ORIGINAL STUDIES
The Use of Dengue Nonstructural Protein 1 Antigen for the
Early Diagnosis During the Febrile Stage in Patients With
Dengue Infection
Ampaiwan Chuansumrit, MD,* Wathanee Chaiyaratana, MSc,† Viroj Pongthanapisith, MSc,‡
Kanchana Tangnararatchakit, MD,* Sarapee Lertwongrath, BSc,§ and Sutee Yoksan, MD, PhD¶
Background: To evaluate the use of dengue nonstructural protein 1
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(NS1) antigen for the early diagnosis during the febrile stage in
patients with dengue infection.
Methods: A total of 445 sera obtained from 165 patients 关dengue
fever (DF): 42, dengue hemorrhagic fever (DHF) grade I: 50, II: 63,
III and IV: 10兴 and 8 other febrile illnesses 5–15 years of age, were
assayed for the NS1 antigen, dengue-specific Ig M and Ig G
antibodies.
Results: The positive rates of NS1 antigen among patients with
either DF or DHF was 100% (7 of 7) on day 2, 92.3% (12 of 13) on
day 3, 76.9% (40 of 52) on day 4, 56.5% (61 of 108) on day 5 of
fever; and declined to 43.1% (59 of 137) on day 6 with deferves-
cence and 29.8% (25 of 84) on day 7 (1 day after defervescence).
The positive rates of patients with DF were higher than those with
DHF but no statistically significant difference was found. However,
patients with primary DHF infection had significantly higher posi-
tive rates than those with secondary DHF infection. The positive
rates of Ig M antibodies were in reverse proportion to those of NS1
antigen. The additional Ig M antibody determination increased the
positive rates to 90.4% (47 of 52) on day 4, 83.3% (90 of 108) on
day 5 of fever; 95.6% (131 of 137) on day 6 with defervescence, and
88.1% (74 of 84) on day 7.
Conclusions: Dengue NS1 antigen testing is suggested as a helpful
tool for the early diagnosis of dengue infection after the onset of
fever. The additional Ig M antibody determination increased the
diagnostic rates.
Key Words: dengue nonstructural protein 1 antigen, NS1 antigen,
dengue fever, dengue hemorrhagic fever, early dengue diagnosis
(Pediatr Infect Dis J 2008;27: 43– 48)
Accepted for publication July 10, 2007.
From the *Department of Pediatrics, †Research Center, ‡Department of
Pathology, §Nursing, Faculty of Medicine, Ramathibodi Hospital,
Bangkok, and ¶Center for Vaccine Development, Institute of Science and
Technology for Research and Development, Nakhonpathom, Mahidol
University, Thailand.
Address for correspondence: Ampaiwan Chuansumrit, MD, Department of
Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University,
Rama VI Road, Bangkok 10400, Thailand. E-mail: raajs@mahidol.ac.th.
Copyright © 2007 by Lippincott Williams & Wilkins
ISSN: 0891-3668/08/2701-0043
DOI: 10.1097/INF.0b013e318150666d
The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008
D engue virus infection is the most common mosquito-
borne viral disease of public health significance1 caused
by any of the 4 serotypes (dengue 1– dengue 4). Clinical man-
ifestations range from asymptomatic infection, undifferential
fever, and influenza-like symptoms known as dengue fever (DF)
to the more severe manifestations of dengue hemorrhagic fever
(DHF). There are 3 stages of clinical manifestations, namely
febrile, toxic, and convalescent. The febrile stage lasts 2–7 days
followed by an abrupt fall to normal or subnormal body tem-
perature; the toxic stage lasts 24 – 48 hours; and finally, rapid
clinical recovery without sequelae in the convalescent stage. The
toxic stage is the most critical period requiring intensive sup-
portive care. DHF is characterized by similar signs and symp-
toms of DF, but it is followed by increased vascular permeability
inducing plasma leakage, and hemorrhage caused by vasculopa-
thy, thrombocytopenia, and mild coagulopathy. Patients may
end up with circulatory failure, shock, and death.
No specific antiviral treatment for DHF exists. Therapy
is symptomatic and is designated to correct pathophysiologic
disturbances and control the clinical manifestations of shock
and hemorrhage. Early diagnosis by laboratory testing is
essential to identify dengue virus infection during the febrile
stage and to adjust patient management. The major diagnostic
methods currently available are viral RNA detection by
reverse transcription PCR (RT-PCR)2– 4 or serologic testing
of dengue virus-specific Ig M antibodies determined by
enzyme-linked immunosorbent assay (ELISA)5,6. RT-PCR is
expensive and its routine use in clinical diagnostic laborato-
ries is difficult. Ig M antibodies do not become detectable
until 5–10 days after the onset of illness in cases of primary
infection and until 4 –5 days after the onset of illness in
secondary infection.
Young et al7 were the first to describe the development
of a nonstructural protein 1 (NS1) capture ELISA for the
detection of dengue NS1 antigen in the sera during the acute
febrile stage of patients with dengue infection. NS1 is one of
7 NS proteins produced during viral replication. All NS
proteins are intracellular proteins with the exception of NS1
protein, a secreted protein. A number of publications con-
cerning the detection of NS1 antigen in patients’ sera have
attempted to correlate or predict the outcome of disease
severity or and serve as a supplementary assay for early
dengue diagnosis.8 –11
In this study, we were interested in assessing the
dengue NS1 antigen in sera obtained from patients with
either DF or DHF during the entire clinical course of
43
Chuansumrit et al The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008

TABLE 1. Demographic Data of the Studied Patients

No. Patients
Diagnosis Median Age (yr)*
Total Male Female Primary† Secondary†

Dengue fever 42 24 18 5 37 10.6 (7.8 –12.7)

DHF grade I 50 32 18 12 38 11.2 (8.6 –12.9)
DHF grade II 63 34 29 13 50 12.4 (10.4 –14.2)
DHF grades III and IV 10 4 6 0 10 9.6 (6.4 –12.2)
Other febrile illnesses 8 3 5 — — 12.0 (6.5–14.6)
Total 173 97 76 30 135 11.3 (8.9 –13.5)
*Numbers in parentheses indicate interquartile range.
†
Type of dengue antibody response.

febrile, toxic, and convalescent stages. The usefulness of
NS1 antigen for the early diagnosis of dengue infection
was evaluated.
MATERIALS AND METHODS
Patients. One hundred sixty-five Thai children with dengue
virus infection and 8 other febrile illnesses 5–15 years of age
admitted at the Department of Pediatrics, Faculty of Medi-
cine, Ramathibodi Hospital, Thailand from 2002 to 2005
were included in the study. All patients survived. Ethical
approval was obtained from the Faculty Ethics Committee,
and informed consent was obtained from the parents. Chil-
dren had a venous blood sample drawn daily during hospi-
talization and at an outpatient clinic at 3– 4 weeks after
discharge from the hospital. The details for the diagnosis,
gender, type of dengue antibody response, median age, and
the number of patients are shown in Table 1.
D0 was the day of defervescence, when temperature
dropped below 37.5°C without a subsequent elevation. One
and 2 days before defervescence were designated as D-1,
D-2, and so on. Also, one and 2 days after defervescence were
designated as D⫹1 and D⫹2, respectively.
Diagnostic Criteria. Clinical diagnosis of DHF is based on 4
major characteristic manifestations defined by the World
Health Organization12: (1) sustained high fever lasting 2–7
days; (2) hemorrhagic tendency such as a positive tourniquet
test or petechiae; (3) thrombocytopenia (platelets ⱕ100,000/
␮L); and (4) evidence of plasma leakage caused by increased
vascular permeability manifested by hemoconcentration (an
increase in hematocrit of ⱖ20%) or pleural effusion. The
severity of DHF is categorized into 4 grades; grade I, without
overt bleeding but positive tourniquet test; grade II, with
clinical bleeding diathesis such as epistaxis and ecchymosis;
grade III, circulatory failure manifested by a rapid, weak
pulse and narrowing pulse pressure or hypotension with the
presence of cold, clammy skin and restlessness; and grade IV,
profound shock in which pulse and blood pressure are not
detected. In this study, patients without evidence of hemocon-
centration had a right lateral decubitus view of chest radio-
graph performed on the day after defervescence. All patients
with DF or DHF had a serologic confirmation of dengue
specific Ig M and Ig G antibodies in the acute and convales-
cent sera. Patients with other febrile illnesses who had no
dengue specific Ig M and Ig G antibody response were
presumed to most likely have self-limited viral illnesses and
were included as controls.
Laboratory Testing. A total of 445 serum specimens were
included. The number of consecutive specimens from each
patient was as follows: 1 (n ⫽ 18), 2 (n ⫽ 63), 3 (n ⫽ 55),
4 (n ⫽ 20), and 5 (n ⫽ 9). A total of 354 specimens were
collected from 135 patients with secondary infection (37 DF,
98 DHF) and 80 specimens were collected from 30 patients
with primary infection (5 DF, 25 DHF). An additional 11
specimens were collected from 8 patients with other febrile
illnesses (7 patients each had 1 specimen and 1 patient had 4
consecutive specimens). The full array of serum specimens
tested is shown in Table 2. All serum specimens were
aliquoted and stored at ⫺70°C until testing. Half of the
specimens had been previously thawed once or twice while
the remaining specimens were not thawed. All specimens
were examined for the dengue NS1 antigen, Ig M and Ig G
antibodies.
Dengue NS1 Antigen. The NS1 antigen was determined using
the Platelia Dengue NS1 Ag kit (Bio-Rad Laboratories,
Marnes La Coquette, France). It is a one-step sandwich-
format microplate enzyme immunoassay for detecting dengue
virus NS1 antigen in human plasma or serum. It makes use of
murine monoclonal antibodies (Mab) for capture and detec-

TABLE 2. The Distribution of Studied Sera According to the Day of Defervescence and the Day of Illness After the
Onset of Fever

Day of Defervescence D-4 D-3 D-2 D-1 D0 D⫹1 D⫹2 D⫹3

Total
Day of Illness Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9

DF 2 5 13 33 34 18 6 2 113
DHF 5 8 39 75 103 66 22 3 321
Other febrile illnesses 0 0 2 3 5 1 0 0 11
Total 7 13 54 111 142 85 28 5 445
Day 0 and Day 6 were the day of defervescence.

44 © 2007 Lippincott Williams & Wilkins

The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008 Dengue Infection

tion. Tests were carried out on sera from patients and controls
as described previously.13
Serologic Assay. The dengue-specific Ig M and Ig G antibod-
ies were determined by capture ELISA technique. Results
were expressed by the ratio of the optical density reading of
the sample and reference serum. Dengue infection was de-
fined as a primary infection when the ratio of dengue specific
Ig M to Ig G was ⬎1.8:1 and the ratio of ⬍1.8:1 was defined
as a secondary infection. Ig M antibody determination was
defined as a positive result when the level was ⱖ0.5 and ⱖ1
in association with the simultaneous Ig G antibody response
of ⱖ3 and ⬍3, respectively.14
Statistical Analysis. ␹2 of Fisher exact test was used for
discrete data, where appropriate. Mann–Whitney U or Wil-
coxon Signed Ranks tests were used for continuous data. P ⬍
0.05 was considered statistically significant.
RESULTS
The retrospectively studied sera were obtained from
larger number of males than females. No difference in age,
sex, and day of illness on study entry was found among the
DF, DHF, and control groups. The median duration of febrile
stage was 5 days (minimum 1 day, maximum 10 days) with
a interquartile range of 4 – 6 days. By analyzing the number of
days in relation to defervescence with the day of illness after
the onset of fever among 165 patients with dengue infection,
the results revealed that D-4 was the second day of illness
(day 2), D-3 was the third day (day 3), D-2 was the fourth day
(day 4), and D-1 was the fifth day (day 5) of illness with
fever. Then, D0, the day of defervescence was the sixth day
(day 6) of illness. Finally, D⫹1, D⫹2, D⫹3 were the seventh
day (day 7), eighth day (day 8), and ninth day (day 9) of
illness without fever, respectively.
One hundred forty-eight out of 173 patients (85.5%)
had 2–5 consecutive serum samples for simultaneously de-
termining NS1 antigen, Ig M and Ig G antibodies. The
positive rates of dengue NS1 antigen according to the day of
illness are shown in Table 3. The positive rates among
patients with DF were comparable with the published data13
and were higher than those with DHF but no statistically
significant difference was found. The positive rates among
patients with grades I and II were higher than those with
grades III and IV but no statistically significant difference
occurred. Furthermore, the positive rates of dengue NS1
antigen were analyzed by the status of primary and secondary
infection as shown in Figure 1. The patients with primary DF
infection had higher positive rates than those with secondary
DF infection but no statistically significant difference was
found. However, patients with primary DHF infection had
significantly higher positive rates than those with secondary
DHF infection on the day 5 of fever (88.2% versus 41.4%),
day 6 with defervescence (90.9% versus 25.9%), and day 7
(100% versus 12.5%) with P values of 0.001, 0.0001, and
0.0001, respectively. Most of the patients had positive results
followed by equivocal or negative results. Only 2 patients
with DF had negative results followed by positive results. Of
particular note, 11 sera obtained from patients with other
febrile illnesses were completely negative.
The positive rates of the Ig M antibody were in reverse
proportion to those of the NS1 antigen as shown in Figure 2.
The addition of the Ig M antibody determination for early
dengue diagnosis starting for day 4 of fever was tabulated.
The results revealed that the positive rates of the combination
were significantly higher than those of NS1 antigen alone
from day 4 and day 5 with fever to day 6 with defervescence
and day 7 without fever (day 4, 90.4% versus 76.9%; day 5,
83.3% versus 56.5%; day 6, 95.6% versus 43.1%; and day 7,
88.1% versus 29.8%) as shown in Figure 2.
Of note, the Ig M antibody determination in this study
was defined as a positive result when the level was ⱖ0.5 and
ⱖ1 in association with the simultaneous Ig G antibody
response of ⱖ3 and ⬍3, respectively. If the Ig M antibody
alone was determined, it was defined as a positive result when
the level was ⱖ1. Thus, the positive rates of Ig M antibodies
were slightly lower than those of Ig M interpretated with Ig
G antibodies (day 4, 34.6% versus 42.3%; day 5, 59.2%
versus 65.7%; day 6, 79.6% versus 92.0%, day 7, 77.4%
versus 84.5%). As a result, the positive rates of the combi-
nation of NS1 antigen and Ig M antibodies were slightly
lower than those of the NS1 antigen combined with Ig M
interpretated with Ig G antibodies (day 4, 84.6% versus
90.4%; day 5, 77.8% versus 83.3%; day 6, 83.9% versus
95.6%; and day 7, 83.3% versus 88.1%).

TABLE 3. Positive Dengue NS1 Antigen in Patients DISCUSSION

With Dengue Virus Fever (DF) and Dengue Hemorrhagic The designated date of D-4, D-3, D-2, D-1, D0, D⫹1,
Fever (DHF) Grades I–IV According to the Day of Illness D⫹2, and D⫹3 in relation to defervescence was essential for
After the Onset of Fever including patients with the same pathophysiological status
into the same group since the febrile stage lasts 2–7 days. If
Day of DF DHF Grade I DHF Grade II
DHF Grades III the days of illness such as day 1 to day 8 without concerning
and IV the relation to defervescence was used, an error in enrolling
Illness (n ⫽ 113) (n ⫽ 119) (n ⫽ 177)
(n ⫽ 25)
patients would occur. For instance, on day 5 of illness, some
Day 2 2/2 1/1 4/4 ND patients had fever, but other patients had no fever symptoms.
Day 3 5/5 2/3 5/5 ND
Day 4 12/13 8/12 17/23 3/4 However, the subsequent conversion of the number of days in
Day 5 22/33 14/28 24/43 1/4 relation to defervescence to the days of illness starting from
Day 6 18/34 17/44 23/52 1/7 the onset of fever will be conveniently applied for the clinical
Day 7 8/18 7/24 10/34 0/8
Day 8 3/6 3/7 5/13 0/2 practice.
Day 9 1/2 ND 0/3 ND The determination of the dengue NS1 antigen in this
ND indicates no data; Day 6, day of defervescence. study was performed by using an enzyme immunoassay

© 2007 Lippincott Williams & Wilkins 45

Chuansumrit et al The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008

FIGURE 1. Comparison of positive

dengue NS1 antigen between pri-
mary and secondary infection
among 165 patients with dengue
infection (A) which were classified as
dengue fever 42 patients (B) and
dengue hemorrhagic fever 123 pa-
tients (C) according to the day of
illness after the onset of fever.

representing a qualitative or semiquantitative detection. The
positive rates in patients with DF were comparable with the
previous study13 and were slightly higher than those with
DHF. High levels of the dengue NS1 antigen in patients with
DF and DHF have been reported by Libraty et al8 in 2002.
Additionally, positive rates were significantly higher in pa-
tients with primary infection compared with those with sec-
ondary infection especially in patients with DHF. Further-
more, the positive rates were slightly higher in patients with
DHF grades I and II compared with those with DHF grades
III and IV. Patients with DHF grades III and IV commonly
found with secondary infection. The absence of the NS1
antigen may be related to the circulation of antibodies. Ig G
dengue antibodies are produced early in the course of illness
during secondary infection while Ig M dengue antibodies
appear later during the course of a primary infection.
Apart from the history, physical examination and clin-
ical laboratory results of leucopenia and thrombocytopenia,15
dengue NS1 antigen testing is suggested as a supplementary
laboratory tool for the early diagnosis of dengue virus infec-

46 © 2007 Lippincott Williams & Wilkins

The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008
tion during the febrile stage especially the first 5 days of
illness. It is more likely to be positive the sooner it is
performed after the onset of fever. Patients with positive
results may end up with the manifestations of DF or DHF.
The addition of Ig M antibodies increases the early dengue
diagnostic rate. Moreover, the determination of a positive Ig
M antibody result at ⱖ0.5 in association with the simulta-
neous Ig G antibodies of ⱖ3 is helpful especially in patients
with secondary infection14. Their Ig G antibodies are rapidly
produced during the early course of illness while Ig M
antibodies are not as numerous as Ig G antibodies.
Patients with consecutive serum specimens should
show positive results followed by equivocal and negative
© 2007 Lippincott Williams & Wilkins
Dengue Infection
FIGURE 2. Comparison of positive
dengue NS1 antigen, Ig M antibod-
ies and combination of NS1 antigen
and Ig M antibodies among 165 pa-
tients with dengue virus infection (A)
which were classified as dengue fe-
ver 42 patients (B) and dengue
hemorrhagic fever 123 patients (C)
according to the day of illness after
the onset of fever.
results. However, 2 patients with DF in this study who had
negative results followed by positive results might be ex-
plained by a false negative study caused by the effect of
stored or thawed sera before testing. The process of storage
and thawing may decrease the sensitivity of the test.
The limitation of this study was that no determination
of dengue serotype was made among the studied patients. In the
previous study13, the sensitivity of dengue NS1 antigen testing
ranged from 87.9% to 90.5% among dengue serotypes 1– 4. In
addition, the number of patients with DHF grade IV and the
control group of other febrile illnesses was rather small. Thus,
the specificity of the test could not be concluded. Further
prospective study with serotype identification is warranted.
47
Chuansumrit et al The Pediatric Infectious Disease Journal • Volume 27, Number 1, January 2008
ACKNOWLEDGMENTS
This work was supported by the Thailand Research
Fund-Senior Research Scholar 2006 (A.C.)
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© 2007 Lippincott Williams & Wilkins