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DOI: 10.4103/2224-3151.115828
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Co-circulation of dengue virus

serotypes with chikungunya virus in
Madhya Pradesh, central India
Pradip V Barde1, Mohan K Shukla1, Praveen K Bharti1,
Bhupesh K Kori1, Jayant K Jatav2, Neeru Singh1

Abstract
Background: Dengue and chikungunya present with very similar signs and
symptoms in the initial stage of illness and so it is difficult to distinguish them
clinically. Both are transmitted by Aedes aegypti and Aedes albopictus mosquitoes.
This study was conducted with the aim to explore the co-circulation of dengue and
chikungunya viruses in central India.
Materials and methods: Samples from suspected dengue cases were subjected
to dengue immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA)
and dengue-negative samples were tested with chikungunya-specific IgM ELISA.
The samples collected in acute phase of illness were tested by nested reverse
transcription polymerase chain reaction (nRT-PCR). Chikungunya virus (CHIKV)
sequences were analysed to determine their genotype.

Regional Medical Research Centre

for Tribals (ICMR), Jabalpur,
Madhya Pradesh, India.
2
Netaji Subhash Chandra Bose
Medical Collage, Jabalpur,
Madhya Pradesh, India.
1

Address for correspondence:

Dr N Singh, Regional Medical
Research Centre for Tribals (Indian
Council of Medical Research),
Nagpur Road, Post Garha,
Jabalpur 482003, Madhya Pradesh,
India.
Email: neeru.singh@gmail.com;
rmrctjabalpur@rediffmail.com

Results: Of 138 samples screened for dengue, 21 (15.2%) were positive, and of
119 samples screened for chikungunya, 13 (10.9%) were positive. Dengue viruses
1 and 4 were found co-circulating with chikungunya virus in Jabalpur, central
India. The chikungunya virus detected belonged to the East Central South African
genotype.
Conclusion: Accurate and timely diagnosis would help in patient management
and use of resources. It is advocated to simultaneously test samples for these two
diseases in endemic areas. This will also aid in understanding the epidemiology
of chikungunya.
Key words: Central India, chikungunya, co-circulation, dengue

Introduction
Arthropod-borne viral infections cause major disease burden in
tropical and subtropical countries worldwide. The incidence of
dengue has increased more than 30-fold in the last 50 years.1 It
is estimated that about 100 million dengue cases and over 390
million infections occur worldwide annually.1,2 India contributes
about 34% of these cases.2 Four antigenically related serotypes
of dengue virus (DENV), with different genotypes, are known
to be circulating in India.3 All four serotypes can cause simple
febrile illness (DF), which may lead to more complex clinical
outcomes such as dengue hemorrhagic fever (DHF), dengue
shock syndrome (DSS) or death.4 Chikungunya re-emerged in
2005, after a gap of 32 years, with about 1.4 million cases, and

36

continues to circulate in India,4 with a predominance of the East

Central South African genotype of virus.5, 6 Dengue-confirmed
and chikungunya-suspected cases have been reported from
this part of the country in the recent past.4 Infection with the
chikungunya virus is generally self-limiting but in a few cases
it may cause severe incapacitating arthralgia in small joints,
lasting up to 6 months or more.4
DENV and CHIKV are both transmitted by Aedes aegypti
and Aedes albopictus mosquitoes. In the initial stage of
illness, both diseases present with a similar set of clinical
symptoms, such as sudden onset of high-grade fever, muscle
and joint pain, headache, nausea and vomiting, and rash,4
making differential diagnosis difficult clinically. Chikungunya

WHO South-East Asia Journal of Public Health | January-March 2014 | 3 (1)

Barde et al.: Co-circulation of dengue and chikungunya viruses

has low/no mortality, whereas for dengue, mortality may go

up to 35% in outbreak situations and serious cases, though
early referral and good management can reduce this. In the
absence of any licensed vaccine for either of these diseases,
timely intervention by prompt accurate diagnosis, along with
mosquito control, are the best tools for controlling or avoiding
outbreaks in endemic areas.

Ethical approval
Samples were referred to this virology laboratory for diagnosis
of diseases and thus considered as part of the public health
response; nonetheless, the project Establishment of Grade II
Virology Laboratory had ethical clearance from the institution
ethics committee of RMRCT, Jabalpur, India.

Chikungunya can spread with ease and causes a high

percentage of clinical cases with a very high attack rate in an
immunologically naive population.7 However, chikungunya
is overshadowed by dengue in outbreak situations and in
dengue-endemic areas.8 Diagnosis of chikungunya is equally
important, as it would help to estimate the disease burden
and, in turn, help with designing intervention strategies. Cocirculation and coinfections of these two viruses are reported.5
This study sought to confirm the co-circulation of the two
viruses in central India, using serological and molecular tools.

Samples and testing

This year-round (April 2011 to March 2012) study was
conducted in Madhya Pradesh. Blood samples of patients
reporting to health-care units of Jabalpur and the adjoining
14 districts, with symptoms of dengue and chikungunya,
as defined by NVBDCP,4 were collected by clinicians and
referred for diagnosis to the virology laboratory of RMRCT,
along with clinical and demographic information in the World
Health Organizations (WHOs) predesigned format.9 Upon
receipt, serum was separated by brief centrifugation at 4oC
and was preferably tested on same day by immunoglobulin M
(IgM) enzyme-linked immunosorbent assay (ELISA) or nested
reverse transcription polymerase chain reaction (nRT-PCR).

Materials and Methods

The virology laboratory of the Regional Medical Research
Centre for Tribals (RMRCT), Jabalpur is recognized as the
Apex Referral Laboratory by the National Vector Borne Disease
Control Program (NVBDCP) for dengue and chikungunya, for
Madhya Pradesh and Chhattisgarh.

The testing sequence for the samples is shown in Figure 1.

All the samples were tested by dengue-IgM-specific ELISA;
DENV-ELISA-negative samples were tested by chikungunya
IgM ELISA, using kits manufactured by the National Institute

Total
sample
n=138

Acute phase
sample n=25
DEN nRT-PCR

DENV IgM
ELISA n=138

n=19
Positive

n=119
Negative

n=06
Positive

n=19
Negative

CHIK RT-PCR
n=19

CHIK IgM
ELISA n=119

n=06
Positive

n=113
Negative

n=09
Positive

n=10
Negative

Figure 1: Flowchart depicting the methodology of processing the samples

ELISA: enzyme-linked immunosorbent assay; CHIKV: chikungunya virus; DENV: dengue virus; IgM:
immunoglobulin M; nRT-PCR: nested reverse transcription polymerase chain reaction

WHO South-East Asia Journal of Public Health | January-March 2014 | 3 (1)

37

Barde et al.: Co-circulation of dengue and chikungunya viruses

of Virology, Pune, India, as described by manufacturer. A

subset of samples, which were collected in the acute phase of
illness, were also subjected to DENV nRT-PCR,10 for detection
of DENV RNA and the DENV serotype. Of these, samples for
which the DENV nRT-PCR result was negative were subjected
to CHIKV-specific RT -PCR,11 with minor modifications. The
PCR products were extracted and sequenced as described
earlier.12 The sequences were analysed for their homologies,
using the basic local alignment search tool, and representative
sequences were submitted to GenBank. The partial nucleotide
sequence of CHIKV envelop gene 1 (E1) was compared with
14 sequences of CHIKV strains from the National Center for
Biotechnology Information (NCBI) database from Asia and
Africa. Multiple sequence analysis was done using CLUSTAL
W software and the p-distance Neighbor Joining phylogenetic
tree was generated using Molecular Evolutionary Genetics
Analysis (MEGA), version 5, with application of 1000
bootstrap replicates.13
The data were double-key entered into Microsoft Excel.
Single-sample proportion testing and logistic regression were
done using SPSS version 20.

Results
One hundred and thirty-eight samples from 15 districts of
Madhya Pradesh were screened for dengue (see Figure 1). Of
these, 63 (46%) patients were from rural areas; 85 (62%) were
male and 53 (38%) were female. The results for age, sex and
screening tests for diagnosis of dengue and chikungunya are
given in Table 1. A total of 15.2% (21/138) were positive for
dengue and 10.9% (13/119) were positive for chikungunya.
Single-sample testing confirmed that there was no significant
difference between these two proportions (P=0.117).
Most of the patients, where infection was suspected 112
(81%) had fever (100103oF) for more than 2days, with
associated symptoms such as headache, body ache, joint pain,
fatigue and nausea. Hepatomegaly was seen in 18 (13%) cases.
The platelet count was available for 42 patients, of whom 26%
had thrombocytopenia (platelet count <105/mm3). No cases of
DHF, DSS or death were noted. Statistical analysis revealed
no significant difference in initial symptoms such as fever in
confirmed cases of dengue and chikungunya.
Out of 138 samples tested, 21 (15.2%) were found to be positive
for dengue, from four districts, namely Jabalpur (DENV-

1 and DENV-4), Katni (DENV-4), Panna and Narsinghpur

(DENV-4). Of the 21 dengue-positive cases, 15 were only
IgM ELISA positive, two were only RT-PCR positive and four
samples were positive on both tests.
The nRT-PCR and sequencing results confirmed that DENV-1
(n=1) and DENV-4 (n=6) were circulating in and around
Jabalpur, central India. Most of the confirmed cases of dengue
and chikungunya were detected in the monsoon and postmonsoon periods.
DENV-1 was first identified in central India and the sequence
showed that it was closely related to the DENV identified from
New Delhi (JF 815209.1). DENV-4 identified in this study
had sequence homology with DENV-4 identified in 2010
(GenBank: JF929180).12
Out of 119 dengue-negative samples, 13 (10.9%) were positive
for chikungunya. All chikungunya-positive samples (n=13)
were from Jabalpur district. Six samples were IgM ELISA
positive and nine were RT-PCR positive; of these, two samples
were positive on both tests. The phylogenetic analysis carried
out using nucleotide sequences established that CHIKV from
Jabalpur belonged to the East Central South African (ECSA)
genotype (see Figure 2), with close sequence homology with
isolate from Delhi (GenBank: JN048826).
It was noted that, in the case of dengue, the positivity was higher
among males (16/85; 19%) as compared to females (5/53; 9%),
though the difference was not statistically significant. In the
case of chikungunya, no such difference was observed. After
adjusting for the sex of cases, the positivity of dengue was
significantly higher among adults (age 16 years and above)
when compared to children (15 years; odds ratio [OR]=31.1;
95% confidence interval [CI]=6.8 to 142.8; P<0.01) but in
the case of chikungunya, this difference was not significant
(OR=1.5; 95% CI=0.44 to 5.5; see Table 1).

Discussion
DENV-3 was detected during an outbreak that occurred
at Jabalpur in 1966.14 However, dengue and chikungunya
remained neglected diseases in this part of country, with very
few studies focusing on these infections.4,12,15 Because of the
emergence of different serotypes and their genotypes, dengue
epidemiology is continually changing, posing challenges to
clinicians and health authorities. CHIKV re-emerged in 2005,

Table 1: Age and sex distribution of the suspected and confirmed cases of dengue and chikungunya
Age group
(years)
0 to 15
16 and above
Total

Suspected casesa
M
54
31
85

F
38
15
53

T
92
46
138

Dengue positive
by sex
M
F
0
2
16
3
16
5

Dengue positive by
screening method
ELISA nRT-PCR
T
2
nil
2
17
6
23
19
6
21b

Chikungunya
positive by sex
M
F
4
5
3
1
7
6

Chikungunya positive
by screening method
T
ELISA RT-PCR
3
6
9
3
3
6
6
9
13b

ELISA: enzyme-linked immunosorbent assay; F: female; M: male; nRT-PCR: nested reverse transcription polymerase chain reaction; T: total.
a
Dengue-negative (n=119) samples were tested for chikungunya by ELISA and 19 by RT-PCR.
b
Four samples of dengue and two samples of chikungunya were positive by both tests.

38

WHO South-East Asia Journal of Public Health | January-March 2014 | 3 (1)

Barde et al.: Co-circulation of dengue and chikungunya viruses

India-06-HQ702750
64 India-11-JQ740183

India-10-JN048826
94

India-11-JX911895
India-06-FJ000068

83

68 Reunion-06-AM258993

Central Africa-84-HM045784

100

99 India-63-EF027140

Thailand-58-HM045810
Thailand-95-HM045787

84
92

Indonesia-10-AB678693

ECSA Genotype

Asian Genotype

} West African Genotype

Onyong-nyong-96-AF079456 } Outgroup
Senegal-66-AF192891

0.25

0.20

0.15

0.10

0.05

0.00

Figure 2: Phylogenetic tree of chikungunya viruses generated by the NJ method, using the p-distance model based on the partial nucleotide
sequence (266bp) of the E1 gene. Tree showing chikungunya virus detected in this study () belonging to East Central South African genotype.
Each strain is labelled with the country and year of isolation followed by GenBank accession number. Onyongnyong virus was used as the
outgroup

after a gap of almost 32 years, and made its presence felt in

India with more than one million cases.4 However, after the
epidemic, the medical fraternity neglected CHIKV infections,
probably because chikungunya is considered to be a disease
with mild morbidity and no mortalities. In contrast, dengue
is a disease with significant morbidity and mortality and is
endemic in the country.2 As reported earlier,4 and also seen in
this study, the initial symptoms of chikungunya are similar to
those of dengue and both are transmitted by same vectors Aedes
aegypti and Aedes albopictus. Probably because of the severity
of dengue, both clinicians and public health officials focus on
this disease, and samples are sent to laboratories with requests
for diagnosis of dengue only. The present study demonstrates
that DENV and CHIKV are co-circulating in Jabalpur, and
there is no significant difference in the positivity for the two
diseases. Although this study has a limited number of samples,
it emphasizes the importance of chikungunya diagnosis, as
there is no significant difference in the positivity of samples
for the two diseases.
Serological tests (ELISA) have demonstrated the presence
of dengue and chikungunya from central India in the recent
past.4,12,15 With establishment of the Viral Diagnostic Laboratory,
it was possible to provide molecular diagnosis of DENV, with
identification of the serotype, and to conduct characterization
studies on CHIKV, which lead to identification of DENV
serotype 1 and CHIKV ECSA genotype (see Figure 2).
It is known that, before 1973, the Asian genotype was
circulating in India and was subsequently replaced by the
ECSA genotype.4,5 This study documents, for the first time,
circulation of the ECSA genotype in this part of India. It will be

interesting to isolate and further characterize these circulating

viruses for better epidemiological understanding.
In an endemic area like India, where multiple DENV serotypes
are circulating with CHIKV, the possibility of concurrent
infection occurs.4 An earlier study reported the presence
of DENV serotype 4 in central India;12 detection of another
serotype (DENV-1) is alarming. There is some evidence
that infection with multiple DENV serotypes, or coinfection
with other pathogens, may produce a severe outcome of the
disease.16,17 An early and accurate diagnosis can help clinicians
to decide the course of treatment.
The presence of IgM for DENV and CHIKV can only be
detected around the fifth day of infection; the tests such as
nonstructural protein (NS1) detection for DENV and RT-PCR
for both these viruses to detect virus antigen/RNA should be
preferred. As apparent in the present study, although based
on a small sample size, the early symptoms cannot clinically
distinguish between dengue and chikungunya; thus, we
advocate early and simultaneous testing of samples for DENV
and CHIKV.

Acknowledgements
The authors are grateful to the Secretary to the Government
of India, Department of Health and Research, Ministry of
Health and Family Welfare, and The Director-General, Indian
Council of Medical Research, for financial support under the
project Establishment of Grade II Virology Laboratory.
The financial support and the kits provided by the National

WHO South-East Asia Journal of Public Health | January-March 2014 | 3 (1)

39

Barde et al.: Co-circulation of dengue and chikungunya viruses

Vector Borne Disease Control Programme, New Delhi, India

is acknowledged. Help from Dr RK Sharma for statistical
analysis and technical help from Virology Laboratory staff
of the Regional Medical Research Centre for Tribals is also
acknowledged

2.

3.
4.
5.
6.

7.

8.
9.

10.

40

12.
13.

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How to cite this article: Barde PV, Shukla MK, Bharti PK, Kori
BK, Jatav JK, Singh N. Co-circulation of dengue virus serotypes
with chikungunya virus in Madhya Pradesh, central India. WHO
South-East Asia J Public Health 2014; 3(1): 3640.
Source of Support: The study was funded by the Indian Council of
Medical Research (ICMR), under ICMRs Viral Diagnostic Laboratory
network project, and the Directorate of National Vector Borne Disease
Control Program, New Delhi provided kits for diagnosis of dengue and
chikungunya. Conflict of Interest: None declared. Contributorship: PVB
and NS designed the study and did literature search and writing. BKK and
PVB did data collection, data analysis, data interpretation and writing. JKJ
did sample collection, clinical diagnosis and treatment. PVB and MKS did
data analysis and interpretation (ELISA, nRT-PCR). MKS and PKB did
data analysis and interpretation (sequencing and sequence analysis) and
writing. All authors read and agreed upon the manuscript.

WHO South-East Asia Journal of Public Health | January-March 2014 | 3 (1)