Journal of Vector Borne Diseases

: 2019  |  Volume : 56  |  Issue : 4  |  Page : 295--302

Involvement of dual serotypes during a severe dengue outbreak in Wadi area, Nagpur district, Maharashtra 2017

AB Sudeep1, P Shil1, MM Charmode2, S Mohandas1, S Bansod3, MD Gokhale1, M Jagtap4, PS Shah1,  
1 ICMR–National Institute of Virology, Pune, Maharashtra, India
2 District Malaria Officer, Malaria Department, Nagpur Division, Nagpur, Maharashtra, India
3 District Medical Officer, Vyahad PHC, Wadi, Nagpur, Maharashtra, India
4 State Entomologist, Maharashtra, India

Correspondence Address:
Dr. A B Sudeep
ICMR–National Institute of Virology, Microbial Containment Complex, Sus Road, Pashan, Pune–411 021


Background & objectives: An outbreak of dengue-like illness was reported from Wadi area within the Nagpur Municipal Corporation during September–October 2017 with five deaths. Major symptoms reported were high fever (103–106 oF), acute joint pains, myalgia, drowsiness, breathlessness, etc. An investigation was conducted to confirm the etiological agent, its characterization and the vectors involved in the outbreak. Methods: Serological analysis was conducted to detect dengue (DEN)/chikungunya IgM antibodies in 158 sera samples. Nested-PCR was carried out to serotype eight ELISA positive samples. Adult and larval mosquito collections were conducted in the affected areas to determine species composition and mosquito density. Results: Dengue IgM antibodies were detected in 44 sera samples. Molecular typing revealed involvement of DEN-2 and DEN-3 serotypes. Dengue hemorrhagic fever symptoms were observed in two patients. Aedes aegypti breeding was found rampant with Breteu index and house index ranging from 23 to 70 and 17 to 56, respectively. Major breeding habitats encountered were, used tyres, cement tanks and refrigerator trays. Interpretation & conclusion: Clinical symptoms, detection of anti-DEN IgM antibodies in high number of samples and heavy breeding of Ae. aegypti confirmed it was a dengue outbreak.

How to cite this article:
Sudeep A B, Shil P, Charmode M M, Mohandas S, Bansod S, Gokhale M D, Jagtap M, Shah P S. Involvement of dual serotypes during a severe dengue outbreak in Wadi area, Nagpur district, Maharashtra 2017.J Vector Borne Dis 2019;56:295-302

How to cite this URL:
Sudeep A B, Shil P, Charmode M M, Mohandas S, Bansod S, Gokhale M D, Jagtap M, Shah P S. Involvement of dual serotypes during a severe dengue outbreak in Wadi area, Nagpur district, Maharashtra 2017. J Vector Borne Dis [serial online] 2019 [cited 2021 Feb 26 ];56:295-302
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Full Text


Dengue (DEN) has emerged as an important arboviral disease in the tropical countries with ~390 million cases and one million deaths annually[1]. The disease is caused by dengue virus (DENV), an RNA virus of family Flaviviridae and is transmitted by Aedes mosquitoes, in particular Aedes aegypti[2]. Four distinct serotypes (DEN-1, DEN-2, DEN-3 and DEN-4) of the virus exist in nature and all the four have the potential to cause outbreaks[3]. According to WHO estimate, ~40% of the global population lives in dengue endemic areas[4]. An upsurge in dengue cases was seen in recent years and it is estimated that approx.1.8 billion people in southeast Asian countries are at risk of dengue (http: // hotspot-area.php). In India, dengue has become an ongoing concern as the country continues to experience sporadic fatal outbreaks every year from almost every part of the country[5],[6]. In 2015, an estimated 100,000 cases and 180 deaths were reported from across the country[7]. All the four serotypes either singly or in combination were involved in the outbreaks and are showing an increasing trend in the number of fatal cases[5],[8],[9],[10],[11],[12],[13],[14]. In 2017 alone, 188,401 cases and 325 dengue related deaths were reported in India, mainly from Tamil Nadu, Maharashtra and Kerala (NVBDCP, Government of India).

An outbreak with dengue like clinical manifestations was reported from a suburban township in Wadi area within the Nagpur Municipal Corporation (NMC) during September–October 2017. Cases were reported mainly from different wards of Vyahad Primary Health Centre (PHC). The patients complained of high fever (103–106 °F) on and off with chills, acute joint pains, myalgia, etc. Other major characteristic symptoms reported were drowsiness, breathlessness and loss of appetite. Two patients developed dengue haemorrhagic fever/shock syndrome and had symptoms, viz. gastro intestinal bleeding, fulminant hepatic failure, acute renal failure and respiratory failure. In total five deaths were reported during the outbreak. Initial serological studies demonstrated dengue IgM antibodies in the patient serum. Entomological investigations demonstrated heavy Ae. agypti (Diptera: Culicidae) breeding in all the affected wards. The present report describes the entomological, virological and epidemiological studies carried out as part of outbreak investigation.

 Material & Methods

Study area, population and climate

Wadi is a census town situated between 21.1553°N and 79.0109°E within the NMC, Nagpur, India. It is a transport hub with a large number of godowns, warehouses, etc. Nagpur-Amravati national highway passes through Wadi and a number of small business establishments (mainly shops) occupy both sides of the highway. It comprises 25 wards with a population of ~54,000. Majority of the population depends on agriculture and a few on small-scale business. One side of the highway represents the suburban township while the other side is occupied by a slum. The outbreak was reported from the suburban area comprising Indrayani Nagar, Bakshi Layout, Suraksha Nagar, Samarth Nagar, Dhammakirthi Nagar, Shivaji Nagar wards of Vyahad PHC with a population of 9617. The area is characterized by bungalow type houses with well-managed drainage systems. The area is comparatively clean with very less garbage and other discarded containers. However, due to limited hour water supply, water storage was common among the residents. The main storage container is cement tanks of varying capacities.

Wadi experiences extremes of climate during summer and winter. In May–June the maximum temperature reaches >47 °C and in winter season the minimum temperature goes below 8 °C. However, during South West Monsoon (July–September/October), the weather is pleasant having an average maximum and minimum temperature of 34 and 23 °C, respectively, which is ideal for mosquito breeding. Nagpur receives 1092 mm rainfall annually.

Serological investigations

Blood samples were collected by the Medical Officer; Vyahad PHC and IgM analysis was carried out at the Indira Gandhi Government Medical College, Nagpur and Government Medical College, Nagpur, using the National Institute of Virology (NIV), Pune MAC-ELISA kits as per manufacturer’s instructions. Sera were considered positive when the optical density exceeded 2.1 times that of the negative control.

PCR detection and serotyping of dengue positive samples

Eight sera samples from acute cases were processed for virus detection and serotyping at NIV, Pune. In brief, RNA was extracted from the samples using viral RNA extraction kit (QIAGEN) according to the manufacturer’s instructions. The RNA was subjected to one step real time RT-PCR for detection of dengue, chikungunya and Zika virus using CDC trioplex real time RT-PCR kit according to the manufacturer’s instructions. CT value < 38 for each target was considered as positive. Depending on the target for which the Ct value was < 38, the sample was assigned as dengue or chikungunya or Zika positive.

The samples which were tested positive for dengue were serotyped using nested Multiplex RT-PCR as described by Lanciotti et al[15]. In brief, 10 μl of RNA was reverse transcribed using AMV reverse transcriptase (Promega Corporation, Madison, WI, USA) in the presence of 50 pm of group-specific reverse primer (DENV REV). Ten μl of this cDNA was used in the first cycle PCR with group-specific forward and reverse primers.

Entomological investigation

Adult collection: Mosquito adult collections were carried out in all the affected wards during 30th September to 31st October 2017. Oral consents from the house owners were taken before inspection of houses (Indoor adult collections). Efforts were made to conduct larval survey in all the houses of the clinically sick patients. Collections were made using hand-held mouth aspirators and the mosquitoes were pooled according to species, location, and gender and brought to the NIV for virus isolation.

Larval collection: Single larval survey method was employed to study the prevalence of Aedes mosquitoes and the nature of key breeding habitats. Both indoor and outdoor containers with water such as cement tanks, buried mud pots, broken earthenware, discarded metal ware/ plastic ware, etc. were searched in the affected areas for mosquito larvae. Efforts were made to conduct larval survey in all the houses of the clinically sick patients. The different entomological indices, viz. Breteau Index (BI: number of containers positive for Aedes larvae per 100 houses searched), Container Index (CI: percentage of containers infested with larvae or pupae) and House Index (HI: Number of houses positive for Aedes larvae per 100 houses searched) were calculated to determine the Aedes mosquito incidence.

Virus detection and isolation: Head of adult mosquitoes (n = 12) were severed, squashed and screened for detection of DENV and chikungunya virus (CHIKV) by immunofluorescent assay (IFA) using anti-dengue and anti-CHIKV serum raised in mouse as described by Sudeep et al[16]. The severed bodies were triturated in one ml minimum essential medium (MEM) using handheld battery operated homogenizer (Sigma USA), filtered (0.22Um, Millipore) and inoculated the filtrate in unfed Ae. aegypti mosquitoes for virus propagation as described earlier[17]. Second passage was done in the same mosquito on the 10th day post-infection (PI) and screened for both the viruses by IFA as described earlier. The severed bodies of the 2nd passage were processed in Vero E6 cell line for virus isolation as described by Sudeep et al[16].

Estimation of epidemiological parameters: Growth rate, extrinsic incubation period (EIP) and basic reproduction number

Based on the epidemiological records and patient interviews, the dates of onset were determined. Number of confirmed cases per day was obtained and cumulative cases per day were estimated. During the initial phase of the outbreak, assuming exponential increase in the cumulative cases, the growth rate of the epidemic (r) was calculated from the estimates of cumulative number of confirmed cases (y) and the estimated start date and size of the outbreak (t0 and y0), respectively, using the following formula:


Any outbreak of dengue occurs under environmental conditions favouring mosquito abundance and effective viral transmission by Aedes mosquitoes. The EIP for dengue is defined as the time period between the virus entry into mosquito body and virus being readily available in saliva for transmission. This is dependent on the mean day temperature[18],[19]. EIP for dengue was calculated as:


Where, T is the mean temperature. Meteorological parameters for the initial phase of the outbreak (September–October 2017) for Nagpur city (including Wadi) was obtained from online records at (accessed on February 7, 2018). Mean temperature, T was computed daily and average value for the initial phase was used in determination of EIP.

The basic reproduction number is defined as the number of secondary cases generated by introduction of an infective person in a susceptible population over the course of infection[20],[21]. For dengue, this was computed by:


Where, r is the growth rate of the outbreak, 1/γH is the mean infectious period in host, τi is the Intrinsic Incubation period (incubation period of dengue in human), τe is the dengue EIP, and 1/μV is the mean lifespan of the vector[22].

Software used

Mathematical calculations were performed in MATLAB software package.

Ethical statement

Since the study was conducted during the outbreak/ emergency, ethical approval was not taken.


Confirmation of dengue outbreak

Monthly incidence of confirmed dengue cases (IgM +ve) in the Vyahad and adjoining PHCs in NMC showed dengue activity since January 2017 [Figure 1]. In the Vyahad PHC, four cases of laboratory confirmed dengue were reported in August 2017. However, a spurt in the number of dengue positive cases was seen from 17 September to 23 October 2017 resulting in 44 laboratory confirmed cases and five deaths. In the other PHCs, only a few isolated cases were reported during the period [Figure 1]. Clinical manifestations, high number of dengue IgM positive cases and heavy breeding of Ae. aegypti mosquitoes confirmed the existence of a dengue outbreak in Wadi area in the NMC during the period.{Figure 1}

Clinical manifestations observed among the diseased dengue patients: A comparison

During the outbreak, five patients succumbed to dengue infection within 3–8 days post-onset of symptoms. Among the five, one had dual infection with DEN-2 and DEN-3 while the rest four had only DEN-2 infection. The former, a female patient aged 8.5 yrs, complained of high grade fever and got admitted on 26 September 2017. Despite the supportive treatment provided by the hospital, the girl expired on the 3rd day of admission. The cause of death was shown as myocarditis with GI bleeding and shock syndrome. An adult male patient aged 37 yrs died of dengue haemorrhagic fever and had metabolic acidosis, fulminant hepatic failure, acute renal failure, respiratory failure, etc. Details of the patients, clinical manifestations and the cause of death are given in [Table 1].{Table 1}

IgM antibody status among the patients: Screening of the 158 serum samples collected from suspected patients from 17 September 2017 to 23 October 2017 from Vyahad PHC using MAC-ELISA kits (NIV) yielded 44 (28%) samples positive for anti-DEN IgM antibodies. None of the samples tested positive for IgM antibodies to CHIKV.

Serotyping of dengue strains: Eight ELISA positive sera samples screened for detection of dengue, chikungunya and Zika virus by one step real time RT-PCR using CDC trioplex real time RT-PCR kit demonstrated the eight samples positive only for dengue. Nested-PCR analysis of the eight specimens has demonstrated the involvement of DEN-2 and DEN-3 viruses. Five cases had DEN-2 serotype, two had dual infection, i.e. infection with DEN-2 and DEN-3 serotypes and one sample, though dengue positive in the first PCR, serotype could not be determined.

Majority of the positive cases belonged to 0–40 yr age group with maximum cases coming from 0–10 and 11–20 yr age groups, respectively. Age-wise distribution of IgM positive patients is given in [Figure 2]. Male female ratio among the IgM positive cases was found almost equal and the maximum affected age group was found to be 0–10, 11–20 and 21–30 yr in the decreasing order. Gender-wise distribution of dengue IgM antibody positive case s is given in [Figure 2]. The attack rate was 0.45% (44/9617), approximately 5 per 1000 persons and case fatality of 0.05% (5/9617) that is, approximately 5 per 10,000 persons.{Figure 2}

Adult mosquito collections: Despite heavy fogging and source reduction activities, adult indoor mosquito survey yielded 135 mosquitoes belonging to three genera during the mosquito survey in the worst affected areas [Table 2]. Culex mosquitoes were the predominant genus followed by Anopheles and Aedes. Aedes genera was represented only by Ae. aegypti and constituted 26% of the total catch. Ward-wise distribution of Ae. aegypti mosquitoes (n = 35) collected during 30 September to 31 October 2017 is given in [Table 2] (data of only Ae. aegypti are shown).{Table 2}

Larval collections and mosquito indices: Aedes. aegypti larvae collection was conducted in 157 houses in 10 wards and 30 houses were tested positive (11.6%). Ward-wise HI ranged from 3.85 to 55.6 in different wards. HI was found lowest in Dhammakirti Nagar while the highest was observed in Suraksha Nagar (55.6) followed by Indrayani Nagar (46.2) as both these wards had highest breeding of Ae. aegypti. A total of 317 containers were searched during the survey of which 39 tested positive for Aedes larvae. Highest CI was seen from 30 September to 4 October and ranged from 8.54 to 29.23. BI ranged from 4 to 70.4 in different wards, the maximum during the peak of the outbreak, i.e. 30 September 2017 to 4 October 2017. The highest BI was detected in Suraksha Nagar and Indrayanai Nagar. Data on houses and containers searched and the entomological indices are given in [Table 3].{Table 3}

Breeding habitats: Aedes breeding was mainly observed in cement tanks, discarded tyres, mud pots and discarded metal and plastic-wares [Table 4]. Tyres contributed for the highest percentage of Aedes breeding (66.7%). Cement tanks constituted 33% (13 out of 39) of the total positive containers but might have contributed for maximum emergence of Aedes mosquitoes as they provided ample space for very heavy breeding. The most favoured breeding habitat for Ae. aegypti was cement tanks followed by refrigerator trays, though discarded tyres contributed for the highest percentage of positive containers [Table 4]. Breeding was also seen in half buried mud pots and discarded plastic/metal containers. An interesting observation during the larval survey was the detection of Aedes larvae in the refrigerator trays (tray provided on the backside of refrigerators for collecting condensed water). Nine refrigerators were found positive for Aedes breeding among 72 searched (12.5%).{Table 4}

Epidemiological parameters: Growth rate, EIP and basic reproduction number: Based on the growth of cumulative number of cases during the initial phase of the epidemic [Figure 3], the intrinsic exponential growth rate was estimated as 0.12 per day.{Figure 3}

Since the EIP for dengue is linked to mosquito metabolism and is known to be dependent on the mean temperature at any geographic location, we estimated the average value of mean temperature T for the initial growth phase from meteorological data and used the average value (T = 28 °C) in Eqn (2) to obtain the EIP. EIP was estimated to be 9.7 days for the outbreak period.

Estimation of the basic reproduction number (R0) was based on Eqn 3. Assuming the intrinsic incubation period of dengue in humans to be τi = 5.9 days[23], the average infectious period in humans to be 1/γH = 5 days[22], mean lifespan of Aedes mosquito (vector) to be 15 days and using the computed values of EIP and growth rate r = 0.12, the R0 was estimated as 29.12.

Virus detection and isolation: Virus could not be isolated from the mosquitoes. No virus positivity either for dengue or chikungunya could be detected by IFA in the head squashes despite two passages in Ae. aegypti mosquitoes. Vero E6 cells inoculated with mosquito body suspensions also failed to exhibit any cytopathic effect even after two consecutive passages suggestive of the absence of virus.


In the Vyahad PHC, dengue activity was first detected in August 2017 with 4 confirmed cases, which later blossomed into a massive outbreak with five case fatalities during September–October 2017. The virus might have reached Vyahad PHC from Kamptee and Ramtek areas of Nagpur district which showed isolated DEN activity during the early months of 2017 (Weekly dengue report, Nagpur Division). No history of migration of the cases could be ascertained as most of the patients were the inhabitants of the locality only. Clinical manifestations, presence of IgM antibodies to dengue virus in 28% of patients and the extensive breeding of the principal vector, Ae. aegypti mosquitoes clearly confirmed a dengue outbreak in Vyahad PHC of Wadi. Though 158 cases were reported with dengue like symptoms, only 44 samples were found positive for dengue IgM antibody. None of the samples tested positive for chikungunya IgM antibody. Etiology of the remaining cases could not be confirmed. Since only IgM antibody detection test was performed for mass screening in the study, many of the early POD samples might have tested negative. It could also be possible to have a few flu cases due to similarity in symptoms. Molecular typing of eight samples has confirmed the involvement of DEN-2 serotype predominantly though two sera samples had dual infection of DEN-2 and DEN-3 serotypes. The exact proportion of the two serotypes involved in the outbreak could not be determined due to low number of samples tested for sero-typing. Though the outbreak affected all the age groups, maximum cases were reported among the 0–20 and 30–40 yr age groups. Similarly, an almost equal proportion of male and female populations were affected during the outbreak though their age groups varied. In the present outbreak, it is difficult to correlate gender and age groups due to limited data. It has been reported earlier also that dengue affects all the age groups irrespective of gender though the percentage vary from outbreak to outbreak depending on host factors[11],[24]

Two of the five deaths reported during the outbreak presented with typical dengue hemorrhagic fever (DHF) manifestations. One of the diseased cases, a girl aged 8 and a half year, had dual infection with DEN-2 and DEN-3 serotypes while the other (M, 37 yr) had only DEN-2 infection. This shows the patients already contacted dengue infection with one or other serotype earlier and the recent outbreak could have lead to DHF. India, being endemic to dengue, the probability of getting a dengue infection, though subclinical, is high. In a recent study conducted in Vadu village, Pune, Maharashtra showed dengue antibodies among all the age groups including children in the age group of 0–5 yr[25]. Among the five fatal cases, three were children aged between 4 months to 14 yr while other two were adults aged in their mid-30s.

Extensive mosquito control activities such as source reduction and aerial fogging by the NMC before commencement of the investigation have considerably reduced the adult density. Still the team could collect adult mosquitoes from the houses. However, virus could not be isolated might be either due to the low number of mosquitoes processed or the delay in mosquito collections (almost after the peak of the outbreak). Heavy Ae. aegypti breeding was detected in Indrayani Nagar, Bakshi Layout and Suraksha Nagar and the major breeding source was the large cement tanks used for water storage. Almost all the households in the area had one large underground cement tank for collection of piped water, from where the water is pumped to the cement tanks located at the ground level. Though majority of the ground level tanks were covered, a few underground tanks remained open and this had lead to enhanced Ae, aegypti breeding during the outbreak. Another novel breeding habitat for Ae. aegypti observed during the survey was the refrigerator trays in the households. The trays are situated in the backside of the refrigerator meant for collection of the condensed water. Though breeding of Aedes mosquitoes in refrigerator trays were reported earlier, the presence of 12.5% of refrigerators positive for Ae. aegypti breeding is a matter of concern. It is a unique place for Aedes breeding and most of the health workers or members in the family miss this habitat. The cement tanks which are located just outside the house and the refrigerators are so close to people could be the possible reason for large outbreak in the area. Aedes breeding was also observed in other discarded items such as rubber tyres, mud pots, metal wares, plastic wares, etc.

Entomological investigation has revealed high mosquito indices in the area. In the worst affected wards, viz. Indrayani Nagar and Suraksha Nagar the HI and BI were so high (61 to 70) which reflected in the increased number of dengue cases. In other affected areas, BI was below the threshold and ranged from 4 to 8. However, number of dengue cases was high and correlated to the indices. This is in agreement with studies conducted in Singapore where explosive dengue outbreaks were reported despite very low BI[26].

Vector control seems to be the only effective method for the control of dengue today as no licensed vaccine or therapeutic is available. Unlike Anopheles mosquitoes, which transmit malaria, controlling Ae. aegypti mosquitoes by source reduction is easier as they breed mainly in clean stored waters in and around houses. During outbreaks, adult mosquito control by aerial spraying is initiated in large-scale; however, it is less effective for Ae. aegypti control[2]. Studies have demonstrated that source reduction helps to bring down the entomological indices drastically along with virus transmission during dengue outbreaks[2],[26]. House-to-house survey for larval breeding and source reduction has been accepted as the major dengue control programme in many countries. In the present outbreak also, dengue transmission was contained effectively within a month by the collective efforts of the health workers.

The study also included estimation of epidemiological parameters based on the available data. The cumulative confirmed cases increased sharply indicating a rapid spread. This is evident from the exponential growth rate of the outbreak, r = 0.12 per day. During the initial phase of the outbreak (mid September 2017), the mean temperature was 29 °C resulting in an associated EIP of 9.7 days. Thus, a favourable EIP, clubbed with a low diurnal temperature range (DTR) of ~8 °C, which is the difference between daily maximum and minimum temperatures during the period, has favoured increased metabolic activity (abundance) of the mosquitoes and helped enhanced virus transmission[27]. Our estimation of the basic reproduction number, R0 at 29.12, which is in agreement with earlier studies in dengue outbreak investigations[22] considering an EIP associated with mean temperature for the early phase, small population size and no interventions during the early phase. Favier et al[28] have reported reproduction number in the range from 2.0 to 103.0 for dengue outbreaks in varied tropical settings in Brazil.


Data on the present investigations comprehensively points towards a DENV outbreak with the involvement of two serotypes in Wadi, Nagpur during September–October 2017. Water storage in the close vicinity of houses contributed to heavy Ae. aegypti breeding that has enhanced virus transmission. Timely identification of the etiological agent (by IgM antibody detection) has helped to contain the outbreak by effective source reduction. One of the major drawbacks of the investigation was that virus could not be isolated from mosquitoes as isolation efforts were made only at a very late stage. Also the number of mosquitoes processed for isolation was also very small (n = 35). The investigators could not determine the IgG status of the patients due to limitations in resources and this could also be a drawback in the study. Nagpur, being an endemic area for dengue virus, continuous monitoring of the virus/mosquitoes is needed especially when circulation of different dengue serotypes was detected in the present outbreak.

Conflict of interest: None to declare.


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