Pediatric Infectious Diseases Revisited PDF

Birkhäuser Advances in Infectious Diseases
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Pediatric Infectious
Diseases Revisited
Edited by Horst Schroten and Stefan Wirth
Birkhäuser Verlag
· ·
Basel Boston Berlin
Editors
Horst Schroten Stefan Wirth

Pediatric Infectious Diseases Children’s Hospital
Department of General Pediatrics HELIOS Klinikum
University Children’s Hospital Witten-Herdecke University
Moorenstr. 5 Heusnerstr. 40
40225 Düsseldorf 42283 Wuppertal
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Contents
List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface ................................................................... 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 Pedi-

atrics, University Children’s Hospital, Moorenstrasse 5, 40225 Düsseldorf,
Germany; e-mail: adam@med.uni-duesseldorf.de
Ian A. Clark, School of Biochemistry and Molecular Biology, Australian Na-
tional University, Canberra ACT 0200, Australia;
e-mail: ian.clark@anu.edu.au
Susanna Cunningham-Rundles, Host Defenses Program, Department of
Pediatrics, Weill Medical College of Cornell University, 1300 York Ave-
nue, New York, NY 10021, USA; e-mail: scrundle@mail.med.cornell.edu
Sieghart Dittmann, Hatzenporter Weg 19, 12681 Berlin, Germany;
e-mail: sd.internat.immun.consult@t-online.de
Rudolf Eggers, Expanded Programme on Immunization Plus, World Health
Organization, Geneva, Switzerland; e-mail: eggersr@who.int
Patrick Gerner, Zentrum für Kinder- und Jugendmedizin, HELIOS Klini-
kum Wuppertal, Heusnerstr. 40, 42283 Wuppertal, Germany;
e-mail: patrick.gerner@web.de
Michael J. Griffiths, Department of Paediatrics, Newcastle General Hospital,
Newcastle upon Tyne, U.K.; e-mail: griffmj@stanford.edu
Andreas H. Groll, Infectious Disease Research Program, Center for Bone
Marrow Transplantation and Department of Pediatric Hematology/On-
cology, Children’s University Hospital, Albert-Schweitzer-Str. 33, 48129
Münster, Germany; e-mail: grollan@ukmuenster.de
Ronald de Groot, Department of Pediatrics, Radboud University Nijmegen
Medical Centre, and the Nijmegen University Center for Infectious Dis-
ease, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands; e-
mail: r.degroot@cukz.umcn.nl
Deborah Ho Lin, Department of Pediatrics Host Defenses Program, Weill
Medical College of Cornell University, New York, NY 10021, USA;
e-mail: debbyholin@aol.com
Hien Q. Huynh, Department of Pediatrics, Stollery Children’s Hospital, Ab-
erhart Centre #1, Room 9222, 11402 University Avenue, Edmonton, AB,
Canada T6G 2J3; e-mail: hien.huynh@ualberta.ca
Matthew Jukes, Harvard Graduate School of Education, Appian Way, Cam-
bridge, 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: jukesma@gse.harvard.edu
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: kwangkim@jhmi.edu
Shigenobu Kimura, Department of Oral Microbiology, Iwate Medical Uni-
versity School of Dentistry, 1-3-27 Chuodori, Morioka, Iwate 020-8505,
Japan; e-mail: kimuras@iwate-med.ac.jp
Julia Koehler, Children’s Hospital Boston, Harvard Medical School, Divi-
sion of Infectious Diseases, 300 Longwood Avenue, Boston, MA 02115,
USA; e-mail: julia.koehler@childrens.harvard.edu
Wilbert Mason, Los Angeles Children’s Hospital, 4650 Sunset Boulevard,
Los Angeles, CA 90027, USA; e-mail: wmason@chla.usc.edu
David Nadal, Abteilung für Infektiologie und Spitalhygiene, Kinderspital
Zürich, Universitäts-Kinderkliniken, Steinwiesstrasse 75, 8032 Zürich,
Switzerland; e-mail: david.nadal@kispi.unizh.ch
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 Uni-
versity Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Naga-
saki, Japan
Axel Schmidt, Institute of Microbiology and Virology, Faculty of Medicine,
University Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany;
e-mail: axel780961@t-online.de
Horst Schroten, Pediatric Infectious Diseases, Department of General Pedi-
atrics, University Children’s Hospital, Moorenstr. 5, 40225 Düsseldorf,
Germany; e-mail: schroten@uni-duesseldorf.de
Nanette B. Silverberg, Department of Dermatology, St. Luke’s-Roosevelt
Hospital Center, 1090 Amsterdam Avenue, Suite 11D, New York, NY
10025, USA; e-mail: nsilverberg@juno.com
Duncan Steele, Initiative for Vaccine Research, Department of Immunisa-
tion, Vaccines and Biologicals, World Health Organisation, Geneva, Swit-
zerland; e-mail: steeled@who.int
Rudolf H. Tangermann, Polio Eradication Initiative, World Health Organi-
zation, 20, Avenue Appia, 1211 Geneva 27, Switzerland;
e-mail: tangermannr@who.int
Thomas J. Walsh, Immunocompromised Host Section, National Cancer In-
stitute, National Institutes of Health, Bethesda, Maryland, USA;
e-mail: walsht@mail.nih.gov
Adilia Warris, Department of Pediatrics, Radboud University Nijmegen
Medical Centre, and the Nijmegen University Center for Infectious Dis-
ease, Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands;
e-mail: a.warris@cukz.umcn.nl
John V. Williams, Division of Pediatric Infectious Diseases, Vanderbilt Uni-
versity Medical Center, D-7235 Medical Center North, 1161, 21st Avenue
South, Nashville TN 37232-2581, USA;
e-mail: john.williams@vanderbilt.edu
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 diseas-
es 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 socio-
cultural issues as well as clinical and molecular biological aspects of pedi-
atric infectious diseases. New insights into the pathogenesis of infection are
presented and an update on diagnostics, prevention and treatment of pedi-
atric 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 col-
leagues 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 amphotericin B colloidal dispersion

ABLC amphotericin B lipid complex
ACIP Advisory Committee for Immunization Practices
AD auto-disable (syringe-needle unit)
ADIP accelerated development and introduction plan
AEFI adverse events following immunization
AGA appropriate for gestational age
ANCA antibodies to neutrophil cytoplasmic antigens
ARDS adult respiratory distress syndrome
ARF acute renal failure
ARI acute respiratory infection
ART Anti-retroviral therapy
AZT zidovudine
BBB blood-brain barrier

BMEC brain microvascular endothelial cell
BMI body mass index
CAA coronary artery abnormalities

CAG cyctotoxin-associated gene
CD Crohn’s disease
CF cystic fibrosis
CFR case fatality rate
CGD chronic granulomatous disease
CIS Commonwealth of Independent States
CLEAR Collaborative Exchange of Antifungal Research (registry)
CM cerebral malaria
CMA cow’s milk allergy
CNS central nervous system
COPD chronic obstructive pulmonary disease
CP choroid plexus
CSF cerebrospinal fluid
CVI Children’s Vaccine Initiative
CVO circumventricular organ
DAMB amphotericin B deoxycholate

DQ Development quotient
DTP diphtheria/tetanus/pertussis (vaccine)
xii Glossary
EAE experimental autoimmune encephalomyelitis

ECC early childhood caries
ECD early childhood development
EDV epidermodysplasia verruciformis
EM electron microscopy
EMEA European Medicines Evaluation Agency
EMS emergency medical service
EPI Expanded Program on Immunization
FC fluorocytosine
FDA Food and Drug Administration (USA)
FTT failure to thrive
GAVI Global Alliance for Vaccine and Immunization

GCF gingival crevicular fluid
GERD gastroesophageal reflux disease
GIVS Global Immunization Vision and Strategy
GMT geometric mean titer
GTF glucosyltransferase
GVHD graft-vs.-host disease
HA hemagglutinin
HAART highly active antiretroviral treatment
HBIG hepatitis B immunoglobulin
HBMEC human brain microvascular endothelial cell line
HbS sickle cell haemoglobin
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCV hepatitis C virus
Hib Haemophilus influenzae type b
HMGB1 high mobility group box1 (protein)
hMPV human metapneumovirus
HPAI highly pathogenic avian influenza
hPIV human parainfluenza virus
HPV human papilloma virus
HPV human papillomavirus
HRCT high-resolution computed tomography
HSCT hematopoietic stem cell transplant/transplantation
HUVEC human umbilical vein endothelial cell
IBD inflammatory bowel disease

ICC Interagency Coordinating Committee
ICP intracranial pressure
IDO indoleamine 2,3-dioxygenase
IE infected erythrocyte
IFFIm International Finance Facility for Immunization
IMCI Integrated Management of Childhood Illnesses
IPN infantile periarteritis nodosa
IPV inactivated poliovirus vaccine
Glossary xiii
ITP idiopathic thrombocytopenia

IUGR intrauterine growth retardation
IVIG intravenous immunoglobulin
JORRP juvenile onset-recurrent respiratory papillomatosis
KD Kawasaki disease
LAMB liposomal amphotericin B

LPS 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 neuraminidase
NICU neonatal intensive care unit
NID National Immunization Day
NIS Newly Independent States
NT neonatal tetanus
OME otitis media with effusion

OPC oropharyngeal candidiasis
OPV oral polio vaccine
OR odds ratio
ORT oral rehydration therapy
PAF platelet-activation factor

PAMP pathogen-associated molecular pattern
PATH Program for Applied Technology in Health
PBMEC porcine brain microvascular endothelial cell line
PCM protein-calorie malnutrition
PCV Pnc conjugate vaccine
PCZ posaconazole
PEM protein-energy malnutrition
pIgR polymeric immunoglobulin receptor
PMNL polymorphonuclear leukocyte
Pnc pneumococcus
PPI proton pump inhibitor
PPV Pnc polysaccharide vaccine
PRGP proline-rich glycoprotein
RBC red blood cell

xiv Glossary
RED Reach Every District (vaccination strategy)

RES reticuloendothelial system
RRV rhesus rotavirus
RSV respiratoy syncytial virus
RTI respiratory tract infection
SA superantigen
SAE sepsis-associated encephalopathy
SAGE Strategic Advisory Group of Experts
SARS severe acute respiratory syndrome
SCID severe combined immunodeficiency
SGA small for gestational age
SIA supplementary immunization activities
SIDS sudden infant death syndrome
SIGN Safe Injection Global Network
SNP single-nucleotide polymorphism
SSPE subacute sclerosing panencephalitis
SUV small unilamellar vesicle
SVCC shell vial centrifugation culture
TNF tumor necrosis factor

TSST toxic shock syndrome toxin
TT tetanus toxoid
UC ulcerative colitis
UCI Universal Child Immunization
UNICEF United Nations Children’s Fund
VEGF vascular endothelial growth factor

VLP virus-like particle
VVM vaccine vial monitor
WHIM warts/hypogammaglobulinemia/recurrent bacterial infections/

myelokathexis (syndrome)
Pediatric Infectious Diseases Revisited 1
ed. by Horst Schroten and Stefan Wirth
© 2007 Birkhäuser Verlag Basel/Switzerland
Global control of infectious diseases

by vaccination programs
Rudolf H. Tangermann1, Hanna Nohynek2 and Rudolf Eggers1

1WorldHealth 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 nec-
essarily 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 immuniza-
tion 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.
2 Rudolf H. Tangermann et al.
Introduction
In both industrialized and developing countries, child immunization has

become one of the most important and cost-effective public health inter-
ventions [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 mea-
sured by the reported infant coverage with the third dose of diphtheria–tet-
anus–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 under-
used in developing countries. The estimated number of deaths (from mea-
sles, 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 exist-
ing 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 vac-
cines 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 recog-
nize 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 chil-
dren < 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, meningococ-
cal disease, poliomyelitis, and yellow fever. In older age groups, approximately 600 000 HepB
deaths are preventable by routine immunization.
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 vac-
cine antigens available, many of which have entered clinical phases of test-
ing for safety, immunogenicity and eventually efficacy. Pressures are grow-
ing 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 indus-
trialized 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 elabo-
rated 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 develop-
ing and industrialized countries, and provides short updates on the current
status of priority and new vaccines.
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 coun-
tries to reach at least 80% infant coverage (Universal Child Immunization,
UCI). Following renewed efforts in developing countries and by immuniza-
tion 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 acceler-
ated 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 immuni-
zation 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)
Table 1. Routine immunization schedule for infants recommended by the EPI
Vaccine Age
Birth 6 weeks 10 weeks 14 weeks 9 months
BCG X
Oral polio X† X X X
DTP X X X
Hepatitis B* Scheme A X X X
Hepatitis B Scheme B X X X
Haemophilus infl. type B X X X
Yellow fever X**
Measles X***
†In polio-endemic countries.
*Scheme A 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 chil-
dren. This may be done either as part of the routine schedule or in a campaign.
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 coun-
tries 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 industri-
alized countries vary considerably, for historical, epidemiological, and eco-
nomical reasons (compare the 2006 U.S. Child and Adolescent Immunization
Schedule, Table 2). WHO keeps track of and publishes national immuniza-
tion 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 vaccina-
tion 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
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 contribut-
ing to low coverage.
Booster doses and ‘second opportunity’ for measles vaccination
Few vaccines give life-long protection after the primary series. To main-
tain 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 recom-
mended, 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 epidemio-
logical 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 commu-
nity 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 cov-

erage – now often referred to as supplementary immunization activities
(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 dis-
ease eradication and elimination initiatives – to achieve global polio eradica-
tion, 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 sup-
port 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 detri-
mental 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 long-
term synergies exist between polio eradication and health systems [29] (build-
ing 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 freez-
ing 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 col-
laboration with manufacturers, have set standards [31] for technologically
appropriate cold-chain equipment and helped to develop such equipment,

such as ice-lined refrigerators, which can maintain appropriate storage tem-
perature 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 inappropri-
ate freezing of freeze-sensitive vaccines is also a problem in many countries,
potentially affecting the potency of vaccines with adjuvants (HepB, combi-
nation 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 pro-
duce 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 coinci-
dental 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 associ-
ated with vaccination. In cases where the vaccine of the vaccination pro-
gram 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 seri-
ous adverse events related to program error are vaccine reconstitution with
the wrong diluent, administration of dangerous drugs for vaccines, contami-
nation 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, particu-
larly in industrialized countries, about possible risks attributed to vaccines.
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 vac-
cines, 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 disease-
specific 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 immu-
nization 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 injec-
tion safety. The Safe Injection Global Network (SIGN)6, a global partner-
ship 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/
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 inju-
ries and reduce risk to communities [39]. There is also progress in develop-
ing 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 immuniza-
tion 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 nation-
al, sub-national and particularly at the district level. For immunization pro-
grams, main quality indicators include immunization coverage for the vac-
cines 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 opportu-
nities for immunization. Other program components monitored include
injection and immunization safety, cold-chain maintenance and social mobi-
lization 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 annu-
ally, 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 continu-
ously declining discrepancies.
Many middle- and high-income countries have better demographic data
available for more precise estimation of coverage: total or sample popula-
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 what-
soever 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 ‘numera-

tor data’ are available, which allow evaluatation of timeliness of vaccina-
tions 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 surveil-
lance data, which enables program managers to take decisions on planning,
implementation and evaluation of immunization programs. High-qual-
ity 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 transmis-
sion. 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
including primary virus isolation from stool specimens, intratypic differ-

entiation 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 rota-
virus 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 lab-
oratory (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 com-

mitment 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 immu-
nization 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
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, includ-
ing 12.1 million in South Asia (8 million in India alone), 8.7 million in sub-
Saharan Africa, and 4.6 million in East Asia and the Pacific (Fig. 4).
Also, regional averages conceal variations in coverage between coun-
tries. 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 cov-
erage 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-emer-
gence 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
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 aver-
ages, 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 low-
income 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 support-
ive supervision, strengthening of district immunization management, regu-
lar 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 immuniza-
tion, 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 oppor-
tunities 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 vaccina-
tion 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
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 com-
munity 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 immu-
nization infrastructure – buildings, vehicles and vaccine cold-chain equip-
ment – 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 ser-
vices, 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 insecti-
cide-treated bed nets to prevent malaria, has considerable impact on child
health and reducing child mortality.
Immunization services can also assist through establishing ‘best prac-

tices’, 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 establish-
ment of a global virological laboratory network (see Fig. 3), and in strength-
ening 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; microplan-
ning 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 nei-
ther 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.
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 manufac-
turers of vaccines for mainly developing country markets, which are consid-
ered ‚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 develop-
ing world, see above).
New vaccines go through a lengthy and very costly research and devel-
opment 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 patent-
protected 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 develop-
ing 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 criti-
cal 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 nation-
al 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, particular-
ly 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 develop-
ment and introduction of new vaccines, such as the ‘accelerated devel-
opment 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
mainly on disease burden and vaccine efficacy) early in the vaccine cycle,
i.e., before manufacturers begin the lengthy development and scale-up pro-
cess. 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 vac-
cines 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 low-
income 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 responsibil-
ity.
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 capac-
ity 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 devel-
oping 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 commit-
ted to directly support countries, with US$ 603 million disbursed.
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 coun-
tries 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 address-
ing fundamental barriers to improved vaccination coverage and program
efficiency. In addition to HepB, Hib and yellow fever vaccines, it is antici-
pated 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 vac-
cines 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 long-
term commitments by existing public and private funding entities and new
long-term financial mechanisms, to support research, development, produc-
tion 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
Table 3. Current and future vaccines and supportive technologies.
from [8].
available but under-used, new vaccines against rotavirus, certain pneumo-

coccal 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.
Poliomyelitis: progress towards eradication
Following significant progress towards interrupting wild poliovirus trans-

mission in the Americas, all Member States of WHO passed a resolution
in 1988 to eradicate polio globally by the year 2000 [55]. The global eradi-
cation 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 interna-
tional polio eradication partnership spearheaded by Rotary International,
WHO, UNICEF and the U.S. CDC, and involving millions of health work-
ers 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 stop-
ping 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 out-
breaks 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 sub-
national level, ‘sub-NIDs’.
12 Countries where circulation of indigenous wild poliovirus has never been interrupted
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 fre-
quency 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 aware-
ness 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 worsen-
ing 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 esti-

mated > 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
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 informa-
tion
– 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 elimi-
nated 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 miss-
ing the global elimination goal are continued relatively low TT coverage of
13 < 1 NT case per 1000 live births at district level.

Figure 5. Countries using Hib vaccine in routine immunization program, by Hib vaccine cover-
age, 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 immuniza-
tion 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 child-
bearing 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
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 pri-
orities. More recently, the WHO Immunization Strategic Advisory Group
of Experts (SAGE) recommended global implementation of Hib vaccina-
tion 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 introduc-
tion. 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-prevent-
able 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
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 can-
cer 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 child-
hood will become long-term HBsAg carriers, at high risk for chronic liver
disease and liver cancer. An estimated 600 000 deaths every year are attrib-
uted 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 coun-
tries [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 pro-
grams 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 aware-
ness 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 pro-
grams, 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 pro-
portion 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.
As of 2005, 158 of 192 WHO Member States have introduced HepB vac-
cination 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 vac-
cination, 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 rou-
tine 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 vaccina-
tion campaigns nearly eliminated yellow fever during the 1950s, but sub-
sequently 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 underes-
timate 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 epi-
demics [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 laborato-
ry 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
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 cover-
age, 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 particu-
larly 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
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 diph-
theria toxoid as carrier protein has been licensed (in 76 countries by early
2007), but formally introduced into immunization programs in only 15 coun-
tries. 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 estima-
tions 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, all-
cause 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 underestima-
tion 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 con-
jugate 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 epidem-
ics 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,
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 mon-
ovalent 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 vac-
cine 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 sero-
group 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 coun-
tries 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 representa-
tives have highlighted the need to select countries based on specific criteria,
for example, burden of meningococcal disease, epidemiological and labora-
tory 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
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 preva-
lent 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 infec-
tion 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 man-
aged; 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 vac-
cines [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-medi-
ated 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
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 ben-
efit of preventing a very common, albeit usually harmless, sexually transmit-
ted infection that has only a remote possibility many years in the future of
progressing to cervical cancer. The impact of a vaccine, particularly if admin-
istered 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 vac-
cination 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 socio-
cultural 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 imple-
mented in national programs, will have a major impact on public health glob-
ally. 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 anti-
microbial resistance to existing treatments in the worst-affected countries.
However, current levels of investment in vaccine research and develop-
ment 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
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 vaccine-
induced 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 vari-
able [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 develop-
ment 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 sta-
bility, 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 strat-
egy 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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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 multina-
tional 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 indi-
cates 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 devel-
oping 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 potential-
ly having a significant impact on reducing childhood mortality and contribut-
ing to achieving Goal 4 (i.e., reducing childhood mortality) of the Millennium
44 Duncan Steele
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 child-
hood 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 out-
come of diarrheal diseases, due to such global interventions as oral rehydra-
tion 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 infec-
tion. 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 approxi-
mately 25 million clinic or emergency room visits and about 2 million hospi-
talizations in children less than 5 years of age annually [8]. This tremendous
burden of disease and associated mortality is concentrated in the develop-
ing countries of Africa and Asia, where over 82% of the rotavirus-associ-
ated 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-
Potential impact of rotavirus vaccination on the mortality of children… 45
cines completed large-scale safety and efficacy evaluation in Latin America,

Europe and the USA [14, 15]. These vaccines have been licensed by the mul-
tinational 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 asso-
ciated with this, as well as reduce the limited rotavirus-associated deaths in
these regions [16, 17]. Nevertheless, the true effectiveness of rotavirus vac-
cines 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 infec-
tion in developing countries. Regional rotavirus networks have been estab-
lished 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 conse-
quences 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 protec-
tion 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-
46 Duncan Steele
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 Developed Developing Implications for

country country vaccine trials
Age of first infection
- Percent infected by 12 40% 75% Vaccine must be given earlier.
months
- Median age of infection 12–18 month 6–12 months Potential interference of maternal
antibody with vaccine take.
Seasonality Winter peak All year Year round exposure to infants,
round earlier age of acquisition. Need for
earlier vaccination.
Case fatality Low High
Mixed infection with other Uncommon Common Outcomes and measurements in
enteropathogens vaccine trial design.
Multiple virus serotypes One major More than May limit vaccine take, and affect
type one type diarrhea outcomes of trial design.
circulating circulating Necessitate additional vaccine
doses. Vaccine efficacy may be
challenged in trial design. Vaccine
candidates may need different for-
mulations
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 subse-
quent 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 re-
infection. 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
confirmed that a primary rotavirus infection, whether associated with symp-

tomatic 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 symp-
tomatic 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 hyper-
immune sera could distinguish rotavirus serotypes [34]. However, genotyp-
ing 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 vac-
cine-related efficacy, strain diversity and surveillance studies are a prime
research tool for ongoing studies [36, 38].
48 Duncan Steele
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 charac-
teristics [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 cen-
tered on genotyping of the VP4 gene due to difficulties of generating immu-
nological 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 com-
monly. 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 configura-
tions [35, 37, 38, 47].
Thus, there is a complex array and diversity of rotavirus strains circu-

lating 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 etio-
logical 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 par-
ticles 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 recov-
ered 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 devel-
op 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 diar-
rhea 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 rota-

virus disease occurs is unknown, whether after natural infection [31] or
50 Duncan Steele
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 protec-
tion.
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], vac-
cine 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 differ-
ences in different settings. In Danish children, IgA correlated with protec-
tion 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 immunoglobu-
lins 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 circulat-
ing 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 neutral-
izing antibody [59, 63, 64].
However, several studies in human populations have indicated that neu-
tralizing 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
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-spe-
cific neutralizing antibody does have a role to play.
Studies investigating the T cell responses in humans have been lim-
ited. 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 virus-
specific 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 con-
cept of the need for serotype-specific neutralizing antibody for protection
has resulted in the approach of the multivalent reassortant rotavirus vac-
cines, 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 com-
mon human rotavirus VP7 serotypes have been developed as discussed
below. An alternative approach is based on the premise that natural rota-
virus 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 dis-
covery 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 rota-
virus disease was mediated primarily by intestinal immunity [72].
52 Duncan Steele
Table 2. Live oral rotavirus vaccine candidates which are currently licensed or in clinical
development
Vaccine strain Type of vaccine Company/ Status Inventor

developer
Licensed vaccines
RotaShield® Quadrivalent reassortant Wyeth Licensed in AZ Kapikian,
rhesus rotavirus strain Ayerst USA (1998) NIH
with human rotavirus (USA)
VP7 genes for G1-G4
LLR Monovalent lamb Lanzhou Licensed in ZS Bai, Lanzhou
rotavirus Institute China (2000) Institute
(China)
RotaTeq® Pentavalent reassortant Merck & Licensed in HF Clark,
bovine strain with human Co., Inc. USA (2006) Wistar Institute
rotavirus VP7 and VP4 (USA)
genes to G1–G4 and P[8]
Rotarix® Monovalent human GlaxoSmith Licensed in R Ward, Gamble
rotavirus strain G1P[8] Kline Mexico and Institute
(Belgium) in Europe
(2006)
Clinical development
UK Multivalent reassortant NIH with Phase 2 data AZ Kapikian,
bovine strain with human vaccine available NIH
rotavirus genes producers in
Brazil, China
and India
RV3 Monovalent human University of Phase 2 data RF Bishop,
neonatal rotavirus strain Melbourne available. University of
with High titer Melbourne
BioFarma, strains
Indonesia
116E and I321 Monovalent human- Indian/USA Phase 1 data MK Bhan,
bovine reassortant consortium available RI Glass,
strains HB Greenberg,
CD Rao et al.
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 vac-
cines that have been developed by the multinational pharmaceutical indus-
try 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).
Reassortant rotavirus vaccines using animal rotavirus strains
The protection observed by the primary rotavirus infection and the antigen-
ic relatedness of animal and human rotaviruses, stimulated the “Jennerian”
approach to rotavirus vaccination, which relied on immunization with ani-
mal 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 vac-
cine development currently. The early rotavirus vaccine candidates included
bovine strains (RIT4237 and WC3) and the monovalent parent rhesus rota-
virus (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 vac-
cine 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 vac-
cine 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 hospi-
talizations or rehydration of 70–100% [82–84]. The protective efficacy was
54 Duncan Steele
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 coun-
tries. Questions remain whether the vaccine should have finished clinical
evaluation in the developing world due to the high risk-benefit of a rotavi-
rus vaccine where mortality is high due to rotavirus disease [87, 88].
The debate about the actual risk of the RotaShield® vaccine with intus-
susception 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 intussuscep-
tion, and it is likely that this can only be addressed in large post-marketing
surveillance studies once the vaccines are introduced. This has been specifi-
cally 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 reas-
sortant 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 immu-
nogenic 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.,
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 blind-
ed, 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 fol-
low-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 uti-
lized 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 immu-
nogenic 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
56 Duncan Steele
concomitant vaccines and 95% of the infants developed neutralizing anti-

body 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 sea-
son. [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 clini-
cal trial of the attenuated 89-12 vaccine strain was seen to offer 89% protec-
tive 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 immu-
nogenicity 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
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-con-
trolled 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 wild-
type 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 repre-
sents 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 com-
pleted 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
58 Duncan Steele
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 com-
munication).
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-struc-
tural 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 internation-
ally and on the verge of being introduced in multiple countries in the
Americas and in Europe? Certainly, these vaccines will reduce the tremen-
dous 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 intro-
duced 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].
Efficacy in developing countries in Africa and Asia
The WHO has recommended consistently that the efficacy of the new gen-
eration 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) differenc-
es 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 vac-
cine (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 situa-
tions (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 cluster-
ing 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®
60 Duncan Steele
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 follow-
ing 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 placebo-
controlled studies with no association identified between receipt of the vac-
cine 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 post-
licensure surveillance studies. Therefore, it is likely that post-licensure surveil-
lance 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 vaccina-
tion, 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-
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 vac-
cines 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 coun-
tries 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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350–353
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 some-
times controversially discussed by parents, the media and even by some parts of the medi-
cal 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/contraindica-
tions 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 con-
traindications 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
72 Sieghart Dittmann
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 con-
troversial 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 vac-
cine-preventable diseases already disappeared due to the improved socio-
economic conditions before vaccines were introduced.
Counter-argument
Improved socioeconomic conditions as well as the development of anti-

biotics have undoubtedly had a great impact on disease incidence, dis-
ease-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 immuni-
zation 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 out-
break 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].
Controversially discussed indications for immunization 73
Figure 1. Implementation of polio immunization (OPV) in Germany. Start of massive cam-

paigns in East (1960) and West (1962).
Figure 2. Polio outbreak in Albania – Outbreak control with OPV, April–December 1996.
In a country where a disease no longer exists, there is no longer an indica-

tion to continue with immunization against this disease.
Figure 3. Success of immunization program with conjugated meningococcal serogroup C vac-

cine, 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
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 immu-
nized 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 dis-
eases.
– Pertussis: In the 1970s in Japan, a near boycott of the vaccine due to sus-
pected vaccine complications caused the recurrence of epidemic pertussis
with hundreds of deaths. Similarly, because of aggressive publicity concer-
ning central nervous system damage following immunization using whole-
cell pertussis vaccine in UK, coverage rates fell from 75% to about 25%
during the mid-1970s, and major epidemics re-emerged. The re-implemen-
tation 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 popu-
lation 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 immu-
nization 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 imp-
lementation 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, popu-
lation movement, deteriorating health infrastructure, initial shortages of
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%, Rarely, allergic reactions, anaphylax-
nerve paralysis and myocarditis is and peripheral neuritis following
often occur DTP may occur
Hepatitis B 1% (Western Europe. North In rare cases, allergic reactions or

America) to 10% (Asia, Pacific, anaphylaxis may occur
sub-Saharan Africa) of population
are chronically infected; about 1 in
4 chronic carriers develop cirrho-
sis or liver cancer
H. influenzae CFR: meningitis, 5%; epiglottiditis, In case of fever young children may
disease 1%; about 1 in 4 survivors has develop febrile seizures, allergic
permanent brain or nerve damage reactions are very rare
Influenza Causes increased hospitalization In rare cases allergic reactions, vas-

rates and excess mortality in high culitis or thrombocytopenia may
risk groups, particularly the elderly occur; Guillain-Barré syndrome is
and the chronically ill reported in about 1 in 1 million vac-
cinees
Measles 4% of patients develop pneumo- In rare cases, allergic reactions or

nia, and 1 in 1000 encephalitis; anaphylaxis may occur; in case of
CFR of measles encephalitis, 10%; fever young children may develop
and 40% have permanent brain febrile seizures
damage; rarely subacute sclerosing
panencephalitis (SSPE) occurs
Meningococcal Meningitis, septicemia; CFR Following conjugated MenC vaccine

disease, invasive ~10%; ~10–20% permanent only local and systemic reactions
damage (CNS, physical sequelae reported
range from necrosis to amputa-
tion following extensive gangrene,
bone lesions and skeletal growth
disturbances
Mumps 4% of patients develop meningitis, Allergic reactions are rare; in case

occasionally mumps causes of fever young children may develop
deafness; 1 in 5 males past puberty febrile seizures; meningitis may
develop inflammation of testes occur but has not reported follow-
ing mumps vaccines based on the
‘Jeryl Lynn’ and ‘Jeryl Lynn’-derived
strains
Pertussis CFR (due to pneumonia or In rare cases, allergic reactions, ana-

encephalopathy) in infants about phylaxis, hypotonic-hyporesponsive
1% following DTP may occur; following
whole-cell pertussis vaccine, rare
cases of encephalopathy occurred
Pneumococcal Meningitis, septicemia, bacteremia; Following conjugated pneumococcal

disease in CFR ~5%; ~ 20% permanent vaccine only local and systemic reac-
children, invasive damage (CNS, hearing loss, tions reported, allergic reactions are
learning disabilities) very rare
Table 1 (continued)
Disease Severity of disease Complications following

immunization
Poliomyelitis CFR 5%; 1 in 2 survivors is per- Vaccine-associated paralytic polio-

manently paralyzed myelitis may occur following OPV;
IPV has an excellent safety profile
Rubella 50% of adolescents and adults In case of fever young children may
develop arthritis/arthralgia; develop febrile seizures, allergic
very rarely thrombocytopenia reactions are very rare; arthritis/
or encephalitis; 9 of 10 babies arthralgia may rarely occur in (pref-
infected during the first 10 weeks erably female) adolescents/adults
of pregnancy will develop major
congenital abnormalities (CRS)
Tetanus CFR: 10%, much higher in older In rare cases allergic reactions, ana-
individuals phylaxis and peripheral neuritis may
occur
vaccine, and delays in implementing control measures. Since 1995, aggres-

sive 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].
Natural immunity is better than vaccine-induced immunity.
Counter-argument
While vaccine-induced immunity may diminish with time, ‘natural’ immu-

nity, 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).
Many vaccines are not indicated because they are often useless and many
people get the disease despite being immunized.
Counter-argument
First, no vaccine is 100% effective. For reasons related to the individual,

not all immunized individuals develop immunity. Most vaccines are effec-
tive 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 under-
stood 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 cir-
cumstances 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 individu-
als is usually much less severe than in those who were not immunized.
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 pos-
sible 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 differ-
ent organisms. In the face of these normal events, it seems unlikely that the
number of separate antigens contained in childhood vaccines would rep-
resent an appreciable added burden on the immune system that would be
Table 2. Content of immunogenic proteins and polysaccharides in vaccines – 1900 vs 2000
1900 1960s 2000

Vaccine Proteins Vaccine Proteins Vaccine Proteins/
polysaccharides
Smallpox ~200 Smallpox ~200 Measles-mumps- 24

rubella
Diphtheria- 2 Diphtheria-tetanus 2
tetanus
Pertussis ~3000 Pertussis (acellular) 2–5
(whole-cell)
Poliomyelitis 15
H. influenzae type b 2
Hepatitis B 1
Total ~200 Total ~3200 Total Western 46-49
European countries
Varicella 69
Plus additional vaccines included in the Pneumococcal 8
US vaccination schedule conjugate vaccine
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 vac-
cine 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 evi-
dence 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 combi-
nation 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 administra-
tion 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
office visits, which saves parents both time and money and may be less trau-
matic for the child [5–8].
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 immu-
nization, local and/or systemic reactions may develop such as redness, swell-
ing 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 protec-
tive 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].
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?
Asthma
There is no evidence that vaccination causes or worsens asthma. It is espe-

cially 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 thimero-
sal-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 com-
mittees 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 includ-
ing 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 influ-

enzae 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
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 sig-
nificantly 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 immu-
nization 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 relation-

ship is simply an incidental association, as SIDS tends to occur in babies of
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 addi-
tion, 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 contraindica-
tion to the use of standard vaccines. With regard to live vaccines, only small-
pox 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 immuniza-
tion during pregnancy is not an indication for termination. To date, con-
genital 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
– 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 pre-
gnancy [8, 17].
Immunization of pre-term babies
Despite their immunological immaturity, pre-term babies should be immu-

nized 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 pre-
mature 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 chrono-
logical 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 moth-
ers 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, begin-
ning when the infant is age 1 month. The optimal timing of the first dose
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 chrono-
logical 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, congeni-
tal 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 pre-
dispose 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-pre-
ventable 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 immuni-
zation 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 cer-
tain 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 lym-
phocyte) immunodeficiencies, cell-mediated (T lymphocyte) immunodefi-
ciencies, disorders of the complement and phagocytic function. Secondary
disorders are acquired and occur in individuals with HIV infection, asplenia,
malignant neoplasms, transplantation(s) or immunosuppressive or radiation

therapy [18].
The immunization of individuals with impaired immune systems pres-
ents several problems. Firstly, the immune response to vaccines may be
inadequate and, secondly, there is a risk that some live vaccines may them-
selves cause progressive infection. Degrees of immunodeficiency vary from
insignificant to profound, and this should be taken into account when con-
sidering 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, effec-
tiveness and safety of vaccines administrated to the immunocompromised,
whereas experience with immunization in persons with other specific dis-
orders 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 immuniza-
tion, the number of vaccine doses received, the interval between immuniza-
tion and assay, the type of antibody assay used and the degree of immuno-
suppression. In general, seroconversion rates and geometric mean titers are
lower in HIV-infected children than in uninfected children and infected chil-
dren 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 admin-
istered 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 immuniza-
tion 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.
Table 3. Immunogenicity and effectiveness of immunization in HIV-infected children (adapted

from [19])
Vaccine Sero- Geometric Antibody per- Booster Effectiveness

conversion mean titer sistence response Studie Field
rate (GMT)
Diphtheria- 40–100% Lower than More rapid

tetanus uninfected decline than
toxoid children in uninfected
children
Pertussis Lower than
(wP/aP) in uninfect-
ed children
Hepatitis B 25–50% More rapid No effect no long-
decline than in children term fol-
in uninfected after extra low-up
children or higher studies
doses
Hib 37–86% Lower than More rapid Rapid
conjugated uninfected decline than antibody
children in uninfected increase
children due to
immuno-
logical
memory
Meningococcal No data available
Pneumo- Better antibody response In Ugandan adults
coccal to conjugated vaccine 23-valent PS vaccine
than that of 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 vac- > 90% after No studies; polio
cine 3 doses eliminated from
several high HIV
prevalence countries
Measles 17–100%, More rapid Generally
median decline than poor
value 60% in uninfected
children
Yellow fever Much lower
than in
uninfected
children
– Immunodeficient travelers should not be given live oral cholera or typho-

id 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
should obtain exemption certificates of immunization ratified by health

authorities and immigration departments where international immunizati-
on 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 vari-
cella-zoster vaccines to prevent them from infecting their immunodefi-
cient 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 pneu-
mococcal conjugate vaccine or 23-valent pneumococcal polysaccharide
vaccine, depending on their age; although the immune response to pneu-
mococcal 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 associ-
ated 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 recom-
mended vaccines as early in life as possible, according to the nationally
Table 4. WHO/UNICEF recommendations for immunization of HIV-infected children and

women of childbearing age [20]
Vaccine Asymptomatic Symptomatic Optimal timing of

HIV infection HIV infection immunization
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 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 immuniza-
tion 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 recommen-
dations 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 polysaccha-

ride vaccines. There is clear evidence that these patients have an increased
risk of complications following natural influenza and pneumococcal dis-
ease, whereas the administration of these vaccines is not associated with an
increased risk of exacerbations of MS [8, 17].
Table 5. Contraindications for childhood vaccines – ACIP [6, 7]
Vaccines Immunize?
Immunodeficiency
Family history OPV See Note 1

Note 1: Do not give OPV to a member of a household with a Varicella See Note 2
family history of immunodeficiency until the immune status All others Yes
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
In household contact OPV No
All others Yes
In recipient OPV No
(hematological and solid tumors, congenital immunodeficiency, MMR No
long-term immunosuppressive therapy, including steroids) Varicella See Note 3
Note 3: Varicella vaccine should not be administered to per- All others Yes
sons 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 lym-
phoblastic leukemia (ALL).
HIV infection
In recipient (asymptomatic) OPV No
Note 4: Varicella vaccination should be considered for Varicella See Note 4
asymptomatic or mildly symptomatic HIV infected children MMR See Note 5
with age-specific T cell percentages of 25% or higher All others Yes
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
In recipient (symptomatic) OPV No
Note 6: MMR vaccination should be considered for all Varicella See Note 4
symptomatic HIV-infected persons who do not have evidence MMR See Note 6
of severe immunosuppression or of measles immunity All others Yes
In household contact OPV No
All others Yes
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-pre-
ventable diseases. When hepatitis B or any other vaccine is indicated for a
patient with a bleeding disorder or a person receiving anticoagulant therapy,
the vaccine could be administered intramuscularly if, in the opinion of a

physician familiar with the patient’s bleeding risk, the vaccine can be admin-
istered 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 recom-

mended 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-
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 con-
traindication. 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 spe-

cifically.
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 tempe-
rature < 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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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 coun-
tries. 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 neonato-
rum 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 opposi-
tion 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 elo-
quence 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 introduc-
ing this method, Credé saved or improved the eyesight of millions of people – a signifi-
cant 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 infec-
tions is still in the future, because its full range of activity remains to be
elucidated.”
Q.L. Feng et al., 2000 [1]
96 Axel Schmidt
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 neona-
torum. 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 obstet-
rics by introducing a new and safer method for the delivery of the placenta
(“Credé’scher Handgriff”/Credé’s method) [16, 17]. Credé was a consistent-
ly 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 depart-
ment became very prestigious. He personally focused on obstetrics being
convinced that improvements in obstetrics are a key parameter to reduc-
ing 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
Credé’s eye prophylaxis… 97
Figure 1. Carl Siegmund Franz Credé (1819–1892)
“Senckenberg Preis/Senckenberg Award” due to his outstanding achieve-

ments 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.
98 Axel Schmidt
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 sum-
marized 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 obste-
tricians and midwives. I shall confine my remarks exclusively to the practical
question of prophylaxis.
(…) My request for further testing of the prophylaxis I am recommend-
ing 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 inflamma-
tory 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-
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 infec-
tious 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 chil-
dren 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 imme-
diately 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 moder-
ate inflammation of the conjunctiva of the left eye, without swelling of the
100 Axel Schmidt
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 conjunc-
tiva 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 dis-
infection 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 dis-
eases 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 dis-
ease observed in this maternity hospital in recent years. (…).
Year Number of Number of cases of Percentage

births inflammatory eye disease
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 200 1* 0.6
8 December)
*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
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 secre-
tion 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 proce-
dure 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 break-
ing with a prestigious medical privilege in obstetrics by apparently by-pass-
ing 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 neona-
torum 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 experi-
ences 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 con-
sidered 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-
102 Axel Schmidt
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 caus-
ative 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 ophthal-
mia 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.
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 reflec-
tion 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 dermatol-
ogy, 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 gon-
orrhea”) [33]. In this paper he was the first person to describe that a very typi-
cal 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
104 Axel Schmidt
in genital smears of patients suffering from symptomatic gonorrhea. He

mostly observed this “Micrococcus” topologically associated with inflam-
matory 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 recommenda-
tion for a scientific proof, based on something like the later-discussed, com-
municated 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 scien-
tists 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 repeat-
edly 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 exag-
gerated 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
community at that time, which also put Neisser under an extreme pressure
concerning the validity of his insight that the “Micrococcus” was the caus-
ative 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 prophy-
laxis 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 infec-
tion 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 ophthal-
mia neonatorum from approximately 10% to 0%. Adverse effects of chemi-
cal 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% solu-
tion 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 microscopi-
cally [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
106 Axel Schmidt
speed up and to enhance the pressure for an establishment of his prophy-

lactic 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 scien-
tific 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 hos-
pitalized 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 physi-
cians/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 neonato-
rum 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-
cantly. Despite cases of ophthalmia neonatorum due to Neisseria gonor-

rhoeae 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 conjunc-
tivitis 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 oph-
thalmia 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 medi-
cine and specialized as a surgeon in Dresden, Germany, thereafter.
Scientifically, Credé followed the steps of his father in so far as perform-
ing research activities on silver in colloidal form, which he introduced into
medical practice in 1897. This was possible by collaboration with the com-
pany “Chemische Fabrik von Heyden”, and led to the development of the
“Collargolum Credé” (“Collargol”) [73, 74] for systemic, parenteral thera-
py. 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 found-
ed the Archiv für Gynäkologie, of which almost every volume contained a
108 Axel Schmidt
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 fur-
ther 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 prophy-
laxis). 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 aus-
schließlich 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.
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 lästi-
gen 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 geburtshülfli-
chen Polikliniken thätig sind, und, gleich mir, häufige Erkrankungen zu
beobachten haben.
Wohl von den meisten Geburtshelfern wird meine Ansicht geth-
eilt 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 mit-
tels 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 erkrank-
ten 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 ausge-
dehnte 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 kom-
menden Schwangeren und Gebärenden reinigende Ausspülungen der Vagina
110 Axel Schmidt
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 nitri-
cum (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 bli-
eben 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 BEHAN-
DELTE 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 mässi-
gen 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.
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 hof-
fentlich 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 eigenthümli-
che, von vielen, auch grösseren Städten abweichende Verhältnisse besonders
in Betracht gezogen werden müssen.
Jahr Zahl der Zahl der Procentsatz

Geburten Augenerkrankungen
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 200 1* 0,6
bis 8 Decbr.)
*Es ist dies der Fall, bei welchem die Augen nicht desinficirt wurden; also
eigentlich sind 0,0% zu verzeichnen.
112 Axel Schmidt
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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 mal-
nutrition is not uncommon in wealthy industrialized societies due to poverty, lack of edu-
cation, 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 dis-
eases 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 malnutri-
tion 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 activa-
tion of the acute phase response that is more difficult to resolve in malnutri-
118 Susanna Cunningham-Rundles and Deborah Ho Lin
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 indus-
trialized 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 cir-
cumstances and have less access to health care. Such children are especially
vulnerable to the effects of nutrient deficiency. For infants and toddlers, ade-
quate 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 dispro-
portionate effect on development, immunity, and susceptibility to infection.
For children with secondary malnutrition, specific macronutrient and micro-
nutrient 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 devel-
oped 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 malnutri-
tion 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.
Malnutrition and infection in industrialized countries 119
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, mal-

absorption, 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 rel-
evant genetic mechanisms can be helpful in approaching the clinical mani-
festations. 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 under-
lying 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
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 com-
mon 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 constitu-
ent 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 fre-
quently 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 increas-
ingly common in children, raising concerns about type 1 diabetes, cardiovas-
cular 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 cyto-
kine-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 infec-
tions are enhanced in obesity [26]. A role for fetal programming that links
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 malnutri-
tion. 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 infec-
tion. 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), lyso-
zymes, and defensins. Loss of barrier function due to malnutrition pro-
motes 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 IL-
10 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 poten-
tially impaired in malnutrition through reduced production of key media-
tors 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 chil-
dren 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].
The development of immune response in the neonate occurs in the con-

text 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 1,25-
dihydroxyvitamin 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 secre-
tion 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 stimula-
tory effect on postnatal development of immune responses, and metabolic
processes central to nutrition, and the development of mucosal (oral) toler-
ance. 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 imbal-
ance, 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 insulin-
like growth factor-1 (IGF-1) in marasmus and kwashiorkor [50] may com-
promise 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-
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 com-
mon. The T cell functional defects resemble those of congenital thymic
aplasia as in Di George syndrome [50, 56]. The selective effect of malnu-
trition on the thymus gland is due to apoptosis-induced thymocyte deple-
tion, 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 mal-
nutrition. 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 nega-
tive tuberculin skin test, and have an increased risk for developing dissemi-
nated 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 some-
what 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].
Specific micronutrient deficiencies
Micronutrients have a major impact on immune response, through antioxi-

dant 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 cutane-
ous 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 includ-
ing fetal alcohol syndrome, sickle cell disease due to hyperzincuria, celiac
disease, enteritis and diarrhea. Zinc deficiency due to loss occurs in epider-
molysis 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, glu-
tathione 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 func-
tion [77]. Selenium is emerging as a critical micronutrient in host defense
against viral infection since selenium deficiency is associated with progres-
sion in HIV disease and in viral shedding [78, 79].
Vitamin A has long been appreciated as a significant factor in the sever-
ity of infection such as measles, rotavirus diarrhea, and HIV in the mal-
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 supplementa-
tion in vivo led to improved immune function in vitro.
Vitamin C is a free radical scavenger that serves as an important antioxi-
dant. Vitamin C concentrations in the plasma and leukocytes decline during
infections and stress. Supplementation with antioxidant vitamins includ-
ing 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 sever-
ity 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 tod-
dlers [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 sug-
gests 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
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 prema-
turity. 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 immuni-
ty, 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 comprehen-
sive 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].
Table 2. Mechanisms of nutrient action
Nutrient Target Effect Mechanism
Zinc T, B, NK cells, Deficiency increases Deficiency causes cytokine shifts,

GALT (gas- infection, impairs lym- activation of the HPA axis, T cell
trointestinal- phopoiesis apoptosis
associated
lymphoid
tissue)
Antioxidant Monocytes, Deficiency causes oxi- Anti-inflammatory effects
vitamins, C, E, T cells, dative stress, increases Decreases PGE2 production
and carotenoids neutrophils, reactive oxygen species, Increases IFN-a, IL-4 production
NK cells DNA damage Enhances phagocytosis
Vitamin A Monocytes, Deficiency leads to Decreased Th-2 cytokine and IgA
neutrophils, infections, morbidity, response, NK, B cells, Decreased
T, B, NK cells, mortality cilia, microvilli, mucin, abnormal
GALT keratin
Glutamine T cells, Increased thymus Increases lymphocyte prolif-
monocytes, weight, T cell response, eration, cytokine response, phago-
GALT IgA, autoimmunity? cytosis, MHC class II expression
Arginine T cells. Increased immune Increases thymocyte proliferation,
monocytes, response thymic modulates TNF-_ production,
GALT weight, phagocytosis increases production of ROS.
and killing phagocyte function
Saturated/ T cells, NK, NK, T cells response, Regulation of adhesion
unsaturated monocytes phagocytosis molecules, membrane fluidity
fatty acid ratio
Essential fatty Monocytes, Cellular immunity Deficiency or excess: decreases
acids n-6, n-3 neutrophils chemotaxis, phagocytosis, NK
T cells activity
Eicosapentanoic Monocytes, Anti-inflammatory High levels decrease IL-2, IFN-a,
acid T, and NK ICAM-1, superoxide production
cells
Decosahexanoic Monocytes, Anti-inflammatory Signal transduction MHC class II
acid T cells
Gastrointestinal disease
Short bowel syndrome (congenital atresia or surgical resection), and inflam-

matory 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-
infections of the GI tract, which provokes inflammation, motility abnormali-

ties 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 tol-
erance 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 caus-
ative. 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 intes-
tinal 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 coun-
tries, 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 intes-

tinal 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-
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 pancre-
atic 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-regu-
late 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 inges-

tion 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 aller-
gies 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
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 sys-
temic pro-allergenic IL-4 responses on intestinal antigen contact [117].
Lactose intolerance is most common cause of carbohydrate malabsorp-
tion, 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 child-
hood 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 susceptibil-
ity 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].
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, expendi-
ture 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 path-
ways 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 polysac-
charides, 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 defi-
ciency 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 ill-
nesses 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 infec-
tious 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 met-
abolic 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
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 defi-
ciency [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 feed-
ing 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 per-
sons. Micronutrient impairment is causally associated with the course of
HIV infection and immune dysfunction. This occurs due to malabsorp-
tion, 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 breastfeed-
ing mothers with B, C, and E reduces child mortality and HIV-1 transmis-
sion 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 vac-
cinated 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 dif-
ferent 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 infec-
tion. 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 myco-
bacterial colonization and lesion formation compared to wild-type mouse.
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, regard-
less 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 NO-
independent 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 bacil-
lary 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 hel-
minths 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 ele-
ment 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 imbal-
ance 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 con-
sequences of any major parasite. It infects the placenta and compromises
blood flow to the fetus, causing low birth weight. It also causes PCM in preg-
nant 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, vita-
min 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].
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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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 devel-
opment, 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, psy-
chosocial 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 gener-
ally larger than for school-age children. Effects are seen in all dimensions of school readi-
ness – 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 prior-
ity 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 chap-
ter 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.
146 Matthew Jukes
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 esti-
mated 600 million preschool children worldwide [3] and they have several-
fold 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 develop-
ing 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 dis-
eases, 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 develop-
ment persist as cognitive impairments or emotional problems throughout
the school-age years.
Better education through improved health and nutrition 147
School readiness refers to a range of competencies that preschool chil-

dren 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 develop-
ment, 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 nutri-
ents 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 devel-
opmental 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 developmen-
tal 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 undernu-
trition 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 coun-
tries steps have been taken to prevent malnutrition in children by begin-
ning 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
148 Matthew Jukes
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 nutri-
tional supplementation and maternal education program worked synergisti-
cally: 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 find-
ing 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 implica-
tions 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 nutri-
tional 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 supple-
ments, 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 sup-
plementation 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 nour-
ished children [13].
Long-term effects on cognition
The above studies show that undernutrition leads to impaired school readi-
ness 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 long-
term effects.
Beginning with the most profound nutritional insults, severe malnutri-
tion 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 substan-
tially 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 under-
nourished 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
150 Matthew Jukes
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 abili-
ties 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 child-
hood. 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 chil-
dren, 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 achieve-
ment [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
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 pro-
gram of nutritional supplementation and/or psychosocial stimulation, last-
ing 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 informa-
tion 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 develop-
ment. 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
152 Matthew Jukes
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 begin-
ning 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 supplemen-
tation 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 nutri-
tional 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 evi-

dence on mental and motor development. This is due in part to the difficulty
in measuring development in this domain and the time-consuming observa-
tion 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, under-
nourished 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 emo-
tional behavior can be improved by interventions. The program in Mexico
[9], which gave nutritional supplements from shortly after birth and through-
out 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
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 poor-
er verbal comprehension scores at 5 years [24]. However, poor socio-emo-
tional 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 pro-
gram 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 supplementa-
tion as part of this program.
It is not clear from this study how such long-term effects arose. It is pos-
sible that they represent the continuation of social and emotional benefits
of the psychosocial intervention, which were already evident in early child-
hood. 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 find-
ings 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
154 Matthew Jukes
such as this is a reminder of the difficulty in interpreting findings related to

timing effects of nutritional deficiencies on cognitive development. At pres-
ent, 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 cru-
cial for development so is the active engagement of others in their environ-
ment. 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 malnutri-
tion [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 mother-
child interactions [32].
These findings have clear implications for children’s development. We
have seen that psychosocial stimulation is perhaps the most important fac-
tor 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
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 devel-
opment 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 develop-
ment. 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 differ-
ences [38]. In general, the evidence is not conclusive but is strongly sugges-
tive 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].
156 Matthew Jukes
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 substan-
tial 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, chil-
dren 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 supple-
mentation can improve the development of children under 2 years if sus-
tained 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 per-
formed 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 supplemen-
tation, 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 supplementa-
tion improved preschoolers’ language outcomes by 0.14 SD.
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 settle-
ments 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 long-
term 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 treat-
ment. 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 abili-
ties 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-econom-
ic 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 back-
ground 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.
158 Matthew Jukes
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 sul-
fate) 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 emo-
tional development. In Costa Rica [40], infants with iron-deficiency anemia
were found to maintain closer contact with caregivers; to show less plea-
sure and delight; to be more wary, hesitant, and easily tired; to make fewer
attempts at test items; to be less attentive to instructions and demonstra-
tions; and to be less playful. In addition, adults were found to behave differ-
ently 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 for-
merly anemic group was more likely to have a number of behavioral prob-
lems. 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
hypothyroidism (a deficiency in maternal thyroid activity), which affects

development of the fetal brain during the third trimester, and hypothyroid-
ism 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 impair-
ments. Nearly 50 million people suffer from iodine-deficiency disorder-
related 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 find-
ings of impaired cognitive function in adults and children living in iodine-
deficient 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 met-
als in their sweat and hair [58]. Animal studies show that zinc deficiency in
offspring causes impaired learning, which can be corrected by zinc supple-
mentation [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-
160 Matthew Jukes
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 hos-
pitalization. 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 impair-
ment [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 preven-
tion 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 per-
formance at age 17–21 years. For those who had received the longest pro-
tection 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.
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 associ-
ated 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
162 Matthew Jukes
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 non-
encephalopathic 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 edu-
cation. 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 mea-
sure 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 func-
tion [80]. When infected children are given deworming treatment, immedi-
ate educational and cognitive benefits are apparent only for children with
heavy worm burdens or with nutritional deficits in addition to worm infec-
tions [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 infec-
tion with whipworm (Trichuris trichiura). However, for most children, treat-
ment 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 cogni-
tive 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
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 demon-
strated cognitive improvements in preschool children following combined
treatment for worm infections and iron-deficiency anemia. However, nei-
ther study was able to disentangle the effects of the two treatments.
Other parasitic infections
Infection with Giardia lamblia has been associated with mental develop-
ment. Giardia is a protozoan parasite that is ingested and inhabits the gas-
trointestinal 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 environ-
ment [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.
164 Matthew Jukes
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 men-
ingitis 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 edu-
cation programs during early childhood, moving to school health programs
during the school-age years. However, the focus of this report is on interven-
tions that can be delivered through ECD programs.
Interventions: What works?
Table 1 summarizes the impact of health interventions on cognitive devel-

opment in early childhood. Only studies giving strong evidence from experi-
mental interventions are included. Such evidence is available for four types
of intervention: iron supplementation, iron supplementation and deworm-
ing, 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.
Table 1. Impact of health interventions on development during early childhood
Study Country Intervention Age Sample Effect Outcomes

characteristics size
Jukes et al. India Iron (30 d) + 2–6 yrs ECD pupils 0.18 SD Attention
[48] deworming
Seshadri and India Iron (60 d) 5–8 yrs Anemic vs. +ve IQ
Golpadas non-anemic
no. 1 [46]
Seshadri and India Iron (60 d) 5–6 yrs Anemic vs. 0.33 SD Verbal IQ
Golpadas non-anemic 0.67 SD Performance
no. 2 [46] boys IQ
Soewondo Indonesia Iron (56 d) 4 yrs Anemic vs. +ve Learning task
et al. [45] non-anemic No 3 cognitive
effect tests
Stoltzfus Zanzibar 12 mo iron + 6–59 Community 0.14 SD Language
et al. [88] deworming mo
McKay et al. Columbia Nutrition + 84 mo Malnourished 0.80 SD Cognitive
[12] education ability
from 42 mo
Grantham- Jamaica Psychosocial 9–24 Malnourished 0.67 SD Mental devel-
McGregor stimulation mo opment
et al. [13]
Grantham- Jamaica Nutritional 9–24 Malnourished 0.79 SD Mental devel-
McGregor supplemen- mo opment
et al. [13] tation
Vermeersch Kenya School 4–6 yrs ECD pupils 0.4 SD Educational
and Kremer feeding (for sub achievement
[11] 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 inter-
ventions that have worked. Rather, it is a summary of all experimental inter-
ventions 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 remark-
ably 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 inter-
ventions and assessed their cognitive impact in the long term. Three of the
studies tracked participants to adolescence and found that improvements
166 Matthew Jukes
Table 2. Long-term impact of health interventions in early childhood on cognitive and edu-
cational outcomes
Study Country Intervention Age Sample Effect size Outcomes

character-
istics
Grantham- Jamaica Maternal 14 Severely 0.68 IQ

McGregor education mal-
et al. [14] nourished
Walker Jamaica Stimulation 11–12 Stunted 0.38 IQ
et al. [17]
Chang et al. No effect Education
[25] tests
Pollitt et al. Indonesia Nutritional 8 Initially > +ve Working
[21] supplements 18 months memory
Jukes et al. Gambia Malaria 14–19 Community 0.25–0.4 Cognitive
[66] prevention cohort 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 child-
hood: 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 addi-
tional cases of mental retardation (IQ< 70).
All three conditions are easily preventable. The following section out-
lines current best practice for addressing these conditions.
Undernutrition
The most obvious way in which ECD programs can address chronic under-
nutrition 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
Table 3. Estimated global impact of malaria, anemia and stunting on cognitive development
Devel- Total Effect size Equivalent loss in Additional cases of

oping cases IQ points per child mental retardation
country (mil- (millions)2
pre- lions)1
valence Lower Upper Lower Upper Lower Upper
estimate estimate estimate estimate estimate estimate
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 on a developing country population of under-5s of 548 million [34].

2Using 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 signifi-
cant impact on preschool attendance and achievement [11].
Beyond school feeding, current recommendations are for counseling
mothers and caretakers to improve feeding practices and improved man-
agement 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 con-
sequences 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-
168 Matthew Jukes
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 impor-
tant than dealing with its consequences for development, as sure as preven-
tion 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 cogni-
tive 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 litera-
ture 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 interven-
tion 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 supple-
mentation 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.
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 varia-
tions in nutritional intake or in the transmission of disease, or where com-
munities 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 program-
matic 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 preced-
ing review we saw examples where lack of breast feeding, or otitis media
infection led to cognitive impairments only for children of the least edu-
cated 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 edu-
cational 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 func-
tion on the same day to a greater extent for children with chronic malnutri-
tion [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 interven-
tions have the largest impacts for the preschool age group, but are also
170 Matthew Jukes
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 nutri-
tion 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 pro-
grams 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 valu-
able comments on earlier drafts of this chapter.
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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 resi-
dent 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 micro-
flora (“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 dis-
tribution 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
178 Shigenobu Kimura and Yuko Ohara-Nemoto
tooth and crevicular epithelial surfaces) [2]. Among these, the latter two
habitats are important for etiology of dental caries and periodontal dis-
eases, 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 epi-
thelial 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 bac-
teria 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 periodon-
tal 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 car-
bohydrates, 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 indus-
trialized 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 coloniza-
tion (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 fermenta-
tion 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.
Early childhood caries and childhood periodontal diseases 179
Figure 1. Structure of tooth and periodontal tissues. The structure of tooth and periodontal tis-
sues 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 cario-
genicity 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 microflo-
ra. 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 forma-
tion 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

Table 1. Oral streptococcal phylogenetic groups and species
Mutans streptococci
Phylogenetic group Species Serotypes Natural habitat
mutans group S. mutans c/e/f human

S. sobrinus d/g human
S. cricetus a hamster
S. rattus b rat
S. dewnei h monkey
S. ferus c rat
S. macacae c 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 irre-
versible. 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 synthe-
size the adhesive water-insoluble glucan, producing lactate by homolactic
fermentation, which accounts for the augmented virulence of these bac-
teria in hosts that frequently consume high-sucrose diets. Epidemiological
observations support the sucrose-caries-mutans streptococci association; an
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 car-
ies among patients who were urged to reduce their frequency and level of
sucrose consumption [10]. Thus, mutans streptococci are the primary cario-
genic 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 demineral-
ization of enamel. The model of ‘sucrose-caries-mutans streptococci associa-
tion’ 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 strepto-

cocci 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 regard-
ing relationship between pH and growth of plaque bacteria in a mixed cul-
ture 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 predomi-
nance 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 associa-
tion, 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 rele-
vant, and that frequent metabolism of fermentable carbohydrates in plaque
induces the critical pH condition, which leads to the growth of acid-tolerat-
ing 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 strepto-

cocci 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 includ-
ing the colonization of non-mutans streptococci in plaque. Although the
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-sali-
varius 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 coloniza-
tion 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, spe-
cies-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 correla-
tion 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 etio-
logical 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 develop-
ment of cariogenic and non-cariogenic microflora can be monitored. The
cross-sectional survey using species-specific PCR assays indicated that
many oral streptococcal species including mutans streptococci can colo-

nize 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 pri-
mary 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 antimicro-
bial agents, glucan-hydrolyzing enzymes, and vaccination and passive immu-
nization 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 par-
tially 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 carbohy-
drates. 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 elimina-
tion cariogenic mutans streptococci in the plaque [30].
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 effec-
tive 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 organ-
isms entering the blood stream can potentially attach and grow, and then
cause the infective endocarditis. Furthermore, the glucan-synthesizing abil-
ity 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-promot-
ing 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 periodonti-

tis, 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, gin-
givitis is characterized by inflammation confined to the gingiva, and peri-
odontitis 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
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 child-
hood periodontal diseases.
The following sections address: (i) clinical and etiological aspects of
childhood periodontal diseases, (ii) pathogenic bacteria, (iii) onset of peri-
odontal diseases – host-parasite relationship, and (iv) transmission of peri-
odontal bacteria.
Clinical and etiological aspects of childhood periodontal diseases
Plaque-induced gingivitis without other local contributing factors is a bac-

terially elicited inflammation of the marginal gingiva, and is the most com-
mon 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 accumulat-
ed supragingival plaque, and that gingivitis was eliminated by the removal
of the bacterial plaque. The early finding of a strong cause-and-effect rela-
tionship 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 gin-
givitis 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 demon-
strates 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 accumu-
lation 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, steroid-
addicted 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.
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 dif-
ference 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 periodon-
titis in children for some reason.
The term ‘early-onset periodontitis’ has now been renamed to ‘aggres-
sive 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 etio-
logical 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 catego-
rized 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 inci-
sors 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 peri-
odontitis 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.
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 peri-
odontitis. Quantitative analyses using a flow cytometer revealed that about
50% of the patients with localized and generalized aggressive periodon-
titis, but not chronic periodontitis (formerly adult periodontitis), exhib-
ited depression of phagocytic function of peripheral blood PMNL (Tab. 2)
[52]. The depressed phagocytic responses could be due to cell-associated
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 periodon-

tal treatments, suggesting that the depression of PMNL phagocytosis in both
types of aggressive periodontitis may not be a transient phenomenon associ-
ated 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 find-
ings 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 mani-
festations 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 devel-
opment and prevalence of anaerobic culture methods were required to
isolate and identify the periodontopathic bacteria. Clinical and basic
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 dis-
eases. These include Gram-negative obligatory anaerobic rods: P. gingivalis,
Prevotella intermedia, Prevotella nigrescens and Tannerella forsythensis (for-
merly 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 periodon-
titis in adults and generalized (but not localized) aggressive periodontitis
[55]. This microorganism possesses several virulence factors for periodon-
topathogenicity, including fimbriae, proteolytic enzymes and lipopolysac-
charide (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, fibro-
nectin 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 immuno-
logical activities in the infectious process [60].
Large amounts of the proteolytic enzymes of gingipains and collage-
nase 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 lev-
els 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 dem-
onstrates that not all A. actinomycetemcomitans can produce leukotoxin,
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 chil-
dren; 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 pro-
posed as possible periodontophatic pathogens in some types of periodonti-
tis, 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-
ice (sulcus) is a groove between the tooth surface and the sulcular epithe-
lium 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 crev-
ices 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 avail-
able on periodontopathic bacterial infection in their plaque. Our recent
longitudinal investigations by means of PCR method using the periodon-
topathic bacterial species-specific primers for 16S rRNA genes indicated
that seven out of ten bacteria, i.e., C. rectus, E. corrodens, A. actinomy-
cetemcomitans, 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,
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 signifi-
cant, number of the periodontopathic bacteria are detected from periodon-
tally 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 bac-
teria 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 malfunc-
tion of PMNL due to premature senescence. Our microbial observation in
children with Down’s syndrome indicated that the seven putative periodon-
topathic 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 chil-
dren [70]. Furthermore, the cluster group characterized by the additional
infections with P. gingivalis, T. denticola and P. intermedia to the seven puta-
tive 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 demon-
strated that children whose parents were colonized by the BANA-positive
periodontpathic species including P. gingivalis, T. denticola, and T. forsythen-
sis 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 transmis-
sion 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 (parents-
to-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
members was considerable, it was suggested that P. gingivalis commonly

colonizes in an established oral microbiota. According to these observa-
tions, 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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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 menin-
gitis remains an important cause of high morbidity and mortality in the pediatric popula-
tion with no significant improvement in the outcome in recent years. Further ameliora-
tion 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 develop-
ments 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 blood-
cerebrospinal 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 neuro-
psychological sequelae. To improve present strategies and to develop new
options in diagnostic, prevention and therapy, knowledge and understand-
ing of pathogenesis and pathophysiology of bacterial meningitis is of utmost
importance. It is well established that most cases of bacterial meningitis
200 Rüdiger Adam et al.
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-cerebrospi-
nal 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 con-
tinues to be an important cause of morbidity and mortality in children. The
causative organism varies with age, immune function and immunization sta-
tus. 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 antibi-
otics, 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.
Role of the blood-brain barrier and blood-CSF barrier… 201
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 run-
ning “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 gastrointesti-

nal tract are colonized by bacterial pathogens. The bacteria must attach to
the mucosal epithelium and resist clearance by mechanical and immuno-
logical mechanisms. All meningeal bacterial pathogens seem to express a
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 patho-
gens 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 den-
sity 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 opsono-
phagocytosis 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 nota-
bly ineffective in eliminating invading bacterial pathogens. Bacterial multi-
plication 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` (IL-
1`) 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
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, lipopoly-
saccharide (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 del-

eterious events leads to maximal leptomeningeal inflammation. A substan-
tial 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 men-
ingitis [17] (Fig. 2).
The blood-CNS barriers
The homeostasis in the brain is an unconditional prerequisite for correct neu-

ron 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
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 proper-
ties 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 menin-

gitis 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 cir-

culation and the brain, protecting it and maintaining its homeostasis. Its
anatomical base constitutes a complex system of brain microvascular endo-
thelial cells (BMECs) (Fig. 3A). These cells are ensheathed by astrocytic
outgrowths, which are referred to as astrocytic end-feet, necessary to main-
tain 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 pino-
cytotic capacity and lack endothelial fenestrations [12].
The BBB eliminates (toxic) substances from the endothelial compart-
ment and supplies the brain with nutrients and other (endogenous) com-
pounds, 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.
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 proc-
esses extensively abut the outside surface of the endothelium. The darkened area is the inter-
stitial 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 mem-
brane 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 per-
fusion 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 epithe-
lial 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
actively regulate the concentration of molecules within the CSF by numer-

ous 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 CNS-
to-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 rec-
ognized as important sites for blood-brain communication as neurosecreto-
ry 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 interme-
diate 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 initi-
ate 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 inva-
sion to be drawn. It has to be kept in mind, though, that multiple routes into
the CNS compartment may be used simultaneously.
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, sev-
eral 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 inci-
dence 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 enter-
ing 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].
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 mod-
els are available. One reason for this emphasis on the BBB is its assumed
preponderance regarding surface area in comparison with the other blood-
CNS 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 microvas-
culature 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 tak-
ing 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 exami-
nations 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 cere-
bral 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 sig-
nificant inflammatory infiltrate predominantly composed of polymorpho-
nuclear 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 menin-
geal vessels and the perivascular spaces within the cerebral cortex [35].
Histological examination of brain tissue from a fatal case of meningococ-
cal disease revealed attachment of N. meningitidis on the CP and microvas-
cular 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 blood-
CNS barriers, there remains a distinct paucity of studies clearly pointing
towards the cerebral vasculature as the primary site of CNS invasion for
certain pathogens. Probably due to this dilemma, some authors indepen-

dently 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 recep-
tors for various meningeal pathogens are present on cells of cerebral capil-
laries potentially mediating attachment or penetration of the BBB [52, 53].
The blood-CSF barrier
For many important meningitis pathogens certain experimental data sug-

gests 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 capil-
laries (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 stud-
ies 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 experimen-
tal 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 ven-
tricular fluid suggests entry via the CPs.
Another pathogen, Streptococcus suis, which accounts for both human

and porcine meningitis cases, is also suspected of entering the CNS primar-
ily 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 endo-
thelial cells of the CP core besides vascular endothelial cells and ependymal
cells. In this work, which was performed on cryostat brain sections of neo-
natal rats, pre-treatment of the slices with neuraminidase or a fimbrial ana-
logue 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 peri-
vascular 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 subcu-
taneous 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. monocyto-
genes within phagocytic cells. It is suggested that choroiditis and meningitis
developed as a consequence of hematogenous dissemination of L. monocy-
togenes 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 informa-
tion 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
events on “the brain side”. Notwithstanding bypassing the microbial perme-

ation 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 inva-
sion genes, intracellular signaling cascades and modes of cellular perme-
ation. 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 devel-
opment 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 men-
ingitis models are performed with adult animals by direct systemic or intra-
cerebral 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 per-

meation 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 inter-
ference by possible contributions of other cell types such as neuroglia or resi-
dent 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
models is the BMEC. BMECs are usually harvested from brain homog-
enates, 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 appro-
priate targets for meningitic bacteria [52].
Extending the potential of cell monolayers, several coculture systems
have been developed. Bilayer systems consisting of endothelial and epithe-
lial 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 astro-
cytes 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 endo-
thelial 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 invalu-
able 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
paracellular tracers (e.g., inulin, sucrose, mannitol or dextran) and to ions,

resulting in low permeability coefficients and high transendothelial electri-
cal 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 prepara-
tion 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 includ-
ing other species: rabbit [103, 104], rat [105–107], cow [102] and swine [108].
However, many primary cultures have been problematic regarding contami-
nating 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 compart-
ment 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 cyto-
skeleton 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)
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. meningiti-
dis, 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 pneumo-
cocci 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 cere-
bral endothelial cells. They adhere to and invade HBMEC using several
capsular and fimbrial epitopes and can be found within intracellular vacu-
oles of HBMEC [67]. Bacterial proteins necessary for bacterial invasion
have been identified, i.e., IbeA, IbeB, YijP and CNF1 [52, 65, 67]. Using the
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 trans-
cellular 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 suggest-
ed 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 intra-
axonal 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
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 devel-
opment 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 inter-
action 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 inva-
sion, 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 phosphoryl-
choline and the BMEC PAF receptor [117]. This has been shown by partial
inhibition of pneumococcal invasion of BMEC by a PAF receptor antago-
nist. Phosphorylcholine decoration was found to be up-regulated in pneu-
mococci 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.
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 fibro-

nectin-binding proteins of other streptococcal species, was identified as a
pneumococcal adhesin for fibronectin. In an experimental mouse menin-
gitis 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 compro-
mising 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. menin-

gitidis 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 encapsu-
lated 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
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 associ-
ated 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 microtu-
bule elements of the eukaryotic cytoskeleton. The streptococcal polysaccha-
ride 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 coor-
dinate set of genes known to orchestrate neutrophil recruitment, activation
and enhanced survival (e.g., CXC family chemokines IL-8, Gro-_ and `, IL-
6, 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 integ-
rity 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
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 blood-
CNS 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, transmigra-
tion) are involved in this cascade.
The initial capture, the ‘tethering’ of leukocytes as well as subsequent roll-
ing 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 ade-
quate 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
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 neutro-
phils, 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 leuko-
cyte 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 menin-
gitis 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 epithe-
lial 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 proin-
flammatory 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 stud-
ies 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. menin-
gitidis, the endothelial cells were also shown to respond with IL-8 production
with the p38 mitogen-activated (MAP) kinase being centrally involved [93].
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 perivascu-
lar astrocytes and microglia either diffuse or are transported to the endothe-
lial 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 MIP-
1` [169].
Cytokine release at the blood-CNS-barrier
Various studies have demonstrated that BMECs are well capable of produc-
ing 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 resi-
dent macrophages in the CNS that by virtue of their strategic location at
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 peri-
vascular macrophages by intraventricular injection of mannosylated clo-
dronate 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 phago-
cytes [178]. Immunohistochemical studies on rat brains have revealed exten-
sive 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 comple-
ment 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, IL-
1_ and IL-1` as well as IL-1 receptor antagonist mRNA expression were
noted primarily within the CPs and the CVOs. Interestingly, characteriza-
tion 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 micros-
copy MHC class II-positive cells were found in abundance in the CPs of rat
brains. The dendriform morphology and large size of these epiplexus/mac-
rophage-like cells led to the assumption that these cells could indeed rep-
resent “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 sys-

temic bacterial/viral infection. This inflammatory response, characterized
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 pro-
gressively 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 molecu-
lar 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 constitu-
tive 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 intracel-
lular 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 addi-
tion, 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 bac-
teria 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. men-
ingitidis 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 antimicro-
bial 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 2,3-
dioxygenase (IDO) with subsequent degradation of the essential amino
acid L-tryptophan has been found to be the principle antimicrobial mecha-
nism. 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-
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 astro-
cytes, 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 simi-
larities 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 vascu-
lar 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 mito-
chondrial 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 molecu-
lar 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 fal-

ciparum, 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].
240 Ian A. Clark and Michael J. Griffiths
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 overlap-
ping) 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 varia-
tion 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 fun-
damental 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 understand-
ing 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 comple-
mentary.
The molecular basis of paediatric malarial disease 241
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
Defining criteria
Coma* 10.0 16.8 17.1 41.7
Severe anemia** 17.6 4.7 10 0
Pulmonary oedema
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
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 mortal-
ity in other continents particularly South East Asia [6]. In malaria-endemic
regions (e.g. sub-Saharan Africa), where the resident population have con-
tinuous 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,
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 pathophys-
iology 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 lev-
els 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 multi-
factorial, involving mechanical, haemodynamic, and immunological factors
[15]. The observation that ARF is more frequently observed as a complica-
tion of trauma in adults than children [12] suggests that age-related varia-
tions 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 metabo-
lism may also occur [19]. Such functional impairments may, in part, be influ-
enced 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, capil-
lary 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 alter-
native 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-
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 polymor-
phisms 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-_ recep-
tor-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
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 inflamma-

tion [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 falci-
parum 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 pro-
inflammatory 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].
Cytokines as a disease mechanism extends beyond malaria
As noted above, the idea that excessive production of inflammatory cyto-

kines 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 pro-
vide 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 diag-
nosis 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 individu-
als has not been ascertained. Consequently, whether sequestration is the
principal instigator of local pathology, or whether sequestration is an asso-
ciated 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 repeat-
edly 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
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), vascu-
lar 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 adhe-
sion, 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 infec-
tion [60].
Sequestration during falciparum malaria appears to be concentrated
in the brain and placenta. There is some evidence to suggest that the pro-
pensity 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 activa-
tion of the coagulation cascade. Therefore vasculature with lowest throm-
bomodulin 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 infect-
ed 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
inflammatory response itself, so anaemia and mitochondrial dysfunction

(see Mitochondrial dysfunction section below), both consequences of sys-
temic 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 flex-
ible enough to squeeze through fenestrations in specialised vessels in the
red pulp of the spleen, and thus avoid phagocytosis by adjacent macro-
phages. 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+ accumu-
lates 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 deform-
ability 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 reduc-
tion 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 fal-
ciparum 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 eryth-
rocyte deformability, replacement by new recruits is vital to avoid anaemia.
Unfortunately, the same inflammatory cytokines that shorten lifespan also
retard replacement. Some years ago researchers began to stress the con-
tribution 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, how-
ever, 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 differ-
entiation 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 consider-
ably 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 respira-

tion. Cellular respiration requires oxygen and pyruvate, as well as multiple
cofactors and active transport molecules. Within the matrix of the mitochon-
drion organelle, pyruvate is catabolised via the Krebs cycle and oxidative
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 sep-
sis (or exposure to pro-inflammatory cytokines), despite sufficient oxygen
supply [97, 100, 101]. More recently, impairment of enzyme activity associ-
ated 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 oxida-
tive damage that accompanies sepsis could be both a cause and a conse-
quence 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 infec-
tions [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-
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 buffer-
ing 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 mitochon-
drial 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 respira-
tion. 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 thera-
peutically 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].
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 capac-
ity, 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 mito-
chondrial function (in response to severe systemic inflammation) the out-
come 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 exuber-
ant, 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 para-
sites on peripheral blood smears, and exclusion of other causes of encepha-
lopathy [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
(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 cyto-
skeletal-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 contain-
ing 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 unconscious-
ness 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 previ-
ously discussed, this type of cell swelling involves disturbance of the “pump-
leak 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 imbal-
ance in supply and demand of energy within the cells. Several mechanisms,
such as sustained increase in neuronal activation, impaired substrate deliv-
ery (structural and functional) and impaired mitochondrial utilisation of
available substrates, including oxygen, may coexist to generate this imbal-
ance. 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 out-
come [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-occlu-
sive/ischaemic (i.e. stroke-like) events that manifested clinically as CM.
However, CM demonstrates several features that are atypical for stroke. In
children, focal neurological signs do not tend to accompany coma, although

a sub-set of patients do exhibit hemiparesis or focal brainstem deficits dur-
ing 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 swell-
ing 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, his-
tological 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 (HO-
1) [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 dif-
fuse 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 detoxi-
fication 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 imbal-
ance. 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 concentra-
tion in CSF was observed across patient outcome groups of increasing
severity in African children [162].
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 inflam-
mation, 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-infec-
tions. 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. menin-
gitis, macroscopic cerebritis and brain abscesses are excluded). SAE is asso-
ciated 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
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 impair-
ment 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 impair-
ment [174]. These parallel changes are set out in Table 3. It is clear, there-
fore, 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.
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 falci-
parum parasitaemia is associated with the generation of specific antibodies
for voltage-gated calcium channels directed against neurones. Higher anti-
body 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 hypoth-
esis’, 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 demonstrat-
ing 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 defi-
ciency [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 sev-
eral 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 dis-
tribution 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 addi-
tion to IE rosetting (the adhesion of IEs to uninfected erythrocytes) and IE
agglutination by sera. These findings provide the prospect that HbC pro-
tects against severe malaria by mitigating the obstruction and inflammation

caused by the PfEMP1-mediated adherence of IEs [186]. However, seques-
tration 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 mecha-
nism is appealing, since it would be equally plausible across a range of hae-
moglobinopathies, including HbC.
Variations in erythrocyte membrane proteins also have a profound influ-
ence 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 par-
allel alterations in other haematinic indices leaves the mechanism of this
process unclear.
Malarial protection within individuals exhibiting multiple RBC abnor-
malities 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 approach-
es could help rectify this – addressing public-health problems resulting
in delayed presentation, and identifying the physiological processes and
molecular pathways that lead to these early deaths, with a view to develop-
ing 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 falci-
parum 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 mac-
rophages, 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 mitochon-
drial 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 con-
sciousness. However, inflammatory cytokines control these changes, as well
as inhibit the capacity of mitochondria to use oxygen. Thus, as described
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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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
274 Wilbert Mason
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 epidemi-
ology 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 abnormali-
ties (CAA). With increasing experience, it became apparent that a signifi-
cant minority of infants and children were not identified by the classic diag-
nostic 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 echocar-
diographic 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 con-

ducted in Japan (i.e., every 2 years) under the auspices of the Ministry of
Epidemiology and etiology of Kawasaki disease 275
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 Asia China

Ireland Bejing
Sweden Taiwan
Finland Shanghai
Germany Hong Kong
France Korea
Portugal Thailand
Italy India
Iran
Oman
Africa Nigeria South America Argentina
South Africa Brazil
Egypt Chile
Senegal
Tunisia Oceania Australia
Sudan New Zealand
Health, Labor and Welfare. Questionnaires were sent to hospitals with pedi-
atric 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 ques-
tions 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.
276 Wilbert Mason
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 any-
time assistance is needed. (1) Infants ) 6 months old on day * 7 of fever without other expla-
nation 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 posi-
tive 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 func-
tion, 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 inflamma-
tion. Taken from [5]. Copyright © 2004 American Heart Association.
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 con-
ducted by specialty societies and more recently through the use of adminis-
trative 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 sur-
vey 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 previ-
ously 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 inci-
dence 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-
278 Wilbert Mason
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 coun-
tries 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,
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, inci-
dences 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
280 Wilbert Mason
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 sea-
sonal 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 fre-
quent 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-
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 epide-

miologically 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 mater-
nal antibody) and peak occurrence early in childhood is another character-
istic 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 cen-
tury, 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 patho-
gens 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 chil-
dren developed coronary artery aneurysms as a sequela [2].
282 Wilbert Mason
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 medium-
sized 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 vari-

ety 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+ help-
er cells and a proliferation of circulating activated B cells spontaneously
secreting IgG and IgM. Furukawa et al. [56], in Japan, demonstrated activa-
tion 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 facili-
tate the vascular injury.
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 patho-
genesis of the disease. Macrophage activation syndrome has been observed
with KD [65]. CD25+ CD4+ regulatory T cells, which maintain immunologi-
cal 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 pri-
mary growth factor for formation of blood vessels and is a vascular perme-
ability factor. It may also be a driver of inflammation as it enhances mono-
cyte 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 immu-
nopotent 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 appar-
ent greater tendency for CAA to occur in some children but not in others
284 Wilbert Mason
Table 5. Gene polymorphisms and single nucleotide polymorphisms (SNPs) in KD
Polymorphism or SNP Susceptibility or risk factor Reference
HLA Bw22J Associated with KD in Japanese children 74, 75

HLABw51 Associated with KD in United States 76, 77
and Israel population
HLABw44 Associated with KD during Boston 78
epidemic
IFN-a gene Over production of TNF-_ 79
Monocyte chemoattractant Over production of MCP-1 80
protein 1 gene (MCP-1
TNF-_ gene A/A at LT-x + 250 Over production of TNF-_ 81
SLC11A1 gene 51-promoter (GT)n Possible dysregulation of IL-1B or 82
TNF-_
Genotype 1/11 fa IL1-Ra Higher susceptibility of KD 83
Angiotensin converting enzyme Associated with KD 84
genotype ID
Vascular endothelial growth Associated with KD 85
factor (VEGF) CGCC
Angiotensin/converting enzyme Associated with development of CAA 86
genotype I/I
MHC-class-1-chain related gene A Protective for development of CAA 87
(MICA) allele 4A
Methylenetetrahydrofolate reductose Protective for females and risk factor 88
(MTHFR) TT genotype for males for CAA
CD14/-159 TT genotype Associated with CAA 89
CD40 ligand (CD40L) SNP in Associated with CAA in males 90
intron 4
VEGF G allele Associated with CAA 91
Matrix metalloproteinase-3 gene Associated with CAA 92
6A/6A genotype
Fc x RIIa HR and RR alleles May predict failure of IVIG therapy 93
Mannose-bending lectin (MBL)gene May predict failure of IVIG therapy 94
MBL2 genotype in < 1 year olds
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.
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
286 Wilbert Mason
Table 7. Comparison of the Effect of Conventional and Superantigens
Conventional antigen Superantigen
Processed by antigen presenting all (APC). Directly bind to class II MHC molecules on
Presented as a peptide on the APC surface in the APC and TCR.
association with MHC II molecule.
Interacts with the variable (V), joining (J) Binding restricted to the specificity of the
and diversity (D) portions of the _ and ` variable regions of the ` chain (V`) of TCR.
chains of the T cell receptor (TCR).
Recognized by the few sensitized T-cells with Activates a specific set of V` families
receptors for the antigen resulting in a resulting in activation of a large portion
limited more specific immune response. 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 secret-
ing 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 differ-
ent statistically. A subset of patients with organisms expressing superanti-
gens that stimulate V`2+ T cell receptor families of T cells were found sig-
nificantly 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 conven-
tional 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 cor-
onary arteries taken from infants and young children who had died in the
acute phase of KD initiated this line of investigation. [109, 110]. IgA-secret-
ing plasma cells were found infiltrating the vascular wall, pancreatic ducts
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 pre-
dominantly 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 round-
to-oval intracytoplasmic perinuclear inclusion bodies in medium-sized
bronchi. They stained with both eosin and hematoxylin, suggesting they con-
tained 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 corona-
virus [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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288 Wilbert Mason
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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 infec-
tion 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 dis-
ease, gastric cancer and mucosal associated lymphoma with H. pylori infection. The asso-
ciation 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 devel-
opment 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 win-
ning 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].
298 Hien Q. Huynh
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 bac-
terium. 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 com-
pared 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 com-
monly 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 protect-
ing 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 (cytotoxin-
associated 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
Helicobacter pylori infection in children 299
protein into epithelial cells [13, 14]. This protein is phosphorylated intracel-
lularly; 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, form-
ing 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 patho-
genesis 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 asymp-
tomatic. Most infected patients with H. pylori develop gastritis, particularly
nodular gastritis in children [17, 18]. Those with antral predominant gastri-
tis 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 HLA-
DQA1 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
300 Hien Q. Huynh
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 gas-
tric cancer was found in those not infected or in a subgroup of patients who
received eradication therapy for H. pylori [28]. However, in another pro-
spective 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 inci-
dence 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
[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 pre-
cancerous 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 gas-
tric 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 cri-
teria 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.
302 Hien Q. Huynh
H. pylori and gastroesophageal reflux disease
Currently there is no evidence that H. pylori eradication worsen gastro-

esophageal 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 infec-
tion 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
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 administra-

tion was associated with an increase in platelet count in patients with chron-
ic 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 occur-
ring in approximately 50%. Subsequently, there has been a randomized con-
trol 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
304 Hien Q. Huynh
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 ver-
sus H. pylori eradication therapy in the treatment of ITP [58]. The potential
explanation for this is likely molecular mimicry, where anti-platelet antibod-
ies 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 con-
ditions. 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-to-
test variability [33].
Urea breath test
The urea breath test utilizes the essential enzyme urease, which is pro-
duced 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 chil-
dren. 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/base-
line (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
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 specific-
ity 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 speci-
ficity 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 dif-
ficulty, 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
306 Hien Q. Huynh
studies in which the monoclonal was compared to the polyclonal stool anti-
gen 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 mono-
clonal 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 infec-
tion 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 anti-
gen 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-
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 nod-
ular 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 clini-
cal 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].
308 Hien Q. Huynh
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 iso-
lates 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 previ-
ous 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 fac-
tor 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 place-
bo 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, consis-
tent with results from adult studies [85, 86]. This sequential treatment needs
to be further studied in different populations to determine its efficacy and
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 addi-
tion, 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 fre-
quency 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 need-
ed. Improving the social situation of children such as better housing, sanita-
tion and hygiene remain one of the key pillars in reducing the prevalence
of this infection in childhood.
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Human metapneumovirus infection
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 symp-
toms 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 associat-
ed 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 pres-
ence 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 tech-
nology 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-
318 Adilia Warris and Ronald de Groot
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 pre-
sumably 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 syn-
thesis 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 tur-
keys, and the virus is unlikely to be a zoonotic source.
RT-PCR analyses using primer sets for specific paramyxoviruses (para-
influenza 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-spe-
cific antisera did not react in immunofluorescence (IF) assays with cells
infected with a panel of paramyxoviruses and orthomyxoviruses (parainflu-
enza 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 micros-
copy revealed paramyxovirus-like pleiomorphic particles of 150–600 nm,
Figure 1. Classification of viral pathogens of the Paramyxoviridae family that infect humans.
319
with short envelope projections of 13–17 nm, indistinguishable from RSV

[1]. hMPV is chloroform sensitive, and replicates optimally in a trypsin-
dependent 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 deter-
mined 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 sequenc-
es 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 pro-
tective 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
Human metapneumovirus infection 321
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 varia-
tion, 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 clas-
sification, 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 phy-
logenetic 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 predomi-
nant 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 vari-
ous 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
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 signifi-
cantly 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 RT-
PCR protocols.
hMPV has circulated in humans for at least 50 years; a 100% seropreva-
lence 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 peri-
ods 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 cat-
egories 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-immunocom-
promised 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 infec-
tions account for at least 3% of children who visit a general practitioner or
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 patho-
gen, 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%, respec-
tively 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, includ-

ing 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 cor-
responding with a suppurative rhinitis at pathological examination. In addi-
tion, 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 histo-
pathological 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
Table 1. Incidence of human metapneumovirus (hMPV) infections in hospitalized children with respiratory tract disease
324
Country Study period Population Method hMPV-positive/ Prevalence Peak age

total number of
patients
Regev et al. [15] Israel Nov. 02–May 03 < 5 years, RTI RT-PCR(1) 42/338 10.8% 1–2 year
Nov. 03–May 04
Wilkesmann et al. [23] Germany Oct. 02–May 03 Children; RTI RT-PCR(2) 114/637* 17.9% <24 months
Oct. 03–May 04
Foulongne et al. [24] France Nov. 03–Oct. 04 < 5 years; RTI RT-PCR(2) 50/589 8.5%
Bouscambert-Duchamp et France Sept. 01–June 02 Infants; <24 months RT-PCR(2) 6/94 6.4% 2–6 months
al. [25]
IJpma et al. [22] South Africa June–Aug. 2002 Children; RTI RT-PCR(2) 8/137 5.8% 2–24 months
König et al. [26] Germany Nov. 99–Oct. 01 < 3 years; RTI PCR 15/87 18%
< 6 months; apneu
admitted to ICU
McAdam et al. [27] USA Oct. 00–Sept. 02 ) 18 years RT-PCR(1) 54/868 6.2% 3–24 months
Jartti et al. [28] Finland Sept. 00–June 02 3 months–16 years; acute RT-PCR(2) 12/291 4% 3–11 months
expiratory wheezing
Døllner et al. [29] Norway Nov. 02–Apr. 03 Children; RTI PCR(2) 50/236 21% ) 12 months
Mullins et al. [30] USA Aug. 00–Sept. 01 <5 years; RTI RT-PCR(2) 26/641 4% 6–24 months
Esper et al. [31] USA Nov. 01–Nov. 02 <5 years RT-PCR(3) 54/668 8.1% < 12 months
Madhi et al. [8] South-Africa Mar. 00–Oct. 00 Infants RT-PCR(3) 14/196 7.1%
van der Hoogen et al. [32] Netherlands Oct. 00–Feb. 02 All ages; RTI RT-PCR(1) 48/685* 6.5% 4–6 months
Viazov et al. [18] Germany Jan. 02–May 02 <2 years; RTI RT-PCR(2) 11/65 17.5%
Maggi et al. [33] Italy Jan. 00–May 02 <2 years; RTI RT-PCR(4) 23/90 25% ) 3 months
Peiris et al. [9] Hong Kong Aug. 01–Mar. 02 ) 18 years; RTI RT-PCR(2) 32/587 5.5%
Thanasugarn et al. [34] Thailand Mar. 01–Sept. 02 < 14 years; RTI RT-PCR(2) 5/120 4.2%
Rawlinson et al. [35] Australia 2 summers & 2 < 12 years; URTI PCR(2) 9/150 6%
winters 00-02 < 17 years; asthma PCR(2) 3/179 2%
Freymuth et al. [36] France Nov. 00–Mar. 01 Children RT-PCR(3) 19/337* 6.6% < 1 year
Nov. 01–Feb. 02
(U)RTI: (upper) respiratory tract infection.

(1) All respiratory specimens obtained; (2) nasopharyngeal aspirates; (3) on common respiratory viruses negative nasopharyngeal aspirates; (4) nasal
swabs.
* Number of samples.
325
Table 2. Incidence of hMPV infections in non-hospitalised children with RTI
326
Country Study period Population Method hMPV-positive/ Prevalence Peak age

total number of
patients
Williams et al. [16] USA 1982–2001 <5 years; URTI RT-PCR(3) 118/2384 5%
König et al. [26] Germany Oct. 00–Apr. 01 <3 years; RTI <6 months; PCR(3) 2/620 <1%
apneu
Laham et al. [37] Argentina June 02–Sept. 02 <1 year; RTI RT-PCR(4) 22/373 6%
Principi et al. [38] Italy Nov. 02–Apr. 03 <15 years; RTI PCR 41/1331 3.1%
Williams et al. [39] USA 1976–2001 <5 years; RTI or AOM RT-PCR(3) 49/248 20% 6–12 months
Bastien et al. [40] Canada Oct. 01–Apr. 02 RTI RT-PCR(1) 66/445 14.8% <5 years,
>50 years
Falsey et al. [41] USA Nov. 99–Apr. 00 Fit elderly > 65 years;RTI RT-PCR(2)
Nov. 00–Apr. 01 Young adults;RTI serology 4/233 11/167 1.7% 6.6%
(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.
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 use-
ful 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 con-
trast, 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 respira-
tory 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 respira-
tory 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 mono-
nuclear 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 contrib-
ute 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 infec-
tion, 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
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 associ-
ated 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 paramyxo-
virus infections early during childhood can be associated with the develop-
ment 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
stimulation by RSV [56]. In a study using BALB/c mice, it was shown that
the indolent pulmonary inflammatory response was characterized by mini-
mal 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) activ-
ity [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 respon-
sible 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 chil-
dren < 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 elu-
cidate the exact mechanisms of illnesses caused by hMPV.
The fusion F surface glycoprotein has been identified as a major cross-
protective antigen [48, 49]. In addition to the F protein, the subfamily
Pneumovirinae of the paramyxoviruses also have a separate surface gly-
coprotein 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 glyco-
proteins did not induce detectable neutralizing antibodies, and the contribu-
tions to protection were minor or negligible, respectively. This is in contrast
with other paramyxoviruses (including RSV) in which the G protein stimu-

lates 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 sug-
gest 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 infec-
tions: 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 diag-
nostic 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 amplifica-
tion 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
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 lineag-
es [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 infec-
tion. 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 immu-
nofluorescence antibody (IFA) detecting whole proteins of hMPV. Western
blot analysis using recombinant P protein was not successful due to nonspe-
cific 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 pro-
tein [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 Bac-
F 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
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 seroepi-
demiological 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 glycopro-
teins 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 cen-
trifugation cultures (SVCC) are methods commonly used in clinical virol-
ogy 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 mAb-
8 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 back-
ground 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
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-posi-
tive 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-posi-
tive 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 dis-
tress (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 percent-
age 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 exac-
erbations 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
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 pres-
ence of both RSV and hMPV has been detected [23, 33, 39, 72]. Dual infec-
tion 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 mechani-
cal ventilation [26, 72]. Foulongne et al. [24] showed that another respira-
tory 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 supple-
mental 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 diag-
nosed 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 detect-
able. 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 chil-
dren 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 detec-
tion 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 shed-
ding 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
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 impor-
tant 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 cul-
ture-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 sub-
set 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 iden-
tity; consequently, antisera generated against either RSV or hMPV do not
neutralize across the Pneumoviridae group [77].
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 vac-
cines. Studies in cynomolgous macaques showed that re-infection is sup-
pressed by high titers of virus neutralization antibodies against the homolo-
gous 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 mon-
ovalent 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 para-
myxoviruses 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 gener-
ate 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 mon-
keys 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-
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 rep-
resent 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 clini-
cal 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 antibod-
ies 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 vac-
cinations were timed appropriately [86].
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 infec-
tions, with recurrent epidemics during the winter months. hMPV is the sec-
ond 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 stud-
ies, 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 RT-
PCR 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 influ-
enza 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 infec-
tions 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.
346 John V. Williams
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 neuramini-
dase (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 addi-
tional sources of viral mutation, to evade host immunity or introduce com-
pletely 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 ani-
mals 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 require-
ments for a pandemic: (1) novel HA or NA types (thus no pre-existing
Avian influenza viruses: a severe threat of a pandemic in children? 347
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) world-
wide 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 mortal-
ity 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 mecha-
nisms 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 influen-
za 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 recre-
ated in a highly secure biocontainment facility at the CDC using the tech-
nique 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 reas-
sortant 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-
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, reassort-
ment with human strains could allow a recombinant virus to emerge that is
both highly pathogenic and highly infectious for human hosts. This reassort-
ment 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 human-
to-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 determi-
nants 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
Table 1. Cumulative number of confirmed human cases of avian influenza A/(H5N1) reported
to the WHO
Country 2003 2004 2005 2006 Total

Cases Deaths Cases Deaths Cases Deaths Cases Deaths Cases Deaths
Azerbaijan 0 0 0 0 0 0 8 5 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 migrato-

ry 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, Serbia-
Montenegro, 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
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 reas-
sortant 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 analy-
sis 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 chil-
dren 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 con-
firmed 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 hospi-
talization. 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, rhinor-
rhea and cough [39]. Both patients fully recovered. There was also a large
outbreak of H7N7 in the Netherlands in 2003 on poultry farms, with infec-
tion 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 con-
tact 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-con-
firmed 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
myalgia, rash or conjunctivitis. Similar to the Thailand cases, the most com-
mon 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 includ-
ing 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, head-
ache, 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 fail-
ure 5 days after admission. During his hospitalization, he developed lym-
phopenia, 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 pre-
dominance 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 fac-
tors 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 dis-
tress, 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,
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 symp-

toms 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 expo-
sure. The median duration of symptoms prior to hospitalization was 4 days
(range 0–18 days).
Prominent laboratory findings include leukopenia (especially lympho-
penia), 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
Table 3. Laboratory and radiological findings of H5N1 avian influenza in children
Reference [36] [42] [41, 43]

Leukopenia (%) 29 100 100
Thrombocytopenia (%) 29 86 44
Elevated transaminases (%) 43 80* 71
Radiographic infiltrates (%) 29 100 100
*Transaminases not reported for two patients.
is reminiscent of the mortality associated with the highly virulent 1918 pan-
demic 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 necrotiz-
ing diffuse alveolar damage with patchy and interstitial paucicellular fibro-
sis [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 experi-
mental 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 infec-
tion, but will not differentiate between human and avian influenza A virus
subtypes. Specimens from cases of potential avian influenza should be for-
warded 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
Table 4. Pediatric cases of H5N1 infection and mortality in countries other than Thailand and
Vietnam
Country Total no. of cases No. of pediatric Pediatric mortality

cases (%) (%)
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
Turkey 21* 19 (90) 21
*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 condi-
tions 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 effec-
tive 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
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 cur-
rent 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 dura-
tion 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 osel-
tamivir 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 vac-
cines 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 influ-
enza 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 attenu-
ate 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
strain HA or NA proteins, and the potential to modify virus genes to alter

virulence or replication characteristics [62, 64–67]. However, major limita-
tions 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 influ-
enza vaccines may alleviate some of these obstacles [62, 68–72].
Clinical trials have been conducted with an inactivated H5N1 vac-
cine 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 protec-
tive 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 inacti-
vated 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 com-
ponents 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 pro-
ducing 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.
Summary
The emergence of avian influenza viruses in the human population pro-

motes 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-to-
human transmission is rare. Most children present with fever, rhinorrhea
and cough, and lymphopenia, thrombocytopenia and elevated transami-
nases 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 contin-
ued 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 high-
risk group that should be targeted for early vaccine testing.
Additional information on influenza, including avian influenza, is avail-
able 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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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 com-
mon 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 condy-
loma 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
366 Nanette B. Silverberg
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 pri-
marily 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 mate-
rial (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 nourish-
ment of the verrucae [1, 3, 4].
HPV gene expression is noted as cellular differentiation of the kerati-
nocytes 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].
Human papillomavirus infections in children 367
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 success-
fully 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 immu-
nity, 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 cel-
lular 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 includ-
ing 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 gym-
nasium are 27 times more likely to catch warts, obviating the need for pool
shoes or sandals when using communal pools and showers [17].
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 micro-
scopically, 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 mea-
sure 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
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) 3, 5*, 8*, 10, 12, 14, 15, 17, 25–30, 41
4. Donut warts 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 phenom-
enon) 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 papu-
losis 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].
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 versi-
color. This appearance is uncommon and is usually limited to patients with
EDV, HIV infection, or other immune deficiency [23].
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 immunosup-
pression [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-
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, con-
dyloma 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 treat-
ing 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-onco-
genic 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 com-
monly result from vertical transmission from a virally infected genital tract
or caretakers with hand warts. When condylomata are observed in children,
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 aver-
age, 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 vac-
cination 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 vac-
cination with a hspE7 linked to E7 gene of HPV 16 [33].
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 charac-

terized 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 inflamma-
tory 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 con-
sists of three arms: (1) protective skin barrier, (2) innate immunity and (3)
acquired immunity [1, 3, 19].
Table 2. Summary of the categorizations of warts
Type of wart Mode of regression

1. Myrmecia (palmoplantar warts)/ Humoral vascular reaction
Butcher’s 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 cuta-
neous immunity and thus the skin barrier may not be the only factor influ-
encing 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 produc-
tion 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-medi-
ated 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 cell-
induced apoptosis of virally infected cells. Satellite cell necrosis or apop-
tosis 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].
Table 3. Host response to warts [19]
Type of response Role in HPV infection
1. Humoral immunity (antibody Prevents re-infection

production) Reduces infectivity
Prevents dissemination
2. Recruitment of immune cells to the area Increased local blood flow Homing leukocytes
Nitric oxide ICAM, VCAM, MAdCam-1 to site infection
upregulation
3. Antigen presentation Keratinocytes MHC class II restricted
May promote tolerance of HPV
Langerhans’ cells Presentation to T cells, MHC class II restricted
4. Cytokine production by keratinocytes Down-regulation of HPV infection
and mononuclear cells Inhibition of growth of HPV infected cells
TNF-_, IL-1, TGF-`, IFN-_, IFN-a, EGF Up-regulation of MHC and adhesion
and regulatory genes (e.g. c-myc) Molecule expression
Autocrine growth inhibitory effects
IL-6 (Increased levels) Triggers leukocyte anti-HPV activation
EGF, TGF-_, Amphiregulin Promoting HPV infected cell growth
Persistent sTNF-RI TNF-R reduces TNF-_ activity
Promotes persistent infection
5. Lymphocytes (T helper cells) Regulate cell mediated/ humoral reactions
Th1 cells Produce IL-2, IFN-a, TNF-`; Promotes CMI
Th2 cells Produce IL-4, IL-5, IL-10, IL-13
Cytotoxic T cells MHC restricted
Killing of virus infected/tumor cells including
via release of granzyme and perforin
Co-stimulatory molecules B7, ICAM, LFA-3
Natural killer cells 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 Cancers
MHC class II-HLA-DQw3 Cervical cancer
HLA-DR6, DR13 Lower prevalence cervical cancer
HLA-DR7 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 pro-

longed 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].
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 dysmor-
phic 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/dis-

ease, collagen vascular disorders, and diabetes mellitus) should not be treat-
ed 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 syn-
drome. Of the acquired conditions, renal transplantation, because it often
occurs in younger patients, is associated with a high degree of morbidity
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 devel-
opment [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 immunodeficien-
cies, particularly those with defects of cell-mediated immunity, the most
prevalent being common variable immunodeficiency [50]. The WHIM syn-
drome (an acronym for warts, hypogammaglobulinemia, recurrent bacte-
rial 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 caus-
ative 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-
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 3, 5, 8, 10, 16, 33

Squamous cell carcinoma (head and neck)
7. Malignant proliferating trichilemmal tumor (EDV) 21
8. Carcinoma of internal organs
Esophageal carcinoma 16, 54
Anal carcinoma 16, 18, 31, 33, 34
Adenoid cystic carcinoma 33
Ovarian carcinoma 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 adoles-
cence 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 process-
ing of HPV is atopic dermatitis, although some studies have not supported
an increased incidence. A recent study from the United Kingdom demon-
strated 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.
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 ini-
tially 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 com-
pelling, 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 wide-
spread 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 lit-
erature. 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 verru-
cous 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
Table 6. Overview of treatments for extra-genital and genital HPV infections

Extra-genital Genital
Destructive
Cryotherapy X X
Cantharidin X X*
Duct tape X
Garlic X
Podophyllin X
Photodynamic therapy X
Salicylic acid X X*
Surgery X X
Immunotherapy
Antigen injection (Candida, mumps) X
Cimetidine X X
Imiquimod X+ X+,*
Squaric acid immunotherapy X X*
Interferon X
Vaccination X
Vascular
Bleomycin X
Pulsed dye laser X X
Psychological 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 dis-
ease
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 docu-
menting 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.
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 re-
applied 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 IL-
8. 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 twice-
daily 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 gen-
erally 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 injec-
tions 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), eventu-
ally 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 epidemio-
logical studies of adult women [1, 3, 5]. Pap smears are cytological sam-
pling of the cervical mucosa and has been able to detect many cases of
invasive HPV and has sampling errors, false positives and false negatives
[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 muco-
sal 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 three-
dose 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 vac-
cine [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, vac-
cination 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 sched-
ule as well [100].
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 ran-
domized to receive three doses of vaccine or placebo and additional treat-
ment 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 preven-
tion of genital HPV-related morbidity and mortality.
Pulse dye laser destroys the extensive vascular supply required to main-
tain 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 inter-
feres 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 papil-
lomatosis.
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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 amino-
transferase 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 inter-
feron-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) serocon-
version 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 riba-
virin 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.
392 Patrick Gerner
Hepatitis B
Epidemiology
Worldwide, an estimated one million people die annually due to the con-
sequences 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 unnec-
essary. 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 anti-
HBe 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
New treatments for hepatitis B and C in children and adolescents 393
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 clini-
cally 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 child-

hood: these include interferon-alpha, the nucleoside analogue lamivudine
(Zeffix®), and adefovir dipivoxil (Hepsera®). The therapeutic goal is com-
plete 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 favor-
able prerequisite therefore is seroconversion to anti-HBe and reduction
in viral replication. Treatment of chronic hepatitis is indicated when ami-
notransferase 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 transami-
nase levels.
394 Patrick Gerner
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 anti-

viral 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 anti-
HBe, and with it the transition from the first to the second phase of infec-
tion, 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 geno-
types, especially the genotypes A and D predominant in Europe and North
America, treatment success is only marginal.
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 significant-

ly 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-inter-
feron-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 re-
treatment has not proven to be effective. Instead, after unsuccessful treat-
ment 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 anti-
HBe, 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.
396 Patrick Gerner
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-_ can-
not be tolerated or the patient has advanced liver cirrhosis, are the nucleo-
side analogues. Based on many years of experience, currently lamivudine
is the best choice for children. Under certain circumstances, adefovir dip-
ivoxil 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 con-
ventional 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 serocon-
version (Fig. 3). In the largest published trial [12], it was evident that anti-
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 lamivu-
dine.
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, stud-
ies 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, includ-
ing adefovir dipivoxil, and most recently, entecavir or telbivudine. An inter-
national 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 resis-
tant 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.
398 Patrick Gerner
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 serocon-
version. 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-
Table 2. Advantages and disadvantages of antiviral medications for chronic hepatitis B
Advantages Disadvantages
IFN-_ Anti-HBs seroconversion ~5% Significant side effects
Anti-HBe seroconversion 26–38% Expensive
Lamivudine Few if any complications No anti-HBs seroconversion

In most patients HBV-DNA and Anti-HBe seroconversion only 10–15%
GPT decrease during therapy
Available in juice form Higher than spontaneous development
Resistance development Less long-term experience
Adefovir Like lamivudine but fewer More expensive and little experience
resistances with children
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 infec-
tion for HCV is steadily increasing.
Serological diagnosis
Screening tests to determine the levels of anti-HCV antibodies are conduct-

ed 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 geno-
typing. 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 influ-
ence 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 inter-
course, 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].
400 Patrick Gerner
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 toler-
ated 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
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 hypo-
thyroidism 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 anoth-

er 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 treat-
ment, 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.
402 Patrick Gerner
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
Diarrhea 25 <1
Pharyngitis 27
Weight loss 25
Alopecia 23
Inflammation at place of injection 19 <1
Emotional instability 16
Depression 13 <1
Pruritis 12
Arthralgia 15
The future
High hopes for the treatment of chronic hepatitis C are being pinned on
two new groups of substances: protease inhibitors and polymerase inhibi-
tors 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 anti-
viral substances.
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Invasive fungal infections in children: advances

and perspectives
Andreas H. Groll1, Julia Koehler2 and Thomas J. Walsh3

1InfectiousDisease 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 immuno-
compromised children. The past two decades have seen a dramatic increase in both num-
ber 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 algo-
rithms 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 mortal-

ity 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 interven-
tions, 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.
406 Andreas H. Groll et al.
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 there-
fore 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 particu-

larly 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 vol-
umes, and the functional immaturity of hepatic metabolism and renal excre-
tion 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 conse-
quences on drug penetration, may also be one reason for the enhanced risk
of neonates to develop meningoencephalitis, an otherwise unusual compli-
cation 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 lympho-
cytes [18] as well as a possibly increased susceptibility to the immunosup-
pressive effects of glucocorticosteroids [4], which may render them suscep-
tible to nosocomially acquired opportunistic mycoses. The yet-developing
cellular immunity may also explain the occurrence of overwhelming infec-
tions 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]
Invasive fungal infections in children: advances and perspectives 407
or of chronic granulomatous disease [24, 25]. In older children and adoles-

cents, 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 fre-
quently encountered yeasts and molds. In contrast, deficiencies or imbalanc-
es 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 pres-
ence 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 immunodefi-
ciencies such as HIV infection, cancer, hematopoietic stem cell transplan-
tation (HSCT) or solid organ transplantation, and immunosuppressive
treatment with corticosteroids; children of all age groups beyond the neo-
natal 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 mucocutane-
ous 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, partic-
ularly in very low birth weight infants. Candida spp. now account for 9–13%
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 candidia-
sis 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 colo-
nization, vascular catheters, the use of broad-spectrum antibiotics and cor-
ticosteroids, 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 infec-
tions 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 candi-
demia and poses a particular challenge to the infectious disease specialist
[51, 57]. Numerous outbreaks have been reported, which underscores the
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 rele-
vant. However, these organisms also may gain access to the bloodstream via
percutaneous vascular catheters to cause a potentially fatal systemic infec-
tion 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 con-
taminated 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 neo-
nates. 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
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 detect-
ed 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 physiologi-
cal factors and topical antifungal treatment [28].
Children with congenital immunodeficiencies
Among the phagocyte-defect syndromes, myeloperoxidase (MPO) deficien-

cy 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 infec-
tions reported in some patients with this disorder [67, 68]. Chronic granu-
lomatous 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 dis-
order 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 aspergil-
losis, 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 fre-
quency 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].
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 defi-
cits 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 aspergil-
losis [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 trans-
plantation, 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 associ-
ated 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 polymor-
phonuclear 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) follow-
ing HSCT [28, 85, 86].
Cancer
While current treatment for pediatric cancers is curative in most instances,

highly dose-intensive chemotherapy regimens and aggressive support-
ive 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
cancer [87, 88]. Other well-known, but notable risk factors include chemo-
therapy-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 chil-
dren 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 con-
spicuous OPC [28], and Candida epiglottitis and laryngeal candidiasis may
emerge in neutropenic children as life-threatening causes of airway obstruc-
tion [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 leu-
kemias 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-associ-
ated 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 cuta-
neous 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 quad-
rant 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
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 cutane-
ous 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 aspergil-
losis 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 medi-
cal 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 organ-
isms 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 fungi-
cidal 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, pulmo-
nary, 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 organ-
ism is highly angioinvasive and leads to hemorrhagic infarction. Fusarium
is among the few filamentous fungi that cause detectable fungemia and
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 mortal-
ity 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 associ-
ated with the emergence of fluconazole-resistant Candida strains [140]; it
has been shown that such resistant strains can be exchanged among HIV-
infected 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 outpa-
tient treatments, candidemia may present as a community-acquired infec-
tion 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 polymorpho-
nuclear leukocytes [145, 146] makes a major contribution to the increased
susceptibility of patients with advanced HIV infection to invasive aspergil-
losis [147–149]. Invasive aspergillosis has also been reported in HIV-infect-
ed 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
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 experi-
ence 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 crypto-
coccal 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 chil-
dren. 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 dif-
fuse 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 deferox-
amine 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 keto-
acidosis, and congenital aminoaciduria [28]. For example, among 41 reported
cases of rhinocerebral zygomycosis in children beyond the neonatal age,
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 sinopul-
monary infection and lung destruction, as it may be associated with con-
genital 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 pre-
dominate in this setting [166]. However, invasive pulmonary mould infec-
tions 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 micro-
biological 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-cen-
trifugation 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].
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 inva-
sive 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 bron-
choalveolar lavage or transcutaneous bioptic measures is greatly encour-
aged. 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 neutrope-
nic patients, while avoiding empirical therapy, has been demonstrated in a
single-center study: Preemptive therapy based on serial galactomannan test-
ing 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 pro-
tocols appear to be less useful in children than in adults, and reliance on inva-
sive 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 micro-
biologically and clinically resistant [101].
After IV administration of the deoxycholate formulation, amphotericin
B rapidly dissociates from its vehicle and becomes highly protein bound
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 hetero-
genicity 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 asso-
ciated with the use of DAMB and often limit successful therapy. Infusion-
related 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]. Infusion-
related 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, hyper-
tension, flushing and vestibular disturbances; bronchospasm and true ana-
phylaxis 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 azote-
mia, 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 wast-
ing 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 tacro-
limus, 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-
Table 2. Medical management of invasive infections by opportunistic yeast
Fungal disease Management
Uncomplicated candidemia or – Amphotericin B deoxycholate (0.6–1.0 mg/kg/day) (A-I)

invasive candidiasis – 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 – Amphotericin B deoxycholate (0.7–1.0 mg/kg/day) plus
hemodynamic instability flucytosine*** (100 mg/kg/day in 3–4 dosages) (B-III)
Second line therapy of – Liposomal amphotericin B (3–5 mg/kg/day) (A-II)
– refractory infections – Amphotericin B lipid complex (5 mg/kg/day) (B-II)
– limiting toxicity – 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 deoxy-
cholate: 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 usu-
ally 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 antifun-
gal treatment of opportunistic mycoses with conventional deoxycholate
amphotericin B (Tabs 2–4). These include candidemia and acute dissemi-
nated candidiasis, particular in neonates, and induction therapy for crypto-
coccal 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
Table 3. Medical management of invasive infections by opportunistic molds
Invasive aspergillosis – Voriconazole (4 mg/kg IV bid, day 1: 12 mg/kg) (A-I)*

– First line – Liposomal amphotericin B (5 mg/kg/day) (A-II)**
– Second line for – Liposomal amphotericin B (* 5 mg/kg/day) (A-II)
– refractory infections – Amphotericin B lipid complex (5 mg/kg/day) (A-II)
– limiting toxicity – 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 life- – Liposomal amphotericin B (* 5 mg/kg/day) plus caspo-
threatening infections fungin (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 hyalo- – Voriconazole (4 mg/kg bid IV; day 1: 12 mg/kg) (B-III)*
hyphomycetes – Liposomal amphotericin B (5–10 mg/kg/day IV) (C-III)
– Amphotericin B lipid complex (5 mg/kg/day) (C-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 – Voriconazole (4 mg/kg bid; day 1: 12 mg/kg) (C-III)*
filamentous fungi – Liposomal amphotericin B (* 5 mg/kg/day) (C-III)
– Amphotericin B Lipid Complex (5 mg/kg/day) (C-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,
Table 4. Medical management of invasive infections by endemic molds
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)
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)
* 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 concentra-
tions 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 fea-
tures of each formulation translate into different pharmacodynamic proper-
ties 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-
Table 5. Physicochemical properties and multiple-dose pharmacokinetic parameters of the

four currently marketed amphotericin B formulations
DAMB ABCD ABLC LAMB

Lipids (molar ratio) Deoxycholate Cholesteryl- DMPC/DMPG HPC/CHOL/
sulfate (7:3) DSPG (2:1:0.8)
Mol% AMB 34% 50% 50% 10%

Lipid configuration Micelles Micelles Membrane- suv
like
Diameter (+m) 0.05 0.12–0.14 1.6–11 0.08
Dosage (mg AMB/kg) 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 phosphati-

dylglycerol; DMPC, dimiristoyl phosphatidylcholine; DMPG, dimiristoyl phosphatidylglyc-
erol; 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%, respec-
tively, 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 param-
eters in these children were not significantly different from those obtained
in a dose-matched cohort of adult patients [216]. Forty-nine children with

febrile neutropenia were treated in a prospective, randomized trial compar-
ing 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 hepato-
splenic candidiasis; ABLC was effective and well tolerated, and no pharma-
cokinetic 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, emer-
gency 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 ces-
sation of ABLC therapy (1.32 ± 0.12 mg/100 mL) over 6 weeks. No signifi-
cant differences were observed between baseline and end-of-therapy levels
of serum potassium, magnesium, hepatic transaminases, alkaline phospha-
tase, and hemoglobin. However, there was an increase in the mean total bili-
rubin (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 antifun-
gal 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
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 stud-
ies indicate that the disposition of LAMB in pediatric patients is not fun-
damentally 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 sepa-
rately 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 empiri-
cal 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, respec-
tively. 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 immunodefi-
ciencies [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 maxi-
mum dosage of 2.5 mg/kg for various indications to pediatric cancer/HSCT
patients revealed a low rate of adverse events (4%) necessitating discontin-
uation. While mean GOT, GPT and AP values were slightly higher at end of
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 antifun-
gal 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 transami-
nase 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 infu-
sion-related reactions of ABCD and ABLC appear to be similar to those
of DAMB. Several individual cases of substernal chest discomfort, respira-
tory distress and of sharp flank pain have been noted during infusion of
LAMB [230, 231]. Similarly, in comparative studies, hypoxic episodes associ-
ated 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
DAMB, and, limited to LAMB, for empirical therapy of persistently neu-

tropenic 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 therapeu-
tic 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 recom-
mended by the manufacturer.
Antifungal triazoles
The antifungal triazoles have become an important component of the anti-

fungal 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 inhibit-
ing 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 thera-

py. 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. gla-
brata 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 intra-
gastric 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
Table 6. Pharmacokinetic parameters of fluconazole in pediatric patients
Age group VD (L/kg) Cl (L/h·kg) T1/2 (h)

Preterm <1500 g
day 1 1.18 0.010 88
day 6 1.84 0.019 67
day 12 2.25 0.031 55
Term neonates 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 inter-
actions. 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-neutrope-
nic 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
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 flu-
conazole 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 prevent-
ing invasive Candida infections in patients undergoing bone marrow trans-
plantation [279, 280]. Fluconazole has been shown to reduce Candida infec-
tions 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-insolu-
ble, 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 bio-
availability [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
interference with mammalian cytochrome P450-dependent drug metabo-

lism 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 antifun-
gal 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 adoles-
cents, 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 pub-
lished 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 resis-
tance to fluconazole [292, 293, 298]. The clinical efficacy of itraconazole in
candidemia and deeply invasive Candida infections has not been systemati-
cally 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 investi-
gated oral itraconazole for treatment of proven or probable invasive asper-
gillosis suggest a response rate comparable to that reported for amphoteri-
cin 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 paracoccidioi-
domycosis, 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
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 immunocompro-
mised patient and those with life-threatening infections by the endemic
fungi [201, 214] (Tab. 4).
Prophylactic itraconazole may reduce the incidence of proven or sus-
pected invasive fungal infections in patients with hematological malignan-
cies [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 clear-
ance and distribution volume has been reported [196]; separate pharmaco-
kinetic 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
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 infec-
tions 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 exemp-
tion 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 bioavailabil-
ity 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 primar-
ily 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
Figure 2. Structural formulas of voriconazole and posaconazole and, for comparison, those of
fluconazole and itraconazole.
liver enzyme abnormalities (10–20%), skin reactions (< 10%), hallucina-

tions 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 vori-
conazole 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 fol-
lowed 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 compara-
tive 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
Table 7. Principal activity in vitro of new antifungal agents

Vori- Posa- Caspo- Anidula- Micafungin
conazole conazole fungin fungin
Aspergillus spp. + + + + +
Candida spp. + + + + +
C. glabrata + + + + +
C. krusei + + + + +
Cryptococcus + + – – –
neoformans
Non-Aspergillus hyalo- +/– +/– – – –
hyphomycetes
Fusarium spp. +/– +/– – – –
Scedosporium spp. +/– +/– – – –
Phaeohyphomycetes + + +/– +/– +/–
(‘black moulds’)
Zygomycetes – + – – –
Dimorphic (‘endemic’) + + +/– +/– +/–
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
Vori- Posa- Caspofungin Anidula- Micafungin

conazole conazole fungin
Formulation 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 2 >5 n/a 0.7–0.9 0.24
(L/kg)
Elimination half-life (h) 6 25 8–10 24 15
Substrate / inhibitor of 3A4, 2C9, 3A4 n/a n/a n/a
CYP450 2C19
Elimination
– via feces (%/% <20/? 77/– Degrad- Degrad- Meta-
metabolites) ation/meta- ation only, bolization,
– urine (%/% 80/78 14/14 bolization, feces feces >
metabolites) urine > feces urine
n/a, not applicable

meet the prespecified statistical endpoint for non-inferiority in a composite

endpoint , but was associated with significantly fewer breakthrough inva-
sive 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, fusari-
osis, 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 pharmacokinet-
ics 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 load-
ing 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 com-
plete 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 adoles-
cents 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 tran-
sient 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].
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, posacon-
azole 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 distribu-
tion in the order of 5 L/kg and a prolonged elimination half-life of approxi-
mately 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 abnor-
mal 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 coadminis-
tered 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, pre-
liminary 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 ran-
domized 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 inva-
sive 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
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 retrospec-
tive 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 flu-
conazole 400 mg/day, respectively, with the start of immunosuppression for
a total of 16 weeks. Treatment with posaconazole led to a decreased inci-
dence 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 asper-
gillosis (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 randomiza-
tion, 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 pre-
ventative 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 coccidioi-
domycosis 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
food, a dosage of 200 mg four times a day (qid) is recommended, prefer-

entially 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 lipo-

peptides that are composed of a cyclic hexapeptide core linked to a vari-
ably 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 pharma-
cokinetic 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 dif-
ferent with respect to spectrum, pharmacokinetics, safety and antifungal
efficacy [201, 214].
Caspofungin
Caspofungin (Cancidas™) was the first licensed compound of the echino-

candin class of antifungal agents. In vitro, caspofungin has broad-spectrum
antifungal activity against Candida and Aspergillus spp. without cross-
resistance 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 dissemi-
nated and pulmonary aspergillosis, both in normal and in immunocompro-
mised animals [348].
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 single-
dose 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 moni-
toring [351–353]. Caspofungin has no significant potential for drug interac-
tions 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, dexametha-
sone, and carbamazepine may reduce caspofungin concentrations [348].
The clinical efficacy of caspofungin against Candida spp. has been dem-
onstrated first in Phase II and III studies in immunocompromised patients
with esophageal candidiasis [354–356]. A multicenter, randomized, double-
blind Phase III clinical trial investigated the efficacy of caspofungin for
primary treatment of invasive Candida infections in 224 mostly non-neutro-
penic 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 thera-
pies. 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 cat-
egory 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 caspo-
fungin 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-find-
ing study in 39 children and adolescents, none of the patients developed a
serious drug-related adverse event or was discontinued for toxicity [360].

A similarly favorable safety profile has also been reported in immunocom-
promised pediatric patients who received the compound for various indica-
tions, 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 immunocompro-
mised patients with esophageal candidiasis and candidemia. Anidulafungin
had equivalent efficacy to fluconazole in esophageal candidiasis in a ran-
domized, double-blind, international multicenter study with success docu-
mented 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 under-
going hemodialysis. Mild to moderate hepatic impairment (Child-Pugh class
A and B) does not cause clinically significant changes in the pharmacoki-
netics 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
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 stud-

ies 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 differ-
ence 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 asper-
gillosis 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 can-
didiasis 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
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; pharmacoki-
netics 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 dos-
ages 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 consider-
able 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 predomi-
nantly by C. albicans and C. parapsilosis. They are associated with intravas-
cular catheters, intracranial shunt systems, use of broad-spectrum antibacte-
rial 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 excel-
lent safety and tolerance and broad spectrum, mostly cidal activity against
Candida spp., the new class of echinocandin lipopeptides may offer alterna-
tive 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
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 dysfunc-
tions, 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 inva-
sive 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 inva-
sive 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, sta-
bilization 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 infec-
tions 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
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 tis-
sue 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 pul-
monary 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 con-
tinuous 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 discon-
tinuation 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-colony-
stimulating 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 evalu-
ated [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 pre-
sentation of fungal infections relative to the adult population. Over the past
decade, major advances have been made in the field of medical mycology.
Most importantly, an array of new antifungal agents has entered the clini-
cal 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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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 con-
cerned 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 ini-

tial 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-
474 Kwang Sik Kim
Table 1. CDC classification of bioterrorism diseases agents
Category A – Diseases Category A – Agents
The U.S. public health system and primary – Bacillus anthracis (anthrax)
healthcare providers must be prepared to – Clostridium botulinum
address various biological agents, including – Yersinia pestis
pathogens that are rarely seen in the U.S. – Variola major (smallpox) and other pox
High-priority agents include organisms that viruses
pose a risk to national security because they: – Francisella tularensis (tularemia)
– Can be easily disseminated or transmitted – Viral hemorrhagic fever
from person to person – Arenaviruses
– Result in high mortality rates and have the – LCM, Junin virus, Machupo virus,
potential for major public health impact Guabarito virus
– Might cause public panic and social – Lassa fever
disruption – Bunyaviruses
– Require special action for public health – Hantaviruses – Rift Valley fever
preparedness – Flaviruses
– Dengue
– Filoviruses
– Ebola – Marburg
Category B – Diseases Category B – Agents
– Are moderately easy to disseminate – Burkholderia pseudomallei

– Result in moderate morbidity rates and low – Coxiella burnetii (Q fever)
mortality rates – Brucella species (brucellesis)
– Require specific enhancements of – Burkholderia Mallei (glanders)
diagnostic capacity and enhanced disease – Ricin toxin (from Ricinus communis)
surveillance of the CDC – 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
Pediatric aspects of bioterrorism 475
Table 1. (continued)
Category C – Diseases Category C – Agents
The third highest priority agents include Emerging infectious disease threats such as
emerging pathogens that could be engineered Nipah virus and additional hantaviruses
for dissemination in the future because of
– availability
– Ease of production and dissemination
– Potential for high morbidity and mortality
rates and major health impact
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 engi-
neered 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 consider-
ations 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 equip-
ment and interventions, e.g., children cannot be decontaminated in adult
decontamination units, (5) children have special susceptibilities to dehydra-
tion 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 encoun-
ters a patient with symptoms suggestive of the possibility of illness attribut-
able to Bacillus anthracis and variola major.
476 Kwang Sik Kim
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 com-
monly, direct contact with contaminated material leads to cutaneous dis-
ease. 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 clini-
cal 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 men-
ingitis and inhalation anthrax due to the intentional release of B. anthracis
spores. The discovery of other infected people at several sites quickly fol-
lowed. 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 duster-
type 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 sep-
ticemia, 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].
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 Laboratory- ILI from other

(n = 10) confirmed influenza 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 90% 84–93% 72–80%
nonproductive)
Shortness of breath 80% 6% 6%
Chest discomfort or 60% 35% 23%
pleuritic chest pain
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 mediasti-
num 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 (approxi-
mately 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 inha-
lation anthrax in monkeys have demonstrated meningeal involvement by
478 Kwang Sik Kim
pathology in 40–50% of cases [7, 8]. In contrast, in the 2001 bioterrorism-

related 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], pre-
sumably 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 devel-
oped 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 hemateme-
sis. 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
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 inhala-
tion 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
480 Kwang Sik Kim
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
aAllchildren 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 consid-
eration of anthrax may be given and its management will follow the guide-
lines 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 con-
tagious. The last known non-laboratory case of smallpox occurred in 1977
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 small-
pox 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 maculopapu-
lar 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 approxi-
mately 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 pus-
tular 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
482 Kwang Sik Kim
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 gen-
erally distributed centrifugally.
Management
A suspected case should prompt immediate consultations with health

authorities. Strict airborne, droplet, and contact precautions should be insti-
tuted immediately for victims and should continue until all scabs separate.
All contacts should be interviewed, vaccinated, and placed under surveil-
lance. 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 expo-
sure, 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 immu-
nocompromised 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 peo-
ple 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 individu-
als at high risk because of occupational exposure is immunization every 3
years. People with multiple immunizations during childhood may have long-
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 bio-
terrorism 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 popu-
lations 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 American Academy of Pediatrics (2000) Chemical-biological terrorism and its
impact on children: A subject review. Pediatrics 105: 662–670
2 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
3 Inglesby TV, Otoole T, Henderson DA, Bartlett JG, Ascher MS, Eitzen E,
Friedlander AM, Gerberding J, Hauer J, Hughes J (2002) Anthrax as a bio-
logical weapon, 2002: updated recommendations for management. JAMA 287:
2236–2252
4 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
5 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
6 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
7 Fritz DL, Jaax NK, Lawrence WB, Davis KJ, Pitt ML, Essell JW, Friedlander
AM (1995) Pathology of experimental inhalation anthrax in the rhesus mon-
key. Lab Invest 73: 691–702
8 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
484 Kwang Sik Kim
9 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
10 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 biologi-
cal weapon: medical and public health management. Working group on Civilian
Biodefense. JAMA 281: 2127–2237
11 American Academy of Pediatrics – Policy Statement (2002) Smallpox vaccine.
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 con-
solidation and careful extension of existing activities aiming at conducting high level
research, offering high standard teaching, and providing high quality patient manage-
ment. This can only be accomplished by exquisitely dedicated individuals with extraordi-
nary 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 medi-

cal 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, pediatri-
cians are considerably involved in the diagnosis, treatment and prevention
of infectious diseases. In consequence, every pediatrician must be consid-
ered also an infectious disease specialist. This, in turn, has been a downside
for the development of pediatric infectious diseases as a medical disci-
pline 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 pediat-
ric infectious disease specialists providing profound knowledge, expertise
and quality assurance. Accordingly, pediatric infectious disease specialists
486 David Nadal
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, epi-
demiology, 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 interac-
tions, the position of the pediatric infectious disease specialists differ from
those of specialists for adult infectious diseases. These differ not only in rela-
tion 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 infec-
tious diseases in children may substantially differ from those in adults.
One important example of the uniqueness of pediatric infectious diseas-
es 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 rea-
son for up to 60% of the hospitalization of children. Etiological diagnosis of
these infections may be hampered by the limited volumes of biological sam-
ples 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
Pediatric infectious diseases – Quo vadis 2015? 487
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 gen-

eral practitioners, are in charge of the management of children with com-
mon 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 microbio-
logical 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 fre-
quent communicable diseases and relevant guidelines for the community.
Pediatric infectious disease specialists are involved in the care of both out-
patients 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 infec-
tious disease or infection control issues requested by both experienced
488 David Nadal
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 anti-
microbials, 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 develop-
ment 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 immuno-
suppression 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
interactions with colleagues from adult medicine taking over the care of
these patients before, during and after the transition process are indispens-
able 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 consider-

ation of the past and the current situation. Thus, recent changes in the spec-
trum 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 infec-
tious 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, vaccina-

tion 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 man-
ageable 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 preven-
tive measures are strictly applied, vertical transmission of HIV has virtually
vanished [10]. This success story, however, evolved at the expense of intra-
uterine 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-
490 David Nadal
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, how-
ever, 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 wait-
ing 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, con-
jugate 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. pneu-
moniae 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 lack-
ing. Thus, the reduction of S. pneumoniae or N. meningitidis-induced disease
will not be as impressive as for H. influenzae type b. In consequence, pedi-
atric infectious disease specialists will have to explore modalities to improve
surveillance and treatment of these prominent and potentially deadly bacte-
rial 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
increase in the prevalence of antibiotic-resistant bacteria and the reemer-

gence of previously eradicated pathogens as agents of bioterror. Among the
most feared and serious antibiotic-resistant bacteria are methicillin-resis-
tant 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 infec-
tious 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 infec-
tious 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 dif-

ficulties. Vaccines can be developed following the principles of Pasteur, i.e.,
isolating, inactivating and injecting causative microorganisms. Such devel-
opment, 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 fol-
lowed for other pathogens representing a threat to infants and children.
492 David Nadal
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 vac-
cine. 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 protec-
tion. 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 organ-
isms – 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 lipo-
polysaccharide 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 automa-
tion, 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-
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 antici-
pated to become highly solicited. Thus, intra- and interdisciplinary interac-
tions will be more than ever crucial for pediatric infectious disease special-
ists in the years to come. Continued extensive communication, and close col-
laboration 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 indispens-
able 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-
494 David Nadal
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 reli-
able 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 devel-
opment 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 societ-
ies 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 multi-
center 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 infec-
tious 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/paediat-
rica/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 conduct-
ing high-level research, offering high-standard teaching, and providing high-
quality 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
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 priori-
ties. 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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19 Rappuoli R (2001) Reverse vaccinology, a genome-based approach to vaccine
development. Vaccine 19: 2688–2691
20 Danzig L (2006) Reverse vaccinology – in search of a genome-derived menin-
gococcal vaccine. Vaccine 24 (Suppl) 2: S2–11–2
21 Krieg AM (2006) Therapeutic potential of Toll-like receptor 9 activation. Nat
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23 Abel L, Casanova JL (2006) Human genetics of infectious diseases: Fundamental
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Index 497
Index
Abbe, Ernst 103 entry 209

abdominal pain, recurrent 301 booster dose 8
acquired heart disease 274 borax solution 99
Actinobacillus actinomycetemcomitans 189 breast feeding 155
acute respiratory illness 317 bronchiolitis 332, 333
adefovir dipivoxil 393 bronchopneumonia 334
adenovirus 323
adverse events following immunization 10, Candida 406–411
11 Candida albicans 408
amphotericin B 417–424 Candida laryngitis 406
anaemia 246 Candida parapsilosis 408
aneurysm 281 Candida thrombophlebitis 406
anidulafungin 438, 440, 441 Candida tropicalis 412
anthrax 476–479 carbolic acid 99
anthrax management guidelines for caspofungin 437–440
children 478–479 CD40 ligand 283
antibiotic resistance 307, 491 celiac disease 128
antifungal agents 405 Chediak-Higashi syndrome 187
antifungal chemotherapy 405 chickenpox, see varicella
antifungal triazoles 426 child mortality 44, 146
antigen, conventional 284, 285 chlamydial eye infection 107
anti-TNF antibodies 244 chronic disseminated candidiasis 412
Archiv für Gynäkologie 107 chronic granulomatous disease 187, 407, 410
Argentum nitricum 97 chronic mucocutaneous candidiasis 411
Aspergillus species 411 chronic obstructive pulmonary disease
asthma 81, 333 (COPD) 333
autism 81 circumventricular organs 206
avian influenza 345–357 cocidioidomycosis 410
Cohn, Ferdinand 103
basic immunization schedule 6 co-infections, bacterial-viral 335
bioterrorism 473–483, 491 co-infections of respiratory viruses 333
bioterrorism, pediatric considerations 475 cold chain 9, 10
blastomycosis 410 collagenase 189
blindness 96 Collargolum Credé 107
blood-brain barrier, anatomy of 204 combination vaccines 78
blood-brain barrier, cell culture models community pediatrics 487
211 complications following immunization 76
blood-brain barrier, microbial port of condyloma acuminata 372, 373
entry 208 congenital T cell immunodeficiency 406
blood-CSF barrier, anatomy of 205 Conheim, Julius Friedrich 103
blood-CSF barrier, cell culture models 213 conjugate vaccine 490
blood-CSF barrier, microbial port of consensus interferon 401
498 Index
contraindications for immunization 72, 91, Expanded Program on Immunization 5

92 eye prophylaxis 99–107
coronary artery, abnormalities of 274 eyesight, impairment of 96
coronary artery, lesions of 274
cost-effectiveness calculation of failure to thrive 126
immunization program 18 false contraindications for immunization
cows milk allergy 129 72, 92
Credé, Benno Carl 107 “Father of Gonococcus“, see Neisser,
Credé, Carl Siegmund Franz 95–111 Ludwig
Credé´s eye prophylaxis 95–111 fimbriae 189
Credé’s method 96 fluconazole 426–428, 432
Credé’sche Prophylaxe, see Credé´s eye fluoride 183
prophylaxis 5-fluorocytosine (5-FC) 430, 431
Credé’scher Handgriff, see Credé’s method fulminant hepatitis B 398
Credé-Hoerder, Carl 108 fungal infections 405–445
Crohn’s disease 81 fungal infection, adjunctive immuno-
croup 333 therapy 444
cryptococcal meningoencephalitis 415 fungal infection, epidemiology 407
cryptococcosis 413 fungal infection, neonate 407
Cryptococcus neoformans 407 Fusarium 413
cyclic neutropenia 187
cystic fibrosis 128 galactomannan antigen, serial monitoring
cytokines 282 of 417
cytotoxic oedema 252 galactomannan ELISA 417
cytotoxin-associated gene (cag) gastric cancer 299
pathogenicity island 298 gastritis 299
gastroesophageal reflux disease 302
demineralization 178 gastrointestinal endoscopy 306
dengue fever 34 Giardia 163
dental caries, alternative etiology of 181 gingipain 189
dental plaque 178 gingival crevice 184
dental plaque, local environment in 183 gingival crevicular exudate 186
diabetes 81, 82 gingival inflammation 187
diabetes mellitus 185, 187 gingivitis 184
dietary carbohydrates 178 GlaxoSmithKline 45, 56, 60
DiGeorge syndrome 411 Global Alliance for Vaccines and Immuniza-
Diplococcus Neisser 102 tion (GAVI) 4, 5, 43, 57, 60, 61
Down’s syndrome 192 Global Immunization Vision and Strategy
dyserythropoiesis 247, 248 (GIVS) 4, 16, 17
global poliovirus lab network 13
early childhood caries (ECC) 178 glucosyltransferase (GTF) 179
ECC, preventive strategies 183 gonorrhea 96
early maternal-infant attachment 107
eating disorders 130 habitats within the mouth 177
echinocandin 437 haemoglobinopathies 256
Ehrlich, Paul 103 Haemophilus influenzae type b 490
enamel 178 Haemophilus influenzae type B vaccine 26,
endocrine disorders 185 27
Erwärmungswanne 97 health and nutrition interventions 164–169
Escherichia coli, blood brain barrier health nutrition and gender equity 169
translocation 213 Helicobacter pylori infection 297–309
Escherichia coli, CNS invasion 216 Helicobacter pylori, flagella of 298
esophageal candidiasis 414 Helicobacter pylori, test 304–307
exotoxin 189 Helicobacter pylori, treatment 308
Index 499
Helicobacter pylori, triple therapy 308 IFN-_-2b 400

Helicobacter pylori, virulent factors 298 IFN-a 282
hemagglutinin 346 IgA 286
hemorrhagic meningitis 477 IgM antibodies 283
hepatitis B 391–398 IL-1 282
hepatitis B, clinical course 392 IL-6 282
hepatitis B, fulminant 397 immune defect, adaptive 122
hepatitis B, seroconversion to anti-HBe 392 immune defect, innate 121
hepatitis B, serological diagnosis 392 immunity, natural 77
hepatitis B, therapy response 395 immunity, vaccine-induced 77
hepatitis B treatment 393–398 immunization
hepatitis B treatment, complications 396 and chronic diseases 85
hepatitis B treatment, contraindications 396 and impaired immunity 85–90
hepatitis B treatment, resistant during pregnancy 83, 84
mutations 397 of MS patients 89
hepatitis B vaccine 28, 29 of preterm babies 84, 85
hepatitis B virus, genotype 394 immunization, indications for 72
hepatitis C 398–402 immunization program, funding of 20
hepatitis C, serological diagnosis 399 immunization safety 10, 11
hepatitis C, transmission 399 infantile periarteritis nodosa (IPN) 274
hepatitis C treatment 400–402 infective endocarditis and oral strepto-
hepatitis C treatment, contraindications 401 cocci 184
hepatitis C treatment, side effects 401, 402 inflammatory cytokines in malaria 243
hepatitis C virus RNA 401 inflammatory eye disease 100
high mobility group box 1 (HMGB1) 244 influenza 477
high-resolution computed tomography influenza encephalopathy 255
(HRCT) 416 influenza pathogenesis 243
histoplasmosis 410 influenza virus type A 323
HIV 17, 131, 161, 162, 491 influenza virus type B 323
hMPV vaccine 336 innate immune defense 492
home delivery 95 intensive care units 415
hospital pediatrics 106, 277, 487 interferon-alpha 393
host-parasite relationship 185 intracellular oxidative burst 188
Howe, Lucien 106 intrauterine growth retardation 126
human papillomavirus (HPV) 372, 373 intravenous immunoglobulin (IVIG) 281,
HPV, condyloma acuminata 380 283
HPV, differential diagnosis of 380 intussusception 54
HPV, E6 gene 367 invasive aspergillosis 409, 412
HPV, E7 gene 367 invasive candidiasis 408
HPV, genital sub-types 382, 383 iodine deficiency, effect on cognitive
HPV, immune response to 376 development 158
HPV, malignant conversion of 379 iron deficiency anemia 155–158, 167, 302
HPV, mode of regression 375 iron deficiency anemia, maternal
HPV, spontaneous 368 behaviour 157
HPV, subclinical 371 itraconazole 428–430, 432
HPV treatment 380, 381
HPV vaccination 383, 384 juvenile onset-recurrent respiratory
HPV vaccine 32–34 papillomatosis (JORRP) 365, 373
human brain microvascular endothelial cells
(HBMEC) 212 Kawasaki disease 273–287
human metapneumovirus (hMPV) 317–328 Kawasaki disease, diagnosis 274
hyperlactataemia 250 Kawasaki disease, diagnosis algorithm 276
Kawasaki disease, epidemiology 274–280
idiopathic thrombocytopenia (ITP) 302–304 Kawasaki disease, etiology 285–287
500 Index
Kawasaki disease, global distribution 277, meningitis, pathogenesis 201

278 meningocephalitis 406
Kawasaki disease, pathogenesis 281–285 meningococcal vaccines 31, 32
Kawasaki disease, pathology 281, 282 Merck 45, 56, 60
Kawasaki disease, risk factors 278–281, 283, metabolic acidosis in malaria 249
284 Metapneumovirus 317–319
Koch, Robert 103 micafungin 438, 441, 442
Koch-Henle postulates 104 Micrococcus 102–106
kwashiorkor 119 micronutrients 119, 120
micronutrient deficiencies, effect on mental
laboratory automation 492 development 159
lactoferrin 191 midwife education 95, 98, 101
lactose intolerance 130 Millennium Development Goals 43, 61
lamivudine 393, 398 mitochondrial dysfunction 248, 249
lazy leukocyte syndrome 187 molecular biology techniques 492
Leopold, Gerhard 96 Monatsschrift für Geburtshilfe und Frauen-
leptin 131 krankheiten 107
leukotoxin 189 Morbillivirus 319
lipopolysaccharide (LPS) 189 mouth, habitat 177
liposomal amphotericin B 424–426 mucosal associated lymphoid tissue (MALT)
lochial discharge 99 lymphoma 301
low birth weight 126, 154, 155, 410 multiferon 399
lysozyme 191 multiple immunization 78
multiple sclerosis 82
macrophage activation syndrome 283 mumps 319
magnetic resonance imaging (MRI) 416 mutans streptococci 179–183
malaria 34, 133, 239–259, 491 mutans streptococci, cariogenic
malaria, effect on mental development 159, potential 183
160 mutans streptococci, transmission of 184
malaria, effect on socioemotional Mycobacterium tuberculosis 131, 132
development 160 myeloperoxidase (MPO) deficiency 410
malaria, inflammatory cytokines 243
malaria, intervention programs 167, 168 Neisser, Ludwig Sigesmund Albert 103–105
malaria, neurological involvement 251–253 Neisseria meningitidis 217, 490
malaria pathogenesis 243 Neisseria meningitidis, CNS invasion 217
malarial disease, cytokine theory of 240 neonatal invasive candidiasis 442
malarial disease, mechanical theory of 240 “Nestor of German midwifery”, see Credé,
malnutrition syndromes 125 Carl Siegmund Franz
malnutrition, see also undernutrition neuronal excitotoxins 253
marasmus 119 neutropenic children 412
maternal and neonatal tetanus non-cariogenic sweetener 183
elimination 25, 26 nucleoside analogues 396
maternal education programs 148 nutrient action, mechanisms of 127
matrix metalloproteinases 283 nutrition, long term effects on cognition 149
measles 319 nutritional interventions, timing of 153, 154
measles mortality reduction/elimination nutritional supplementation 147–152
24, 25
measles vaccine 24 obesity 120, 131
mediastinal lymphadenopathy 477 obligatory anaerobes 188
meningitis, cognitive effects of 164 ophthalmia neonatorum 95
meningitis, innate immunity 222, 223 oral microflora, development of 177
meningitis, indoleamine 2,3-dioxygenase oral poliovaccine (OPV) 9, 23
(IDO) 223 oral streptococci (table) 180
meningitis, leukocyte recruitment 219, 220 oropharyngeal candidiasis (OPC) 412
Index 501
orphanhood 162 Rotarix® 45, 52, 60

otitis media 163, 333 RotaShield® 52, 54, 59
outpatient 95, 101, 106 RotaTeq® 45, 52, 54, 60
routine infant immunization 6
palivizumab 335 rubelavirus 319
pandemic influenza 346
parainfluenza virus 317, 319, 323 safe injection 11
Paramyxoviridae 317–320 salicylic acid 99
Paramyxovirinae 318, 319 school readiness 146
parasite sequestration 245 secretory IgA 191
pediatric infectious disease specialist’s Senckenberg Award 97
activities 486, 487 sepsis-associated encephalopathy
pediatricians, career perspectives 494 (SAE) 254
pediatricians, interdisciplinary severe acute respiratory syndrome 334
networking 493, 494 severe combined immunodeficiency
PegIntron 401 (SCID) 411
periodontal bacteria, transmission of 192 shell vial centrifugation cultures
periodontal destruction 187 (SVCC) 332
periodontal diseases 184 sialic acid 346
periodontal diseases, onset of 190 silver acetate 106, 107
periodontitis 184, 186 single-nucleotide polymorphisms 284, 285
periodontopathic bacteria 184, 188, 190 skin cancer 374
peroxydase 191 smallpox 479–483
pityriasis 409 sorbitol 183
plasma cells 286 Spiegelberg, Otto 107
Plasmodium falciparum 239, 242 Staphylococcus aureus 286
pneumococcal vaccines 30, 31 steroid-addicted subjects 185
pneumonia 332–334 stool antigen test 305
Pneumovirus 318, 319 streptococcal bacteria 286
poliomyelitis eradication 23, 24 Streptococcus agalactiae, CNS invasion
polymerase chain reaction (PCR) 182, 366 218
polymorphonuclear leukocytes Streptococcus mutans 179, 180, 182
(PMNL) 186 Streptococcus pneumoniae 490
PMNL disorder 187 Streptococcus pneumoniae, CNS
PMNL phagocytosis, depression of 188 invasion 216
poor red cell membrane deformability 247 Streptococcus sobrinus 179, 180, 182
Porphyromonas gingivalis 189 Streptococcus suis, CNS invasion 218
posaconazole 432, 434–436 sucrose-caries-mutans streptococci
povidone-iodine 107 association 180, 181
protein-calorie malnutrition 119 sudden death 273
sudden infant death syndrome 82, 83
Reach Every District (RED) strategy 16 sugar substitutes 183
respiratory distress 333 superantigen 285
respiratory synctial virus (RSV) 317–320, supplementary immunization activities
329, 334 (SIAs) 8, 9
respiratory tract infection 317, 318 surveillance for vaccine-preventable
respiratory virus 317 diseases 12
respirovirus 319 surveillance and containment, smallpox 483
retractions 333
reverse genetics technology 317, 336 T cells 282, 283, 285, 286
rhinitis 333 thalassaemia 257
rhinovirus 323 thrombomodulin 246
ribavirin 335 thymic atrophy 122
ring immunization, smallpox 483 TNF-_ 282
502 Index
tooth and periodontal tissues, structure Vero cell clone 118 330
of 179 viral glycoprotein 332
Trichosporon beigelii 413 virus-like particles 367
tuberculosis 491 VISION 2020 107
tuberculosis vaccine 34 vitamin deficiency 125, 132
tumour necrosis factor (TNF) 243 vitamin E 395
vivax malaria 254
undernourished/malnourished children, voriconazole 431–435
educational and psychosocial stimulation VP4 54, 55
of 148–152 VP4 P type 47, 48
undernutrition, effect on cognitive develop- VP7 G type 47, 48, 57
ment 147–151 VP7 gene 53
undernutrition/malnutrition, effect on motor V`2+ T cell 285
development 151, 152 V`8.1+ T cell 285
undernutrition/malnutrition, effect on
socio-emotional development 152, 153 warts, see also HPV
undernutrition/malnutrition, maternal common warts 368
behaviour 150, 152, 155 digitate warts 370
urea breath test 304 flat warts 369
urease 298 palmoplantar warts 372
periungal warts 372
vaccines 24–35 plantar warts 368, 369
vaccine, avian influenza 355 warts, excessive number of 377
vaccine, hMPV 336 warts classification system (table) 369
vaccine, HPV 383, 384 water-insoluble glucan 180
vaccine, oral polio 9, 23 Weigert, Carl 103
vaccine, vector-based 337 wheezing 333
vaccinology 489 World Health Organization(WHO) 43, 45,
vacuolating cytotoxin 299 49, 57, 59, 107
vaginal catarrh 98 worms 162
vaginal contact during birth 102
varicella 481 xylitol 183
vascular endothelial grown factor
(VEGF) 283 yellow fever vaccine 29, 30
vasculitis 274, 281, 282
vasogenic oedema 252 zygomycetes 413
vector-based vaccine 337 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 patho-
gens 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

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Birkhäuser Advances in Infectious Diseases

Axel Schmidt Manfred H. Wolff

Stefan H.E. Kaufmann

Edited by Horst Schroten and Stefan Wirth

Horst Schroten Stefan Wirth

Library of Congress Control Number: 2007920789

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© 2007 Birkhäuser Verlag, P.O. Box 133, CH-4010 Basel, Switzerland

List of contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Rudolf H. Tangermann, Hanna Nohynek and Rudolf Eggers

Susanna Cunningham-Rundles and Deborah Ho Lin

Shigenobu Kimura and Yuko Ohara-Nemoto

Rüdiger Adam, Kwang Sik Kim and Horst Schroten

Ian A. Clark and Michael J. Grifﬁths

Adilia Warris and Ronald de Groot

Andreas H. Groll, Julia Koehler and Thomas J. Walsh

Kwang Sik Kim

Rüdiger Adam, Pediatric Infectious Diseases, Department of General Pedi-

The fifth volume of Birkhäuser Advances in Infectious Diseases is focused

Düsseldorf/Wuppertal, Germany, December 2006 Horst Schroten

ABCD amphotericin B colloidal dispersion

BBB blood-brain barrier

CAA coronary artery abnormalities

DAMB amphotericin B deoxycholate

EAE experimental autoimmune encephalomyelitis

GAVI Global Alliance for Vaccine and Immunization

IBD inflammatory bowel disease

ITP idiopathic thrombocytopenia

JORRP juvenile onset-recurrent respiratory papillomatosis

LAMB liposomal amphotericin B

MALT mucosa-associated lymphoid tissue

OME otitis media with effusion

PAF platelet-activation factor

RBC red blood cell

RED Reach Every District (vaccination strategy)

TNF tumor necrosis factor

VEGF vascular endothelial growth factor

WHIM warts/hypogammaglobulinemia/recurrent bacterial infections/

Global control of infectious diseases

Rudolf H. Tangermann1, Hanna Nohynek2 and Rudolf Eggers1

In both industrialized and developing countries, child immunization has

Figure 1. Annual third dose of diphtheria-tetanus-pertussis vaccine (DTP3) coverage globally

potential life years saved through vaccination in cost-effectiveness analyses.

Immunization policies and strategies

The ‘Expanded Program on Immunization’

Established in 1974 [3], the EPI targeted to achieve 80% immunization

3 GAVI partners include governments in industrialized and developing countries, UNICEF,

Table 1. Routine immunization schedule for infants recommended by the EPI

Routine infant immunization

Table 1 shows the ‘basic’ immunization schedule recommended by the EPI/

Booster doses and ‘second opportunity’ for measles vaccination

Supplementary immunization activities

Immunization campaigns to supplement routine programs to increase cov-

The vaccine cold chain

EPI programs established a system of vaccine transport and storage at

appropriate cold-chain equipment and helped to develop such equipment,

Immunization safety and adverse events following immunization

The goal of immunization is to protect the individual and the community

5 Position papers on immunization safety can be found at http://www.who.int/vaccine_safety/en/

disease by reducing the number of unnecessary injections, and ensuring

Program monitoring and surveillance for vaccine-preventable diseases

The main aim of an immunization program is to reduce the incidence of,