12 Dengue Seroprevalence Comparison

Volume - III, Issue-1
National Journal of Basic Medical Sciences
DENGUE: SEROPREVALENCE, COMPARISON OF RAPID TEST

WITH ELISA
Jayasimha V.L1, Thippeswamy M.T.R2, Yogesh Babu K.V3, Vinodkumar C.S4, Niranjan H.P5,
6
7
Raghukumar K.G , Basavarajappa K.G.
ABSTRACT
INTRODUCTION
Introduction: - Dengue is a major health problem in many

parts of the tropical world. It is a mosquito borne illness
caused by one of the serotypes of dengue viruses.
Dengue is a major health problem in many parts of

tropical world. Dengue is caused by infection with one of
the four serotypes of dengue virus (DEN 1- 4) which are
1,2
Arboviruses belonging to the Flaviviridae family
and
are transmitted by mosquito principally Aedes aegypti.
Infection with dengue virus may be clinically apparent or
may be present as a nonspecific febrile illness, Classic
3
dengue fever or Dengue Hemorrhagic Fever (DHF) .
Aims and objectives: - The present study was done to

know the common clinical features and Seroprevalence
of dengue in our region. An attempt was made to
compare rapid test SD dengue duo (IgM, IgG & NS-1 Ag
detection) with capture ELISA (IgM, IgG Microlisa
Dengue).
The major diagnostic methods currently available are

viral culture, viral RNA detection by reverse transcriptase
PCR (RT-PCR) & serological tests such as IgM Capture &
IgG Capture ELISA. However early dengue diagnosis still
remains a major problem as all these assays have their
own pitfalls. The first two assays have restricted scope as
4
a routine diagnostic procedure . Viral isolation by
Immunoflourescence though a gold standard cannot be
used as a routine diagnostic procedure due to its low
sensitivity, laborious procedure & time consumption. The
MAC- ELISA which is a commonly used assay has low
5-7
sensitivity in first few days of illness.
Materials & Methods: - 226 serum samples were tested

in patients clinically suspected Dengue. All the 226
samples were subjected to IgG, IgM Microlisa test. The
same were put on rapid SD bioline Dengue duo rapid test
and was compared with ELISA.
Results:- 226 serum samples were tested in patients
clinically suspected Dengue before noting common
clinical signs and symptoms. 150 samples were tested
positive with ELISA (either positive for IgG, IgM or both).
Seroprevalence of 66% were reported. When compared
with ELISA, Rapid test showed sensitivity of 80.6%
specificity and positive predictive value of 100% & zero
false positive rates. Efficiency of the test was 87.16%
Now- a- days detection of NS-1 Ag on rapid tests offer an

even faster route to a presumptive dengue diagnosis.
NS-1 (Non structural protein) is a highly conserved
glycoprotein that is essential for the viability of Dengue
virus & is produced both in membrane associated &
8
secretary forms by the virus . The detection of secretary
NS-1 protein represents a new approach to the diagnosis
of dengue infection.
Conclusion:- High prevalence rate in our region

particularly in premonsoon & monsoon season gives an
alarm to the doctors regarding early and accurate
diagnosis of dengue virus infection. SD Dengue duo rapid
test should be a valuable screening test for dengue fever
which can be interpreted easily. Results were comparable
to ELISA. It provides additional diagnostic investigation
that compliments NS-1 antigen detection.
The present study aims to determine the common clinical

features and Seroprevalence of Dengue virus infection in
Davangere & an attempt was made to compare the rapid
test SD dengue duo (IgG, IgM, and NS-1 Ag detection)
with Capture ELISA (IgG, IgM) (Microlisa Dengue)
Key words: Dengue, ELISA, rapid test, NS-1 antigen
Associate Professor, Dept of Microbiology, 2Professor, Dept of Pathology,

Associate Professor, Dept of Microbiology, 4Assistant Professor, Dept of Microbiology,
5
Assistant Professor, Dept of Microbiology, 6Assistant Professor, Dept of Microbiology,
7
Professor & Head, Dept of Microbiology, SSIMS&RC Davangere
57
Website : www.njbms.com

MATERIAL AND METHODS
RESULTS
Case Definition:- In children experiencing febrile illness

consistent with dengue fever and clinically suspected
cases of dengue fever (according to World Health
Organization criteria)
Total of 226 samples from June 2009 to May 2010 were

studied. We observed maximum number of clinically
suspected cases of dengue in August, September,
October months (Graph-1). There was male patient
predominance over female with maximum cases
between the ages 6 10 yrs.
Serological Definition:- Primary dengue virus and

Secondary dengue virus infections were defined as those
serum samples positive to IgM antibodies and IgG
antibodies respectively.
We tried to note the symptamatology of dengue cases in

our region. The most common symptoms apart from
fever were vomiting, Abdomen pain, Rashes, Malaena
and Coldness of feet & common signs were
Hepatomegaly, Splenomegaly, and Jaundice. ( Graph -2)
Patients and study design:- Blood samples from 226

clinically suspecting cases of dengue were screened from
June 2009 to May 2010 in Paediatric OPD SSIMS & RC
Davangere. Two serum samples were collected from each
patient, one at the day of enrollment and second 7-14
days after the fever onset.
All the 226 samples were subjected to IgM, IgG Microlisa

test. In this 124 samples were positive to IgM (Primary
Dengue infection) and 26 samples were positive to IgG
(Secondary Dengue infection). Total of 150 seropositive
cases were detected. (124 IgM + 26 IgG).
The samples were subjected to Dengue IgM and IgG

Microlisa and SD Bioline Dengue Duo rapid test.
Serum samples were tested for IgM and IgG dengue
antibodies by IgM and IgG capture Microlisa. The ELISA
was performed as per the manufacturer's instructions.
Out of 150 seropositive cases from ELISA, the rapid

Bioline Dengue duo test showed 93 IgM positives and 22
IgG positives. 8 samples were positive to both IgG and
IgM. 6 samples were positive to NS-1 antigen. 29 samples
were negative to all IgM, IgG and NS-1 antigen.(Graph3&4)
SD Bioline dengue Duo rapid test is an invitro

immunochromatographic, one step assay designed to
detect IgM and IgG antibodies to dengue virus in human
serum & NS-1 antigen. The test was read after 20
minutes.
When rapid test results were compared with ELISA test,

out of 226 samples 121 were true positives, 76 were true
negatives whereas there were 29 false negatives but no
false positives in rapid test. (Table-1)
Interpretation of the SD Bioline Dengue duo rapid test :

The presence of each one color line (control) within the
result window indicates a negative result.
The control line (C) and IgM line (M) are visible on the test
device. This is positive for IgM antibodies to Dengue virus
and indicates primary dengue infection.
40
The control line and IgG line (G) are visible on the test
device. This is positive for IgG antibodies and indicates of
secondary or past dengue infection.
35
The control line, IgM line (M) and IgG line (G) are visible
on the test device. This is positive for both IgM and IgG
antibodies and indicates late primary or early secondary
dengue infection.
15
30
25
20
Positives
10
5
Ju
ne
Ju
A ly
Se ugu
pt st
em
Oc ber
No tob
ve er
De mb
ce er
m
Ja ber
nu
Fe ary
br
ua
r
M y
ar
ch
Ap
ril
M
ay
The control line, NS-1 Ag line is visible on the test device.

This is positive for NS-1 antigen and indicates of early
acute dengue infection.
Suspected Cases
Graph 1 : Seasonal variation of dengue
58

Rapid test
Sensitivity = 80.66%
Specificity = 100%
Positive Predictive Value = 100%
Negative Predictive Value = 72.4%
False Positive Rate = 0.
Efficiency of the test = 87.16%
100
50
Fe
Ab Vom ver
do it
m ing
en
Ar Pa
th in
ra
lg
M ia
ya
Su
Pe lgia
bc
t
Ec ech
on
ch ia
ju
ym e
ct
iva
o
l h M sis
ae ala
Co ma ena
ld rrh
ne a
ss ge
o
Al Co f F
te nv ee
re ul t
d
s
Pu sen ion
ffi so s
n
e
Sw s rium
el s o
lin f f
g i ac
n e
lim
b
He Jau s
pa nd
i
Sp tom ce
Ly le eg
m no al
ph m y
ad eg
en aly
op
at
hy
DISCUSSION
In the present study, maximum number of cases were
between 6- 10 yrs. Gomber S et al in a similar study had
similar findings and this could be attributed to the health
care seeking behavior of the patients, endemic nature of
the infection 10.
Graph 2 : Clinical Features in dengue suspected cases

160
140
120
100
80
Sero positives
60
Sero Negatives
12-18 % of the positive cases occurred during the months

of July, August, September. This is in comparison with
similar pattern of month-wise case distribution seen with
authors Rasul CH et al in 2002, Narayan et al in 2002,
11-14
Gomber et al in 2001 . The reasons may be due to the
geographical region with prime occupation of the people
being agriculture. July and August months are paddy
sowing months which needs large stores of water. Also
the breeding of Aedes aegypti is highest during pre and
post monsoon period. But sporadic cases extend up to
December which indicates endemicity of the infection up
to December.
40
20
0
Rapid Test
ELISA
Graph 3 : Seropositivity in ELISA and Rapid test

8
22
IgM positves
ELISA test showed 82.6% of cases as primary dengue and

17.3% as secondary dengue. High dengue primary
infection is due to the virulence of the infecting serotype
of the virus.
IgG Positives
Igm+IgG Positives
NS-I
93
We made a comparative evaluation of both ELISA and

Rapid test. Rapid test had a sensitivity of 80% and
specificity 100% when compared to ELISA. The variations
in sensitivity and specificity are comparable with
previously published data14 & this might be caused by
different principles of the assays, different antigens,
conjugates.
Graph 4 : Pie diagram showing

Seropositivity in rapid test (SD Bioline Dengue Duo test)
Rapid test
(IgG, IgM & NS-1)
Positives
Negatives
Total
Cases
121
Positives
(TRUE
POSITIVES)
(FALSE
POSITIVES)
29
76
Negative
(FALSE
NEGATIVES)
(TRUE
NEGATIVES)
105
Total
150
76
226
121
A rapid and accurate method for the diagnosis of the

dengue fever is important for both the clinician and the
patient. The commercially available dengue rapid test is
suitable for the detection of anti-dengue IgM and IgG
antibodies and NS-1 antigen with results available in just
20 mins, with a positive predictive value and negative
predictive value of 100%, 72.4% respectively4. Caution
Table 1 : Comparison of RAPID test with ELISA test

ELISA (IgG & IgM)
59

should be applied in interpreting tests that are positive to
dengue virus IgM or IgG only in areas where dengue virus
co circulates with other flavi viruses 15. This might be the
probable reason for the false negative rate (19.3%) &
negative predictive value (72.4%) of rapid test in our
study.
The role of NS1 Ag for early detection of Dengue virus
infection is currently being evaluated by many
investigators. As there was time lag in the patients who
were referred to our hospital which is a tertiary care
hospital from the peripheries, the chance of detecting
NS-1 antigen was low. In this regard NS-1 antigen
detection by ELISA method may be useful5. The mean
duration of illness of patients in our study was between
5-14 days after fever onset.
Efficiency of the rapid test in our study is 87.16%. The use
of IgM & IgG test parameters with NS-1 antigen detection
is rational as it would likely provide improved
presumptive diagnostic coverage towards the end of
acute illness when NS-1 levels are declining but dengue
5
virus specific IgM & IgG titres are climbing .
3.
Burke, D.S., A. Nisalk, and C.H. Hoke, Jr. 1986. Fireld trial of a Japanese
encephalitis diagnostic kit. J. Med. Virol. 18:41-49.
4.
Innis, B.L., A. Nisalak, S. Nimmnannitya, S.Kusalerdchariay, V.

Chongswasdi, S. Suntayakorn, P. Puttisri, and C.H.Hoke. 1989. An
enzyme-linked immunosorbent assay to characterize dengue
infections where dengue and Japanese encephalitis co-circulate. Am.
J. Trop. Med. Hyg. 40; 418-427.
5.
Vianney Tricou et al. BMC infectious Diseases 2010,10:142
6.
Shu PY, Huang JH. Current advances in dengue diagnosis. Clinical and
Diagnostic Laboratory Immunology 2004;11:642-50
7.
Alcon S, Talarmin A, Debruyne M, Falconar A, Duebel V, Flam and M.

Enzyme-Linked Immunosorbent Assay Specific to Dengue Virus Type 1
Nonstructural Protein NS1 Reveals Circulation of the Antigen in the
Blood during the acute Phase of Disease in Patients Experiencing
Primary or Secondary Infections. J Clin Microbiol 2002; 40:376-81.
8.
Dussart P, Labeau B, Lagathu G, Louis P, Nunes MRT, Rodrigues SG, et

al. Evaluation of an Enmzyme Immunoassay for detection of dengue
virus NS1 antigen I human serum. Clin Vaccine Immunol 2006;
13:1185-9.
9.
Rao, MCVR, Dengue fever in India, Indian Journal Pediatrics 1987, 54

(1):11-14
10.
Rasul CH. Ahasan HAMN, Epidemiological factors of dengue

hemorrhagic fever in Bangladesh Indian pediatrician 2002 39:369372.
11.
Narayan Manjit, Aravind MA. Dengue fever outcome in channi A

study of clinical profile & outcome Indian Pediatric 2002 Nov
17:39:1027-33.
12.
Goumber.S. Kumar. S. Hematological observations as diagnostic

markers in DHF A repraissal Indian Patrician 2001 May 17:38:477-81.
13.
Cazzubbo, A.J., et al. 1999 comparison of Panbio dengue duo ELISA

and MRL dengue fever virus IgM capture ELISA for diagnosis of a
dengue in south east asia. Clin. Diagn. Lab. Immunol 6:705-12.
14.
Lam.S., et al. Detection of specific IgM in dengue infection. J. Trop

Med. (1987) : 18:532:38.
15.
Russel.P., et al. Antibody response in dengue hemorrhagic fever. J.

Med. Sci. Biol. (1987) : 20 : 103 - 108.
CONCLUSION
High prevalence rate in our region particularly in pre
monsoon and monsoon season gives an alarm to the
doctors regarding early and accurate diagnosis of dengue
virus infection and its complications. Prompt diagnosis of
index cases can facilitate vector control activities in the
community so as to mitigate further transmission. The
commercially available SD dengue Duo rapid test
described in the study should be a valuable screening test
for dengue fever. It is rapid, easily be performed,
interpreted early and has a extended shelf life. The
strength of the SD dengue duo rapid test is that dengue
IgM and IgG test windows provides additional diagnostic
investigation that compliments NS-1 antigen detection.
We conclude that rapid test is an effective tool, if when
used in combination with NS-1 MAC ELISA in single
sample of suspected cases, has the ability to improve the
diagnostic algorithm contributing significantly to clinical
treatment and to control dengue viral infections.
REFERENCES
1.
Gazman Mg, Kouri G (2002) Dengue; an update. Lancet Infect Dis 2:33-42
2.
Monath TP (1994) Dengue; the risk to developed and developing

countries. Proo Natl Aead Sci U.S.A. 91;2395-2400
60

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Volume - III, Issue-1

National Journal of Basic Medical Sciences

DENGUE: SEROPREVALENCE, COMPARISON OF RAPID TEST

Introduction: - Dengue is a major health problem in many

Dengue is a major health problem in many parts of

Aims and objectives: - The present study was done to

The major diagnostic methods currently available are

Materials & Methods: - 226 serum samples were tested

Now- a- days detection of NS-1 Ag on rapid tests offer an

Conclusion:- High prevalence rate in our region

The present study aims to determine the common clinical

Key words: Dengue, ELISA, rapid test, NS-1 antigen

Associate Professor, Dept of Microbiology, 2Professor, Dept of Pathology,

Volume - III, Issue-1

National Journal of Basic Medical Sciences

Case Definition:- In children experiencing febrile illness

Total of 226 samples from June 2009 to May 2010 were

Serological Definition:- Primary dengue virus and

We tried to note the symptamatology of dengue cases in

Patients and study design:- Blood samples from 226

All the 226 samples were subjected to IgM, IgG Microlisa

The samples were subjected to Dengue IgM and IgG

Out of 150 seropositive cases from ELISA, the rapid

SD Bioline dengue Duo rapid test is an invitro

When rapid test results were compared with ELISA test,

Interpretation of the SD Bioline Dengue duo rapid test :

The control line, NS-1 Ag line is visible on the test device.

Graph 1 : Seasonal variation of dengue

Volume - III, Issue-1

National Journal of Basic Medical Sciences

Graph 2 : Clinical Features in dengue suspected cases

12-18 % of the positive cases occurred during the months

Graph 3 : Seropositivity in ELISA and Rapid test

ELISA test showed 82.6% of cases as primary dengue and

We made a comparative evaluation of both ELISA and

Graph 4 : Pie diagram showing

A rapid and accurate method for the diagnosis of the

Table 1 : Comparison of RAPID test with ELISA test

Volume - III, Issue-1

National Journal of Basic Medical Sciences

Innis, B.L., A. Nisalak, S. Nimmnannitya, S.Kusalerdchariay, V.

Vianney Tricou et al. BMC infectious Diseases 2010,10:142

Alcon S, Talarmin A, Debruyne M, Falconar A, Duebel V, Flam and M.

Dussart P, Labeau B, Lagathu G, Louis P, Nunes MRT, Rodrigues SG, et

Rao, MCVR, Dengue fever in India, Indian Journal Pediatrics 1987, 54

Rasul CH. Ahasan HAMN, Epidemiological factors of dengue

Narayan Manjit, Aravind MA. Dengue fever outcome in channi A

Goumber.S. Kumar. S. Hematological observations as diagnostic

Cazzubbo, A.J., et al. 1999 comparison of Panbio dengue duo ELISA

Lam.S., et al. Detection of specific IgM in dengue infection. J. Trop

Russel.P., et al. Antibody response in dengue hemorrhagic fever. J.

Monath TP (1994) Dengue; the risk to developed and developing

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