Susceptibility to dengue hemorrhagic fever in Vietnam: Evidence of an association with variation in the vitamin D receptor and FC gamma receptor IIA genes

Abstract

Dengue is an increasingly important cause of morbidity and mortality in the tropics, with more than a billion people at risk each year. Immunologic enhancement is thought to contribute to disease pathogenesis. Only a very small proportion of infected individuals develop life-threatening dengue hemorrhagic fever (DHF). In a large case-control study with 400 DHF patients and 300 matched controls, we have assessed five polymorphic non-HLA host genetic factors that might influence susceptibility to DHF. The less frequent t allele of a variant at position 352 of the vitamin D receptor (VDR) gene was associated with resistance to severe dengue (P = 0.03). Homozygotes for the arginine variant at position 131 of the Fc gammaRIIA gene, who have less capacity to opsonize IgG2 antibodies, may also be protected from DHF (one-tailed P = 0.03). No associations were found with polymorphisms in the mannose binding lectin, interleukin-1 (IL-4), and IL-1 receptor antagonist genes. Further studies to confirm these associations are warranted.

Full text

SUSCEPTIBILITY TO DENGUE HEMORRHAGIC FEVER IN VIETNAM: EVIDENCE
OF AN ASSOCIATION WITH VARIATION IN THE VITAMIN D RECEPTOR AND
FC␥ RECEPTOR IIA GENES
HSIN LOKE, DELIA BETHELL, CAO XUAN THANH PHUONG, NICK DAY, NICHOLAS WHITE, JEREMY FARRAR,
AND ADRIAN HILL
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; Centre for Tropical Diseases, Nuffield
Department of Clinical Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; Dong Nai Paediatric
Centre, Dong Nai Province, Vietnam; Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; University of
Oxford-Wellcome Trust Clinical Research Unit, Centre for Tropical Diseases, Ho Chi Minh City, Vietnam
Abstract. Dengue is an increasingly important cause of morbidity and mortality in the tropics, with more than a
billion people at risk each year. Immunologic enhancement is thought to contribute to disease pathogenesis. Only a very
small proportion of infected individuals develop life-threatening dengue hemorrhagic fever (DHF). In a large case-
control study with 400 DHF patients and 300 matched controls, we have assessed five polymorphic non-HLA host
genetic factors that might influence susceptibility to DHF. The less frequent t allele of a variant at position 352 of the
vitamin D receptor (VDR) gene was associated with resistance to severe dengue (P ⳱ 0.03). Homozygotes for the
arginine variant at position 131 of the Fc␥RIIA gene, who have less capacity to opsonize IgG2 antibodies, may also be
protected from DHF (one-tailed P ⳱ 0.03). No associations were found with polymorphisms in the mannose binding
lectin, interleukin-1 (IL-4), and IL-1 receptor antagonist genes. Further studies to confirm these associations are war-
ranted.
INTRODUCTION
Dengue fever has become one of the most important ar-
thropod-borne diseases. Any of the four serotypes of dengue
virus can result in dengue fever (DF), an acute viral infection
characterized by fever, rash, headache, muscle and joint pain,
and nausea. Occasionally, DF progresses to dengue hemor-
rhagic fever (DHF), a potentially life-threatening illness as-
sociated with vascular leakage, hemorrhage, and shock.
1
More than 1 billion people are at risk of dengue infection and
every year, there are approximately 100 million cases of DF
and approximately 500,000 cases of DHF.
2
The pathogenesis of DHF is poorly understood. The anti-
body-dependent enhancement theory of DHF pathogenesis
draws support from epidemiologic studies, which show that of
the presence of circulating dengue-specific IgG antibodies
constitutes the largest risk factor for DHF and, in vitro, den-
gue-specific IgG can enhance viral entry and infection of
cells.
3–5
Recently, cross-reactive T cells were proposed to play
a pathogenic role by causing tissue damage and secreting per-
meability enhancing cytokines.
6
Pathogenesis has also been
linked to viral virulence factors; in particular the Southeast
Asian dengue viruses may be specifically associated with
DHF.
7
Host genetic factors may also be relevant and predispose
some individuals to DHF. It is known that only a small pro-
portion of antibody-positive individuals who experience a
dengue infection actually develop DHF.
8
In addition, there
may be racial differences in susceptibility to DHF.
9
Our group
and others have shown that polymorphisms at the major his-
tocompatibility complex (MHC) class I loci are associated
with an altered risk of DHF.
10–12
Studies in other infectious
diseases have found associations with polymorphic non-MHC
genes, which are linked with immune responses.
13
In this
study, we investigated whether susceptibility to DHF is asso-
ciated with polymorphisms within five non-HLA candidate
genes.
The vitamin D receptor (VDR) mediates the immunoreg-
ulatory effects of 1,25-dihydroxyvitamin D
3
(1,25D
3
), which
include activating monocytes, stimulating cellular immune re-
sponses, and suppressing immunoglobulin production and
lymphocyte proliferation.
13
Recently, the tt genotype of a
single nucleotide polymorphism (SNP) at position 352 of the
VDR gene has been associated with tuberculoid leprosy, en-
hanced clearance of hepatitis B infection and resistance to
pulmonary tuberculosis.
14,15
Expression of VDR may affect
susceptibility to DHF since activated B and T lymphocytes
express VDR, and 1,25D
3
affects monocytes, the main sites of
dengue virus infection and replication.
16
The Fc␥ receptor II (Fc␥RII) is a widely distributed recep-
tor for all subclasses of IgG, and is able to mediate antibody-
dependent enhancement in vitro by binding to virus-IgG com-
plexes.
17,18
An arginine (R) to histidine (H) substitution at
position 131 of the Fc␥RIIA gene has been associated with
meningococcal disease and recurrent respiratory tract infec-
tions.
19,20
This polymorphism changes the IgG binding
affinity of the receptor, with reduced opsonization of IgG2
antibodies causally associated with the arginine variant.
21
Therefore, it seemed reasonable to investigate whether ho-
mozygosity for the arginine variant might be associated with
a reduced risk of DHF caused by antibody-dependent en-
hancement.
Dengue shock syndrome (DHF grades III and IV) is asso-
ciated with marked changes in vascular permeability poten-
tially due to inflammatory mediators and complement activa-
tion.
6
Interleukin-4 (IL-4) is primarily produced by Th2 sub-
set of CD4+ T cells. It regulates B cell growth and IgG class
switching, as well as suppresses Th1-type responses.
22,23
It is
a good candidate gene for DHF since it affects both antibody
responses and inflammatory responses during disease. An
SNP identified within the IL-4 promoter has been reportedly
associated with increased levels gene transcription in vitro.
24–26
IL-1RA is involved in the regulation of IL-1-mediated inflam-
matory responses by competitive binding to IL-1 receptors.
27
A two-repeat allele (IL-1RA2) of an 86-basepair variable
number tandem repeat in the IL-1RA gene is associated with
increased serum levels of IL-1RA, and has also been associ-
ated with a number of autoimmune diseases, including
Am. J. Trop. Med. Hyg., 67(1), 2002, pp. 102–106
Copyright © 2002 by The American Society of Tropical Medicine and Hygiene
102

systemic lupus erythematosus, psoriasis, and Graves dis-
ease.
28,29
Intrinsic differences in levels of IL-1RA due to ge-
netic polymorphisms could affect regulation of inflammatory
responses, and thus DHF pathogenesis.
Mannose-binding lectin (MBL) mediates carbohydrate-
dependent activation of the classical complement path-
way.
30,31
Several mutations in the MBL gene, including a G →
A substitution at codon 54 (MBP54) have been associated
with marked reduction in serum MBL levels and MBL-
mediated complement activation.
32,33
The MBL mutations
have been associated with susceptibility to hepatitis B infec-
tion and recurrent childhood infections.
34,35
Thus, MBL is a
candidate gene for DHF since complement activation may
contribute to DHF pathogenesis, and dengue virus has glyco-
sylated envelope and non-structural (NS1) proteins that may
be opsonized by MBL.
36,37
We have assessed these five candidate genes in the largest
genetic susceptibility study of dengue yet reported. We con-
ducted this initial study as a classical case-control study with
age-, sex-, and ethnically matched healthy children as the con-
trol group and the most severe form of DHF as the cases.
MATERIALS AND METHODS
This study was carried out at Dong Nai Paediatric Centre,
a provincial pediatric hospital 40 km north of Ho Chi Minh
City in Vietnam. The DHF grade III and Grade IV patients
were classified according to criteria of the World Health Or-
ganization.
1
Controls were selected from children admitted to
the minor surgical unit in the same hospital and were age, sex,
and ethnically matched.
DNA was extracted from 3–5 mL of blood from each indi-
vidual using Nucleon II DNA extraction kits (Anachem Ltd
Luton LU2OEB UK). The polymerase chain reaction (PCR)
was carried out to amplify the genetic region of interest using
the following oligonucleotide primers: 5⬘-CAG AGC ATG
GAC AGG GAG CAA G-3⬘ and 5⬘-GGT GGC GGC AGC
GGA TGT A-3⬘ for VDR; 5⬘-CAA GCC TCT GGT CAA
GGT C-3⬘ and 5⬘-GAA GAG CTG CCC ATG CTG-3⬘ for
Fc␥RII; 5⬘-ACT AGG CCT CAC CTG ATA CG-3⬘ and 5⬘-
GTT GTA ATG CAG TCC TCC TG-3⬘ for IL-4; 5⬘-CTC
AGC AAC ACT CCT AT-3⬘ and 5⬘-TCC TGG TCT GCA
GGT AA-3⬘ for IL-1RA; and 5⬘-GCA CCC AGA TTG TAG
GAC AGA G-3⬘ and 5⬘-CAG GCA GTT TCC TCT GGA
AGG-3⬘ for maltose-binding protein.
The SNPs were typed by dot-blotting PCR products onto a
nylon membrane, hybridization with 3⬘ digioxigenin-labeled
sequence-specific oligonucleotides, and visualization of the
probes using an anti-digioxigenin chemiluminescence system
(Boehringer Mannheim, Indianapolis, IN). Oligonucleotide
probes used were 5⬘-GCG CTG ATT GAG GCC ATC-3⬘ for
VDR T and 5⬘-GCG CTG ATC GAG GCC ATC-3⬘ for
VDR t; 5⬘-ATT CTC CCG TTT GGA TC-3⬘ for Fc␥RII R
and 5⬘-ATT CTC CCA TTT GGA TC-3⬘ for Fc␥RIIH;5⬘-
GAA CAT TGT CCC CCA GTG-3⬘ for IL-4 C and 5⬘-GAA
CAT TGT TCC CCA GTG-3⬘ for IL-4 T, and 5⬘-CGT GAT
GGC ACC AAG GGA-3⬘ for MBL G and 5⬘-CGT GAT
GAC ACC AAG GGA-3⬘ for MBL A. The IL-1RA repeat
polymorphisms were visualized by electrophoresis of the PCR
products on a standard 2% agarose gel. Results were ana-
lyzed for any differences in genotype or allele frequencies
between the cases and controls in a stepwise fashion. The
StatCalc statistical analysis package (EpiInfo, Centers for
Disease Control and Prevention, Atlanta, GA) was used to
carry out the standard contingency table chi-square test and
the chi-square test for trends.
Informed consent was obtained from the cases and controls
recruited into the study or from their parents or guardians.
The study was approved by the Ethical and Scientific Com-
mittee of the Centre for Tropical Diseases in Ho Chi Minh
City and the Ethical Committee of the Dong Nai Paediatric
Centre in the Dong Nai province of Vietnam.
RESULTS
Three hundred fifteen DHF grade III patients, 37 grade IV
patients, and 251 healthy controls were recruited into the
study. All patients and controls originated from Dong Nai
Province in Vietnam, and cases and controls were ethnically
matched as a group. It was not possible as part of this study to
determine the serotype of the dengue virus. All four sero-
types continuously circulate in southern Vietnam. Recruit-
ment of subjects took place in 1994–1996 when the dominant
serotypes circulating in southern Vietnam were dengue II and
dengue III.
Genotype frequencies for the VDR polymorphism did not
differ between DHF cases and controls (3 × 2 chi-square
analysis, degrees of freedom [df] ⳱ 2, P ⳱ 0.154; Table 1).
However, allele frequency analysis showed that there was an
association between VDR polymorphism and DHF disease
severity (chi-square test for trend analysis, df ⳱ 1, P ⳱ 0.033;
Table 2). This result suggests that the t allele may be protec-
tive against severe DHF.
Genotype frequencies (3 × 2 chi-square analysis, df ⳱ 2,
P ⳱ 0.178; Table 3) and allele frequencies (2 × 2 chi-square
analysis, df ⳱ 1, P ⳱ 0.107; Table 3) for the Fc␥RII poly-
morphism were not significantly different between DHF pa-
tients and controls. However, based on the hypothesis that
TABLE 1
Vitamin D receptor (VDR) polymorphism genotype frequencies in a
dengue hemorrhagic fever (DHF) case-control study*
VDR genotype
DHF cases (n ⳱ 327) DHF controls (n ⳱ 247)
Number Frequency (%) Number Frequency (%)
TT 316 96.6 231 93.5
Tt 11 3.4 15 6.1
tt 00 10.4
* Genotype frequencies for DHF cases vs. controls: 3 × 2 chi-square analysis, degrees of
freedom ⳱ 2, P ⳱ 0.154.
TABLE 2
Vitamin D receptor (VDR) polymorphism allele frequencies in a
dengue hemorrhagic fever (DHF) case-control study*
VDR
allele
DHF Grade IV cases DHF Grade III cases Controls
Number
(n ⳱ 70)
Frequency
(%)
Number
(n ⳱ 584)
Frequency
(%)
Number
(n ⳱ 494)
Frequency
(%)
T 70 100 573 98.1 477 96.6
t 0 0 11 1.9 17 3.4
* DHF cases (Grades III and IV) vs. controls: 2 × 2 chi-square test, degrees of freedom (df)
⳱ 1, P ⳱ 0.056.
chi-square test for trend analysis, df ⳱ 1, P ⳱ 0.0326.
SUSCEPTIBILITY TO DHF IN VIETNAM 103

homozygosity for the lower-affinity binding arginine variant
may be protective against antibody-dependent enhancement–
associated immunopathogenesis, a 2 × 2 chi-square analysis
comparing R/R individuals who possess only the lower-
affinity binding allele with individuals who possess copies of
the higher-affinity binding allele (R/H and H/H) found a pos-
sible protective effect (2 × 2 chi-square analysis, one-tailed P
⳱ 0.036, odds ratio ⳱ 0.57, 95% confidence interval ⳱ 0.29–
1.11; Table 3). The observed frequency of the HH genotype in
this Vietnamese population, 54.6% in DHF cases, and 50% in
controls, is similar to those found in Japanese (61%) and
Chinese (50%) populations and higher than those found in
whites.
38
There were no significant differences in MBL genotypes (3
× 2 chi-square analysis, df ⳱ 2, P ⳱ 0.187; Table 4) or allele
frequencies (2 × 2 chi-square test, df ⳱ 1, P ⳱ 0.072) between
the cases and controls. However, the relatively low frequency
of the variant allele in this population limits the statistical
power of this analysis.
No differences between cases and controls were observed
for the IL-4 promoter polymorphism, either at the genotypic
level (3 × 2 chi-square analysis, df ⳱ 2, P ⳱ 0.688; Table 5)
or the allelic level (2 × 2 chi-square analysis, df ⳱ 1, P ⳱
0.624). Interestingly, the T allele, which is infrequently found
(0.02) in whites, is more common (0.45) in African popula-
tions, and is the dominant allele in the Vietnamese population
(0.77).
39
There was no difference in genotype frequencies of the
IL-1RA repeat polymorphisms between DHF cases and con-
trols (6 × 2 chi-square analysis, df ⳱ 5, P ⳱ 0.833; Table 6).
Allele frequencies were also not significantly different be-
tween the two groups (4 × 2 chi-square analysis, df ⳱ 3, P ⳱
0.94). The frequency of IL-1RA2 (7%) was intermediate be-
tween that observed in southern Indians (25%) and European
whites (24%), and that observed in West Africans (2.5%).
40
DISCUSSION
In this study of genetic susceptibility to DHF, variation in
two of the five genes assessed showed evidence of association
with altered risk of severe dengue. The IL-4 and the IL-1RA
gene variants were not associated with altered risk, but there
is no compelling evidence that the polymorphisms assessed in
these genes are of functional significance. In contrast, het-
erozygotes and particularly homozygotes for MBL variants
clearly have reduced serum levels of this lectin, indicating that
MBL deficiency does not appear to affect the risk of severe
dengue significantly.
41
The less frequent t allele of a dimorphism at position 352 of
the VDR gene was associated with dengue disease severity.
This t allele was initially associated with susceptibility to os-
teoporosis, and some evidence of higher expression of recep-
tor alleles of this type has been presented.
42
However, sub-
sequent studies of bone density association and some limited
studies of the functionality of this variant have provided con-
flicting results.
43
This variant has now been studied in several
infectious diseases. In case-control studies of Gambians, ho-
mozygotes for the t allele were at reduced risk of tuberculosis
and of persistent hepatitis B virus infection.
15
Further evi-
dence of a tuberculosis association was obtained in a study in
the United Kingdom,
44
and the tt genotype was associated
with tuberculoid rather than lepromatous leprosy in Indi-
ans.
14
These associations led to the suggestion that the tt
genotype may be associated with a relatively stronger TH1-
type cellular immune response than the TT genotype; inter-
estingly, dihydroxyvitamin D has recently been found to alter
IL-12 expression and dendritic cell maturation.
14,45,46
By ex-
tension it might be inferred that the association of the t allele
with resistance to severe dengue might reflect a protective
role for enhanced cellular immunity in this disease. However,
further work will be required both to confirm this association
and to explore possible mechanisms.
TABLE 3
Fc␥RII polymorphism genotype frequencies in dengue hemorrhagic
fever (DHF) cases and controls*
CD32
genotype
DHF cases (n ⳱ 302) DHF controls (n ⳱ 238)
Number Frequency (%) Number Frequency (%)
RR 19 6.3 25 10.5
RH 118 39.1 94 39.5
HH 165 54.6 119 50.0
* Comparison of RR vs. ‘RH + HH’ genotypes: 2 × 2 chi-square analysis, P ⳱ 0.036
(1-tailed test).
Genotype frequencies for DHF cases vs. controls: 3 × 2 chi-square analysis, degrees of
freedom (df) ⳱ 2, P ⳱ 0.178.
Allele frequencies for DHF cases vs controls: 2 × 2 chi-square analysis, df ⳱ 1, P ⳱ 0.107.
TABLE 4
MBP54 polymorphism genotype frequencies in dengue hemorrhagic
fever (DHF) cases and controls*
MBP54
genotype
DHF cases (n ⳱ 340) DHF controls (n ⳱ 264)
Number Frequency (%) Number Frequency (%)
GG 241 70.9 202 76.5
GA 88 25.9 58 22.0
AA 11 3.2 4 1.5
* Genotype frequencies for DHF cases vs. controls: 3 × 2 chi-square analysis, degrees of
freedom (df) ⳱ 2, P ⳱ 0.187.
Allele frequencies for DHF cases vs. controls: 2 × 2 chi-square test, df ⳱ 1, P ⳱ 0.072.
TABLE 5
Interleukin-4 (IL-4) promoter polymorphism genotype frequencies in
dengue hemorrhagic fever (DHF) cases and controls
IL-4 promoter
genotype
DHF cases* (n ⳱ 147) DHF controls* (n ⳱ 147)
Number Frequency (%) Number Frequency (%)
CC 12 8.2 8 5.4
CT 46 31.3 49 33.3
TT 89 60.5 90 61.2
* Only a subset of the cases and controls were studied for this polymorphism.
Genotype frequencies: 3 × 2 chi-square analysis, degrees of freedom (df) ⳱ 2, P ⳱ 0.688.
Allele frequencies: 2 × 2 chi-square analysis, df ⳱ 1, P ⳱ 0.624.
TABLE 6
Interleukin-1 repeat allele (II-1RA) polymorphism genotype fre-
quencies in dengue hemorrhagic fever (DHF) cases and controls*
IL-1RA genotype
DHF cases (n ⳱ 280) DHF controls (n ⳱ 229)
Number Frequency (%) Number Frequency (%)
11 232 82.9 195 85.2
12 42 15.0 28 12.2
13 20.7 20.9
14 31.1 20.9
22 00 10.4
44 10.4 10.4
* Genotype frequencies: 6 × 2 chi-square analysis, degrees of freedom (df) ⳱ 5, P ⳱ 0.833.
Allele frequencies: 4 × 2 chi-square analysis, df ⳱ 3, P ⳱ 0.94.
LOKE AND OTHERS104

There is also evidence of a protective effect of homozygos-
ity for the arginine variant at amino acid position 131 of
Fc␥RII against DHF. Studies have reported a role for this
variant in several infectious disease, and there is preliminary
evidence that homozygotes for the arginine variant are more
susceptible than homozygotes for the histidine variant to in-
fections with encapsulated bacteria.
20,47,48
This is consistent
with the lower opsonic capacity of this variant for IgG2 and
the evidence that IgG2 is protective against disease caused by
many encapsulated bacteria.
17
Therefore, the association of
the genotype encoding homozygosity for the arginine variant
with resistance to DHF may relate to a pathogenic role for
disease-enhancing IgG2 antibodies in this disease.
The frequency of the RR polymorphism varies signifi-
cantly. It is much less common in Asian populations (RR
polymorphism ⳱ 6–10%) than in white and African popula-
tions from Europe, India, and North and South America (RR
polymorphism ⳱ 23–37%).
38,47,49
The variation in the fre-
quency of this polymorphism between Asian (Japanese, Chi-
nese, and Vietnamese) and South American populations
(Brazil) is of particular interest, given the apparent protective
effect we have demonstrated in this study and the increased
incidence of severe dengue hemorrhagic disease in Asia com-
pared with South America. Further assessments in compara-
tive studies of the geographic variation in this gene might be
important.
The availability of an increasing number of defined poly-
morphisms throughout the human genome will greatly in-
crease the potential power of genetic susceptibility studies in
dengue and should provide further insights into possible
mechanisms of pathogenesis and protection. This large study
demonstrates potentially important associations with two host
genes and susceptibility to the severe form of DHF and finds
no link with three other candidate genes. We used healthy
children, rather than children with milder forms of dengue, as
controls because we were primarily interested in what factors
led healthy children to develop severe dengue, and wanted to
avoid errors associated with assessment of the clinical state;
the World Health Organization classification of dengue has
been developed on clinical criteria and may not accurately
reflect the underlying pathogenesis. It is possible that a com-
ponent of susceptibility to severe dengue involves an increase
in the risk of acquiring a primary dengue infection, and fur-
ther studies in a separate cohort are underway to compare
DHF with DF to identify factors associated solely with the
severity of disease. We believe the current study is a first step
in piecing together the human factors that may underlie DHF.
Acknowledgments: We are very grateful to the Director and staff of
the Dong Nai Paediatric Centre for help and support during the
study. Dr. Cao Xuan Thanh Phuong, who supervised the clinical
aspects of this study, died tragically at the end of October 2000. Her
dedication to the care of sick children and enthusiasm for clinical
research are greatly missed by friends, colleagues, and the local com-
munity.
Financial support: This study was funded by the Wellcome Trust of
Great Britain.
Authors’ addresses: Hsin Loke, Wellcome Trust Centre for Human
Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 9DL,
United Kingdom. Delia Bethell and Nick Day, Centre for Tropical
Diseases, Nuffield Department of Clinical Medicine, John Radcliffe
Hospital, University of Oxford, Oxford, United Kingdom. Cao Xuan
Thanh Phuong, Dong Nai Paediatric Centre, Dong Nai Province,
Vietnam. Nicholas White, Centre for Tropical Diseases, Nuffield De-
partment of Clinical Medicine, John Radcliffe Hospital, University of
Oxford, Oxford, United Kingdom and Faculty of Tropical Medicine,
Mahidol University, Bangkok, Thailand. Jeremy Farrar, Centre for
Tropical Diseases, Nuffield Department of Clinical Medicine, John
Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
and University of Oxford-Wellcome Trust Clinical Research Unit,
Centre for Tropical Diseases, 190 Ben Ham Tu Quan 5, Ho Chi Minh
City, Vietnam, Telephone: 84-8-836-2225, Fax: 84-89238904, E-mail:
JEREMYJF@HCM.VNN.VN. Adrian Hill, Wellcome Trust Centre
for Human Genetics, University of Oxford, Roosevelt Drive, Oxford
OX3 9DL, United Kingdom and Centre for Tropical Diseases, Nuf-
field Department of Clinical Medicine, John Radcliffe Hospital, Uni-
versity of Oxford, Oxford, United Kingdom.
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