Dengue, a vector-borne viral infection transmitted by Aedes mosquito, has recently become a major public health concern in the tropical regions of the world. Forty percent of global population is at risk of dengue virus infection, and about 2.5% people affected with severe dengue die each year. In new 5-year strategic plan of the World Health Organization announced in January 2019, dengue infection was identified among the ten highest priority health issues threatening global health. India contributes to a third of the total global burden of dengue. Dengue epidemic occurs in India almost every year. Dengue virus was first isolated in 1946, and the first dengue hemorrhagic fever (DHF) was reported in Kolkata in 1963. Since then, several outbreaks have been reported from different parts of India which become more frequent from mid-1990s.
Dengue virus, a member of Flaviviridiae family, has five serotypes – DEN 1, DEN2, DEN3, DEN4, and DEN5 (isolated in 2013).[6,7] It is transmitted to human being by Aedes mosquito. All the serotypes can cause dengue fever, a self-limiting febrile illness. The sequential or secondary infection by different serotypes of dengue virus induces the two major life-threatening complications – DHF and dengue shock syndrome (DSS) which are due to cross-reactive nonneutralizing antibodies from previous dengue virus infection.[9,10]
In the past 50-year cocirculation of different serotypes was reported from different parts of India which makes this country as hyperendemic for dengue virus. Kolkata is also a dengue-endemic zone where all the four serotypes are circulating with changes occurring in the leading serotypes. Since dengue cases are increasing at an alarming rate, the identification of predominant serotypes can guide in forecasting dengue outbreaks. It shall be immensely helpful for future prevention and disease control.[11,13]
In this context, a retrospective study was formulated to perform serological analysis, molecular detection, and serotyping of dengue virus to identify the serotype/s presently prevelant in the region.
The descriptive observational retrospective study was conducted in the Department of Microbiology, ID and BG Hospital, Kolkata, for 1-year period between November 2017 and October 2018. Clinically suspected dengue fever cases presented with high fever along with headache, retro-orbital pain, nausea, vomiting, malaise, joint pain, generalized skin rashes, and/or hemorrhagic manifestations who were attending outpatient department or admitted in inpatient department of ID and BG hospital were selected for this study. All the cases were nondiabetic and had no other physiological complications. After taking informed consent, 5240 blood samples were collected in clot vials. Fever cases other than dengue infection caused by common infective pathogens such as malaria parasite, enteric fever, and viral hepatitis were excluded by respective laboratory investigations.
As per National Vector Borne Disease Control Program guidelines, the blood samples were screened for dengue-specific immunoglobulin M (IgM) antibody by MAC-ELISA (InBios ELISA kit) if the duration of fever in patients was more than 5 days, and the blood samples were screened for NS1 antigen by NS1 ELISA (InBios ELISA kit) in case of acute dengue fever (duration of fever ≤5 days).
Blood samples of 70 NS1 reactive admitted patients who showed features of DHF such as positive tourniquet test, petechiae, gum bleeding, malena, epistaxis, thrombocytopenia (platelet <100,000 mm3), and hemoconcentration >20% and/or features of DSS such as narrow pulse pressure or profound hypotension were sent in maintained cold chain to ICMR-NICED Virus Laboratory, Kolkata, for molecular detection and serotyping.
Viral RNA was extracted from NS1 positive serum samples by QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) using 140 μl of serum sample, and RNA was eluted in 50 μL of elution buffer according to manufacturer’s protocol and was stored in − 80°C for molecular dengue virus detection. Nested reverse transcription-polymerase chain reaction (RT-PCR) (Lanciotti et al., 1992) was done for serotyping of dengue viruses. The PCR products were visualized under gel documentation to distinguish the 4 dengue serotypes, namely DENV-1 to 4. The entire process includes first-round RT-PCR using highly conserved D1 (forward) and D2 (reverse) primers which are homologous for all 4 serotypes. Serotype-specific amplification of DENV was performed by semi-nested PCR. Amplicon of first-round RT-PCR was diluted 1:100 times as template in second-round semi-nested PCR. The primers used are forward and reverse primers (D1, TS1, TS2, TS3, and TS4). The expected size of PCR products was 485bp for DENV-1, 119bp for DENV-2, 290bp for DENV-3, and 392bp for DENV-4. Electrophoresis of amplified products was run on 1.5% agarose gel. Ethidium bromide-stained products were visualized under Gel Documentation System (Bio-Rad, USA) [Figure 1].
Total 5240 samples were tested for dengue. Nearly 30.48% (1597) were serologically reactive for dengue infection (either IgM or NS1) [Figure 2]. Among 1597 dengue reactive cases, 482 cases were from IPD and 1115 cases were from outpatient department, and among 482 IPD cases, 70 cases admitted with hemorrhagic complications. About 80.52% (1286/1597) of dengue cases were NS1 reactive and 19.47% (311/1597) were IgM reactive [Figure 3]. Male sex was predominantly (66.37%) more affected than females [Figure 4]. Male: female ratio of dengue infected patients was found 1.9:1 in this study. There was a rise of dengue cases from the months of August and September, and a postmonsoon season peak was noted in October and November in our study followed by decline in December [Figure 5].
Among 70 NS1 reactive samples from indoor patients who were suffering from DHF and/or DSS, 67 samples were analyzed as being RNA positive. DEN2 was found in 61 samples, DEN3 was found in 3 samples (4.28%), and DEN4 was found in 3 samples (4.28%). DEN2 (87.14%) was found as most prevalent serotype presently circulating in the region [Figure 6].
Dengue fever has re-emerged as a major health problem in tropical and subtropical regions. There is a constant change in the serotype pattern of dengue virus circulating in different geographical regions. During outbreaks from different parts of India, different serotypes have been reported.
In this study, 30.48% (1597/5240) of patients were serologically reactive for dengue virus. This is concordant with many studies reports from different states of India such as Uttar Pradesh, Andhra Pradesh, Odisha, Delhi, and Kolkata.[3,4,8,11,16]
In the current study, 80.52% (1286/5240) were reactive for NS1 antigen whereas 19.47 (311/5240) samples showed reactivity for the presence of dengue IgM antibody.
In our study, postmonsoon period recorded the largest proportion of serologically positive cases. Pan-India picture was almost similar with reports published from Amritsar and Delhi in the north, Davangere and Kerala in the south, [3,6,9,13] and by Sarkar et al. from Kolkata. Heavy rainfall leads to decrease in climate temperature during the monsoon and postmonsoon period in the months of September and October. Humidity also increases. Hence, optimum temperature, high humidity, and abundant freshwater stagnation provide the perfect environment for rapid mass breeding and propagation of vector and transmission of viruses. Hence, to prevent the spread of dengue, full-fledged preventive measures are an absolute necessity at the beginning of the rainy season.
The prevalence of dengue infection was higher among males than females. This correlates well with other studies from Delhi, Amritsar, and Lucknow.[3,6,15] However, female predominance was also evident in many studies. This gender variation may be because of household orientation and less migration of women.
Published reports so far suggest that all four dengue virus serotypes are circulating simultaneously in India. All of them are identified in Kolkata. In our study, predominance of DEN2 serotype (88.57%) observed followed by DEN3 and DEN4. This proves that multiple serotypes are circulating at the same point in time at a particular pocket of endemicity. DEN1 serotype was not detected in any of the cases. Simultaneous coinfection with multiple serotypes in a particular patient was not found. In Kolkata, dengue was first documented in 1824, and in the year 1945, the first serotype isolated in India was DEN1 from Kolkata.[3,4] DHF was also first reported from Kolkata in 1963-63. Past studies from Kolkata have shown that there is an increasing trend of infection with multiple serotypes which suggests that the city is becoming hyperendemic for dengue virus. Sarkar et al. report the presence of all the four dengue serotypes in Kolkata with DEN2 being most predominant.
Our study revealed that DEN-2 serotype is more commonly associated with severe dengue disease, although serotyping was done only for all admitted DHF/DSS cases. Previous studies also suggested that DEN2 causes more large and severe disease than other serotypes in India.[3,11] Studies in Thailand and Taiwan also found that DEN-2 caused more severe disease among hospitalized patients. Speculation is there that enhanced pathogenicity of DEN-2 might be due to greater virus replication for DEN-2 in primed host.
Hence, the data on serotypes are very important which will help to develop laboratory-based surveillance that can forecast impending dengue outbreak. It shall be beneficial to increase our resource and preparedness at the administrative and decision-making level to combat repeated occurrence of dengue epidemic in India.
As our study finds cocirculation of 3 serotypes (DEN-2, DEN-3, and DEN-4) with predominance of DEN-2 serotype during the study period, it highlighted the extent of hyperendemicity of dengue serotype in this region. The greatest limitation in this study was that serotyping could not be done for all dengue IgM/NS1 reactive cases. Hence, the correlation between serotypes and severity of dengue infection was not established significantly, but the overall evidence of DEN-2 infection among severe DHF/DSS cases is a suggestive trend.
Laboratory-based active surveillance system is required which can forecast dengue epidemics by increased grass root level detection of dengue cases and detect the changing pattern in the prevalent serotype. As a specific serotype (namely DEN2) is associated with higher incidence of complications, this study will help to alert the public and health-care providers to diagnose properly and treat cautiously the complicated dengue cases.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
We acknowledge all staffs of Department of Microbiology and Dr. Pravash Chandra Sadhukhan and his team of National Institute of Cholera and Enteric Disease, Kolkata, for their support.
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