Clinical and Virological Factors Influencing the

Performance of a NS1 Antigen-Capture Assay and

Potential Use as a Marker of Dengue Disease Severity
Veasna Duong1, Sowath Ly1, Patrich Lorn Try2, Anne Tuiskunen3, Sivuth Ong1, Norith Chroeung2, Ake
Lundkvist3, Isabelle Leparc-Goffart4, Vincent Deubel1, Sirenda Vong1, Philippe Buchy1*
1 Institut Pasteur in Cambodia, Reseau International des Instituts Pasteur, Phnom Penh, Cambodia, 2 Paediatric Department, Kampong Cham Provincial Hospital,
Kampong Cham, Cambodia, 3 Swedish Center for Infectious Disease Control, Stockholm, Sweden, 4 Unite de Virologie, Institut de Medecine Tropicale du Service de Sante
des Armees, Marseille, France

Abstract
Background: Detection of dengue NS1 antigen in acute infection has been proposed for early diagnosis of dengue disease.
The aim of this study was to evaluate the clinical and virological factors influencing the performance of the Platelia NS1 Ag
kit (BioRad) and to assess the potential use of NS1 antigen and dengue viral loads as markers of dengue disease severity.
Methodology/Principal Findings: Blood specimens were collected from patients hospitalized at the Kampong Cham
hospital during the 2006 and 2007 dengue epidemics in Cambodia. Dengue infection was confirmed in 243/339
symptomatic patients and in 17 asymptomatic individuals out of 214 household members tested. Overall sensitivity and
specificity of Platelia NS1 Ag kit were 57.5% and 100% respectively. NS1 Ag assay combined with IgM antibody capture
ELISA significantly increased the sensitivity for dengue diagnosis. NS1 Ag positivity rate was found significantly higher in DF
than in DHF/DSS, in primary than in secondary infections, in patients with a high viremia (.5 log/mL) and in patients
infected with DENV-1. In asymptomatic individuals, the NS1 Ag capture sensitivity tends to be lower than that in
symptomatic patients. Milder disease severity was observed independently in patients with RNA copy number .5 log10
cDNA equivalents/mL or in high level of NS1 antigen ratio or in DENV-1 infection.
Conclusions: Overall sensitivity of NS1 Ag detection kit varied widely across the various forms of dengue infection or
disease. Sensitivity was highest in patients sampled during the first 3 days after onset of fever, in patients with primary
infection, DENV-1 infection, with high level of viremia and in DF rather than DHF/DSS. In asymptomatic patients, RT-PCR
assay has proved to be more sensitive than NS1 antigen detection. The NS1 antigen level correlated significantly with
viremia and a low NS1 antigen ratio was associated with more severe disease.
Citation: Duong V, Ly S, Lorn Try P, Tuiskunen A, Ong S, et al. (2011) Clinical and Virological Factors Influencing the Performance of a NS1 Antigen-Capture Assay
and Potential Use as a Marker of Dengue Disease Severity. PLoS Negl Trop Dis 5(7): e1244. doi:10.1371/journal.pntd.0001244
Editor: Maria G. Guzman, Tropical Medicine Institute Pedro Kour (IPK), Cuba
Received October 23, 2010; Accepted June 3, 2011; Published July 19, 2011
Copyright: 2011 Duong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the European Union under the DENFRAME project (6th Framework Programme: 2005-INCO-DEV2-nu517711). The funders
had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: pbuchy@pasteur-kh.org

rapid urbanization, failure to control vector mosquitoes and rapid

progress in air transportation have contributed to the emergence
of endemic dengue in over 100 countries [2,4].
In Cambodia, there is a high incidence of reported diseases (DF,
DHF) of 10,00012,000 cases annually during 20022006 and the
case-fatality rate was 12% over the past 5 years [5].
Commonly used diagnosis methods are often unable to confirm
dengue infection during the acute febrile stage in a timely manner
and at a reasonable cost [3,6]. Virus isolation is a time-consuming
and fastidious process that requires specialized laboratory
equipments and experienced personnel. The development of
reverse transcriptase polymerase chain reaction (RT-PCR) and
recently real time RT-PCR techniques have significantly reduced
the processing time and permitted the detection of the virus in the
early stage of the infection [7]. However, these methods remain
expensive and technically difficult, particularly in laboratory

Introduction
Dengue virus (DENV), a mosquito-borne virus (family Flaviviridae, genus Flavivirus) is an enveloped, single stranded positive-sense
RNA virus. There are 4 serologically related but antigenically
and genetically distinct dengue viruses (DENV-1, -2, -3, and -4)
causing disease in human. While most infections result in asymptomatic response or mild febrile illness (dengue fever or DF), all 4
serotypes are capable of producing the more severe and potentially
fatal dengue hemorrhagic fever (DHF) and dengue shock
syndrome (DSS) and non-specific complication of systemic diseases
(e.g., encephalitis, hepatitis) [1,2,3].
With over 2.5 billion people living in area at high risk for
infection and an estimated 50100 million cases of dengue
infection every year, DENV has become the most important
arthropod-borne virus affecting human [2]. Several factors such as
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Dengue NS1 Capture Test and Disease Severity

believed to constitute an important reservoir of the virus in

countries where dengue is highly endemic [18,19].

Author Summary
Dengue is the most prevalent arthropod-borne disease in
tropical regions. The clinical manifestation may vary from
asymptomatic to potentially fatal dengue shock syndrome.
Early laboratory confirmation of dengue diagnosis is
essential since many symptoms are not specific. Dengue
non-structural protein 1 (NS1) may be used in simple
antigen-capture ELISA for early detection of dengue virus
infection. Our result demonstrated that the Platelia NS1
antigen detection kit had a quite low overall sensitivity.
However, sensitivity rises significantly when used in
combination with MAC-ELISA. When taking into account
the various forms of dengue infection, the NS1 antigen
detection was found relatively high in patients sampled
during the first 3 days of fever onset, in patients with
primary infection, DENV-1 infection, with high level of
viremia and in mild form of dengue fever. In asymptomatically infected individuals, RT-PCR assay has proved to be
more sensitive than NS1 antigen detection. Moreover, the
NS1 antigen level correlated significantly with high viremia
and low level of NS1 antigen was associated with more
severe disease.

Materials and Methods

Patients recruitment and samples processing
Patients were enrolled in the pediatric ward of the Kampong
Cham provincial hospital during 2 consecutive dengue epidemics
between May and October in 2006 and 2007. This study was
approved by the National Cambodian Ethics Committee and
patients enrolment was subject to obtaining a written consent
signed by the patients or the under 16 year old patients legal
representatives.
Patients clinically diagnosed with dengue infection and who
fulfilled the following inclusion criteria were enrolled: age $$24
months, fever .38uC and having at least one of the symptoms:
rash or severe headache or retro-orbital pain or myalgia or joint
pain or bleeding symptom. Patients information and clinical data
were collected by the physicians using a specific case report form
and blood samples were taken on hospital admission and
discharge. Patients diagnosed for other infections beside dengue
and patients hospitalized with a non-infectious disease (e.g., cranial
trauma, etc.) were recruited as control group.
Blood samples were tested for haematocrit and platelet count as
well as for other biological parameters necessary for patients followup. Sera were tested for dengue using serology and molecular
methods at Institut Pasteur - Cambodia.
Patients diagnosed for dengue infections were classified as DF,
DHF or DSS using the former WHO criteria [20] as recommended
at the time of the study.
In order to identify non-symptomatic cases, family members
of dengue-infected patients were visited the next day following
laboratory confirmation of the dengue infection (which usually took
approximately 24 hours). Their body temperature was followed for 7
days. Blood samples were taken at the first and 7th day of follow-up
and if a family member developed fever. A non-symptomatic dengue
case was defined as a household member who tested positive for
dengue infection but did not display any of the symptoms of the
inclusion criteria.

settings of the developing world. Serological diagnosis of dengue

infection has many advantages including more flexibility, wide
availability of reagents, lower cost, and less equipments required
[3]. Unfortunately, cross-reactivity between flaviviruses, antibody
half-life, need of paired sera, and inability to be detected in acute
phase of infection make the serological diagnosis more complicated [3] .
Due to the drawbacks of serological methods to reliably
diagnose acute infections, a number of alternative options have
been explored [7] and one of the most promising methods was the
detection of nonstructural protein 1 (NS1) . NS1, produced in both
membrane-associated and secreted forms, may play an essential
role in viral replication. The amount of secreted NS1 (sNS1) in the
serum of individuals infected with DENV has been shown to
directly correlate with viremia and the pathogenesis of dengue
infection [8,9,10,11,12].
The NS1 protein is detectable by enzyme-linked immunosorbent assay (ELISA) as early as the first day of fever and can be
found up to 9 days in serum even after RT-PCR detection has
become negative [9,13,14,15]. Given all these advantages, NS1based ELISAs may be an important diagnostic tool for those acute
samples in which IgM is not detectable and for which PCR is not
available. Thus, NS1 antigen might be useful in early detection
and a potential prognosis parameter for severe dengue infections.
Several NS1 antigen commercial kits are now available and most
of them have been evaluated for their sensitivity and specificity in
patients experiencing clinically apparent infections. The sensitivity
observed for these assays varied from 63% to 94% [8,14,16,17].
In this study, we evaluated the clinical and virological factors
influencing the performance of NS1 antigen-capture assay and
assessed the potential association of the level of NS1 antigenemia
(using simple semi-quantitative estimation) and the level of viremia
with dengue disease severity using well characterized sera from
patients presenting at hospitals. NS1 antigen test could be an easy
tool to confirm dengue infection in such individuals on a single
blood sample during the acute phase of the disease rather than by
indirect methods that require at least 2 samples. We also evaluated
the test in asymptomatically dengue-infected individuals. Indeed,
asymptomatic individuals and unreported patient with mild febrile
disease who represent the vast majority of dengue infection are
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Laboratory diagnosis
A confirmed dengue infection ("gold standard algorithm") was
defined by the detection of anti-dengue virus (DENV)-specific IgM
or a 4 fold increase of hemagglutination inhibition (HI) titer in the
pair of sera collected with an interval of minimum 7 days and the
detection of NS1 antigen in serum by the NS1 Platelia test
(BioRad, Hercules, CA) and/or the isolation of DENV after
inoculation into mosquito cell lines and/or the detection DENV
RNA by RT-PCR or real time RT-PCR assay.
An in-house IgM capture Enzyme-Linked Immuno-Sorbent
Assay (MAC-ELISA) was used to detect DENV and JEV IgM as
the 2 viruses co-circulate in the country [21,22]. A result was
considered positive when the optical density (OD) was . mean
OD of three negative control specimens +3 standard deviations.
When the anti-JEV result was higher than the anti-DENV result,
the subject was not considered to have DENV infection.
HI test was carried out according to the method described
by Clarke and Casals [23] adapted to 96-well microtitre plate.
Due to the serological cross-reactivity between arboviruses, paired
specimens were tested for DENV and JEV hemagglutinationinhibiting antibodies. Primary or secondary acute dengue infection
was determined by a fourfold increase in HI titer between the
first and second sample according to criteria established by the
WHO [20].
2

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Dengue NS1 Capture Test and Disease Severity

During the household investigation, 17 (8%) dengue-infected

individuals who did not experience any symptoms and 2 (1%)
symptomatic household members of 15 dengue index cases (DIC)
were identified among 214 household members (Table S1).
Figure 1 shows the overall positive rate of NS1-antigen capture
assay, RT-PCR and MAC-ELISA in relationship with the day after
onset of fever. During the first 2 days in the course of the disease the
sensitivity of NS1 assay and RT-PCR reached the highest values
(81% and 90.5%, respectively) and then decreased to less than 20%
and 45.5% respectively by day 78. On the contrary, the number of
specimens positive by MAC-ELISA increased steadily from less
than 20% at day 12 to 100% by day 7.
The sensitivity and specificity of the NS1-capture assay were
57.7% (95% CI: 51.463.8%) and 100% respectively (Table 2).
When diagnosed solely by RT-PCR, the sensitivity was 77.3%
(95% CI: 71.782.2) and the specificity 100%. When samples were
collected during the first 3 days of illness, the sensitivity of the NS1
commercial test improved: 74% (95% CI: 62.883.4) in the
dengue-confirmed case group and 82.3% (95% CI: 69.590.0) in
the group of individuals who tested positive by RT-PCR only.
The NS1 antigen kit combined with MAC-ELISA detected a
significantly higher number of acute dengue cases than NS1
antigen kit alone (overall sensitivity: 85.7% vs. 57.7%;
p,0.001, Table S2). An increased sensitivity was also observed
when combining RT-PCR and MAC-ELISA results (overall
sensitivity: 95.4% vs. 77.3% for RT-PCR alone, p,0.001,
Table S2). The comparison with DOF subgroups was detailed
in Table S2.
When analyzing all dengue-infected individuals, the NS1
antigen ratio correlated with the RNA load of cDNA equivalents
per milliliter (r = 0.540, p,0.001; Fig. 2).
The overall sensitivity of the NS1 detection kit was significantly
higher in DF (72.3%; 95% CI: 63.581%) than in DHF/DSS
(40.2; 95% CI: 29.8551.3). But the difference in NS1-capture
assay sensitivity was significant only for samples collected after day
3 of fever and not for specimen obtained during the very early
phase of the disease (Table 3).
The sensitivity of the NS1-capture assay was significantly
higher in primary dengue infection (87.5%; 95% CI: 70.096.5)
than in secondary infection (53.5%; 95% CI: 46.160.7)
(p,0.001). The difference was also significant in the DOF 48
group (p = 0.002) and at the limit of significance in the DOF 13
group (p = 0.055).
The sensitivity of the test also varied with the virus serotype. It
was significantly higher in DENV-1-infected patients (80%; 95%
CI: 6789.6) than in DENV-2- (40%; 95% CI: 12.273.8,
p = 0.008), DENV-3- (63.6%; 95% CI: 54.472.2, p = 0.03) and
DENV-4- (53.3%; 95% CI: 26.678.7, p = 0.03) infected patients
although the difference was only significant for the DOF 48
group (Table 4).
DF was significantly more frequent after infection with DENV-1,
compared to other dengue virus infections (38/47, [80.85%, 95%
CI: 66.790.8] vs. 4/8 [50%,95% CI: 15.784.3] for DENV-2, 42/
89 [47.2%, 95% CI: 36.558] for DENV-3 and 5/9 [55.6%, 95%
CI: 21.286.3] for DENV-4; p = 0.002). The proportion of primary
infections in DENV-1-infected patients was 20% versus 12.3% for
the overall studied population (p = 0.13).
Of 201 RT-PCR positive samples, 189 were tested for RNA
quantification using real-time RT-PCR. Among these, 70 samples
were containing RNA levels lower than the detection limit of the
real time RT-PCR. During clinical classification, 44 cases were
excluded from the analysis because the clinical information was
not sufficiently precise to allow a classification according the
WHO criteria. NS1 antigen-capture assays sensitivity was

The NS1 Platelia antigen detection (BioRad, Marnes-laCoquette, France) was performed on patients sera according to
the manufacturers instructions. Samples with equivocal result
were repeated and if they were still equivocal they were considered
as negative. The optical density (OD) reading obtained with a
spectrophotometer at 450/620 nm is proportional to the amount
of NS1 antigen present in the sample [13]. The assay provides
qualitative and semi-quantitative results in human serum or
plasma. The semi-quantitative results were expressed as the ratio
calculated by dividing the absorbance measured on the sample by
the mean value of the optical densities of 2 cut-off controls. The
cut-off value corresponds to the mean value of the OD of the cutoff control provided and tested in duplicate.
The isolation of DENV was performed using mosquito cell line
(clone C6/36 of Ae. Albopictus cells). Briefly, each acute serum was
diluted 1:20 with L15 Leibovitz Medium (Sigma Aldrich,
Steinheim, Germany) in which 2% of fetal calf serum was added.
Diluted sera were inoculated into 12-well plate containing 100%
confluent C6/36 cells and then incubated for 7 days at 28uC. Cells
were harvested, and DENV infection was confirmed by an
immunofluorescence assay using dengue serotype-specific monoclonal antibodies as described previously [21,22].
Viral RNA was extracted from acute phase serum samples using
the QIAmp Viral RNA Mini kit (Qiagen, Hilden, Germany). The
DENV serotype was determined by RT-PCR based on the
technique developed by Lanciotti et al. [24] and modified by
Reynes et al. [25]. The positive samples by conventional RT-PCR
were then tested for dengue viral loads by a serotype-specific realtime RT-PCR assay targeting NS5 gene using quantified internal
controls [26]. The results were expressed as cDNA equivalents per
milliliter of serum. The limit of detection for this assay was 500
cDNA equivalent/mL.

Statistical analysis
All statistical analyses were performed using Stata/SE version
9.0 (StataCorp, TX, USA). Significance was assigned at P,0.05
for all parameters and 95% of interval confidence was used.
Categorical variables between groups were compared by Pearsons
Chi-squared and Fishers exact test. T-test and Kruskal-Wallis
rank test were used for continuous variables. The correlation
coefficients between 2 continuous variables were calculated by
Spearmans rank correlation test. For multivariate analyses, we
identified independent determinants using a logistic regression
model. For clarity, adjustments by the day after onset of fever
(DOF) was performed and presented using DOF as a categorical
variable: #3 days and 48 days. In some analyses, DHF and DSS
were grouped to increase statistical power.

Results
A total of 134 and 205 patients were enrolled in 2006 and 2007,
respectively, of which 243 patients were diagnosed with acute
dengue infection, 62 with non-dengue infection and 17 as having
non infectious disease. The summary of patients characteristics,
clinical and virological data of this study is shown in Table 1.
Using the former WHO criteria [20], 101 dengue patients were
classified as DF, 42 as DHF, 45 as dengue with DSS and 72 as
indeterminate. The inability to classify these 72 patients was due to
the lack of clinical and laboratory data necessary for the
classification or they did not meet all the four WHO criteria (see
materials and methods). After measuring HI titers on paired sera,
dengue cases were classified in primary infections (n = 32, 14.5%),
secondary infections (n = 189, 85.5%) but in 39 cases it was not
possible to determine the immune status.
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Dengue NS1 Capture Test and Disease Severity

Table 1. Summary of demographic, clinical and virological information of studied population.

Variables

Years
2006

2007

Total

Acute dengueSymptomatic Asymptomatic

107#90/117 (77%)17/214 (8%)

153#153/205 (75.5%)0

260#243/322 (75.5%)17/214 (8%)

Non-dengue infection

27

35

62

Non infectious disease

17

17

Age (median, iqr*)

8 (511)

6 (48)

7 (49)

Sex (female, %)

50 (46.7%)

86 (56%)

136 (52.3%)

Median of day of illness (range)

4 (26)

5 (18)

4 (18)

Dengue diagnosis (n = 260)

Virus isolation

48 (45%)

46 (30%)

94 (36%)

RT-PCR

91 (85%)

110 (72%)

201 (77%)

NS1 antigen assay

73 (68.2%)

77 (50.3%)

150 (57.7%)

MAC-ELISA Positive in acute

serumSeroconversionNegative

97 (90.6%) 21 (19.5%)
76 (71%)10 (9.4%)

148 (96.7%)92 (60%)

56 (36.7%)5 (3.3%)

245 (94.2%)113 (43.5%)

132 (50.7%)15 (5.8%)

Hemagglutination-Inhibition assay (titer)Fourfold

rise in antibodies on pair seraNo change or less
than fourfold riseData not available

87 (81.3%)
7 (6.5%)
13 (12.2%)

134 (87.6%)
7 (4.6%)
12 (7.8%)

221 (85%)
14 (5.4%)
25 (9.6%)

DENV-1

40 (37%)

15 (10%)

55 (21%)

DENV-2

2 (2%)

8 (5%)

10 (4%)

DENV-3

47 (44%)

74 (48.5%)

121 (46.5%)

DENV-4

2 (2 %)

13 (8.5 %)

15 (5.8%)

Unknown serotype

16 (15%)

43 (28%)

59 (22.7%)

DF

73 (68%)

28 (18%)

101 (39%)

DHF

17 (16%)

25 (16%)

42 (16%)

DSS

45 (29.5%)

45 (17%)

Indeterminate clinical status

17 (16%)

55 (36%)

72 (28%)

Primary

24/87 (28%)

8/134 (6%)

32/221 (14.5%)

Secondary

63/87 (72%)

126/134 (94%)

189/221 (85.5%)

Indeterminate or unknown

20

19

39

DENV serotypes

Clinical manifestation

Serological status

*interquartile range.
#
Numbers used as denominator for each column, otherwise indicated.

Insufficient serum volume or no second serum.

doi:10.1371/journal.pntd.0001244.t001

significantly higher in patients with viremia .5 log cDNA

equivalents/mL than ,5 log cDNA equivalents/mL regardless
the clinical severity and day of sample collection after onset of
fever (Table 5). The overall NS1 antigen-capture sensitivity in
patients with viremia .5 log cDNA equivalents/mL was 91%
versus 45% in patients with viremia ,5 log equivalents/mL
(p,0.001).
In asymptomatic individuals the sensitivity of NS1 test was
significantly lower than that in DIC (35.3% versus 86.7%,
p = 0.003; Table S1) and at the limit of significance when
compared to the sensitivity observed in all symptomatic cases
(59.3%, p = 0.053). Seventy three percent (8/11) of the asymptomatic individuals experienced secondary infection which was
lower than in DIC (100%, p = 0.063). The levels of viremia
expressed in log10 cDNA equivalents/mL in asymptomatic
individuals was significantly lower than in DIC (2.72, SD: 2.72,
n = 13 vs. 4.96, SD: 2.37, n = 15; p = 0.043) but the difference was
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not significant if compared with the level of viremia in all dengue

confirmed cases (3.79, SD: 3.06, n = 176; p = 0.145).
In these asymptomatic individuals, nested RT-PCR detection
was significantly more sensitive than NS1 antigen-capture assay
(76.5% vs. 35.3%, p = 0.015).
In multivariate analysis, DHF/DSS were independently associated with secondary infection (adjusted OR = 6.6, p = 0.01) when
controlled with age, day of fever onset, DENV serotypes and
immunity status (primary/secondary infection). Out of 77 DHF/
DSS patients, 74 (96%) had secondary dengue infection. Milder
disease severity was associated with high NS1 antigen level
(adjusted OR: 0.21, p = 0.002) (Table S3A and Fig. 3A) or DENV1 infection (adjusted OR: 0.083, p = 0.006). Similar results were
found in multivariate analysis when using the number of cDNA
copies instead of NS1 antigen OD ratio: association persisted
between DHF/DSS and secondary infection (adjusted OR = 6.03,
p = 0.01) and milder disease severity was observed in patients with
4

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Figure 1. Sensitivity of Platelia NS1, MAC-ELISA and RT-PCR depending on DOF* (n = 239). *DOF: Day after onset of fever.
doi:10.1371/journal.pntd.0001244.g001

cDNA copy number .5 log10 cDNA equivalents/mL (adjusted

OR = 0.33, p = 0.019) (Table S3B and Fig. 3B).

Platelia kit. The relatively low sensitivity NS1 antigen detection in

the current study is probably due to the high number of secondary
infections (85.5%) which reflects of the true situation in Cambodia
and other dengue hyper-endemic countries [14,29,30]. Indeed, we
recorded a lower sensitivity of the NS1 antigen-capture assay in
secondary infections in comparison to primary infections (87.5%
vs. 53.5%). Anti-NS1 antibodies are more frequently detected in
dengue secondary infection [31] and the antibody-antigen
complex impedes the tests ability to detect free NS1 [9,12]. A
dissociation of NS1 antibody-antigen immune complexes would
increase the sensitivity of NS1 antigen detection [31] but such a
method is unfortunately not offered in the commercial kits and was
not performed in our study.
When the NS1 antigen assay was coupled with MAC-ELISA,
the overall sensitivity increased by 28%. This combination of NS1antigen capture assay and IgM antibody detection for dengue
diagnosis showed higher sensitivity than RT-PCR alone and a
slightly lower sensitivity than RT-PCR combined with IgM
antibody detection. When performed together, NS1 antigencapture and IgM assays appear to be highly sensitive and
complementary, allowing a sufficiently good presumptive (IgM)
or definitive (NS1) diagnosis during the acute and the convalescent
phase of the disease. This advantage of the combination was

Discussion
DENV NS1 antigen is detected in the blood circulation as early
as viral RNA [8,9,12,13]. Thus its detection is useful for early
dengue diagnosis and could be used as an easy, fast and feasible
alternative to RT-PCR in developing countries. For this reason,
the sensitivity of a commercial NS1 antigen detection kit was
studied in context of several factors: severity of the infection
including asymptomatic dengue-infected individuals, time of
sampling, serological status (primary or secondary infection),
DENV serotype and level of viremia in acute sample.
The overall sensitivity of Platelia Dengue NS1 Ag kit (58%) is
slightly lower than that observed in previous studies (6394%)
[8,14,16,27,28], although, the excellent specificity reported here is
in agreement with results provided by other authors (98.4100%)
[8,14,16,27,28]. The sensitivity of the test was better during the
early stage of the illness (before day 4). The modest overall
sensitivity reported here was comparable to that of a recent multicountry NS1 antigen assay evaluation [28] which showed a 66%
(range: 34% to 76%) sensitivity of the NS1 antigen detection by

Table 2. Sensitivity, specificity, positive and negative predictive values of Platelia NS1 assay against dengue-confirmed cases.

Sensitivity
% [CI95%]

Specificity
% [CI95%]

NPV %
[CI95%]

p
value$

100

41.8 [34.749.2]

p,0.001

100

62.3 [47.975.2]

p,0.001

29.7 [21.439.1]

p,0.001

Studied
population

Acute dengue
infection

NS1
positive

PPV %
[CI95%]

Total

339*

260#

150{

57.7 [51.463.8]

100

DOF 13

110

77

57

74 [62.883.4]

100

DOF 48

196

163

85

52.2 [44.260.0]

100

100

*33 cases with imprecise DOF were excluded.

#
20 cases with imprecise DOF were excluded.
{
8 cases with imprecise DOF were excluded.
$
P values refer to 262 contingency comparison between % of NS1 positive cases and % of dengue confirmed cases.
PPV: positive predictive value.
NPV: negative predictive value.
doi:10.1371/journal.pntd.0001244.t002

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Dengue NS1 Capture Test and Disease Severity

Figure 2. Comparison of NS1 antigen OD* ratio and viral load (log 10 cDNA equivalents/mL). *OD: optical density.
doi:10.1371/journal.pntd.0001244.g002

DENV-1 infection than that in patients infected with any of the

three other DENV serotypes, although the difference was
significant only when DENV-1 was compared with DENV-3
(data not shown). Other factors like a better affinity of the NS1
probe and monoclonal antibodies used in the assay for the DENV1 strains circulating in Cambodia or the variations in the
performances of the RT-PCR method used to establish the
diagnosis [28] could also explain this observation. A multi-country
evaluation of NS1 antigen capture assay has shown that the
sensitivity was highest in DENV-1 infection and lowest in DENV2 [28]. Additionally, two Vietnamese studies has also shown that

positively demonstrated in the multi-country study by Guzman

et al. [28]. Moreover, both assays are easy to perform, fast, require
limited equipments and expertise, and are affordable. The
combination of NS1 antigen and dengue IgM/IgG used in rapid
diagnostic test (RDT) format for dengue infection detection has
shown in previous studies to be more sensitive than NS1 antigen
detection alone and can be used as a point of care diagnosis
[30,32].
The sensitivity of NS1 antigen-capture assay was significantly
higher for DENV-1 than for the three other serotypes. This could
be explained by the higher level of viremia measured during

Table 3. Sensitivity of Platelia NS1 assay in DFand DHF/DSS patients according to timing of sample collection after DOF*.

No. of sera tested

NS1 positive

DF (n = 101)

DHF/DSS (n = 87)

No. of Positive/
total tested

Sensitivity
% [95% CI]

No. of positive/
total tested

p value$
Sensitivity
% [95% CI]

Total

188

108#

73/101

72.3 [63.581]

35/87

40.23 [29.8551.3]

p,0.001

DOF 13

55

43

31/41

75.6 [59.787.6]

12 /14

85.7 [57.298.2]

p = 0.407

DOF 48

119

60

38/49

77.5 [63.488.2]

22/70

31.4 [20.843.6]

p,0.001

*DOF: Day after onset of fever.

14 cases with imprecise DOF were excluded.
5 cases with imprecise DOF were excluded.
$
P values refer to the comparison between NS1 positive rate of DF vs. DHF/DSS for total and for each DOF subgroup.
CI: confidence interval.
doi:10.1371/journal.pntd.0001244.t003

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Dengue NS1 Capture Test and Disease Severity

Table 4. Sensitivity of NS1 assay for each DENV serotype.

DENV-1

DENV-2

DENV-3

DENV-4

NS1 positive/ Sensitivity%

total tested# [95% CI]

NS1 positive/ Sensitivity%

total tested
[95% CI]

NS1 positive/
total tested{

Sensitivity%
[95% CI]

NS1 positive
/total tested

Sensitivity
% [95% CI]

p value$

Total

44/55

80.0 [67.089.6]

4/10

40.0 [12.273.8]

77/121

63.6 [54.472.2]

8/15

53.3 [26.678.7]

p,0.05

Day 13

21/25

84 [63.995.5]

2/3

66.6 [9.499.2]

27/32

84.3 [67.294.7]

3/5

60.0 [14.794.7]

p.0.05

Day 48

21/25

84.0 [63.995.5]

2/7

28.6 [3.770.9]

48/79

60.7 [49.1 71.6] 5/10

50.0 [18.781.3]

p,0.05

5 cases with imprecise DOF were excluded.

10 cases with imprecise DOF were excluded.
P values refer to comparison between NS1 positive cases in DENV-1 and DENV-2, DENV-3 or DENV-4 groups.
CI: confidence interval.
doi:10.1371/journal.pntd.0001244.t004

{
$

the sensitivity in DENV-1 infection was significantly higher than in

DENV-2 but not in DENV-3 infected patients and data on
DENV-4 was not available [8,33]. The lowest sensitivity of NS1
antigen capture assay observed in DENV-2 infections (40%) particularly at DOF 48 - might only be a bias due to the limited
number of DENV-2 cases included in our study.
Unlike other self limited viral diseases, dengue infection may
develop into the life threatening DSS form in a few days. A test
allowing early diagnosis, which can predict a risk of subsequent
evolution to the severe form is desirable in order to improve the
clinical management of dengue infection. This could reduce
unnecessary use of antibiotics, hospitalization of patients with
milder disease in countries with limited resources and allow early
hospitalization and supportive care of those developing potentially
life threatening DHF. Quantification of viremia by real-time RTPCR methods might be useful in this regard [9,10] but is expensive
and not readily available in endemic regions, which hampers its
use in clinical practice.
This present study demonstrates a moderate correlation of the
semi-quantitative result of NS1 antigen-capture assay with the
level of viremia quantified by real time RT-PCR. This finding is in
agreement with previous studies in which NS1 antigen-capture
assay was demonstrated in vitro to be applicable as an easy and fast
method for semi-quantification of DENV in cell culture [12,13,34]
and NS1 levels were found in vivo to correlate with viremia level
[8,9]. However, as also stated by Ludert et al. [34], a limitation of
the use of Platelia NS1 antigen capture kit as a semi-quantitative
test was that we did not use quantified NS1 protein as internal
control and our sera were not serially diluted.
As expected and already largely described, DHF/DSS cases are
more frequently observed in secondary infection with an adjusted
odd ratio of 6.6 [10,31,35,36]. The apparent lowest severity of

DENV-1 infections observed in our study is partially in agreement

with data published by Vaughn et al. [10] who reported that this
serotype caused less severe pleural effusion than DENV-2 but not
than DENV-3 and DENV-4 secondary infections. Due to the low
number of DENV-2 and also DENV-4 cases recruited in our study
but also at the country level (with DENV-2 representing 9.2% and
9.1% and DENV-4 accounting for 2.9% and 3.1% of the
serotypes isolated out of the 16,635 and 39,618 dengue cases
reported in 2006 and 2007, respectively) [5], we cannot discuss it
further.
Interestingly, the mildest dengue infection was also associated
with high NS1 antigen level semi-quantitatively measured by the
Platelia Dengue NS1 Ag kit (OR = 0.21, p = 0.002) and in patients
infected with DENV-1 (OR = 0.083, p = 0.006). These findings
contrasted with those of studies which showed conversely that a
higher viremia titer [10] and NS1 plasma levels were associated
with more severe disease [9]. Of note, these studies were
conducted on fewer cases and measured the viremia in patients
recruited less than 72 hours after fever onset while our results were
based on more patients, although only 14/84 DHF/DSS cases
were included before DOF 4, and we included additional
characteristics that were controlled in the multivariate analysis
(i.e. patients age, day of sample collection after fever onset, DENV
serotypes and anti-DENV immune status with well characterized
clinical and biological data from hospitalized patients). In addition,
in our series, all ambiguous data in regard to severity or primary/
secondary dengue infection classification were excluded. The
multivariate analysis in principle would avoid the confounding
factor introduced by the higher proportion of DENV-1 infections,
associated with higher NS1 titers, observed among mild DF cases.
Nonetheless, in a multi-country study, Guzman et al. did not find
any association between the NS1 detection and disease severity

Table 5. Sensitivity of NS1 test compared with level of viral RNA in serum (log10 cDNA equivalents/mL).

DF (n = 88*)NS1 positive/total tested [sensitivity; 95% CI]

DHF/DSS (n = 57#)NS1 positive/total tested [sensitivity; 95% CI]

,5 log/ml

.5 log /ml

P value

,5 log/ml

.5 log /ml

p value

Total

19/33 [57.6%; 39.274.5]

51/55 [92.7%; 82.498]

p,0.001

14/41 [34% ; 20.150.6]

15/16 [93.8%; 69.899.8]

p,0.001

DOF 13

7/11 [63.6%; 30.889.1]

24/26 [92.3%; 74.899]

p = 0.035

1/2[50%; 1.298.7]

10/10 [100%; 69.2100]

p.0.05

DOF 48

12/17 [70.6%; 4489.7]

24/25 [96%; 79.699.9]

p = 0.021

13/38 [34.2%; 19.651.4]

5/5 [100%; 47.8100]

p = 0.009

*9 cases with imprecise DOF were excluded.

#
2 cases with imprecise DOF were excluded.
CI: confidence interval.
doi:10.1371/journal.pntd.0001244.t005

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Dengue NS1 Capture Test and Disease Severity

Figure 3. Level of NS1 antigen and viremia by disease severity. Shown are the median, interquartile and 95 percent range of OD ratio of NS1
antigen (A) and log10 cDNA equivalents/mL (B) distributed by disease severity (DF, DHF/DSS). The number of patients is shown under the X axis bar.
NS1 antigen OD ratio was significantly higher in DF group than in DHF/DSS group (p,0.001) at DOF 48 (A). Log10 cDNA equivalents/ml was
significantly higher in DF group than in DHF/DSS group at DOF 48 (p,0.001) (B).
doi:10.1371/journal.pntd.0001244.g003

enrolled during the period of this study. The enhanced antiDENV immune response associated with the severity of the disease
[37] and leading to an increased infected cell mass at the early
stages of the disease may afterwards accelerate the virus clearance
from the serum. Since the Platelia NS1 assays sensitivity is
enhanced after immune complexes dissociation [14,31], the lower
antigenemia or viremia observed in the severe cases could be the
result of a higher anti-NS1 immune response.
Indeed, it has already been suggested that the sNS1 can be
trapped within immune complexes which impedes the detection
by the antigen-capture assays by preventing the plate-bound or

[28]. Since our study was the first to find this association and
considering the limitations in the use of Platelia kit for a semiquantification of NS1 antigen, a more explicit study will be needed
to confirm our results.
The same multivariate analysis but using viral load found that
patients with viremia lower than 5 log10 cDNA equivalents/mL
experienced more severe dengue infection (28% vs. 62.5%,
adjusted OR = 0.33, p = 0.019). The result suggests that low level
of viral load and NS1 antigen increases the likelihood of
developing severe dengue infection at least in the context of
Cambodian DENV strains in circulation and/or population
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Dengue NS1 Capture Test and Disease Severity

probe monoclonal antibodies to access the NS1 target epitopes

[31]. Koraka et al. have shown that the dissociation of NS1
antigen-antibody immune complexes improved the sensitivity of
their in-house test, particularly in sera collected during secondary
infections [31]. Lapphra et al. observed an increase of sensitivity of
the Platelia kit by 10% using acid treatment [14]. Unfortunately,
immune complex dissociation was not performed in our study to
confirm these findings on Cambodian samples.
Immune complex formation with sNS1 [12,38] and sNS1
binding to endothelial cells [39] have been proposed as potential
factors in DHF pathogenesis. In addition, antibodies directed
against NS1 cross-react with human platelets and endothelial cells
[40]. Anti-NS1 antibodies induce endothelial cells to undergo
apoptosis and in vitro experiments demonstrated that these
antibodies were responsible for an increased endothelial cell
monolayer permeability [40]. NS1 may also activate complement
by alternative pathway and this might explain the complement
activation observed in infants with DHF during primary
infections [41].
Our two former hypotheses in their attempts to explain the low
antigenemia and viremia observed in the severe cases do not take
into consideration the role of the virus. Some strains might be
more virulent than others and we cannot rule out the possibility
that a lower antigenemia and viremia could be at least partially
also the consequence of a lower virus replication. Indeed, our
observation is supported by in vitro experiments conducted by
Tuiskunen et al (personal communication) using the DENV strains
collected in this study during the same epidemic year. The in vitro
study demonstrated that DENV-1 strain isolated from severe
dengue infection (DSS) had lower level of replication in
mammalian Vero cells than strains isolated from DF and DHF
patients. Nonetheless, a non significant relationship between
disease severity and level of NS1 antigen or DENV serotype
detected in patients was reported elsewhere [8,28], Hence, our
findings might be relevant only for the DENV-1 strains circulating
in Cambodia.
Another interesting aspect of this study was the recruitment of
individuals asymptomatically infected. In this group, NS1 antigencapture assay was significantly less sensitive than in the DIC
(p = 0.003). The lower sensitivity of NS1 antigen-capture in
asymptomatic patients might be explained by the lower level of
viremia in these individuals (Table S1). Along the same line, RTPCR detection was more sensitive than NS1 antigen-capture assay
in detecting infection in apparently healthy individuals (76.5% vs.
35.3% respectively, p = 0.01) but the difference was not significant
in DIC. The level of viremia in asymptomatic cases was not
significantly lower than in all dengue confirmed cases (p = 0.145).
Since asymptomatic individuals did not experience more primary
infections than DIC, this observation is probably not related to the
presence of more anti-NS1 antibodies in one group rather in the
other. In addition, the positivity rate of RT-PCR or real time RTPCR was not significantly different between the two groups.
Additional evaluations using greater numbers of asymptomatic
cases would probably be helpful to address more explicitly the

question of the mechanism of the lower sensitivity of NS1 antigen

capture test in this particular group.
In conclusion, we have shown the usefulness of qualitative result
of NS1 antigen detection assay in early recognition of dengue
infection particularly in combination with IgM test. The point of
care rapid diagnostic tests including NS1 antigen and IgM/IgG
detection would be probably a helpful tool for early dengue
infection diagnosis in clinical practice but these tests need to be
further extensively evaluated.
The evaluation of the Platelia NS1 Ag detection kit exhibited a
quite low overall sensitivity. These data suggest that the NS1
antigen results should be interpreted with caution when used
alone. However, its sensitivity was relatively high in patients who
were sampled during the first 3 days after the onset of fever, in
patients with primary infection, in patients with DENV-1
infection, in patients experiencing a high level of viremia and in
patients with dengue fever forms. In asymptomatic patients, RTPCR assay has proved to be more sensitive than NS1 antigen
detection.
Moreover, using the semi-quantitative approach of the test, we
have demonstrated that the NS1 antigen level was significantly
correlated to the level of viremia and that the low level of NS1
antigen was associated with more severe disease.

Supporting Information
Table S1 Virological results and clinical features of
dengue index cases and household members (in 74
households).
(DOC)
Table S2 Comparison of NS1 kit or RT-PCR sensitivity
against the combination of each assay with MAC-ELISA.
(DOC)
Table S3 A. Multivariate analysis of factors* associated
with DHF/DSS. B. Multivariate analysis of factors*
associated with DHF/DSS.
(DOC)
Checklist S1 STARD checklist.

(DOC)

Acknowledgments
We would like to express our sincere thanks to patients, nurses, doctors
who participated in this study at Kampong Cham reference hospital,
Cambodia and lab technicians in Virology Unit and administrative teams
of Institut Pasteur in Cambodia who made this work possible. Finally, we
thank Dr Monica Naughtin for editorial assistance.

Author Contributions
Conceived and designed the experiments: PB V. Duong V. Deubel.
Performed the experiments: V. Duong SO. Analyzed the data: V. Duong
SL SV PB. Contributed reagents/materials/analysis tools: PLT AT NC
AL ILG. Wrote the paper: V. Duong AL ILG V. Deubel SV PB.

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