it after the onset of illness protects well in experimental sepsis [39, 40]. It accumulates, in proportion to degree of illness, in serum from African children infected with falciparum malaria [41]. Once neutralising anti-TNF antibodies became available for human use, they were tested for efficacy against malarial disease. Unfortunately, a central tenet of the cytokine concept of infectious disease (that the proinflammatory cytokines that cause disease are the same mediators that, in lower concentrations, are responsible for the innate immunity that controls parasite growth) was not taken into consideration. TNF has been shown to inhibit a mouse malarial parasite in vivo [42], and P. falciparum in vitro, provided white cells to generate the next down-stream mediator, possibly nitric oxide (NO) [43], were present [44]. This is consistent with findings in human subjects [45]. Thus, it is not surprising that anti-TNF antibody, by removing inhibitory pressure from the pathogen, can enhance the disease in falciparum malaria [46], as shown 5 years earlier in human sepsis [47].

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Cytokines as a disease mechanism extends beyond malaria As noted above, the idea that excessive production of inflammatory cytokines underlies the pathology of illness is used widely, from malaria across a range of conditions, infectious or otherwise. As reviewed recently [48], this now includes the illnesses caused by rickettsias, protozoa other than malaria, and viruses. Increased circulating levels of these cytokines have been detected in the serum very soon after onset of illness in virtually all those infectious diseases in which they have been sought. Some cytokine increased, and consequences are shown in Table 2. When rTNF was under trial in volunteers as an anti-tumour agent [49, 50] nearly 20 years ago, virtually all of the symptoms and signs they share were reproduced as side effects. This includes headache, fever and rigours, nausea and vomiting, diarrhoea, anorexia, myalgia, thrombocytopaenia, immunosuppression, and central nervous system manifestations, all of which have been shown to be caused by a mechanism involving inflammatory cytokines. The rate, timing and intensity of cytokine release vary in different disease states, and provide them with somewhat individual clinical pictures, but the fundamentals remain. Nevertheless, the clinical patterns generated are remarkably close, in that, at least in some populations, clinical features cannot predict a diagnosis of malaria from other causes of fever [51].

Inflammatory cytokines acting indirectly to cause disease Vascular occlusion Mature erythrocytic forms of P. falciparum are not seen in peripheral blood smears, and cause the erythrocytes they inhabit to adhere to the walls of venules and capillaries. From this observation arose the widely held view that much of the pathology following malarial infection is explained through parasite sequestration causing impairment of microvascular flow. Sequestration certainly occurs, since the life cycle dictates this. However, whether the temporal and anatomical patterns of sequestration are the same in both individuals with fatal disease and in parasite tolerant individuals has not been ascertained. Consequently, whether sequestration is the principal instigator of local pathology, or whether sequestration is an associated feature of all malarial infections with local pathology determined by other factors in the host response to the infection, e.g. a local imbalance of inflammatory mediators, has not been fully elucidated. Erythrocyte cyto-adherence (irrespective of whether this adhesive process is directly or indirectly due to parasite sequestration) has repeatedly been shown to be mediated through a series of host-derived ligands. CD36 and thrombospondin were the first described endothelial receptors that bound infected red blood cells (RBCs) [52, 53], with most studied wild

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parasite isolates demonstrating adhesion to CD36 [54]. More recently, it has been shown that P. falciparum also interacts with other host adhesion receptors, i.e. intercellular adhesion molecule-1 (ICAM-1 CD54), vascular cell adhesion molecule-1 (VCAM-1 CD106) and E-selectin [55, 56]. Certain adhesive phenotypes, such as rosetting (the spontaneous tethering of infected and non-infected RBCs) and clumping (tethering of infected RBCs through platelets) have been preferentially associated with severe malarial disease [57, 58]. CD36 is involved in both mechanisms of adhesion, and a non-sense mutation in the gene encoding for CD36 has also been associated with protection from severe malaria [59]. Polymorphisms in the gene encoding ICAM-1 have also been associated with susceptibility to severe disease [27]. Furthermore, ICAM-1, together with VCAM and E-selectin, are up-regulated by TNF, with circulating levels of these ligands shown to be increased in severe malaria compared to uncomplicated infection [60]. Sequestration during falciparum malaria appears to be concentrated in the brain and placenta. There is some evidence to suggest that the propensity of inflammatory cytokines to up-regulate cell adhesion molecules, secondary to local variation in the density of thrombomodulin, is potentially higher in the microvasculature of the brain and placenta compared to other tissues. As reviewed [61], TNF and IL-1 increase tissue factor expression on endothelial cells, thereby initiating pathways that generate thrombin [62]. When thrombin binds to thrombomodulin on the endothelial cell surface, protein C is activated, which in turn can lead to further downstream activation of the coagulation cascade. Therefore vasculature with lowest thrombomodulin densities on the endothelial cell surface (brain least, placenta next least, and other organs more [63]) will have more unbound thrombin available for its other functions on activated endothelium. These other functions include up-regulation of adhesion molecules such as selectins, ICAM-1, VCAM-1 [64] and monocyte chemotactic protein-1 (MCP-1) [65]. Therefore, up-regulation of adhesion molecules within the cerebral vessels may occur as a local endothelial response to systemic inflammation and may not necessarily be precipitated by parasite sequestration.

Anaemia Anaemia is another obvious way in which too little oxygen reaches cells, and thus their mitochondria [66]. As recently reviewed [67], critical illness associated with an inflammatory response invariably causes multifactorial anaemia. Obviously a high parasite load in malaria indicates that the infected RBCs will soon burst when the next generation of erythrocytic forms escapes, but anaemia does not correlate with parasitaemia, and sometimes is extreme when very few parasites are, or have been, present. The severe anaemia in transgenic mice expressing human TNF [68] incriminates the

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inflammatory response itself, so anaemia and mitochondrial dysfunction (see Mitochondrial dysfunction section below), both consequences of systemic inflammation, can be expected to coexist, and both contribute to total energy depletion. Poor red cell membrane deformability The lifespan of an RBC is, in part, limited by how long it can remain flexible enough to squeeze through fenestrations in specialised vessels in the red pulp of the spleen, and thus avoid phagocytosis by adjacent macrophages. Normally this loss is balanced by erythropoiesis, and haematocrit remains normal. If RBCs develop a premature loss of deformability they are removed from the circulation earlier. This loss of deformability happens to both infected and non-infected red cells in malaria, whether caused by P. vivax or P. falciparum. Under physiological conditions, erythrocytes (and other cells) control the passive influx of osmotic active solutes (especially Na+) via an active, energy-dependent elimination of these solutes using Na+/K+-ATPase. This prevents intracellular accumulation of osmotically active solutes, preventing a subsequent influx of water, cell swelling and loss of cell integrity. During human [69] and monkey [70] malaria infection, intracellular Na+ accumulates within erythrocytes (both parasitised and non-parasitised) implying that this Na+/K+ pump is impaired during the disease process. Parallel changes in the ionic content of erythrocytes have been documented in a sepsis model of infection [71]. Similarly, reduction in erythrocyte deformability was shown to be associated with increased NO, an inhibitor of this membrane pump [72], in another sepsis model [73]. Since inhibition of the Na+/K+ pump in vitro correlates with both reduced red cell deformability and decreased red cell filterability [74], any factor that inhibits the Na+/K+ pump could potentially worsen anaemia. Identification of inducible NO synthase (iNOS) activity, as one factor influencing red cell deformability, suggests that a pro-inflammatory milieu [75] may again govern the reduction in red cell deformability observed during malaria infection. Originally observed in uraemic patients, poor red cell deformability was recognised in a small pilot study of malaria patients in 1985 [76]. It was reported soon afterwards in sepsis [77, 78], and subsequently studied in falciparum malaria with a view to understanding both circulatory obstruction [79] and anaemia [80]. It seems clear that a short life (poor deformability), and a slow replacement rate (dyserythropoiesis, below) can combine to cause severe anaemia in various diseases, particularly in chronic infections such as malaria. Dyserythropoiesis When red cells have a shortened lifespan, e.g. secondary to reduced erythrocyte deformability, replacement by new recruits is vital to avoid anaemia. Unfortunately, the same inflammatory cytokines that shorten lifespan also

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retard replacement. Some years ago researchers began to stress the contribution of bone marrow dyserythropoiesis to the anaemia of falciparum malaria [81, 82]. A group in Oxford [83], seeking an explanation for this dyserythropoiesis through an electron microscopy study of bone marrow, observed sequestration of parasitised red cells and argued that this caused the bone marrow dysfunction in falciparum malaria by restricting blood flow and thus inducing hypoxic changes. This idea proved inadequate, however, when this same group subsequently reported dyserythropoiesis and erythrophagocytosis in vivax malaria, in which parasitised red cells do not sequester [84]. Some time ago an undefined product in macrophage supernatants [85], later identified as TNF [86], was found to inhibit the growth and differentiation of erythroid progenitor cells. When rTNF became available, the dyserythropoiesis and erythrophagocytosis seen in terminal Plasmodium vinckei-infected mice was reproduced by giving a single injection early in the course of the infection [87]. Phagocytosis of erythroblasts in bone marrow, a phenomenon also reported by Wickramasinghe et al [83, 84] in human malaria, also occurred. Decreased erythropoiesis was subsequently reported in mice receiving continuous TNF infusions via implanted osmotic pumps, and mice expressing high levels of human TNF have been shown to become markedly anaemic during malaria infections [68], even though parasite numbers, and therefore red cell loss post-schizogony, are considerably reduced. The past decade has seen an expansion of this line of enquiry into human malaria, and also the number of cytokines, both pro-inflammatory and anti-inflammatory [88, 89] in absolute amounts and ratios [90, 91], that have been investigated in this context. Investigations have been extended to include other pro-inflammatory cytokines, such as IL-12 [92] and FasL [93], and examined the role in anaemia of the persistence of cytokine production during malaria infection [94]. Another inflammatory cytokine, macrophage inhibitory factor (MIF) that is increased in malaria, and induced by TNF, has been shown to cause dyserythropoiesis in in vitro studies on bone marrow cells [95, 96]. Thus, inflammatory cytokines generated during malaria are a major determinant of the degree to which anaemia influences the amount of oxygen that reaches tissues in malaria.

Inflammatory cytokines acting directly to cause disease Mitochondrial dysfunction Mitochondria are vital to energy (ATP) generation through cellular respiration. Cellular respiration requires oxygen and pyruvate, as well as multiple cofactors and active transport molecules. Within the matrix of the mitochondrion organelle, pyruvate is catabolised via the Krebs cycle and oxidative

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phosphorylation (involving NADH and FADH2) to generate ATP. When this series of reactions are 100% efficient (unlikely in vivo), 1 molecule of glucose generates 2 molecules of pyruvate, which are further catabolised to water and carbon dioxide with the concomitant generation of 36 molecules of ATP. In comparison, during anaerobic glucose catabolism, pyruvate is converted to lactate with the concomitant generation of 2 molecules of ATP, a process that also facilitates regeneration of NADH and FADH2. Evidence is accumulating that inflammatory cytokines, as released in malaria, sepsis, and viral diseases, induce mitochondrial dysfunction and dysregulate cellular respiration, resulting in the incomplete catabolism of pyruvate. The process, termed ‘cytopathic hypoxia’[97], mimics cellular hypoxia, in that it results in the incomplete catabolism of pyruvate and accumulation of lactate. Awareness of this mechanism began with oxygen tension being shown to be increased in septic rats [98] and patients [99]. A cytokine model of mitochondrial dysfunction has since been developed in which impairment of cellular respiration occurs following induction of sepsis (or exposure to pro-inflammatory cytokines), despite sufficient oxygen supply [97, 100, 101]. More recently, impairment of enzyme activity associated with the mitochondrial complexes has been demonstrated in muscle biopsies retrieved from rodent models of sepsis [102] and septic patients [103, 104]. The observation that the inflammatory cytokines implicated in mitochondrial shutdown are prominent in both sepsis and malaria [105, 106] supports such organelle dysfunction being equally plausible in malaria. Researchers are also becoming aware that, beyond energy production, mitochondria also play a vital role in cell homeostasis through generation and detoxification of reactive oxygen species [107]. The accelerated oxidative damage that accompanies sepsis could be both a cause and a consequence of cytokine-induced mitochondrial dysfunction. Interestingly, the ultrastructural damage reported to accompany mitochondrial dysfunction in sepsis [102] reflects Maegraith’s observations in monkey malaria [108– 110] decades ago.

Metabolic acidosis in falciparum malaria Metabolic acidosis, often associated with hyperlactataemia, has been described in African children with severe falciparum malaria [111, 112]. It is not unique to this disease, being seen in viral, rickettsial and bacterial infections [113] as well as acute gastroenteritis, where its prevalence is higher than in malaria [114]. The terms hyperlactataemia and lactic acidosis are often mistakenly used interchangeably in the malaria literature. As often reviewed in the basic literature [115–118], protons (H+, the basis of acidosis) are not formed when ATP and lactate are generated during glycolysis, but on the subsequent hydrolysis of ATP in tissues. Every time a molecule of ATP undergoes hydrolysis, a proton is released. If this occurs under aero-

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bic conditions, these protons are consumed within ATP regeneration from ADP, and pH remains normal, i.e. acidosis does not occur. In contrast, if the mitochondria are not functioning adequately, whether through insufficient oxygen supply or an inability to use it, ATP regenerates under anaerobic condition, and the protons are not consumed. Hence, once the buffering capacity of the body is exceeded, acidosis occurs. In short, metabolic acidosis requires the ratio of glycolytic (i.e. anaerobic) ATP hydrolysis to mitochondrial (i.e. aerobic) ATP hydrolysis to reach a point at which the buffering systems can no longer cope. Pathological changes in the buffering system can be a major determinant of when this occurs.

Is hyperlactataemia a cause or marker of the acidosis of malaria? High lactate levels have traditionally been seen not only as a marker for poor oxygen delivery in disease states, but also a consequence of it, and the cause of the acidosis. For some time hyperlactataemia has been regarded as a functionally relevant marker for a poor prognosis in both sepsis [119] and malaria [66, 112, 120]. Although the sepsis world now discusses several origins for the lactate increase, including inflammation-induced mitochondrial dysfunction [97], in falciparum malaria it is still generally attributed to a reduced oxygen supply, mostly through microvascular occlusion by sequestered parasitised erythrocytes [121]. Other mechanisms are known to contribute to acidosis in malaria, independent of lactate production, e.g. acute renal failure [8]. Impaired hepatic clearance [8, 112], production by parasites, and, in some areas, thiamine deficiency [122] are also argued to contribute to lactate accumulation independent of impaired cellular respiration. Thus, as described below, although acidosis and hyperlactataemia can be associated, they are independent cellular mechanisms. Lactate anion has complex roles in biology. Hyperlactataemia may be associated with acidosis, a normal pH, or alkalosis [123]. A recent editorial in Critical Care Medicine [124] has lucidly summarised the key points of the mechanism of metabolic acidosis in sepsis, a condition that shares systemic inflammation and a range of its consequences with severe malaria (Tab. 2). These authors argue against lactate as the cause of the acidosis associated with hypoxia. Instead, they note the evidence that during hypoxia, be it from limited oxygen supply or utilisation, the unconsumed protons that cause acidosis arise from the hydrolysis of non-mitochondrial ATP. Since these reactions are independent of lactate levels, it is difficult to see how therapeutically reducing levels of this anion, as has been proposed [125], could increase survival rate in falciparum malaria any more than in sepsis [126]. Indeed, in theory it could harm comatose patients, since there is evidence that lactate helps brain tissue survive hypoxic and hypoglycaemic episodes [127–129], and the lactate shuttle is proving to be how astrocytes protect neurons from metabolic stress [130].

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Even when considerable lactate is generated in acute inflammatory states, other, unidentified, anions contribute much more than it does to the strong ion difference that, through influencing the body’s buffering capacity, influences acidosis in sepsis [131, 132] and falciparum malaria [114, 133]. Thus, lactate accumulation can only partially account for the high anion gap observed during the metabolic acidosis associated with severe malaria. In summary, lactate is an imprecise but useful marker for metabolic acidosis in malaria. In turn, acidosis is an imprecise but useful marker of impaired cellular respiration. Whether impaired cellular respiration arises from (a) poor supply of oxygen to mitochondria (through vaso-occlusion, low circulating volume, anaemia or cardiac insufficiency) or impaired mitochondrial function (in response to severe systemic inflammation) the outcome is essentially the same. The resulting high anion gap metabolic acidosis is strongly predictive of death in severe malaria. Greater understanding of the multiple factors influencing the metabolic acidosis could provide further insight into the underlying pathophysiological process and may provide additional therapeutic options.

Hypoglycaemia in paediatric malaria When glycolysis is enhanced for any period glycogen stores are soon depleted, and gluconeogenesis supervenes. However, its substrate supplies are limiting [134], and the hypoglycaemia often reported in severe malaria [135] and sepsis [19, 136] occurs. Hypoglycaemia is therefore a secondary cause of harm in these diseases, and is an inevitable consequence of exuberant, mostly anaerobic, glycolysis.

Neurological involvement in malaria CM is a clinical syndrome characterised by coma (inability to localise a painful stimulus) at least 1 h after termination of a seizure or correction of hypoglycaemia, detection of asexual forms of P. falciparum malarial parasites on peripheral blood smears, and exclusion of other causes of encephalopathy [137].

Energy depletion and cerebral oedema A relatively consistent feature of acute CM in children is raised intracranial pressure (ICP). Studies in African children have demonstrated a raised cerebrospinal fluid (CSF) opening pressure during lumbar puncture in 80% of CM children [138], raised ICP during intracranial pressure monitoring

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(23/23 ICP > 10 mmHg) [139]and papilloedema (a late sign of raised ICP) in 44% of CM patients who died [140]. Where computer tomography has been performed, there was evidence of diffuse brain swelling in 40% of patients [139]. The cause of the raised ICP is likely to be multi-factorial and has been postulated to involve both vasogenic and cytotoxic patterns of cerebral oedema. Vasogenic oedema is characterised by accumulation of interstitial fluid within the brain secondary to increased permeability of the blood-brain barrier (BBB). It has been demonstrated in bacterial cerebral infections, but evidence of significant disruption of the BBB is not conclusive in CM [141]. Others have proposed that ICAM-1 binding by infected erythrocytes may generate a cascade of intracellular signalling events that disrupt the cytoskeletal-cell junction structure and cause focal disruption to the BBB [142]. Adult post-mortem analysis has shown cerebrovascular endothelial cell activation (increased ICAM-1 endothelial staining, reduction in cell junction staining, and disruption of junction proteins), particularly in vessels containing infected erythrocytes [143]. However, disruption of intercellular junctions is not associated with significant leakage of plasma proteins (fibrinogen, IgG, or C5b-9) into perivascular areas or CSF [143]. In Thai adults, transfer of radioactively labelled albumin into CSF was not raised during unconsciousness compared with convalescence [144]. Similarly, the albumin index (ratio of concentrations of albumin in CSF to those in blood) was not altered significantly in Vietnamese adults [145] or significantly different between Malawian children with CM who died and those who survived [143]. Cytotoxic oedema is increasingly being recognised as an important mechanism of cerebral oedema in traumatic brain injury [146]. As previously discussed, this type of cell swelling involves disturbance of the “pumpleak equilibrium” maintained, under physiological conditions via active elimination of osmotically active solutes through the energy-dependent Na+/K+-ATPase. Thus, cytotoxic oedema can occur secondary to an imbalance in supply and demand of energy within the cells. Several mechanisms, such as sustained increase in neuronal activation, impaired substrate delivery (structural and functional) and impaired mitochondrial utilisation of available substrates, including oxygen, may coexist to generate this imbalance. All these mechanisms could contribute to ATP depletion and Na+/K+ ATPase failure, leading to cytotoxic oedema in CM. CM is clearly associated with increased neuronal activity. A recent review identified that 80% of African children with CM have a history of seizures, with prolonged and recurrent seizures associated with a poor outcome [147]. Impaired vascular flow during acute CM may limit substrate delivery within the brain and contribute to energy imbalance. In the past, a common premise was that parasite sequestration precipitated cerebral vaso-occlusive/ischaemic (i.e. stroke-like) events that manifested clinically as CM. However, CM demonstrates several features that are atypical for stroke. In

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children, focal neurological signs do not tend to accompany coma, although a sub-set of patients do exhibit hemiparesis or focal brainstem deficits during the agonal period [148]. The incidence of residual neurological deficits following recovery from coma is relatively low (11% [147]) when compared to childhood stroke (93% had residual neurological deficit [149]). Where computer tomography has been performed in children, diffuse brain swelling was observed [150] rather than focal lesions more typical of stroke. Although retinal haemorrhages have been observed in 46% of Malawian children with CM (and in 63% of patients who died), these lesions were also seen in 30% of children with SMA in the same study [140]. Consequently, although associated with CM, retinal haemorrhages do not confirm that focal cerebral vaso-occlusive/ischaemic events underlie CM. Similarly, histological examination of 32 fatal CM cases of African children at autopsy demonstrated that one third had little or no evidence of local vascular change in the brain, as indicated by sequestered parasites, monocyte clusters, micro-haemorrhages, local vascular iNOS [151] or haemoxygenase -1 (HO1) [152] staining. Accepting that CM may occur without ischaemia does not exclude temporary or less severe reductions in vessel flow occurring during acute CM (associated or independent of parasite sequestration) that may contribute to impaired substrate delivery and lead to energy imbalance. As previously discussed, energy imbalance may also be impaired due to the uncoupling action of inflammatory cytokines on mitochondrial ATP production. In Gambian and Ghanaian children, concentrations of TNF and its receptor were higher in those with CM than in those with mild or uncomplicated malaria [153, 154]. Polymorphisms in the TNF promoter region have also been associated with increased risk of CM and death [155] or neurological sequelae [156]. Cytokines may also up-regulate iNOS in brain endothelial cells, increasing production of NO, which could then diffuse into brain tissue and disrupt neuronal (and/or mitochondrial) function [157, 158]. In the brain, mitochondrial function may also be influenced by neuronal excitotoxins. Within the simplified model of dissociated neuronal culture, mitochondria appear to play a critical role in neuronal homeostasis during excitotoxin exposure. Mitochondria are not only involved with maintaining ATP production but also calcium homeostasis, and generation and detoxification of reactive oxygen species [107]. Excitotoxin production may also be influenced by cytokine release. TNF administration has been shown to alter brain metabolism of tryptophan to produce more kynurinine [159, 160]. Thus, as part of a general inflammatory reaction, increased excitotoxin generation during acute malaria may contribute to cellular energy imbalance. Elevated levels of neuronal excitotoxins (quinolinic and picolinic acid) in the CSF have been associated with a fatal outcome in Malawian children with CM [161]. Similarly, a graded increment of quinolinic acid concentration in CSF was observed across patient outcome groups of increasing severity in African children [162].

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Encephalopathy with systemic inflammation but without sequestration Although a subset of the Malawaian autopsy patients [163] demonstrated negligible histological change in their brains, they did demonstrate inflammation, as indicated by iNOS, MIF [151] and HO-1 [152], staining in other tissues. These systemic changes were shared with the comatose sepsis cases in the study, and therefore are consistent with the premise that coma may in part be secondary to a host inflammatory response to systemic infection. Below are further examples of systemic responses to infection that present with diffuse cerebral syndromes, including coma.

Cerebral malaria manifesting with P. vivax infection In the past, the term CM has been restricted to falciparum malaria, and patients with P. vivax infection exhibiting symptoms of severe malaria, including coma, have been dismissed as undiagnosed falciparum co-infections. However, the use of more sensitive diagnostic techniques makes such dismissal less tenable. Two such studies report adults exhibiting severe malaria with P. vivax (but not P. falciparum) infection detectable on PCR and serological and testing [142, 143]. The patients exhibited multiple organ failure including cerebral symptoms, renal failure, circulatory collapse, severe anaemia, haemoglobinuria, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Vivax malaria has been associated with a strong systemic inflammatory response [164], but this was not investigated in the above studies.

Sepsis-associated encephalopathy Sepsis-associated encephalopathy (SAE) syndrome has multiple features that resemble CM. It is characterised by a diffuse disturbance of cerebral function (typically impairment of consciousness) that occurs in the context of systemic response to infection without direct neuroinvasion (i.e. meningitis, macroscopic cerebritis and brain abscesses are excluded). SAE is associated with generalised slow waves on the electroencephalogram (EEG), with the depth of coma linked with mortality. Mild SAE cases often recover completely, while survivors of severe SAE may have persistent neurological deficit [165]). In line with adult CM, the severity of encephalopathy parallels the severity of systemic organ failure [141]. Inflammatory cytokines have been demonstrated to be higher in the serum than in the CSF, suggesting that sepsis encephalopathy is a consequence of the systemic inflammatory response to infection [141]. An animal model in which prior administration of a neutralising antibody to TNF prevented the sepsis encephalopathy of pancreatitis [166] is consistent with this. Further postulated reversible

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Table 3. Influenza encephalopathy

Cerebral malaria

Seizures/coma after high grade fever

+

+

Metabolic acidosis

+

+

Hyperlactataemia

+

+

Serum TNF, IL-6, sTNFRI up

+

+

Serum nitrite/nitrate up

+

+

CSF TNF, IL-6, sTNFRI up

+

+

Multiple organ failure

+

+

Residual neurological deficit

+

+

Thrombocytopaenia

+

+

Damage to vascular endothelial cells

+

+

Brain oedema/damage to BBB

+

+

Apoptosis in neurons/glial cells

+

+

Evidence of active caspase-3 (brains)

+

+

Caspase-cleaved PARP (brains

+

+

mechanisms of pathogenesis include changes in regional cerebral blood flow, neurotransmitter imbalance, mitochondrial dysfunction, BBB impairment and oxidative stress [167].

Influenza encephalopathy Severe influenza infection can present with encephalopathy, yet as in malaria, the pathogen is not neuroinvasive [168]. Seizures and coma occur after high fever [169], commonly accompanied by thrombocytopaenia [169], with metabolic acidosis and hyperlactataemia in severe cases (T. Ichiyama, personal communication). Similar to adult malaria, neurological sequelae occur concurrently with multiple organ failure [170]. TNF, IL-6, sTNFRI, and soluble E-selectin are increased in serum and CSF [171, 172], and serum nitrite/nitrate levels are increased [173]. Detailed examination of brain has revealed apoptosis of neurons and glial cell, histological evidence of active caspase-3 and caspase-cleaved PARP, cerebral oedema, and BBB impairment [174]. These parallel changes are set out in Table 3. It is clear, therefore, that the presence of sequestering parasitised red cells is not necessary to generate these changes, which are also demonstrable in the falciparum malaria encephalopathy. Notably, high levels of inflammatory cytokine are present in each disease.

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Seizures and malaria Seizures are a very common component of acute malaria illness in children. A recent review documented that 80% of African children had a history of seizures, with 60% exhibiting seizures during hospital admission [175]. The molecular basis of the seizures is unclear. Multiple mechanisms have been postulated, including fever, hypoxia and/or cytokine stimulation leading to an imbalance of neurotransmitters and excitotoxins or neuronal damage [11, 148]. Recently, Lang and co-workers [176] demonstrated that falciparum parasitaemia is associated with the generation of specific antibodies for voltage-gated calcium channels directed against neurones. Higher antibody concentrations were detectable in sera from patients exhibiting CM or malaria with seizures than uncomplicated malaria, suggesting that these antibodies may influence seizure propensity.

Red cell abnormalities and malaria Only the erythrocytic form of malaria is associated with disease, so valuable information about which African children are likely to have more, or less, severe malaria has inevitably been obtained from examining the inborn RBC abnormalities that endemic malaria has selected across the tropics. The coinciding geographic distributions of malaria transmission and the thalassaemias prompted Haldane to put forward the ‘malaria hypothesis’, which proposed that common erythrocyte abnormalities are selected because of the fitness advantage they confer against malaria [177]. Sickle cell haemoglobin (HbS) has also been repeatedly shown to be associated with malaria resistance, with heterozygotes for the HbS trait demonstrating 10% of the population at risk for severe malaria in certain populations [178]. Other haemoglobinopathies (e.g. HbC [179, 180] and HbE [181]) and deficiencies in RBC enzymes (e.g. glucose-6-phosphate dehydrogenase deficiency [182]) have also been linked with protection against severe malaria. The mechanisms of protection afforded by haemoglobinopathies are likely to be multi-factorial. Studies have demonstrated evidence to support several independent mechanisms including: reduced parasite invasion of RBCs and diminished intraerythrocytic growth of parasites in patients with the HbS trait [183], enhanced phagocytosis of parasite-infected erythrocytes (IEs) [184] and enhanced immune responses against IEs [185]. Recent in vitro studies observed that HbC modifies the quantity and distribution of the variant antigen P. falciparum erythrocyte membrane protein 1(PfEMP1) on the IE surface. PfEMP1 has been implicated in numerous IE adhesive interactions. In the latter study the authors demonstrated that HbC reduces the level of IE adhesion to endothelial monolayers, in addition to IE rosetting (the adhesion of IEs to uninfected erythrocytes) and IE agglutination by sera. These findings provide the prospect that HbC pro-

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tects against severe malaria by mitigating the obstruction and inflammation caused by the PfEMP1-mediated adherence of IEs [186]. However, sequestration is believed to enhance parasite survival by enabling IEs to avoid splenic clearance, so any reduction of sequestration by HbC can be expected to limit parasite fitness. Multiple epidemiology studies (e.g. [179, 187, 188]) have failed to identify any significant impact of HbC on the frequency or density of parasitaemia in naturally exposed populations. Consequently, the influence of the changes in IE surface conformation needs to be confirmed and further examined in vivo [189]. A recent study re-confirmed that African children with _-thalassaemia trait are significantly less likely to be hospitalised with severe malaria, particularly with coma or severe anaemia (Hb < 5 g/100 ml). It is intriguing that the _-thalassaemia patients did not demonstrate a lower incidence of uncomplicated malaria nor any reduction in peripheral parasite density [190]. Thalassaemia has also been associated with increased incidence of clinical vivax and falciparum malaria during early life [191]. The findings raise speculation that the trait may indirectly afford enhanced immunity through increased non-lethal exposure to malarial parasites. Such a mechanism is appealing, since it would be equally plausible across a range of haemoglobinopathies, including HbC. Variations in erythrocyte membrane proteins also have a profound influence on malaria susceptibility. Most notably the absence of Duffy antigen protein confers absolute protection to P. vivax infection. More recently, the Duffy antigen has also been associated with a protection against falciparum malaria [192]. Enzymes involved with iron handling may also have a critical influence on malaria morbidity. A recent study from the Gambia demonstrated that children in an endemic malaria area possessing the haptoglobin 2,2, isotype had a significantly increased risk of anaemia [193]. However, a lack of parallel alterations in other haematinic indices leaves the mechanism of this process unclear. Malarial protection within individuals exhibiting multiple RBC abnormalities appears even more complex. A recent study observed that the concurrent presence of sickle cell and _-thalassaemia trait among African children had a negative influence on the risk of malaria infection [194]. The results warn geneticists that gene epistasis may have a profound influence on overall malarial susceptibility.

Potential therapies directed at disease mechanisms In tropical countries many hospital deaths from falciparum malaria happen before anti-malarial drugs have had time to kill the parasites. Two approaches could help rectify this – addressing public-health problems resulting in delayed presentation, and identifying the physiological processes and

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molecular pathways that lead to these early deaths, with a view to developing evidence-based adjunct therapies. Therapies being explored in sepsis, and based on disease pathogenesis data common to sepsis and malaria, may prove to be transferable from either of these diseases to the other. As noted above, circulating levels of a late-appearing inflammatory cytokine, HMGB1, are increased in falciparum malaria [41] as well as in sepsis. Results from animal models on the role of HMGB1, although untested in humans, have inspired enthusiasm for inhibition of this molecule as a potential intervention for human sepsis. For instance, anti-HMGB1 antibodies provided dose-dependent protection [37] and reduced mortality [195] against experimental sepsis in mice. Late administration of ethyl pyruvate, which inhibits HMGB1 release from macrophages, also conferred protection against endotoxaemia in mice [196]. Treatments directed towards critical downstream consequences of malaria infection and inflammation, such as those intended to limit acidosis, are also a focus of investigation. One current approach is to identify which acute malaria patients most benefit from early volume expansion [197]. Controlling lactic acidosis via sodium dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenate kinase (maintaining pyruvate dehydrogenase in its active form), is also being examined. DCA reduced lactate levels in acute malaria patients [198], although the study was unable to determine whether treatment improved outcome. An earlier large sepsis study also demonstrated that DCA reduced lactate, but again with no improvement in outcome [126]. As outlined in the section ‘Is hyperlactataemia a cause or marker of the acidosis of malaria?’, some researchers argue, in view of the strong ion difference contributing to acidosis and the postulated mitochondrial dysfunction during acute malaria infection, that lactate reduction per se may have limited impact on prognosis. Other adjunct therapies are also being examined. Improving RBC deformability provides one potential therapeutic approach. In vitro studies with N-acetylcysteine (NAC), reported to scavenge free radicals, showed improvement in red cell deformability through in vitro studies [199]. Unfortunately, an initial in vivo trial of NAC in malaria patients had no effect on mortality [200]. Blocking endothelial activation is also a focus of research, with initial in vitro studies providing some encouraging results [201]. In conclusion, continuing to identify the host responses to malaria infection that lead to disease is providing insights into novel molecular mechanisms. This information is beginning to guide the design of much needed additional therapies against this disease. There is little doubt that poor oxygen supply through vascular occlusion or anaemia could contribute to the body relying on excessive glycolysis to generate energy, resulting in hyperlactataemia, hypoglycaemia, and metabolic acidosis, and altered consciousness. However, inflammatory cytokines control these changes, as well as inhibit the capacity of mitochondria to use oxygen. Thus, as described

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Figure 1. The wide-ranging influences of inflammatory cytokines in severe malaria.

throughout this review, inflammatory cytokines are likely to have various pivotal roles across the multiple pathological processes involved in malarial disease (Fig. 1).

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Olivieri A, Calissano C, Paganotti GM et al (2001) Haemoglobin C protects against clinical Plasmodium falciparum malaria. Nature 414: 305–308 Hutagalung R, Wilairatana P, Looareesuwan S, Brittenham GM, Aikawa H, Gordeuk VR (1999) Influence of hemoglobin E trait on the severity of falciparum malaria. J Infect Dis 179: 283–286 Ruwende C, Khoo SC, Snow AW, Yates SNR, Kwiatkowski D, Gupta S, Warn P, Allsopp CEM, Gilbert SC, Peschu N et al (1995) Natural selection of hemi- and heterozygotes for G6PD deficiency in Africa by resistance to severe malaria. Nature 376: 246–249 Pasvol G, Weatherall DJ, Wilson RJ (1978) Cellular mechanism for the protective effect of haemoglobin S against P. falciparum malaria. Nature 274: 701–703 Cappadoro M, Giribaldi G, Obrien E, Turrini F, Mannu F, Ulliers D, Simula G, Luzzatto L, Arese P (1998) Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency. Blood 92: 2527–2534 Abu-Zeid YA, Abdulhadi NH, Hviid L, Theander TG, Saeed BO, Jepsen S, Jensen JB, Bayoumi RA (1991) Lymphoproliferative responses to Plasmodium falciparum antigens in children with and without the sickle cell trait. Scand J Immunol 34: 237–242 Fairhurst RM, Baruch DI, Brittain NJ, Ostera GR, Wallach JS, Hoang HL, Hayton K, Guindo A, Makobongo MO, Schwartz OM et al (2005) Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 435: 1117–1121 Ringelhann B, Hathorn MK, Jilly P, Grant F, Parniczky G (1976) A new look at the protection of hemoglobin AS and AC genotypes against Plasmodium falciparum infection: a census tract approach. Am J Hum Genet 28: 270–279 Mockenhaupt FP, Ehrhardt S, Cramer JP, Otchwemah RN, Anemana SD, Goltz K, Mylius F, Dietz E, Eggelte TA, Bienzle U (2004) Hemoglobin C and resistance to severe malaria in Ghanaian children. J Infect Dis 190: 1006–1009 Duffy PE, Fried M (2006) Red blood cells that do and red blood cells that don’t: how to resist a persistent parasite. Trends Parasitol 22: 99–101 Wambua S, Mwangi TW, Kortok M, Uyoga SM, Macharia AW, Mwacharo JK, Weatherall DJ, Snow RW, Marsh K, Williams TN (2006) The effect of alpha+thalassaemia on the incidence of malaria and other diseases in children living on the coast of Kenya. PLoS Med 3: e158 Williams TN, Maitland K, Bennett S, Ganczakowski M, Peto TEA, Newbold CI, Bowden DK, Weatherall DJ, Clegg JB (1996) High incidence of malaria in alpha-thalassaemic children. Nature 383: 522–525 Oguariri RM, Borrmann S, Klinkert MQ, Kremsner PG, Kun JFJ (2001) High prevalence of human antibodies to recombinant Duffy binding-like alpha domains of the Plasmodium falciparum-infected erythrocyte membrane protein 1 in semi-immune adults compared to that in non-immune children. Infect Immun 69: 7603–7609 Atkinson SH, Rockett K, Sirugo G, Bejon PA, Fulford A, O’Connell MA, Bailey R, Kwiatkowski DP, Prentice AM (2006) Seasonal childhood anaemia

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in West Africa is associated with the haptoglobin 2–2 genotype. PLoS Med 3: e172 Williams TN, Mwangi TW, Wambua S, Peto TEA, Weatherall DJ, Gupta S, Recker M, Penman BS, Uyoga S, Macharia A et al (2005) Negative epistasis between the malaria-protective effects of alpha(+)-thalassemia and the sickle cell trait. Nat Genet 37: 1253–1257 Yang H, Ochani M, Li J, Qiang X, Tanovic M, Harris HE, Susarla SM, Ulloa L, Wang H, DiRaimo R et al (2004) Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci USA 101: 296–301 Ulloa L, Ochani M, Yang H, Tanovic M, Halperin D, Yang R, Czura CJ, Fink MP, Tracey KJ (2002) Ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation. Proc Natl Acad Sci USA 99: 12351–12356 Maitland K, Pamba A, English M, Peshu N, Marsh K, Newton C, Levin M (2005) Randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit. Clin Infect Dis 40: 538–545 Krishna S, Agbenyega T, Angus BJ, Beduaddo G, Oforiamanfo G, Henderson G, Szwandt ISF, Obrien R, Stacpoole PW (1995) Pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria. Q J Med 88: 341–349 Dondorp AM, Omodeo Sale F, Chotivanich K, Taramelli D, White NJ (2003) Oxidative stress and rheology in severe malaria. Redox Rep 8: 292–294 Watt G, Jongsakul K, Ruangvirayuth R (2002) A pilot study of N-acetylcysteine as adjunctive therapy for severe malaria. Q J Med 95: 285–290 Wassmer SC, Cianciolo GJ, Combes V, Grau GE (2005) Inhibition of endothelial activation: a new way to treat cerebral malaria? PLoS Med 2: 885–890

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Epidemiology and etiology of Kawasaki disease Wilbert Mason Los Angeles Children’s Hospital, 4650 Sunset Boulevard, Los Angeles, California 90027, USA

Abstract Kawasaki disease was first reported in Japan in 1967 by Dr. Tomisaku Kawasaki. It has since been recognized worldwide, and in at the United States and Japan is the most important cause of acquired heart disease in children, surpassing other more recognized conditions such as rheumatic fever, endocarditis and myocarditis. It is primarily a disease of children less than 5 years of age but has been reported in older children and adults. Risk factors for the illness include Asian ancestry, male gender and certain familial predispositions. Observations such as similarity to certain exanthematous infectious diseases, temporal-geographic clustering of cases and seasonality in incidence favors an infectious etiology. Pathology and pathogenesis of the disease indicate that it is a medium-sized artery vasculitis that results from a dramatic immune activation that in most cases reversed by immune modulating agents such as intravenous immunoglobulin. Unfortunately, the etiology of the illness remains obscure, although recent studies favor a possible viral etiology.

Introduction Doctor Tomisaku Kawasaki first described Kawasaki disease (KD) in 1967 based on 50 cases he had observed over the preceding 6 years at the Tokyo Red Cross Hospital [1]. He termed the illness mucocutaneous lymph node syndrome because of characteristic changes of the mucous membranes and skin, which seemed to characterize the illness. During the first few years following its description, it appeared to be a self-limited disease without sequelae. However, following the first nationwide survey of the illness in Japan in 1970, sudden death due to coronary artery disease was firmly linked to the illness [2]. KD was independently described in the United States in 1974 by Melish and colleagues [3], and following consultation with Dr. Kawasaki, there was agreement that the illnesses were clinically the same. Following the recognition of cardiac complications in both Japan and the United States, pathologists in both countries observed similarities

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between coronary artery lesions seen in KD patients and those in patients who had died of infantile periarteritis nodosa (IPN), a rare vasculitis of infancy [3]. The question arose as to whether the two were the same disease. The issue was resolved by Landing and Larson in 1976 [4], who performed blinded evaluations of autopsy cases of children from both Japan and the United States who had died with a diagnosis of KD and IPN. They found that the two illnesses were pathologically indistinguishable. In subsequent years KD has been recognized worldwide, and in all age groups, although 85% of cases occur in children < 5 years of age. It is now recognized as the most common cause of acquired heart disease in children in the United States. We attempt here to describe the current understanding of the epidemiology of KD and the most recent findings regarding its pathogenesis and etiology.

Diagnostic criteria and diagnostic approach The diagnostic criteria described by Dr. Kawasaki have been used, with some modification, since the original description of the disease [5] (Tab. 1). Children with four or more principal criteria and at least 4 days of fever can be diagnosed on day 4. If fewer than four principal criteria are observed, KD may be diagnosed with the appearance of coronary artery abnormalities (CAA). With increasing experience, it became apparent that a significant minority of infants and children were not identified by the classic diagnostic criteria. This was especially true for infants < 6 months of age who often presented with less than the required criteria in what became known as “atypical” or more properly “incomplete” KD. The most recent guidelines have included an algorithm for the evaluation of suspected incomplete KD that incorporates refined clinical assessment, laboratory tests and echocardiographic results into the diagnostic equation [5] (Fig. 1).

Epidemiology While reports of the occurrence of KD have come from every continent (not including Antarctica), most epidemiological data comes from Japan and the United States and Canada with increasing reports coming from Taiwan, China and Korea in recent years (Tab. 2).

Japan Since 1970, a total of 17 retrospective incidence surveys have been conducted in Japan (i.e., every 2 years) under the auspices of the Ministry of

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Table 1. Clinical and laboratory features of Kawasaki disease Epidemiological case definition (classic clinical criteria)* Fever persisting at least 5 days Presence of at least 4 principal features:† Changes in extremities Acute: Erythema of palms, soles; edema of hands, feet Subacute: Periungual peeling of fingers, toes in weeks 2 and 3 Polymorphous exanthem Bilateral bulbar conjunctival injection without exudates Changes in lips and oral cavity: Erythema, lips cracking, strawberry tongue, diffuse injection of oral and pharyngeal mucosae Cervical lymphadenopathy (> 1.5 cm diameter), usually unilateral Exclusion of other diseases with similar findings * Patients with fever at least 5 days and < 4 principal criteria can be diagnosed with Kawasaki disease (KD) when coronary artery abnormalities detected by 2-D echocardiography or angiography are present. † In presence of * 4 principal criteria, KD diagnosis can be made on day 4 of illness. Experienced clinicians who have treated many KD patients may establish diagnosis before day 4.

Table 2. Global distribution of KD beyond Japan and North America Europe

England Ireland Sweden Finland Germany France Portugal Italy

Asia

China Bejing Taiwan Shanghai Hong Kong Korea Thailand India Iran Oman

Africa

Nigeria South Africa Egypt Senegal Tunisia Sudan

South America

Argentina Brazil Chile

Oceania

Australia New Zealand

Health, Labor and Welfare. Questionnaires were sent to hospitals with pediatric departments and a bed capacity of at least 100, or hospitals with a bed capacity of less than 100 beds but specializing in pediatrics. The survey questions were created by the Japan Kawasaki Disease Research Committee. Response to the surveys has been about 70% [6]. The last reported surveys included the years 1999–2002 [6]. Since the inception of the epidemiological study 186 069 KD patients have been reported.

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Figure 1. Evaluation of suspected incomplete Kawasaki disease (KD). In the absence of gold standard for diagnosis, this algorithm cannot be evidence-based, but rather represents the informed opinion of the expert committee. Consultation with an expert should be sought anytime assistance is needed. (1) Infants ) 6 months old on day * 7 of fever without other explanation should undergo laboratory testing and, if evidence of systemic inflammation is found, an echocardiogram, even if the infants have no clinical criteria. (2) Patient characteristics suggesting KD are listed in Table 1. Characteristics suggesting diseases other than KD include exudative conjunctivitis, exudative pharyngitis, discrete intraoral lesions, bullous or vesicular rash, or generalized adenopathy. Consider alternative diagnoses. (3) Supplemental laboratory criteria include albumin ) 3.0 g/100 ml, anemia for age, elevation of alanine aminotransferase, platelets after 7 days * 450 000/mm3, white blood cell count * 15 000/mm3, and urine * 10 white blood cells/high-power field. (4) Can treat before performing echocardiogram. (5) Typical peeling begins under nail bed of fingers and then toes. (6) Echocardiogram is considered positive for purposes of this algorithm if any of three conditions are met: z score of LAD or RCA * 2.5, coronary arteries meet Japanese Ministry of Health criteria for aneurysms, or * 3 other suggestive features exist, including perivascular brightness, lack of tapering, decreased LV function, mitral regurgitation, pericardial effusion, or z scores in LAD or RCA of 2–2.5. (7) If the echocardiogram is positive, treatment should be given to children within 10 d of fever onset and those beyond day 10 with clinical and laboratory signs (CRP, ESR) of ongoing inflammation. Taken from [5]. Copyright © 2004 American Heart Association.

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During the most recent study period, 32 266 patients were reported with an annual incidence of 137.7 per 100 000 children < 5 years old in 1999 and 151.2 per 100 000 in 2002. The male to female ratio was 1.30 [6]. The annual incidence of KD in Japan has increased progressively from 1987 to 2002 from 73.8 to 151.2 per 100 000 < 5 years of age [6–10]. Over the 32-year period of surveillance, three nationwide epidemics of KD have been observed, in 1979, 1982 and 1986 [11]. The incidence rate in the last epidemic in 1986 was 176.8 per 100 000 children < 5 years of age. No national epidemic outbreaks have been reported since 1987 but regional outbreaks continue to occur.

United States Surveillance of KD in the United States is through passive reporting of cases to the Centers for Disease Control and Prevention, where a database has been maintained since 1984. Unfortunately, only a fraction of cases are identified through this system. More robust estimates of the incidence of KD has come from reports from regional investigators [12–14], surveys conducted by specialty societies and more recently through the use of administrative databases [15] of childhood hospitalizations. Taubert [16] conducted surveys of 440 general hospitals with at least 400 beds that included a pediatric section and of 63 children’s hospitals. The survey periods covered the years 1984–1987, 1988–1990 and 1991–1993. During the latter period only the children’s hospitals were surveyed. The surveys yielded rates of 7.6 cases per 100 000 children < 5 years of age and 9.2 cases per 100 000 for the first two periods. Following the third survey, the reported cases throughout the 10-year period were totaled and a minimum estimate of the annual rate for the period was calculated which was 8.9 cases per 100 000 children < 5 years of age. This rate was similar to rates found previously reported in regional studies [13, 14]. More recently, investigators from the Centers for Disease Control and Prevention utilized data from a large inpatient database to determine incidence rates in the United States. The database was designed to generate robust national estimates of pediatric hospitalizations [15]. The rates were determined for the years 1997 and 2000, which were 17.6 per 100 000 < 5 years of age and 17.1 per 100 000 < 5 years, respectively. These rates were comparable to those determined from the regional studies using State health or health maintenance organization data [18–20]. Based on data from this study and others published previously during the decade, the authors concluded that incidence rate for KD in the United States had been stable. A national epidemic of KD involving ten regions in the United States occurred between August 1984 and January 1985 [21]. Several other regional outbreaks have been reported over the years as well [16]. Similar incidence rates were reported from Canada based on a national health statistic data-

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base. From 1990–1991 to 1995–1996 the mean rates for children < 5 years across Canada were 13.8 per 100 000 children [22].

Global distribution KD has been reported from every continent and several island groups across the globe (Tab. 2). While the incidence rates in Japan remain the highest in the world, several other Asian nations have posted high rates as well. Several reports from China (Beijing [23], Hong Kong [24], Shanghai [25]), Taiwan [26] and Korea [27] documented rates intermediate between those of Japan and North America (Tab. 3). All but Taiwan appeared to have increasing rates over the study periods. Case reports or case series have been reported from many countries in Europe, Oceania, Africa and South Africa as well as other Asian countries (Tab. 2). Of those countries reporting incidence rates, only those from Ireland are comparable to those in North America [28]. To summarize the global experience with KD, the highest incidence rates are found in Japan followed by Korea, China, North America and Europe. Local or regional outbreaks have been documented in both Japan and the United States, and national epidemics have been observed in both countries as well as Finland [29]. Incidence rates have trended upward in several countries and have remained stable in others. The effect of ascertainment bias on apparent increases in incidence is not known.

Race As suggest by higher incidence rates in Asian countries, KD occurs in higher frequency in Asian populations. Numerous studies from the Unites States have shown KD to be over-represented among Asian children [12, 14–16, 18, 19]. An interesting study of the epidemiology of KD in Hawaii dramatically demonstrated this predominance [30]. A retrospective analysis of the State Inpatient Database for Hawaii was performed for KD patients hospitalized during 1996 through 2001. Race classification provided by Census 2000 indicated race listed alone or in combination with other races. This race-specific numerators and denominators could be determined. The average annual incidence for KD was 45.2 per 100 000 children < 5 years of age, highest in the United States. Japanese-American children < 5 years had the highest incidence (197.7 per 100 000) followed by Native Hawaiian (99.1), Chinese (81.3) and Filipino (64.8). Caucasian children < 5 years old had a rate of 35.3 per 100 000 children. These findings suggest there may be true differences in incidence of KD among Asian populations. Since the populations in Hawaii came from relatively similar social and physical environments and have similar access to healthcare and diagnostic practices,

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Table 3. Incidence rate of KD in different areas Incidence rate Location

Surveillance period

Overall

Range

Beijing [23]

1995–1999

22.9

18.2–30.6

Taiwan [26]

1996–2002

66.0

59.0–76.0

Hong Kong [24]

1997–2000

39.0

–

Shanghai [25]

1998–2002

–

16.8–36.8

Korea [27]

2000–2002

86.4

73.7–95.5

the socioeconomic and environmental factors would not introduce bias to the ascertainment. Incidence rates among racial groups in the United States (2000) were Caucasian (non-Hispanic) 11.4 per 100 000 children < 5 years, African American (non-Hispanic) 19.7 per 100 000 children, Hispanic 13.6 per 100 000 and Asian/Pacific Islander 39.0 per 100 000 [15]. In a separate study of American Indian/native Alaskan children the rate was 4.2 per 100 000 [31].

Age and gender Most series from diverse geographic and racial populations have shown approximately 85% of children with KD are < 5 years of age [2]. Thus, incidences are expressed generally as a proportion of children < 5 years old. The most recent population-based study in the United States indicated 76% and 77% of patients were < 5 years of age in 1997 and 2000. The median age of KD patients in the United States is 2 years. In Japan, the peak age is 9–11 months and 88.9% of KD patients were < 5 years of age [6]. KD is relatively uncommon in children < 6 months old and above 5 years of age [6, 15, 17–19]. Studies have suggested that CAA are more common in these two age groups possibly because the illness is less typical and thus diagnosis is delayed [6, 32–36]. While KD is overwhelmingly a disease of children, rare cases have been reported in adults [37]. KD occurs in males more frequently than females [5–16]. Males are at greater risk of developing CAA as well [33]. In the United States the male: female ratio is 1.5:1 while in Japan it is 1.3:1.

Seasonality and temporal-geographic clustering of KD In the United States, KD hospitalizations are more frequent in the winter months [16, 38]. The seasonality in Hawaii is less obvious, although fewer cases were seen during April through June [30]. In a 5-year period of active

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surveillance in San Diego County, California, KD incidence was inversely associated with average monthly temperature and positively associated with average monthly precipitation [39]. In Japan, excluding epidemic years, there appears to be a bimodal seasonal occurrence with peaks in January and early summer and a nadir in October [6, 40]. In China and Korea seasonal peaks appear to be more frequent in spring and summer [23, 26, 29]. Temporal-geographic clustering has been frequently observed in both the United States [15, 32, 38, 41] and Japan [6, 40]. The Japanese experience has been especially well described with “hot spots” occurring in various prefectures on a rotating basis [6, 40].

Familial cases There appears to be an observable enhanced risk of KD within certain families. In Japan, there is a tenfold increased risk of the illness in siblings of an index case [42, 43]. Parents of children with KD are twice as likely to have had the disease as compared to the general population [42]. In families where parents had KD, sibling cases among children are significantly more common [44]. Similar findings have been reported from the United States [45].

Recurrence of KD Recurrent cases of KD have been reported in both the United States and Japan [42, 46]. The estimated rate of recurrence in Japan is 3%, while that in the United States is < 1 to slightly over 1% [46, 47].

Socioeconomic factors KD patients in the United States come from families with a higher median household income and are more likely to have private insurance [15, 39]. An analysis of hospitalization costs for KD in the United States for children < 5 years of age showed that the median cost was $6189 [48]. The average annual total estimated cost associated with hospitalization for KD patients < 18 years of age was $38.6 million [48].

Other risk factors Several other risk factors for KD have been reported in the past. An ante-

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cedent respiratory illness has been a significant association with KD patients as compared to controls in outbreak situations in the United States [32]. Shampooing or spot-cleaning carpets within 30–45 days of onset of KD has been a risk factor in some studies but not others [49, 50]. Other factors associated with KD include use of a humidifier [49], living near a body of water [38] and having preexisting eczema [51].

Synthesis of epidemiological data KD is an acute self-limited illness of children that is characterized epidemiologically by seasonality and occurring in geographic clusters. It shares many clinical characteristics with known infectious diseases such as scarlet fever, toxic shock syndrome, measles and adenovirus infections. It has been associated with antecedent viral-like illnesses in some epidemic situations. All of these factors suggest an infectious etiological agent or agents as a cause. The relative rarity in the first 3 months of life (possibly due to maternal antibody) and peak occurrence early in childhood is another characteristic shared by many common childhood infections. Host factors also appear to be important in the disease. Susceptibility to the disease is clearly influenced by ethnicity, familial risk factors and possibly preexisting conditions such as atopy. A genetic predisposition is suggested by these factors. Finally, environmental influences cannot be ruled out as suggested by apparently recent emergence of the disease in the last half of the 20th century, a socioeconomic bias toward more affluent lifestyle, possibly climatic associations and less well established associations such as rug cleaning. The exposure of a predisposed host to an infectious pathogen or pathogens with possible environmental contributing factors is a reasonable model to propose for KD.

Pathology Pathologically KD is a vasculitis of medium-sized vessels [52]. Studies from Japan have described four stages of pathology in the heart [53] (Tab. 4). The classification was based on the careful evaluation of 20 hearts taken from patients who had died of KD. Stages were based on the duration of illness at the time of death. The pathological description is considered unique and is distinguished from other vasculitis in the “medium-sized vasculitis” group, polyarteritis nodosa, in that the arterial inflammation does not affect vessels smaller than arteries [52]. Prior to the introduction and the availability of intravenous immunoglobulin (IVIG) for treatment of KD, 20–25% of children developed coronary artery aneurysms as a sequela [2].

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Table 4. Pathology of the heart in KD [52] Stage I (0–9 days):

Acute perivasculitis and vasculitis of microvessels (arterioles, capillaries and vessels) and small arteries Acute perivasculitis and end-arteritis of the three major coronary arteries (MCAs) Pericarditis myocarditis Inflammation of the atrioventricular conductor system Endocarditis with valvulitis

Stage II (12–25 days):

Panvasculitis of the MCAs and aneurysm with thrombus in the stems Myocarditis, coagulation necrosis, lesions of the conduction system Pericarditis Endocarditis with valvulitis

Stage III (28–31 days):

Disappearance of inflammation in the microvessels Granulation of the MCAs Myointimal proliferation in the coronary and other mediumsized arteries

Stage IV (40 days to 4 years):

Scarring with severe stenosis in the MCAs Fibrosis of the myocardium Coagulation necrosis Lesions of the conduction system Endocardial fibroelastosis

Pathogenesis The vasculitis of KD is clearly immunologically mediated and a wide variety of immunoregulatory abnormalities have been documented during the acute phase [54]. In a study of 21 children in the acute phase of KD, Leung and colleagues [54, 55] demonstrated a significant reduction in circulatory T8-positive (T8+) suppressor-cytotoxic T cells, increased activated T4+ helper cells and a proliferation of circulating activated B cells spontaneously secreting IgG and IgM. Furukawa et al. [56], in Japan, demonstrated activation of CD23+ monocytes/macrophages in the peripheral blood of patients with acute KD. Immunopotent cellular activation is associated with a broad array of proinflammatory cytokines including TNF-_ [57, 58], IL-1 [59], IFN-a [60] and IL-6 [61]. These mediators undoubtedly contribute to the high fever, discomfort and inflammatory changes during the acute phase but also facilitate the vascular injury.

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Vascular endothelial cells become activated by cytokine stimulation and may induce or increase expression of endothelial cell surface antigens that promote functional changes such as leukocyte adhesion and antigen presentation. These changes may make endothelial cells more vulnerable to attack by cytotoxic IgM antibodies present in acute phase serum of KD [62]. Further evidence of immunological injury to coronary arteries derives from immunohistochemical studies demonstrating transmural infiltration of artery walls with CD45RO T lymphocytes (activated/memory T cells) with CD8 lymphocytes predominating over CD4 T cells [63]. Remarkably, T cell activation, cytokine excretion and other immunological perturbations are reversed by IVIG [64]. Numerous other immunoregulatory abnormalities have been observed during KD or have been suggested as possible contributors to the pathogenesis of the disease. Macrophage activation syndrome has been observed with KD [65]. CD25+ CD4+ regulatory T cells, which maintain immunological self tolerance and control immune responses to microbial invasion, have been shown to be reduced in acute KD patients more than normal controls, suggesting this might play a role in the disease [66]. Wang et al. [67] proposed that CD40 ligand (CD40L) might play a role in the pathogenesis of KD because they found CD40L expression on CD4+ T lymphocytes in patients with the acute disease. CD40L is a potent activator of the immune system and might enhance endothelial cell inflammation and vascular damage [67]. Several recent reports implicate vascular endothelial grown factor (VEGF) in the pathological findings associated with KD. VEGF is the primary growth factor for formation of blood vessels and is a vascular permeability factor. It may also be a driver of inflammation as it enhances monocyte chemotaxis in humans and increased the production of B cells in mice [68]. Several reports have documented significantly elevated levels of VEGF in the acute and subacute phases of KD [69–71]. VEGF induces expression of matrix metalloproteinases (MMP), which degrade extracellular matrix and basement membrane proteins such as collagen and elastin [68]. Gavin et al. [72] demonstrated MMP-2 and MMP-9 in the damaged arterial walls of children who died of KD. The same group subsequently demonstrated angiogenesis in acute KD aneurysms much earlier than previously reported, probably related to several angiogenesis factors including VEGF. Thus, through such highly complex and intricate interactions of immunopotent cells, cytokines and other mediators, the inflammatory process results in vascular damage in KD.

Genetic influence of the pathogenesis of KD Racial differences in incidence, families with multiple cases, and the apparent greater tendency for CAA to occur in some children but not in others

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Table 5. Gene polymorphisms and single nucleotide polymorphisms (SNPs) in KD Polymorphism or SNP

Susceptibility or risk factor

HLA Bw22J

Associated with KD in Japanese children

74, 75

HLABw51

Associated with KD in United States and Israel population

76, 77

HLABw44

Associated with KD during Boston epidemic

78

IFN-a gene

Over production of TNF-_

79

Monocyte chemoattractant protein 1 gene (MCP-1

Over production of MCP-1

80

TNF-_ gene A/A at LT-x + 250

Over production of TNF-_

81

Possible dysregulation of IL-1B or TNF-_

82

Genotype 1/11 fa IL1-Ra

Higher susceptibility of KD

83

Angiotensin converting enzyme genotype ID

Associated with KD

84

Vascular endothelial growth factor (VEGF) CGCC

Associated with KD

85

Angiotensin/converting enzyme genotype I/I

Associated with development of CAA

86

MHC-class-1-chain related gene A (MICA) allele 4A

Protective for development of CAA

87

Methylenetetrahydrofolate reductose (MTHFR) TT genotype

Protective for females and risk factor for males for CAA

88

CD14/-159 TT genotype

Associated with CAA

89

CD40 ligand (CD40L) SNP in intron 4

Associated with CAA in males

90

VEGF G allele

Associated with CAA

91

Matrix metalloproteinase-3 gene 6A/6A genotype

Associated with CAA

92

Fc x RIIa HR and RR alleles

May predict failure of IVIG therapy

93

Mannose-bending lectin (MBL)gene MBL2 genotype

May predict failure of IVIG therapy in < 1 year olds

94

SLC11A1 gene

51-promoter

(GT)n

Reference

are all observations that suggest there may be a genetic influence in the pathogenesis of KD. Many investigators are attempting to identify genetic factors that predispose to acquiring KD or its complications. Many single-nucleotide polymorphisms (SNPs) have been identified that seem to be associated with susceptibility to KD or a risk factor for developing CAA. Table 5 lists 21 studies that identify polymorphisms or SNPs possibly associated with these or other risks.

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Table 6. Proposed etiological agents of KD Infections

Human herpes virus 6 & 8 Epstein-Barr virus Human parvovirus B-19 Retrovirus Adenovirus TT virus Propionibacterium acnes Streptococcus mitis Leptospira Erlichia species Staphylococcus aureus (TSST-1 positive) Chlamydia pneumoniae

The numerous known or candidate SNPs identified on multiple separate gene locations suggest that the genetic influence on pathogenesis, like the inflammatory process itself is highly complex [68]. New genetic markers will undoubtedly be identified in the future.

Etiology After almost 40 years of investigation following description of KD, we know a great deal about the epidemiology, pathology, pathogenesis of the disease and there is a fairly effective, although less than elegant, therapy available in the form of IVIG. The etiology of KD, however, remains an enigma. As discussed previously, clinical and epidemiological factors favor an infectious etiology, but, as yet, a single microbial pathogen has not been consistently associated with the disease. A very abridged list of microbial and environmental agents that have been proposed as causes for KD is found in Table 6. The two most heavily investigated areas in the last 10–15 years have been a bacterial toxin-mediated cause versus a viral pathogen etiology.

Superantigen-mediated etiology The possibility of a superantigen (SA) being implicated in the cause of KD was prompted by the observation that the illness is associated with marked activation of T lymphocytes and monocytes/macrophages. The differences between SA and conventional antigen are compared in Table 7. Early studies showed significantly elevated levels of V`2+ and V`8.1+ T cells in patients with KD [95, 96]. Subsequently, Leung et al. [97] published a case

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Table 7. Comparison of the Effect of Conventional and Superantigens Conventional antigen

Superantigen

Processed by antigen presenting all (APC). Presented as a peptide on the APC surface in association with MHC II molecule.

Directly bind to class II MHC molecules on the APC and TCR.

Interacts with the variable (V), joining (J) and diversity (D) portions of the _ and ` chains of the T cell receptor (TCR).

Binding restricted to the specificity of the variable regions of the ` chain (V`) of TCR.

Recognized by the few sensitized T-cells with receptors for the antigen resulting in a limited more specific immune response.

Activates a specific set of V` families resulting in activation of a large portion of T-cells causing a much more intense immune response.

Adapted from: Curtis et al. [95].

control study on the presence of bacterial colonization with Staphylococcus aureus organisms capable of producing toxic shock syndrome toxin (TSST). They found a significant association between colonization with toxin secreting S. aureus and the KD patients. Subsequent studies seemed to confirm the association [95, 98, 99]. A prospective multicenter trial assessing KD patients and controls for SA-producing staphylococcal and streptococcal bacteria (TSST-1, staphylococcal enterotoxins B and C, and streptococcal pyrogenic exotoxins A and C) were undertaken. Overall, isolation rates of SA-producing bacteria between KD patients and controls were not different statistically. A subset of patients with organisms expressing superantigens that stimulate V`2+ T cell receptor families of T cells were found significantly more often in the KD group [100]. Other investigations have not confirmed the SA hypothesis, however [101–104]. More recent serological studies suggest a role in the pathogenesis of KD for TSST-1 staphylococcal enterotoxin B and streptococcal pyrogenic exotoxins A and C [105–107]. The role of SA in the etiology of KD remains controversial [108].

Conventional antigen/viral etiology An alternative hypothesis to the superantigen theory is that KD results from infection with an as yet unidentified viral pathogen. Central to this idea is that the immunological response is oligoclonal in response to a conventional antigen rather than a polyclonal response as seen with a challenge by SA. A series of studies have compiled evidence to support this proposition. An unexpected observation from an immunohistochemical study of coronary arteries taken from infants and young children who had died in the acute phase of KD initiated this line of investigation. [109, 110]. IgA-secreting plasma cells were found infiltrating the vascular wall, pancreatic ducts

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and kidneys of 100% of KD patients compared to none of the age-matched control patients. This observation was intriguing in view of the relative immaturity of the systemic IgA response in infancy as compared to a fully developed and more robust secretory IgA response at this age. A polyclonal response to an SA might engender infant B cells to respond with a predominantly IgM reaction. A viral or other microbe presented to a mucosal site in a similar patient might stimulate vigorous IgA response [111]. Also observed was a heavy infiltration of IgA-secreting plasma cells in the upper respiratory tract of KD patients as compared to controls [112]. Subsequently, the same investigators demonstrated that the vascular IgA response was oligoclonal in nature and, therefore, probably resulted from stimulation by a conventional antigen, not a superantigen [112]. As a next step, the group developed synthetic antibody from prevalent IgA gene sequences found in acute-phase KD arterial tissue. They then exposed the tissues to the antibody and detected antigen in the respiratory epithelium of proximal bronchi from lungs and in subsets of invading macrophages from the myocardium of other inflamed tissues. The strength of the antigen signal paralleled the concentration of IgA plasma cell infiltration. These findings were not present in tissue from non-KD control patients [113]. Spheroid bodies were seen in the region between the nucleus and apical surface of ciliated epithelium of the proximal bronchi. Similar bodies were seen in the splenic and lymph node tissues. Evaluation of these tissues was undertaken using light microscopy (LM) and transmitting electron microscopy (TEM) focusing on areas containing the spheroid bodies [114]. LM revealed roundto-oval intracytoplasmic perinuclear inclusion bodies in medium-sized bronchi. They stained with both eosin and hematoxylin, suggesting they contained both protein and nucleic acid. TEM showed regular electron-dense inclusion bodies in the perinuclear region of ciliated bronchial epithelial cells resembling aggregates of viral proteins and nucleic acids that are found in respiratory tissues during infection with RNA viruses [114]. A recent report of association between newly described human coronavirus [115, 116] and KD raised hope that the etiology of KD had finally been identified [117]. Unfortunately, a series of reports from Japan and Taiwan and the United States found no consistent association between the new RNA respiratory virus and KD [118–121]. Thus, the search for the etiology of KD continues. Hopefully, with the application of histochemical and molecular techniques described above, the cause will soon be identified, possibly among the many new viral agents being identified at an increasing rate [122, 123].

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Helicobacter pylori infection in children Hien Q. Huynh Department of Pediatrics, Stollery Children’s Hospital, Aberhart Centre #1, Room 9222, 11402 University Avenue, Edmonton, AB, Canada T6G 2J3

Abstract Helicobacter pylori is generally acquired in childhood, and the prevalence of this infection varies between and within populations and is decreasing in the developed world. The clinical manifestation of diseases is dependent on the interaction between host, environmental and bacterial factors. The mode of transmission is likely person to person. Strong evidence has accumulated, establishing the causal link between peptic ulcer disease, gastric cancer and mucosal associated lymphoma with H. pylori infection. The association with refractory iron deficiency anemia and idiopathic thrombocytosis purpura are compelling but need more studies. New indications for the eradication of H. pylori are emerging – such as those with strong family history of gastric cancer. Prevention of gastric cancer may require eradication of this bacterium in childhood prior to the development of precancerous lesions. A test-and-treat strategy is not indicated for those with recurrent abdominal pain. In addition, the rate of antibiotic resistance has increased in some populations. Novel eradication strategies need to be developed. Improving the children socioeconomic situation, such as better housing, sanitation and hygiene, remains one of the major pillars in reducing the prevalence of H. pylori children and its diseases burden.

Introduction Since discovery of Helicobacter pylori in 1983, this bacterium has become the most studied bacteria over the last two decades culminating in the winning of the Nobel Prize in Medicine in 2005 by two medical practitioners, Drs. Barry Marshall and Robin Warren, who were the first to culture the bacterium and established the link between peptic ulcer disease and H. pylori infection [1].

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Epidemiology It is now well established that H. pylori infection is typically acquired in childhood [2]. Without eradication therapy in general, this infection will remain with the host for life. H. pylori remains a very common infection worldwide, with up to 50% of the world’s population colonized with this bacterium. However, over the last century there has been a significant decline in the prevalence of this infection in the developed world [3]. The prevalence of this infection in Canadian children is approximately 5%. Interestingly, the prevalence is quite varied even within a population. For example, the Aboriginal or Native populations of Canada and children of immigrants from the Third World have a much higher prevalence of this infection compared to the rest of the population [4, 5]. It has also now become increasingly clear that H. pylori infection is a marker of poverty, and is found more commonly in individuals of low socioeconomic status and in areas where there is household overcrowding and poor sanitation [6–8]. Humans are the natural reservoir for H. pylori; however, the route of transmission is unclear. This could either be oral-oral or oral-fecal, although successful cultures of this bacterium from stool have proven to be extremely difficult [9]. The rate of acquisition of H. pylori over the last few decades in the industrialized world has decreased substantially and the observed increase in the prevalence in this infection with age is likely to be a cohort effect, with the old age groups being more likely to acquire the infection, and the younger age group being less likely to acquire the infection in childhood [10].

Pathogenesis H. pylori is a gram-negative spiral bacterium that has developed the unique ability to colonize the gastric mucosa, which is usually well protected against bacterial infections. The H. pylori genome has been sequenced and contains a number of virulent factors that permits it to colonize the gastric mucosa [11]. The flagella of H. pylori allow it to be mobile in the mucous layer of the gastric epithelium. It also possesses urease, which has the ability to hydrolyze urea into carbon dioxide, and hydrate ammonia, thereby protecting the bacterium from an acid environment with an ammonia envelop. This enzyme activity is regulated by the pH in the immediate environment of the bacterium via a unique pH-gated urea channel (UREI). H. pylori also has a number of outer membrane proteins such as the BabA, HopZ and AlpAB proteins, which can mediate adhesions to gastric epithelial cells. The BabA protein has been shown to bind to Lewis B blood group antigen [12]. A significant proportion of H. pylori strains also possess the cag (cytotoxinassociated gene) pathogenicity island (837 kDa), which is a foreign piece of DNA that was acquired during its evolution. It is thought to encode for a type 4 secretion apparatus, a “molecular syringe” that translocates the CAG

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protein into epithelial cells [13, 14]. This protein is phosphorylated intracellularly; its function is unknown but is thought to modify cellular response and cytokine production in host cells. In certain populations, the presence of this pathogenic island is associated with more severe disease. Also, a significant proportion of H. pylori strain secret vacuolating cytotoxin like an exotoxin. This toxin was named because it causes vacuous formations in epithelial cells infected with H. pylori strain producing this toxin. This cytotoxin has the ability to insert itself into epithelial cell membranes, forming channels in which bicarbonate and other organic ions can be released. This vacuolate toxin also targets mitochondrial membranes and may induce apoptosis via release of cytochrome c in certain populations, particularly in Western countries. Certain vacuolating gene variants are associated with more severe disease; however, this association has not been found in the Far East. Currently, the pathogenic role of these toxins is still not clear. It is now established that vacuolate is not essential for colonization. The pathogenesis of H. pylori has been reviewed by Hatakeyama and Brzozowski and by Kusters et al. [15, 16].

Clinical presentation The clinical course or natural history of the infection is quite variable and is likely to be dependent on host, environmental and bacterial factors. The majority of children and adults infected with this bacterium are asymptomatic. Most infected patients with H. pylori develop gastritis, particularly nodular gastritis in children [17, 18]. Those with antral predominant gastritis are more likely to develop duodenal ulcers and have a reduced risk of gastric cancer compare to those with corpus predominant atrophic gastritis who have an increase risk of gastric cancer (Fig. 1) [19]. As an example of a host factor that determine disease outcome, polymorphism in the HLADQA1 gene results in resistance to atrophic gastritis and thus lower the risk of gastric cancer in Japanese but not in Italian individuals [20, 21], whereas polymorphisms of IL-1` gene give rise to corpus gastritis and thus increase the risk of gastric cancer [22, 23]. The lifetime risk of peptic ulcer disease is approximately 3–25%, depending on the population [19, 24]. In children, the risk for peptic ulcer is low; in one Japanese study, the prevalence of H. pylori in duodenal ulcer, and gastric ulcer was 83.0%, and 44.2%, respectively [18, 25]. In those with H. pylori infection and peptic ulcer disease, there is now little doubt that eradication of H. pylori is superior to ulcer-healing drugs for duodenal ulcers; for gastric ulcers, eradication achieves similar result to ulcer-healing drugs. In terms of preventing recurrence of peptic ulcer disease, eradication is superior to placebo [26]. The lifetime risk of gastric cancer though is approximately 1% based on large epi-immunological case-controlled studies. The association between

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Figure 1. Schematic representation of gastric pathology and disease outcome. Adapted from [19], with kind permission of the Massachusetts Medical Society

gastric cancer and H. pylori infection has been confirmed further in animal studies using the Mongolian gerbil [27]. In a prospective cohort study of Japanese patients, gastric cancer was shown to develop in 2.9% of 1246 adult patients infected with H. pylori. Mean follow-up was 7.8 years. No gastric cancer was found in those not infected or in a subgroup of patients who received eradication therapy for H. pylori [28]. However, in another prospective randomized placebo-controlled population-based primary study of 1630 healthy Chinese patients, carriers of H. pylori infection in a region of China with a high prevalence of gastric cancer, 817 received eradication therapy and 813 a placebo. No difference was found in terms of the incidence of gastric cancer development between the two groups over a period of 7.5 years. In a subgroup analysis of patients without precancerous lesions, such as atrophic gastritis and intestinal metaplasia, eradication seemed to decrease the development of gastric cancer [29]. A recent trial suggests that eradication of H. pylori may reduce the incidence of precancerous lesions

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[30]. Eradication of H. pylori in the older individuals with precancerous lesions appeared not to be effective in preventing gastric cancer. Since these precancerous lesions are rarely seen in children [31], perhaps eradication should take place in childhood to prevent the development of these precancerous lesions later in life. Currently, the effect of H. pylori eradication on the incidence of gastric cancer is unknown and conclusive trials will take many years. Most experts favor eradication in first-degree relatives of gastric cancer patients [32–34]. Mucosal associated lymphoid tissue (MALT) lymphoma is rare in those infected with H. pylori, with a lifetime risk of less than 1%.

Helicobacter pylori and abdominal pain The association between recurrent abdominal pain and H. pylori infection remains controversial. Some studies have supported the link and others have not [35]. Of interest, a recent study published by Malaty et al. [36] demonstrates that younger children suffering from recurrent abdominal pain are more likely to be infected with H. pylori than older children with recurrent abdominal pain. Another study from Taiwan found that H. pylori infection is more commonly found in children with short-term (2 weeks to 3 months) recurrent abdominal pain, suggesting that perhaps short-term abdominal pain may be a feature of acute H. pylori infection [37]. On the other hand, a recent community-based cross-sectional study from Sweden of 695 children between ages 10 and 12 years showed that 18% of children were infected with H. pylori based on positive anti-H. pylori antibody tests, and that there was no increase in recurrent abdominal pain reported in this age group of children with H. pylori infection [38]. In a double-blind randomized placebo-controlled trial, symptomatic response to H. pylori eradication was determined in children with recurrent abdominal pain. The control group was put on Omeprazole and the treatment group received eradication triple therapy; there were 10 children in each group. Bacteria eradication was achieved an 8 out of 10 children in the treatment group and none in the placebo group. After 52 weeks, there was a similar reduction in the symptom index observed in both groups. A limitation of this study was the small number of patients enrolled [39]. A recent Japanese study showed that children with recurrent abdominal pain that fulfilled the Room II criteria are more likely to have H. pylori infection and a psychiatric disorder [40]. All these studies suggest that recurrent abdominal pain of childhood is a heterogeneous syndrome with unclear etiology. H. pylori infection is likely to represent only a very minor cause of recurrent abdominal pain, perhaps affecting those who are younger and have recent onset of abdominal pain. There is therefore no indication for the test-and-treat strategy for H. pylori in children with recurrent abdominal pain.

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H. pylori and gastroesophageal reflux disease Currently there is no evidence that H. pylori eradication worsen gastroesophageal reflux disease (GERD) in adults [41]. Limited data are also available in children. However, there is a theoretical risk, even though the data are conflicting, that long-term proton pump inhibitor (PPI) treatment could increase the development of H. pylori-associated atrophic gastritis and increase the risk of gastric cancer [42, 43]. Some experts recommend testing and eradicating H. pylori if the child or adolescent is undergoing endoscopy for GERD, but not for those with clinically diagnosed GERD [44].

Extragastric manifestations of H. pylori infection in children With the increasing awareness among clinicians of H. pylori infection over the last decade, there have been a number of reports on the consequences of adverse effects from H. pylori infections outside the gastrointestinal tract (Tab. 1). Studies purporting the association between these manifestations of H. pylori infections are weak in terms of design and are not reviewed in this chapter. However, there are two manifestations that need to be mentioned: the first is refractory iron-deficiency anemia and the second is idiopathic thrombocytopenia (ITP) [45].

Refractory iron-deficiency and H. pylori infection There are a number of potential biological explanations for iron deficiency observed in H. pylori infection apart from bleeding secondary to peptic ulcer disease. Currently, there is no evidence to support that chronic gastritis secondary to H. pylori results in occult blood loss. However, H. pylori infection in some settings might give rise to hypochlorhydria, low ascorbic acid levels, and increased lactoferrin (a host iron-binding protein) sequestration by the organisms. Also, H. pylori possesses multiple iron acquisition systems in its genome, which makes it an avid competitor for iron uptake with the host in the gastric microenvironment [46]. There are a number of case reports as well as case control studies, and recently a population study, supporting the role of H. pylori infection as a potential cause of otherwise unexplained refractory iron deficiency [47–49]. H. pylori infections, especially those with atrophic gastritis, are more likely to have unexplained refractory iron-deficiency anemia compared to the age- and sex-matched controls without iron deficiency [50]. Baysoy et al. [51] describe a group of children undergoing investigation for upper gastrointestinal symptoms and found that those with H. pylori were more likely to have lower iron stores compared those without infection. However, eradication of H. pylori has not consistently increased hemoglobin levels

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Table 1. Purported extragastric manifestations of H. pylori infection Manifestations Cardiovascular

Atherosclerotic heart disease, stroke

Neurological

Parkinson disease, migraine

Autoimmune

Autoimmune thrombocytopenia purpura, Reynaud’s phenomenon, Sjögren’s syndrome, diabetes mellitus

Dermatological

Chronic urticaria, angioedema, rosacea, alopecia areata

Others

Refractory iron deficiency, halitosis, hyperemesis gravidarum, anorexia, glaucoma, oral ulcers, urethritis

in control studies. One study confirmed an increase in hemoglobin level in Korean female athletes [52], whereas a large control household randomized open-label trial involving 290 Alaskan children with iron deficiency and H. pylori infection in a population that has a high prevalence of this infection, failed to show that H. pylori eradication improved isolated iron deficiency or mild anemia up to 14 months after treatment initiation. The limitation is that this study was not designed to detect small effects, with only 2 patients had hemoglobin less than 100 g/L [53]. Further randomized control trials in different populations, with age groups with varying degree of anemia are still needed to confirm the association between H. pylori and refractory iron deficiency. Despite this, the Canadian consensus conference on H. pylori still recommends, in the absence of other causes of iron deficiency, H. pylori should be tested for and treated [33].

Idiopathic thrombocytopenia and H. pylori infection In 1998, Akiyama and Onozawa [54] demonstrated that a PPI administration was associated with an increase in platelet count in patients with chronic idiopathic thrombocytopenia (ITP). Subsequently, a Lancet paper in a pilot study by Gasbarini et al. [55] described a significant increase in platelet count in 8 of 11 patients with H. pylori, which was successfully eradicated. Since then, there have been numerous case reports and case series reporting that eradication of H. pylori is accompanied by platelet increase in adults patients with ITP. A review by Franchini and Vener [56]i summarized the adult literature. Of a total of 1126 patients with ITP, 64% were infected with H. pylori, eradication occurred in 81% with the platelet response rate occurring in approximately 50%. Subsequently, there has been a randomized control trial looking at the effect of H. pylori eradication in adult patients with chronic ITP involving 36 Japanese patients, 25 of whom were positive for H. pylori; 13 of these 25 were randomized to the eradication group and 12 to the non-eradication group. Of the 13 patients in the eradication group, 6 had

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either partial or complete response in their platelet count, whereas none of the patients in the non-eradicated group responded [57]. In another small randomized control trial there was no difference when comparing PPI versus H. pylori eradication therapy in the treatment of ITP [58]. The potential explanation for this is likely molecular mimicry, where anti-platelet antibodies in the serum recognize the CAG protein of H. pylori [59]. The data are conflicting for children [60, 61]. There are a number of case reports of children with ITP and increased platelet count following H. pylori eradication [62–64]. Most of these reports came from the Far East. Other reports demonstrated no association between these two conditions [65]. These reports suggest that much larger randomized placebo-controlled trials need to be performed in children from different ethic backgrounds to determine whether in fact there is an association between the two conditions. This study needs to be conducted in areas with both low and high prevalence of H. pylori infection.

Investigations Non-invasive test Currently, there are two non-invasive tests that are becoming extremely reliable in detecting H. pylori infection. The more established one is the urea breath test and the other is the stool antigen test. Serology test is not recommended as diagnostic tool because of its poor sensitivity and test-totest variability [33].

Urea breath test The urea breath test utilizes the essential enzyme urease, which is produced by H. pylori. Urease converts urea to ammonia and CO2. If the CO2 is labeled with a stable isotope, this can be detected in the expired air (Fig. 2). In the non-infected individual, urea will leave the stomach unchanged. This test is essentially a detection of urease activity, which can also be produced by other bacteria in the oral cavity, as in the setting of bacterial overgrowth. 13C and 14C are the two isotopes that are well validated [66]. Only the 13C urea breath test has been extensively tested in children [67]. 14C is radioactive and is not acceptable to be used in children. The 13C test is more expensive than the 14C test, because it requires mass spectrometry or infrared spectroscopy equipment for analysis of the expired breath. The results of the urea breath test are reported as 6/baseline (DOB), which is a measure of the ratio of 13C CO2, to 12C CO2. If the DOB exceeds a certain point, the patient is considered to be infected with H. pylori infection. The test is best performed when the patient has fasted

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Figure 2. Carbon in Urea is labeled with either 13C or 14C and exhaled after being converted to label CO2 by urease produced by Helicobacter pylori in the gastric mucosa.

and has not been on a PPI for at least 2 weeks. The patient should not have taken antibiotics for 4 weeks prior to testing because this can reduce the H. pylori load. The use of an acid solution as part of the test solution, either citric acid or orange or apple juice, is ideal because urea activity is highest in an acid environment. Expired breath can be collected between 15 and 45 min depending on the laboratory. Despite the variability in the dosage of urea used and the different cut off points, this study consistently shows that the urea breath test has a sensitivity of over the 96% and a specificity of over 90% [68]. Infants and toddlers are much more likely to have a positive result in comparison to school-age children and adolescents [69]. Reducing the tracer dose and changing the DOB value increase the specificity of this test in younger children [70, 71]. Also, technically, it is much more difficult to collect reliable expired air from infants and toddlers compared to school age children.

Stool antigen test This is a non-invasive test for H. pylori antigen in stool in the pediatric population. Unlike with the urea breath test, there is no collection difficulty, particularly in the younger age group. There are two types of stool antigen tests on the market: one is polyclonal, and the other a monoclonal, antibody enzyme immunoassay. In a recent meta-analysis of 22 studies including 2499 patients, the monoclonal stool antigen test was found to have sensitivity and specificity of 94% and 97%, respectively. In 13 of the

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studies in which the monoclonal was compared to the polyclonal stool antigen tests, the monoclonal test had a higher sensitivity of 95% versus 83% for the polyclonal test. In terms of eradication, analyzing 12 studies with 957 patients post treatment, the sensitivity and specificity for the monoclonal test were 93% and 96%, respectively. In 8 of the studies where both monoclonal and polyclonal tests were used, sensitivity was higher for the monoclonal (91%) than for the polyclonal test (76%), demonstrating that the monoclonal stool antigen test is a much more accurate, non-invasive test for both diagnosis and confirmation of eradication of H. pylori infection post treatment [72]. A recent European multi-centered study comparing urea breath tests with stool and serology tests, as well as antibody detection in urine, found that the urea breath test is the most sensitivity and specific. The stool antigen test used in this study was polyclonal (Meridian). The urea breath test had a sensitivity and a specificity exceeding 96%, whereas the stool test has sensitivity and specificity of 92%. This study only had a small number of children under the age of 6 (48 children accounted for 15% of the study population) [73]. However, another study performed in Egypt compared the urea breath test, monoclonal stool antigen test, and serology test to endoscopy with biopsy and rapid urease test. In this population 53 of the 108 children tested were under the age of 6. Overall, the sensitivity and specificity of the urea breath test was 98% and 89%, respectively, and those of the monoclonal stool antigen test were 94% and 81%, respectively. Interestingly, the urea breath test sensitivity was not affected by age but the specificity was lower in those under the age of 6 (86% versus 95%). With regard to the monoclonal stool antigen test, those performed on children under the age of 6 showed a sensitivity of 94% and specificity of 81%. However, above the age of 6, the sensitivity remained about the same at 92% but the specificity increased to 100%. Serology had the worst outcome in this study, giving a sensitivity of 50% and specificity of 80% [69]. Overall, the urea breath test remained the most sensitive and specific non-invasive test for H. pylori. There is still some conflicting data on how reliable this test is in the younger age group. The monoclonal stool antigen test is also proving to be quite a sensitive and specific test , and, again, its reliability in the younger age group requires further study. The use of these diagnostic tests needs to be interpreted in the local context, particularly whether these tests have been validated to the population that is under investigation, in particular with regard to the population age group as well as ethnicity and geography.

Invasive tests Upper gastrointestinal endoscopy with mucosal biopsy remains the gold standard for the diagnosis of H. pylori infection in children. It has the advan-

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tage of being able to detect upper gastrointestinal pathology including the complications of H. pylori infection such as nodular gastritis, peptic ulcer disease, gastric cancer, and MALT lymphoma. In pediatrics, the primary indication for upper GI endoscopy is the presence of persistent, severe upper abdominal symptoms and not simply the presence of H. pylori [33]. It is difficult to differentiate symptoms secondary to the complication of H. pylori infection such as peptic ulcer disease and functional dyspepsia. The most common endoscopic finding in children with H. pylori infection is nodular gastritis, which is seen most commonly in the antrum with an irregular (cobblestone) appearance, which is highlighted with blood from a bleeding biopsy site. When nodular gastritis is found, it has high specificity (98%) for H. pylori infection, and therefore a high predictive indicator for H. pylori infection, but it has low sensitivities (44%) [17, 74]. In naïve patients, antral biopsy had the highest yield, particularly in the mid antrum region of the lesser curvature [75]. For a patient who has been on acid suppression therapy or antibiotics, a biopsy from the transitional zone and body are also required to improve the yield [76, 77]. For patients who have developed complications from H. pylori infection such as peptic ulcer disease, multiple biopsies from different regions of the stomach are required. H. pylori can often be seen using hematoxylin and eosin staining; immunohistochemistry using polyclonal and H. pylori antibodies is likely to be the most reliable detection method on biopsy sections, although this method is expensive and time consuming. Among other stains that are often used is the Giemsa stain, which is less reliable but widely available and affordable [78]. The optimal staining method is often guided by local expertise. With a biopsy of gastric mucosa, a rapid urease test can also be used. This test essentially detects the presence of urease produced by H. pylori. The test is highly specific and sensitivity in adults, but less so in children [79]. The accuracy of this test also depends on the number of biopsies taken, site of biopsy and the use of antibiotics and PPIs. However, one of the major advantages of biopsy in H. pylori infection is the ability to culture this bacterium. In clinical trials, the success rate is up to 80% of infected children [80]. Bacterial culturing is time consuming and expensive, but it does allow for antibiotic sensitivity to be determined. This is particularly useful for those who failed previous eradication therapy. In addition, for a positive culture, the genotype of clinical isolate can be investigated for specific bacterial virulent factors. There are now a number of molecular techniques that can be used to detect the presence of H. pylori and the presence of a number of point mutations in the bacterial genome that determines antibiotic resistance genotypes. For example, the predominant cause of clarithromycin resistance is a point mutation in the peptidyl transferase of the 23S rRNA gene. There are also a number of inactivating mutations involving some reductase genes that convert Metronidazole from a harmless product to an anti-bacterial agent, inactivating the gene responsible for a portion of H. pylori resistance to Metronidazole [81].

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Antibiotic resistance Studies of antibiotic resistance in children are small in number; an US network for antibiotic resistance that tracked the national incidence rate of H. pylori and microbial resistance reported 340 clinical H. pylori isolates collected over a period of 4 years that demonstrated a 29% rate of antibiotic resistance to at least one antimicrobial agent and 5% resistant to two or more antimicrobial agents: 25% were resistant to Metronidazole and 12.9% were resistant to Clarithromycin. Only a very small number of cases (0.9%) were resistant to Amoxicillin [82]. However, more recently, the results of a larger prospective multi-center study from Europe on the rate of antibiotic resistance in H. pylori strain in 1233 children have been reported. These patients came mostly from western and southern Europe. Most of the isolates were obtained prior to any treatment, and overall the resistance rate for Clarithromycin was 24%. This increased to 42% in those who had previously received treatment that had failed. The resistance rate to Metronidazole was 25% and higher (35%) in those who received previous failed treatment. Resistance to both antibiotics only occurred at 6.9%; however, this increased to 15.3% in those who received previous failed treatment. Resistance to Amoxicillin was exceptionally low at 0.6%. These results confirm that antibiotic usage in children represents a major risk factor for developing treatment resistance [80].

Treatment Unfortunately, there have not been many randomized placebo-controlled studies in the pediatric population looking at eradication of H. pylori. One study involving 73 children with dyspeptic symptoms demonstrated an eradication rate of 74% using Amoxicillin and Clarithromycin with a PPI, and 9.4% using dual therapy of Amoxicillin and Clarithromycin for 7 days using intention to treat analysis [83]. Another study by Oderda et al. [84] used Lansoprazole, Amoxicillin and Tinidazole triple therapy versus placebo plus Amoxicillin and Tinidazole dual therapy for 1 week; after 6 months the eradication rate was 72% for triple therapy and remained at 71% for dual therapy, showing no difference between the two treatments. A recently developed 10-day sequential treatment for H. pylori eradication was studied in 78 children. They were either randomized to receiving Omeprazole plus Amoxicillin for 5 days, followed by Omeprazole plus Clarithromycin and Tinidazole for another 5 days, compared to triple therapy of Omeprazole plus Amoxicilline and Tinidazole for 1 week. Sequential treatment had an eradication rate of 97.3% and triple therapy an eradication rate of 75.7%, demonstrating that sequential treatment is superior to triple therapy, consistent with results from adult studies [85, 86]. This sequential treatment needs to be further studied in different populations to determine its efficacy and

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safety, and to confirm its higher eradication rate in comparison to a 2-week course of triple therapy. A recent Canadian Helicobacter Study Group Consensus Conference still recommends the use of triple therapy for 2 weeks using a PPI with Clarithromycin and Amoxicillin or Metronidazole given for 14 days. The duration of treatment of 2 weeks is likely to be optimal, but not conclusive; there is a 7–9% increase in the eradication rate with 14 days of treatment versus 7 days [87]. Tetracycline should be avoided in children under the age of 12 because it may cause staining of the children’s enamel. In addition, treatment failure is increased with antibiotic resistance [88]. A recent Russian study by Nijevitch et al. [89] treated 76 children, who had failed triple therapy, using quadruple therapy. These children were randomized to receive a 2-week course of bismuth subcitrate, Amoxicillin with Nifuratel or Furazolidone plus Omeprazole. The eradication rate was 89% for Nifuratel and 87% for Furazolidone. Nifuratel is preferred because of a lower frequency of side effects. Potentially, this could be a treatment of choice for those who have failed eradication. It is vital that reference laboratories are available to monitor the population H. pylori antibiotic sensitivity and test those with treatment failure.

Conclusion H. pylori is generally acquired in childhood and the prevalence in developed countries is now decreasing. The clinical manifestations of disease are a result of the host, bacteria and environment interaction, and are only seen in a subset of infected individuals. Its association with peptic ulcer disease, gastric cancer and MALT lymphoma is beyond dispute. A test-and-treat strategy is not indicated for children with recurrent abdominal pain. New indications in children are now emerging advocating its eradication, such as refractory iron deficiency, ITP and a strong family history of gastric cancer, although further studies are needed. Stool antigen tests and urea breath tests have emerged as some of the best non-invasive tests for H. pylori. Antibiotic resistance is on the rise, and novel treatment strategies are needed. Improving the social situation of children such as better housing, sanitation and hygiene remain one of the key pillars in reducing the prevalence of this infection in childhood.

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Yetgin S, Demir H, Arslan D, Unal S, Kocak N (2005) Autoimmune thrombocytopenic purpura and Helicobacter pylori infection effectivity during childhood. Am J Hematol 78: 318 Pathak CM, Bhasin DK, Khanduja KL (2004) Urea breath test for Helicobacter pylori detection: present status. Trop Gastroenterol 25: 156–161 Niv Y, Abuksis G, Koren R (2003) 13C-urea breath test, referral patterns, and results in children. J Clin Gastroenterol 37: 142–146 Koletzko S (2005) Noninvasive diagnostic tests for Helicobacter pylori infection in children. Can J Gastroenterol 19: 433–439 Frenck RW Jr, Fathy HM, Sherif M, Mohran Z, El Mohammedy H, Francis W, Rockabrand D, Mounir BI, Rozmajzl P, Frierson HF (2006) Sensitivity and specificity of various tests for the diagnosis of Helicobacter pylori in Egyptian children. Pediatrics 118: e1195–1202 Canete A, Abunaji Y, Alvarez-Calatayud G, DeVicente M, Gonzalez-Holguera JA, Leralta M, Pajares JM, Gisbert JP (2003) Breath test using a single 50–mg dose of 13C-urea to detect Helicobacter pylori infection in children. J Pediatr Gastroenterol Nutr 36: 105–111 Dondi E, Rapa A, Boldorini R, Fonio P, Zanetta S, Oderda G (2006) High accuracy of noninvasive tests to diagnose Helicobacter pylori infection in very young children. J Pediatr 149: 817–821 Gisbert JP, de la Morena F, Abraira V (2006) Accuracy of monoclonal stool antigen test for the diagnosis of H. pylori infection: a systematic review and meta-analysis. Am J Gastroenterol 101: 1921–1930 Megraud F (2005) Comparison of non-invasive tests to detect Helicobacter pylori infection in children and adolescents: results of a multicenter European study. J Pediatr 146: 198–203 Bahu Mda G, da Silveira TR, Maguilnick I, Ulbrich-Kulczynski J (2003) Endoscopic nodular gastritis: an endoscopic indicator of high-grade bacterial colonization and severe gastritis in children with Helicobacter pylori. J Pediatr Gastroenterol Nutr 36: 217–222 Elitsur Y, Lawrence Z, Triest WE (2002) Distribution of Helicobacter pylori organisms in the stomachs of children with H. pylori infection. Hum Pathol 33: 1133–1135 Van Zanten SJ, Dixon MF, Lee A (1999) The gastric transitional zones: neglected links between gastroduodenal pathology and helicobacter ecology. Gastroenterology 116: 1217–1229 Logan RP, Walker MM, Misiewicz JJ, Gummett PA, Karim QN, Baron JH (1995) Changes in the intragastric distribution of Helicobacter pylori during treatment with omeprazole. Gut 36: 12–16 Rotimi O, Cairns A, Gray S, Moayyedi P, Dixon MF (2000) Histological identification of Helicobacter pylori: comparison of staining methods. J Clin Pathol 53: 756–759 Chu C, Yu YJ, Kong MS, Ou JT (2003) Rate of Helicobacter pylori infection in children and clonality of Taiwan strains. Microbiol Immunol 47: 813–821 Koletzko S, Richy F, Bontems P, Crone J, Kalach N, Monteiro ML, Gottrand F, Celinska-Cedro D, Roma-Giannikou E, Orderda G et al (2006) Prospective

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multicentre study on antibiotic resistance of Helicobacter pylori strains obtained from children living in Europe. Gut 55: 1711–1716 Huynh HQ (2005) Invasive tests for Helicobacter pylori in children. Can J Gastroenterol 19: 429–432 Duck WM, Sobel J, Pruckler JM, Song Q, Swerdlow D, Friedman C, Sulka A, Swaminathan B, Taylor T, Hoekstra M et al (2004) Antimicrobial resistance incidence and risk factors among Helicobacter pylori-infected persons, United States. Emerg Infect Dis 10: 1088–1094 Gottrand F, Kalach N, Spyckerelle C, Guimber D, Mougenot JF, Tounian P, Lenaerts C, Roquelaure B, Lachaux A, Morali A et al (2001) Omeprazole combined with Amoxicillin and Clarithromycin in the eradication of Helicobacter pylori in children with gastritis: A prospective randomized double-blind trial. J Pediatr 139: 664–668 Oderda G, Marinello D, Lerro P, Kuvidi M, de’Angelis GL, Ferzetti A, Cucchiara S, Franco MT, Romano C, Strisciuglio P et al (2004) Dual vs. triple therapy for childhood Helicobacter pylori gastritis: a double-blind randomized multicentre trial. Helicobacter 9: 293–301 Francavilla R, Lionetti E, Castellaneta SP, Magista AM, Boscarelli G, Piscitelli D, Amoruso A, Di Leo A, Miniello VL, Francavilla A et al (2005) Improved efficacy of 10–day sequential treatment for Helicobacter pylori eradication in children: a randomized trial. Gastroenterology 129: 1414–1419 Graham DY, Lu H (2006) Is there a role for sequential in sequential anti-H. pylori therapy? Gastroenterology 130: 1930–1931; author reply 1931 Calvet X, Garcia N, Lopez T, Gisbert JP, Gene E, Roque M (2000) A metaanalysis of short versus long therapy with a proton pump inhibitor, clarithromycin and either metronidazole or amoxycillin for treating Helicobacter pylori infection. Aliment Pharmacol Ther 14: 603–609 Khurana R, Fischbach L, Chiba N, Veldhuyzen van Zanten S (2005) An update on anti-Helicobacter pylori treatment in children. Can J Gastroenterol 19: 441–445 Nijevitch AA, Shcherbakov PL, Sataev VU, Khasanov R, Al Khashash R, Tuygunov MM (2005) Helicobacter pylori eradication in childhood after failure of initial treatment: advantage of quadruple therapy with nifuratel to furazolidone. Aliment Pharmacol Ther 22: 881–887

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Human metapneumovirus infection Adilia Warris and Ronald de Groot Department of Pediatrics, Radboud University Nijmegen Medical Centre, and the Nijmegen University Center for Infectious Disease, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

Parts of this chapter have been published in: Hot topics in infection and immunity in children III, edited by A.J. Pollard and A. Finn. Adv Exp Med Biol 2006; 582: 251–264. With kind permission of Springer Science and Business Media.

Abstract Initially, human metapneumovirus (hMPV) was isolated from children with clinical symptoms of respiratory syncytial virus (RSV) infection in whom RSV could not be detected. Since then, numerous reports have described the detection of hMPV in clinical specimens from children, adults and the elderly (both immunocompetent and immunocompromised patients), diagnosed with an acute respiratory illness all over the world. hMPV is associated with a substantial number of respiratory tract infections in otherwise healthy children, with clinical illnesses similar to those associated with other common respiratory viruses. Serological surveys have shown that hMPV is a ubiquitous virus that infects all children by the age of 5–10 years and has been circulating in humans for at least 50 years. hMPV is a member of the Metapneumovirus genus of the Paramyxoviridae family, a group of negative-stranded RNA viruses. Genetic studies on hMPV have demonstrated the presence of two distinct hMPV serotypes each divided in two subgroups. Diagnosis is made by RT-PCR assays on respiratory secretions. Rapid antigen detection tests are not yet available and its growth in cell cultures is fastidious. No vaccines, antibodies (monoclonal or polyclonal), or chemotherapeutic agents are currently licensed for use to prevent or treat hMPV infections. The contribution of hMPV to pediatric respiratory tract infections suggests that it will be important to develop a vaccine against this virus in combination with those being developed for RSV and parainfluenza viruses. Reverse genetics technology is currently used to develop multivalent vaccines against hMPV and a variety of other important respiratory viruses such as RSV. Additional research to define the pathogenesis of this viral infection and the host’ specific immune response will enhance our knowledge to guide the search for preventive and therapeutical strategies.

Background In 2001, a new infectious agent, human metapneumovirus (hMPV), was isolated from nasopharyngeal aspirates of young children with respiratory tract illness from The Netherlands [1]. Initially, hMPV was isolated from children with clinical symptoms of respiratory syncytial virus (RSV) infec-

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tion in whom RSV could not be detected. Since then, numerous reports have described the detection of hMPV in clinical specimens from children, adults and the elderly (both immunocompetent and immunocompromised patients), diagnosed with an acute respiratory tract infection (RTI) all over the world. hMPV is an enveloped virus with a genome that is a single strand of RNA of approximately 13 kb [1]. Its genome contains eight genes that presumably code for nine different proteins [2, 3]. The genomic organization for hMPV is similar but not identical to that for RSV being a member of the Pneumovirus. In contrast to the Pneumovirus, the Metapneumovirus lacks the NS1 and NS2 genes and has a different positioning of the other common genes, i.e., the N (nucleocapsid RNA binding protein), P (phosphoprotein), M (matrix protein), F (fusion glycoprotein), L (major polymerase subunit), G (major attachment protein), M2 (transcription elongation and RNA synthesis regulatory factor), and SH (small hydrophobic surface protein). The absence of open reading frames (ORFs) between the M and F genes in the hMPV virus and the lack of NS1 and NS2 genes is in agreement with it being the first identified non-avian member of the Metapneumovirus genus [2, 4]. Genetic analysis of the N, M, P and F genes revealed that hMPV showed a higher sequence homology to the Metapneumovirus genus (average of 66%) as compared to the genus Pneumovirus (average of 30%) [1, 5]. On the basis of the organization of the viral genome and sequence identity to the Metapneumovirus avian pneumovirus, also known as turkey rhinotracheitis virus, hMPV was assigned to be a member of the Metapneumovirus genus of the Paramyxoviridae family. The Metapneumovirus and the Pneumovirus genera are two genera within the subfamily of Pneumovirinae (Fig. 1). The Pneumovirinae and the Paramyxovirinae belong to the Paramyxoviridae family, a group of negatively stranded RNA viruses including several major pathogens of humans and animals. hMPV does not infect chickens or turkeys, and the virus is unlikely to be a zoonotic source. RT-PCR analyses using primer sets for specific paramyxoviruses (parainfluenza virus, mumps virus, measles virus, RSV, simian virus type 5, Sendai virus and Newcastle disease virus) did not react with the newly identified hMPV, indicating no close genetic relatedness to these viruses. hMPV-specific antisera did not react in immunofluorescence (IF) assays with cells infected with a panel of paramyxoviruses and orthomyxoviruses (parainfluenza viruses, influenza virus A and B, RSV) [1]. Although genetically not closely related, hMPV shares many biological properties with RSV. The hMPV isolates replicate slowly in tertiary monkey kidney (tMK) and rhesus monkey kidney (LLC-MK2) cells, very poorly in Vero cells and A549 cells, and could not be propagated in Madin Darby canine kidney (MDCK) cells or chicken embryo fibroblasts (CEF) [1]. The cytopathic effects are indistinguishable from those caused by RSV, although they occurred slightly later, 10–17 days post inoculation. Electron microscopy revealed paramyxovirus-like pleiomorphic particles of 150–600 nm,

Figure 1. Classification of viral pathogens of the Paramyxoviridae family that infect humans.

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with short envelope projections of 13–17 nm, indistinguishable from RSV [1]. hMPV is chloroform sensitive, and replicates optimally in a trypsindependent manner, in contrast to RSV, in tMK cells. No hemagglutinating activity with turkey, chicken or guinea pig erythrocytes was displayed. These combined virological data indicate that the hMPV is indeed a member of the Paramyxoviridae family. The mode of transmission has not been formally studied, but is likely by large particle respiratory secretions and fomites, based on its relatedness to other pneumoviruses. Nosocomial transmission does occur [6] and warrants contact isolation and scrupulous hand washing by health care providers.

Epidemiology Genetic studies on hMPV have demonstrated the presence of two distinct hMPV groups each divided in two subgroups [4, 5, 7–9]. Representative strains of the four subgroups are hMPV/NL/1/00 (subgroup A1), hMPV/ NL/1/99 (subgroup B1), hMPV/CAN/97/83 (subgroup A2), and hMPV/ CAN/98/75 (subgroup B2). A nearly complete genome sequence was determined for the prototype NL/1/00 strain of hMPV, and complete genome sequences were determined for two Canadian strains, CAN97/83 and CAN98/75 [2, 3]. These studies also confirmed that the 3’–5’ hMPV gene order is N-P-M-F-M2,1-M2,2-SH-G-L. None of these proteins have been identified or characterized by direct biochemical means, and their functions still need to be confirmed [10]. Bastien and colleagues [5] determined the complete nucleotide sequences of the N, P, M, and F genes of Canadian hMPV isolates. Comparison of the deduced amino acid sequences for the N, M, and F genes of the different isolates revealed that all three genes were well conserved with 94.1–97.6% identity between the two distinct clusters. The P gene showed more diversity with 81.6–85.7% amino acid identity for isolates between the two clusters, and 94.6–100% for isolates within the same cluster. The Canadian cluster 1 isolates show over 96% amino acid identity with the NL/1/00 isolates for all the viral proteins analyzed [2]. Analysis of the F and G protein genes of the four subgroups show that the F protein is highly conserved and demonstrated low variability within the four groups [7]. With an amino acid identity of 93–96% between the subgroups, the F protein becomes attractive as a principal target of protective antibodies. In contrast, the G gene shows high sequence diversity. Furthermore, these phylogenetic analyses showed that the hMPV strains obtained from different years and from different countries were randomly distributed over all four sublineages. To address the antigenic relationship between the different lineages, virus neutralization assays were performed showing a difference in antigenicity between lineage A and B. On the basis of these results it was proposed to define the two main lineages of hMPV as

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serotypes A and B. Although each serotype can be divided into two genetic subgroups, these subgroups did not reflect major antigenic differences. To characterize the extent of genetic diversity among hMPV strains in Australia and worldwide, comparative nucleotide- and predicted amino acid-sequence studies were performed with the N and P genes [11]. Comparison of aligned sequences revealed an 11.9–17.6% nucleotide variation, which divided the viral strains into two main lineages. In addition, two distinct subtypes were apparent within each lineage, which were defined as hMPV types A1, A2, B1, and B2. The variability of the P gene permitted a reliable classification of hMPV into its four subtypes, indicating that the P gene is a valuable target for phylogenetic studies. To confirm that this classification, based on both the N and the P gene, agreed with that proposed in other studies, similar sequencing and analyses of all available M, F, G, and L gene sequences were performed. The same lineages were found and thus the P gene seems a useful single target for genotyping and for the creation of a global classification scheme for hMPV. A large community-based phylogenetic study of hMPV for both surface glycoproteins F and G provides the evidence for the presence of multiple genotypes within each subgroup of hMPV [12]. This evidence came from the topology of the phylogenetic trees and bootstrap values in which sequences were arbitrarily considered a genotype if they clustered together with bootstrap values of 70–100%. This resulted in nine genotypes and six possible genotypes in the four subgroups together. Strains from both hMPV groups may co-circulate in a particular year as shown in South Africa, but at the same time not all four subgroup viruses are detected in a single year [12]. Limited data indicate that both hMPV groups can circulate in a single season with the possibility of the predominant group switching in successive seasons [4, 11, 13]. Agapov et al. [13] showed furthermore that, within each genotype, the F and N genes were conserved, but that the G and SH genes showed marked variation. Despite the genetic variability, no difference in the severity of illness caused by various hMPV isolates was noted. In contrast, a recent study [14] suggests that genotype A causes a more severe acute RTI in small children compared to genotype B. Although the number of cases was small, but comparable to the study of Agapov et al. [13], significant differences were found in parameters reflecting greater severity (diagnosis of pneumonia) as well as the severity index combining clinical data (hypoxemia, intensive care admission) [14]. These different results might be related to the patient groups studied. Many studies reported the detection of hMPV serogroup 1 as the only or the predominant serotype circulating. In an Israeli study, hMPV serogroup 1 also had the highest circulation rate (92% of the sequenced samples). Of the four subgroups, only three were identified (1A 65%, 1B 25%, and 2B 10%) [15]. Williams et al. [16] showed that the four genetic lineages of hMPV have persisted over the last 20 years in the community. More than one lineage

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was present concurrently during some seasons, whereas a single lineage dominated others. In their study the B2 lineage was most common, whereas the B1 lineage seems to circulate periodically. This is in contrast with others studies, were the A1 and A2 lineage accounts for the majority of clinical isolates. This might be due to primers that have shown to be less sensitive for detection of the B lineages. The circulation of multiple lineages and the changes of the dominant group of virus may suggest an attempt at evasion of preexisting immunity, as has been seen also for RSV. Studies performed in very different geographic areas showed that specific strains coexist across geographic areas [11, 12]. Preliminary evidence of the existence of hMPV other than the four known major genetic lineages of hMPV comes from the isolation of hMPV in a child with an acute asthma exacerbation [17]. Positive PCR results were obtained using primers derived from the N gene [18] and this amplified fragment was cloned in a plasmid vector and sequenced. This confirmed the specificity of the PCR, although the nucleotide sequence differed significantly from the representative hMPV strains of the known four lineages. This might indicate that the heterogenicity of hMPV is higher than recorded till now, and this has important consequences in the optimization of RTPCR protocols. hMPV has circulated in humans for at least 50 years; a 100% seroprevalence was found in 72 serum samples obtained from individuals 8–99 years old, collected in 1958 in the Netherlands [1]. There appears to be two periods of acquisition of hMPV in childhood [19]. The first period occurs within the first 3 years of life, the second period occurs in children > 48 months of age. The percentage of seropositive children increases in these age categories from 35–45% to about 75%, while it is > 90% in children > 5 years of age. Seroprevalence studies in children from Japan and Israel showed a 100% seropositivity in children above 8–10 years of age, while only 52% of children up to 2 years of age had hMPV-specific antibodies [15, 20, 21]. In the Netherlands, the seroprevalence of hMPV in children reaches 100% by the age of 5 years [1]. In South African children a lower seropositivity rate was observed up to the age of 10 months (22%), which can be partially explained by the clearance of maternal-derived antibodies. From 10 months onwards the seropositive rate increased to 92% in children aged 24–36 months [22]. The seroprevalence studies indicate that virtually all children are infected in early childhood. Since the first description of hMPV infection in children by van den Hoogen et al. [1], hMPV has been found in most parts of the world (Tabs 1 and 2): North America, Europe, Asia, and Australia [8, 9, 15, 16, 18, 23–41]. The virus has also been identified in HIV-infected and non-immunocompromised children from South Africa [8]. hMPV infections account for at least 4–8% of RTI in hospitalized children, although some studies report much higher prevalences (Tab. 1). In the general community hMPV infections account for at least 3% of children who visit a general practitioner or

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outpatient clinic for RTI (Tab. 2). The relative role of hMPV in respiratory syndromes of adults has not been well studied. In a large study of patients with RTI, the diagnostic outcomes for 685 specimens sent specifically for respiratory pathogen testing were compared. RSV was detected most frequently, in 126 (18%) of 685 samples obtained with patients with RTI. hMPV was the second-most-detected viral pathogen, found in 7% of the samples, and was isolated more frequently than parainfluenza viruses, adenovirus, rhinovirus, and influenza viruses types A and B [9]. In almost 200 premature infants and young children < 2 years of age with chronic lung disease or congenital heart disease in Buenos Aires, the impact of hMPV among other respiratory viruses causing RTIs was only 2%. RSV and parainfluenza virus were detected in 25% and 4%, respectively in this patient group [42]. In spite of the low number of infections caused by hMPV, severe lung disease was seen in some cases. hMPV has been isolated in 51–55% of patients with severe acute respiratory syndrome (SARS), but its contribution to that illness remains uncertain [43, 44].

Pathogenesis and host response Experimental animal models of hMPV infection have been reported, including both primates and rodents. The first published experimental hMPV infection model in cynomolgus macaques (Macaca fascicularis) confirmed that hMPV is a primary pathogen of the respiratory tract in primates [45]. The hMPV-infected macaques showed mild clinical signs of rhinorrhea corresponding with a suppurative rhinitis at pathological examination. In addition, mild erosive and inflammatory changes in the mucosa and submucosa of conducting airways, and an increased number of alveolar macrophages in bronchioles and pulmonary alveoli were observed. A close association between these lesions and the specific expression of hMPV antigen was shown by immunohistochemistry. Based on the antigen expression, viral replication mainly took place at the apical surface of ciliated epithelial cells throughout the respiratory tract. Pharyngeal excretion of hMPV showed a peak at day 4 post infection (p.i.) decreasing to zero by day 10, concomitant with a reduction in the number of infected epithelial cells. The mild upper respiratory tract disease as observed in these macaques corresponds to that in immunocompetent adults. Due to the fact the hMPV can replicate in the lower respiratory tract of cynomolgus macaques, more severe disease can be expected in immunocompromised patients. Some investigators have also shown that hMPV can replicate in the lungs of hamsters and cotton rats without producing recognizable clinical signs, although transient histopathological pulmonary changes were noted in cotton rats [46–50]. hMPV infection in other small-animal models such as ferrets and rabbits has been reported to induce a strong immune response [10], but the level of virus replication in these animals has not been reported. The study of

South-Africa

Netherlands

Germany

Madhi et al. [8]

van der Hoogen et al. [32]

Viazov et al. [18]

Jan. 02–May 02

Oct. 00–Feb. 02

Aug. 00–Sept. 01

Nov. 02–Apr. 03

Sept. 00–June 02

Mar. 00–Oct. 00

Finland

Jartti et al. [28]

Oct. 00–Sept. 02

Nov. 01–Nov. 02

USA

McAdam et al. [27]

Nov. 99–Oct. 01

June–Aug. 2002

USA

Germany

König et al. [26]

Esper et al. [31]

South Africa

IJpma et al. [22]

Sept. 01–June 02

Norway

France

Bouscambert-Duchamp et al. [25]

Nov. 03–Oct. 04

USA

France

Foulongne et al. [24]

Oct. 02–May 03 Oct. 03–May 04

Døllner et al. [29]

Germany

Wilkesmann et al. [23]

Nov. 02–May 03 Nov. 03–May 04

Study period

Mullins et al. [30]

Israel

Regev et al. [15]

Country

RT-PCR(1)

) 18 years

<2 years; RTI

All ages; RTI

Infants

<5 years

<5 years; RTI

Children; RTI

RT-PCR(2)

RT-PCR(1)

RT-PCR(3)

RT-PCR(3)

RT-PCR(2)

PCR(2)

3 months–16 years; acute RT-PCR(2) expiratory wheezing

PCR

RT-PCR(2)

RT-PCR(2)

RT-PCR(2)

RT-PCR(2)

RT-PCR(1)

Method

< 3 years; RTI < 6 months; apneu admitted to ICU

Children; RTI

Infants; <24 months

< 5 years; RTI

Children; RTI

< 5 years, RTI

Population

11/65

48/685*

14/196

54/668

26/641

50/236

12/291

54/868

15/87

8/137

6/94

50/589

114/637*

42/338

hMPV-positive/ total number of patients

Table 1. Incidence of human metapneumovirus (hMPV) infections in hospitalized children with respiratory tract disease

17.5%

6.5%

7.1%

8.1%

4%

21%

4%

6.2%

18%

5.8%

6.4%

8.5%

17.9%

10.8%

Prevalence

4–6 months

< 12 months

6–24 months

) 12 months

3–11 months

3–24 months

2–24 months

2–6 months

<24 months

1–2 year

Peak age

324 Adilia Warris and Ronald de Groot

France

Freymuth et al. [36]

Nov. 00–Mar. 01 Nov. 01–Feb. 02

2 summers & 2 winters 00-02

Mar. 01–Sept. 02

Aug. 01–Mar. 02

Jan. 00–May 02

Children

< 12 years; URTI < 17 years; asthma RT-PCR(3)

PCR(2) PCR(2)

RT-PCR(2)

RT-PCR(2)

) 18 years; RTI < 14 years; RTI

RT-PCR(4)

<2 years; RTI

19/337*

9/150 3/179

5/120

32/587

23/90

6.6%

6% 2%

4.2%

5.5%

25%

< 1 year

) 3 months

* Number of samples.

(1) All respiratory specimens obtained; (2) nasopharyngeal aspirates; (3) on common respiratory viruses negative nasopharyngeal aspirates; (4) nasal swabs.

(U)RTI: (upper) respiratory tract infection.

Thailand

Australia

Thanasugarn et al. [34]

Hong Kong

Peiris et al. [9]

Rawlinson et al. [35]

Italy

Maggi et al. [33]

Human metapneumovirus infection 325

Germany

Argentina

Italy

USA

Canada

USA

König et al. [26]

Laham et al. [37]

Principi et al. [38]

Williams et al. [39]

Bastien et al. [40]

Falsey et al. [41]

Nov. 99–Apr. 00 Nov. 00–Apr. 01

Oct. 01–Apr. 02

1976–2001

Nov. 02–Apr. 03

June 02–Sept. 02

Oct. 00–Apr. 01

1982–2001

Study period

Fit elderly > 65 years;RTI Young adults;RTI

RTI

<5 years; RTI or AOM

<15 years; RTI

<1 year; RTI

<3 years; RTI <6 months; apneu

<5 years; URTI

Population

RT-PCR(2) serology

RT-PCR(1)

RT-PCR(3)

PCR

RT-PCR(4)

PCR(3)

RT-PCR(3)

Method

4/233 11/167

66/445

49/248

41/1331

22/373

2/620

118/2384

hMPV-positive/ total number of patients

1.7% 6.6%

14.8%

20%

3.1%

6%

<1%

5%

Prevalence

<5 years, >50 years

6–12 months

Peak age

(U)RTI: (upper) respiratory tract infection; AOM: acute otitis media. (1) All respiratory specimens obtained; (2) nasopharyngeal aspirates; (3) on common respiratory viruses negative nasopharyngeal aspirates; (4) nasal swabs.

USA

Williams et al. [16]

Country

Table 2. Incidence of hMPV infections in non-hospitalised children with RTI

326 Adilia Warris and Ronald de Groot

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Skiadopoulos et al. [49] extended these observations to show that members of both hMPV lineages replicated efficiently in hamsters and that infection induced a high level of neutralizing antibodies and resistance to challenge that was effective against both homologous and heterologous strains. In addition, two species of nonhuman primates were also identified as useful models for the development of respiratory tract disease (chimpanzees) and for viral replication (African green monkeys). Chimpanzees developed a robust immune response, although the level of virus shedding was low. They were protected from disease following re-challenge with either strain. Therefore, chimpanzees may provide a useful nonhuman primate model for hMPV disease but are less ideal for studying virus replication. In contrast, rhesus macaques are not ideal animal models for the quantitation of hMPV replication, although they developed serum neutralizing antibodies following hMPV infection. hMPV replicated most efficiently in the respiratory tract of African green monkeys and the infected animals developed high level of hMPV serum-neutralizing antibodies effective against both lineages. A high degree of genetic relatedness and cross-protection was shown mediated by immunity to the highly conserved F protein. An human parainfluenza virus 1 (hPIV1) vector bearing the hMPV F protein provided protection against hPIV1 as well as both lineages of hMPV, indicating that such vectors might be useful as vaccines to protect against disease caused by both hPIV1 and hMPV. BALB/c mice and cotton rats are considered a good and convenient experimental model to study the pathogenesis of human RSV, another paramyxovirus. For hMPV, the BALB/c mouse has been described as a convenient animal model, with efficient viral replication and significant histopathological changes in the lungs associated with systemic and respiratory signs when large intranasal inocula are used [50–52]. A small animal experimental model of hMPV infections in BALB/c mice was developed to study mechanisms contributing to immunity and disease pathogenesis [51]. A biphasic kinetics of hMPV replication in lung tissue was shown with peak titers on days 7 and 14 p.i. Viable virus could be recovered from the lungs up to 60 days p.i., while genomic hMPV-RNA was detected up to 180 days p.i. The lung histopathology was modest and characterized by mononuclear cell infiltration in the interstitium starting on day 2, peaking on day 4 and decreasing on day 14 p.i, associated with bronchial and bronchiolar inflammation. This low pulmonary inflammatory response may contribute to the persistence of the virus. Hamelin et al. [50] did not detect any infectious virus in the lungs of BALB/c mice by day 21 after hMPV infection, although histopathological changes were still significant at that time, compared with those in sham-infected mice. Both duration and severity of inflammation around the alveoli was more limited in cotton rats compared to the BALB/c mice. Clinical symptoms of respiratory distress and weight loss were observed between days 4 and 10 p.i. in mice, but not in infected cotton rats. Recently, Alvarez and Tripp reported that hMPV RNA could

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still be detected * 180 days p.i. in the lungs of hMPV-infected mice and that such persistence results in an aberrant immune response [53]. The duration of pulmonary inflammation associated with a single hMPV challenge and the characterization of the consequences of this viral infection with respect to respiratory functions was further evaluated by Hamelin et al. [54]. The results showed that small amounts of viral RNA are still present in 33% in the lungs of hMPV-infected mice for at least 154 days p.i. and are associated with significant peribronchiolitis and perivasculitis. During the first 2–3 weeks, the inflammation mostly consisted of interstitial inflammation and the presence of alveolitis, as reported previously [50]. Over time, the inflammation became characterized by a prominent peribronchiolar and perivascular infiltrate, which was still significant on day 154. An increased number of PAS-positive cells in the central and peripheral airways up to day 12 p.i. were seen, suggesting increased mucus production. Concurrently with the time of maximal viral replication and histopathological score, the airway obstruction was most severe, followed by a gradually decrease but was still significant on day 70 p.i. Such inflammation seems to be responsible for chronic obstruction and hyperresponsiveness of the airways, which persist for > 2 months. These results reinforce the concept that severe paramyxovirus infections early during childhood can be associated with the development of asthma in children. Overall, these data suggest that BALB/c mice are more susceptible to hMPV infection than cotton rats on the basis of higher virus titers and levels of lung inflammation, combined with the absence of clinical signs. The absence of clinical signs has also been reported in hamsters and ferrets. These experimental models of hMPV infection show similarities with the pathogenesis, as far as studied, of RSV infection in humans. Histopathological assessment of hMPV infection on lung tissue obtained by open or transbronchial biopsies from five immunocompromised patients showed acute and organizing lung injury [55]. More specifically, areas of diffuse alveolar damage with hyaline membrane formation and foci of bronchiolitis obliterans/organizing pneumonia-like reactions were seen. In each sample, enlarged type II pneumocytes with smudged hyperchromatic nuclei resembling smudge cells found in adenovirus infection were detected. In contrast, smudge cells were not detected in lung tissue samples of four patients with lower RTIs due to RSV, rhinovirus, or parainfluenza virus. This might be a characteristic histopathological pattern of hMPV lower RTI. The histopathological pattern shown in this study with humans was distinct from those found in experimental infection of nonhuman primates, in which erosive and inflammatory changes were confined to the conducting airways [45]. Little is known about the nature of cytokine responses to hMPV. Human peripheral blood mononuclear cells in culture stimulated by hMPV revealed that classical CD4 T cell activation depending on antigen presentation and CD86-mediated co-stimulation occurred, comparable to

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stimulation by RSV [56]. In a study using BALB/c mice, it was shown that the indolent pulmonary inflammatory response was characterized by minimal innate immune and CD4 T cell trafficking, with low-level interferon (IFN)-a expression, induction of Th2-type interleukin (IL)-10 expression later during the infection, and delayed cytotoxic lymphocyte (CTL) activity [53]. Peak expression of macrophage inflammatory protein 1_, IFN-a, IL-4 and RANTES (regulated upon activation, normal T cell expressed and secreted) was related to the severity of the pulmonary inflammation in BALB/c mice [50]. hMPV was a weaker inducer of IFN-a, IL-10 and CCL5 than RSV, but induced higher levels of IL-6 instead. When looking at cytokine releases at the respiratory epithelial surfaces, hMPV, in contrast with RSV, seemed to be a poor inducer but elicited identical symptoms of similar severity [37]. Levels of the inflammatory cytokines IL-1`, TNF-_, IL-6, IL-8, IL-10, and IL-12 in respiratory secretions of infants < 1 year with an acute RTI, were two- to sixfold lower in those infected with hMPV compared to RSV. The higher levels of IL-6, inhibiting Th1 differentiation, combined with the lower levels of IFN-a induced by hMPV, are responsible for a weaker antiviral response leading to lower memory cells upon viral recall. This mechanism underlies the life-long, typically symptomatic re-infection with hMPV. IL-8 and RANTES in nasal secretions of children < 16 year admitted to hospital with acute expiratory wheezing were different from that reported in infections with RSV [57]. Patients with RSV infection had high concentrations of RANTES and varying levels of IL-8, whereas children with hMPV infection had lower concentrations of RANTES and higher levels of IL-8. It seems that mechanisms other than those known for RSV elicit symptomatic disease after infection with hMPV. Other mechanisms may include, although they are not limited to, (1) direct viral damage to the airways; (2) Th1 vs. Th2 polarization of the pulmonary immune response, leading to different clinical symptoms; and (3) chemokine-mediated inflammation. Further research is needed to elucidate the exact mechanisms of illnesses caused by hMPV. The fusion F surface glycoprotein has been identified as a major crossprotective antigen [48, 49]. In addition to the F protein, the subfamily Pneumovirinae of the paramyxoviruses also have a separate surface glycoprotein that is involved in attachment and is called the G protein. The F and G surface glycoproteins are the only significant neutralization antigens, and are major independent protective antigens [58]. hMPV virions appear to have three surface glycoproteins, the F, G and SH protein [59]. To analyze the contribution of these three glycoproteins in neutralizing and protective antibodies, hamsters were immunized intranasally with recombinant PIV type 1 expressing each glycoprotein individually from an added gene [60]. The F glycoprotein was shown to be the major contributor to the induction of neutralizing antibodies and protective immunity. The G and SH glycoproteins did not induce detectable neutralizing antibodies, and the contributions to protection were minor or negligible, respectively. This is in contrast

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with other paramyxoviruses (including RSV) in which the G protein stimulates high levels of neutralizing and protective antibodies. Cleavage of the precursor of the F glycoprotein is a prerequisite for infectivity and is an important determinant of virulence for most Paramyxoviridae. The contribution of the trypsin-dependent cleavage site R-Q-S-R in hMPV to its growth in vitro is well known. This requirement for trypsin in vitro raises the possibility that hMPV virulence is restricted by the inefficient cleavage of the F protein. Using recombinant hMPV in which the naturally occurring cleavage sequence was replaced by sequences not depending upon added trypsin in vitro, it was shown that replication in hamsters and African green monkeys was not changed. These results suggest that cleavage activation is not a major determinant of hMPV virulence [61]. Similar results were reported by others using a point mutation in the F gene that conferred intracellular cleavability of hMPV in a hamster model [62].

Diagnosis Four principal methods are used for the diagnosis of respiratory virus infections: virus isolation by culture, antigen detection, RNA or DNA detection, and serological study. For a virus that is not easily detected by virus isolation in the laboratory, it is of great importance to develop rapid, sensitive and reproducible diagnostic tests. The identification of the two hMPV serotypes, A and B, with each serotype divided into genetic sublineages, 1 and 2, has implications for the development of RT-PCR assays and serological diagnostic tests. Because of the unavailability of rapid antigen detection tests and because of its fastidious growth in cell cultures, RT-PCR has become the method of choice. RT-PCR procedures have proved to be more sensitive than virus isolation, and can detect genetically distinct hMPV strains [32]. The cytopathic effect is variable, with RSV-like syncytia formation or focal rounding and cell destruction. The search by van den Hoogen et al. [63] of a cell line with similar susceptibility for the four hMPV lineages and with enhanced detection of the virus by cytopathic effects, resulted in the generation of a subclone of Vero cells (Vero cell clone 118). This cell line is now used routinely for virus isolation in the Netherlands. Commercially available antibodies are not yet available. Monoclonal antibodies (mAb) recognizing conserved epitopes will be useful for rapid viral diagnostics using immunofluorescence (IF) or direct IF techniques as currently used for diagnosing RSV. Confirmation of hMPV causing the cytopathic effect is achieved by RT-PCR testing of the viral culture. Most RT-PCR protocols reported to date have relied on amplification of the L, N, or F gene with primer sequences mainly derived from the prototype strain 001 from the Netherlands. A comparative evaluation of RT-PCR assays performed in a LightCycler instrument for detection of

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331

hMPV in infected cell cultures showed positivity rates of 100%, 90%, 75%, 60%, and 55% using primers for the N, L, M, P, and F genes [64]. A second evaluation in the same study on nasopharyngeal aspirates positive for the hMPV N gene, the PCR positivity rate for the L, M, P, and F genes were 90%, 60%, 30% and 80%, respectively. From this study it can be concluded that RT-PCR assays aimed at amplifying the N and L genes, which code for two internal viral proteins and seemed to be more conserved regions of the genome, appear particularly suitable for detecting hMPV from both lineages [32, 64, 65]. Rapid and sensitive RT-PCR assays for the N gene (detection limit of 100 copies) have been developed allowing rapid amplification and detection of hMPV sequences directly from clinical samples in < 2 h [64, 65]. However, if inadequate primers are selected for PCR amplification, the hMPV detection might be underestimated. Serological testing only permits a retrospective diagnosis. Because infection is almost universal in childhood, a seroconversion or a * fourfold increase in antibody titers must be demonstrated to confirm recent infection. The serological survey performed in the Netherlands was based on an indirect IF assay using hMPV-infected cells [1]. A homemade ELISA method has also been developed using cell lysates of hMPV [41]. To conduct large serological surveys, simpler ELISA tests using viral proteins possibly derived from the two serotypes will be needed. mAbs used for diagnostic purposes can be directed against whole hMPV proteins or against individual proteins. Ishiguro et al. [66] used specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples, and these were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. Results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1% N protein; 40.8% M protein) when compared with immunofluorescence antibody (IFA) detecting whole proteins of hMPV. Western blot analysis using recombinant P protein was not successful due to nonspecific binding to human sera. The hMPV IFA-positive sera reacted with the F protein of hMPV by SDS-PAGE, but the signal was weak, suggesting that they were probably directed to conformational-type epitopes of the F protein [67]. Most of the antibodies detected by hMPV IFA were suspected to reaction with the F protein. These authors developed a baculovirus (Bac)expressed hMPV protein IFA and showed that it was more sensitive than hMPV IFA. An ELISA using the N protein of hMPV has been developed recently [50] and was reported to detect in 58 (81.6%) of 71 adults antibodies against the N protein of hMPV. In previous studies, 20 (100%) of 20 adults aged > 20 years had antibodies detected by both hMPV IFA [1, 20], and BacF IFA [67]. In this Bac-F IFA study, 192 of 200 serum samples of Japanese subjects between 1 month and 41 years of age showed concordant results with conventional IFA based on hMPV-infected LLC-MK2 cells [67]. The titers obtained by Bac-F were equal or higher than those obtained by the conventional IFA. From the Bac-F IFA study it can be concluded that the

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availability of large quantities of Bac-expressed hMPV F protein offers an opportunity to use this recombinant protein as a diagnostic reagent (EIA, IFA, immunoblot) and to study antigenic and immunogenic characteristics of the F protein. Studies like these are important and urgently needed to be able to develop an hMPV vaccine in the near future. Leung et al. [19] used vesicular stomatitis virus (which infects animals and seldom humans) to produce recombinant hMPV F protein in a seroepidemiological study. The ELISA-based system has many advantages over the methods used in previous studies. The amount of hMPV-specific antigen can be standardized for each assay, antibody to genotype-specific viral glycoproteins can be measured, and the results are based on defined criteria rather than subjective determinations of a positive result in an IFA. Two rapid antigen detection methods are available: an IFA test and an ELISA. This study compared the rate of virus detection in nasopharyngeal secretions by an indirect IFA with that by RT-PCR, and showed that the IFA with an anti-hMPV mouse mAb could detect hMPV in nasopharyngeal secretions with 73.3% sensitivity and 97.0% specificity compared with the results of RT-PCR [20]. ELISA is easier to perform in daily clinical practice and provides results that are more objective than IFA. Immunofluorescence staining of clinical specimens and shell vial centrifugation cultures (SVCC) are methods commonly used in clinical virology laboratories for rapid diagnosis, but need sensitive and specific mAbs. Landry et al. [68] evaluated mAb-8 to hMPV M protein for its utility in the rapid diagnosis of hMPV by both IF and SVCC methods. Detection of hMPV was similar in A549, Hep-2, and LLC-MK2 SVCC, and mAb8 staining was optimal on day 2 post inoculation. The ability to detect positive results by 1 or 2 days after inoculation is a great advantage over present conventional culture methods. The use of mAb-8 in IF staining of clinical specimens was, however, not successful due to nonspecific background staining. mAb-8 is commercially available (MAB8510, Chemicon International, Temecula, CA) and the results of its utility in the diagnosis of viral RTIs are awaited.

Clinical characteristics The first description of hMPV in children with lower RTI has been reported by a Dutch group that identified the virus in respiratory secretions [1]. Clinical symptoms were similar to those caused by RSV, ranging from upper RTI, severe bronchiolitis and pneumonia during the winter season. All 28 children observed were < 5 years of age, and 46% were < 1 year old. Asymptomatic carriage seems to be rare in children; no hMPV was detected in 400 infants without respiratory symptoms. The prevalence and clinical symptoms of hMPV-infected patients, identified by RT-PCR in respiratory samples obtained from patients in a

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university hospital, indicated that the prevalence and clinical severity due to hMPV infections are slightly lower than those of RSV infections during the winter season [32]. Most of the hMPV-positive patients were children < 2 years old without any underlying illnesses. hMPV was found significantly less frequently than RSV in children < 2 months old. Of the 31 hMPV-positive children < 2 years old, only 4 (31%) were < 2 months old, whereas 43 (35%) of the 122 hRSV-positive children < 2 years old were also < 2 months old. Others have found that the mean age of patients infected with hMPV was slightly lower than that compared to RSV [39]. Of the hMPV-positive patients who were > 5 years old, most had other diseases (e.g., cystic fibrosis, leukemia, and non-Hodgkin lymphoma) or had recently received bone marrow or kidney transplantation, indicating an association with immunosuppression. Two severely immunocompromised patients died due to progressive respiratory failure with hMPV as the sole pathogen detected [69]. In studies involving young and elderly adults, hMPV caused more severe disease in fragile elderly than in healthy elderly or young adults [4, 41]. Clinical symptoms in children < 10 years of age (n = 238) due to hMPV infection include cough (82%), rhinitis (67%), fever (72%), respiratory distress (71%), wheezing (59%), and retractions (54%) [29, 30–32, 37, 39, 69]. Specific clinical syndromes caused by hMPV seem to differ from that caused by other respiratory viruses. Williams et al. [39] tested respiratory specimens over a 25-year period in the US from previously healthy children. Infection due to hMPV was more likely to be associated with bronchiolitis and less likely to be associated with croup than infection due to (para)influenza virus. hMPV infection was less likely to be associated with pneumonia than was infection with RSV or influenza virus. Various studies show frequent involvement (16–24%) of hMPV in acute bronchiolitis in infants, a percentage only second to RSV [18, 33, 70]. hMPV is associated with a substantial number of URTI episodes in otherwise healthy outpatient children with clinical illnesses similar to those associated with other common viruses, including frequent acute otitis media [16]. Studies examining the role of hMPV with respect to exacerbations of asthma have yielded conflicting results [35, 39, 57]. Two studies in adult patients with chronic obstructive pulmonary disease (COPD) showed that hMPV could be detected in 2.5% of the hospitalized COPD patients with an acute exacerbation [14, 71], while no hMPV was detected in stable COPD patients. Although there is no doubt that some patients with asthmatic exacerbations have hMPV infection, whether or not the virus is associated more frequently than other respiratory viruses with these exacerbations is not yet clear. Remarkably, a history of asthma or a family member with asthma was more often associated with hMPV (16% and 67%, respectively) than with RSV (0% and 30%, respectively) [9]. The similar seasonality and susceptible population shared by several respiratory viral infections will result in prevalent co-infection of hMPV with other respiratory viruses. This might lead to an underestimation of the

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percentage of hMPV-positive samples identified in studies in which only samples negative for other respiratory viruses were tested (see also Tabs 1 and 2). Co-infection rates of 5–10% with one or more respiratory viruses have been demonstrated in several studies searching for the causative pathogen of RTI. Because the epidemic seasonality for RSV coincides with that for hMPV, the potential exists for RSV/hMPV co-infections. Several studies have identified cases of lower RTI in which evidence for the presence of both RSV and hMPV has been detected [23, 33, 39, 72]. Dual infection with RSV and hMPV was more frequent in infants with severe disease (i.e., those who needed supplementary oxygen) and even more frequent in infants with severe disease admitted to the intensive care unit for mechanical ventilation [26, 72]. Foulongne et al. [24] showed that another respiratory virus was detected in 32% of hMPV-positive samples obtained from children < 5 years of age with RTI, and all but one of these co-infections involved RSV. Duration of hospitalization and requirement for supplemental oxygen was significantly higher in hMPV/RSV co-infected children. Greensill et al. [73] collected non-bronchoscopic bronchoalveolar samples from 30 infants < 48 weeks of age ventilated with RSV bronchiolitis diagnosed by antigen testing. Detection of hMPV was performed by RT-PCR of the M, F, and N genes. In 16 of the 24 infants with a positive RT-PCR for RSV in the bronchoalveolar lavage sample, genomic hMPV was also detectable. This high rate of co-infection raises the possibility that co-infection with RSV and hMPV is a determinant of disease severity. These results were confirmed by others studying the association between severe bronchiolitis and dual infection by RSV and hMPV in children < 2 years of age who were admitted to the hospital. Co-infection with both viruses conferred a tenfold increase in relative risk of admission to a pediatric intensive care unit for mechanical ventilation. A high case incidence (52%) of hMPV infection has been described in association with hospital admission of patients with severe acute respiratory syndrome in Hong Kong [43]. In contrast, others found a similar rate of bronchopneumonia in infants infected with hMPV alone as in dual infections [33]. Wilkesmann et al. [23] did not find a lower illness severity when comparing hMPV-infected children with matched RSV-infected children without hMPV co-infections. On the other hand, the seasonal distribution of hHMP and RSV may differ in specific geographic areas as demonstrated in studies from Argentina and Hong Kong where co-infections were not or infrequently observed [9, 37]. The peak of hMPV in these countries becomes prevalent in late winter and early spring. It is likely that by the development of more sensitive detection methods, dual or mixed infections will be increasingly recognized, and do not necessarily result in more severe infection. A positive RT-PCR test result does not differentiate between active infection and prolonged shedding after a recent acute infection that has been terminated. It is currently not known whether hMPV infection leads to an increased susceptibility to secondary bacterial infections. The absence of sensitive

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tools to diagnose bacterial pneumonia has been an obstacle to defining the role of bacterial co-infection in children with virus-associated pneumonia. In a hypothesis-generating study involving a cohort of children randomized to receive the 9-valent pneumococcal vaccine or placebo, children were tested for the presence of hMPV by a nested RT-PCR when admitted to the hospital with a lower RTI. In both HIV-uninfected and HIV-infected children the incidence of hMPV-associated lower RTI was reduced by 46%, and the incidence of clinical pneumonia was reduced by 58% [74]. These data, combined with comparable finding for other respiratory viruses [75], suggest that respiratory viral infections as caused by hMPV predispose to pneumococcal co-infection and that bacterial-viral co-infections are important in the pathogenesis of virus-associated pneumonia in children. The socioeconomic impact of hMPV infection on children and their households is not well known. It is reported that household contacts of hMPV-infected children, like influenza-infected children, fell ill significantly more frequently, required more medical visits, received more anti-pyretic prescriptions, and were also absent more frequently from work or school, than those of RSV-infected children [76]. These findings suggest that hMPV infection in children considerably affects their families.

Vaccination No vaccines, antibodies (monoclonal or polyclonal), or chemotherapeutic agents are currently licensed for use to prevent or treat hMPV infections. However, ribavirin and polyclonal antibody preparations (IVIG), used in the therapy and prevention of RSV infections in children, are known to have broad-spectrum activity and can inhibit different viruses. In tissue culture-based assays ribavarin and IVIG preparations containing high titers of hMPV-neutralizing antibodies were found to inhibit hMPV replication [77]. The clinical utility of these findings needs to be tested. Ulbrandt et al. [78] describe the generation of a panel of neutralizing mAbs that bind to the hMPV F protein (like palivizumab for RSV). A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. Studies so far have not shown that mAbs to the F protein alone can protect animals from virus challenge. This might be the first step to use such an mAb prophylactically to prevent lower RTI caused by hMPV. Two of the antibodies found, mAb 234 and mAb 338, have characteristics comparable to palivizumab that make them appealing for further studies. Despite similarities in structure of hMPV and RSV, the F proteins of these two viruses share only a 33% amino acid sequence identity; consequently, antisera generated against either RSV or hMPV do not neutralize across the Pneumoviridae group [77].

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The contribution of hMPV to pediatric RTIs suggests that it will be important to develop a vaccine against this virus in combination with those being developed for RSV and parainfluenza viruses. The circulation of two serotypes of hMPV might have implications for the development of vaccines. Studies in cynomolgous macaques showed that re-infection is suppressed by high titers of virus neutralization antibodies against the homologous virus and far less by heterologous virus neutralization antibodies [7]. Others report cross-protection and reciprocal cross-neutralization studies in experimental models of hMPV infection, showing that cross-protection is induced at a high level, consistent with a single serotype [10]. The most relevant test of the importance of genetic diversity is whether or not viruses of one genotype induce greater protection against the homologous virus than against the heterologous one. Although difficult to assess, the extent of cross-protection is important to estimate to ultimately develop a monovalent or bivalent vaccine formulation. One of the difficulties in assessing the cross-protection is the occurrence of re-infections. Virus neutralization antibody titers in children > 5 years of age are higher than in those of 1–2 years of age, which suggests that re-infections may occur frequently. Before the discovery of hMPV in 2001, several groups were working with molecular systems that allow the generation of recombinant paramyxoviruses from plasmid DNA copies of virus genes and virus genome. Similar strategies using this technique referred to as reverse genetics, have been rapidly employed to study the replication of hMPV and to generate live attenuated hMPV vaccine candidates. Foreign genes such as the reporter gene for green fluorescence protein were inserted into the hMPV genome and expressed, which effectively defined the transcription start and gene end signals [59]. Reverse genetics has been used to rescue both strains from Canada and the Netherlands entirely from complementary DNA (cDNA). Because the viruses are made from DNA copies, chimeric viruses can be made with the use of the antigenic protein of one virus inserted into the genome of another virus. Neutralizing antibody responses can be induced by such a chimeric virus, protecting the host against challenge with hMPV strains. MacPhail et al. [48] identified both small-animal and primate models for evaluation of vaccine candidates. These kind of models are not only wanted to evaluate the effectiveness and safety of vaccine candidates, but also for future hMPV antiviral drugs, and therapeutic and prophylactic mAbs. Their results showed that Syrian golden hamsters, ferrets and African green monkeys supported hMPV replication in the lower and upper respiratory tract efficiently, resulting in high levels of hMPV neutralizing antibodies. More recent work by Biacchesi et al. [79] investigated the function of the SH and G gene to develop a live-attenuated vaccine. Previously, it was shown that deletion of a number of RSV genes such as the SH and G gene was not deleterious to the virus and such RSV mutants have been evalu-

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ated in primates as putative live attenuated vaccine candidates [80]. The recovered recombinant hMPV was analyzed in vitro and by experimental infection in hamsters [78]. Deletion of a single gene, either SH or G, showed similar replication as wild-type virus in cell culture. This means that the F protein alone is sufficient to mediate attachment and fusion to cells in the absence of the other two surface proteins. In addition, hMPV G and SH are not required for the efficient assembly or release of progeny virus. Mutant hMPV strains lacking the SH and/or G genes were immunogenic and highly protective against hMPV challenge and represent promising vaccine candidates. The mutants lacking G (both 6G and 6SH/G) showed reduced replication, in contrast to the mutants lacking only the SH gene, and represent promising vaccine candidates that needed to be studied further in nonhuman primates such as African green monkeys. This was performed in a following study of the same group. Experiments were performed with recombinant hMPV in which the SH, G, or M2-2 gene or ORF was deleted by reverse genetics [81]. These mutants were evaluated for replication and vaccine efficacy following intranasal and intratracheal administration to the respiratory tract of African green monkeys. Each gene-deletion virus was highly immunogenic and protective against wild-type hMPV challenge. The 6G and 6M2-2 viruses showed a markedly reduced replication, in contrast to 6SH virus, and are promising vaccine candidates appropriate for clinical evaluation. Deletion of the hMPV M2-2 protein resulted in a decrease in RNA replication and an increase in gene expression in cell culture [82]. The consequence of this might be that this mutant provides greater antigen synthesis and immunogenicity in vivo. Tang et al. [83] used a different approach to generate an hMPV vaccine candidate. They utilized an attenuated PIV type 3 (PIV3) vector to deliver the hMPV F protein with the aim of inducing both humoral and cellular immunity against hMPV infection. The use of this vector is not new, and has been used in the development of RSV and other respiratory pathogens vaccine candidates [84, 85]. In this study, the chimeric bovine/human PIV3/ hMPV F2 was shown to elicit hMPV-specific as well as virus-specific antibodies and T cell responses in African green monkeys. The bovine/human PIV3 vectored hMPV vaccine might, therefore, function as a bivalent vaccine for immunization against both hMPV and PIV3 infections. The development of a bovine/human PIV3 vector-based vaccine expressing both the F protein of hMPV and RSV should provide protection against these three respiratory pathogens that cause significant disease in young children. However, the genetic stability of such a vaccine should be addressed first. The possibility of the host developing immunity to the vector itself is a matter of concern especially when there is a need to boost the primary vaccination. A recent trial in young infants showed that multiple doses of an attenuated PIV3 did not result in inhibitory vector immunity when the intervals between the vaccinations were timed appropriately [86].

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Conclusions The epidemiology and clinical manifestations associated with hMPV have been found to be reminiscent of those of the RSV, with most severe RTI occurring in young infants, elderly subjects, and immunocompromised hosts. The seasonal distribution resembles that of RSV and influenza virus infections, with recurrent epidemics during the winter months. hMPV is the second most important cause, after RSV, of viral lower RTI in children. hMPV infections account for at least 4–8% of the RTI in hospitalized children. In the general community, hMPV infections account for at least 3% of patients who visit a general practitioner for RTI. Interestingly, the rates of detection of hMPV have been generally higher in retrospective than prospective studies, an observation consistent with some selection bias. Larger prospective studies, not limited to the typical respiratory virus season, not limited to testing respiratory samples negative for the other respiratory viruses, and using appropriate controls need to be conducted. Diagnosis is made by RTPCR assays aimed at amplifying the N or L gene. Additional research to define the pathogenesis of this viral infection and the host’ specific immune response will enhance our knowledge to guide the search for preventive and therapeutical strategies. The development of a simple direct IF assay on nasopharyngeal samples in the near future will certainly enhance our understanding of the role of hMPV in RTIs in humans. Reverse genetics technology is currently being used to develop multivalent vaccines against hMPV and a variety of other important respiratory viruses such as RSV.

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Avian influenza viruses: a severe threat of a pandemic in children? John V. Williams Division of Pediatric Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN, USA

Abstract Influenza virus is a leading cause of human respiratory illnesses, causing significant annual morbidity and mortality. The greatest severity of illness due to seasonal influenza occurs in infants less than 6 months of age and the elderly. In recent years, avian influenza virus infections with high mortality have occurred in humans. Many of these avian influenza virus infections have occurred in children, and unlike seasonal influenza, the most severe disease and highest death rates have occurred in children and young adults. Treatment and prevention options for avian influenza viruses are limited at present, although much research effort is directed toward these areas. Avian-derived influenza viruses are potential causes of pandemic influenza that could have a dramatic impact on children worldwide.

Introduction Influenza virus is a leading cause of acute respiratory infection (ARI) worldwide and is associated with substantial morbidity and mortality [1–3]. Influenza is an important respiratory pathogen in young children, with the greatest morbidity and rates of hospitalization in young infants [4, 5]. Several features of the biology of the influenza virus allow novel viruses to emerge into the human population, causing pandemics such as the 1918 “Spanish flu” pandemic. In recent years, highly pathogenic avian influenza viruses (HPAI) have crossed the species barrier and caused human infections with very high mortality. Of major concern to pediatricians is the fact that severe disease has occurred even in previously healthy children, a phenomenon quite distinct from seasonal influenza. HPAI viruses have the potential to cause a pandemic of virulent influenza that would have far greater effects on children than either seasonal influenza or historical pandemics.

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Influenza virus Influenza viruses are enveloped viruses, containing a segmented RNA genome, and are members of the family Orthomyxoviridae. Influenza viruses are divided into three types: A, B, and C. Types A and B cause most human influenza in annual winter epidemics. Influenza A viruses are further divided into subtypes based on the hemagglutinin (HA) and neuraminidase (NA) genes. The WHO nomenclature for classification of influenza strains is as follows: Type (A, B, or C)/Geographic origin/Isolate #/Year of isolation/subtype (HA and NA); e.g. A/Sydney/5/97 (H3N2). There are 16 HA subtypes and 9 NA subtypes; HA 1, 2, and 3, and NA 1 and 2 typically circulate in humans. HA is the viral protein that binds to sialic acid on host cells and is a major determinant of species tropism. The HA proteins of the types that usually circulate in humans (H1, H2 and H3) preferentially bind to the particular sialic acid moieties present on human respiratory epithelial cells. Conversely, HA in viruses that circulate in birds bind with far greater affinity to sialic acid moieties present in avian cells. The other HA and NA subtypes primarily circulate in migratory shore birds, with a few subtypes occurring naturally in horses. This provides a reservoir of novel influenza HA and NA subtypes in nature to which humans have no pre-existing immunity. The mechanisms of influenza virus replication allow for two additional sources of viral mutation, to evade host immunity or introduce completely novel subtypes. The influenza virus genome consists of segmented, single-stranded RNA molecules. The RNA polymerase enzyme that copies the genome to produce progeny virions is error-prone and, unlike DNA polymerases, has no inherent proofreading activity to correct mistakes. This leads to point mutations and progressive variation in protein sequences called ‘antigenic drift’. Furthermore, viruses with segmented genomes can exchange or reassort genome segments when two different viruses infect the same cell. Reassortment leads to a complete change of the HA or NA proteins. If the new type has not circulated in humans recently, there is no pre-existing immunity in the population. The encoding of HA and NA by separate RNA molecules facilitates the reassortment of these genes in animals simultaneously infected by two different subtypes. For example, H3N1 virus has been recovered from pigs simultaneously infected with swine flu virus (H1N1) and the Hong King virus (H3N2) [6]. This abrupt change of a major immune target is called ‘antigenic shift’ and is a major source of pandemic strains of influenza.

Pandemic influenza Pandemic influenza is defined as virulent human influenza that causes a global outbreak, or pandemic, of serious illness. There are three requirements for a pandemic: (1) novel HA or NA types (thus no pre-existing

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immunity in population); (2) a highly virulent strain of influenza virus; and (3) easy human-to-human spread. A worldwide influenza pandemic occurred in 1918. At least 21 million people (possibly 50–100 million) worldwide died from the ‚Spanish flu’ – the most devastating plague in human history. Many of the deaths were in healthy young adults and two-thirds occurred during a 4-month period [7, 8]. The propensity for high mortality in previously healthy young adults was a very unusual feature of the 1918 influenza virus. Routine seasonal influenza viruses cause the highest mortality in the very young (< 6 months of age) and the elderly (> 65 years old). The increased virulence of the 1918 virus in healthy young persons has been hypothesized to be due to a “cytokine storm”, but the biological mechanisms are not known. This has disturbing parallels to the mortality pattern exhibited by the recent H5N1 avian influenza viruses (see below). Two pandemics of influenza have swept the world since the “Spanish flu“ of 1918 (H1N1): the “Asian“ flu pandemic of 1957 (H2N2) and the “Hong Kong“ flu pandemic of 1968 (H3N2). These pandemics were milder, with an estimated 2 million deaths in 1957 and 1 million deaths in 1968. These data suggest that flu pandemics occur when the virus acquires a new HA and/or NA. The pandemic of 1957 probably infected more people than 1918. However, the availability of antibiotics to treat the secondary infections that are the usual cause of death resulted in a much lower death rate. In addition, the 1918 influenza virus was likely more virulent than the viruses from the 1957 and 1968 pandemics. In 1997, Taubenberger et al. [9] reported partial sequences of five influenza genes recovered from the preserved lung tissue of a U.S. soldier who died from influenza in 1918. Continued work by this group led to the sequencing of the entire genome of the 1918 virus [10–16]. Phylogenetic analysis of the genomic sequence data suggests that the 1918 virus was derived from an avian-like influenza virus a short time (perhaps a few years) before the start of the pandemic, but the origin is still not known. This virus has been recreated in a highly secure biocontainment facility at the CDC using the technique of reverse engineering [15]. Studies of this recreated 1918 virus in mice suggested that the HA and NA from the 1918 strain are major determinants of virulence [15]. The contribution of other genes to virulence has not been completely determined [12, 17–20]. However, this work suggests that a reassortant human influenza virus containing only an HA and/or NA gene from a highly virulent strain could cause severe disease and high mortality similar to that caused by the 1918 virus. This has important implications for the possible outcome of a pandemic that could occur due to avian influenza viruses.

Avian influenza virus Avian H5N1 influenza is an emerging pathogen in both avian and human populations. Highly pathogenic strains of H5N1 have caused numerous out-

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breaks in commercial poultry flocks in recent years, with major economic consequences [21–23]. Avian influenza viruses carry novel HA types such as H5, H7 and H9 but generally do not replicate efficiently in humans. Part of this species barrier is due to the distinct preferences for HA binding to mammalian or avian sialic acids, as mentioned above. However, reassortment with human strains could allow a recombinant virus to emerge that is both highly pathogenic and highly infectious for human hosts. This reassortment between human and avian strains is thought to occur primarily in pigs, which are susceptible to infection by both strains [6]. The close proximity of humans, swine and birds in areas with endemic HPAI is of major concern as a potential source of a pandemic strain. It is also possible for avian influenza viruses to directly infect humans. Numerous outbreaks of such novel avian influenza viruses in humans have been reported in recent years. Almost all have been epidemiologically linked to close contact with poultry, chiefly chickens or ducks, and humanto-human transmission has rarely been documented. There have been over 200 cases of human disease due to H5N1 influenza to date, with an overall 57% mortality (Tab. 1). Most of the deaths have been in previously healthy young adults and children, suggesting that H5N1 possesses significantly greater virulence than usual seasonal influenza. Again, for H5N1, virtually all cases have occurred in those with close contacts to poultry, with only a few likely cases of person-to-person transmission [24–26]. Viral determinants of virulence of the H5N1 strain have been established in birds and mice, and include a polybasic HA cleavage site (containing multiple basic amino acids) and point mutations in HA and the RNA polymerase [27]. Influenza HA must be cleaved by host proteases to be active, and normally is cleaved only by enzymes present in the respiratory tract. HPAI viruses have a polybasic HA cleavage site that is cleaved by enzymes present in many cells, thus allowing spread beyond the respiratory tract. The polybasic cleavage site also determines virulence in ferrets and cats [28–31]. These changes have not been proven to be determinants of virulence in humans, but it is likely that they are important for highly pathogenic strains.

Spread of H5N1 influenza in avian populations Since the current strains of H5N1 influenza emerged in poultry in Southeast Asia, continuous spread to both neighboring and distant countries has been observed. Migratory waterfowl and shorebirds are carriers of avian influenza viruses, and often intermingle with domesticated fowl in open-air farms and markets. Poultry industry and governmental efforts to control the spread of H5N1 in avian populations is critical and often consists of culling large numbers of birds. These efforts have significant economic effects and have been resisted in some locations. Transmission between geographic areas has also occurred due to importation (legal and illegal) of exotic birds

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Table 1. Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported to the WHO 2003

Country

2004

2005

2006

Total

Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases 0

0

0

0

0

8

5

Deaths

Azerbaijan

0

8

5

Cambodia

0

0

0

0

4

4

2

2

6

6

China

0

0

0

0

8

5

11

7

19

12

Djibouti

0

0

0

0

0

0

1

0

1

0

Egypt

0

0

0

0

0

0

14

6

14

6

Indonesia

0

0

0

0

17

11

34

28

51

39

Iraq

0

0

0

0

0

0

2

2

2

2

Thailand

0

0

17

12

5

2

0

0

22

14

Turkey

0

0

0

0

0

0

12

4

12

4

Vietnam

3

3

29

20

61

19

0

0

93

42

Total

3

3

46

32

95

41

84

54

228

130

Source: http://www.who.int/csr/disease/avian_influenza/country/cases_table_2006_06_20/en/ index.html ; accessed 26 June 2006.

[32]. As of 13 June 2006, influenza A (H5N1) had been reported in migratory birds or poultry flocks in Africa (Burkina Faso, Cameroon, Côte d’Ivoire, Djibouti, Egypt, Niger, Nigeria, and Sudan), Asia (Afghanistan, Azerbaijan, Cambodia, China, Georgia, Hong Kong, Kazakhstan, India, Indonesia, Iraq, Iran, Israel, Jordan, Malaysia, Mongolia, Myanmar, Palestinian Autonomous Territories, Pakistan, Thailand, Turkey, and Vietnam), and Europe (Albania, Austria, Bosnia-Herzegovina, Bulgaria, Croatia, Czech Republic, Denmark, France, Germany, Greece, Hungary, Italy, Poland, Romania, Russia, SerbiaMontenegro, Slovakia, Slovenia, Sweden, Switzerland, Ukraine, and the United Kingdom) [33]. The spread of the virus has been associated with the migration of wild birds from Asia [34], suggesting that apparently healthy birds can carry the virus over long distances [35]. Most experts consider it highly likely that H5N1 viruses will reach North and South America and intense surveillance activity is being conducted in wild bird populations. The spread of human cases closely parallels the spread in birds (Tab. 1).

Epidemiology and clinical features of avian influenza in children Avian influenza viruses have caused a spectrum of disease in humans, including typical influenza-like illness, conjunctivitis and severe respiratory disease. More recent outbreaks, and the H5N1 virus in particular, have been

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more severe and more frequently associated with fatal illness. In general, avian influenza virus infections in children have been no less severe than in adults. Mild respiratory disease was reported in 2 Dutch children due to reassortant human-avian influenza viruses in 1994 [6]. In 1997, a 3-year-old boy in Hong Kong died of acute respiratory failure and multiorgan system dysfunction due to an H5 influenza strain. Genomic sequencing and analysis of the virus showed that it was an H5N1 avian strain [36]. During that outbreak, 5 other children under the age of 18 were infected. A 13-year-old girl died of acute respiratory failure and multiorgan system dysfunction, a 2-year-old boy was hospitalized for 3 days with pneumonia and 3 other children experienced uneventful upper respiratory infection. The children who died were previously healthy. A total of 12 cases were reported, with more severe disease and higher fatality rate in the adults [37]. Outbreaks of avian influenza have continued since 1997, and have spread to broader geographic areas, particularly H5N1. There were 2 confirmed and 1 probable H5N1 cases in Hong Kong in February of 2003 [38]. A 33-year-old man developed fatal progressive respiratory failure and his 8-year-old son recovered from respiratory disease after a prolonged hospitalization. Both had profound lymphopenia, hypoxia and consolidation of chest radiographs. The family also had a 7-year-old daughter who had died of a febrile pneumonia 1 week prior to the father and brother’s illnesses, but she had not been tested for influenza. Two cases of H9N2 avian influenza infection of humans occurred in Hong Kong, one a child, with typical influenza symptoms of fever, rhinorrhea and cough [39]. Both patients fully recovered. There was also a large outbreak of H7N7 in the Netherlands in 2003 on poultry farms, with infection of both pigs and humans [40]. There were a total of 89 human cases, primarily among poultry workers. Most of the illnesses were conjunctivitis, with only a few typical influenza-like illnesses. There was one fatality, a veterinarian who visited one of the farms and developed acute respiratory distress syndrome (ARDS). Most of the cases were attributed to direct contact with infected poultry, although there were three possible instances of person-to-person transmission. During 2003 and 2004, there were 34 cases of confirmed human H5N1 infection in Thailand and Vietnam [41–43]. Seven of the 12 laboratory-confirmed cases in Thailand were boys age 2–13, all of whom presented with fever, cough and tachypnea. Lymphopenia and elevated transaminases were noted in most. All 7 boys had abnormalities on chest radiograph consisting of focal or multifocal consolidation, and all required mechanical ventilation. Five of these 7 children died, and overall mortality in the Thailand outbreak was 8/12 (67%). In January 2004, 10 human H5N1 infections were reported in Vietnam. Seven patients were less than 18 years old, with a mean age of 12 and the youngest 5 years. All patients presented with fever, tachypnea, cough, and hypoxia. Five also had diarrhea, but none of the children had

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351

myalgia, rash or conjunctivitis. Similar to the Thailand cases, the most common laboratory abnormalities noted were lymphopenia, thrombocytopenia and elevated transaminases. All had extensive consolidations on chest radiographs, which progressed despite aggressive therapy. All developed respiratory failure requiring mechanical ventilation, and 7/8 children died, despite aggressive supportive care and treatment with oseltamivir, ribavirin and/or steroids for ARDS. Two other children were identified with probable or confirmed H5N1 infection during the same outbreak [44]. A 9-year-old girl presented with fever, watery diarrhea, shock and lethargy. Initial laboratory tests including cerebrospinal fluid were normal. She had fulminant shock, became comatose and died within 24 hours. No influenza tests were performed. However, 8 days later, her 4-year-old brother presented with fever, headache, vomiting and profound watery diarrhea. His initial laboratory values were remarkable only for elevated transaminases. However, he developed pneumonia and lethargy, progressing to coma, and died of respiratory failure 5 days after admission. During his hospitalization, he developed lymphopenia, thrombocytopenia, and bilateral infiltrates on chest radiograph. Cerebrospinal fluid was remarkable only for elevated protein. He was diagnosed with unexplained encephalitis, but postmortem testing detected H5N1 influenza by RT-PCR in cerebrospinal fluid, serum, throat and rectal swabs, and culture of cerebrospinal fluid grew H5N1 influenza virus. Thus, it is highly likely that his sister had been infected with H5N1. Notably, neither child initially presented with respiratory symptoms and the sister never had respiratory disease. Both had had frequent exposure to ducks and chickens at home and there were no other cases in the family. As of April 30, 2006, a total of 205 cases of human H5N1 infection had been reported to the WHO [45]. One-half of these occurred in patients less than 20 years old, with a range from 3 months to 72 years. Twenty-one cases (10%) were in children < 5 years, 32 (16%) were in children from 5 to 9 years, and 49 (24%) were in 10–19-year olds. There was a male predominance in the younger cases, with a male:female ratio of 1.5 in the 53 cases < 10 years old. The sex ratio was equal in all other age groups. It is not known whether this finding reflects gender-specific epidemiological risk factors or biological differences.

Clinical presentation and outcome The reported symptoms of avian influenza in children have ranged from typical influenza-like symptoms (e.g., fever, cough, sore throat, and muscle aches) to eye infections (conjunctivitis), pneumonia, acute respiratory distress, viral pneumonia, and other severe and life-threatening complications (Tab. 2). The majority of children have presented with fever and respiratory symptoms, although in the Vietnam cases, diarrhea was prominent. Notably,

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Table 2. Clinical features of H5N1 avian influenza in children Reference

[36]

[42]

[41, 43]

Number of patients

7

7

9

Male (%)

57

43

56

Previously healthy (%)

71

100

87

Fever (%)

100

100

100

Cough (%)

43

100

90

Rhinorrhea (%)

71

–*

35

Dyspnea (%)

–*

100

69

GI symptoms (%)

29

57

25

Pneumonia (%)

29

100

100

Ventilated (%)

29

86

100

Mortality (%)

29

86

90

*Not reported.

a number of pediatric cases have presented without any respiratory symptoms but with severe gastrointestinal (GI) or neurological symptoms. A key historical element of virtually all cases is a recent exposure to domestic or wild birds. A high index of suspicion is necessary to consider the diagnosis. In most cases, the onset of symptoms occurs within 1 week of the bird exposure. The median duration of symptoms prior to hospitalization was 4 days (range 0–18 days). Prominent laboratory findings include leukopenia (especially lymphopenia), thrombocytopenia and elevated liver transaminases (Tab. 3). Most pediatric patients do not manifest hemoconcentration; this finding and the prominent respiratory symptoms help distinguish the illness from dengue virus infection in dengue-endemic areas. Renal failure, hyperglycemia and hemophagocytosis have been noted in some patients. Most have abnormal chest radiographs at presentation. Many patients develop complications such as respiratory failure requiring assisted ventilation, ARDS, shock and multiorgan system dysfunction. Severe infections have typically progressed rapidly, with a median duration of symptoms prior to death of 9 days (range 2–31 days). The proximate cause of death is usually respiratory failure. The overall mortality in the cumulative human H5N1 cases reported to date is 59% (Tab. 1). However, the highest mortality rates occurred in patients age 10–19 (73%, n = 49), 20–29 (65%, n = 45), 30–39 (61%, n = 33) and 40–49 years (45%, n = 11). Very high mortality rates were also observed in children < 5 years (43%, n = 21) and 5–9 years (41%, n = 32). The lowest rates were in the patients older than 50 (18%, n = 11) [45]. This distribution

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Table 3. Laboratory and radiological findings of H5N1 avian influenza in children [36]

[42]

[41, 43]

Leukopenia (%)

29

100

100

Thrombocytopenia (%)

29

86

44

Elevated transaminases (%)

43

80*

71

Radiographic infiltrates (%)

29

100

100

Reference

*Transaminases not reported for two patients.

is reminiscent of the mortality associated with the highly virulent 1918 pandemic virus and, again, is quite unlike the mortality curve associated with seasonal influenza. Pediatric mortality in cases reported outside of Thailand and Vietnam vary widely (Tab. 4). Autopsy examination reveals severe lung pathology, including necrotizing diffuse alveolar damage with patchy and interstitial paucicellular fibrosis [46, 47]. H5N1 has been detected in lung tissue by RT-PCR up to day 17 of illness. H5N1 has been isolated in respiratory specimens, blood, GI tract, and cerebrospinal fluid. However, it is not clear whether viral replication and direct cytopathology occurs in tissues outside of the respiratory tract, or whether the major systemic effects are due to cytokine responses. Virus replication was not detected outside of the lungs and tonsils during experimental infection of macaques [48]. However, the same investigators recently reported that experimental H5N1 infection of cats led to virus replication in multiple extra-respiratory tissues, including brain, liver, kidney, heart and GI tract [28]. Further studies in humans are needed to further elucidate the mechanisms of H5N1 pathogenesis.

Diagnosis Timely diagnosis of avian influenza virus infections is critical to limit spread, initiate early therapy and alert health authorities. The usual diagnostic methods for detecting seasonal influenza A and B include rapid antigen tests, viral culture, immunofluorescent antibody assays and RT-PCR [49]. In countries where avian influenza activity has been identified or suspected, the critical issues are laboratory safety and the need to distinguish avian influenza viruses from human A/H1, A/H3 and B infections. The use of rapid antigen assays may rapidly identify influenza A or B virus infection, but will not differentiate between human and avian influenza A virus subtypes. Specimens from cases of potential avian influenza should be forwarded to a national or a WHO H5 Reference Laboratory for confirmatory testing. Since limited data exist describing shedding of avian influenza virus in humans, several respiratory specimens should be collected on different

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Table 4. Pediatric cases of H5N1 infection and mortality in countries other than Thailand and Vietnam Country

Total no. of cases

No. of pediatric cases (%)

Pediatric mortality (%)

Azerbaijan

8

6 (75)

17

Cambodia

6

3 (50)

100

China

19

7 (37)

43

Djerbouti

1

1 (100)

0

Egypt

14

7 (50)

14

Indonesia

52

23 (44)#

70

Iraq

2

1 (50)

100

21*

19 (90)

21

Turkey

*Confirmed by laboratory testing in Turkey; 9 cases not yet confirmed by WHO testing. #Four additional untested pediatric deaths in siblings of confirmed cases. Source: Weekly Epidemiological Record (WER) 2005-2006, World Health Organization. Accessible at: http://www.who.int/wer/en/

days for testing. Rapid tests for the diagnosis of avian influenza infection should be used only in combination with clinical findings and exposure history, due to the unknown sensitivity of these assays for avian influenza viruses. A negative rapid test result does not exclude human infection with avian influenza viruses. Specimens from highly suspect cases should not be cultivated under routine conditions in the clinical virology laboratory, but transported to a reference laboratory under appropriate biosafety conditions for confirmatory RT-PC testing.

Treatment The adamantane drugs, amantadine and rimantadine, block a viral ion channel protein required for cell entry and traditionally have been effective for treatment and prophylaxis of seasonal type A influenza. However, more than 90% of seasonal H3N2 viruses in the US are now resistant to the adamantanes, and in January 2006, the Centers for Disease Control and Prevention (CDC) recommended against the use of the adamantane class of antivirals for the treatment and prophylaxis of influenza in the United States until susceptibility to adamantanes has been reestablished among circulating influenza A isolates [50]. Avian H5N1 influenza strains currently circulating are frequently resistant to these agents [51, 52]. This resistance has been shown to develop during therapy for both seasonal influenza as well as avian influenza, and it has been noted de novo in clinical and field isolates of H5N1 influenza [51, 52]. These drugs reportedly have been

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355

widely used in poultry flocks and it is hypothesized that this has selected for resistant isolates in the field. The resistance appears to be stable in the current H5N1 strains and it is unlikely that these drugs will have a role either in prophylaxis or treatment of avian influenza. Neuraminidase inhibitors include oseltamivir and zanamavir; these agents inhibit the release of new viruses from infected cells and limit spread of infection from cell to cell. These drugs can reduce the severity and duration of illness caused by seasonal influenza, but are most effective when administered early in the course of illness, preferably within 48 h after symptom onset. Most strains of the H5N1 virus tested have been susceptible to the neuraminidase inhibitors, although resistance to oseltamivir has been reported [53, 54]. There are no good clinical data to support the efficacy of these drugs against H5N1 influenza, but they are generally safe and well tolerated. The reported case series from Thailand showed a nonsignificant trend towards better outcome with earlier oseltamivir treatment [41]. The major limitations to the use of neuraminidase inhibitors is likely to be unavailability due to limited production capacity, and prohibitive price for under-resourced countries. The manufacturing process for oseltamivir is complex and time consuming. Although the manufacturing capacity of oseltamivir has recently quadrupled, it will take a decade to produce enough oseltamivir to treat 20% of the world’s population. The majority of H5N1-related human deaths have been due to severe pneumonia, multiorgan system dysfunction and shock resulting directly from the virus, and thus cannot be prevented with antibiotics. However, influenza is often complicated by secondary bacterial pneumonia, and antibiotics could be life saving in the case of late-onset pneumonia. The mainstay of therapy is likely to be early detection and aggressive supportive care.

Vaccines HPAI virus outbreaks in commercial poultry flocks have spurred research into several forms of influenza vaccines. Recombinant viral-vectored vaccines encoding influenza HA have been constructed from fowlpox and vaccinia viruses [55–59]. These vaccines have shown efficacy in chickens against both low- and high-pathogenicity strains. However, safety concerns makes translation of these results to human trials difficult. Traditional influenza vaccines grown in eggs and chemically inactivated (‘killed’ vaccine) have been the mainstay of preventive strategies in commercial poultry [60, 61]. This is essentially the same method used to produce human influenza vaccines. Recent studies have reported the use of reverse engineering to produce vaccine strains in cultured cells that bear modified genes to attenuate virulence [62, 63]. The reverse engineering technique is very promising in that it allows vaccines to be ‘tailor-made’ to respond to variation in field

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strain HA or NA proteins, and the potential to modify virus genes to alter virulence or replication characteristics [62, 64–67]. However, major limitations of both traditional and reverse-engineering approaches are: (a) the requirement to develop vaccine seed strains that replicate to high titers in embryonated eggs; (b) the necessity for vaccine production in eggs, where one egg yields approximately one dose; and (c) the purification required for egg-produced vaccine and concerns regarding poultry-associated infectious agents, such as Salmonella. Recent studies of cell-culture produced influenza vaccines may alleviate some of these obstacles [62, 68–72]. Clinical trials have been conducted with an inactivated H5N1 vaccine produced using a combination of traditional and reverse-engineering methods [73]. Reverse engineering was used to modify the polybasic HA cleavage site of an H5N1 strain. This virus was then grown in eggs and chemically inactivated. Healthy adult volunteers received two doses of the vaccine at varying dosages. Protective antibody responses were produced in slightly over half of adults who received two immunizations with 90 +g HA (seasonal influenza dose 15 +g HA). While this trial showed some protective efficacy, the requirement for such high dosing presents a major obstacle, given the production problems and limitations of traditional egg-grown vaccines. A more recently published European trial found that a similar inactivated vaccine adjuvanted with alum (30 +g HA) induced protective antibody responses in 67% of adults [74]. Further clinical trials of inactivated H5N1 vaccines administered with different adjuvants are underway in several sites. Recombinant protein subunit vaccination is a strategy that has been highly successful for hepatitis B vaccine, which is produced in yeast [75]. Recombinant production allows strict quality control of all vaccine components and more straightforward quantitation of lot-to-lot variation. Recombinant influenza hemagglutinin has been produced in insect cells [76– 78]. Insect cell-expressed HA proteins have been tested in mice and chickens and were immunogenic and protective [79–82]. In subsequent human clinical trials, insect cell-expressed HA stimulated humoral immune responses in human vaccine trials, but required high doses [83–87]. One trial tested insect cell-expressed H5 HA and detected neutralizing antibody responses to a titer of 1:80 or greater in 52% of subjects after two doses of 90 mg. The requirement for such high doses (45–135 +g HA) compared to inactivated seasonal influenza vaccine (15 +g HA) presents a barrier to producing sufficient vaccine for large populations in the event of a pandemic. Similar to the inactivated H5N1 vaccine trial described above, the reason for the decreased immunogenicity of the insect cell-expressed protein is not clear. It may be due to a lack of previous exposure to H5 subtype virus in the subjects, who therefore would have experienced a primary rather than a primed memory response. Alternative adjuvants may be more effective at inducing robust responses to novel antigens and clinical trials of the insect cell-expressed HA with alternative adjuvants are also ongoing.

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Summary The emergence of avian influenza viruses in the human population promotes high concern for a potential pandemic. Avian influenza viruses have extreme virulence in children, with multiorgan disease and high mortality. The majority of cases have exposure to domestic poultry and human-tohuman transmission is rare. Most children present with fever, rhinorrhea and cough, and lymphopenia, thrombocytopenia and elevated transaminases are common. Some children can present with GI disease alone. The complications of illness are severe, including respiratory failure, shock and death. Aggressive supportive care is the mainstay of treatment, although neuraminidase inhibitors may have some efficacy if used early. Suspicion for the presence of avian influenza relies heavily on epidemiological risk factors such as exposure to poultry or travel to endemic regions. The continued spread of these viruses in wild and domestic bird populations requires regular checking of institutional or governmental sources to keep abreast of rapidly changing endemic or epidemic regions. Suspected cases should be kept in strict isolation and appropriate testing obtained with the aid of local or national health departments. Preventive strategies including vaccines are in development, and unlike seasonal influenza, children appear to be a highrisk group that should be targeted for early vaccine testing. Additional information on influenza, including avian influenza, is available at: http://www.cdc.gov/flu Updates on the worldwide avian influenza situation are available from WHO at: http://www.who.int/csr/disease/avian_influenza/en WHO H5 Reference Laboratory: http://www.who.int/csr/disease/avian_ influenza/guidelines/referencelabs/en/

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World Health Organization (2006) Acute Respiratory Infections: Influenza. World Health Organization, Geneva Neuzil KM, Mellen BG, Wright PF, Mitchel EF Jr, Griffin MR (2000) The effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children. N Engl J Med 342: 225–231 Neuzil KM, Zhu Y, Griffin MR, Edwards KM, Thompson JM, Tollefson SJ, Wright PF (2002) Burden of interpandemic influenza in children younger than 5 years: a 25-year prospective study. J Infect Dis 185: 147–152 Glezen WP, Couch RB (1978) Interpandemic influenza in the Houston area, 1974–76. N Engl J Med 298: 587–592 Glezen WP (1980) Considerations of the risk of influenza in children and indications for prophylaxis. Rev Infect Dis 2: 408–420 Claas EC, Kawaoka Y, de Jong JC, Masurel N, Webster RG (1994) Infection of children with avian-human reassortant influenza virus from pigs in Europe. Virology 204: 453–457

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18

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Human papillomavirus infections in children Nanette B. Silverberg Department of Dermatology, St. Luke’s-Roosevelt Hospital Center and Beth Israel Medical Center, New York, NY, USA; Columbia University College of Physicians and Surgeons, New York, NY, USA

Abstract Human papillomavirus (HPV) is a ubiquitous double-stranded DNA virus that infects human squamous cells causing a variety of clinical diseases ranging from plantar or common warts to genital warts to neoplasia of the cervix and genitalia. Over 200 HPV types have been characterized, but only about 20 are commonly identified in pediatric skin lesions. Once infected, the host requires an extended time period to produce antibodies and a cell-mediated immune response against HPV. Two out of three patients will achieve natural immune clearance by 2 years and three out of four by 3 years. Therapy of HPV infections includes agents that destroy the lesion, agents that induce immune response by the host, and removal techniques. For genital HPV, prevention of initial HPV infection is now the therapeutic gold standard and can be achieved by vaccination with a quadrivalent HPV 6, 11, 16, 18 vaccine in three doses introduced before an adolescent’s sexual debut. Another problem that may be alleviated long-term by HPV vaccination is the vertical transmission of genital HPV, which can result in pediatric condyloma or juvenile onset recurrent respiratory papillomatosis (juvenile laryngeal papillomatosis). Genital warts in childhood that cannot be documented to have occurred via vertical transmission from an infected mother must be sexually transmitted, the result of sexual abuse in elementary school children. Until vaccination has become widespread, genital HPV infections must be carefully screened through papanicolaou screening, HPV screening and cytology.

Introduction Human papillomavirus (HPV) is a ubiquitous intracellular DNA virus whose primary host is children and adolescents. The HPV is the viral cause of common and plantar warts as well as the sexually transmitted condyloma acuminata and juvenile onset-recurrent respiratory papillomatosis (JORRP). The epidemiology of warts has changed over the past 30 years due to a rise in sexually transmitted HPV, which has not been completely prevented by condom usage due to the presence of lesions of the labia, scrotum and inner thighs. Despite papanicolaou (Pap) smears, which screen for HPV-related oncogenesis of the cervix, cervical cancer cases continue to

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occur with consequent health-care costs, morbidity and mortality. Similarly, male genital carcinomas occur as a result of long-term HPV infection. As a result, vaccination against HPV has been thought to represent the best route to decrease HPV-related genital carcinomas. This chapter focuses on cutaneous HPV infections in children and treatment strategies for genital and extra-genital disease.

Biology of the HPV Warts (or papillomas) are benign epithelial tumors of the skin and mucous membranes caused by HPV infection, of which there are more than 200 subtypes. The HPV is a coiled, double-stranded DNA virus. Only about 20 subtypes are of clinical significance in childhood and adolescence, some of which are extra-genital (HPV 1–5, 7, 8, 10, 12, 13) and some of which are primarily associated with genital infection (HPV 6, 11, 16, 18, 31–34, 68) [1–3]. When the HPV viruses enter the keratinocytes or squamous cells (in the mucosa) infection occurs through the insertion of viral genetic material (HPV DNA) into the host genome of the basal layer cells. HPV enters the basal layer of the skin through small full-thickness abrasions, which allow contact of the virus with the basal layer of the skin. These types of small abrasions often occur in situations where the skin is moist, such as in a locker-room shower and poolside, or in the genital mucosa. Exposure to HPV is further enhanced by the fact that the virus is extremely hardy and difficult to eradicate from surfaces (such as pool tiles) because it resists freezing, inactivation and desiccation. When the HPV virus infects the basal layer of skin, it may either infect proliferating stem cells or it may infect resting cells [4]. At the time that the resting cells are turned on and proliferate, the virus will become active in causing excess keratinocyte growth and proliferation. HPV can also have an extremely prolonged incubation or latency period between infection and the appearance of clinical lesions. Once proliferation of the virally infected keratinocytes begins, blood flow is promoted locally with resultant nourishment of the verrucae [1, 3, 4]. HPV gene expression is noted as cellular differentiation of the keratinocytes occurs. Hence immunoperoxidase stains show viral gene expression only in suprabasal cells, although polymerase chain reaction can detect viral DNA through the full thickness of the skin. HPV causes a variety of clinical lesions. In general, the benign tumors of the skin and other epithelial tissue caused by HPV infection are termed warts or papillomas. While it is impossible to determine the subtype causing human infection with the naked eye, certain clinical types of HPV infection are associated with specific lesional morphologies or locations of infection. Acral warts represent the most common lesion type in humans. Children are the most common host of common warts [1–3].

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Each HPV subtype has a genetically distinct capsid composed of 72 pentamers of the L1 major capsid protein also known as virus-like particles (VLP). VLP are highly immunogenic proteins and have been used successfully as targets of vaccination [5]. Because the virus infects intracellularly, it is difficult to develop an immune response against HPV. Typically two out of three children will spontaneously clear their warts by 2 years and three out of four by 3 years. This is also true for genital warts. What occurs on the long term for the residual patients is unknown [1, 6–8]. A variety of immunological mechanisms including cell-mediated immunity, anti-HPV immunoglobulin production, and antibody-mediated cellular immunity contribute to wart clearance. In addition localized production of interferon and nitrous oxide may promote wart clearance. Warts may be more widespread and difficult to eradicate in immunosuppressed patients [3, 9]. The genome of HPV contains certain genes that promote abnormal cellular proliferation when inserted into the genome. The E6 and E7 genes are primarily responsible for the proliferation and oncogenesis noted with HPV. The E6 and E7 genes inactivate the tumor suppressor proteins p53 and pRb, respectively, thus allowing excessive keratinocyte proliferation. T cells are the primary cause of HPV-infected keratinocyte apoptosis through local release of granzyme B and perforin. The E6 and E7 oncogenes may alter the balance between cell growth and apoptotic loss of virally infected cells through a variety of pathways [10, 11].

Demographics and epidemiology Warts are a very common illness worldwide. In the United States, children are the most likely targets of the common wart viruses. Warts follow acne and atopic dermatitis in frequency of diagnosis in pediatric dermatology clinics [12, 13]. It is thought that 10–20% of children will at sometime be infected with warts [1, 3, 14]. The peak incidence of disease varies from study to study with some studies showing a peak age grouping of 8–9 years and others pointing to a peak age range between 12 and 18 year olds [1, 14–16]. The incidence of plantar warts has doubled from its incidence in 1968, which was then found in 1.8–2.9% of primary and secondary school children, to currently 4.5% [16]. Females and males are equally affected by HPV infections. The leading sites of HPV infection are the extremities, face and body. Hand warts are often transferred to other cutaneous sites including the lips, nose, and face, via autoinoculation. Autoinoculation is generally the route of disease extension or spreading; however, some patients may be exposed to the HPV in multiple sites at the same time. As noted previously, HPV infection is more easily acquired through wet or moist skin. Studies have shown that users of communal showers at a gymnasium are 27 times more likely to catch warts, obviating the need for pool shoes or sandals when using communal pools and showers [17].

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Natural history Warts will generally spontaneously involute or regress due to host immune response. Thirty percent will clear in 6 months, two-thirds by 2 years and three-quarters by 4 years of clinically apparent disease. Based on these data, it appears that the likelihood of spontaneous clearance wanes significantly with time. The course of 25% of warts that are not resolved by 3 years after wart appearance is unclear [1, 7].

Diagnosis of warts Diagnosis of warts is made by the classic physical examination features, including absence of normal dermatoglyphics of the skin and presence of pinpoint areas of bleeding when the lesion is pared. These latter features distinguish warts from calluses. Because of the verrucous appearance microscopically, warts are often rough to the touch, distinguishing them from other viral skin conditions, such as molluscum. Furthermore, mollusca have central punctae [1, 3]. If the diagnosis is questioned one can perform a lesional skin biopsy for immunoperoxidase stains against HPV. In situ hybridization can also be done to detect specific viral types [18].

Defining wart types Warts are usually defined by morphology, location and host immune response, which are not mutually exclusive [3, 19].

Definition by morphology (Tab. 1) Common warts (Fig. 1) Common warts are rough, verrucous plaques of the skin that usually measure 3–10 mm in diameter. HPV type 2 is the most common immunotype; type 1 warts may be indistinguishable. These lesions are often located on the dorsal surface of the hands, but the knees and other areas of the body may also be affected. Common warts on the soles or heels, “plantar warts”, often develop a thick overlying callus. Mosaic warts are agminated common warts, which take on the appearance of a single wart. Often these grouped warts are located on the soles and are covered by a single callus. The scalloped edge, which is seen due to the grouping of papules, may mimic cutaneous herpetic whitlow. The overlying thick callus may be painful, and paring may

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Table 1. Warts classification systems Morphology

Associated HPV viruses

1. Common warts

1–4, 7, 10

Mosaic warts

1–4, 7, 10

Callous-like warts

1–4, 7, 10

2. Filiform warts

1–4, 7, 10

3. Flat warts (tinea versicolor-like) 4. Donut warts

3, 5*, 8*, 10, 12, 14, 15, 17, 25–30, 41 Various+

5. Epidermal cyst, punctate type or pigmented warts

60, 63, 65

5. Sub-clinical infection

Various

Location

Associated HPV viruses

1. Common warts

2–4, 7, 10, 16, 29, 57

2. Palmoplantar warts

1, 2–4, 7, 10, 60, 63

3. Respiratory papillomatosis

6, 11, 16, 18

4. Periungual warts

1–4, 7, 10, 16*, 34*

5. Condyloma acuminatum

6, 11, 16*, 18*, 31*, 33*, 34*, 35, 39, 42–45, 51–53, 55, 56, 58, 59, 63, 66, 68

6. Oral papillomatosis (Heck’s disease)

13, 24, 32*

7. Verrucous carcinoma

1–4, 6, 11, 18

*Types associated with malignant transformation +Associated with prior therapy of warts

give a high degree of relief to the patient and reveal the true number of lesions [3, 19, 20].

Flat warts (verruca plana) Flat warts are flesh- to tan-colored papules, which are smaller and smoother than common warts, usually 2–4 mm in size. These lesions are common on the face and neck. Flat warts may be spread by shaving (Koebner phenomenon) in adolescents, causing a linear appearance. Flat warts are also seen in the genetic immunodeficiency syndrome, epidermodysplasia verruciformis (EDV). In EDV, warts spread rapidly, and may progress to bowenoid papulosis or Bowen’s disease (squamous cell carcinoma in situ). Such malignant conversion has been reported in children as young as toddler age both with and without immunodeficiency [21, 22].

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Figure 1. Common warts on the fingers in a teenage female.

Filiform warts/digitate warts Filiform warts are long thin warts with a narrow base and verrucous tip. Digitate warts are a slightly larger version of filiform warts. These warts usually bleed excessively when cut, due to tortuous blood vessels in the stalk of the wart. Filiform warts often appear on the face in children, specifically around the nares and on the lips. Digitate warts are common on the scalp. These warts are an exophytic version of the common wart.

Tinea versicolor-like warts/extensive flat warts When flat warts spread over a wide surface area and are either slightly hyper- or hypopigmented, they may mimic the appearance of tinea versicolor. This appearance is uncommon and is usually limited to patients with EDV, HIV infection, or other immune deficiency [23].

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Doughnut or ring warts (many types) Ring warts occur around the site of a wart that has been treated previously. These warts are seen after using destructive therapies, particularly liquid nitrogen or cantharidin [24].

Epidermal cyst-type warts (HPV 60, 63, 65) Occasionally a wart virus can be associated with epidermal cyst formation, including keratinous contents. These generally occur on the sole of the foot [25].

Punctate warts (HPV 60, 63, 65) Punctate warts are localized endophytic hyperkeratotic papules, seen on the palms. Clinically, these lesions mimic the palmar pits of basal cell carcinoma syndrome and punctate keratoderma. These types of warts are rare [19].

Pigmented warts (HPV 60) Occasionally warts can present with a high degree of pigmentation, so as to mimic a primary melanocytic process.

Keratoacanthoma (HPV 37) Keratoacanthomas have been associated with HPV 37, and can progress to squamous cell carcinoma, especially in patients with EDV or immunosuppression [26].

Subclinical or latent infection As the incidence of HPV 1 antibodies in select populations can be as high as 50%, clinically inapparent HPV infection is very likely the most common form of infection. Subclinical or latent infections are particularly common as well for genital warts caused by non-oncogenic HPV types.

Definition by location (Tab. 1) Wart location is an important descriptor in defining and describing warts. Wart location may dictate appearance, biological behavior, therapeutic con-

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cerns (e.g., scarring from treatment), and response to therapy. Warts may infect the normal skin or the mucosa. Mucosal warts usually appear as finely verrucous papules and plaques, often grouped on a common base, taking on a grape-like appearance. Mucosal warts are often characterized by extensive subclinical infection in the surrounding mucosa. Thus, treatments are less a microscopic cure than a cure of clinical appearance. There are two benign types of significance, condyloma and Heck’s disease, and two malignant types, verrucous carcinoma and bowenoid papillomatosis.

Palmoplantar warts (HPV 1-4, 7, 10, 60, 63) Palmoplantar warts, also known as myrmecia, are characterized as thick warts with a large overlying callus. These warts are often difficult to treat due to their thickness and the difficulty of eradicating wart at the base [1, 3].

Periungual warts (benign HPV 1–4, 7, 10/premalignant 16, 34) Periungual warts are warts that involve the periungual skin, the cuticle, and/or the subungual skin. These warts present a treatment difficulty due to the physical blockade created by the nail itself. Furthermore, when treating a wart in this location, accidental injury to the nail matrix may occur. Induction of wart immunity is often best when risk of nail matrix injury exists, or for subungual warts.

Condyloma acuminatum/giant condyloma of Buschke and Ollendorf (HPV 6, 11, 16, 18, 31, 33, 34, etc.) HPV infection of the mucosal surfaces is often asymptomatic, but may result in lesions, which are termed condylomata (condyloma singular) or papillomas. Condylomata are characterized as grouped papules that are usually smooth, unlike other wart subtypes. Condylomata may result from non-oncogenic types (HPV 6 and 11) and oncogenic types. When non-oncogenic lesions are very large, they are termed giant condyloma of Buschke and Ollendorf. Oncogenic virus types (HPV 16, 18, 31, 33, 34, etc.) may cause cutaneous, cervical, and penile dysplasia and/or neoplasia [1, 27]. Condylomata also develop when common wart HPV, such as HPV type 2, are transferred to the genitalia. Condyloma with onset in children under the age of 4 years most commonly result from vertical transmission from a virally infected genital tract or caretakers with hand warts. When condylomata are observed in children,

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careful social and physical evaluation to search for the possibility of sexual abuse is required. It is estimated that nearly 10% of the adult population of the United States are infected with genital warts. Despite the probable high rate of perinatal exposure, pediatric condyloma is uncommon. Recently, a British study demonstrated HPV DNA in a cohort of girls with and without vulvar disease [28]. About a quarter of these girls carried HPV DNA in vulvar skin or urine samples, suggesting that vertical HPV transmission is more common than previously thought, but is usually subclinical or latent. This may relate to transplacental transfer of neutralizing antibodies for HPV [29]. Teenagers are at risk for condyloma through unprotected sexual contact, whether penetration is involved or not. The younger the age at first sexual contact and the greater the number of lifetime partners, the greater the risk of cervical intraepithelial neoplasia. Consequently, sex education and usage of condoms should be encouraged in teenagers. Of HPV genital infections, 82.9% can be diagnosed by dermatologists in the form of external genital disease. External genital disease does not imply cervical disease, which is seen in only 53.4% of patients [30]. Recently common warts have been found to be statistically linked to the development of cervical cancer later in life [31]. One study suggested that oral condyloma in young children is unlikely to be vertically transmitted. The patients examined in this study had mothers with condyloma of the genital area, but the mother’s genital HPV infection and the child’s oral infection were found to be of different HPV genotypes [32].

Juvenile onset-recurrent respiratory papillomatosis (HPV 6, 11, 16, 18) A dreaded complication of vertical HPV transmission is JORRP, in which HPV infects the upper and occasionally lower respiratory tract including the tracheal and bronchial trees of children whose mothers were infected with HPV and transferred the virus via vertical transmission. The disease occurs in 7 offspring per 1000 women with genital warts and is 231 times higher in children of women with a condyloma history than in those without. This disease presents with hoarseness, stridor, cough and dyspnea and is often mistaken early on for asthma or laryngeal hemangiomas. Laryngoscopy and bronchoscopy may be required for proper diagnosis and sampling for pathological confirmation. The incidence is estimated at 1.7–2.6 per 100 000 children in the US, but the medical cost is $100 million per year!!! On average, 5.1 surgeries per year are required to ameliorate symptoms in young children [33, 34]. Because HPV types transmitted are primarily 6, 11, 16, and 18, HPV vaccination of women may ultimately help eliminate or reduce the morbidity, mortality and excessive cost of this illness [35]. Phase II study demonstrated a 93% increase in time between episodes of surgery with therapeutic vaccination with a hspE7 linked to E7 gene of HPV 16 [33].

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Heck disease (HPV 13, 24, 32) Heck disease or oral papillomatosis is the appearance of many papules of the oral mucosa caused by viral infection. The lesions are mucosa colored and when stretched fade into the background mucosa. The disease is seen in many ethnic groups, but is most common in Native Americans and Eskimo girls. The lower lip, upper lip, buccal mucosa and tongue can be affected. Cryotherapy or carbon dioxide laser therapy has been shown helpful in these patients. Spontaneous regression follows a similar time schedule to standard wart resolution and aggressive therapy should be reserved for cases with a protracted course [36, 37].

Bowenoid papulosis (HPV1, 16, 18, 33, 34)/verrucous carcinoma (HPV 1–4, 6, 11, 16, 18) Bowenoid papulosis is a form of squamous cell carcinoma in situ characterized by enlarging plaques of the genital region. It has been reported in immunocompetent and immunosuppressed children. Although sexual abuse need be suspected, acquisition of warts can occur through non-sexual manners and with viral types that are not considered oncogenic, e.g., HPV 1. This is particularly true for girls with human immunodeficiency virus. Verrucous carcinoma is usually seen on the external genitalia or digits, the latter representing the source of genital infection in some cases. Inoculation of hands through contact with genital lesions has been reported as a source of oncogenic virus types in the digits [38–42].

Definition by mode of regression A recent article categorized warts via immune response. The details overlap with the categorizations above and are summarized in Table 2. Two subtypes add new information, HPV type 4 and intermediate warts. HPV type 4 is associated with a specific mode of regression, which produces, in addition to the usual koilocytes, signet-ring vacuolized keratinocytes on microscopy. Intermediate warts (HPV types 10, 27, 28, 29) are essentially common warts seen in patients with depressed cellular immunity, hence variable inflammatory cellular infiltrate and rates of regression can be seen [19].

Host response (Tab. 3) The human clearance of HPV is a complex and variable process, which consists of three arms: (1) protective skin barrier, (2) innate immunity and (3) acquired immunity [1, 3, 19].

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Table 2. Summary of the categorizations of warts Type of wart

Mode of regression

1. Myrmecia (palmoplantar warts)/ Butcher’s

Humoral vascular reaction cellular extravasation

2. Common wart

Infiltrating cytotoxic T cells natural killer cells, macrophages

3. HPV-4 induced warts

Signet-ring vacuolized cells

4. Plane warts

Simultaneous appearance of inflammatory cells (cytotoxic T cells) and anti-viral cytokines

5. Intermediate warts

Same as plane warts, but at a slower pace due to depression of cell mediated immunity

The protective skin barrier is a vital factor in preventing the HPV from accessing the basal layer of the skin. Skin diseases in which barrier is impaired (e.g., atopic dermatitis and Darier’s disease) predispose to HPV infection. However, many of these conditions also feature abnormal cutaneous immunity and thus the skin barrier may not be the only factor influencing the risk of HPV infections. Innate immunity to warts is that aspect of the immune system that works actively against pathogens without prior exposure. These include nitric oxide production, mobilization of natural killer cells and neutrophils, the phagocytic response, and the local production of cytokines and chemokines. Acquired immunity is the adaptive aspect of the immune system. It can take months to years for acquisition of specific anti-HPV immunity. Antibodies to HPV are associated with wart regression; however, the role of antibodies is thought to be in containment and reduction of infectivity of HPV infection. Antibodies will also help prevent re-acquisition of warts through immune surveillance. On the other hand, they are not the limiting factor in wart regression, and in fact it is patients with reduced cell-mediated immunity who have the greatest difficulty clearing wart infections [19, 39, 43]. T cell tolerance to E6 and E7 is often seen with prolonged low level keratinocyte expression of these oncogenes. Furthermore, defective MHC class I expression may prevent immune induction against viral epitopes. Secondary phenomena in acquired immunity include mononuclear cell phagocytosis, localized anti-viral cytokine production and immune cellinduced apoptosis of virally infected cells. Satellite cell necrosis or apoptosis of wart-infected keratinocytes can be seen histologically as a marker of cellular immune destruction [9]. It is known that E6 and E7 genes inactivate the tumor suppressor proteins p53 and pRb, respectively. It is also thought that the E6 and E7 oncogenes may alter the balance between cell growth and apoptotic loss of virally infected cells through a variety of pathways [10]. T cells are the primary cause of HPV-infected keratinocyte apoptosis through local release of granzyme B and perforin [10, 11].

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Table 3. Host response to warts [19] Type of response

Role in HPV infection

1. Humoral immunity (antibody production)

Prevents re-infection Reduces infectivity Prevents dissemination

2. Recruitment of immune cells to the area Nitric oxide ICAM, VCAM, MAdCam-1 upregulation

Increased local blood flow Homing leukocytes to site infection

3. Antigen presentation Keratinocytes

MHC class II restricted May promote tolerance of HPV Presentation to T cells, MHC class II restricted

Langerhans’ cells 4. Cytokine production by keratinocytes and mononuclear cells TNF-_, IL-1, TGF-`, IFN-_, IFN-a, EGF and regulatory genes (e.g. c-myc) IL-6 (Increased levels) EGF, TGF-_, Amphiregulin Persistent sTNF-RI 5. Lymphocytes (T helper cells) Th1 cells Th2 cells Cytotoxic T cells Co-stimulatory molecules Natural killer cells

Down-regulation of HPV infection Inhibition of growth of HPV infected cells Up-regulation of MHC and adhesion Molecule expression Autocrine growth inhibitory effects Triggers leukocyte anti-HPV activation Promoting HPV infected cell growth TNF-R reduces TNF-_ activity Promotes persistent infection Regulate cell mediated/ humoral reactions Produce IL-2, IFN-a, TNF-`; Promotes CMI Produce IL-4, IL-5, IL-10, IL-13 MHC restricted Killing of virus infected/tumor cells including via release of granzyme and perforin B7, ICAM, LFA-3 MHC unrestricted Killing of virus-infected/tumor cells

6. Monocytes/macrophages

Cytocidal activity/Present antigens to T cells

7. Genetic polymorphisms of MHC MHC class I-HLA-A11, B14, B7 MHC class II-HLA-DQw3 HLA-DR6, DR13 HLA-DR7

Cancers Cervical cancer Lower prevalence cervical cancer Renal transplant skin cancers

8. Apoptosis of virally infected cells

Mechanism unknown

Host side effects of note Risk of injury for diabetics Diabetics with palmoplantar warts may experience greater injury or prolonged ulceration post treatment related to diabetic neuropathy. On the other side, diabetics with untreated plantar warts may develop ulcerations from the chronic pressure of the wart. While treatment should be given for all diabetics’ warts, aggressive therapy is contraindicated in diabetics with neuropathic disease. In the pediatric age group, older adolescents with an infantile onset of insulin-dependent diabetes mellitus are most at risk [44].

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Table 4. Conditions associated with excessive numbers of warts 1. Immunosuppression*

Cardiac allograft Chemotherapy HIV Renal allograft

2. Genetic syndromes

Common variable immune deficiency Epidermodysplasia verruciformis* Immunodeficiency syndromes WHIM syndrome (chemokine receptor gene CXCR4)

3. Activity

Butcher Locker room bathers Participants in sports Sexual intercourse*

*Conditions associated with malignant conversion of HPV infected skin

Psychological disturbance Patients are often highly distressed by the appearance of their warts, particularly when they are on the dorsum of hands [45]. When patients are highly distressed by their warts, an underlying psychiatric condition should be considered, including obsessive compulsive disorder and body dysmorphic condition. These diagnoses are rare in early childhood but become more common in the teenage years. Aggressive therapies, such as CO2 laser, may be needed in patients with severe psychological distress.

Vascular insufficiency Patients with vascular insufficiency (e.g., from Raynaud phenomenon/disease, collagen vascular disorders, and diabetes mellitus) should not be treated with cryotherapy at sites overlying the digital vasculature, nor should they receive injectable bleomycin in any site.

Conditions with excessive numbers of warts In most patients, immunosurveillance and innate immunity contain HPV infection. Excessive numbers of warts are seen in a variety of genetic and acquired conditions (Tab. 4), and may be associated with an increased local risk of cutaneous oncogenesis. Acquired immunosuppression (Fig. 2) is exceedingly common, whether from transplantation, chemotherapy or acquired immunodeficiency syndrome. Of the acquired conditions, renal transplantation, because it often occurs in younger patients, is associated with a high degree of morbidity

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Figure 2. 13 year-old boy with ataxia telangiectasia and extensive warts on the right elbow.

from wart viruses. In fact, estimates on the incidence of warts in allograft recipients range from 17% to 87% [46]. Furthermore, appearance of warts after transplantation is correlated with increased risk of skin cancer development [47]. The incidence of warts in HIV patients ranges from 3.3% to 11.2%. Wart infections in HIV tend to be difficult or impossible to eradicate and quicker to spread [48, 49]. Only a handful of genetic conditions are defined by the presence of viral warts, although viral warts may be seen in almost all immunodeficiencies, particularly those with defects of cell-mediated immunity, the most prevalent being common variable immunodeficiency [50]. The WHIM syndrome (an acronym for warts, hypogammaglobulinemia, recurrent bacterial infections, and myelokathexis) is a form of severe chronic neutropenia with hyperplasia of the mature myeloid compartment in the bone marrow. Recently, a chemokine receptor, CXCR4 has been found to be the causative gene. WHIM syndrome is associated with a heterozygous truncating mutation of the CXCR4 gene. The CXCR4 receptor is bound by CXCL12, a chemokine that regulates cardiogenesis and hematopoiesis among others [51, 52]. EDV is an autosomal recessive genetic condition (2p21-24, 17q25) characterized by disseminated flat warts, which can take on the appear-

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Table 5. Malignant conversion of HPV Malignant diseases caused by HPV infection

Associated HPV viruses

1. Cervical intraepithelial neoplasia Anogenital intraepithelial neoplasia cervical cancer

16, 18, 31, 33, 34

2. Bowen’s disease

16

3. Bowenoid papulosis

16, 18, 31, 33, 51

4. Verrucous carcinoma-digital

16

5. Keratoacanthoma

5, 9, 10, 14, 19, 20, 21, 38, 49, 80

6. Actinic keratoses Squamous cell carcinoma (head and neck)

3, 5, 8, 10, 16, 33

7. Malignant proliferating trichilemmal tumor (EDV)

21

8. Carcinoma of internal organs Esophageal carcinoma Anal carcinoma Adenoid cystic carcinoma Ovarian carcinoma

16, 54 16, 18, 31, 33, 34 33 16

ance of tinea versicolor-like macules. Rare patients experience neurological changes or ocular squamous cell carcinoma [53]. Malignant conversion of HPV-infected skin, due to UV exposure, is usually seen in early adolescence and continues through the patient’s lifetime. Polymorphisms of IL-10 gene promoter causing reduced IL-10 production have been reported in Brazilian EDV patients. These polymorphisms are believed to promote skin cancer development [54]. The most common cutaneous illness associated with abnormal processing of HPV is atopic dermatitis, although some studies have not supported an increased incidence. A recent study from the United Kingdom demonstrated that cervical cancer is more common in eczema patients and patients who acquire common warts. However, this study suggests that non-atopic eczemas, such as seborrheic dermatitis, may be the type associated with cervical cancers, as hay fever, an illness commonly co-morbid with atopic eczema, was not statistically correlated to cervical cancer [31].

Malignant conversion of HPV Although uncommon in childhood and adolescence, occasional malignant conversion of HPV infection may be seen, particularly in the setting of acquired immunodeficiency. As this is uncommon, a brief overview of these malignancies and their associated HPV viral types is included in Table 5.

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Role of HPV in non-wart skin conditions HPV can often be detected in common dermatoses, leading to speculation that HPV may play a role in the development of conditions such as psoriasis vulgaris. It has been postulated that EDV HPV types may play a role in the hyperproliferation of skin in psoriasis vulgaris. Anti-HPV 5 antibodies have been demonstrated in epidermal repair processes, including second degree burn and autoimmune skin diseases, including bullous disorders. Thus, it initially appeared that antibodies to the EDV HPVs were artifactual [55]. More recently, a French group looked at the presence of EDV HPV types 5 and 36 in scrapings from adults and children with psoriasis vulgaris. More than 42% of children and adults demonstrated HPV 5 DNA sequences. The most compelling, although anecdotal, piece of evidence was demonstration of HPV 5 DNA in an 18-month-old girl and a boy with a 1-week history of disease [56]. The mechanism by which hyperproliferative HPV types may trigger a widespread epidermal disorder like psoriasis is yet unknown, although it is clear that it is only one of many plausible putative etiologies of psoriasis.

Differential diagnosis Warts can be mistaken for any other benign or malignant overgrowths/ tumors, and vice versa, benign and malignant overgrowths or tumors of the skin and mucosa may take on the appearance of warts. Thus, warts can be mistaken for a callus, a nevus, acrochordons, seborrheic keratoses, actinic keratoses, a squamous cell carcinoma or a melanoma (when pigmented). On the other hand, cases of depigmented and verrucous melanomas and warty-appearing squamous cell carcinomas have been reported in the literature. Perianal verrucous epidermal nevi can be mistaken for perianal warts [57]. Pseudoverrucous nodules, seen in association with incontinence, resemble condyloma as well [58]. Although these entities are disparate, the association of the wart virus with tumor promotion may mean that verrucous carcinomas and warts can exist side by side in a lesion. Thus, biopsy for confirmation is required in a normal-appearing wart if biological behavior or response to therapy is atypical.

Treatment options (Tab. 6) [2, 3] A variety of drug options exist for HPV infections in childhood. Choice of treatment depends on the age of the patient, the location of the warts, the number of warts, duration of infection, underlying illnesses and patient preference. For warts, there are six types of treatments that can be used: destructive, immunological, psychological, sclerosant, antiviral and anti-mitotic. Often

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Table 6. Overview of treatments for extra-genital and genital HPV infections Extra-genital

Genital

Cryotherapy

X

X

Cantharidin

X

X*

Duct tape

X

Garlic

X

Destructive

Podophyllin

X

Photodynamic therapy

X

Salicylic acid

X

X*

Surgery

X

X

Immunotherapy Antigen injection (Candida, mumps)

X

Cimetidine

X

X

Imiquimod

X+

X+,*

X

X*

Squaric acid immunotherapy Interferon

X

Vaccination

X

Vascular Bleomycin

X

Pulsed dye laser

X

Psychological

X

X

Anti-mitotic 5 Flourouracil

X

X*

*Use of these substances in the genital area in young children should be limited and observed closely by a physician +Treatment regimen and efficacy differ significantly between genital and extra-genital disease

the clinician will choose moderately effective methods of therapy that have few side effects or no pain over a very effective but painful regimen of therapy. Among the destructive methods, salicylic acid has the best clinical trial data supporting its usage, with a number of placebo-controlled trials documenting a 50–75% rate of cure with 6 weeks usage [59]. On the other hand, liquid nitrogen application every 2–3 weeks is generally 60–76% effective [60, 61]. Liquid nitrogen is painful and may induce hemorrhagic blisters [62–64]. Nerve block may reduce the pain associated with liquid nitrogen [65]. Cantharidin has been described as a fairly effective therapy for warts. Donut warts around the lesion tend to be very common with this drug.

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Cantharidin is about as effective as liquid nitrogen and is best in young children as a therapy because its use is rarely associated with pain. Rarely, lymphedema can be seen with cantharidin application [24, 66]. Usage of cantharidin mixed with other agents such as podophyllin is inadvisable in pediatric practice. Topical garlic has been used on a nightly basis as a homeopathic method of wart therapy and works over a 6-week time period. It is well tolerated and very cheap [67]. Garlic is a nitric oxide releaser and a vesicant, and may be effective due to both of these properties [68, 69]. Occlusion of wart therapies is a standard mechanism of enhancing drug efficacy. Recently, a small study documented the efficacy of weekly reapplied duct tape alone as a therapy for non-acral warts. The success rate was 85% in 6 weeks with limited side effects [61]. Immunotherapies hasten immune recognition of HPV by the body. Imiquimod 5% cream is an immune response modifier approved in the United States for the treatment of genital warts. When applied to the skin, Imiquimod induces production of interferon-_, TNF-_, IL-1, IL-6, and IL8. Many small case series or single case reports have anecdotally reported a variety of successful regimens of Imiquimod application for common warts in children. The most effective regimen reported has been a twicedaily application. Usage under a diaper is inadvisable, as severe ulceration may result [70–76]. Other topical immunotherapies used in children include diphencyclopropenone (DCP) and squaric acid (SADBE) [77–79]. SADBE has been described for office or home usage, while DCP is generally used in-office [80–82]. Clearance rates in published studies have varied from 58–90% with eczematous side effects being common and rare urticaria [79]. Oral cimetidine in standard pediatric dosage can enhance wart clearance, but works in only about half of patients treated [83, 84]. Genital warts can also respond to cimetidine [85]. Intralesional injections of mumps and Candida antigen are painful and cause flu-like side effects but may induce more than 60% wart clearance [86–90]. Interferon injections can also be used as immunotherapy for warts and condyloma [91, 92].

Genital HPV infection Almost all strains of genital HPV are oncogenic, whether of high potential (e.g., HPV 16, 18, 31, 33, 34) or low potential (e.g., HPV 6, 11), eventually potentially causing invasive disease such as cervical intraepithelial neoplasia, cervical cancers, genital cutaneous and mucosal squamous cell carcinomas (e.g., Bowen’s disease) including penile carcinomas. Cervical cancer is the second or third leading cause of cancer deaths in epidemiological studies of adult women [1, 3, 5]. Pap smears are cytological sampling of the cervical mucosa and has been able to detect many cases of invasive HPV and has sampling errors, false positives and false negatives

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[93]. Viral release from the capsid allows entry of HPV into the DNA of the host, thereby exposing the host to a potentially oncogenic event. Hence, prevention of release of HPV DNA from its capsid is required for complete disease prevention. While abstinence is completely preventive, it is unrealistic as a lifetime prevention strategy. Condoms, as they do not cover the entire genital tract, do not completely prevent the mucosal to mucosal or skin-to-skin contact required for transmission of HPV. Vaccination is therefore the best method for reduction of HPV-related cervical disease in future generations. As HPV 6 and 11 are the most common genital types and 16 and 18 are the most common oncogenic strains, these four strains have been targeted for vaccine development. Unfortunately, there are so many HPV types that complete prevention of genital HPV may require addition of at least another ten strains in the vaccine strategy (e.g., HPV 31, 33, 34). The first HPV vaccine Phase III trial reported was a monovalent threedose HPV 16 trial. This trial of 2392 college-aged women (defined as females 16–23 years of age) was the first to demonstrate the capabilities of HPV vaccines. While 3.8 cases of HPV were noted per 100 women treated with placebo, none were noted in vaccinated women. Moreover, 9 cases of cervical intraepithelial neoplasia were noted in placebo and zero with vaccine [94]. Prolonged immunity has been noted with this vaccine [95]. Bi-valent HPV 16, 18 vaccination (Cerviarix®) was also efficacious in a trial of 1113 women aged 15–25 years. This trial noted 100% efficacy against persistent infection, but only 91.6% efficacy (95% CI 64.5–98.0) against incident infection [96]. In the intention-to-treat analyses, vaccine efficacy was 95.1% (63.5–99.3) against persistent cervical infection with HPV 16/18 and 92.9% (70.0–98.3) against cytological abnormalities associated with HPV 16/18 infection. Vaccination against HPV is now FDA approved in the United States with the introduction of the quadrivalent three-dose Gardasil®, a vaccine against HPV 6, 11, 16 and 18, approved for prevention in women aged 9–26 years old . The target group for vaccination is young women who may acquire oncogenic strains of HPV through sexual intercourse. Phase II trial of Gardasil in 277 young women (mean age 20.2 years) who received doses at 0, 2 and 6 months demonstrated a 90% fall in combined incidence of persistent infection or disease with vaccine [97]. Injection site reactions and fever were the most common vaccine side effects [98]. However, vaccination was described as ineffective after acquisition of HPV infection, hence vaccination is targeted at women who have not yet had their sexual debut. Vaccination of a single sex is more cost effective and is thought to be adequate for disease reduction, despite not taking advantage of herd immunity [99]. Unfortunately, the vaccine is not approved for males and will not prevent homosexual male HPV transmission. Some authors believe that ideally young males should be added to the vaccination schedule as well [100].

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The vaccines are immunogenic against the VLP whether delivered bronchially or intramuscularly, as Gardasil® [101]. Each HPV sub-type has a unique VLP, which is expressed in the capsid. Vaccine against capsid elements kills the VLP prior to intercalation of HPV DNA into the host genome. VLP vaccines induce a high titer of virion neutralizing antibodies and require little adjuvant. Vaccination using HPV 6 has been proven ineffective in a clinical trial of HPV type 6L2E7 vaccine in patients with condyloma acuminata. In a controlled trial in which 320 patients with HPV 6 or 11 infection were randomized to receive three doses of vaccine or placebo and additional treatment with podophyllin or ablative therapy, a trend toward better clearance was noted in HPV 6-infected patients, but was not statistically significant. Genital HPV typing showed most patients were infected with multiple HPV types, perhaps accounting for the poor response to monovalent vaccine [102]. Overall vaccine prevention strategies hold great promise at prevention of genital HPV-related morbidity and mortality. Pulse dye laser destroys the extensive vascular supply required to maintain rapid cellular proliferation in warts. Pulse dye laser can be effective for common and anogenital warts in children [103]. Photodynamic therapy and CO2 laser are two other destructive techniques of wart removal; however, they are painful and can scar [104–106]. There are no anti-HPV medications, but patients with concurrent HIV will have less HPV-related morbidity when treated with anti-retrovirals. Two anti-mitotic chemotherapeutic agents have been described as being helpful in wart therapy. Podophyllin can be used in-office or at home as the extracted podophyllotoxin. 5-Flourouracil in a cream base can be used for common and genital wart therapy [107]. This pyrimidine metabolite interferes with DNA and RNA synthesis. The medication is used locally on a daily basis [108]. Ulceration is a common side effect of 5-flourouracil.

Conclusions HPV causes a variety of skin lesions in pediatric patients with a wide range of associated morbidity. Careful history and physical examination allow for better diagnosis and treatment of each individual patient. Vaccination holds promise to reduce the most dreaded complications of HPV, namely condyloma, genital carcinomas and vertically transmitted respiratory papillomatosis.

References 1

Cobb MW (1990) Human papillomavirus infection. J Am Acad Dermatol 22: 547–566

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6 7 8 9 10

11 12 13 14 15 16

17 18

19 20 21

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(2003) High frequency of detection of human papillomaviruses associated with epidermodysplasia verruciformis in children with psoriasis. Br J Dermatol 149: 819–825 Bandyopadhyay D (2003) Perianal verrucous epidermal naevus mimicking perianal warts. Sex Transm Infect 79: 424–429 Coppo P, Salomone R (2002) Pseudoverrucous papules: an aspect of incontinence in children. J Eur Acad Dermatol Venereol 16: 409–410 Gibbs S, Harvey I, Sterling JC, Stark R (2003) Local treatments for cutaneous warts. In: The Cochrane Review, Issue 4. John Wiley and Sons, Chichester Bunney NH, Nolan MW, Williams DA (1976) An assessment of methods of treating viral warts by comparative treatment trials based on a standard design. Br J Dermatol 94: 667–679 Focht DR, Spicer C, Fairchok MP (2002) The efficacy of duct tape vs. cryotherapy in the treatment of verruca vulgaris (the common wart). Arch Pediatr Adolesc Med 156: 971–974 Ichiki Y (1999) Lidocaine tape for reducing pain in the cryotherapy of warts. Pediatr Dermatol 16: 481–482 Buckley D (2000) Cryosurgery treatment of plantar warts. Ir Med J 95: 140– 143 Hancox JG, Graham GF, Yosipovitch G (2003) Hemorrhagic bullae after cryosurgery in a patient with hemophilia A. Dermatol Surg 29:1084–1086 Wagner AM, Suresh S (1998) Peripheral nerve blocks for warts: taking the cry out of cryotherapy and laser. Pediatr Dermatol 15: 238–241 Stazzon AM, Borgs P, Witte CL, Witte MH (1998) Lymphangitis and refractory lymphedema after treatment with topical cantharidin. Arch Dermatol 134: 104–106 Silverberg NB (2002) Garlic cloves for verrucae vulgaris. Pediatr Dermatol 19: 183 Kim-Park SA, Ku DD (2000) Garlic elicits a nitric oxide-dependent relaxation and inhibits pulmonary vasoconstriction in rats. Clin Exp Pharmacol Physiol 780–786 Rafaat M, Leung AK (2000) Garlic burns. Pediatr Dermatol 17: 475–476 Grussendorf-Conen EI, Jacobs S (2002) Efficacy of imiquimod 5% in the treatment of recalcitrant warts in children. Pediatr Dermatol 19: 263–266 Oster-Schmidt C (2001) Imiquimod: a new possibility for treatment resistant verrucae. Arch Dermatol 137: 666–667 Micali G, Dall’Oglio F, Nasca MR (2003) An open label evaluation of the efficacy of imiquimod 5% cream in the treatment of recalcitrant subungual and periungual cutaneous warts. J Dermatol Treat 14: 233–236 Hengge UR, Goos M, Arndt R (2000) Topical treatment of warts and mollusca with imiquimod. Ann Intern Med 32: 95 Hengge UR, Esser S, Schiltewolter T, Behrendt C, Meyer T, Stockfleth E, Goos M (2000) Self-administered topical 5% imiquimod for the treatment of common warts and molluscum contagiosum. Br J Dermatol 143: 1026–1031 Majewski S, Pniewki T, Malejczyk M, Jablonska S (2003) Imiquimod is highly effective for extensive, hyperproliferative condyloma in children. Pediatr Dermatol 5: 440–442

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Wenzel K, Saka B, Zimmerman R, Gundlach KKH, Barten M, Gross G (2003) Malignant conversion of florid oral and labial papillomatosis during topical immunotherapy with imiquimod. Med Microbiol Immunol 192: 16 Buckley DA, du Vivier AW (1999) Topical immunotherapy in dermatology. Int J Clin Pract 53: 130–137 Mastrolonardo M, Lopalco PL, Diaferio A (2002) Topical immunotherapy with contact sensitizers: a model to study the natural history of delayed hypersensitivity. Contact Dermatitis 47: 210–214 Silverberg NB, Lim JK, Paller AS, Mancini AJ (2000) Squaric acid immunotherapy for warts in children. J Am Acad Dermatol 42: 803–808 Micali G, Dall’Oglia F, Tedeschi A, Pulvirenti N, Nasca MR (2000) Treatment of cutaneous warts with Squaric acid dibutylester: A decade of experience. Arch Dermatol 136: 556–557 Lee AN, Mallory SB (1999) Contact immunotherapy with squaric acid dibutyl ester for the treatment of recalcitrant warts. J Am Acad Dermatol 41: 595–599 Upitis JA, Krol A (2002) The use of diphencyclopropenone in the treatment of recalcitrant warts. J Cutan Med Surg 6: 214–217 Orlow SJ, Paller AS (1993) Cimetidine for multiple viral warts in children. J Am Acad Dermatol 28: 794–796 Gooptu C, Higgins CR, James MP (2000) Treatment of viral warts with cimetidine: an open label study. Clin Exp Dermatol 25: 183–185 Franco I (2000) Oral cimetidine for the management of genital and perigential warts in children. J Urol 164: 1074–1075 Phillips RC, Ruhl TS, Pfenninger JL, Garber MR (2000) Treatment of warts with Candida antigen injection. Arch Dermatol 136: 1274–1275 Signore RJ (2002) Candida albicans intralesional injection immunotherapy for warts. Cutis 70: 185–192 Signore RJ (2001) Candida immunotherapy of warts. Arch Dermatol 137: 1250–1251 Clifton M, Johnson SM, Roberson PK, Kincannon J, Horn TD (2003) Immunotherapy for recalcitrant warts in children using intralesional mumps or candida antigens. Pediatr Dermatol 3: 268–271 Johnson SM, Roberson PK, Horn TD (2001) Intralesional injection of mumps or candida skin test antigens. Arch Dermatol 137: 451–455 Nimura M (1990) Application of beta-interferon in virus-reduced papillomas. J Invest Dermatol 95: 149S-151S Klutke JJ, Bergman A (1995) Interferon as adjuvant treatment for genital condyloma acuminatum. Int J Gynaecol Obst 49: 171–174 Gharoro EP, Ikeanyi EN (2006) An appraisal of the level of awareness and utilization of the Pap smear as a cervical cancer screening test among female health workers in a tertiary health institution. Int J Gynecol Cancer 16: 1063– 1068 Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB Chiacchierini LM, Jansen KU, Proof of Principle Study Investigators (2002) A controlled trial of a human papillomavirus type 16 vaccine. N Engl J Med 347: 1645–1651 Harper DM, Franco EL, Wheeler C, Ferris DG, Jenkins D, Schuind A, Zahaf T,

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New treatments for hepatitis B and C in children and adolescents Patrick Gerner Children’s Hospital, Heusnerstrasse 40, HELIOS Klinikum Wuppertal, Witten-Herdecke University, 42283 Wuppertal, Germany

Abstract The treatment of chronic viral hepatitis is a rapidly evolving field. Therapy for chronic hepatitis B is indicated at times of high viral replication, as long as the patient’s aminotransferase levels are increased by more than twice the norm, and when hepatitis B e antigen (HBeAg) is positive. The treatment options for chronic hepatitis B include interferon-alpha and the nucleoside analogues lamivudine and adefovir dipivoxil. Between 26% and 38% of patients respond to treatment with interferon-alpha and nucleoside analogues; from 17% to 36% respond with antibodies to HBeAg (anti-HBe) seroconversion after 1 year. With seroconversion, HBeAg disappears and there is a dramatic decrease in HBV-DNA and usually in the aminotransferases. Further development of nucleoside analogues promises to increase the effectiveness of the therapy. Complete recovery, with conversion to antibodies to hepatitis B surface antigen (anti-HBs), occurs in about 5% of patients only after interferon-alpha therapy. The success of treatment is influenced by factors such as the origins of infection, the viral load before therapy, and the intensity of liver inflammation. Without therapy, the rate of seroconversion to anti-HBe ranges from 2.5% to 11% a year. It is becoming evident that patients with fulminant hepatitis B benefit from treatment with lamivudine. In contrast to hepatitis B, the treatment goal for chronic hepatitis C is the patient’s full recovery. Currently, depending on the HCV genotype, the combination therapy of interferon-alpha and ribavirin administered for 6–12 months has proven effective. Approximately 80% of children are infected with genotype 1a or 1b. They have a recovery rate of 45%. Genotypes 2 or 3 respond much better to treatment. More than 84% of patients can be successfully treated. Genotype 4 is relatively rare and appears to respond to treatment like genotype 1. Under certain circumstances, unsuccessfully treated patients can be treated a second time, after a number of years, with another interferon-alpha, e.g., natural human alpha interferon (Multiferon®) or consensus interferon (Inferax®) plus ribavirin. In addition, new medications such as protease and polymerase inhibitors are currently being tested in adult patients and should be available in the next few years.

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Hepatitis B Epidemiology Worldwide, an estimated one million people die annually due to the consequences of hepatitis B virus (HBV) infection. Almost half of the world’s population will contract HBV, and approximately 350 million people are chronic virus carriers. The prevalence of chronic hepatitis B in children and young people ranges from 0.1% to 8%. In Europe and Northern America the prevalence of chronic hepatitis B in adults is about 0.4–0.8% but lower during childhood. Among adults in some ethnic groups, for example in Egypt, prevalence may be as high as 25% [1, 2].

Serological diagnosis With HBV infection the following parameters are relevant: hepatitis B surface antigen (HBsAg), antibodies to HBsAg (anti-HBs), hepatitis B e antigen (HBeAg), antibodies to HBeAg (anti-HBe), and antibodies to the hepatitis B core antigen (anti-HBc) (both IgG and IgM). Once infection is confirmed, quantitative measurement of HBV-DNA is also useful. These levels are not only an indicator of viral load, and therefore the degree of infectiousness, but also a help to determine the probability of therapy response. Under therapy, the decline in viral replication indicates a positive response. HBcAg is expressed on the membrane of hepatocytes and can therefore only be detected in liver tissue using special dyes. HBV-DNA is the most sensitive marker of HBV infection. It can often be detected before the rise in antibodies even after the disappearance of antigens. In most cases a liver biopsy for histological examination is unnecessary. In the future, determination of HBV genotypes may become more important as a marker of successful therapy.

Serological and clinical course Serologically, chronic hepatitis B can be divided into three major phases (Fig. 1). In the first phase, HBeAg is positive and anti-HBe is negative. Typically, there is a high level of viral replication and the transferases can also be increased. The second phase begins once seroconversion to antiHBe has occurred; HBeAg is then no longer detectable. This seroconversion is not predictable in the individual case and usually occurs together with a dramatic reduction in the viral load and the transferases return to normal. The seroconversion to anti-HBs signals the third phase of infection and is generally equated with recovery from hepatitis. However, highly sensitive polymerase chain reactions (PCR) can detect HBV-DNA sequences in

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Figure 1. Three phases (I–III) of chronic hepatitis B.

roughly 10% of these patients [3]. HBV-DNA is detectable in liver tissue in as many as 30–80% of patients. Whether these low viral levels are clinically relevant is still unclear. However, it is known that the infection may be reactivated in the case of immune suppression.

Therapy Three drugs are available for the treatment of hepatitis B in childhood: these include interferon-alpha, the nucleoside analogue lamivudine (Zeffix®), and adefovir dipivoxil (Hepsera®). The therapeutic goal is complete recovery from hepatitis. However, this occurs in roughly 5% of those patients treated with interferon-alpha. Thus, the primary goal becomes prevention of life-threatening complications such as liver cirrhosis and hepatocellular carcinoma. Most importantly, this is achieved through a reduction in the histological inflammatory activity in liver tissue. A favorable prerequisite therefore is seroconversion to anti-HBe and reduction in viral replication. Treatment of chronic hepatitis is indicated when aminotransferase levels are more than double upper normal limits, and when HBeAg is detectable in serum. If the transaminases remain high in spite of anti-HBe seroconversion and the loss of HBeAg, treatment can still prove to be helpful. It is important that every HBV-infected patient has been tested, to rule out a co-infection with hepatitis C or D virus, especially if the patient exhibits anti-HBe seroconversion and still has high transaminase levels.

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Figure 2. Seroconversion to anti-HBe after therapy with IFN-_ (black column) or without therapy (gray column) in percentages. Columns from left to right: (1)+(2) [5] (n = 29); (3)+(4) [6] (n = 149); (5) [4] (n = 107); (6)+(7) [7] (n = 166); (8)+(9) [8] (n = 74).

Interferon-_ Interferon-_ (IFN-_) has immune-modulating, anti-proliferating, and antiviral properties. There are a variety of genetically manufactured as well as naturally derived IFN-_ preparations available, and all need to be injected subcutaneously. During a 6-month treatment period, seroconversion to antiHBe, and with it the transition from the first to the second phase of infection, is achieved in 26–38% of children (see Figs 1 and 2). The spontaneous rate of seroconversion is clearly exceeded with this therapy. Seroconversion to anti-HBs is achieved in approximately 5% of children. The success of anti-HBe or anti-HBs-seroconversion is influenced by a series of factors (Tab. 1). Prognostically, the most important as well as the most favorable factor is a high level of inflammatory activity in the liver. New research suggests that the HBV genotype may influence response to therapy in a way similar to that for hepatitis C. Genotypes A and B also respond better than genotypes C and D [2]. However, with HBV genotypes, especially the genotypes A and D predominant in Europe and North America, treatment success is only marginal.

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Table 1. Predictors for therapy response Positive predictors

Negative predictors

Horizontal transmission

Vertical transmission

Transaminases increased > 2 ×

Normal transaminases

Clear evidence of inflammatory activity in liver tissue

Minimal hepatitis

HBV-DNA < 1000 pg/ml serum

Immune suppression

HBV genotypes A and B

HBV-DNA > 1000 pg/ml HBV genotypes C and D

Although the rate of anti-HBe seroconversion using IFN-_ is significantly higher in comparison to the spontaneous course of development, it must be noted that according to a number of long-term studies, seroconversion is probably just delayed in those who do not receive treatment. According to a study conducted by Bortolotti et al. [4], there was no difference (in a second study only a small difference) between those who received treatment and those who did not after 5 years (Fig. 2). Further studies following patients over the long-term course after therapy do not exist. In most studies the combination of IFN-_ with lamivudine, in comparison to monotherapy with IFN-_ does not improve anti-HBe seroconversion and is thus not worthy of consideration [2]. In conclusion, successful treatment accelerates anti-HBe seroconversion in the individual patient but does not increase the overall rate by a statistical significant proportion. To date there are no data in children with PEG-interferon-alpha therapy. According to recent data from adults it seems quite reasonable to assume that the treatment results are comparable.

Second line treatment The majority of patients do not respond to treatment with IFN-_ and retreatment has not proven to be effective. Instead, after unsuccessful treatment with interferon, and if the transaminases are increased by at least 1.5 times upper normal limit, treatment with nucleoside analogues is indicated. Personal experience and the still unpublished results of a double-blind study indicate an increased anti-HBe seroconversion after 4 months of treatment with vitamin E (200–600 IE/day according to body weight). From a total of 92 patients treated with vitamin E, 23% seroconverted to antiHBe, seroconversion occurred in only 8% of the placebo group. However, the difference between the two groups is not significant and represents a trend that must be tested on a larger group of patients. A second treatment with IFN-_ is not useful.

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Treatment IFN-_ for 24 weeks, 5 MU/m2 3 ×/week s.c. or PegIntron* 1.5 +g/kg 1 ×/week s.c. is used for treatment (* as of yet there are no published data regarding treatment of hepatitis B in childhood).

Complications The following are complications in IFN-_ treatment of hepatitis B : – Flu symptoms – Loss of hair (often minimal and reversible) – Depression (seldom) – Neutropenia, thrombopenia – Delayed growth (usually compensated for once therapy ends) – Autoimmune thyroiditis.

Contraindications The following are contraindications for treatment : – Leukopenia, thrombopenia – Autoimmune illnesses (autoimmune hepatitis, thyroiditis) – Decompensated liver cirrhosis – Acute psychosis, depression – Epilepsy – Immune suppression

Nucleoside analogues The second best choice for therapy or when the side effects of IFN-_ cannot be tolerated or the patient has advanced liver cirrhosis, are the nucleoside analogues. Based on many years of experience, currently lamivudine is the best choice for children. Under certain circumstances, adefovir dipivoxil can be given, especially as resistance is less likely to develop under this drug. Essentially, nucleoside analogues are effective due to the misarranged inclusion of a nucleoside during viral replication. Initially, in 50–80% of patients the HBV-DNA level drops and may no longer be detectable in conventional PCR, and in 50–70% the transaminase levels return to normal. In addition, liver inflammation is suppressed in over 50% of patients. However, these improvements appear to be restricted to the period of therapy, and 17–36% of those who do not receive treatment achieve anti-HBe seroconversion (Fig. 3). In the largest published trial [12], it was evident that anti-

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Figure 3. Seroconversion to anti-HBe (%) after 1 year of treatment with lamivudine (black column) or without therapy (gray column). Columns from left to right: (1)+(2) [9] (n = 58); (3)+(4) [10] (n = 106); (5) [11] (n = 20); (6)+(7) [12] (n = 288).

HBe seroconversion under lamivudine treatment strongly correlated with the pre-treatment aminotransferase levels, which is very similar to that seen for IFN-_ treatment. Beside patients with chronic infection, there is now strong evidence that patients with fulminant hepatitis B benefit from treatment with lamivudine. One disadvantage in treatment is the selection of resistant mutations. This occurs in approximately 19% of patients over the course of 1 year and often corresponds to an increase in transaminases. However, studies in adults show that long-term therapy with lamivudine over 5 years increases the rate of seroconversion to approximately 60%. On the other hand, 70% of these patients develop resistant mutations [7]. In the USA, lamivudine has been approved for the treatment of chronic hepatitis B in children. In addition to lamivudine, there are other nucleoside analogues, including adefovir dipivoxil, and most recently, entecavir or telbivudine. An international multi-centered study is currently considering whether or not to approve the treatment of children with adefovir. Adefovir does not appear to be more effective than lamivudine, but induces significantly fewer resistant mutations. According to a study by Hadziyannis et al. (unpublished), no resistant mutations arose in the first year of treatment, only 3% in the second, and at most, 18% after 4 years.

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Course of therapy Lamivudine is given for 12 months, 15 mg/kg per day p.o. (max. 100 mg/ 100 ml). Adefovir dipivoxil is given for 12 months 0.3–0.5 mg/kg per day p.o., max. 10 mg.

Side effects Problems with kidney function are rare.

Contraindications Renal insufficiency/failure.

Summary of treatment options (Tab. 2) Summarizing treatment options in chronic hepatitis B in children and adolescents, it can be stated that both IFN-_ and nucleoside analogues have no very high effectivity in terms of induction of anti-HBe seroconversion. IFN-_ exceeds nucleoside analogues by roughly 10%. Effectivity increases with pre-treatment aminotransferases levels of more than 80– 100 U/l. It has to be pointed out that the drugs are not generally approved for this age group (e.g., in Europe) and they have to be used “off label” in most cases.

The future In the coming years, further development of nucleoside analogues (e.g., entecavir, telbivudine, emtricitabine, clevudine) should expand treatment options. In adults, more patients are undergoing long-term treatment with nucleoside analogues. To date it remains open whether these developments prove to be useful in the treatment of children.

Hepatitis C Epidemiology Chronic hepatitis C occurs only infrequently in childhood. Based on our own research, the estimated rate is about 1/2000 children in Germany [13]. Many of these patients were found to have received blood or blood prod-

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Table 2. Advantages and disadvantages of antiviral medications for chronic hepatitis B

IFN-_

Advantages

Disadvantages

Anti-HBs seroconversion ~5%

Significant side effects

Anti-HBe seroconversion 26–38% Expensive Lamivudine

Adefovir

Few if any complications In most patients HBV-DNA and GPT decrease during therapy Available in juice form Resistance development

No anti-HBs seroconversion Anti-HBe seroconversion only 10–15%

Like lamivudine but fewer resistances

More expensive and little experience with children

Higher than spontaneous development Less long-term experience

ucts, in other words a treatment via parenteral injection in countries where inadequate measures of sterilization were practiced. While this method of infection is becoming increasingly less important, the vertical route of infection for HCV is steadily increasing.

Serological diagnosis Screening tests to determine the levels of anti-HCV antibodies are conducted using commercial tests, whereas HCV-RNA is detected via PCR. Usually a PCR is conducted to quantify the “viral-load”, which provides the number of circulating HCV genomes. A positive finding is followed by HCV genotyping. Characteristically, the transaminases progress in an often unsteady and fluctuating manner, and the discovery of normal levels is not unusual. A liver biopsy is usually not necessary and should only be performed if there is any reason to suspect significant liver damage or as a means of ruling out other liver diseases.

Transmission The risk of vertical infection is 3–8%. The method of birth does not influence the risk of transmission. Transmission via blood or blood products still occurs, and remains an important consideration. Blood transmissions took place either before the introduction of screening tests for HCV in the early 1990s, or the child originated from a country without adequate screening methods. Although transmission is possible during sexual intercourse, it seldom occurs. With monogamous partners, the transfer of HCV is so rare that a concern about such transmission does not even justify condom use [14].

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Figure 4. Continuing response to therapy (%) (HCV-RNA negative 6 months after end of treatment). Black, genotype 1; gray, genotype 2 or 3. Columns from left to right: (1)+(2): [15], review (n = 366); (3)+(4) [16] (n = 41); (5)+(6) [17] (n = 61); (7)+(8) [18] (n = 118).

Therapy In comparison to hepatitis B, at least 50% of patients with chronic hepatitis C can be cured. IFN-_-2b (Intron A®) plus ribavirin (Rebetol®) is the approved treatment for children. Ribavirin can be obtained in capsule form as well as in juice form for smaller children. Based on numerous studies conducted in pediatric patients, successful treatment is assured with these two drugs. With the combination therapy of IFN-_ and ribavirin, approximately 45% of patients infected with genotype 1 and 4 and about 90% of those with genotypes 2 and 3, can be cured (Fig. 4). The rate of successfully treated pediatric patients therefore corresponds to those of adults [19]. To avoid late complications and to reduce the chances of contagion, and to maintain the high rates of success, treatment for all chronically infected children should be considered. Treatment with IFN-_ is much better tolerated in childhood than in adulthood and, therefore, it is best to think about treatment before puberty, as long as there are no contraindications. Children also experience the typical side effects of interferon, (see above), but usually they are significantly milder than in adults. Side effects are particularly present in the early phase of treatment, and can be treated

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with paracetamol given as a prophylaxis before each injection. After the first weeks many children get used to the side effects of interferon, which is a further argument for the treatment during childhood. In about 10% of children, specific thyroid antibodies are produced, which may cause hypothyroidism and in some instances must be treated (yet unpublished).

Therapy regimen IFN-_ is given over 12 weeks, 3 MU/m2 3 ×/week s.c. plus ribavirin 15 mg/kg per day. In the eventuality that HCV-RNA decreases by more than 99% after 3 months, treatment should either be continued for the total of 48 weeks or ceased. Treatment must be terminated if HCV-RNA continues to rise in spite of treatment. PegIntron® is given over 12 weeks 1.5 +g/kg 1 ×/week s.c. plus ribavirin 15 mg/kg per day. In case that HCV-RNA decreases by more than 99% after 3 months treatment should either be continued for the total of 48 weeks or ceased. Treatment must be terminated if HCV-RNA continues to rise in spite of treatment. Due to good response to therapy, patients with genotypes 2 and 3 need only be treated for 6 months as a whole.

Contraindications and side effects The most common side effects are flu-like symptoms, which often subside after a few weeks. For the most part they arise after an injection with IFN-_ and can be mitigated by paracetamol given as a prophylaxis (for details see Tab. 3). Side effects are definitively less pronounced before puberty. The induction of autoimmune thyroiditis is possible. Ribavirin may induce an anemia, which has little clinical relevance in most cases.

Second line treatment Basically, unsuccessful treatment can be re-attempted at a later time with another interferon (for example, Multiferon® or consensus interferon). However, no experience has been documented on repetition of treatment in children. We are currently conducting a study in previously treated children who are now receiving Multiferon® plus ribavirin. It is expected that patients who were free of the virus for a short time and became positive again during or after treatment, could profit from re-treatment. For patients who did not become HCV RNA negative during the first treatment period, re-treatment will most likely have less benefit. In any case, the rate of recovery with re-treatment is expected not to exceed 20% for patients infected with genotype 1.

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Table 3. Side effects of interferon-alpha therapy plus ribavirin [19] Side effect

%

Serious %

Headaches

69

3

Fever

61

Abdominal pain

39

Vomiting

42

Myalgia

32

2 <1

Diarrhea

25

Pharyngitis

27

Weight loss

25

Alopecia

23

Inflammation at place of injection

19

Emotional instability

16

Depression

13

Pruritis

12

Arthralgia

15

<1

<1

The future High hopes for the treatment of chronic hepatitis C are being pinned on two new groups of substances: protease inhibitors and polymerase inhibitors that suppress HCV replication. These have been around for a number of years. Some are presently being tested in Phase II studies on patients. The most promising drugs are SCH 503034, VX 950 and valopicitabine (NM 283). It is expected that at least one of these substances will be employed for the treatment of chronic hepatitis C in adults, at least in drug trials. Presumably, these new drugs will be used in combination with other antiviral substances.

References 1 2 3

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Thomas HC, Lemon S, Zuckerman AJ (eds) (2005) Viral hepatitis, 3rd edn. Blackwell Publishing, Oxford Shapiro CN, Margolis HS (1990) Hepatitis B epidemiology and prevention. Epidemiol Rev 12: 221–227 Grob P, Jilg W, Bornhak H, Gerken G, Gerlich W, Gunther S, Hess G, Hudig H, Kitchen A, Margolis H et al (2000) Serological pattern “anti-HBc alone”. J Med Virol 62: 450–455 Bortolotti F, Jara P, Barbera C, Gregorio GV, Vegnente A, Zancan L, Hierro

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L, Crivellaro C, Vergani GM, Iorio R et al (2000) Long term effect of alpha interferon in children with chronic hepatitis B. Gut 46: 715–718 Sokal EM, Wirth S, Goyens P, Depreterre A, Cornu C (1993) Interferon alfa-2b therapy in children with chronic hepatitis B. Gut 34: 87–90 Sokal EM, Conjeevaram HS, Roberts EA, Alvarez F, Bern EM, Goyens P, Rosenthal P, Lachaux A, Shelton M, Sarles J, Hoofnagle J (1998) Interferon alpha therapy for chronic hepatitis B in children: a multinational randomized controlled trial. Gastroenterology 114: 988–995 Lai CL, Dienstag J, Schiff E, Leung NW, Atkins M, Hunt C, Brown N, Woessner M, Boehme R, Condreay L (2003) Prevalence and clinical correlates of YMDD variants during lamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis 36: 687–696 Vo Thi Diem H, Bourgois A, Bontems P, Goyens P, Buts JP, Nackers F, Tonglet R, Sokal EM (2005) Chronic hepatitis B infection: long term comparison of children receiving interferon alpha and untreated controls. J Pediatr Gastroenterol Nutr 40: 141–145 Janssen HL, van Zonneveld M, Senturk H, Zeuzem S, Akarca US, Cakaloglu Y, Simon C, So TM, Gerken G, de Man RA et al (2005) Pegylated interferon alfa-2b alone or in combination with lamivudine for HBeAg-positive chronic hepatitis B: a randomised trial. Lancet 365: 123–129 Figlerowicz M, Kowala-Piaskowska A, Filipowicz M, Bujnowska A, MozerLisewska I, Sluzewski W (2005) Efficacy of lamivudine in the treatment of children with chronic hepatitis B. Hepatol Res 31: 217–222 Hartman C, Berkowitz D, Shouval D, Eshach-Adiv O, Hino B, Rimon N, Satinger I, Kra-Oz T, Daudi N, Shamir R (2003) Lamivudine treatment for chronic hepatitis B infection in children unresponsive to interferon. Pediatr Infect Dis J 22: 224–249 Jonas MM, Mizerski J, Badia IB, Areias JA, Schwarz KB, Little NR, Greensmith MJ, Gardner SD, Bell MS, Sokal EM (2002) International Pediatric Lamivudine Investigator Group. Clinical trial of lamivudine in children with chronic hepatitis B. N Engl J Med 346: 1706–1713 Gerner P, Wirth S, Wintermeyer P, Walz A, Jenke A (2006) Prevalence of hepatitis C virus infection in children admitted to an urban hospital. J Infect 52: 305–308 Vandelli C, Renzo F, Romano L, Tisminetzky S, De Palma M, Stroffolini T, Ventura E, Zanetti A (2004) Lack of evidence of sexual transmission of hepatitis C among monogamous couples: results of a 10-year prospective follow-up study. Am J Gastroenterol 99: 855–859 Jacobson KR, Murray K, Zellos A, Schwarz KB (2002) An analysis of published trials of interferon monotherapy in children with chronic hepatitis C. J Pediatr Gastroenterol Nutr 34: 52–58 Wirth S, Lang T, Gehring S, Gerner P (2002) Recombinant alfa-interferon plus ribavirin therapy in children and adolescents with chronic hepatitis C. Hepatology 36: 1280–1284 Wirth S, Pieper-Boustani H, Lang T, Ballauff A, Kullmer U, Gerner P, Wintermeyer P, Jenke A (2005) Peginterferon alfa-2b plus ribavirin treatment

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in children and adolescents with chronic hepatitis C. Hepatology 41: 1013– 1018 Gonzalez-Peralta RP, Kelly DA, Haber B, Molleston J, Murray KF, Jonas MM, Shelton M, Mieli-Vergani G, Lurie Y, Martin S et al (2005) Interferon alfa-2b in combination with ribavirin for the treatment of chronic hepatitis C in children: efficacy, safety, and pharmacokinetics. Hepatology 42: 1010–1018 Ahn J, Flamm S (2004) Peginterferon-alpha(2b) and ribavirin. Expert Rev Anti Infect Ther 2: 17–25

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Invasive fungal infections in children: advances and perspectives Andreas H. Groll1, Julia Koehler2 and Thomas J. Walsh3 1Infectious Disease Research Program, Center for Bone Marrow Transplantation and Department of Pediatric Hematology/Oncology, University Children’s Hospital, Münster, Germany; 2Division of Infectious Diseases, Children’s Hospital Boston, Boston, Massachusetts, USA; 3Immunocompromised Host Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

Abstract Invasive fungal infections are important causes of morbidity and mortality in immunocompromised children. The past two decades have seen a dramatic increase in both number and overall relevance of invasive fungal infections in the hospital. At the same time, however, improved microbiological and imaging techniques together with an increased awareness have shifted the diagnosis of fungal infections from the autopsy theatre to the bedside. Major advances have been made in the definition of fungal diseases, the algorithms of antifungal interventions, the design and implementation of clinical trials and the development of standardized in vitro susceptibility testing. Most importantly, however, an array of new antifungal agents has entered the clinical arena and has made antifungal therapy more safe, more effective, but also more complicated. This article reviews some unique features of invasive fungal infections in infants and children and provides an update on the pharmacology of antifungal therapeutics in the pediatric population.

Introduction Invasive fungal infections are important causes of morbidity and mortality in immunocompromised children. These infections remain difficult to diagnose and the outcome depends critically on the prompt initiation of appropriate antifungal chemotherapy and restoration of host defenses. The past two decades have seen a dramatic increase in both number and overall relevance of invasive fungal infections in the hospital. At the same time, however, improved microbiological and imaging techniques together with an increased awareness have shifted the diagnosis of fungal infections from the autopsy theatre to the bedside. Major advances have been made in the definition of fungal diseases, the algorithms of antifungal interventions, the design and implementation of clinical trials and the development of standardized in vitro susceptibility testing. Most importantly, however, an array of new antifungal agents has entered the clinical arena and has made antifungal therapy more safe, more effective, but also more complicated.

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Children, in particular neonates and young infants, represent a special population not only in a pharmacological sense but also with regard to epidemiology and manifestations of fungal infections. This review therefore focuses on unique features of invasive fungal infections in infants and children and the pharmacology of antifungal therapeutics in the pediatric population.

Host biology: Aspects unique to pediatric patients Anatomical considerations are important throughout infancy, but particularly in preterm neonates. Due to the reduced thickness of the skin, the use of medical devices and the moist environment, preterm neonates have a particular susceptibility to developing primary cutaneous aspergillosis and zygomycosis [1, 2]. Similarly, the extremely tenuous gastrointestinal wall structures lead to a unique propensity to primary invasive gastrointestinal mold infections with precipitous perforation, a pattern that is relatively uncommon in other settings [3, 4]. The comparably small diameter of blood vessels provides a nidus for catheter-associated Candida thrombophlebitis, -thrombosis, and endocarditis [5–8]; life-threatening Candida laryngitis and epiglottitis may occur in immunocompromised infants and young children for similar anatomical reasons [9–12]. In neonates, physiological differences such as the larger fractional water content, the smaller plasma protein fraction, relatively larger organ volumes, and the functional immaturity of hepatic metabolism and renal excretion result in considerable differences in drug distribution, metabolism, and elimination as compared to a standard healthy adult [13–15]. The still incomplete blood-brain barrier, in addition to its pharmacological consequences on drug penetration, may also be one reason for the enhanced risk of neonates to develop meningoencephalitis, an otherwise unusual complication of invasive Candida infection [16, 17]. Infants and younger children continue to exhibit differences in the relative proportion of body water, adipose tissue, and organ volumes; of note, as compared to the age-related, decreasing organ function in adult individuals, the functional reserve of both hepatic and renal function is generally larger [15]. Specific immunological characteristics in neonates include a functional immaturity of mono- and polymorphonuclear phagocytes and T lymphocytes [18] as well as a possibly increased susceptibility to the immunosuppressive effects of glucocorticosteroids [4], which may render them susceptible to nosocomially acquired opportunistic mycoses. The yet-developing cellular immunity may also explain the occurrence of overwhelming infections by Histoplasma capsulatum [19, 20] and possibly other endemic fungi in infants [21]. The pediatrician may also become confronted with neonates and infants who present with superficial or invasive fungal infections as one of the first manifestations of a congenital T cell immunodeficiency [22, 23]

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or of chronic granulomatous disease [24, 25]. In older children and adolescents, genetic illnesses such as cystic fibrosis or B cell disorders, which lead to chronic recurrent airway infection and lung destruction, may result in fungal airway disease including allergic bronchopulmonary aspergillosis, aspergilloma, and sometimes, invasive mould infections [26, 27].

Pediatric populations at risk for invasive infections The pediatric populations at risk can be defined by specific predisposing defects in host defenses and several additional, non-immunological factors. In general, deficiencies in the number or function of phagocytic cells are associated with invasive infections by opportunistic fungi, such as Candida spp., Aspergillus spp., zygomyces spp. and a large variety of other, less frequently encountered yeasts and molds. In contrast, deficiencies or imbalances of T lymphocyte function are linked to mucocutaneous candidiasis and invasive infections by Cryptococcus neoformans and the dimorphic moulds (Fig. 1). Non-immunological factors include the necessary exposure to the organism, preexisting tissue damage, and, limited to Candida spp., the presence of indwelling vascular catheters, colonization of mucous membranes, the use of broad-spectrum antibiotics, parenteral nutrition, and complicated intra-abdominal surgery [28]. In extension of this classification, the pediatric populations at risk for invasive fungal infections include neonates, in particular preterm neonates; pediatric patients with congenital immunodeficiencies involving phagocytic or T lymphocyte functions; pediatric patients with acquired immunodeficiencies such as HIV infection, cancer, hematopoietic stem cell transplantation (HSCT) or solid organ transplantation, and immunosuppressive treatment with corticosteroids; children of all age groups beyond the neonatal period that are hospitalized for severe acute illnesses; and those with chronic-destructive lung disease (Tab. 1).

Epidemiology and presentation of invasive fungal infections in pediatric patients The neonate Candida spp. colonize the vaginal tract of approximately 30% of pregnant women; very rarely, they can become the cause of chorioamnionitis and intrauterine infection [29, 30]. Candida rapidly colonizes the mucocutaneous surfaces [31, 32]; in healthy infants, this colonization may result in thrush and diaper dermatitis [31]. In hospitalized, ill neonates, however, Candida has evolved as important cause of life-threatening invasive infections, particularly in very low birth weight infants. Candida spp. now account for 9–13%

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Figure 1. Clinical classification of fungal pathogens observed in humans.

of all bloodstream isolates in neonatal intensive care units (NICUs) [33, 34]. In the U.S., Candida spp. currently are the third most common cause of late onset sepsis, and second only to polyresistant Enterobacter spp. in mortality [35, 36]. Case series indicate that invasive candidiasis occurs in up to 5% of infants with a birth weight of < 1500 g and in 8–28% of infants with a birth weight of < 1000 g; the crude mortality associated with these infections ranges from 15% to 30% with an attributable mortality of 6–22% despite appropriate therapy [37–50]. Moreover, a recent large analysis showed that 73% of extremely low birth weight infants (< 1000 g) with invasive candidiasis did not survive or had significant neurodevelopmental impairment [51]. Invasive candidiasis in preterm infants is most commonly due to C. albicans and C. parapsilosis [43, 47] and associated with prior mucocutaneous colonization, vascular catheters, the use of broad-spectrum antibiotics and corticosteroids, and parenteral hyperalimentation [47, 52–55]. Most neonates with systemic candidiasis are symptomatic at the onset of their disease and present with signs and symptoms that are virtually identical to those of non-fungal etiological agents. Among deeply invasive infections, cutaneous, renal, pulmonary, and cerebral involvement are disproportionally common [28], and Candida is increasingly recognized as causative agent of infections associated ventricular shunts and drains [56]. Fungemia persisting for 14 days and longer despite appropriate management has been reported to occur in as much as 10% of extremely low birth weight infants with candidemia and poses a particular challenge to the infectious disease specialist [51, 57]. Numerous outbreaks have been reported, which underscores the

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Table 1. Pediatric populations at risk for invasive fungal infections – Neonates – Infants – Children with congenital immunodeficiencies – defects of phagocytic host defenses – defects of specific cellular host defenses – Children with acquired immunodeficiencies – iatrogenic immunosuppression – treatment for cancer – HIV infection – Children with acute illnesses – Children with chronic airway diseases

importance of appropriate infection control measures for prevention of these infections [44, 53]. Malassezia spp. are lipophilic commensal yeasts that colonize the human skin and may cause pityriasis, a skin infection that is only cosmetically relevant. However, these organisms also may gain access to the bloodstream via percutaneous vascular catheters to cause a potentially fatal systemic infection in premature infants receiving parenteral nutritional lipid supplements [58, 59]. Similar to Candida, the most probable mode of acquisition is via the hands of health care workers [60], but direct contamination through contaminated intravenous (IV) solutions and catheters has also been reported [61]. Special media containing olive oil are required for isolation [58]. Infections by Aspergillus species and zygomyces are very rare in the neonatal setting. They tend to have a predilection for the skin, and, in the case of the zygomycetes, for the gastrointestinal tract, resulting in necrotizing skin lesions and devastating necrotizing gastroenterocolitis, respectively. Potential sources of the organism are contaminated water, contaminated ventilation systems and contaminated dressing materials or infusion boards [1–4, 62]. A large literature review in the late 1990 found 44 cases of invasive aspergillosis that were reported in children of ) 3 months of age. Most of these infants had either invasive pulmonary (23%), primary cutaneous (25%), or disseminated aspergillosis (32%). Prematurity, chronic granulomatous disease, and a complex of diarrhea, dehydration, malnutrition, and invasive bacterial infections accounted for the majority of underlying conditions (82%). Only few patients were neutropenic, but at least 41% had received corticosteroids. While all other forms of the disease mainly occurred in term infants, cutaneous as well as alimentary tract aspergillosis occurred almost exclusively in preterm neonates. Disseminated disease was uniformly fatal, but patients who received appropriate therapy had over 70% survival [4]. Invasive mould infections in the setting of neonatal medicine should be considered in infants with

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expanding, necrotizing skin lesions or gastrointestinal perforation. Surgical debridement is essential in most cases [3, 4].

The infant Disseminated histoplasmosis is a classical example for the potentially dismal course of a primary infection by an endemic fungus in apparently healthy infants that were exposed to the organisms. The disease is fatal if not detected and treated. Its clinical manifestations include prolonged fevers, failure to thrive, hepatosplenomegaly, pancytopenia, and ultimately, disseminated intravascular coagulation and multiorgan failure [19, 20, 63]. Not much is known about blastomycosis and cocidioidomycosis in this age group, but ultimately fatal cases have been reported [21, 64, 65]. Conceptually, primary infection by endemic fungi during infancy is reminiscent of the infantile form of pulmonary pneumocystosis, which is associated with young age, malnutrition, and endemic exposure [66]. Candida albicans is a ubiquitous agent of diaper dermatitis, which may be precipitated by moisture, occlusion, fecal contact and urinary pH. Its classical presentation is that of an erythema bordered by a collarette of scale with satellite papules and pustules. Concomitant dermatophytosis may occasionally be present. Treatment consists of the correction of physiological factors and topical antifungal treatment [28].

Children with congenital immunodeficiencies Among the phagocyte-defect syndromes, myeloperoxidase (MPO) deficiency is the most common entity. While MPO-deficient cells have only minor microbicidal abnormalities against bacteria in vitro, killing of Candida spp is highly deficient and may serve as explanation for invasive Candida infections reported in some patients with this disorder [67, 68]. Chronic granulomatous disease of childhood (CGD) is a genetically diverse congenital disorder of the NADPH oxidase complex that is associated with an inability of phagocytic cells to provide antimicrobial oxidants and to kill ingested microorganisms [69]. It is the prime example for an inherited immune disorder with a high risk of invasive mycoses; at the same time, it serves as a paradigm for the importance of phagocytosis in the defense of infections by opportunistic moulds. Invasive mycoses, particularly invasive aspergillosis, may repeatedly complicate the course of this disorder, accounting for an estimated lifetime incidence of between 16% and 40% [24, 25, 70, 71]. Interferon-a (IFN-a) or prophylactic antifungal triazoles may reduce the frequency of these infections [72, 73]. Treatment is protracted and consists of antifungal chemotherapy, IFN-a, and appropriate surgical interventions; the precise role of gene therapies and HSCT has yet to be defined [28, 74–76].

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The role of immunoglobulins in host defenses against fungi is important against cryptococcosis and possibly mucosal and invasive candidiasis [77], but it is not well understood for other mycoses. Children with inherited deficits of B lymphocytes appear to be not at increased risk for fungal infection, unless there is a concomitant disorder of T lymphocytes or phagocytosis. This includes individuals with the x-linked hyper-IgM syndrome [78], and patients with the hyper-IgE syndrome, which is associated with chronic mucocutaneous candidiasis, and possibly with cryptococcosis and aspergillosis [79]. Inherited immunodeficiencies involving the number or function of T lymphocytes predispose to mucocutaneous and, occasionally, invasive candidiasis, and conceptually, to cryptococcosis and histoplasmosis [22, 77]. Severe combined immunodeficiency (SCID) and severe types of thymic hypoplasia (DiGeorge syndrome) are medical emergencies of the neonatal period that can be managed successfully only with HSCT or thymus transplantation, respectively [80–82]. Refractory mucocutaneous candidiasis is a hallmark of these disorders and can therefore be an important clue to the appropriate immunological work-up. Chronic mucocutaneous candidiasis is a less severe congenital immunodeficiency with an impaired T cell response to Candida antigens [83]. It is characterized by chronic recurrent candidiasis of nails, skin, perineum, and oropharynx and may be idiopathic or associated with either the polyendocrinopathy syndrome type I or the hyper-IgE syndrome [79, 84].

Children with acquired immunodeficiencies Iatrogenic immunosuppression Treatment with pharmacological dosages of glucocorticosteroids rapidly provides a functional impairment of phagocytosis by mono- and polymorphonuclear leukocytes. Similar to adults, such therapy is one of the most important reasons for the increased susceptibility to invasive mycoses of children with immunosuppressive therapy for immunological disorders, solid organ transplantation, and for graft-vs.-host disease (GVHD) following HSCT [28, 85, 86].

Cancer While current treatment for pediatric cancers is curative in most instances, highly dose-intensive chemotherapy regimens and aggressive supportive care measures also result in profound impairments of host defenses. Prolonged, profound granulocytopenia is the single most important risk factor for opportunistic fungal infections in children and adolescents with

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cancer [87, 88]. Other well-known, but notable risk factors include chemotherapy-induced mucositis, extended courses of broad-spectrum antibiotics, the presence of indwelling central venous lines, and, particularly in children with acute leukemia, the therapeutic use of glucocorticosteroids [89]. Oropharyngeal candidiasis (OPC) may occur in up to 15% of children undergoing intensive chemotherapy or bone marrow transplantation despite various forms of topical or systemic antifungal prophylaxis [90]. Esophageal candidiasis is also not uncommon, even in the absence of conspicuous OPC [28], and Candida epiglottitis and laryngeal candidiasis may emerge in neutropenic children as life-threatening causes of airway obstruction [9, 10, 91]. Similar to the adult cancer population, Candida- and Aspergillus spp are the most common causes of invasive fungal infections in children with cancer [88, 92]. Invasive candidiasis in neutropenic children may present as catheter-associated candidemia, acute disseminated candidiasis, and deep single organ candidiasis. Its overall frequency in children with high-risk leukemias and/or bone marrow transplantation is between 5% and 10%; the crude mortality associated with these infections is at least 20% and close to 100% in patients with persistent neutropenia [88, 93–100]. Catheter-associated fungemia is most commonly caused by C. albicans, but non-albicans Candida spp., particularly C. parapsilosis, and previously uncommon yeast pathogens are increasingly encountered [88, 100–102]. Whether the primary source of fungemia or a target for attachment of circulating organisms, the intravascular catheter serves as a source for continued seeding of the bloodstream and should be removed whenever feasible [103–106]. Acute disseminated candidiasis occurs typically in granulocytopenic children and manifests with persistent fungemia, hemodynamic instability, multiple cutaneous and visceral lesions and high mortality despite antifungal therapy [28, 97]. Candida albicans is the most frequent cause, although C. tropicalis has been increasingly implicated as an important pathogen in neutropenic children. Flynn et al. [107] reported 19 children treated for leukemia in whom C. tropicalis infections developed. Fungemia without meningitis in 11 children was treated successfully, whereas meningitis in 7 children was uniformly fatal, underscoring that meningitis is a critical factor for outcome of this infection. Chronic disseminated candidiasis typically presents with fever despite granulocyte recovery, often coupled with right upper quadrant abdominal pain, and increased alkaline phosphatase levels [108, 109]. Imaging studies demonstrate multiple lesions in liver, spleen, and other organs that correspond morphologically to large granulomas with extensive chronic inflammatory reaction [110]. Treatment is protracted [28], but may not necessarily require the interruption of anticancer therapy, provided that the disseminated infection has stabilized or is resolving [111]. Invasive aspergillosis has emerged as important cause for morbidity and mortality in children with hematological malignancies or undergoing bone marrow transplantation; more recent pediatric series indicate a frequency

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of 4.5–10% in this setting with an associated crude mortality of 40–94% [88, 94, 102, 112–114]. The disease is rather rare in children treated for solid tumors, underscoring the role of prolonged neutropenia and corticosteroid therapy in its pathogenesis [94, 112]. Similar to the adult setting, the lungs are the most frequently affected site, and disseminated disease is found in approximately 30% of cases [113]. While paranasal sinus aspergillosis appears to be less common than in adults [112, 115, 116], primary cutaneous aspergillosis has been preferentially reported in the pediatric setting in association with lacerations by armboards, tape, and electrodes and at the insertion site of peripheral or central venous catheters [115, 117–120]. With combined surgical and medical therapy, primary cutaneous aspergillosis has a comparatively more favorable prognosis [115]. The outcome of invasive aspergillosis children with hematological malignancies may not be as dismal as in adults [88, 112]. In a recent small series, all patients who were treated with amphotericin B for a minimum of 10 days responded to medical or combined medical and surgical therapy, and 64% were cured [112]. Nevertheless, the overall long-term survival was merely 31% after a median follow-up of 5.6 years. Apart from recurrent or refractory cancer, in that study, the main obstacles to a successful outcome were failure to diagnose the invasive aspergillosis during lifetime and, in patients with established diagnosis, catastrophic pulmonary or cerebral hemorrhage. Similar to histoplasmosis [121, 122], cryptococcal meningoencephalitis or pneumonitis are rare opportunistic infections in children with cancer [19]. In patients with pediatric sarcomas, however, pulmonary cryptococcosis may be a differential diagnosis of lung metastasis [123] and case reports such as that from a child with acute leukemia in remission that died suddenly from unrecognized disseminated cryptococcosis may serve as a reminder of the risk for this potentially life-threatening infection [124]. During the last decade, previously uncommon fungal pathogens have been increasingly recognized to cause systemic infection in neutropenic patients [101, 125] (Fig. 1). Particularly notable among the yeast-like organisms is Trichosporon beigelii, a normal human commensal and the agent of White Piedra. Trichosporonosis in neutropenic patients presents in a similar way as systemic candidiasis with fungemia and disseminated infection and carries a high mortality [126, 127]. Tr. beigelii is often resistant to the fungicidal effects of amphotericin B, but may be amenable to antifungal azoles [128–131]. Among the filamentous fungi, the zygomycetes are notorious for their propensity to invade blood vessels, a rapidly deteriorating clinical course, and clinical refractoriness to antifungal therapy; the most common clinical presentations in the neutropenic host are rhinocerebral, pulmonary, cutaneous, and disseminated infection therapy [132, 133]. Fusarium has emerged in some institutions as the second most common filamentous pathogen after Aspergillus [134, 135]. Like the latter, the airborne organism is highly angioinvasive and leads to hemorrhagic infarction. Fusarium is among the few filamentous fungi that cause detectable fungemia and

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metastatic skin lesions are a hallmark of disseminated fusariosis. A clinical stabilization can sometimes be achieved with high-dose amphotericin B or voriconazole, but rapid recovery from neutropenia is always a prerequisite for survival [101, 134, 136].

HIV infection Children are recognized as one of the most rapidly expanding populations worldwide infected with human immunodeficiency virus (HIV); mucosal as well as invasive fungal infections are major causes of morbidity and mortality in advanced stages of the disease [137]. OPC is the most prevalent opportunistic infection in HIV-infected children and, prior to the advent of highly active antiretroviral treatments (HAART), occurred in virtually all patients at some time during the course of their disease. Esophageal candidiasis in the era prior to HAART occurred in approximately 10% of patients and was associated with recurrent OPC, low CD4+ counts, and use of broad-spectrum antibiotics [138]; it may still be observed in the subgroup of patients not responding to HAART and presents without concomitant OPC or typical clinical symptoms [139]. In the absence of significant immunological reconstitution, oropharyngeal and esophageal candidiasis have an exceedingly high propensity to recur. The chronic use of fluconazole under these circumstances has been associated with the emergence of fluconazole-resistant Candida strains [140]; it has been shown that such resistant strains can be exchanged among HIVinfected family members [141]. Children with HIV infection may develop candidemia or disseminated candidiasis as a nosocomial infection during prolonged hospitalization for complicated medical problems [142]. However, with increased use of outpatient treatments, candidemia may present as a community-acquired infection that is associated with ambulatory total parenteral nutrition and IV therapy via indwelling central venous lines [143]. Univariate and multiple logistic regression reveal that the prolonged presence of a central venous catheter is the most important risk factor for fungemia in this setting [144]. Non-albicans spp. account for almost 50% of all isolates. A high rate of survival (95%) from fungemia without post-therapeutic sequelae has been obtained by early detection, appropriate antifungal chemotherapy, and removal of the vascular catheter [143]. HIV-related impairment of phagocytosis by mono- and polymorphonuclear leukocytes [145, 146] makes a major contribution to the increased susceptibility of patients with advanced HIV infection to invasive aspergillosis [147–149]. Invasive aspergillosis has also been reported in HIV-infected children [150–152]. Invasive aspergillosis was diagnosed in 7 (1.5%) of 473 HIV-infected children followed at the Pediatric Branch of the National Cancer Institute from 1987 to 1997 [152]. Invasive pulmonary aspergillosis

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occurred in 5, and aspergillosis of the skin and adjacent soft tissues in 2 patients. All patients had low CD4+ counts (median, 2 /+L; range, 0–338). Neutropenia (< 500/+L) lasting for longer than 7 days or corticosteroid therapy were encountered in only two patients. Consistent with the experience in other immunocompromised children [115], patients with cutaneous aspergillosis were diagnosed during life and successfully treated, whereas diagnosis of pulmonary aspergillosis was made antemortem in only one patient [152]. Compared to adults, HIV-infected children have lower rates of cryptococcal infections, and, with the exception of disseminated penicilliosis [153], data on histoplasmosis and other endemic mycoses are very limited [137]. With an estimated 10-year point prevalence of 1% [154], cryptococcosis appears to be an infrequent opportunistic infection in HIV-infected children. It is associated with low CD4+ counts, and, in the majority of cases, a previous AIDS defining illness and older age; the clinical presentation may be subtle to fulminant, and may include unexplained fever and mostly diffuse central nervous and/or respiratory symptoms [155]. A review of 30 of an approximate total of 50 published cases indicated a crude mortality of 23% within the first month after diagnosis [154].

Children with severe acute illnesses Invasive procedures, indwelling vascular and urinary catheters, use of broad-spectrum antibiotics and corticosteroids, mechanical ventilation and parenteral feeding as well as length of stay and severity of the underlying condition, all contribute to a heightened risk of deeply invasive Candida infections in critically ill patients requiring intensive care [156]. While few data are available for general pediatric intensive care units, recent studies in adults have confirmed the high frequency of nosocomial Candida infections in this setting [156–160]. Candida spp. are among the five most common causes of bloodstream infections in intensive care units (ICUs) [158, 159, 161] and account for up to 17% of microbiologically documented infections [158]. Mirroring the general epidemiological trend, more than half of such infections are now due to non-albicans Candida spp. [159, 162]. In a recent investigation of the distribution and susceptibility of 179 clinical isolates of Candida spp. from four children’s hospitals, C. parapsilosis isolates were identified in 32%; nearly 20% were resistant to amphotericin B [163]. Zygomycosis may develop in the settings of neutropenia, corticosteroid therapy, bone marrow or solid organ transplantation, burn, and deferoxamine therapy for iron and aluminum overload states. Similar to adults [164], zygomycosis in children occurs in other distinct settings as well: Juvenile onset (type I) diabetes mellitus, particularly with uncontrolled diabetic ketoacidosis, and congenital aminoaciduria [28]. For example, among 41 reported cases of rhinocerebral zygomycosis in children beyond the neonatal age,

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20 (49%) had diabetes mellitus [133]. Rhinocerebral zygomycosis usually begins as an infection of the paranasal sinuses, which progresses to invade the orbit, retroorbital region, cavernous sinus and brain. Thus, signs and symptoms of sinusitis along with ocular findings in a diabetic patient should prompt a careful evaluation for rhinocerebral zygomycosis [28, 165].

Children with chronic pulmonary diseases Mycoses may also occur in children and adolescents with chronic sinopulmonary infection and lung destruction, as it may be associated with congenital B cell defects, the hyper-IgE syndrome, and, most commonly, cystic fibrosis. Non-invasive fungal diseases associated with the colonization of the respiratory tract by Aspergillus spp. and other moulds such as allergic bronchopulmonary aspergillosis and aspergilloma formation clearly predominate in this setting [166]. However, invasive pulmonary mould infections have been reported [79, 167, 168], and also, fungemias associated with the presence of indwelling vascular catheters [169].

Recent advances in early diagnosis and preemptive therapy Early diagnosis and rapid initiation of effective antifungal chemotherapy is paramount to the successful management of invasive mycoses. The microbiological diagnosis should be attempted if feasible in all cases of suspected invasive fungal infection, and the organism identified at the species level. Because of the lack of its predictive value in other settings, the performance of in vitro susceptibility testing is currently reserved to Candida species vs. fluconazole and flucytosine, respectively. Additional in vitro testing of other organism/drug combinations may be indicated in refractory infections and within surveillance programs [170]. Improved blood culture detection techniques, such as the lysis-centrifugation and the BacTec Alert system, are able to detect candidemia earlier and more frequently than conventional systems [171]. However, it must be emphasized that candidemia is only one manifestation of invasive candidiasis, and that single organ or early disseminated candidiasis are not reliably detected by blood culture techniques and may therefore require more invasive diagnostic procedures [172]. For such tissue-invasive Candida infections, ultrasound, high-resolution computed tomography (HRCT) and magnetic resonance imaging (MRI) have become indispensable tools for detection, monitoring and as guidance of diagnostic procedures [173–176]. In the future, nonculture techniques, particularly nucleic acid amplification based systems, may complement existing blood culture systems not only for early detection purposes but also for determining resistance patterns to antifungal agents [177].

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Apart from improved detection of invasive mould infections of the paranasal sinuses [178], the advent of modern imaging techniques has also permitted earlier detection of pulmonary infiltrates consistent with invasive pulmonary aspergillosis and early preemptive treatment [179–181]. However, although peripheral nodules, the halo-sign and cavitation are all characteristic of pulmonary aspergillosis, these radiological criteria are not entirely specific, and nonspecific air space consolidation is common in early phases [181]. As previously rare filamentous fungi are becoming more common, a microbiological diagnosis by fiberoptic bronchoscopy with bronchoalveolar lavage or transcutaneous bioptic measures is greatly encouraged. Indeed, amplification of fungal DNA from biopsy specimens allows for rapid identification of the causative organism of invasive aspergillosis and mucormycosis and may allow guided antifungal treatment in patients with invasive mold infections [182]. Serial monitoring of galactomannan antigen and Aspergillus-specific nucleic acid sequences in blood [183–186] may contribute substantially to the detection of invasive pulmonary aspergillosis, particularly in the neutropenic host. The feasibility of a “preemptive“ approach based on the incorporation of sensitive, noninvasive diagnostic tests for consecutive high-risk neutropenic patients, while avoiding empirical therapy, has been demonstrated in a single-center study: Preemptive therapy based on serial galactomannan testing and high-resolution CT scans reduced the exposure to antifungal drugs and offered effective antifungal control, but it failed to detect non-Aspergillus invasive mycoses [187]. However, both galactomannan ELISA and PCR protocols appear to be less useful in children than in adults, and reliance on invasive procedures such as bronchoalveolar lavage or lung biopsy coupled with molecular diagnostics has been advocated [188]. Carefully designed clinical trials are now needed to determine the value of preemptive strategies at the dawn of more effective chemoprophylaxis in high-risk populations.

Pediatric pharmacology of established antifungal agents Amphotericin B deoxycholate For many years, amphotericin B deoxycholate (DAMB) has been the standard agent for systemic antifungal therapy. Amphotericin B primarily acts by binding to ergosterol in the fungal cell membrane, leading to pore formation and ultimately, cell death [189]. Amphotericin B possesses a broad spectrum of antifungal activity that includes most fungi pathogenic in humans. However, some of the emerging pathogens such as A. terreus, Tr. beigelii, Scedosporium prolificans and certain Fusarium spp. may be microbiologically and clinically resistant [101]. After IV administration of the deoxycholate formulation, amphotericin B rapidly dissociates from its vehicle and becomes highly protein bound

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before distributing predominantly into liver, spleen, bone marrow, kidney, lung and other sites [190]. Elimination from the body is slow; only small quantities are excreted into urine and bile [191, 192]. Due to the heterogenicity in underlying disease conditions and differences in the modes of administration, the reported pharmacokinetics of DAMB in pediatric patients are characterized by a high variability among individual patients [193–197]. Infants and children appear to clear the drug more rapidly than adults, as indicated by a significant negative correlation between patient age and clearance of DAMB [194, 195]. Whether this enhanced clearance from the bloodstream has implications for dosing remains to be elucidated as systematic studies correlating pharmacokinetic parameters with measures of outcome or toxicity have not been performed to date. Infusion-related reactions and nephrotoxicity are major problems associated with the use of DAMB and often limit successful therapy. Infusionrelated reactions (fever, rigors, chills, myalgias, arthralgias, nausea, vomiting, and headaches) are thought to be mediated by the release of cytokines from monocytes in response to the drug [198] and can be noted in up to 73% of patients prospectively monitored at the bedside [199]. In a more recent prospective study in pediatric cancer patients, fever and/or rigors associated with the infusion of DAMB were observed in 19 of 78 treatment courses (24%) [200]. Interestingly, however, these so characteristic adverse effects of DAMB are only rarely observed in the neonatal setting [38]. Infusionrelated reactions may be blunted by slowing the infusion rate, but often require acetaminophen, hydrocortisone (0.5–1.0 mg/kg) or meperidine (0.2–0.5 mg/kg) premedication [28]. Less common are hypotension, hypertension, flushing and vestibular disturbances; bronchospasm and true anaphylaxis are rare [201]. Cardiac arrhythmias and cardiac arrest due to acute potassium release may occur with rapid infusion (< 60 min), in particular if there is preexisting hyperkalemia and/or renal impairment [202, 203]. The hallmarks of amphotericin B-associated nephrotoxicity are azotemia, wasting of potassium and magnesium; tubular acidosis and impaired urinary concentration ability are rarely of clinical significance [201, 204]. As assessed prospectively in a large clinical trials in the setting of empirical therapy in persistently granulocytopenic patients, relevant electrolyte wasting occurs in approximately 12%, and increases in the serum creatinine by more than 100% in 34% of patients [199]. Azotemia can be exacerbated by concomitant nephrotoxic agents, in particular by cyclosporine and tacrolimus, but also by aminoglycosides and glycopeptides [205]. While some data suggest a somewhat lower rate of azotemia in children as compared to adults [206], this has not been a consistent observation [205]. Of note, DAMB-associated azotemia has been reported in only 2% of pediatric cancer patients receiving the drug at 1 mg/kg/day for comparatively short periods as empirical antifungal therapy [200]; in premature neonates, in more contemporary series containing safety data of DAMB (0.5–1.0 mg/ kg), the incidence of azotemia ranged from zero to 15% [38–41], indicat-

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Table 2. Medical management of invasive infections by opportunistic yeast Fungal disease

Management

Uncomplicated candidemia or invasive candidiasis

– Amphotericin B deoxycholate (0.6–1.0 mg/kg/day) (A-I) – Fluconazole (8–12 mg/kg/day; max. 800 mg/day) (A-I) – Fluconazole (16 mg/kg/day plus amphotericin B deoxycholate (0.7 mg/kg/day for days 1–5) (A-I) – Caspofungin (50 mg/day; day 1: 70 mg)* (A-I) – Voriconazole (4 mg/kg bid IV; day 1:12 mg/kg)** (A-I)

Acute dissem. candidiasis with hemodynamic instability

– Amphotericin B deoxycholate (0.7–1.0 mg/kg/day) plus flucytosine*** (100 mg/kg/day in 3–4 dosages) (B-III)

Second line therapy of – refractory infections – limiting toxicity

– Liposomal amphotericin B (3–5 mg/kg/day) (A-II) – Amphotericin B lipid complex (5 mg/kg/day) (B-II) – Voriconazole (4 mg/kg bid IV; day 1:12 mg/kg)** (B-II) – Caspofungin (50 mg/day; day 1: 70 mg)* (B-III)

Cerebral cryptococcosis

– Amphotericin B deoxycholate (0.7 mg/kg/day) plus flucytosine*** (100 mg/kg/day in 3–4 dosages) for * 2 weeks (induction), followed by fluconazole (8–12 mg/kg/day) (consolidation/maintenance) (A-I) – “Second line” for intolerance of amphotericin B deoxycholate: Liposomal amphotericin B (5 mg/kg/day) (B-II); in case of polyene intolerance: Fluconazole (8–12 mg/kg/ day) plus flucytosine*** (B-II)

Extracerebral manifestations

– Amphotericin B deoxycholate (0.7–1.0 mg/kg/day) (C-III) – Fluconazole (8–12 mg/kg/day) (C-III) – Amphotericin B deoxycholate (0.7 mg/kg/day) plus flucytosine*** (100 mg/kg/d in 3–4 dosages) (C-III)

* Adult dosage, not approved for individuals < 18 years; proposed pediatric dosage: 50 mg/m2/ day (day 1: 70 mg/m2, max.: 70 mg/day). ** IV dosage for patients > 11 years; IV dosage for children from 2 to 11 years: 7 mg/kg/day without loading dose. *** Monitoring of plasma concentrations recommended (> 40 to < 100 +g/mL).

ing that DAMB is much better tolerated than previously reported [207]. The renal toxicity associated with DAMB therapy may lead to renal failure and dialysis; however, azotemia most often stabilizes on therapy and is usually reversible after discontinuation of the drug [28]. Avoiding concomitant nephrotoxic agents, and using appropriate hydration and normal saline loading (10–15 mL NaCl/kg/day) [208–210] may lessen the likelihood and severity of azotemia. With the advent of new antifungal agents and following the completion of pivotal clinical Phase III trials, a few indications are left for antifungal treatment of opportunistic mycoses with conventional deoxycholate amphotericin B (Tabs 2–4). These include candidemia and acute disseminated candidiasis, particular in neonates, and induction therapy for cryptococcal meningitis. The recommended daily dosage in these settings ranges from 0.7 to 1.0 mg/kg/day administered over 2–4 h as tolerated. Treatment

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Table 3. Medical management of invasive infections by opportunistic molds Fungal disease

Management

Invasive aspergillosis – First line

– Voriconazole (4 mg/kg IV bid, day 1: 12 mg/kg) (A-I)* – Liposomal amphotericin B (5 mg/kg/day) (A-II)**

– Second line for – refractory infections – limiting toxicity

– Liposomal amphotericin B (* 5 mg/kg/day) (A-II) – Amphotericin B lipid complex (5 mg/kg/day) (A-II) – Caspofungin (50 mg/day IV; day 1: 70 mg) (A-II)*** – Voriconazole (4 mg/kg bid IV; day 1: 12 mg/kg) (A-II)*

Therapy of immediately lifethreatening infections

– Liposomal amphotericin B (* 5 mg/kg/day) plus caspofungin (50 mg/day IV; day 1: 70 mg) (C-III)*** – Voriconazole (4 mg/kg/day IV; day 1: 12 mg/kg)** plus caspofungin (50 mg/day IV; day 1: 70 mg) (C-III)***

Consolidation therapy

– Voriconazole (4200 mg bid PO) (B-III)* – Itraconazole (2.5 mg/kg bid PO) (B-III)# – Posaconazole (400 mg bid or 200 mg qid PO) (B-III)##

Non-Aspergillus hyalohyphomycetes

– Voriconazole (4 mg/kg bid IV; day 1: 12 mg/kg) (B-III)* – Liposomal amphotericin B (5–10 mg/kg/day IV) (C-III) – Amphotericin B lipid complex (5 mg/kg/day) (C-III) – Posaconazole (400 mg bid or 200 mg qid PO) (B-III)##

Zygomyces infections

– Liposomal amphotericin B (5–10 mg/kg/day) (B-II) – Amphotericin B lipid complex (* 5 mg/kg/day) (B-II) – Posaconazole (400 mg bid or 200 mg qid PO) for second line therapy only (B-II)##

Infection by pigmented filamentous fungi

– Voriconazole (4 mg/kg bid; day 1: 12 mg/kg) (C-III)* – Liposomal amphotericin B (* 5 mg/kg/day) (C-III) – Amphotericin B Lipid Complex (5 mg/kg/day) (C-III) – Posaconazole (400 mg bid or 200 mg qid PO) (B-III)## – Itraconazole (2.5 mg bid PO) (C-III)#

* IV dosage for patients >11 years; IV dosage for children of 2–11 years: 7 mg/kg/day without loading dose. PO dosages from 2 years onward: 200 mg bid. ** Based on a recently presented clinical trial [345] *** Adult dosage, not approved for individuals < 18 years; proposed pediatric dosage: 50 mg/ m2/day (day 1:70 mg/m2, max.: 70 mg/day) # Proposed pediatric dosage, monitoring of plasma trough concentrations recommended (target: > 0.5 +g/mL) ## Not approved in pediatric patients; 800 mg/day have been safely given to children > 12 years of age.

should be started at the full target dosage with careful bedside monitoring during the first hour of infusion [28, 106]. While better tolerated, continuous infusion over 24 h is not recommended due to the complete lack of efficacy data [211].

Lipid formulations of amphotericin B During the past decade, three novel formulations of amphotericin B have become available for clinical use: AMB colloidal dispersion (ABCD,

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Table 4. Medical management of invasive infections by endemic molds Fungal disease

Management

Histoplasmosis

– Liposomal amphotericin B (3 mg/kg/day IV) (A-I) – Amphotericin B deoxycholate (0.7 mg/kg/day IV) (B-I) – Itraconazole*,** (2.5 mg/kg bid) (A-II) – Fluconazole*** [(8)–12 mg/kg/day PO/IV] (A-II)

Coccidioidomycosis

– Amphotericin B deoxycholate (0.5–1.0 mg/kg/day IV) (A-III) – Fluconazole*** [(8)–12 mg/kg/day PO/IV] (A-II) – Itraconazole*,** (2.5 mg/kg bid) (A-II) – Posaconazole (400 mg bid or 200 mg qid PO) (B-III)##

Blastomycosis

– Amphotericin B deoxycholate (0.5–1.0 mg/kg/day IV) (A-II) – traconazole*,** (2.5 mg/kg bid) (A-II)

Paracoccidioidomycosis

– Amphotericin B deoxycholate (0.5–1.0 mg/kg/day IV) (A-II) – Itraconazole*,** (2.5 mg/kg bid) (B-III)

Penicilliosis

– Amphotericin B deoxycholate (0.5–1.0 mg/kg /day IV) (A-II) – Itraconazole*,** (2.5 mg/kg bid) (A-II)

* Clinically stable patients with mild to moderate disease outside and no CNS involvement, or as consolidation or maintenance therapy. Dosages refer to the cyclodextrin solution. ** Monitoring of trough plasma concentrations is recommended (target: > 0.5 +g/mL). Intravenous therapy 200 mg BID for 2 days, followed by 200 mg/day for patients > 18 years of age. *** Agent of first choice in (1) consolidation therapy of meningeal coccidioidomycosis; (2) Coccidioides-meningitis; (3) coccidioidomycosis of stable patients with mild to moderate disease or as consolidation or maintenance therapy. ## Second line therapy; not approved in pediatric patients; 800 mg/day have been safely given to children > 12 years of age.

Amphocil™, or Amphotec™) AMB lipid complex (ABLC or Abelcet™), and a small unilamellar vesicle (SUV) liposomal formulation (LAMB, AmBisome™). In comparison to DAMB, the lipid formulations share a reduced nephrotoxicity, which allows for the safe delivery of higher dosages of AmB [212, 213]. Each of the lipid formulations possesses distinct physicochemical and pharmacokinetic properties (Tab. 5). All three, however, preferentially distribute to the reticuloendothelial system (RES) and functionally spare the kidney. While the micellar dispersion of ABCD behaves very similar as compared to DAMB, the unilamellar liposomal preparation is only slowly taken up by the RES and achieves strikingly high peak plasma concentrations and AUC (area under the plasma concentration time curve) values. In contrast, the large ribbon-like aggregates of ABLC are rapidly taken up by the RES, resulting in lower peak plasma and AUC values [212, 213]. Whether and how the distinct physicochemical and pharmacokinetic features of each formulation translate into different pharmacodynamic properties in vivo is largely unknown. Safety and antifungal efficacy of ABCD, ABLC, and LAMB have been demonstrated in an array of phase II and III clinical trials in immunocom-

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Table 5. Physicochemical properties and multiple-dose pharmacokinetic parameters of the four currently marketed amphotericin B formulations

Lipids (molar ratio)

Mol% AMB Lipid configuration Diameter (+m) Dosage (mg AMB/kg)

DAMB

ABCD

ABLC

LAMB

Deoxycholate

Cholesterylsulfate

DMPC/DMPG (7:3)

HPC/CHOL/ DSPG (2:1:0.8)

34%

50%

50%

10%

Micelles

Micelles

Membranelike

suv

0.05

0.12–0.14

1.6–11

0.08

1

5

5

5

Cmax (+g/mL)

2.9

3.1

1.7

58

AUC0–24 (+g/mL·h)

36

43

14

713

VD (L/kg)

1.1

4.3

131

0.22

Cl (L/h·kg)

0.028

0.117

0.476

0.017

HPC, hydrogenated phosphatidylcholine; CHOL, cholesterol; DSPG, disteaoryl phosphatidylglycerol; DMPC, dimiristoyl phosphatidylcholine; DMPG, dimiristoyl phosphatidylglycerol; suv, small unilamellar vesicles; Cmax, peak plasma concentration; AUC0–24, area under the concentration vs. time curve from 0 to 24 h; VD, volume of distribution; Cl, clearance. Data represent mean values, stem from adult patients and were obtained after different rates of infusion. Modified from [213].

promised patients. The overall response rates in these trials ranged from 53% to 84% in patients with invasive candidiasis and 34% to 59%, respectively, in patients with presumed or documented invasive aspergillosis [201, 214]. A few randomized, controlled trials have been completed in which one of the new formulations has been compared to DAMB [199, 205, 215]. These studies have consistently shown at least equivalent therapeutic efficacy but reduced nephrotoxicity of the lipid formulations [214]. A considerable number of pediatric patients have been treated with either ABCD, ABLC or LAMB within the above-mentioned protocols, but separately published pediatric data are limited with the exception of ABLC.

Amphotericin B colloidal dispersion Population-based multiple-dose pharmacokinetic studies with ABCD in bone marrow transplant patients with systemic fungal infections included the compartmental analysis of five children < 13 years of age who received the compound at 7.0 and 7.5 mg/kg/day. Estimated pharmacokinetic parameters in these children were not significantly different from those obtained

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in a dose-matched cohort of adult patients [216]. Forty-nine children with febrile neutropenia were treated in a prospective, randomized trial comparing ABCD with DAMB; an additional 70 children with presumed or proven fungal infection were treated on five different open-label Phase II trials of ABCD. In the randomized trial, there was significantly less renal toxicity in the children receiving ABCD than in those receiving amphotericin B (12.0% vs. 52.4%; p = 0.003); other adverse symptoms were not significantly different. In the additional open-label studies, although 80% of patients receiving ABCD reported some adverse symptom, the majority of these were infusion related, and nephrotoxicity was reported in only 12%; there were no other unexpected severe toxicities [217].

Amphotericin B lipid complex The pharmacokinetics of ABLC have been studied in pediatric cancer patients who received the compound at 2.5 mg/kg over 6 weeks for hepatosplenic candidiasis; ABLC was effective and well tolerated, and no pharmacokinetic differences were observed as compared to those in adults [218]. Safety and antifungal efficacy of ABLC were studied in 111 treatment episodes in pediatric patients (21 days to 16 years of age) refractory of or intolerant to conventional antifungal agents through an open label, emergency use protocol. ABLC was administered at a mean daily dosage of 4.85 mg/kg (range, 1.1–9.5 mg/kg/day) for a mean duration of 38.9 days (range, 1–198 days). The mean serum creatinine for the entire study population did not significantly change between baseline (1.23 ± 0.11 mg/100 mL) and cessation of ABLC therapy (1.32 ± 0.12 mg/100 mL) over 6 weeks. No significant differences were observed between baseline and end-of-therapy levels of serum potassium, magnesium, hepatic transaminases, alkaline phosphatase, and hemoglobin. However, there was an increase in the mean total bilirubin (3.66 ± 0.73–5.13 ± 1.09 mg/100 mL) at the end of therapy (p = 0.054). In 7 patients (6%), ABLC therapy was discontinued because of one or more adverse effects. Among 54 cases fulfilling criteria for evaluation of antifungal efficacy, a complete or partial therapeutic response was obtained in 38 patients (70%) after ABLC therapy [219]. The safety and efficacy of ABLC was also assessed in 548 children and adolescents who were enrolled in the Collaborative Exchange of Antifungal Research (CLEAR) registry of the manufacturer between 1996 and 2000. Most patients were either intolerant of or refractory to conventional antifungal therapy. Response data were evaluable for 255 of the 285 patients with documented single or multiple pathogens. A complete (cured) or partial (improved) response was achieved in 54.9% of patients. There was no significant difference between the rates of new hemodialysis versus baseline hemodialysis. Elevations in serum creatinine of > 1.5× baseline and > 2.5× baseline values were seen in 24.8% and 8.8% of all patients, respectively. The overall response rate and safety

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profile in pediatric patients were consistent with earlier reported findings of smaller trials [220]. A population pharmacokinetic study in 28 mostly immature neonates with invasive Candida infections has demonstrated that the disposition of ABLC in neonates is similar to that observed in other age groups: weight was the only factor that influenced clearance. Based on the results of this study and a cure rate of > 80%, a dosage of 2.5–5.0 mg/kg is recommended for treatment of neonatal candidiasis [221].

Liposomal amphotericin B The pharmacokinetics of LAMB in pediatric patients beyond the neonatal period have been investigated in a formal Phase II dose-escalation trial investigating dosages of 2.5, 5.0, and 7.5 mg/kg in immunocompromised patients and using a population-based approach; the results of these studies indicate that the disposition of LAMB in pediatric patients is not fundamentally different from that in adults and that weight is covariate that determines clearance and volume of distribution [222, 223]. Many pediatric patients have been enrolled on clinical trials with LAMB but were not separately evaluated [199, 224]. Two hundred-four children (mean age, 7 years) with neutropenia and fever of unknown origin were randomized in an open label, multicenter trial to receive either DAMB 1 mg/kg/day (n = 63), LAMB 1 mg/kg/day (n = 70) or LAMB 3 mg/kg/day (n = 71) for empirical antifungal therapy [206]. Twenty-nine percent of patients treated with 1 mg/kg/day LAMB, 39% of patients treated with 3 mg/kg/day LAMB, and 54% of patients treated with DAMB experienced adverse effects (p = 0.01); nephrotoxicity, defined as 100% or more increase in serum creatinine from baseline, was noted in 8, 11, and 21%, respectively (n.s.). Hypokalemia (< 2.5 mmol/L) occurred 10%, 11%, and 26% of patients (p=0.02), increases in serum transaminase levels (* 110 U/L) in 17%, 23%, and 17% and increases in serum bilirubin (* 35 µmol/L) in 11%, 12%, and 10% of patients, respectively. Efficacy assessment by intent-to-treat analysis indicated successful therapy in 51% of children treated with DAMB and 64% and 63% in children treated with LAMB at either 1 or 3 mg/kg/day (p = 0.22). LAMB at either 1 or 3 mg/kg/day was significantly safer and at least equivalent to DAMB with regard to resolution of fever of unknown origin [206]. LAMB was well tolerated and effective in small cohorts of immunocompromised children requiring antifungal therapy for proven or suspected infections, including patients with bone marrow transplant for primary immunodeficiencies [225] and cancer patients [226]. A Phase IV analysis of 141 courses of LAMB administered for a mean of 17 days duration at a mean maximum dosage of 2.5 mg/kg for various indications to pediatric cancer/HSCT patients revealed a low rate of adverse events (4%) necessitating discontinuation. While mean GOT, GPT and AP values were slightly higher at end of

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treatment (p < 0.01), bilirubin and creatinine values were not different from baseline. LAMB had acceptable safety and tolerance and displayed efficacy in prevention and treatment of invasive fungal infections [227]. LAMB (2.5–7 mg/kg/day) was evaluated prospectively in 24 very low birth weight infants (mean birth weight 847 ± 244 g, mean gestational age 26 weeks) with systemic candidiasis. Thirteen infants failed previous antifungal therapy with amphotericin B (with or without 5-flucytosine). Candida spp. were isolated from the blood in all 25 episodes and from skin abscesses and urine in four infants each, respectively. The mean duration of therapy was 21 days; the cumulative LAMB dose was 94 mg/kg. Fungal eradication was achieved in 92% of the episodes; 20 (83%) infants were considered clinically cured at the end of treatment. No major adverse effects were recorded; one infant developed increased bilirubin and hepatic transaminase levels during therapy. Four (17%) infants died; in two of them (8%) the cause of death was directly attributed to systemic candidiasis [228]. In a second study undertaken by the same investigators, high-dose (5–7 mg/kg/day) LAMB was evaluated prospectively in 41 episodes of systemic candidiasis occurring in 37 neonates (36 of the 37 were premature infants with very low birth weights). Candida spp. were isolated from blood in all patients and from urine, skin abscesses and peritoneal fluid in 6, 5 and 1 neonates, respectively; 28, 5 and 8 infants received 7, 6–6.5 and 5 mg/kg/day, respectively. Median duration of therapy was 18 days; median cumulative dose was 94 mg/kg. Fungal eradication was achieved in 39 of 41 (95%) episodes; one patient died due to systemic candidiasis on day 12 of therapy. High-dose LAMB was effective and safe in the treatment of neonatal candidiasis. Fungal eradication was more rapid in patients treated early with high doses and in patients who received high-dose LAMB as first-line therapy [229]. The lipid formulations of AMB have less renal toxicity as defined by development of azotemia than conventional AMB; distal tubular toxicity also may be somewhat reduced. Infusion-related side effects of fever, chills, and rigor appear to be substantially less frequent with LAMB. The infusion-related reactions of ABCD and ABLC appear to be similar to those of DAMB. Several individual cases of substernal chest discomfort, respiratory distress and of sharp flank pain have been noted during infusion of LAMB [230, 231]. Similarly, in comparative studies, hypoxic episodes associated with fever and chills were more frequent in ABCD recipients than in DAMB recipients [205, 232]. Mild increases in serum bilirubin and alkaline phosphatase have been registered with all three formulations, and mild increases in serum transaminases with LAMB. However, no case of fatal liver disease has occurred. Pharmacokinetic and safety data from children so far indicate no fundamental differences in these parameters as compared to those obtained in the adult population. The lipid formulations of amphotericin B are currently licensed for the treatment of patients with invasive mycoses refractory of or intolerant to

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DAMB, and, limited to LAMB, for empirical therapy of persistently neutropenic patients. Evidence-based, but not formally licensed indications for first-line therapy exist for LAMB for treatment of invasive aspergillosis [233], invasive candidiasis [234], and zygomycosis (all formulations) [106]. The currently recommended therapeutic dosages are 3 (to 5) mg/kg/day for LAMB, and 5 mg/kg for ABCD and ABLC, respectively [106]; the therapeutic dosage for treatment of zygomycosis should not be less than 5 mg/kg/day (Tabs 2–4). Similar to conventional amphotericin B (DAMB), treatment should be started with the full calculated dosage at the infusion rate recommended by the manufacturer.

Antifungal triazoles The antifungal triazoles have become an important component of the antifungal armamentarium. They are associated with overall less toxicity than DAMB, possess a suitable spectrum of activity, and have demonstrated clinical efficacy under many circumstances. The triazoles function by inhibiting the cytochrome P450-dependent conversion of lanosterol to ergosterol, which leads to altered membrane properties and inhibition of cell growth and replication. Whereas fluconazole and itraconazole are now available for more than a decade, new triazoles such as voriconazole and posaconazole have entered the clinical arena only recently [201, 214].

Fluconazole The availability of fluconazole has been a major advance in antifungal therapy. Its spectrum of activity includes Candida spp, Cryptococcus neoformans, Trichosporon asahii, and endemic dimorphic fungi, but not Aspergillus spp. and the other opportunistic moulds. C. krusei, and to a lesser extent, C. glabrata are considered intrinsically resistant to fluconazole in vitro [235]. Available as oral and parenteral formulation, fluconazole possesses almost ideal pharmacokinetic properties. Independent of food or intragastric pH, oral bioavailability is > 90%. Due to its free solubility in water, protein binding is low and penetration into CSF and tissues is excellent; most of the drug is excreted in an unchanged form into the urine [236]. The plasma pharmacokinetics of fluconazole in pediatric age groups exhibit changes in the volume of distribution and clearance that are characteristic for a water-soluble drug with minor metabolism and predominantly renal elimination. Except for premature neonates, where clearance is decreased, pediatric patients tend to have an increased normalized plasma clearance and a shorter half-life in comparison to adults [237–242] (Tab. 6). As a consequence, dosages at the higher end of the recommended dosage range are necessary for the treatment of invasive mycoses in children. Because

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Table 6. Pharmacokinetic parameters of fluconazole in pediatric patients Age group

VD (L/kg)

Cl (L/h·kg)

T1/2 (h)

day 1

1.18

0.010

88

day 6

1.84

0.019

67

Preterm <1500 g

day 12 Term neonates

2.25

0.031

55

1.43

0.036

28

Infants > 1–6 months

1.02

0.037

19

Children, 5–15 years

0.84

0.031

18

Adult volunteers

0.65

0.015

30

Data represent mean values and are compiled from six studies; VD, volume of distribution; Cl, total clearance; T1/2, elimination half-life. Modified from [211].

exposure over time appears to be the pharmacodynamic parameter that is most predictive of antifungal activity [243, 244], fractionating the daily dose is not required in infants and children despite the shorter half-life in these age groups. In adults, dosages of up to 1200 mg/kg/day have been safely administered over prolonged periods of time [245]. In pediatric patients of all age groups, at dosages of up to 12 mg/kg/day, fluconazole is generally well tolerated [246]. The most common reported side effects in pediatric patients include gastrointestinal disturbances (8%), increases in hepatic transaminases (5%) and skin reactions (1%); toxicity-related discontinuation of therapy with fluconazole occurs in approximately 3% of patients [246]. Severe side effects, including relevant hepatoxicity and exfoliative skin reactions have been reported anectodically in association with fluconazole therapy [201]. Fluconazole undergoes minimal cytochrome P450 (CYP) metabolism but inhibits CYP3A4 and several other isoforms and interacts with enzymes involved in glucuronidation, thereby leading to numerous drug-drug interactions. Due to a lesser affinity for human CYP450 3A, however, number and frequency of relevant drug-drug interactions are lower than those of ketokonazole or itraconazole [214, 247, 248]. Several controlled studies including both neutropenic and non-neutropenic adult patients have demonstrated that IV fluconazole (400–800 mg/day) is as effective as DAMB (0.5–1.0 mg/kg/day) against candidemia and other forms of documented or suspected invasive candidiasis, and that it is better tolerated [249–252]. Apart from oropharyngeal and esophageal candidiasis [253–256], fluconazole can thus be used for invasive Candida infections caused by susceptible organisms in patients who are in stable condition and who have not received prior azole therapy [106, 257] (Tab. 2). This also applies to the neonatal setting: In six published series including * 10 patients

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with proven invasive Candida infections, treatment with fluconazole at a daily dosage of 5–6 mg/kg was successful in 83–97% and crude mortality ranged from 10% to 33%; in none of the altogether 125 patients was fluconazole discontinued due to toxicity [258–263]. The recommended dosage range for pediatric patients of all age groups is 6–12 mg/kg/day; in view of the faster clearance rate, however, 12 mg/kg/day may be most appropriate dosage for treatment of life-threatening infections in infants and children. Because of an initially decreased clearance in preterm neonates of < 1500 g, we advocate every other day dosing with 6–12 mg/kg during the first week of life in this specific setting. Further potential indications for fluconazole include consolidation therapy for chronic disseminated candidiasis [264, 265] and cryptococcal meningitis [266, 267]. High dose fluconazole has been used for infections by the yeast Tr. beigelii in non-neutropenic hosts; because of the potential for breakthrough infections by other opportunistic fungi, the addition of DAMB is recommended in persistently neutropenic patients [28]. Fluconazole has become the drug of choice for treatment of coccidioidal meningitis [268] and has proven effectiveness in nonmeningeal coccidioidal infections [269]. However, fluconazole appears comparatively less active than itraconazole in the treatment of other endemic mycoses such as paracoccidioidomycosis, blastomycosis, histoplasmosis and sporotrichosis [270–275] (Tab. 4). Fluconazole is also active in preventing mucosal candidiasis in patients with HIV infection or cancer [276–278] and has proven efficacy in preventing invasive Candida infections in patients undergoing bone marrow transplantation [279, 280]. Fluconazole has been shown to reduce Candida infections in low birth weight infants [281–286]. Thus, fluconazole prophylaxis is a valid option for centers with a high frequency (> 10%) of invasive Candida infections in premature infants of < 1000 g birth weight or in the setting of a nosocomial outbreak by a fluconazole-susceptible Candida species.

Itraconazole Itraconazole has antifungal activity comparable to fluconazole but also possesses activity against Aspergillus spp. and certain dematiaceous moulds [201, 214]. In contrast to fluconazole, however, itraconazole is water-insoluble, highly protein-bound and undergoes extensive metabolism in the liver. Absorption from the capsule form is highly dependent on a low intragastric pH, compromised in the fasting state and thus often erratic [201, 247]. The hydroxypropyl-`-cyclodextrin solution of itraconazole improves oral bioavailability [287, 288] and, in conjunction with the IV formulation [289–291], has enhanced the clinical utility of itraconazole. Itraconazole is usually well tolerated with a similar pattern and an approximately identical frequency of adverse effects as fluconazole [247]. However, both propensity and extent of drug-drug interactions through

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interference with mammalian cytochrome P450-dependent drug metabolism appear greater [201, 214]. The safety and pharmacokinetics of cyclodextrin itraconazole solution in immunocompromised pediatric patients have been studied in two Phase II clinical trials [292, 293]. The solution was well tolerated and safe in 26 infants and children with cancer (n = 20) or liver transplantation who received the compound at 5 mg/kg/day for documented mucosal candidiasis or as antifungal prophylaxis for 2 weeks [292]. Treatment with cyclodextrin itraconazole achieved potentially therapeutic concentrations of itraconazole in plasma; these levels, however, were substantially lower than those reported in adult cancer patients [294]. In a cohort of 26 HIV-infected children and adolescents, cyclodextrin itraconazole was safe and effective for treatment of OPC at dosages of 2.5 mg/day or 2.5 mg twice a day (bid) given for at least 14 days. Both dosage regimens resulted in higher peak plasma concentrations and AUC 0–24-h values than reported in the above referenced study in pediatric cancer patients. Based on safety and efficacy, a dosage of 2.5 mg/kg bid was recommended for the treatment of OPC in pediatric patients * 5 years old. [293]. Vomiting (12%), abnormal liver function tests (5%) and abdominal pain (3%) were the most common adverse effects considered definitely or possibly related to cyclodextrin itraconazole solution in an open study in 103 neutropenic pediatric patients who received the drug at 5 mg/kg/day for antifungal prophylaxis; 18% of patients withdrew from the study because of adverse events [295]. No experience with the IV formulation in pediatric patients has been reported. Similarly, only anecdotal reports have been published on the use of itraconazole in the neonatal setting. Itraconazole is a useful agent for dermatophytic infections and pityiasis versicolor [296, 297]. It is effective in the treatment of OPC and esophageal candidiasis including adult and pediatric patients who have developed resistance to fluconazole [292, 293, 298]. The clinical efficacy of itraconazole in candidemia and deeply invasive Candida infections has not been systematically evaluated. However, itraconazole is used for long-term treatment of cryptococcal meningitis in patients with HIV infection [266, 267]. Itraconazole is approved as second line agent for treatment of invasive Aspergillus infections; two separate uncontrolled studies that have investigated oral itraconazole for treatment of proven or probable invasive aspergillosis suggest a response rate comparable to that reported for amphotericin B [299, 300] (Tab. 3). Current experience with the IV formulation for this indication is promising but limited [291]. Itraconazole may also be indicated for treatment of invasive infections by dematiaceous moulds [301], but it has no documented activity against zygomycosis and fusariosis. Itraconazole is the current treatment of choice for lymphocutaneous sporotrichosis [302] and non-life-threatening, nonmeningeal paracoccidioidomycosis, blastomycosis, and histoplasmosis in non-immunocompromised patients [63, 303–305]. It also has established efficacy in both induction and maintenance therapy of mild-to-moderate, non-meningeal histoplasmosis

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in HIV-infected patients [306, 307]. The activity of itraconazole against nonmeningeal and meningeal cocidioidomycosis appears somewhat inferior to that of fluconazole [308–310]. It should be emphasized, however, that amphotericin B remains the treatment of choice for most immunocompromised patient and those with life-threatening infections by the endemic fungi [201, 214] (Tab. 4). Prophylactic itraconazole may reduce the incidence of proven or suspected invasive fungal infections in patients with hematological malignancies [311] and following HSCT [312, 313]. Efficacy in the prevention of invasive aspergillosis is supported by a large meta analysis [314] but not by a randomized, comparative trial. Finally, itraconazole was at least as effective as conventional amphotericin B, and was superior with respect to its safety profile when investigated as empirical antifungal therapy in persistently neutropenic cancer patients [290]. The recommended dosage range for oral itraconzole in pediatric patients beyond the neonatal period is 5–8 (12) mg/kg/day [corresponding to dosages of 200–400 (600) mg/day recommended for adults] with a loading dose of 4 mg/kg three times a day (tid) for the first 3 days. Achievement of adequate plasma levels is important, and drug monitoring is strongly recommended in patients with serious disease. The recommended target level is > 0.5 +g/mL before the next dose, as measured by HPLC [106]. Data on the use of IV itraconazole in pediatric patients are currently lacking; the dosage regimen utilized in the published adult studies is 200 mg bid for 2 days, followed by 200 mg/day for a maximum of 12 days [290, 291].

5-Fluorocytosine (5-FC) 5-Fluorocytosine (5-FC) is a fungus-specific synthetic base-analog that acts by causing RNA-miscoding and inhibition of DNA synthesis. Its antifungal activity in vitro is essentially limited to yeasts and certain dematiaceous fungi [315]. In the U.S., 5-FC is available only as oral formulation; in several European countries, 5-FC is also marketed as IV solution. The low-molecular-weight, water-soluble compound is readily absorbed from the gastrointestinal tract. 5-FC has negligible protein binding and distributes well into all tissues and body fluids, including the CSF. In humans, less than 1% of a given dose of 5-FC is believed to undergo hepatic metabolism; approximately 90% is excreted into the urine in an unchanged form by glomerular filtration with an elimination half-life from plasma of 3–6 h in patients with normal renal function [201]. In neonates, an extreme interindividual variability in clearance and distribution volume has been reported [196]; separate pharmacokinetic data for infants and children are lacking. Due to the propensity of susceptible organisms to develop resistance in vitro [316], 5-FC is traditionally not administered as a single agent. An

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established indication is its use in combination with DAMB for induction therapy of cryptococcal meningitis [317, 318] (Tab. 2). The combination with DAMB may also be recommended for the treatment of Candida infections involving deep tissues, in particular for Candida meningitis, infections by certain non-albicans Candida species, and critically ill patients [28]. 5-FC in combination with fluconazole may be used for cryptococcal meningitis, when treatment with DAMB or LAMB is not feasible [319]. The major potential toxicities of 5-FC are gastrointestinal intolerance and hematopoietic toxicity, which is possibly due to the conversion of 5-FC into fluorouracil by intestinal bacteria [201]. Close monitoring of plasma levels and adjustment of the dosage is recommended, in particular when there is evidence for impaired renal function; peak plasma levels between 40 and 60 +g/mL correlate with antifungal activity but are seldom associated with marrow toxicity [315]. A starting dosage for both adults and children of 100 mg/kg daily divided in three or four doses is currently recommended.

New agents for treatment and prevention and their pediatric development New antifungal triazoles Voriconazole Voriconazole (Vfend™) (Fig. 2) is a recently approved synthetic antifungal triazole with activity against a wide spectrum of clinically important yeasts and moulds, including Candida spp., Cryptococcus neoformans, Aspergillus and other hyaline moulds, dematiaceous moulds as well as dimorphic moulds (Tab. 7), both in vitro as well as in animal models. A notable exemption are the zygomycetes, against which voriconazole is intrinsically inactive. Similar to itraconazole, voriconazole is generally considered fungistatic against Candida but fungicidal against Aspergillus spp. [214, 320]. Voriconazole is available in oral and IV formulations; oral bioavailability exceeds 90% in the fasted state. In adults, the compound has nonlinear pharmacokinetics. Plasma protein binding is 58%, and the mean volume of distribution accounts for 2 L/kg. Tissue and CSF levels exceed those of trough plasma levels several fold. The plasma half-life is 6 h, with elimination primarily occurring by oxidative hepatic metabolism to at least eight metabolites that are eliminated via the urine; less than 2% of a dose of voriconazole are excreted unchanged in urine. The major isoenzyme involved in voriconazole metabolization is CYP2C19, but CYP2C9 and CYP3A4 also contribute. There is a wide between-subject variability in the disposition of voriconazole, that is related to genetic CYP2C19 polymorphism (Tab. 8) [214, 321]. Voriconazole has an acceptable safety profile. The accrued clinical data indicate that side effects include four distinct clinical categories: Transient

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Figure 2. Structural formulas of voriconazole and posaconazole and, for comparison, those of fluconazole and itraconazole.

liver enzyme abnormalities (10–20%), skin reactions (< 10%), hallucinations or confusion (< 10%) and transient, dose-related visual disturbances (altered or enhanced perception of light, blurred vision; 25–45%) [214]. However, drug-related adverse effects requiring the discontinuation of voriconazole were infrequent in comparative clinical trials (2–13%) [322–324]. Voriconazole is both substrate and inhibitor of CYP2C19, CYP2C9, and CYP3A4, and therefore, a number of clinically relevant and potentially hazardous drug-drug interactions need to be considered [214]. Voriconazole has demonstrated excellent clinical efficacy in Phase II and III clinical trials in patients with OPC [325] and esophageal candidiasis [322]. In salvage studies of invasive aspergillosis and other mycoses, responses were observed in 41–55% of patients [326, 327]. A multinational, randomized Phase III clinical trial of voriconazole and conventional amphotericin B followed by other licensed antifungal therapy for primary therapy of invasive aspergillosis revealed superior antifungal efficacy and improved survival of voriconazole-treated patients at week 12 [323]. A randomized comparative study of voriconazole versus conventional amphotericin B followed by fluconazole for treatment of candidemia in non-neutropenic patients showed similar response rates and end of treatment and similar survival at 3 months [328]. In a large international collaborative study of voriconazole versus liposomal amphotericin B for empirical therapy, voriconazole did not

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Table 7. Principal activity in vitro of new antifungal agents Voriconazole

Posaconazole

Caspofungin

Anidulafungin

Micafungin

Aspergillus spp.

+

+

+

+

+

Candida spp.

+

+

+

+

+

C. glabrata

+

+

+

+

+

C. krusei

+

+

+

+

+

Cryptococcus neoformans

+

+

–

–

–

+/–

+/–

–

–

–

Fusarium spp.

+/–

+/–

–

–

–

Scedosporium spp.

+/–

+/–

–

–

–

+

+

+/–

+/–

+/–

Zygomycetes

–

+

–

–

–

Dimorphic (‘endemic’) moulds

+

+

+/–

+/–

+/–

Non-Aspergillus hyalohyphomycetes

Phaeohyphomycetes (‘black moulds’)

+, generally active; +/–, variable activity; –, no known activity as single agent at concentrations achieved in human subjects following standard dosages.

Table 8. Principal pharmacokinetic properties of new antifungal triazoles and echinocandins Voriconazole

Posaconazole

PO/IV

PO (IV)

IV

IV

IV

Dose linearity

No

Yes

Yes

Yes

Yes

Oral bioavailability (%)

> 90

> 50

n/a

n/a

n/a

Protein binding (%)

58

> 95

97

84

99

Volume of distribution (L/kg)

2

>5

n/a

0.7–0.9

0.24

Formulation

Caspofungin

Anidulafungin

Micafungin

Elimination half-life (h)

6

25

8–10

24

15

Substrate / inhibitor of CYP450

3A4, 2C9, 2C19

3A4

n/a

n/a

n/a

<20/?

77/–

80/78

14/14

Degradation/metabolization, urine > feces

Degradation only, feces

Metabolization, feces > urine

Elimination – via feces (%/% metabolites) – urine (%/% metabolites) n/a, not applicable

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meet the prespecified statistical endpoint for non-inferiority in a composite endpoint , but was associated with significantly fewer breakthrough invasive fungal infections, particularly those due to invasive aspergillosis [324]. Finally, several reports also suggest the potential usefulness of voriconazole for treatment of infections by unusual hyaline and dematiaceous fungi [327], and for treatment of cerebral mould infections [329]. Voriconazole is approved for treatment of invasive aspergillosis, fusariosis, and scedosporiosis, and for primary treatment of invasive candidiasis in non-neutropenic patients (Tabs 2–4). The recommended IV dosages for patients of * 12 years are 6 mg/kg bid on day 1, followed by 4 mg/kg bid. The oral dosages in adults are 400 mg bid on day 1 (< 40 kg: 200 mg bid), followed by 200 mg bid (< 40 kg: 100 mg bid). In patients with renal insufficiency, no dosage adjustment is needed for the PO formulation; because of the renal clearance of the IV carrier, patients with a creatinine clearance of < 50 mL/ min should receive voriconazole by the oral route. In patients with mild to moderate hepatic function abnormalities, half of the daily maintenance dosage is recommended after the initial loading dose. Recommendations for severe liver failure are lacking [320]. Pediatric patients of < 12 years have a higher capacity for elimination of voriconazole per kilogram of body weight than adult healthy volunteers, resulting in a lower, potentially non-therapeutic exposure at similar dosages [330]. An intraindividual dosage escalation study exploring pharmacokinetics and safety of higher dosage regimens of voriconazole in this patient population has been completed. Based on that study, an IV dosage of 7 mg/kg bid and an oral dosage of 200 mg bid (oral suspension) without loading dose is recommended for children < 12 years of age [331]. Voriconazole has been administered safely and with success to a number of children < 12 years of age without therapeutic alternative. Of 58 immunocompromised children with proven or probable invasive fungal infection refractory to or intolerant of conventional antifungal therapy, 26 patients (45%) had a complete or partial response. Four patients (7%) were discontinued because of intolerance. A total of 23 patients had voriconazole-related adverse events, most commonly elevation in hepatic transaminases or bilirubin (n = 8), skin rash (n = 8), abnormal vision (n = 3) and photosensitivity reactions (n = 3) [332]. The safety and tolerance of voriconazole were further analyzed in a retrospective cohort study of 37 immunocompromised children and adolescents requiring voriconazole therapy for various indications. Voriconazole was administered intravenously and/or orally at dosages ranging from 2 to 8 mg/kg bid for a mean duration of 174 days (range, 5–998 days). Grade I or II adverse events were observed in 19 patients (51%); the most frequent events included transient increases in hepatic transaminases (19) and transient visual disturbances (5). Four patients (10%) experienced grade III/IV adverse events and 3 (8%) were permanently discontinued. While not a primary endpoint of the analysis, voriconazole showed promising efficacy as preventive and therapeutic modality [333].

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Posaconazole Posaconazole (Noxafil™) (Fig. 2) is a novel lipophilic antifungal triazole with potent and broad-spectrum activity against opportunistic, endemic, and dermatophytic fungi in vitro. This activity extends to organisms that are often refractory to existing triazoles, amphotericin B or echinocandins such as C. glabrata, C. krusei, A. terreus, and Fusarium spp. Importantly, posaconazole also possesses activity against zygomycetes both in vitro and in vivo, distinguishing it from all available azoles (Tab. 7) [334, 335]. Posaconazole is available as oral suspension only and achieves optimal exposure when administered in two to four divided doses given with food or a nutritional supplement. The compound has a large volume of distribution in the order of 5 L/kg and a prolonged elimination half-life of approximately 20 h. Posaconazole is not metabolized through the cytochrome P450 enzyme system but primarily excreted in unchanged form in the feces. It is inhibitory against cytochrome P3A4, but has no effects on 1A2, 2C8, 2C9, 2D6 and 2E1 isoenzymes, and, therefore, a limited spectrum of drug-drug interactions can be expected (Tab. 8) [336, 337]. Posaconazole appears to be well tolerated in a manner comparable to fluconazole. The overall safety of posaconazole has been assessed in more than 400 patients with invasive fungal infections from two open label clinical trials [338]. Treatment-related adverse events occurred in 38% of patients (164/428); the most common were nausea (8%), vomiting (6%), headache (5%), abdominal pain (4%), and diarrhea (4%). Treatment-related abnormal liver function test results were observed in up to 3% of patients. Serious adverse events considered possibly or probably related to PCZ occurred in 35 (8%) patients. The drug-drug interaction potential of posaconazole has been investigated in seven open label, cross-over drug interaction studies. As with other azoles, caution is advised when posaconazole is coadministered with CYP3A4 substrates (increased levels of coadministered drugs) and unspecific enzyme inducers (decreased levels of posaconazole) [334]. Apart from two Phase II clinical trials for first- [339] and second line [340] therapy of HIV-associated OPC and esophageal candidiasis, preliminary results have been presented for the pivotal Phase II salvage study in patients with possible, probable and proven invasive fungal infections refractory to or intolerant of standard therapies [341] and a Phase III randomized clinical trial comparing posaconazole to fluconazole for treatment of OPC [342]. Posaconazole has demonstrated strong antifungal efficacy in Phase II and III clinical trials in immunocompromised patients with OPC and esophageal candidiasis. Posaconazole also showed promising efficacy as salvage therapy in a large Phase II study including 330 patients with invasive fungal infections intolerant to or refractory to standard therapies and a contemporaneous external control of 279 patients [341]. Most patients (86%) were refractory to previous therapy. Successful outcomes at end of treatment in the posaconazole and in the contemporaneous external control

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cohorts were 42% vs. 26% in aspergillosis (107 and 86 patients), 39% vs. 50% in fusariosis (18 vs. 4 patients), 56% vs. 50% in zygomycoses (11 vs. 8 patients), 69% vs. 43% (16 vs. 7 patients) in coccidioidomycosis, 52% vs. 53% in candidiasis (23 vs. 30 patients), 48% vs. 58% in cryptococcosis (31 vs. 64 patients), 81% vs. 0% in chromoblastomycosis (11 vs. 2 patients), and 64% vs. 60% in other invasive fungal infections (30 vs. 20 patients). A retrospective analysis of the manufacturer’s compassionate use program including 91 patients with proven or probable zygomycosis refractory or intolerant to prior antifungal therapy revealed a 60% success rate (complete and partial responses) at 12 weeks after initiation of therapy, supporting the usefulness of posaconazole for second line or consolidation therapy of zygomycosis [343]. Preventative randomized Phase III studies in high-risk patients with HSCT and GVHD [344] and acute leukemias [345] have been completed. In the first study, patients received either posaconazole 200 mg tid or fluconazole 400 mg/day, respectively, with the start of immunosuppression for a total of 16 weeks. Treatment with posaconazole led to a decreased incidence of invasive fungal infections at 16 weeks (5% vs. 9%, p = 0.07), with a statistically significant decrease in invasive Aspergillus infections (2 vs. 7%, p = 0.006). At 7 days after the end of treatment, fewer patients had invasive fungal disease (2 vs. 8%, p = 0.004), and fewer patients had invasive aspergillosis (1 vs. 6%, p = 0.001). There were no differences in overall mortality at 12 weeks, and no differences in the rate of drug discontinuations due to adverse events between the two study arms. In the second study, patients received posaconazole 200 mg tid and either fluconazole 400 mg/day or itraconazole 200 mg bid, respectively. Treatment was started with each cycle following drop of the absolute neutrophil count (ANC) to ) 500 +L for up to 12 weeks. Significantly fewer patients enrolled in the posaconazole arm developed an invasive fungal infection at day 7 after the end of treatment as compared to the comparator arm (2% vs. 8%, p < 0.01); most importantly, treatment with posaconazole resulted in a significant decrease in the rate of invasive aspergillosis (1% vs. 7%, p < 0.001). At day +100 after randomization, the rate of invasive fungal infections was 5% and 11% (p < 0.01), and patients treated with posaconazole had a significantly improved survival probability (p = 0.035). These two landmark studies demonstrate the preventative efficacy of posaconazole in particular against invasive Aspergillus infections in high risk patients, and a survival benefit in patients with acute myeloblastic leukemia/myelodysplastic syndrome undergoing remission induction chemotherapy. Posaconazole has recently been approved in the European Union for treatment of aspergillosis, fusariosis, chromoblastomycosis and coccidioidomycosis refractory to or intolerant of standard therapies; it is approved for prophylaxis in neutropenic patients with AML/MDS and in HSCT patients with GVHD grades II to IV in the U.S. with the European approval expected soon (Tabs 2 and 3). The recommended daily dosage for salvage treatment is 400 mg bid given with food; for patients not tolerating solid

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food, a dosage of 200 mg four times a day (qid) is recommended, preferentially given with a nutritional supplement. Current data indicate no need for dosage adjustments based on differences in age, gender, race, renal or hepatic function [334]. The pharmacokinetics of posaconazole in pediatric patients (< 18 years of age) have not been adequately studied. Very limited data obtained in 12 pediatric subjects * 8 years of age appear to indicate no fundamental differences in trough plasma concentrations as compared to adults [346]. Salvage treatment with posaconazole resulted in successful outcomes in 5 of 11 pediatric subjects (8 to 17 years of age), which appears similar to the outcome in the adult population [347].

Echinocandin lipopeptides The echinocandins are a distinct class of semisynthetic amphiphilic lipopeptides that are composed of a cyclic hexapeptide core linked to a variably configured lipid side chain, and that act by inhibiting the synthesis of 1,3-`-D-glucan. This homo-polysaccharide is a major component of the cell wall of many pathogenic fungi and absent in mammalian cells. It provides osmotic stability and is important for cell growth and cell division. The first compound of this class undergoing preclinical evaluation was cilofungin (LY 121019), a semisynthetic echinocandin B derivative with activity limited to Candida spp. However, clinical development was abandoned in early stages due to toxicity concerns associated with the intravenous polyethylene glycol formulation vehicle [201]. Over the past decade, a second generation of semisynthetic echinocandins with extended antifungal spectrum against Candida and Aspergillus spp., a very favorable safety profile and pharmacokinetic characteristics has been developed: Anidulafungin (Eraxis™), caspofungin (Cancidas™), and micafungin (Mycamine™) (Fig. 3). The data accumulated thus far indicate that these agents are not fundamentally different with respect to spectrum, pharmacokinetics, safety and antifungal efficacy [201, 214].

Caspofungin Caspofungin (Cancidas™) was the first licensed compound of the echinocandin class of antifungal agents. In vitro, caspofungin has broad-spectrum antifungal activity against Candida and Aspergillus spp. without crossresistance to existing agents (Tab. 7). The compound exerts prolonged post antifungal effects and fungicidal activity against Candida species and causes severe damage to A. fumigatus at the sites of hyphal growth. Animal models have demonstrated efficacy against disseminated candidiasis and disseminated and pulmonary aspergillosis, both in normal and in immunocompromised animals [348].

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Figure 3. Structural formulas of echinocandin lipopeptides.

Caspofungin is only available for IV administration. The compound exhibits dose-proportional plasma pharmacokinetics with a ` half-life of approximately 15 h that allows for once daily dosing. It is highly (> 95%) protein bound and distributes into all major organ sites including the brain; however, concentrations in uninfected CSF are low. Caspofungin is metabolized by the liver following degradation and is slowly excreted into urine and feces; only small fractions (< 2%) of a dose are excreted into urine in unchanged form [348, 349] (Tab. 8). At the current dosage, caspofungin is generally well tolerated, and only a small fraction of patients enrolled on the various clinical trials (< 5%) discontinued therapy due to drug-related adverse events. The most frequently reported adverse effects include increased liver transaminases, gastrointestinal upset and headaches [350]. Because of transient elevations of hepatic transaminases in singledose interaction studies in healthy volunteers [348], the concomitant use of cyclosporine is currently not recommended; clinical experience, however, indicates that both drugs can be given concomitantly under careful monitoring [351–353]. Caspofungin has no significant potential for drug interactions mediated by the CYP450 enzyme system. It can reduce the AUC of tacrolimus by approximately 20% but has no effect on cyclosporine levels. Unspecific inducers of drug clearance and/or mixed inducer/inhibitors, namely efavirenz, nelfinavir, nevirapine, phenytoin, rifampin, dexamethasone, and carbamazepine may reduce caspofungin concentrations [348].

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The clinical efficacy of caspofungin against Candida spp. has been demonstrated first in Phase II and III studies in immunocompromised patients with esophageal candidiasis [354–356]. A multicenter, randomized, doubleblind Phase III clinical trial investigated the efficacy of caspofungin for primary treatment of invasive Candida infections in 224 mostly non-neutropenic patients with amphotericin B deoxycholate (DAMB; 0.6–1.0 mg/kg) as comparator agent. Among patients receiving at least one dose of study drug, 73% of patients in the caspofungin cohort and 61.7% of patients in the DAMB cohort had a therapeutic success at the end of IV therapy. Among patients who received five or more doses, the response rates were 80.7% and 64.9%, respectively. There was no difference in relapse or survival, but caspofungin was better tolerated [357]. A multicenter Phase II salvage trial of caspofungin has been completed in 83 patients with definite or probable invasive aspergillosis refractory response or intolerant to standard therapies. As determined by an independent expert panel, a complete or partial response was observed in 45% of patients receiving at least one dose of caspofungin; in patients receiving the drug for > 7 days, the response rate was 56% [358]. Finally, in a large, randomized, double-blind clinical trial including 1095 patients, caspofungin was as effective as liposomal amphotericin B for empirical antifungal therapy in persistently febrile granulocytopenic patients but better tolerated. The proportion of patients who survived at least 7 days after therapy was greater in the caspofungin group (92.6% vs. 89.2%) [359]. Currently, caspofungin is licensed in the European Union and the United States in patients * 18 years of age for second line therapy of definite or probable invasive aspergillosis, for primary therapy in non-neutropenic patients with invasive Candida infections, and for empirical antifungal therapy in granulocytopenic patients with persistent fever (Tabs 2 and 3). The recommended dose regimen consists of a single 70-mg loading dose on day 1, followed by 50 mg daily thereafter, administered over 1 h. No dosage adjustment is required in patients with renal insufficiency. In patients with mild hepatic insufficiency (Child-Pugh category A), no adjustments are needed; in patients with moderate hepatic insufficiency (Child-Pugh category B), decreasing the maintenance dose to 35 mg/day is recommended after the loading dose of 70 mg. No recommendations exist for patients with severe hepatic insufficiency (Child-Pugh category C) [348]. In children and adolescents, the pharmacokinetics and safety of caspofungin was investigated using either a weight-based regimen (1 mg/kg body weight/day) or a body surface area regimen (50 mg/m2/day or 70 mg/m2/ day). Compared to adult patients treated with 50 mg/day, the dosage of 1 mg/kg/day achieved suboptimal exposure, whereas a dosage of 50 mg/m2/ day provided similar or slightly higher exposure relative to adults [360]. As a consequence, a dosage of 50 mg/m2/day has been selected for the further pediatric program. Although not approved in this population, caspofungin appears to be well-tolerated in pediatric patients: In a Phase I/II dose-finding study in 39 children and adolescents, none of the patients developed a

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serious drug-related adverse event or was discontinued for toxicity [360]. A similarly favorable safety profile has also been reported in immunocompromised pediatric patients who received the compound for various indications, mostly in combination with other antifungal agents [353, 361], and in neonates with refractory invasive candidiasis [362–364].

Anidulafungin The clinical efficacy of anidulafungin (Eraxis™) against Candida spp. has been demonstrated in Phase II or Phase III studies in immunocompromised patients with esophageal candidiasis and candidemia. Anidulafungin had equivalent efficacy to fluconazole in esophageal candidiasis in a randomized, double-blind, international multicenter study with success documented in 242/249 evaluable anidulafungin patients (97.2%) and 252/255 fluconazole patients (98.8%). Adverse events leading to discontinuation were reported in 29 anidulafungin patients (10%) versus 23 fluconazole patients (8%) [365]. Anidulafungin has also been investigated in patients with invasive candidiasis, including candidemia. In a dose-ranging study in 123 patients, success rates at the end of therapy were 84%, 90%, and 89% in the 50-, 75-, and 100-mg groups, respectively [366]. This study was followed by a randomized, double-blind Phase III study that compared anidulafungin (100 mg once daily) vs. fluconazole (400 mg once daily) in a total of 245 mostly non-neutropenic patients [367]. The preliminary data indicate that more patients receiving anidulafungin had a clinical and microbiological success at end of IV therapy (75.6% vs. 60.2%); similar superiority was found at the 2- and 6-week follow-ups after end of all therapy (64.6% vs. 49.2% and 55.9% vs. 44.1%, respectively). Survival at end of therapy was higher in the anidulafungin group (74% vs. 69%). Anidulafungin is licensed in the U.S. for patients * 18 years of age for primary therapy of esophageal candidiasis and candidemia and select forms of invasive candidiasis in non-neutropenic subjects. The recommended dose regimen for esophagitis is 50 mg/day with 100 mg on day 1, and 100 mg/day with 200 mg on day 1 for candidemia. No dosage adjustment is needed in subjects with mild, moderate and severe renal impairment or in those undergoing hemodialysis. Mild to moderate hepatic impairment (Child-Pugh class A and B) does not cause clinically significant changes in the pharmacokinetics of anidulafungin; dosage recommendations for subjects with severe hepatic impairment (Child-Pugh class C) are pending [368–370]. A pediatric Phase I/II multicenter study of the pharmacokinetics and safety of anidulafungin has been completed in 19 granulocytopenic children with cancer. Patients were divided into two age cohorts (2–11 and 12–17 years) and were enrolled into sequential groups to receive 0.75 or 1.5 mg/kg/ day. No drug-related serious adverse events were recorded. Pharmacokinetic parameters were similar across age groups and dosage cohorts and similar

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relative to adult subjects. Following single and multiple daily doses of 0.75 mg/kg and 1.5 mg/kg, plasma concentration data corresponded to those in adults following a daily 50 and 100 mg dose, respectively. Thus, in pediatric patients, anidulafungin can be dosed based on body weight [371].

Micafungin Micafungin (Mycamine™) has been studied in open label dose-ranging studies of endoscopically proven esophageal candidiasis in HIV patients [372, 373]. A double-blind comparative study investigating 50, 100, 150 mg/day versus fluconazole 200 mg/day for HIV-associated esophageal candidiasis showed similar endoscopic cure rates and safety profiles for micafungin at doses of 100 and 150 mg/day and fluconazole [374]. A further randomized, double-blind comparative trial in 523 patients * 16 years with esophageal candidiasis investigated micafungin (150 mg/day) vs. fluconazole (200 mg/ day) [375]. For the primary end-point of endoscopic cure, treatment difference was -0.3% (micafungin 87.7%, fluconazole 88.0%). A large, Phase III, 1:1 randomized, double-blind non-inferiority trial has been completed that compared micafungin (100 mg/day) and liposomal amphotericin B (3 mg/ kg/day) for first-line therapy of invasive Candida infections in a total of 531 adult patients [376]. The overall success rate in both treatment arms was similar (89.6% vs. 89.5%). There was no difference in survival. Predefined safety parameters showed micafungin to have advantages over liposomal amphotericin B in renal function . The safety and efficacy of micafungin in combination with other antifungal agents for treatment of refractory aspergillosis were investigated in a non-comparative multinational study in 85 patients with bone marrow transplantation. A complete or partial response was reported for 33 patients (39%) [377]. Micafungin (50 mg/day; 1 mg/kg for patients < 50 kg) versus fluconazole (400 mg/day; 8 mg/kg for patients < 50 kg) has been investigated for prophylaxis of invasive fungal infections in 882 patients undergoing HSCT. Prophylaxis was given from the start of the conditioning regimen until 5 days following engraftment. The overall success rate was significantly higher for patients randomized to receive MIF (80.0% vs. 73.5%; p = 0.03). Drug-related adverse events were comparable [378]. Micafungin is licensed only in the U.S for treatment of esophageal candidiasis and for prophylaxis against Candida infections in HSCT recipients. The recommended dose of micafungin for treating esophageal candidiasis is 150 mg/day; the dose of micafungin for prophylaxis of Candida infections in HSCT patients is 50 mg/day. Renal dysfunction (creatinine clearance < 30 mL/min) or dialysis does not alter the pharmacokinetics of micafungin. Subjects with moderate hepatic dysfunction exhibited no differences in weight-normalized clearance [379]. Micafungin has been studied in 70 children and adolescents in an open label, sequential group, dose-escalation study of empirical therapy in febrile

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granulocytopenic children aged 2–17 years. In this study, micafungin was well tolerated at dosages ranging from 0.5 to 3.0 mg/kg/day; pharmacokinetics were linear and pharmacokinetic parameters were similar to those observed in adults [380]. Overall, more than 200 pediatric patients have been included up to now in clinical trials with micafungin and varying dosages and for varying indications without evidence for differences in safety and tolerance as compared to adults. A final pediatric dosage, however, has not been proposed.

Selected management issues of invasive fungal infections Treatment and prevention of neonatal invasive candidiasis As outlined earlier, preterm infants of very low birth weight are at considerable risk to develop invasive Candida infections. In the U.S., Candida spp. currently represent 9–13% of blood culture isolates obtained from NICUs. More recent case series indicate infection rates of ) 5% in infants of < 1500 g birth weight; infection rates in infants < 1000 g, however, are between 8% and 28%. In contrast, the epidemiology of invasive Candida infections in European countries has been less well investigated; however, infection rates appear to be considerably lower than those in the U.S. Invasive Candida infections in preterm infants are caused predominantly by C. albicans and C. parapsilosis. They are associated with intravascular catheters, intracranial shunt systems, use of broad-spectrum antibacterial agents and corticosteroids, mucocutaneous colonization and parenteral hyperalimentation. While most cases present with candidemia, disseminated infection involving skin, kidneys, lungs and in particular the central nervous system is common. Current options for treatment of invasive Candida infections in preterm neonates include amphotericin B deoxycholate (DAMB), amphotericin B lipid complex (ABLC) [221], liposomal amphotericin B [228, 229], and fluconazole [260–263]. The usefulness of amphotericin B may be curtailed by renal adverse events, and that of fluconazole by the compound’s limited spectrum and dosing issues in the first days of life. Based on their excellent safety and tolerance and broad spectrum, mostly cidal activity against Candida spp., the new class of echinocandin lipopeptides may offer alternative options in the future [362, 363]. Independent of the individual choice of treatment, however, removing potentially contaminated intravascular catheters and devices and appropriate supportive care are prerequisites for successful outcome. A randomized, placebo-controlled, double-blind, single-center clinical trial conducted in the U.S. has demonstrated that fluconazole may prevent invasive Candida infections in very low birth weight premature infants without impact on overall survival [279]. Further studies lend support to

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the preventative efficacy of fluconazole in high-risk premature infants [282–286]. Therefore, fluconazole prophylaxis is a valid option for centers with a high frequency (> 10%) of invasive Candida infections in premature infants of < 1000 g birth weight or in the setting of a nosocomial outbreak by a fluconazole-susceptible Candida species.

Choice of antifungal agents and duration of therapy Rational selection of the initial drug of choice is based on the susceptibility of the offending fungus, the type and site of infection, host-based factors such as the severity of immunosuppression and preexisting organ dysfunctions, pharmacokinetic and pharmacodynamic characteristics, and adequate documentation of activity for the particular indication in clinical trials. A guide to the selection of antifungal agents for the treatment of deeply invasive fungal infections agents and pediatric dosage recommendations are provided in the tables. These recommendations are based on the published adult and pediatric literature and the personal expertise of the authors. The duration of therapy is ill defined for the majority of deeply invasive infections. In uncomplicated candidemia, daily blood cultures should be obtained until defervescense of the patient, and a course of 14 days of therapy after sterilization of the bloodstream is given [28, 106, 257]. Similarly, in uncomplicated HIV-associated cerebral cryptococcosis, DAMB, preferentially in combination with 5-FC, is given for a minimum of 2 weeks as induction therapy, to be followed by consolidation and maintenance with fluconazole [266, 267]. For most other infections, however, no uniform recommendations can be made. Responses to treatment in opportunistic fungal infections are difficult to monitor, and in many circumstances, stabilization can be considered a success. For example, pulmonary lesions in invasive aspergillosis may progress during the first week of therapy without necessarily indicating treatment failure [381]. However, the clinical situation needs to be reassessed continuously and alternative agents be considered when there is clear deterioration despite appropriate antifungal treatment. Prolonged, individualized therapy and a multidisciplinary approach are required and treatment should be administered until complete resolution of all signs and symptoms and abatement of the underlying deficiency in host defenses. It is important to realize that patients with invasive mold infections who respond and do not succumb to their underlying condition may require treatment for months and sometimes, years.

Adjunctive interventional therapies Adjunctive interventional therapies for invasive yeast infections include the removal or the exchange of potentially infected catheters, the removal of

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infected artificial implants and, as appropriate, the surgical debridement of focal lesions [106, 257]. For Aspergillus spp. and other opportunistic moulds, surgery is indicated for any infected foreign material, for lesions of the skin or/and adjacent soft tissues, and endocarditis, endophtalmitis, and osteomyelitis. It may be indicated for amenable processes located in the brain and other deep tissue sites. Surgery is also a necessary adjunct in the treatment of invasive sinusitis; however, in the neutropenic host, it should be minimally invasive for aeration and diagnostic purposes only. Indications for surgery in invasive pulmonary aspergillosis include lesions impinging on great vessels or major airways, major hemopthysis from a focal lesion, and lesions progressing into pericardium, thoracic wall, and abdominal cavity [28, 106]. Larger series including neutropenic patients reported minor perioperative morbidity and mortality with pulmonary surgery for mould infections [382–385]. Whether surgery is always indicated for residual lesions in patients who survive a pulmonary mould infection and need to proceed with further myelosuppressive treatment or a bone marrow transplantation, is unclear [384]. However, patients should have had at least a partial response, and should receive continuous and appropriate antifungal chemotherapy.

Adjunctive immunotherapies Reversal of the underlying impairment of host defenses is paramount to successful treatment of invasive fungal infections. This may include discontinuation or at least dose-reduction of concomitant glucocorticosteroids, if feasible. Cytokines, such as granulocyte (G)-colony-stimulating factor and granulocyte-macrophage (GM)-colony-stimulating factor may decrease the duration of neutropenia and increase the function of phagocytic cells [386]. Administration of colony-stimulating factors, such as G- or GM-colonystimulating factors, to neutropenic patients with an invasive fungal infection is strongly advocated, although definite conclusions about efficacy can not be inferred [387, 388]. Other cytokines such as IFN-a, interleukin (IL)-12 and IL-15, and neutralizing antibodies to IL-4 and IL-10 have been shown to have useful effects in certain experimental settings and need to be evaluated [389–391]. Lastly, the indication of growth factor-elicited granulocyte transfusions is still unclear and will have to be defined in controlled clinical studies [392, 393].

Conclusions As demonstrated in this article, pediatric age groups display important differences in host biology, predisposing conditions, epidemiology and presentation of fungal infections relative to the adult population. Over the past

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decade, major advances have been made in the field of medical mycology. Most importantly, an array of new antifungal agents has entered the clinical arena. Although the final pediatric approval of several of these agents remains to be established, the pediatric development is moving forward at steady pace. Invasive fungal infections will remain important causes for morbidity and mortality in immunocompromised pediatric patients. The availability of alternative therapeutic options is an important advance; at the same time, however, antifungal therapy has become increasingly complex. In addition to information on prior antifungal therapies, microbiological data, existing co-morbidities and co-medications, a detailed knowledge of the available antifungal armamentarium and contemporary clinical trials is needed more than ever in the management of the individual patient.

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Candidiasis Study Group (2002) Comparison of caspofungin and amphotericin B for invasive candidiasis. N Engl J Med 347: 2020–2029 Maertens J, Raad I, Petrikkos G, Boogaerts M, Selleslag D, Petersen FB, Sable CA, Kartsonis NA, Ngai A, Taylor A et al (2004) Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin Infect Dis 39: 1563–1571 Walsh TJ, Teppler H, Donowitz GR, Maertens JA, Baden LR, Dmoszynska A (2004) Caspofungin versus liposomal amphotericin B for empirical antifungal therapy in patients with persistent fever and neutropenia. N Engl J Med 351: 1391–1402 Walsh TJ, Adamson PC, Seibel NL, Flynn PM, Neely MN, Schwartz C, Shad A, Kaplan SL, Roden MM, Stone JA et al (2005) Pharmacokinetics, safety, and tolerability of caspofungin in children and adolescents. Antimicrob Agents Chemother 49: 4536–4545 Franklin JA, McCormick J, Flynn PM (2003) Retrospective study of the safety of caspofungin in immunocompromised pediatric patients. Pediatr Infect Dis J 22: 747–749 Natarajan G, Lulic-Botica M, Rongkavilit C, Pappas A, Bedard M (2005) Experience with caspofungin in the treatment of persistent fungemia in neonates. J Perinatol 25: 770–777 Odio CM, Araya R, Pinto LE, Castro CE, Vasquez S, Alfaro B, Saenz A, Herrera ML, Walsh TJ (2004) Caspofungin therapy of neonates with invasive candidiasis. Pediatr Infect Dis J 23: 1093–1097 Odio CM, Pinto LE, Alfaro B, Vasquez S, Castro CE, Hernandez M, Herrera ML, Bi S, Kartsonis N, Stone J (2005) Pharmacokinetics of caspofungin in six premature neonates with invasive candidiasis at a neonatal intensive care unit. In: Abstracts of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, DC, LB16–2005 Krause DS, Simjee AE, van Rensburg C, Viljoen J, Walsh TJ, Goldstein BP, Wible M, Henkel T (2004) A randomized, double-blind trial of anidulafungin versus fluconazole for the treatment of esophageal candidiasis. Clin Infect Dis 39: 770–775 Krause DS, Reinhardt J, Vazquez JA, Reboli A, Goldstein BP, Wible M, Henkel T; Anidulafungin Invasive Candidiasis Study Group (2004) Phase 2, randomized, dose-ranging study evaluating the safety and efficacy of anidulafungin in invasive candidiasis and candidemia. Antimicrob Agents Chemother 48: 2021–2024 Reboli A, Rotstein C, Pappas P, Schranz J, Krause D, Walsh TJ (2005) Anidulafungin vs. fluconazole for treatment of candidemia and invasive candidiasis. In: Abstracts of the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, DC, 19 Thye D, Marbury T, Kilfoil T, Kilfoil G, Henkel T (2002) Anidulafungin: Pharmacokinetics in subjects receiving hemodialysis. In: Abstracts of the 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, DC, 19 Thye D, Marbury T, Kilfoil T, Kilfoil G, Henkel T (2002) Anidulafungin:

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Pediatric aspects of bioterrorism Kwang Sik Kim Johns Hopkins University School of Medicine, 200 North Wolfe Street/Room 3157, Baltimore, MD 21287, USA

Abstract Potential microbes for bioterrorism threats include Bacillus anthrax, Yersinia pestis, Francisella tularemia, Clostridium botulinum, variola virus and hemorrhagic fever viruses such as Ebola. This review covers selective topics associated with anthrax and smallpox, such as epidemiology, pathogenesis, clinical presentation, diagnosis, prevention, and therapy, as well as approaches for clinical management of children in suspected exposure to anthrax and smallpox. Information is lacking regarding weaponized anthrax spores, including LD50, optimal management, alternatives for antibiotic-resistant strains and use of genetically modified strains to escape vaccine protection. The recent US outbreak in 2001 highlights the following features: case fatality rates of 45%, no secondary cases among household contacts of the inhalation anthrax subjects and no cases of anthrax among individuals on antibiotic prophylaxis. Regarding smallpox, discussions have concerned the identification of first response individuals and vaccination of such individuals; however, smallpox vaccine is associated with mortality and morbidity, and current issues include principles and procedures associated with vaccination.

Introduction Illnesses from biological weapons are likely to be unrecognized in the initial occurrence. With highly transmissible agents (e.g., smallpox), the time delay to recognition can result in widespread secondary exposure to others, including health care personnel. A report from the Centers for Disease Control and Prevention (CDC) indicated which biological agents would constitute the potential threats to public health and security, and divided them into three categories (Tab. 1). Category A (highest priority) agents include organisms that pose a risk to national security because they are easily disseminated, resulting in high mortality rates and cause public panic and social disruption. This category includes the causation agents for anthrax, smallpox, plague, tularemia, botulism, and viral hemorrhagic fevers. Category B (second highest pri-

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Table 1. CDC classification of bioterrorism diseases agents Category A – Diseases

Category A – Agents

The U.S. public health system and primary healthcare providers must be prepared to address various biological agents, including pathogens that are rarely seen in the U.S. High-priority agents include organisms that pose a risk to national security because they: – Can be easily disseminated or transmitted from person to person – Result in high mortality rates and have the potential for major public health impact – Might cause public panic and social disruption – Require special action for public health preparedness

– Bacillus anthracis (anthrax) – Clostridium botulinum – Yersinia pestis – Variola major (smallpox) and other pox viruses – Francisella tularensis (tularemia) – Viral hemorrhagic fever – Arenaviruses – LCM, Junin virus, Machupo virus, Guabarito virus – Lassa fever – Bunyaviruses – Hantaviruses – Rift Valley fever – Flaviruses – Dengue – Filoviruses – Ebola – Marburg

Category B – Diseases

Category B – Agents

– Are moderately easy to disseminate – Result in moderate morbidity rates and low mortality rates – Require specific enhancements of diagnostic capacity and enhanced disease surveillance of the CDC

– Burkholderia pseudomallei – Coxiella burnetii (Q fever) – Brucella species (brucellesis) – Burkholderia Mallei (glanders) – Ricin toxin (from Ricinus communis) – Epsilon toxin of Clostridium perfringens – Staphylococcal enterotoxin B – Typhus fever (Rickettsia prowazekii) – Food and waterborne pathogens – Bacteria – Diarrheagenic E. coli – Pathogenic Vibrios – Shigella species – Salmonella – Listeria monocytogenes – Campylobacter jejuni – Yersinia enterocolitica – – Viruses (caliciviruses, hepatitis A) – Protozoa – Cryptosporidium paravum – Cyclospora cayatanensis – Giardia lamblia – Entamoeba histolytica – Toxoplasma – Microsporidia – Additional viral encephalitides – West Nile virus – LaCrosse – California encephalitis – VEE – EEE – WEE – Japanese encephalitis virus – Kyasanur Forest virus

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Table 1. (continued) Category C – Diseases

Category C – Agents

The third highest priority agents include emerging pathogens that could be engineered for dissemination in the future because of – availability – Ease of production and dissemination – Potential for high morbidity and mortality rates and major health impact

Emerging infectious disease threats such as Nipah virus and additional hantaviruses

ority) agents include those that are moderately easy to disseminate and result in moderate morbidity/low mortality rates. This category includes Burkholderia pseudomallei, Coxiella burnetii (Q fever), Brucella species, and food and waterborne pathogens listed in Table 1. Category C (third highest priority) agents include emerging pathogens that could be engineered for mass dissemination in the future because of availability, ease of production and dissemination, potential for high morbidity and mortality rates and major health impact. Some of this category includes Nipah virus and Hantaviruses. As stated by the American Academy of Pediatrics Committee on Environmental Health and Committee on Infectious Diseases [1], the release of biological toxins would disproportionally affect children through several mechanisms. For example, with aerosolized agents (e.g., anthrax), the higher number of respirations per minute in children results in exposure to a relatively greater dosage. There are several unique pediatric considerations that need to be addressed during planning for bioterrorism, which include (1) the developmental abilities and cognitive levels may impede their escape from the site of a biological event, (2) children are vulnerable to psychological injury and special management plans are needed in the event of mass causalities and evacuation, (3) emergency medical service (EMS), medical and hospital personnel require expertise and training to ensure optional care of children, (4) children may require specific equipment and interventions, e.g., children cannot be decontaminated in adult decontamination units, (5) children have special susceptibilities to dehydration and shock from biological agents, and (6) children require different dosages or different antibiotics to many biological agents [1, 2]. This review focuses on two selective illnesses, anthrax and smallpox, and their diagnostic and management options for the pediatrician who encounters a patient with symptoms suggestive of the possibility of illness attributable to Bacillus anthracis and variola major.

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Anthrax Anthrax is a zoonotic disease caused by B. anthracis, a gram-positive spore forming non-motile bacillus able to survive long periods in its spore form without nutrients or moisture. Human infections generally result from exposure to animal products contaminated with B. anthracis spores or from direct exposure to anthrax-infected animals [3]. Due to improved animal vaccination practices and hygiene, anthrax in developed countries including the US is rare. Human infection typically presents in one of three forms. Most commonly, direct contact with contaminated material leads to cutaneous disease. Ingestion of infected meat, however, can result in oropharyngeal or gastrointestinal anthrax, and the inhalation of a sufficient quantity of spores can cause inhalation anthrax. Much of the knowledge related to the clinical course of the disease is derived from the information gathered after an accidental aerosol release in Sverdlovsk in the former Soviet Union in 1979, where there were 66 fatalities among the 77 patients identified and cases occurred up to 6 weeks after exposure [4, 5]. On October 5, 2001, a man from Florida, USA developed anthrax meningitis and inhalation anthrax due to the intentional release of B. anthracis spores. The discovery of other infected people at several sites quickly followed. The anthrax attack was characterized by 22 confirmed or suspect cases (11 inhalational and 11 cutaneous) with 5 deaths, resulting from known or presumed exposure to anthrax-contaminated mail [6]. This means of dispersion represents one mode of attack, but many bioterrorism defense planners fear a wide spread aerosol release (e.g., from a small crop dustertype airplane). The 2001 attack resulted in public anxiety and large demands for medical care and public health resources. All 5 deaths were among the 11 patients with inhalational disease. It remains to be determined whether somewhat improved mortality rate (approximately 45% compared to 86% of the Sverdlovsk incident) was related to improved intensive care, earlier recognition and/or antibiotic therapy.

Inhalation anthrax Inhalation anthrax begins with spore uptake by pulmonary macrophages followed by bacterial germination and toxin production in the regional lymph nodes, leading to hemorrhagic lymphadenitis, mediastinitis and septicemia, with symptoms typically beginning 1–6 days after exposure, but germination may occur up to 60 days after exposure. For example, in the Sverdlovsk incident, cases occurred from 2 to 43 days after exposure [4, 5], and spores have been demonstrated in the mediastinal lymph nodes of experimental monkeys 100 days after exposure [7, 8].

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Table 2. Comparison of clinical findings in inhalation anthrax cases from September– November 2001 (all adults) versus findings in influenza and influenza-like illnesses (ILI) from other causes Symptom/sign

Inhalational anthrax (n = 10)

Laboratoryconfirmed influenza

ILI from other causes

Elevated temperature

70%

68–77%

40–73%

Fever or chills

100%

83–90%

75–89%

Fatigue/malaise

100%

75–94%

62–94%

Cough (minimal or nonproductive)

90%

84–93%

72–80%

Shortness of breath

80%

6%

6%

Chest discomfort or pleuritic chest pain

60%

35%

23%

Headache

50%

84–91%

74–89%

Myalgias

50%

67–94%

73–94%

Sore throat

20%

64–84%

64–84%

Rhinorrhea

10%

79%

68%

Nausea or vomiting

80%

12%

12%

Abdominal pain

30%

22%

22%

From MMWR Vol 50(44), November 9, 2001.

The illness is biphasic and the initial phase consists of a non-specific febrile illness characterized by fever, myalgia, headache, cough, and chest or abdominal pain. The relative lack of rhinorrhea and sore throat help to distinguish this phase from common viral infections such as those caused by influenza (Tab. 2). After this initial phase, the patients will have worsening of fever and chest pain and may develop dyspnea, diaphoresis and shock. At this stage, the illness progresses rapidly to shock and death within 24–36 h. Chest radiograph or computed tomography may reveal a widened mediastinum or prominent mediastinal lymphadenopathy and pulmonary infiltrates or pleural effusion may be seen. Gram stain of peripheral blood smears may reveal the organism at this stage. Prompt treatment is imperative. In the 2001 anthrax attack, all 5 patients with inhalation anthrax who developed signs of fulminant disease before antibiotic administration died. Inhalation anthrax is complicated by hemorrhagic meningitis (approximately 5–50% of cases in adults). For example, in an outbreak of inhalation anthrax following the release of anthrax spores from a military research facility in Sverdlovsk, pathological involvement of the meninges was noted in about 50% of autopsied cases [4, 5]. Also, studies of experimental inhalation anthrax in monkeys have demonstrated meningeal involvement by

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pathology in 40–50% of cases [7, 8]. In contrast, in the 2001 bioterrorismrelated outbreak, 1 of the 11 patients with inhalation anthrax developed anthrax meningitis [6].

Cutaneous anthrax Cutaneous anthrax occurs when organisms or spores gain entry into the skin, particularly through abrasions or cuts. It is characterized by the appearance of a papule at the inoculum site, which progresses over a few days into vesicle, eventually forming an ulcer covered by a black eschar. The surrounding tissue is markedly edematous, but not tender, distinguishing this infection from pyogenic cellulitis. Cutaneous anthrax is amenable to antibiotic therapy and with timely administration of antibiotics it is rarely fatal. In the 2001 attack, all 11 patients with cutaneous anthrax survived. The 1 pediatric patient of the 2001 outbreak was a 7-month-old boy with cutaneous anthrax on his arm [9], presumably contracted after a brief visit to a New York television news studio that had received contaminated mail. He was initially suspected of having a brown recluse spider bite and the diagnosis was confirmed only after the discovery of anthrax contamination at another television studio. He developed evidence of hemolysis, thrombocytopenia, and renal insufficiency, features not typically seen in otherwise uncomplicated cases of cutaneous anthrax, thus raising the possibility of a particular vulnerability of infants.

Gastrointestinal anthrax Gastrointestinal anthrax develops less than 1 week after ingestion of spores in undercooked meat. Symptoms consist initially of fever, nausea, vomiting, and abdominal pain and progress rapidly to bloody diarrhea or hematemesis. Oropharyngeal involvement is manifested by ulcerated lesions at the base of the tongue, dysphagia, and systemic symptoms.

Management of children with suspected anthrax exposure Management of children with suspected anthrax exposure has not been established and is largely extrapolated from experience in adults. The most important predictor of probability of developing anthrax is probability of exposure. Children of high risk groups (e.g., postal workers, mailroom workers, media personnel, government employees, microbiology laboratory personnel, exposure to suspicious dust containing letter or packages, based on the 2001 outbreak of anthrax in the USA) are only high risk if they spend time in the workplace with their parent, have had direct exposure to powder

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Table 3. Clinical management of children with suspected anthrax exposure Asymptomatic 1. No nasal swab 2. Antibiotic prophylaxisa – continue for 60 days if exposure is continued 3. Follow-up Symptomatic with < 5 day history of following symptoms of inhalation anthrax, e.g., fever with or without chills, sweats often drenching, fatigue, malaise, headache, cough (usually non-productive), shortness of breath, chest discomfort, pleuritic pain, nausea, vomiting, diarrhea, abdominal pain 1. Initial labs including complete blood count 2. Obtain blood cultures prior to starting antibiotics 3. Chest x-ray and chest CT 4. Thoracentesis if pleural effusion is present 5. Lumbar puncture 6. Initiate therapeutic antibiotics (see Tab. 5) ªProphylactic regimen: Doxycycline: > 8 years and > 45 kg: 100 mg p.o. bid for 60 days > 8 years and < 45 kg: 2.2 mg /kg p.o. bid for 60 days or Ciprofloxacin: > 8 years, 10–15 mg /kg /dose p.o bid for 60 days or Amoxicillin: (once isolate is confirmed susceptible to penicillin) < 8 years, 80 mg/kg/day p.o. divided tid for 60 days

Table 4. Clinical management of children with possible exposure and suspected cutaneous anthraxa 1. Obtain stain and culture of vesicle fluid, ulcer base and edges or underneath the eschar 2. Consider punch biopsy for anthrax 3. Obtain blood cultures 4. Begin antibiotic treatment (see Tab. 5) 5. If, however, following symptoms and signs develop, then follow the guidelines for inhalation anthrax (see Tab. 3) – fever, headache or regional adenopathy and/or – blood culture positive for anthrax a 1.

Eschar 2. Progression from papule to eschar is 4–9 days 3. Incubation to onset of lesion is up to 14 days from exposure 4. The small papule(s) progresses in 1–2 days to vesicle(s) 5. Vesicle(s) ulcerate to develop a black eschar over 3–7 days 6. The surrounding skin may show extensive cellulitis and brawny edema 7. These lesions typically involve exposed areas such as face, arms, hands 8. The lesions are generally painless

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Table 5. Initial antibiotic for children with suspected anthrax 1. Initial antibiotic treatment for children with cutaneous anthraxa – Ciprofloxacin 10–15 mg /kg bid, not to exceed 1 g/day, or – Doxycycline > 8 years and > 45 kg: 100 mg p.o. bid > 8 years and < 45 kg: 2.2 mg/kg p.o. bid < 8 years: 2.2 mg/kg p.o. bid aAll

children with signs of systemic involvement (fever, pulmonary involvement), extensive edema or lesions on the head and neck should be treated as for inhalation anthrax 2. Initial antibiotic treatment of children with inhalation anthrax – for children with no suspicion of meningitis, include one antibiotic from list A plus * one antibiotic from list B (additional antibiotics may be necessary for other pathogens being considered) List A: Ciprofloxacin 10–15 mg/kg IV every 12 h (max. 400-mg dose) Doxycycline 2.5 mg/kg IV every 12 h (max. 100-mg dose) List B: Penicillin < 12 years, 50 000 U /kg IV every 6 h * 12 years, 4 × 106 U IV every 6 h Ampicillin, Clindanycin, Imipenem, Vancomycin, Rifampin, Clarithromycin, Chloramphenicol – For children with possible meningitis Vancomycin 15 mg /kg IV every 6 h (max. 500-mg dose) plus Ceftiaxone 50 mg /kg IV every 12 h (or cefotaxime 225–300 mg/kg/day div every 6 h) plus Rifampin 10–20 mg/kg daily (max. 600-mg dose) Ciprofloxacin 50 mg/kg IV every 12 h (max. 400-mg dose) 3. Definitive treatment – Definitive anthrax regimen to be based on susceptibilities. – Antibiotic may be changed to oral therapy, usually with a single agent, once the patient has clinically recovered, for penicillin susceptible strains, high-dose amoxicillin (80 mg/kg/ day p.o. divided tid) can be used. – Total antibiotic course (IV and/or p.o.) should be 60 days for anthrax disease or exposure.

or, in the case of adolescent patients, work in these areas directly. Merely household contact or contact with anthrax-exposed people is not considered an exposure. It is, however, important to note that exposure categories and management recommendation may change with new events. If a previously healthy child presents with a wide mediastinum and/or eschar, then consideration of anthrax may be given and its management will follow the guidelines for children with possible exposure to anthrax (Tabs 3–5).

Smallpox Smallpox is a highly contagious infection caused by the DNA virus variola, a member of the genus Orthopoxvirus [10]. Vaccinia virus, the source of the live virus vaccine, also is a member of this genus but is much less contagious. The last known non-laboratory case of smallpox occurred in 1977

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in Somalia. The US discontinued routine childhood immunization against smallpox in 1972 and routine immunization of health care professionals in 1976. In 1980, the World Health Organization (WHO) declared that smallpox had been eradicated successfully worldwide. In recent years, there has been concern that smallpox virus stock may be in the hands of bioterrorists and this concern has been heightened by the terrorist attack on September 11, 2001. Because most of the population is considered to be non-immune, there is debate as to whether smallpox vaccination should be resumed. The disease is highly contagious, and its incubation period permits a terrorist to cause widespread disease through travel and multiple contacts by exposed persons. Case fatality rates of 30% or higher were observed during epidemics of smallpox. In addition, no effective therapy for smallpox exists and modern healthcare providers are unfamiliar with the disease. A single case of smallpox occurring anywhere in the world today would represent a public health emergency.

Pathogenesis and clinical presentation Smallpox infection occurs through respiratory droplets. The incubation lasts approximately 2 weeks (range 7–17 days) and patients commonly present with high fever, malaise, prostration, headache, and backache. A maculopapular rash appears on the oral and pharyngeal mucosa, face and forearms and spreads to the trunk and legs. The rash becomes vesicular within 1–2 days and then become pustules. The pustules are round, dense, and deep. By approximately 8–9 days after onset of the rash, crusts form which eventually scab. In addition to the above-mentioned typical smallpox (more than 90% of cases), there are two forms of variola major, hemorrhagic (characterized by hemorrhage into skin lesions and disseminated intravascular coagulation) and malignant or flat type (in which skin lesions do not progress to the pustular stage but remain flat and soft). Each variant occurred in 5% of cases and was associated with 90–100% mortality rates. Variola minor or alastrim is associated with a longer incubation period, a milder prodromal period, fewer skin lesions, and lower mortality rate than variola major or typical smallpox. In the absence of pre-existing immunity, a favorable prognosis is less likely for infants, the elderly and pregnant women.

Diagnosis The early diagnosis of a sentinel case of smallpox is critical. An important diagnostic tool is the fact that all lesions will progress at the same rate. This contrasts with varicella (chickenpox), in which lesions progress to clusters and all four stages of lesions may be present at the same time (Tab. 6). In addition, varicella lesions are usually concentrated on the trunk rather than

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Table 6. Comparison of characteristics between smallpox and chickenpox Smallpox

Chickenpox

Fever

2–4 days before rash

At time of rash

Rash

Same stage

Several stages

Speed

Slow

Rapid

Locus

Arms/legs

Trunk

Palms/soles

Present

Absent

Death

30%

Rare

the face or extremities and spare palms and soles, whereas smallpox is generally distributed centrifugally.

Management A suspected case should prompt immediate consultations with health authorities. Strict airborne, droplet, and contact precautions should be instituted immediately for victims and should continue until all scabs separate. All contacts should be interviewed, vaccinated, and placed under surveillance. The administration of vaccine within 4 days of exposure may prevent or ameliorate illness. Vaccine immunoglobulin is available for those having reactions to vaccine administration or for immunocompromised patients. Contacts should have daily temperature recordings for 17 days post exposure, and if fever > 38.5 °C is noted, the contact should be isolated at home until it is determined whether the disease has developed. If disease occurs, all contacts of the patient should be vaccinated. A major reason not to initiate universal immunization in the absence of actual cases of smallpox besides the limited availability of vaccine is the risk of serious complications of immunization which include death, post-vaccination encephalitis, progressive vaccinia and eczema vaccinatum. Smallpox vaccine is known to produce significant adverse effects in immunocompromised persons, and patients with chronic skin conditions such as atopic dermatitis. Smallpox vaccine is not recommended for people with eczema or other exfoliative skin disorders, for pregnant women, or for people with immunodeficiency. Before its discontinuation, universal smallpox immunization was recommended in the US for children of 1–2 years of age. Re-immunization was recommended every 5 years and annually to people working in endemic areas. The current recommendation for those individuals at high risk because of occupational exposure is immunization every 3 years. People with multiple immunizations during childhood may have long-

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lasting immunity, but the degree of protection for those immunized before 1972 is unknown. The proposed strategies for smallpox immunization in the face of a bioterrorism threat include mass immunization, voluntary immunization, and ring immunization or surveillance and containment. The ring immunization or surveillance and containment is the current CDC recommendation of the strategy if smallpox were to be introduced in an act of terrorism; this strategy is supported by the American Academy of Pediatrics [11]. Briefly, the strategy comprises the following: Infected patients would be isolated. Contacts of infected individuals and their contacts would then be identified and immunized by specially trained health care professionals. The strategy can control a localized outbreak with minimal exposure of vulnerable populations to the complications of immunization. The ring strategy is based on the knowledge that vaccination can prevent or ameliorate disease severity if given within 3–4 days of initial exposure and can decrease symptoms if given within the 1st week of exposure. It is also desirable to have patients with smallpox cared for by persons who have been immunized.

References 1 2

3

4

5

6

7

8

American Academy of Pediatrics (2000) Chemical-biological terrorism and its impact on children: A subject review. Pediatrics 105: 662–670 Markenson D, Reynolds S (2006) American academy of pediatric committee on pediatric emergency medicine; Task Force terrorism, the pediatrician and disaster preparedness. Pediatrics 117: e340–362 Inglesby TV, Otoole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, Friedlander AM, Gerberding J, Hauer J, Hughes J (2002) Anthrax as a biological weapon, 2002: updated recommendations for management. JAMA 287: 2236–2252 Abramova FA, Grinberg LM, Yampolskaya OV, Walker DH (1993) Pathology of inhalational anthrax in 42 cases from the Sverdlovsk outbreak of 1979. Proc Natl Acad Sci USA 90: 2291–2294 Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, Yampolskaya O (1994) The Sverdlovsk anthrax outbreak of 1979. Science 266: 1202–1208 Guarner J, Jernigan JA, Shieh WJ, Tatti K, Flannagan LM, Stephens DS, Perkins BA, Zaki SR, Inhalational Anthrax Pathology Working Group (2003) Pathology and pathogenesis of bioterrorism-related inhalational anthrax. Am J Pathol 163: 701–709 Fritz DL, Jaax NK, Lawrence WB, Davis KJ, Pitt ML, Essell JW, Friedlander AM (1995) Pathology of experimental inhalation anthrax in the rhesus monkey. Lab Invest 73: 691–702 Vasconcelos D, Barnewall R, Babin M, Hunt R, Estep J, Nielsen C, Carnes R, Carney J (2003) Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis). Lab Invest 83: 1201–1209

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Freedman A, Afonja O, Chang MW, Mostashari F, Blaser M, Perez-Perez G, Lazarus H, Schacht R, Guttenberg J, Traister M, Borowsky W (2002) Cutaneous anthrax associated with microagniopathic hemolytic anemia and coagulopathy in a 7–month-old infant. JAMA 287: 869–874 Henderson DA, Inglesby TV, Bartlett JG, Ascher MS, Eitzen E, Jahrling PB, Hauer J, Layton M, McDade J, Osterholm Mt et al (1999) Smallpox as a biological weapon: medical and public health management. Working group on Civilian Biodefense. JAMA 281: 2127–2237 American Academy of Pediatrics – Policy Statement (2002) Smallpox vaccine. Pediatrics 110: 841–845

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Pediatric infectious diseases – Quo vadis 2015? David Nadal Division of Infectious Diseases and Hospital Hygiene, University Children’s Hospital of Zurich, Steinwiesstrasse 75, 8032 Zürich, Switzerland

Abstract In modern medicine the discipline pediatric infectious diseases is an essential medical specialty. The challenging and complex tasks in the next years include meticulous consolidation and careful extension of existing activities aiming at conducting high level research, offering high standard teaching, and providing high quality patient management. This can only be accomplished by exquisitely dedicated individuals with extraordinary communication and integrative skills following painstaking continued training and formation. Potential careers in the discipline can be envisioned not only in academics, but also in government, public health, and industry, whilst less likely in private practice.

Introduction The discipline pediatric infectious diseases has evolved to an essential medical specialty and faces major challenges in the years to come. One of the most important tasks of pediatricians has always been the management of patients with communicable diseases. The main reason for this is the higher frequency of infectious diseases in infants and young children compared to older children and adults due to the limited adaptive immunity repertoire and thus increased susceptibility to common pathogens. Therefore, pediatricians are considerably involved in the diagnosis, treatment and prevention of infectious diseases. In consequence, every pediatrician must be considered also an infectious disease specialist. This, in turn, has been a downside for the development of pediatric infectious diseases as a medical discipline recognized on its own in many countries. Nevertheless, the multiple technical advances in the recent years have led to substantially improved prevention and treatment success rates in many pediatric disciplines, and a plethora of these success rates are linked to the integral role of pediatric infectious disease specialists providing profound knowledge, expertise and quality assurance. Accordingly, pediatric infectious disease specialists

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nowadays play a pivotal role both for community pediatrics and for clinical pediatrics in highly specialized medical centers. This chapter attempts to summarize the current different activities of pediatric infectious disease specialists, to delineate their interactions with other medical disciplines and to speculate on the near future goals and development of this specialty with the widest scope compared to all the specialties in medicine.

Current activities of the pediatric infectious disease specialist Similarities, overlaps and differences in relation to infectious diseases in adults Pediatric infectious disease specialists are based mainly in hospital settings and have very similar activities in clinics, teaching and research compared to their counterparts in adult medicine. The four disciplines microbiology, epidemiology, immunology, and pharmacology build up the essential basis for both pediatric and adult infectious disease specialists. Nevertheless, despite several overlaps that are beneficial for constructive professional interactions, the position of the pediatric infectious disease specialists differ from those of specialists for adult infectious diseases. These differ not only in relation to the basic training in pediatrics and internal medicine, respectively, but also in relation to distinct focuses in the clinics obviously mandated by many age-related uniqueness of patients in the pediatric age (Tab. 1). Etiology, epidemiology, pathogenesis, management and prevention of infectious diseases in children may substantially differ from those in adults. One important example of the uniqueness of pediatric infectious diseases is the need to deal with infections in newborns. Newborns have distinct pathophysiological characteristics, which mainly relate to the immature immune system. Another example of uniqueness comes from the age-related and more frequent contacts to potential infectious sources or index cases in nurseries, day-care centers or schools. These contacts lead to increased risks to preferentially acquire respiratory or gastrointestinal infections. Similar reasons account for higher frequency of outbreaks of infectious diseases in children compared to adults. Infants and toddlers are often the source of infections within a household, for health care workers or medical personnel as well as for nursery employees and teachers. Infections represent the reason for up to 60% of the hospitalization of children. Etiological diagnosis of these infections may be hampered by the limited volumes of biological samples including blood or cerebrospinal fluid available from young children, often affording rather judicious, and to this-age-group-adapted, diagnostic approaches. Moreover, most of the hospitalized children are prescribed one or more antibiotics [1]. In this context it needs to be underscored that the pharmacokinetics and pharmacodynamics of antimicrobial substances are

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Table 1. Specific clinical tasks of the pediatric infectious disease specialist [4] – Integrative discipline – Provision of primary care and consultative services to patients from all pediatric disciplines – Implementation of quality assurance programs in hospitals and other health care settings, e.g., infection control, hospital epidemiology, antimicrobial management programs – Engagement in preventive efforts through implementation of vaccine strategies and other means; play a significant role in public health programs at all political levels – Conduction of research seeking cures for new diseases as well as preventive measures, such as new vaccines – Teaching and leadership in academic health institutions

rather different in children compared to adults. This may afford the use of distinct preparations or dosages in children. In addition, pharmacology and toxicology of antimicrobial drugs in newborns and specifically in preterm or small-for-date babies are rather special. Accordingly, in pediatrics special knowledge in the distinct uniqueness of newborns and other age groups, other disciplines and on nosocomial infections in neonatal and pediatric nurseries and intensive care units is warranted. Finally, vaccinations make up a larger proportion of the preventive measures in pediatrics than in adult medicine, and this is mirrored by the extraordinary success of general immunization campaigns in children [2].

Relation to community pediatrics and to hospital pediatrics Pediatricians in private practice and, in some countries or regions, also general practitioners, are in charge of the management of children with common and frequent infectious diseases [3]. The quality of this management benefits highly from the continued access to and availability of a pediatric infectious disease specialist during the medical formation and training as well as throughout private practice activities. Pediatric infectious disease specialists provide important recommendations on the use of microbiological and other diagnostic tests, application of antimicrobial drugs, and measures for infection control, which may substantially differ in children compared to in adults. Furthermore, infectious disease specialists possess the required expertise for the establishment of standards of care for frequent communicable diseases and relevant guidelines for the community. Pediatric infectious disease specialists are involved in the care of both outpatients and inpatients [4]. The impact of the pediatric infectious disease specialist within a hospital can easily be deduced from the number of consultations related to infectious disease or infection control issues requested by both experienced

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Figure 1. Communication pathways of pediatric infectious diseases.

and non-experienced physicians within or outside the hospital. Bedside consultations and phone consultations both play an important role [5]. The multiple interactions result, e.g., in a more considerate selection of diagnostic measures and assays, more judicious and less costly use of antimicrobials, and reduction of formal consultations and hospitalizations [6] (Fig. 1). Much of the shared knowledge originates from pediatric infectious diseases research programs, as they substantially contribute to the development of improved diagnostic, treatment and prevention means as well as to the understanding of pathogenesis and epidemiology of infectious diseases. The multifaceted roles of the pediatric infectious disease specialist clearly improve the quality of patient care and teach physicians who are involved in primary health care [4].

Integral and integrative behavior Specialty in pediatric infectious diseases is the paradigm of an integral and integrative discipline providing paramount professional help, advice and support to other pediatric disciplines and to disciplines from adult medicine. Obvious examples are consultations for patients with underlying conditions including congenital heart disease, cystic fibrosis, primary or secondary immunodeficiencies such as due to HIV infection or iatrogenic immunosuppression following allograft transplantation, or tumors. Many of these patients nowadays survive beyond the pediatric age and need to undergo the difficult process of transition to medical care for adults [7]. Thus, close

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interactions with colleagues from adult medicine taking over the care of these patients before, during and after the transition process are indispensable to ensure satisfaction and compliance of the patients with often heavy burdens in addition to the burdens of adolescence. Infectious disease specialists have a considerable number of skills at their disposal [8]. Experienced infectious disease specialists, for example, often reduce the use of expensive diagnostic measures even in the most complex patient situations, apply intravenous antimicrobial treatment also to outpatients and switch from intravenous to apt oral medication on time. Hence, infectious disease specialists increase the satisfaction of patients while ensuring management quality at lowest possible expenses.

New developments for the specialists in pediatric infectious diseases An outlook into the future cannot be undertaken without careful consideration of the past and the current situation. Thus, recent changes in the spectrum of infectious diseases, progress in the field of vaccinology, advances in microbiology, and quantum leap in communication technology are likely to determine new developments and areas of activity for the pediatric infectious disease specialist. The variety of topics covered in the chapters of this book nicely mirrors the wide spectrum of pediatric infectious diseases and the most recent novel developments in the field.

The changing spectrum of infectious diseases Several achievements including clean water, improved sanitation, vaccination and antimicrobial therapies have brought many important infectious diseases under control. Nevertheless, we have had to face the emergence of pathogens that are resistant to antimicrobials and of new pathogens that have not been previously detected in humans. The principal diseases of the last decade can be segregated into three major groups: (i) infections against which significant progresses have been achieved; (ii) newly emerged infections; and (iii) infections on which we had no impact [9]. In industrialized countries, infections with HIV or hepatitis C virus (HCV) have been transformed from diseases with no cure to manageable chronic infections due to newly available treatment or prevention modalities. Most importantly, mother-to-child transmission rates in these countries have fallen from around 15–25% to below 2%, and where preventive measures are strictly applied, vertical transmission of HIV has virtually vanished [10]. This success story, however, evolved at the expense of intrauterine and neonatal exposure to drugs with a considerable toxic potential [11]. Thus, pediatric infectious disease specialists need to conduct long-term surveys on the evolution of these children following exposure to antiretrovi-

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ral drugs in a life period with highest vulnerability, especially of the central nervous system. Testing of blood products has not only virtually abolished transfusion-related HIV infections but also HCV transmission [12]. Poliomyelitis vaccination campaigns have been extremely effective both in industrialized and in non-industrialized countries. Globally, the number of poliomyelitis cases has been reduced by 99% from 350 000 cases in 1988 to less than 800 cases in 2002 [13]. The goal to eradicate poliomyelitis, however, seems to be hurdled by unprecedented reemergence of poliomyelitis due to “escape” variants [14] or due to outbreaks in communities reluctant to vaccination, mainly for religious reasons and in countries where there are governmental obstacles to vaccination campaigns [13]. The tasks waiting the pediatric infectious disease specialists are to promote vaccination at the individual, at the community and at the country levels. This will demand persuasion activities focusing on individuals and on politicians. Similarly, measles, rubella and mumps are three viruses against which we possess excellent vaccines, and thus could be eliminated given that the only host for these viruses is humans. We will eventually defeat theses viruses only if pediatric infectious disease specialists succeed in convincing parents of the necessity of vaccination. Many parents are no longer familiar with the disastrous consequences of these viruses simply because of the decreased circulation of these viruses in the populations due to the fact that a large proportion has been previously vaccinated. But convincing just the parents will not be sufficient, physicians and politicians will need to be convinced too [15]. The general introduction of the conjugate vaccine against Haemophilus influenzae type b for infants and young children early in the 90s has resulted in a dramatic reduction of H. influenzae type b invasive infections including meningitis, epiglottitis, arthritis, and osteomyelitis [16]. More recently, conjugate vaccines against Streptococcus pneumoniae or Neisseria meningitidis type C have also been introduced in general vaccination programs, and it appears that we will again witness a success. Nevertheless, not all S. pneumoniae serotypes are represented in the vaccine and the serotypes against which the vaccine elicits immunity may be replaced by other serotypes. Furthermore, a universal vaccine against N. meningitidis type B is still lacking. Thus, the reduction of S. pneumoniae or N. meningitidis-induced disease will not be as impressive as for H. influenzae type b. In consequence, pediatric infectious disease specialists will have to explore modalities to improve surveillance and treatment of these prominent and potentially deadly bacterial infections. It goes without saying that more research on the elucidation of bacterial and host-related pathogenetic mechanisms is needed to cut the imminent danger from these pathogens [17–19]. We have also witnessed the emergence of an unprecedented number of infections. Most of these infections are of animal origin: avian influenza, severe acute respiratory syndrome (SARS), West Nile, Ebola, and variant Creutzfeldt-Jacob disease. Another unprecedented observation was the

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increase in the prevalence of antibiotic-resistant bacteria and the reemergence of previously eradicated pathogens as agents of bioterror. Among the most feared and serious antibiotic-resistant bacteria are methicillin-resistant Staphylococcus aureus. Multiple antibiotic resistances are a problem also with S. pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa and Mycobacterium tuberculosis [9]. A main challenge for pediatric infectious disease specialists in addition to the challenge faced by their adult counterparts in this context will be the availability of apt antimicrobials in apt formulations. This in turn will demand that pharmacokinetic, efficacy and safety clinical trials for new drugs are conducted in parallel for adults and different age groups of children, to acquire the needed antimicrobial armamentarium on time. Unfortunately, during the last decade we had no impact on tuberculosis, malaria and worldwide HIV, the three leading killer infectious diseases which contribute to half of the global burden of mortality from infectious diseases. In fact, the absolute number of the epidemics has steadily increased. This may cause repercussions in industrialized countries. Thus, we pediatric infectious disease specialists who are in a privileged situation cannot neglect these unsolved medical problems, but rather need to increase our efforts to share our time, knowledge and expertise for the benefit of those who need it most. Vaccines against these three pathogens are, without a doubt, of paramount priority. Finally, another important issue has come up recently: pediatric infectious disease specialists have to deal with aspects of biological terrorism against children (see the chapter by Kwang Sik Kim).

Progress in vaccinology The development of several vaccines has been hampered by technical difficulties. Vaccines can be developed following the principles of Pasteur, i.e., isolating, inactivating and injecting causative microorganisms. Such development, however, is not apt for all pathogens, especially for those which cannot be grown in cultures, including HCV, papillomaviruses 16 and 18 and Mycobacterium leprae or for antigenically hypervariable microorganisms such as N. meningitidis type B, N. gonorrhoea, malaria and HIV [9]. In recent years, many obstacles in the engineering of vaccines have been overcome. Using “reverse vaccinology” [20], a process in which computer analysis, microarrays, proteomics and other genome-based systematic approaches are used to select genomic sequences of microorganisms, antigens likely to confer protective immunity can be identified. Candidate antigens can be expressed by recombinant DNA and be tested in animal models. Reverse vaccinology has enabled the production of vaccines against HCV, human papillomaviruses, and meningococci type B. These examples will be followed for other pathogens representing a threat to infants and children.

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The pediatric infectious disease specialist will have to define priorities, and will have to conceive plans to test the safety and efficacy of these future vaccines, as well as the surveillance of the epidemiology of the targeted pathogens following the introduction of the vaccines on a larger scale. A change in paradigm in vaccinology has come from the recognition that conquering the most difficult infections such as HIV and malaria may require the T cell arm of the immune system. Most vaccines available today work by inducing antibodies, and quantification of these antibodies is often used as a parameter for immunogenicity of and protection by a given vaccine. Unfortunately, protective antibody levels are not clearly defined for every available vaccine. Moreover, as in the example of HBV vaccine, the levels of specific antibodies may not be indicative for the status of protection. The level of specific antibodies may be below the limit of detection but vaccinated individuals may be still protected against HBV infection by the cellular immune responses. The effective stimulation of cytotoxic T cells can be obtained using engineered non-replicating viral vectors, such as modified vaccinia virus, replication-incompetent adenoviruses and DNA vaccines [9]. Another recent quantum jump has been that we – as other living organisms – possess a conserved “innate” immune defense against pathogens. The innate immune defense senses invading microorganisms or their components, and determines the type of adaptive immune response that will eventually result in protection. Toll-like receptors and NOD proteins are involved in this process. An improved knowledge of the pathways of innate immunity, their selectivity and their interactions is likely to improve the efficacy of vaccines, since certain compounds triggering innate immune defenses, e.g., unmethylated CpG, which mimics microbial DNA or lipopolysaccharide as a bacterial cell wall component, could be used as novel vaccine adjuvants to enhance immunity. The field of innate immunity is certainly one of the most promising fields for laboratory and clinic-based research in pediatric infectious diseases [21].

Advances in microbiology, immunology and genetics Among the most important developments resulting in unprecedented insights into pathogenesis, susceptibility and diagnosis of infectious diseases are advances in microbiology, immunology and genetics. Important changes, with introduction of molecular biology techniques and laboratory automation, have increased the accuracy and velocity of microbiological diagnosis (Fig. 2), and new tools are still being developed [22]. The pediatric infectious disease specialist will considerably benefit from close collaborations with microbiologists both at the research and at the routine level. An equally symbiotic relationship between pediatric immunologists and geneticists will help establish the reasons for increased susceptibility to distinct patho-

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Figure 2. Modern techniques used to diagnose infectious diseases.

gens as, for example, mycobacteria or salmonella [23] and novel treatment modalities for immunocompromised children [24].

Increased intra- and interdisciplinary communication, interactions and networking The sick child has the right to receive the best possible medical attention. This includes the caring physicians calling in specialists for consultations, and interdisciplinary consultations can be predicted to become a pivotal component of standard care for patients in the future. Who would dare to prevent a sick child from getting optimal remedial management? Given the growing medical knowledge and the increasing complexity of modern medical care, pediatric infectious disease specialists can be anticipated to become highly solicited. Thus, intra- and interdisciplinary interactions will be more than ever crucial for pediatric infectious disease specialists in the years to come. Continued extensive communication, and close collaboration and partnership with other pediatric infectious disease specialists as well as with experts from pediatric immunology, clinical microbiology, pharmacy, epidemiology and all other pediatric subspecialties will build up the key for pediatric infectious disease specialists to ensure the indispensable optimal patient care, efficient teaching, and prosperous research. The most demanding challenge for pediatric infectious disease specialists will therefore be to comprehensively compile expertise, knowledge and cutting edge research for the ultimate benefit of the patient. Whereas improved communication within the own hospital setting will help to cope with unqualified management of the sick child as much as pos-

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sible, installment of a regular and frequent dialog with other centers will not only provide helpful suggestions from peers for the management of patients, but also facilitate and improve continuous education in the field and ensure exchange of ideas for independent and collaborative patient-related or laboratory-based research. The rapidly evolving communication technology has established excellent and affordable tools to allow for quick and reliable data and digital picture transfer as well as for audiovisual conferences at the national, international and intercontinental levels. Indeed, digital picture documentation of clinical and laboratory findings is advancing and will evolve. The improved communication at the national and international level should pave the way towards standardized training curricula and the development of training quality evaluation programs. In countries where medical specialty units specifically devoted to pediatric infectious diseases await establishment, support from national and international professional societies will be required to promote the specialty, and communication networks will certainly contribute to expediting this process. The goal to install a pediatric infectious disease service at least in every large medical center is justified. Networking will become more and more important to conduct multicenter studies devoted to the pathogenesis, diagnosis or management of less common infectious diseases to enable inclusion of sufficient patients in an appropriate time frame or to adequately respond to emerging infectious diseases [8]. Further, networking that also included experts other than pediatric infectious disease specialists will become increasingly essential to collect and exchange data pertinent to interdisciplinary managed patients as, for example, neonates, cystic fibrosis patients or transplant recipients, to optimize clinical research and management as well as issuing guidelines. Such guidelines will gain importance, e.g. in preventing misuse of highly expensive biologicals or drugs (http://www.swiss-paediatrics.org/paediatrica/vol15/n6/palivizumab2004-ge.htm).

Conclusions The pediatric infectious disease specialist faces many challenging and complex tasks in the next few years. These tasks will include meticulous consolidation and careful extension of existing activities aiming at conducting high-level research, offering high-standard teaching, and providing highquality patient management. These contributions to modern health care and medicine in general and pediatrics in particular can only be accomplished by dedicated individuals with extraordinary communication and integrative skills following painstaking continued training and formation. Potential careers in the discipline can be envisioned not only in academics, but also in government, public health, and industry, although less likely in private

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practice. The diversity of issues and questions to be confronted makes the specialty of pediatric infectious diseases the specialty with the widest scope compared to all the specialties in medicine. Accordingly, commitment to pediatric infectious diseases will be extremely demanding. Since not all imposed tasks can be successfully completed by one person only, it will be of paramount importance to focus the activities and to carefully define priorities. Nevertheless, such demanding commitment will be fully compensated by manifold societal and personal rewards.

Acknowledgement I thank Horst Schroten, Christoph Berger, Christian Kahlert, and Erika Schläpfer for their most valuable comments on this manuscript.

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Index

497

Index

Abbe, Ernst 103 abdominal pain, recurrent 301 acquired heart disease 274 Actinobacillus actinomycetemcomitans 189 acute respiratory illness 317 adefovir dipivoxil 393 adenovirus 323 adverse events following immunization 10, 11 amphotericin B 417–424 anaemia 246 aneurysm 281 anidulafungin 438, 440, 441 anthrax 476–479 anthrax management guidelines for children 478–479 antibiotic resistance 307, 491 antifungal agents 405 antifungal chemotherapy 405 antifungal triazoles 426 antigen, conventional 284, 285 anti-TNF antibodies 244 Archiv für Gynäkologie 107 Argentum nitricum 97 Aspergillus species 411 asthma 81, 333 autism 81 avian inﬂuenza 345–357 basic immunization schedule 6 bioterrorism 473–483, 491 bioterrorism, pediatric considerations 475 blastomycosis 410 blindness 96 blood-brain barrier, anatomy of 204 blood-brain barrier, cell culture models 211 blood-brain barrier, microbial port of entry 208 blood-CSF barrier, anatomy of 205 blood-CSF barrier, cell culture models 213 blood-CSF barrier, microbial port of

entry 209 booster dose 8 borax solution 99 breast feeding 155 bronchiolitis 332, 333 bronchopneumonia 334 Candida 406–411 Candida albicans 408 Candida laryngitis 406 Candida parapsilosis 408 Candida thrombophlebitis 406 Candida tropicalis 412 carbolic acid 99 caspofungin 437–440 CD40 ligand 283 celiac disease 128 Chediak-Higashi syndrome 187 chickenpox, see varicella child mortality 44, 146 chlamydial eye infection 107 chronic disseminated candidiasis 412 chronic granulomatous disease 187, 407, 410 chronic mucocutaneous candidiasis 411 chronic obstructive pulmonary disease (COPD) 333 circumventricular organs 206 cocidioidomycosis 410 Cohn, Ferdinand 103 co-infections, bacterial-viral 335 co-infections of respiratory viruses 333 cold chain 9, 10 collagenase 189 Collargolum Credé 107 combination vaccines 78 community pediatrics 487 complications following immunization 76 condyloma acuminata 372, 373 congenital T cell immunodeﬁciency 406 Conheim, Julius Friedrich 103 conjugate vaccine 490 consensus interferon 401

498

contraindications for immunization 72, 91, 92 coronary artery, abnormalities of 274 coronary artery, lesions of 274 cost-effectiveness calculation of immunization program 18 cows milk allergy 129 Credé, Benno Carl 107 Credé, Carl Siegmund Franz 95–111 Credé´s eye prophylaxis 95–111 Credé’s method 96 Credé’sche Prophylaxe, see Credé´s eye prophylaxis Credé’scher Handgriff, see Credé’s method Credé-Hoerder, Carl 108 Crohn’s disease 81 croup 333 cryptococcal meningoencephalitis 415 cryptococcosis 413 Cryptococcus neoformans 407 cyclic neutropenia 187 cystic ﬁbrosis 128 cytokines 282 cytotoxic oedema 252 cytotoxin-associated gene (cag) pathogenicity island 298 demineralization 178 dengue fever 34 dental caries, alternative etiology of 181 dental plaque 178 dental plaque, local environment in 183 diabetes 81, 82 diabetes mellitus 185, 187 dietary carbohydrates 178 DiGeorge syndrome 411 Diplococcus Neisser 102 Down’s syndrome 192 dyserythropoiesis 247, 248 early childhood caries (ECC) 178 ECC, preventive strategies 183 early maternal-infant attachment 107 eating disorders 130 echinocandin 437 Ehrlich, Paul 103 enamel 178 endocrine disorders 185 Erwärmungswanne 97 Escherichia coli, blood brain barrier translocation 213 Escherichia coli, CNS invasion 216 esophageal candidiasis 414 exotoxin 189

Index

Expanded Program on Immunization 5 eye prophylaxis 99–107 eyesight, impairment of 96 failure to thrive 126 false contraindications for immunization 72, 92 “Father of Gonococcus“, see Neisser, Ludwig ﬁmbriae 189 ﬂuconazole 426–428, 432 ﬂuoride 183 5-ﬂuorocytosine (5-FC) 430, 431 fulminant hepatitis B 398 fungal infections 405–445 fungal infection, adjunctive immunotherapy 444 fungal infection, epidemiology 407 fungal infection, neonate 407 Fusarium 413 galactomannan antigen, serial monitoring of 417 galactomannan ELISA 417 gastric cancer 299 gastritis 299 gastroesophageal reﬂux disease 302 gastrointestinal endoscopy 306 Giardia 163 gingipain 189 gingival crevice 184 gingival crevicular exudate 186 gingival inﬂammation 187 gingivitis 184 GlaxoSmithKline 45, 56, 60 Global Alliance for Vaccines and Immunization (GAVI) 4, 5, 43, 57, 60, 61 Global Immunization Vision and Strategy (GIVS) 4, 16, 17 global poliovirus lab network 13 glucosyltransferase (GTF) 179 gonorrhea 96 habitats within the mouth 177 haemoglobinopathies 256 Haemophilus inﬂuenzae type b 490 Haemophilus inﬂuenzae type B vaccine 26, 27 health and nutrition interventions 164–169 health nutrition and gender equity 169 Helicobacter pylori infection 297–309 Helicobacter pylori, ﬂagella of 298 Helicobacter pylori, test 304–307 Helicobacter pylori, treatment 308

Index

Helicobacter pylori, triple therapy 308 Helicobacter pylori, virulent factors 298 hemagglutinin 346 hemorrhagic meningitis 477 hepatitis B 391–398 hepatitis B, clinical course 392 hepatitis B, fulminant 397 hepatitis B, seroconversion to anti-HBe 392 hepatitis B, serological diagnosis 392 hepatitis B, therapy response 395 hepatitis B treatment 393–398 hepatitis B treatment, complications 396 hepatitis B treatment, contraindications 396 hepatitis B treatment, resistant mutations 397 hepatitis B vaccine 28, 29 hepatitis B virus, genotype 394 hepatitis C 398–402 hepatitis C, serological diagnosis 399 hepatitis C, transmission 399 hepatitis C treatment 400–402 hepatitis C treatment, contraindications 401 hepatitis C treatment, side effects 401, 402 hepatitis C virus RNA 401 high mobility group box 1 (HMGB1) 244 high-resolution computed tomography (HRCT) 416 histoplasmosis 410 HIV 17, 131, 161, 162, 491 hMPV vaccine 336 home delivery 95 hospital pediatrics 106, 277, 487 host-parasite relationship 185 Howe, Lucien 106 human papillomavirus (HPV) 372, 373 HPV, condyloma acuminata 380 HPV, differential diagnosis of 380 HPV, E6 gene 367 HPV, E7 gene 367 HPV, genital sub-types 382, 383 HPV, immune response to 376 HPV, malignant conversion of 379 HPV, mode of regression 375 HPV, spontaneous 368 HPV, subclinical 371 HPV treatment 380, 381 HPV vaccination 383, 384 HPV vaccine 32–34 human brain microvascular endothelial cells (HBMEC) 212 human metapneumovirus (hMPV) 317–328 hyperlactataemia 250 idiopathic thrombocytopenia (ITP) 302–304

499

IFN-_-2b 400 IFN-a 282 IgA 286 IgM antibodies 283 IL-1 282 IL-6 282 immune defect, adaptive 122 immune defect, innate 121 immunity, natural 77 immunity, vaccine-induced 77 immunization and chronic diseases 85 and impaired immunity 85–90 during pregnancy 83, 84 of MS patients 89 of preterm babies 84, 85 immunization, indications for 72 immunization program, funding of 20 immunization safety 10, 11 infantile periarteritis nodosa (IPN) 274 infective endocarditis and oral streptococci 184 inﬂammatory cytokines in malaria 243 inﬂammatory eye disease 100 inﬂuenza 477 inﬂuenza encephalopathy 255 inﬂuenza pathogenesis 243 inﬂuenza virus type A 323 inﬂuenza virus type B 323 innate immune defense 492 intensive care units 415 interferon-alpha 393 intracellular oxidative burst 188 intrauterine growth retardation 126 intravenous immunoglobulin (IVIG) 281, 283 intussusception 54 invasive aspergillosis 409, 412 invasive candidiasis 408 iodine deﬁciency, effect on cognitive development 158 iron deﬁciency anemia 155–158, 167, 302 iron deﬁciency anemia, maternal behaviour 157 itraconazole 428–430, 432 juvenile onset-recurrent respiratory papillomatosis (JORRP) 365, 373 Kawasaki disease 273–287 Kawasaki disease, diagnosis 274 Kawasaki disease, diagnosis algorithm 276 Kawasaki disease, epidemiology 274–280 Kawasaki disease, etiology 285–287

500

Kawasaki disease, global distribution 277, 278 Kawasaki disease, pathogenesis 281–285 Kawasaki disease, pathology 281, 282 Kawasaki disease, risk factors 278–281, 283, 284 Koch, Robert 103 Koch-Henle postulates 104 kwashiorkor 119 laboratory automation 492 lactoferrin 191 lactose intolerance 130 lamivudine 393, 398 lazy leukocyte syndrome 187 Leopold, Gerhard 96 leptin 131 leukotoxin 189 lipopolysaccharide (LPS) 189 liposomal amphotericin B 424–426 lochial discharge 99 low birth weight 126, 154, 155, 410 lysozyme 191 macrophage activation syndrome 283 magnetic resonance imaging (MRI) 416 malaria 34, 133, 239–259, 491 malaria, effect on mental development 159, 160 malaria, effect on socioemotional development 160 malaria, inﬂammatory cytokines 243 malaria, intervention programs 167, 168 malaria, neurological involvement 251–253 malaria pathogenesis 243 malarial disease, cytokine theory of 240 malarial disease, mechanical theory of 240 malnutrition syndromes 125 malnutrition, see also undernutrition marasmus 119 maternal and neonatal tetanus elimination 25, 26 maternal education programs 148 matrix metalloproteinases 283 measles 319 measles mortality reduction/elimination 24, 25 measles vaccine 24 mediastinal lymphadenopathy 477 meningitis, cognitive effects of 164 meningitis, innate immunity 222, 223 meningitis, indoleamine 2,3-dioxygenase (IDO) 223 meningitis, leukocyte recruitment 219, 220

Index

meningitis, pathogenesis 201 meningocephalitis 406 meningococcal vaccines 31, 32 Merck 45, 56, 60 metabolic acidosis in malaria 249 Metapneumovirus 317–319 micafungin 438, 441, 442 Micrococcus 102–106 micronutrients 119, 120 micronutrient deﬁciencies, effect on mental development 159 midwife education 95, 98, 101 Millennium Development Goals 43, 61 mitochondrial dysfunction 248, 249 molecular biology techniques 492 Monatsschrift für Geburtshilfe und Frauenkrankheiten 107 Morbillivirus 319 mouth, habitat 177 mucosal associated lymphoid tissue (MALT) lymphoma 301 multiferon 399 multiple immunization 78 multiple sclerosis 82 mumps 319 mutans streptococci 179–183 mutans streptococci, cariogenic potential 183 mutans streptococci, transmission of 184 Mycobacterium tuberculosis 131, 132 myeloperoxidase (MPO) deﬁciency 410 Neisser, Ludwig Sigesmund Albert 103–105 Neisseria meningitidis 217, 490 Neisseria meningitidis, CNS invasion 217 neonatal invasive candidiasis 442 “Nestor of German midwifery”, see Credé, Carl Siegmund Franz neuronal excitotoxins 253 neutropenic children 412 non-cariogenic sweetener 183 nucleoside analogues 396 nutrient action, mechanisms of 127 nutrition, long term effects on cognition 149 nutritional interventions, timing of 153, 154 nutritional supplementation 147–152 obesity 120, 131 obligatory anaerobes 188 ophthalmia neonatorum 95 oral microﬂora, development of 177 oral poliovaccine (OPV) 9, 23 oral streptococci (table) 180 oropharyngeal candidiasis (OPC) 412

Index

orphanhood 162 otitis media 163, 333 outpatient 95, 101, 106 palivizumab 335 pandemic inﬂuenza 346 parainﬂuenza virus 317, 319, 323 Paramyxoviridae 317–320 Paramyxovirinae 318, 319 parasite sequestration 245 pediatric infectious disease specialist’s activities 486, 487 pediatricians, career perspectives 494 pediatricians, interdisciplinary networking 493, 494 PegIntron 401 periodontal bacteria, transmission of 192 periodontal destruction 187 periodontal diseases 184 periodontal diseases, onset of 190 periodontitis 184, 186 periodontopathic bacteria 184, 188, 190 peroxydase 191 pityriasis 409 plasma cells 286 Plasmodium falciparum 239, 242 pneumococcal vaccines 30, 31 pneumonia 332–334 Pneumovirus 318, 319 poliomyelitis eradication 23, 24 polymerase chain reaction (PCR) 182, 366 polymorphonuclear leukocytes (PMNL) 186 PMNL disorder 187 PMNL phagocytosis, depression of 188 poor red cell membrane deformability 247 Porphyromonas gingivalis 189 posaconazole 432, 434–436 povidone-iodine 107 protein-calorie malnutrition 119 Reach Every District (RED) strategy 16 respiratory distress 333 respiratory synctial virus (RSV) 317–320, 329, 334 respiratory tract infection 317, 318 respiratory virus 317 respirovirus 319 retractions 333 reverse genetics technology 317, 336 rhinitis 333 rhinovirus 323 ribavirin 335 ring immunization, smallpox 483

501

Rotarix® 45, 52, 60 RotaShield® 52, 54, 59 RotaTeq® 45, 52, 54, 60 routine infant immunization 6 rubelavirus 319 safe injection 11 salicylic acid 99 school readiness 146 secretory IgA 191 Senckenberg Award 97 sepsis-associated encephalopathy (SAE) 254 severe acute respiratory syndrome 334 severe combined immunodeﬁciency (SCID) 411 shell vial centrifugation cultures (SVCC) 332 sialic acid 346 silver acetate 106, 107 single-nucleotide polymorphisms 284, 285 skin cancer 374 smallpox 479–483 sorbitol 183 Spiegelberg, Otto 107 Staphylococcus aureus 286 steroid-addicted subjects 185 stool antigen test 305 streptococcal bacteria 286 Streptococcus agalactiae, CNS invasion 218 Streptococcus mutans 179, 180, 182 Streptococcus pneumoniae 490 Streptococcus pneumoniae, CNS invasion 216 Streptococcus sobrinus 179, 180, 182 Streptococcus suis, CNS invasion 218 sucrose-caries-mutans streptococci association 180, 181 sudden death 273 sudden infant death syndrome 82, 83 sugar substitutes 183 superantigen 285 supplementary immunization activities (SIAs) 8, 9 surveillance for vaccine-preventable diseases 12 surveillance and containment, smallpox 483 T cells 282, 283, 285, 286 thalassaemia 257 thrombomodulin 246 thymic atrophy 122 TNF-_ 282

502

tooth and periodontal tissues, structure of 179 Trichosporon beigelii 413 tuberculosis 491 tuberculosis vaccine 34 tumour necrosis factor (TNF) 243 undernourished/malnourished children, educational and psychosocial stimulation of 148–152 undernutrition, effect on cognitive development 147–151 undernutrition/malnutrition, effect on motor development 151, 152 undernutrition/malnutrition, effect on socio-emotional development 152, 153 undernutrition/malnutrition, maternal behaviour 150, 152, 155 urea breath test 304 urease 298 vaccines 24–35 vaccine, avian inﬂuenza 355 vaccine, hMPV 336 vaccine, HPV 383, 384 vaccine, oral polio 9, 23 vaccine, vector-based 337 vaccinology 489 vacuolating cytotoxin 299 vaginal catarrh 98 vaginal contact during birth 102 varicella 481 vascular endothelial grown factor (VEGF) 283 vasculitis 274, 281, 282 vasogenic oedema 252 vector-based vaccine 337

Index

Vero cell clone 118 330 viral glycoprotein 332 virus-like particles 367 VISION 2020 107 vitamin deﬁciency 125, 132 vitamin E 395 vivax malaria 254 voriconazole 431–435 VP4 54, 55 VP4 P type 47, 48 VP7 G type 47, 48, 57 VP7 gene 53 V`2+ T cell 285 V`8.1+ T cell 285 warts, see also HPV common warts 368 digitate warts 370 ﬂat warts 369 palmoplantar warts 372 periungal warts 372 plantar warts 368, 369 warts, excessive number of 377 warts classiﬁcation system (table) 369 water-insoluble glucan 180 Weigert, Carl 103 wheezing 333 World Health Organization(WHO) 43, 45, 49, 57, 59, 107 worms 162 xylitol 183 yellow fever vaccine 29, 30 zygomycetes 413 Zygomycosis 415

Index

503

The BAID-Series Birkhäuser Advances in Infectious Diseases Infectious diseases remain a substantial drain on human well-being and economies despite the availability of modern drugs. New pathogens emerge and known pathogens change their geographical distribution and their susceptibility to the available drugs. An understanding of the structure and function of infectious disease pathogens is a major scientific challenge with important potential applications. This new cross-disciplinary monograph series will provide up-to-date information on the latest developments in infectious disease research. The multi-authored volumes will cover basic biology and biochemistry of pathogens as well as applied medical aspects and implications for public health and policy. The contributions are written by leading infectious disease researchers and pharmaceutical scientists with a wide range of expertise. The envisaged readership includes academic and industrial researchers in medicine and infectious diseases as well as clinicians and others involved in diagnostics and drug development.

Forthcoming volumes: Influenza Vaccines for the Future, R. Rappuoli (Editor), 2007 Available volumes: Coronaviruses with Special Emphasis on First Insights Concerning SARS, A. Schmidt, M.H. Wolff, O. Weber (Editors), 2005 The Grand Challenge for the Future. Vaccines for Poverty-Related Diseases from Bench to Field, S.H.E. Kaufmann and P.-H. Lambert (Editors), 2005 Community-Acquired Pneumonia, N. Suttorp, T. Welte, R. Marre (Editors), 2007 Poxviruses, A. Mercer, A. Schmidt, O. Weber (Editors), 2007