International Journal of Infectious Diseases 74 (2018) 41–46

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International Journal of Infectious Diseases

journal homepage: www.elsevier.com/locate/ijid

An epidemiological study of dengue and its coinfections in Delhi

Deepali Savargaonkara,1, Swati Sinhaa,1, Bina Srivastavaa , B.N. Nagpala, Abhinav Sinhaa,
Arshad Shamima , Ram Dasa , Veena Pandeb, Anupkumar R. Anvikara,* , Neena Valechaa
a
National Institute of Malaria Research, Sector 8 Dwarka, New Delhi, 110077, India
b
Kumaun University, Nainital, 263001, India

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: To study the epidemiology of dengue with reference to serological, demographic profile,
Received 1 February 2018 spatio-temporal distribution, vectors, circulating serotypes and coinfections.
Received in revised form 23 June 2018 Methods: Demographic data and presenting symptoms of fever cases reporting to the clinic were recorded.
Accepted 26 June 2018
Suspected patients were tested for dengue, chikungunya and malaria. Dengue specific RT-PCR was
Corresponding Editor: Eskild Petersen,
Aarhus, Denmark
performed to detect circulating DENV serotypes. Vector surveys were carried out to detect Aedes breeding.
Results: Of the 5536 fever patients tested during 2012 to 2015,1536 (27.7%) had confirmed dengue. The peak
in dengue positivity was observed during September and October. Of the 60 samples analysed, 10 (16.7%)
Keywords:
Dengue
had concurrent infection with multiple dengue serotypes; one of them had all the four serotypes.
Epidemiology Coinfection of dengue with malaria and chikungunya was also observed. The occurrence of dengue and
India malaria was inversely related. Seven percent of the dengue patients required hospitalization. Vector
Serotypes surveys in the draining area revealed Aedes breeding with a high house index.
Conclusion: Delhi being hyperendemic, the occurrence of concurrent infections with multiple DENV
serotypes has become a frequent finding. The study emphasizes the need of epidemiological and
entomological surveillance to monitor trends in dengue distribution, seasonal patterns and circulating
serotypes to guide dengue control activities.
© 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

Introduction
Dengue is the most rapidly spreading mosquito-borne disease
caused by flavivirus. The disease has become a major health
concern in tropical and many subtropical areas. An estimated 96
million clinical dengue infections occur worldwide annually. In
absence of specific treatment, vector control measures form the
mainstay of dengue control activities (WHO).
Occurrence of dengue fever in India was first reported in 1946
(Karamchandani, 1946). It is endemic in most of the states of India,
and after the nationwide outbreak in 1996, many frequent
outbreaks occurred (Gupta and Ballani, 2014; NVBDCP). Delhi,
the capital of India, experienced the first major outbreak in 1967
* Corresponding author.
E-mail addresses: dr.deepali27@gmail.com (D. Savargaonkar),
swati.microbio04@gmail.com (S. Sinha), shbira@gmail.com (B. Srivastava),
b_n_nagpal@hotmail.com (B.N. Nagpal), aspsm2003@yahoo.co.in (A. Sinha),
arshad.samim@yahoo.co.in (A. Shamim), ramdas9@gmail.com (R. Das),
veena_kumaun@yahoo.co.in (V. Pande), Anvikar@gmail.com (A.R. Anvikar),
neenavalecha@gmail.com (N. Valecha).
1
Joint first authors.
(Balaya et al., 1969) and recently, the outbreaks occurred in 2003,
2006, 2010 and 2013 (Gupta et al., 2006; Afreen et al., 2014; Ahmed
and Broor, 2015; Bharaj et al., 2008).
Aedes aegypti is the vector responsible for dengue transmission
in Delhi (Kumar et al., 2015). It is a primary vector and breeds in
manmade containers (Sunyoto et al., 2013). There are four closely
related serotypes of dengue virus (DEN- 1, 2, 3 and 4) (WHO). With
co-circulation of multiple serotypes during outbreaks, Delhi has
been reported to be hyperendemic for dengue (Bharaj et al., 2008).
In 2015, Delhi experienced an outbreak with 15867 cases and 60
deaths (NVBDCP). Along with dengue, other vector borne diseases
reported from Delhi are chikungunya, which is sporadic, and
malaria, for which Delhi falls under a low transmission zone with
API <1 (NVBDCP; NVBDCP, 2016).
We are reporting findings of an epidemiological study of
dengue conducted at ICMR-National Institute of Malaria Research
(NIMR) in Delhi.
Materials & methods
This epidemiological study was carried out at ICMR-NIMR, New
Delhi from January 2012 to December 2015. The fever clinic of

https://doi.org/10.1016/j.ijid.2018.06.020
1201-9712/© 2018 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
42 D. Savargaonkar et al. / International Journal of Infectious Diseases 74 (2018) 41–46
ICMR-NIMR has facilities for diagnosis of dengue, chikungunya and
malaria.
Study population
Fever clinic attendees with a history of fever were included in
the study. Demographic characteristics and presenting symptoms
were recorded.
Serology and microscopy
Serum samples from clinically suspected patients having a
history of fever for 1-5 days were tested for NS1 antigen (Panbio
Dengue Early ELISA, Standard Diagnostics, Inc. Republic of Korea).
Those having fever for more than five days were tested for dengue-
specific IgM antibodies by using IgM antibody capture enzyme-
linked immunosorbent assay (MAC-ELISA) kit (ICMR-National
Institute of Virology, Pune). Fever cases suspected of malaria were
also investigated for malaria by microscopy of blood smear and for
chikungunya by IgM ELISA (ICMR-NIV, Pune). ICMR-NIV dengue
and chikungunya IgM capture ELISA tests are intended for
qualitative detection of IgM antibodies in serum. The tests are
used in the surveillance centres for dengue and chikungunya in the
public health system in India.
The data were entered in Microsoft Excel 2007.
GIS mapping
GIS based mapping of residential areas of dengue cases reported
during the study was performed to identify areas where repeated
transmission of dengue was occuring. The GIS maps were prepared
using the programme ‘ESRI India’s ArcGIS ArcMap 10.5’. The maps
were supplied by ML Infomap.
Serotyping by RT-PCR
Reverse Transcriptase PCR (RT-PCR) was carried out on NS1
positive samples collected in 2015 to detect the serotypes of DENV.
Viral RNA was isolated from serum samples using QIAamp Viral
RNA Mini kit (Qiagen, hidden, Germany) according to manufac-
turer’s instructions. RNA was eluted in 60 ml AVE- Elution buffer
provided with the kit and aliquots were stored at 80  C until use.
Dengue virus strains (serotypes DEN 1, 2, 3 and 4V) were used as
positive controls for RT-PCR assays. These were provided by ICMR-
NIV, Pune.
The extracted RNA template was reverse transcribed into
complimentary DNA (cDNA) copy in a 25 ml reverse transcrip-
tion (RT) reaction mixture using 0.5 mM of gene specific primers
D1 and D2 (Lanciotti et al., 1992), 1 mM dNTPs and 1 ml of RT
enzyme. The reaction mixture was incubated at 45  C for
30 minutes followed by enzyme inactivation at 95  C for
15 minutes, wherein said step of amplification comprised
94  C for 30 seconds, 55  C for 1 minute, 72  C for 2 minutes,
followed by repeating steps 3-5, 35 times and final extension at
72  C for 10 minutes. The amplified RT- PCR products of 511 bp
were subjected to nested PCR for serotyping using D1 and TS1,
TS2, TS3 and TS4 primers (Lanciotti et al., 1992). Samples were
visualised on 2% agarose gel under UV light. All the assays were
repeated by a second operator.
To prevent contamination, experiments were carried out in
biosafety cabinet. Different cabinets were used for processing
controls, clinical samples and PCR master-mix preparation/
template addition. Reagents were kept away from positive
controls. Ethanol (70%) was used to wipe the hands as well as
working space. Filtered tips were used during each step. Universal
safety precautions were followed.
Treatment
Confirmed dengue and chikungunya patients were advised
antipyretics and plenty of fluids and referred to hospital for further
investigations. Malaria cases were given radical treatment with
antimalarials.
Ethical aspects
The study was approved by the Institutional Ethics Committee
of ICMR-NIMR. For performing RT-PCR, written informed consent
and assent (as applicable) was obtained.
Vector survey
Vector survey was carried out from August to October 2015 at the
residential places of confirmed dengue cases. Breeding was checked
in all types of water filled containers present in the houses and their
premises. The larval collections were performed with the help of
flash-light, by dipping and pipetting methods (WHO,1975). Breeding
habitats like overhead tanks (OHTs), water containers, coolers,
curing tanks, Solid Waste, mud pots and other junk materials were
screened for the presence of immature stages of Aedes mosquitoes.
All larvae and pupae were reared to adult stage for species
identification at ICMR- NIMR insectary. Larval indices [House index
(HI), container index (CI) and Breteau index (BI)] were used to record
Aedes aegypti density (World Health Organization, 1997).
Results
During 2012 to 2015, a total of 5536 samples were subjected to
dengue IgM antibody and/or NS1 antigen test, of which 1536
(27.7%) tested positive for dengue. About 61% of these (935/1536)
were reported in the year 2015. Male: female ratio was 1.67.
Distribution of dengue cases had a significant association with the
age of the study population across the study period (2012-2015;
ANOVA; p < 0.00). The most commonly afflicted age group was 11-
30 years. This age predilection for dengue in these specified age
groups was statistically significant (Tukey’s post-hoc test for
pairwise comparison; p < 0.01). The median number of serologi-
cally confirmed dengue cases was estimated to be 28.5% and 29.4%
for the age groups 11-20 years and 21-30 years, respectively. The
next in order was the age group 31-40 years with the median
number of cases of 16.8% (Figure 1). The age range of dengue cases
was 6 months to 85 years and mean age was 26.02 years.
Figure 1. Age distribution of confirmed dengue cases.
 D. Savargaonkar et al. / International Journal of Infectious Diseases 74 (2018) 41–46 43

The distribution of dengue cases across the monsoon and

post-monsoon seasons of the study years showed remarkable
differences in the prevalence of infection which is statistically
significant (chi squared value 15.54; p < 0.05). The peak in
dengue positivity was observed in October of 2012 and 2014 and
in September of alternate years 2013 and 2015. The shape of the
best-fit moving average trend line shows sharp peaks in 2012
and 2013 as compared to the blunt peaks in 2014 and 2015
(Figure 2). During the premonsoon season from January to June
over a period of four years, only three dengue cases were
reported.

Clinical features

All the dengue patients had a history of fever. Other symptoms

recorded were myalgia (77%), headache (23%), vomiting (23%),
itching (13%), pain in abdomen (10%), rash (5.4%) and bleeding
from nose and gum (1%).

Serotypes by RT-PCR

Reverse Transcriptase PCR (RT-PCR) was performed on 60

dengue NS1 positive samples collected in 2015 (Figure 3A). Mono
infection with DENV-2 was found in 47, DEN-3 in one and DEN-4 in
two patients. Concurrent infection of multiple serotypes was
observed in 10 patients. The combinations of the serotypes in these
patients were 1 + 2, 2 + 3, 2 + 4, 3 + 4, 2 + 3 + 4 and 1 + 2 + 3 + 4. One of
these patients had infection with all four serotypes (Figure 3B).
Spatial distribution
GIS mapping of the cases showed that the maximum number of
cases were reported from two areas Raj Nagar II (50%) and Bagdola
(12%) (Figure 4). Multiple (2 to 5) cases were observed from the
same household (217 from 98 households).
Figure 3. The agarose gel images (2%) showing the amplified RT-PCR product of
dengue viral genome. (A) shows a representative DNA gel showing the 511 bp DNA
fragment obtained by RT-PCR using dengue viral RNA as template with D1 (forward)
and D2 (reverse) primers. Lanes 1, 2, 4, 5,6,7,8 and 9 show the presence of dengue
viral genome in the sample as evidenced by a clear DNA band of 511 bp size where
the P lane is for positive control and B is for Blank. However, Lanes 3 and 10 did not
show the presence of dengue viral genome in the corresponding samples. (B) DNA
gel images showing nested PCR products by serotype specific primers with all four
serotypes (Lane 1), Den 2, 3 and 4 in (Lane 2), Den 3 and 4 (Lane 3), Den 2 and 3 (Lane
4), Den 3 (Lane 5), Den 4 (lane 6), Blank (lane 7) and M indicates 100 bp DNA ladder.

Vector survey houses in solid waste (tires and disposable glasses), plastic water
storage containers, overhead tanks and coolers (Table 1). The
Vector surveys were carried out from August to October 2015 in Container Index, House Index and Breteau Index were 1, 10.1 and
houses of 79 dengue cases. Aedes breeding was observed in eight 10.1 respectively, each in surveyed F block of Raj Nagar II.

Figure 2. Distribution of dengue positivity across monsoon & post monsoon seasons of 2012-2015.
44 D. Savargaonkar et al. / International Journal of Infectious Diseases 74 (2018) 41–46

Figure 4. GIS based map showing spatiotemporal distribution of dengue case. ‘A’ shows zones of Delhi. ‘B’ is Najafgarh zone, the area catered by the clinic. ‘C’ shows yearwise
dengue cases reported at NIMR from different areas.

Table 1 trends of percentage positivity for malaria and dengue across four
Containers with Aedes breeding. years. It showed that the positivity of malaria changed in an
inversely proportional manner with that of dengue, based on
Container Checked for breeding Positive for Aedes
actual clinic data from 2012 to 2015.
Overhead Tanks 187 2
Coolers 87 2
Water Containers 130 2 Clinical outcome
Mud pots 103 0
Solid waste 138 2 Dengue patients were followed up to study the clinical
Total 645 8
outcome. Of the 580 cases followed up, 42 (7%) required
hospitalization and no death was reported.

Dengue coinfections Discussion

Of the 334 patients tested for both dengue and chikungunya, 97
had dengue and 40 had chikungunya infection. Coinfection of
dengue and chikungunya was observed in 12 patients. It was
observed that dengue cases had slightly increased probability of
concomitant chikungunya infection (OR 1.01; CI 0.51-2.16) but it
was not statistically significant. A total of 5228 patients were tested
for both dengue and malaria; 1472 tested positive for dengue and
57 for malaria. Coinfection of malaria and dengue was observed in
seven patients. The probability of having dengue in presence of
malaria was 0.35 times lower than that in absence of malaria (95%
confidence interval 0.16-0.78) and was statistically significant. A
similar finding was also observed by comparing the polynomial
Delhi experienced an outbreak of dengue in year 2015, with
15867 reported cases (NVBDCP, 2016). NIMR Fever Clinic reported
1536 dengue cases during 2012 to 2015, with 61% in 2015 and 23%
in 2013.
The most commonly afflicted age group was between 11-30
years. This age predilection for dengue was statistically significant
(Tukey’s post-hoc test for pairwise comparison; p < 0.01). The
median number of serologically confirmed dengue cases was
estimated to be 28.5% and 29.4% for the age groups 11-20 years and
21-30 years, respectively. Being a mosquito-borne disease, dengue
has shown to be common in these age groups since they are more
prone to mosquito bites due to their nature of daily routine, being
 D. Savargaonkar et al. / International Journal of Infectious Diseases 74 (2018) 41–46
more exposed and available to adult mosquito inhabitations. The
findings are comparable to other studies reporting highest
proportion in age groups 11-20 and 15-24 years (Gupta et al.,
2006; Ghouth et al., 2012; Padhi et al., 2014).
The distribution of dengue cases across monsoon and post-
monsoon seasons showed remarkable differences in the preva-
lence and was statistically significant (chi square value 15.54;
p < 0.05). The peak in dengue positivity was observed in October of
2012 and 2014 and in September of alternate years 2013 and 2015.
The shape of the best-fit moving average trend line shows sharp
peaks in 2012 and 2013 as compared to the blunt peaks in 2014 and
2015. This could be attributed to the seasonal fluctuation of
climatic variables, including local rainfall and humidity, which
affect vector density and vectorial capacity of Aedes mosquitoes as
reported by a study from Thailand (Wongkoon et al., 2013).
Variations in temperature, humidity and rainfall have been
reported as significant predictors of number of dengue cases by
a study from central India (Pakhare et al., 2016).
All the four serotypes were found to be circulating in Delhi in
2015, although DENV-2 was predominant (94%). Infection with
more than one serotype was also observed, as reported earlier in
Delhi (Afreen et al., 2014; Bharaj et al., 2008; Tazeen et al., 2016).
One patient had concurrent infection with all the four serotypes.
This is the first such report from Delhi, although all the four
serotypes were found in two patients from Kerala in year 2013
(Reddy et al., 2017). Dengue patients infected with multiple
serotypes of DENV are more prone to have severe manifestations
than with mono infection (Dhanoa et al., 2016). Further, severe
clinical manifestations are commonly seen with DENV -2,
hemorrhagic manifestations with DENV-4, liver involvement with
DENV- 3; while DENV-1 usually has mild clinical presentation
(Kumaria, 2010; Kalayanarooj and Nimmannitya, 2000). Dengue
cases reported in 2015 were followed up for clinical outcome.
Hospitalisation was required in 7% of patients. No death was
reported among these patients. Globally, about 5% of the estimated
cases require hospitalisation annually (Sunyoto et al., 2013).
Although India has seen an increase in the number of cases over the
years, mortality is decreasing (Dayaraj, 2014).
Coinfection of dengue with chikungunya was found though
only sporadic cases of chikungunya were reported. There were
96 chikungunya cases reported during 2012-2015 (NVBDCP). It
was observed that those who had dengue infection had slightly
increased probability of concomitant chikungunya infection, but
this was not statistically significant (OR 1.01; CI 0.51-2.16). Since
both dengue and chikungunya are arboviral diseases transmit-
ted by a common Aedes mosquito, it is highly likely the
transmission of the two viruses occur in the same spatio-temporal
dimension. An increased sample size could bring out the true
probability of their co-circulation. Prevalence of coinfection of
dengue and chikungunya has been reported to be up to 32% and
mortality associated with coinfection has been reported from
Colombia (Edwards et al., 2016; Mercado et al., 2016; Saswata
et al., 2015).
Dengue and malaria coinfection was also found though malaria
transmission is very low in Delhi and falls under category I with API
less than 1 (NVBDCP, 2016). In patients with coinfections of dengue
and malaria, higher chances of presenting with severe disease
compared to patients with mono infection, has been reported
(Epelboin et al., 2012; Magalhães et al., 2014). In areas endemic for
dengue, chikungunya and malaria, there is need to have a higher
degree of suspicion for coinfections in febrile patients to provide
appropriate treatment and close monitoring of such patients to
prevent further complications. An inverse relation was observed
between dengue and malaria positivity. This might be due to the
entomological factors related to preferential breeding of either
Anopheles or Aedes species or due to the host immune factors.
45
Vector surveys were carried out during the transmission season
in 2015. Around 50% of the total dengue cases were reported from
Raj Nagar II, where people store water in different containers for
domestic use due to inadequate water supply, and rain water
stored in discarded containers and vectors breed in those
containers. A study from Ethiopia has reported breeding of Aedes
favored by stored water in containers for domestic use (Getachew
et al., 2015). A study on vector indices has predicted for dengue
transmission of BI at a cut off if 4 and PAHO has described HI >5 as
high risk for dengue transmission (Sanchez et al., 2006; Pan
American Health Organization, 1994). An association of a high
number of dengue cases and larval breeding with a high house
index was observed with the post monsoon period. Positive
correlation between rainfall, larval infestation rate and incidence
of dengue has been shown from Africa (de Souza et al., 2010). The
vector breeding was found in OHT, coolers, solid waste and plastic
containers used for storing water. In other studies in Delhi the
preferred breeding sites were solid waste and plastic containers for
Ae. aegypti during the transmission season, similar to our study
(Kumar et al., 2015), while it was discarded tires and desert coolers
during the monsoon season in central Delhi in 1993 (Katyal et al.,
1996). Some of the dengue cases were members from the same
household with 2.2 patients per household. This indicates
mosquito bites to many members of the family in a short time
and this behaviour of the vector makes it a very efficient epidemic
vector (Gubler, 1998). WHO has recommended the need of
integrated surveillance as one of the elements for control of
dengue (World Health Organization, 2012). Thus laboratory based
surveillance of dengue integrated with vector surveillance in the
residential areas of the cases can be an effective tool for control of
dengue in that area.
Conclusion
Co-circulation of all four serotypes with predominance of
DENV-2 was observed during the 2015 dengue outbreak with
concurrent infection with multiple serotypes. This is the first
report from Delhi of concurrent infection with all four
serotypes in a patient, and implies the need to look for a
clinical outcome.
There is a significant association of peak in dengue positivity
and high larval indices with the postmonsoon period, which
highlights the need of initiating vector control activities in the
premonsoon season. In areas endemic for dengue, chikungunya
and malaria, there is a need to have a higher degree of suspicion of
coinfection to ensure prompt treatment, especially for malaria.
With frequent outbreaks in Delhi, there is a need to conduct
epidemiological and entomological surveillance which will help in
early warning of outbreak and control of dengue.
Acknowledgments
The authors are grateful to the Indian Council of Medical
Research, National Vector Borne Disease Control Programme,
Government of India and South Delhi Municipal Corporation for
facilitating the activities. The technical support as well as support
in the form of IgM ELISA kits and positive controls by ICMR-
National Institute of Virology is also gratefully acknowledged.
Thanks are also due to Mr Sanjeev Gupta for preparing the maps.
Staff of NIMR is also acknowledged.
Authors’ contributions
DS managed the clinic, SS and RD carried out serotyping, BS ran
diagnostic assays, BNN managed vector surveys, AS did the
statistical analyses, AS2 carried out vector collection, VP reviewed
46 D. Savargaonkar et al. / International Journal of Infectious Diseases 74 (2018) 41–46
the manuscript, ARA reviewed the manuscript and managed
laboratory, NV conceived the idea.
Conflict of interest
All the authors declare that they have no conflicts of interest.
Funding source
ICMR-NIMR provided funding for the study. ICMR-NIV provided
the ELISA kits through NVBDCP.
Ethical approval
The study was approved by the Institutional Ethics Committee
of ICMR-NIMR. For performing RT-PCR, written informed consent
and assent (as applicable) was obtained.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.ijid.2018.06.020.
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