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Diagnostic Microbiology and Infectious Disease 60 (2008) 43 – 49
www.elsevier.com/locate/diagmicrobio
Virology
Evaluation of the Panbio dengue virus nonstructural 1 antigen detection
and immunoglobulin M antibody enzyme-linked immunosorbent assays
for the diagnosis of acute dengue infections in Laos☆
Stuart D. Blacksell a,b,c,⁎, Mammen P. Mammen, Jr., d , Soulignasack Thongpaseuth a ,
Robert V. Gibbons d , Richard G. Jarman d , Kemajittra Jenjaroen c , Ananda Nisalak d ,
Rattanaphone Phetsouvanh a , Paul N. Newton a,b , Nicholas P.J. Day a,b,c
a
Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research Collaboration, Microbiology Laboratory, Mahosot Hospital,
Vientiane, Lao PDR
b
Centre for Clinical Vaccinology and Tropical Medicine, Nuffield Department of Clinical Medicine, Churchill Hospital,
University of Oxford, Oxford OX3 7LJ, United Kingdom
c
Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
d
Armed Forces Research Institute of Medical Sciences, Bangkok 10400, Thailand
Received 16 January 2007; accepted 23 July 2007
Abstract
We evaluated 2 commercial enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of dengue infection; one a serologic test for
immunoglobulin M (IgM) antibodies, the other based on detection of dengue virus nonstructural 1 (NS1) antigen. Using gold standard
reference serology on paired sera, 41% (38/92 patients) were dengue confirmed, with 4 (11%) acute primary and 33 (87%) acute secondary
infections (1 was of indeterminate status). Sensitivity of the NS1-ELISA was 63% (95% confidence interval [CI], 53–73) on admission
samples but was much less sensitive (5%; 95% CI, 1–10) on convalescent samples. The IgM capture ELISA had a lower but statistically
equivalent sensitivity compared with the NS1-ELISA for admission samples (45%; 95% CI, 35–55) but was more sensitive on convalescent
samples (58%; 95% CI, 48–68). The results of the NS1 and IgM capture ELISAs were combined using a logical OR operator, increasing
the sensitivity for admission samples (79%; 95% CI, 71–87), convalescent samples (63%; 95% CI, 53–73), and all samples (71%; 95%
CI, 65–78).
© 2008 Elsevier Inc. All rights reserved.
Keywords: Dengue; Serology; Point-of-care; Diagnosis; Immunochromatographic; Laos; NS1; IgM
1. Introduction
The dengue virus is an important cause of acute febrile
illness in much of the tropics, encompassing a spectrum of
☆
The study was funded by the Wellcome Trust of Great Britain. The
Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand, and
the Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicine Research
Collaboration, Vientiane, Lao, PDR, has received test kits from Panbio for
evaluation purposes. The opinions or assertions contained herein are the
private ones of the authors and are not to be construed as official or as
reflecting the view of the US government.
⁎ Corresponding author. Wellcome Trust-Mahidol University-Oxford
Tropical Medicine Programme, Faculty of Tropical Medicine, Mahidol
University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. Tel.: +66-23549172; fax: +66-2-3549169.
E-mail address: [email protected] (S.D. Blacksell).
0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2007.07.011
disease including dengue fever, dengue hemorrhagic fever,
and dengue shock syndrome. Patients with dengue virus
infections present with symptoms and signs similar to those
of other acute tropical febrile illnesses, necessitating
laboratory tests for confirmation of the diagnosis (Gibbons
and Vaughn, 2002; Shu and Huang, 2004; Teles et al., 2005).
Serologic tests are most commonly used and rely on the
detection of dengue virus-specific immunoglobulin M (IgM)
and immunoglobulin G (IgG) antibodies. During the acute
phase, the presence of IgM antibodies alone suggests
primary infection, whereas detection of both IgM and IgG
antibodies is suggestive of secondary or later infection (Shu
and Huang, 2004). Rapid diagnostic tests using immunochromatographic or immunoblot technologies have been
developed for point-of-care serologic testing (Vaughn et al.,
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S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
1998), although these tests have generally demonstrated
limited clinical utility due to poor sensitivity on admission
samples (Blacksell et al., 2006).
Recent studies have shown that the dengue virus
nonstructural 1 (NS1) antigen, a highly conserved glycoprotein produced in both membrane-associated and secreted
forms and abundant in the serum of patients in the early
stages of dengue virus infection (Alcon et al., 2002; Dussart
et al., 2006; Young et al., 2000; Xu et al., 2006), may be an
appropriate marker of acute dengue virus infection. Here, we
evaluate the diagnostic utility of a commercial dengue IgM
antibody detection enzyme-linked immunosorbent assay
(ELISA) and a commercial ELISA kit for the detection of
dengue virus NS1 antigen in patients with a clinical
diagnosis of dengue in the endemic setting of the Lao
People's Democratic Republic (Laos).
2. Methods
2.1. Patient samples
The study was conducted at Mahosot Hospital, Vientiane,
Laos, between September 2004 and September 2005. Ethical
clearance was granted by the Ethical Review Committee of
the Faculty of Medical Sciences, National University of
Laos, Vientiane, Laos, and by the Oxford University
Tropical Ethics Research Committee, United Kingdom.
Patients were recruited to the study if they gave informed
written consent and if the responsible physician suspected
dengue virus infection on clinical grounds based on the
World Health Organization (1997) guidelines (an acute
febrile illness with 2 or more of the following signs:
headache, retro-orbital pain, myalgia, arthralgia, rash,
hemorrhagic manifestations, and leukopenia). Venous
blood samples were collected on the day of admission
(admission specimen) and discharge from hospital (convalescent specimen). Serum was divided for immediate use
and for storage at −80 °C.
2.2. Enzyme-linked immunosorbent assays
Commercial ELISAs for the detection of dengue virus
NS1 antigen (cat. no. E-DEN01A, lot no. 06200; Panbio,
Brisbane, Australia, provided for evaluation purposes)
and dengue virus IgM antibody capture ELISA (cat. no.
E-DEN01M; Panbio) were performed according to the
manufacturer's instructions. Results were calculated as
“Panbio units” with results b9.0, 9.0–11.0, and ≥ 11.0
defined as negative, equivocal, and positive, respectively.
Samples that initially returned an equivocal result were
retested to confirm the result.
2.3. Dengue reference assays
Dengue reference assays were performed at the Armed
Forces Research Institute of Medical Sciences (AFRIMS),
Bangkok, Thailand. Dengue virus infections were confirmed
on an individual patient basis using the results of paired
admission and convalescent specimens tested by the
AFRIMS IgM and IgG antibody capture ELISAs using
the following diagnostic interpretations as previously
described (Innis et al., 1989; Vaughn et al., 1999). For
paired specimens, admission samples with b15 U of
dengue virus IgM antibodies rising to ≥30 U in the
convalescent specimen (with dengue IgM antibodies
greater than Japanese encephalitis virus [JEV] IgM
antibodies) was considered evidence of an acute primary
dengue virus infection. In the absence of IgM N40 U in
the admission specimen, a 2-fold rise in IgG to a value
≥100 U was indicative of a secondary or later dengue
virus infection. In addition, dengue virus primary and
secondary infections were also determined when the ratio
of anti-dengue virus IgM to IgG is N1.8 to b1.8,
respectively. Specimens were considered negative for
serologic evidence for a recent dengue virus infection if a
paired specimen collected at least 7 days apart were
absent of dengue-specific antibody as defined above. The
reverse transcriptase–polymerase chain reaction (RT-PCR)
was used to determine the serotype identity (Blacksell et
al., 2006; Lanciotti et al., 1992). Both reference serology
and RT-PCR assays were performed blinded to the results
of dengue virus NS1 antigen or IgM capture ELISAs. All
samples were labeled using a code that was devoid of
personal identifiers.
2.4. Non-dengue serology
The diagnosis of JEV infection was confirmed by the
detection of specific IgM antibodies by the AFRIMS JEV
IgM capture ELISA (Burke and Nisalak, 1982). Sera were
screened for the presence of Chikungunya virus antibodies
using the hemagglutination inhibition (Clarke and Casals,
1958) method at a 1:10 dilution. The presence of antibodies
against Orientia tsutsugamushi (scrub typhus) and Rickettsia typhi (murine typhus) were assessed with an indirect
micro-immunofluorescence assay (Robinson et al., 1976)
using a 4-fold (or greater) rising titer suggesting acute
infection (Coleman et al., 2002).
2.5. Analysis
Diagnostic accuracy was calculated for the dengue virus
NS1-ELISA and IgM capture ELISAs relative to the final
patient diagnosis (i.e., dengue-positive or dengue-negative)
based on the results of reference serology. To estimate
assay accuracy, NS1-ELISA and IgM capture ELISA
results that were confirmed to be of equivocal status by
retesting were regarded as negative. Diagnostic accuracy
indices of sensitivity, specificity, negative predictive values
(NPVs), and positive predictive values (PPVs) with exact
95% confidence intervals (CIs), interquartile (IQR) ranges
of days of fever, and area under the receiver operating
characteristic curves (AUROCCs) were calculated using
Stata/SE 8.0 (Stata, College Station, TX). Statistically
significant differences (P b 0.05) in the Panbio unit results
S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
45
Table 1
Summary of samples, results, and diagnostic accuracy scores for the NS1 and IgM detection ELISAs
Assay
Results (Panbio unitsa)
Samples
Sample
timing
n
Days with Non-dengue
fever
cases (median
(median
[IQR])
[IQR])
NS1 ELISA Admission
92 5 (4–7)
Convalescent 92 9 (7–12)
All samples 184 7 (5–10)
IgM ELISA Admission
92 5 (4–7)
Convalescent 92 9 (7–12)
All samples 184 7 (5–10)
Combined
Admission
92 5 (4–7)
Convalescent 92 9 (7–12)
All samples 184 7 (5–10)
a
b
c
4.4 (3.7–5.2)
4.3 (3.8–4.8)
4.4 (3.7–4.9)
3.5 (2.9–4.9)
5.1 (2.9–16.6)
3.9 (2.9–7.5)
NA
NA
NA
Diagnostic accuracy (95% CI)
Dengue cases
Sensitivity
(median [IQR])
Specificity
PPVb
NPVc
14.2 (5.0–28.1)
3.6 (3.2–4.4)
4.6 (3.4–15.4)
9.5 (3.7–28.8)
14.1 (3.7–36.6)
12.6 (3.7–34.4)
NA
NA
NA
100
100
100
94.4 (89.8–99.1)
72.2 (63.1–81.4)
83.3 (78.0–88.7)
94.4 (84.8–99.1)
72.2 (63.1–81.4)
83.3 (78.0–88.7)
100
100
100
85.0 (77.7–92.3)
59.5 (49.4–69.5)
68.4 (61.7–75.1)
90.9 (75.7–98.1)
61.5 (51.6–71.5)
75.0 (68.7–81.3)
79.4 (71.2–87.7)
60.0 (50.0–70.0)
68.4 (61.6–75.1)
70.8 (61.6–80.1)
70.9 (61.6–80.2)
70.9 (64.3–77.4)
86.4 (74.4–93.4)
73.6 (64.6–82.6)
89.4 (74.6–86.1)
63.2 (53.4–73.0)
5.3 (0.7–9.8)
34.2 (27.4–41.1)
44.7 (34.6–54.9)
57.9 (47.8–68.0)
51.3 (44.1–58.5)
79.0 (62.7–90.5)
63.2 (53.3–73.0)
71.1 (64.5–77.6)
b9.0, negative; 9.0–11.0, equivocal; ≥11.0, positive.
Positive predictive value.
Negative predictive value.
NS1-ELISA and IgM capture ELISA results for the
diagnosis of dengue? To answer this question, the
results of the NS1-ELISA and IgM capture ELISA
were combined using Boolean operators (i.e., AND,
OR, NOT) to derive a final cumulative result for each
patient sample. A positive combined result was
assigned to those patient samples that were positive
for NS1-ELISA and/or the IgM capture ELISA (i.e.,
either or both assays). The combined patient sample
results were compared to the final patient diagnosis
based on the reference assay results to calculate
accuracy scores.
and positivity rates were calculated using Student t test
and McNemar test, respectively.
2.6. Practical assessment of diagnostic utility
To examine and compare the true diagnostic utility of the
NS1-ELISA and IgM capture ELISA in a clinical setting, the
following 3 questions were posed:
1) In a patient presenting with suspected acute dengue
virus infection, how accurate are the NS1-ELISA and
IgM capture ELISA for making the diagnosis of
dengue? To answer this question, the NS1-ELISA and
IgM capture ELISA results for the admission sample
were compared with the final patient diagnosis (based
on paired reference serology).
2) In a patient who has been recently acutely ill with
dengue-like symptoms and is now recovering (such as
a returning traveler from a dengue virus endemic
region) (Wichmann and Jelinek, 2004), how accurate
are the NS1-ELISA and IgM capture ELISA for
making the diagnosis of dengue? To answer this
question, the NS1-ELISA and IgM capture ELISA
results for the convalescent sample were compared
with the final patient diagnosis (based on paired
reference serology).
3) In a patient presenting with suspected acute dengue
virus infection, how accurate is the combination of
3. Results
3.1. Patient samples and reference diagnosis
Ninety-two patients were recruited into the study and
41% (38/92) were diagnosed with dengue virus infection
(Table 1) as defined by AFRIMS diagnostic criteria. All
4 dengue serotypes were represented (Table 2). Both
admission and convalescent samples from all 92 patients
were tested by NS1-ELISA and IgM capture ELISA
(Table 1). The median IQR number of days between
admission and convalescent sera was 6 (5–7). Of the 92
patients without a reference diagnosis of dengue, 12 were
diagnosed as having scrub typhus (13%), 4 with murine
Table 2
Sensitivity of the dengue virus NS1 antigen and IgM antibody detection ELISAs by infection status and serotype for admission samples
ELISA
format
NS1
IgM
a
% Positivity (n positive/n true dengue cases × 100)
Disease classification (n = 38)a
Serotype (n = 25)
Acute primary
Acute secondary or later
DEN-1
DEN-2
DEN-3
DEN-4
75.0 (3/4)
75.0 (3/4)
60.1 (20/33)
39.4 (13/33)
77.8 (7/9)
22.2 (2/9)
60.0 (3/5)
25.0 (2/5)
0 (0/2)
50.0 (1/2)
66.7 (6/9)
33.3 (3/9)
One dengue confirmed case was of indeterminate infection status.
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S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
typhus (4%), 1 with JEV (1%), and 1 with Streptococcus
pyogenes septicemia (1%). Chikungunya virus antibodies
were not detected, and no diagnosis was available for
39% (36/92) of patients. Clinical information for 87
patients has recently been reported elsewhere (Blacksell
et al., 2007).
3.2. Diagnostic utility in a clinical setting
1) How accurate are the NS1-ELISA and IgM capture
ELISA for the diagnosis of acute dengue? The
sensitivity of the NS1-ELISA on admission specimens
was 63% (24/38) with 100% specificity (Table 1). For
the IgM capture ELISA, the sensitivity for admission
samples was 45% (17/38) and specificity was 94%
(51/54). There was no significant difference in
admission sample results between the 2 ELISAs for
the distribution of results (P = 0.52, McNemar test)
and receiver operating curve analysis (P = 0.51)
(AUROCC: NS1-ELISA = 0.783 and IgM capture
ELISA = 0.751) (Fig. 1).
2) How accurate are the NS1-ELISA and IgM capture
ELISA for the diagnosis of later-presentation dengue?
The sensitivity of the NS1-ELISA and IgM capture
ELISA using convalescent samples were 5% (2/38)
and 58% (22/38) respectively, and were significantly
different (P b 0.00005, McNemar test) (Table 1). The
NS1-ELISA and IgM capture ELISA were 100% and
72% (39/54) specific, respectively.
3) How accurate is the combination of NS1-ELISA
and IgM capture ELISA results for the diagnosis of
acute dengue? The results of the NS1 and IgM
capture ELISAs were combined using a logical OR
to give a final cumulative result that increased the
sensitivity of admission (79%, 30/38) and convalescent (63%, 24/38) samples. The sensitivity
using the cumulative result when all samples were
tested was 71% (54/76) and specificity was 83%
(90/108) (Table 1). All samples positive in both the
NS1-ELISA and IgM capture ELISAs (n = 11)
were taken on admission after 4–8 days of fever
(Fig. 2).
3.3. Comparison of admission and convalescent
sample results
Sample timing (admission and convalescent specimens)
influenced the sensitivity of both ELISAs. For the 38
dengue-confirmed cases, the median Panbio unit result for
the NS1-ELISA was significantly higher for admission
than for convalescent samples (14.2 vs 3.6, P b 0.0005,
Student t test) and the sensitivity was significantly different (P b 0.00005, Student t test) between acute and
convalescent samples. The NS1-ELISA sensitivity for
patients sampled during the first 2 days of fever was
100% (3/3 of true dengue infection patient samples were
positive) (Fig. 3), whereas after 3–4, 5–6, and N7 days, it
was 57% (4/7), 36% (9/25), and 24% (10/41), respectively.
The IgM capture ELISA demonstrated a nonsignificant
(P = 0.1, paired Student t test) increase in median Panbio
unit results (9.5 versus 14.1) between admission and
convalescent samples in confirmed dengue cases. For the
non-dengue cases, the median Panbio unit results for both
assays were all below the b9 units positivity cutoff. The
Fig. 1. AUROCC analysis that compares the accuracy of the dengue virus NS1-ELISA and IgM capture ELISA.
S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
47
Fig. 2. Individual and dual positivity for dengue virus NS1-ELISA and IgM capture ELISA compared with days of fever for dengue virus-confirmed samples
(n = 76 samples).
IgM capture ELISA showed no significant (P = 0.33,
McNemar test) difference in positivity between admission
(45%, 17/38) and convalescent samples (58%; 22/38) in
confirmed dengue cases.
3.4. The influence of infecting dengue virus serotype
Dengue serotype information was available for 66%
(25/38) of the dengue cases, with all 4 dengue serotypes
Fig. 3. Overall positivity for dengue virus NS1-ELISA and IgM capture ELISA compared with days of fever for dengue virus-confirmed samples
(n = 76 samples).
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S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
represented (Table 2). The IgM capture ELISA detected all
serotypes, but the NS1-ELISA was negative for the 2
patients with dengue virus serotype 3 infections.
3.5. The influence of infection status
The majority (87%; 33/38) of dengue patients were
serologically classified as acute secondary or later infections,
with 4 patients (11%) classified as having acute primary
infections with 1 patient having indeterminate infection
status (Table 2). For admission specimens, the NS1-ELISA
detected 75% (3/4) of the acute primary and 60% (20/33) of
the acute secondary (or later) infections. The IgM capture
ELISA detected the same proportion (75%) of acute primary
infections and 39% (13/33) of the acute secondary (or later)
infections. This was not significantly different (P = 0.18
acute primary, P = 0.13 acute secondary, McNemar test)
from those of the NS1-ELISA results.
4. Discussion
Adequate test sensitivity is essential for the accurate
laboratory diagnosis of dengue virus infection (Wichmann
et al., 2006). Results of this study demonstrate that a
commercial NS1 antigen ELISA, especially when used
together with a dengue virus IgM capture ELISA, is
sufficiently sensitive and specific to be clinically informative
in an endemic setting.
Sample timing is an important consideration in the
serologic diagnosis of dengue virus infections. During an
acute primary dengue virus infection, the detection of dengue
IgM antibodies commences approximately 5 days after onset
of fever and 3 days after defervescence (Vaughn et al., 1997).
A sample collected in the febrile phase of dengue virus
infection may have insufficient detectable IgM antibody and
hence result in a false-negative serologic diagnosis, whereas
a second sample collected during the convalescent phase
may improve the accuracy of diagnosis. In contrast, NS1
antigens are detectable in serum from 1 to 9 days after the
onset of fever (Alcon et al., 2002; Huang et al., 1999). In this
study, we posed 3 practical diagnostic questions that relate to
clinical presentation, which in turn dictates sample availability and timing. On acute presentation, the NS1 ELISA
demonstrated higher sensitivity and specificity scores than
the IgM capture ELISA, although these increased values
were not statistically significant. The IgM capture ELISA
first detected antibodies at day 3 after fever onset with the
peak of detectable antibodies at 7 days, but the NS1-ELISA
detected NS1 antigen from day 1 onward followed by a
plateau of NS1 positivity in true dengue virus infection
patients over subsequent days, making it an appropriate test
for the diagnosis of dengue virus infection in the acutely
febrile patient. The high specificity of the NS1-ELISA
demonstrated in this study (100%) is also a major asset in the
tropical setting where many other illnesses serologically
cross-react with other flaviviruses, notably JEV (Teles et al.,
2005). The diagnostic sensitivity was improved for admission samples if the results of the 2 assays were combined in a
logical “OR” manner, although as expected there was a
marginal (6%) drop in specificity and in PPV (9%).
Comparison of the NS1 antigen and IgM capture ELISA
results by days of fever showed periods of exclusive
positivity for one target analyte (NS1 antigen-early infection:
IgM antibody-later infection), with an overlap of dual
positivity at 4–8 days of fever. If an acute specimen tests
negative for NS1 antigen, despite a clinical suspicion of
dengue, the specimen should be tested for the presence of
dengue IgM antibodies.
Recent studies have reported higher sensitivity results for
both the NS1 antigen (Xu et al., 2006; Kumarasamy et al.,
2006; Dussart et al., 2006) and Panbio IgM capture ELISA
(Cuzzubbo et al., 1999; Groen et al., 2000) than those
presented here. The lower sensitivity described here may be
attributable to the stringent patient diagnosis reference
comparator, using paired sera, and the high proportion of
secondary dengue virus infections. The use of individual
sample (admission or convalescent alone) results that are
assigned a dengue reference status after testing with
imperfect reference comparators (i.e., PCR, IgM ELISA,
Haemagglutination Inhibition) can artificially inflate sensitivity scores, especially in the case of admission samples.
That the majority (87%) of the patient samples in this study
were acute secondary infections, with only a small number
(11%) of acute primary dengue infection cases, may also
have affected the accuracy results. The NS1-ELISA had
increased sensitivity for detection of acute primary dengue
virus infections compared to secondary infections, consistent
with a recent assessment of a commercial NS1 ELISA in
Malaysia (Kumarasamy et al., 2006). The high proportion of
secondary infections in our cohort may also account for the
lower than expected sensitivity of the IgM capture ELISA in
later samples, as the IgM antibody response may be
“blunted” after high IgG titer secondary or later infections
(Chanama et al., 2004; Schilling et al., 2004). These results
illustrate the utility of the NS1-ELISA in dengue endemic
settings where multiple serotypes circulate and there is a high
incidence of secondary or later infections. However, the
apparent inability of the NS1-ELISA to detect dengue virus
serotype 3 infections is a concern, although there were
insufficient number of patients (n = 2) infected with this
serotype to determine the true nature of this potential
limitation. While the sensitivity results presented here are
lower than those of other evaluations, these probably reflect
the real-life sensitivity of the assays.
Acknowledgments
We are very grateful to all the patients who participated in
this study, the doctors, nurses, and staff of the Microbiology
Laboratory and Mahosot Hospital, especially Douangdao
Soukaloun, Simmaly Phongmany, Sengmanivong Khounnorath, Khonesavanh Luangxay, Bouachanh Rasachak,
S.D. Blacksell et al. / Diagnostic Microbiology and Infectious Disease 60 (2008) 43–49
Vimone Soukhaseum, Valy Keolouangkot, Konkam
Sisouk, Mayfong Mayxay, Anisone Changthongthip, Olay
Lattana, Manivanh Vongsouvath, Viengmone Davong,
Phonelavanh Phouminh, Sonbandit Duangsy, Sengmani
Symanivong, Viengmala Sihalath, Kai-amporn Keopaseuth,
the pediatric and medical wards, and the AFRIMS staff for
the characterization of the specimens. We thank Professors
Chanpheng Thammavong and Bounkong Syhavong, the
Minister of Health, His Excellency Dr Ponmek Dalaloy
and the Director of the Curative Department, Ministry of
Health of the Lao PDR, and Professor Sommone
Phounsavath, for their support for this study, which was
part of the Wellcome Trust-Mahosot Hospital-Oxford
Tropical Medicine Research Collaboration funded by the
Wellcome Trust of Great Britain.
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