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 ~  Abstract
 ~ Introduction
 ~ Methodology
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 2  |  Page : 236-240
 

Co-circulation of four dengue serotypes at South Eastern Andhra Pradesh, India: A prospective study


1 Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
2 Department of Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

Date of Web Publication7-Aug-2018

Correspondence Address:
Dr. Usha Kalawat
Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati - 517 507, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_109

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 ~ Abstract 

Background: Dengue is one of the most important mosquito-borne viral diseases in the world. The emergence and spread of four dengue viruses (DENVs) (serotypes) represent a global pandemic. The four distinct serotypes are, namely, DENV-1, DENV-2, DENV-3 and DENV-4. Very few dengue serotyping studies have been reported from Andhra Pradesh. In this context, the present study focuses on the circulating serotypes of dengue in South-Eastern Andhra Pradesh. Methodology: Study was done at Sri Venkateswara Institute of Medical Sciences, a teaching hospital in Tirupati, Andhra Pradesh. Acute phase dengue serum samples were collected and tested for NS1 antigen and anti-human IgM antibodies by enzyme-linked immunosorbent assay (ELISA). NS1-positive samples were further serotyped by reverse transcriptase real-time polymerase chain reaction (rRT-PCR). Results: A total of 398 serum samples were received from clinically suspected dengue fever cases. Of these, 150 (37.7%) samples were positive for NS1 and/or IgM ELISA. The 96 NS1 antigen-positive samples were further processed for serotyping, of which 36 were negative by rRT-PCR. DENV-2 (41%) was the predominant serotype, followed by DENV-4 (37%), DENV-3 (12%) and DENV-1 (10%) in descending order. Conclusion: This study reports the all four dengue serotypes' co-circulation. This is the first report from South Eastern Andhra Pradesh. Amongst four, DENV-2 was predominant followed by DENV-4. The information of predominant serotypes can guide in forecasting dengue outbreaks and improving control measures of vectors thus may be helpful in the prevention of outbreaks.


Keywords: Andhra Pradesh, dengue virus, dengue virus-2, dengue virus-4, outbreak, serotypes


How to cite this article:
Racherla RG, Pamireddy ML, Mohan A, Mudhigeti N, Mahalakshmi PA, Nallapireddy U, Kalawat U. Co-circulation of four dengue serotypes at South Eastern Andhra Pradesh, India: A prospective study. Indian J Med Microbiol 2018;36:236-40

How to cite this URL:
Racherla RG, Pamireddy ML, Mohan A, Mudhigeti N, Mahalakshmi PA, Nallapireddy U, Kalawat U. Co-circulation of four dengue serotypes at South Eastern Andhra Pradesh, India: A prospective study. Indian J Med Microbiol [serial online] 2018 [cited 2019 Dec 12];36:236-40. Available from: http://www.ijmm.org/text.asp?2018/36/2/236/238684



 ~ Introduction Top


Dengue infection has emerged as a critical threat to population health globally over the past 50 years. The World Health Organization (WHO) estimates that 50–100 million dengue infections occur each year and that almost half the world's population lives in countries where dengue is endemic. At present, almost three-fourth global populations are exposed to dengue virus (DENV) in Asian-Pacific region.[1] Dengue is one of the most important mosquito-borne viral diseases in the world today. The emergence and spread of four DENVs (serotypes) represent a global pandemic. The four distinct serotypes are, namely, DENV-1, DENV-2, DENV-3 and DENV-4.[2] In India, the first evidence of the occurrence of dengue fever (DF) in the country was reported during 1946 from Madras (now called Chennai) in Tamil Nadu.[3] The first dengue haemorrhagic fever (DHF) outbreak occurred in Calcutta (West Bengal) in 1963,[4] and subsequently, numerous outbreaks have been reported.[2] All these reports suggest that India represents 33% of the worldwide DENV infections.[5],[6] All the four serotypes i.e., DENV-1, 2, 3 and 4 have been isolated in India [4] and were co-circulating or concurrent infections were observed, which makes India one of the hyperendemic countries in the world [5],[7],[8],[9],[10] with frequent outbreaks.

In India, resurgence of epidemic of dengue activity poses a major public health challenge. This upsurge has been associated with the geographical expansion of both the mosquito vectors and the viruses.[1] Dengue infections have spread throughout the country with all serotypes in circulation without clear geographical distribution.[2] Serotyping of dengue is not performed routinely; therefore, data on circulation of different serotypes in various parts of the country are limited.

From South India, serotyping has been reported first from Tamil Nadu.[3] Few reports from Karnataka, Kerala and Telangana are also available. To the best of our knowledge, research articles on serotyping of dengue from this part of Andhra Pradesh are not available from recent past. In this context, the present study was conducted to know about circulating serotypes of dengue in South-Eastern Andhra Pradesh.


 ~ Methodology Top


This prospective study was conducted at Sri Venkateswara Institute of Medical Sciences (SVIMS), a tertiary care teaching hospital, in Tirupati, Andhra Pradesh, India. Institute Ethics Committee approval was obtained for the study. All the patients clinically suspected to have dengue fever as per National Vector Borne Diseases Control Programme [4] case definition presenting to the emergency room, medical outpatient department, medical wards and Medical Intensive Care Unit at SVIMS, a tertiary care teaching hospital, Tirupati; the paediatric medicine wards, paediatric medicine Intensive Care Unit at Sri Venkateswara Ramnarain Ruia Government General Hospital, Tirupati; the teaching hospital attached to Sri Venkateswara Medical College, Tirupati, during October 2017, to November 2017, outbreak were included in the study.

Sample collection

Suspected dengue patients of all age groups with 5 days and less duration of fever were included in the study. Peripheral venous blood (2–3 ml) was collected.[11] The blood was allowed to clot at room temperature (20°C–25°C) and then centrifuged at 3300 rpm for 10 min.[12] If not tested within 2 days, the separated serum was transferred to a sterile vial and stored frozen at −70°C.[13] The cases were classified into dengue (with or without warning signs) and severe dengue according to the WHO [14] based on patients' clinical presentations (only for serotyped cases).

Enzyme-linked immunosorbent assay

Serum was tested for the presence of dengue NS1 antigen and antihuman IgM antibodies using Panbio Dengue Early enzyme-linked immunosorbent assay (ELISA) kit (Standard Diagnostics, Inc., Republic Korea) and National Institute of Virology (NIV) DEN IgM capture ELISA ® kit supplied by NIV, Pune, respectively, by ELISA as per the manufacturer's protocol. Samples tested positive for NS1 antigen were further processed for serotyping.

Dengue serotyping

Viral RNA from NS1 antigen ELISA-positive serum samples was extracted using QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. The extracted RNA was reverse transcribed and real-time polymerase chain reaction (rRT-PCR) was done using Superscript-III one step RTPCR kit (Thermo Fisher Scientific Inc. Waltham, Massachusetts, United States).

Serotyping was done using CDC DENV-1-4 Real-Time RT-PCR Assay for Detection and Serotype Identification of DENV primers'[15] primer-probe set provided by CDC, Atlanta, USA. The reporter dyes used were DENV-1 (FAM), DENV-2 (VIC), DENV-3 (TEXAS RED), DENV-4 (CY5) and Rnase P (endogenous control) (FAM). Briefly, the amplification conditions were reverse transcriptase – 30 min at 50°C, Taq polymerase activation – 2 min at 95°C, 15 s at 95°C, 1 min at 60°C for 45 cycles (after Taq polymerase activation step). The results were interpreted as per kit instructions and analysed.

Statistical analysis

Based on age, patients were divided into three groups: paediatric ≤18 years, adult 19–59 years and geriatric ≥60 years. Normally distributed continuous variables were summarised by mean and standard deviation. Remaining variables were summarised as median (interquartile range). All categorical variables were summarised as percentages. The association between categorical variables was studied by Pearson's Chi-square test. For data analysis, statistical software IBM SPSS Statistics 20.0 for windows version, (NY IBM Corp., USA) was used.


 ~ Results Top


A total of 398 dengue-suspected samples were received during the study period. Of these 398 samples, 150 (37.7%) samples were positive for NS1 and/or IgM ELISA. Of the 150 positive samples, 105 were positive for dengue IgM antibodies and 96 for NS1 antigen [Table 1]. Samples positive for both NS1 and IgM antibodies were 51. NS1 positive but IgM negative samples were 45, whereas 54 samples were IgM positive but NS1 negative. Age-wise test results for NS1 antigen and IgM ELISA are shown in [Table 2].
Table 1: Dengue NS1 and IgM-positive cases

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Table 2: Age-wise distribution of dengue cases

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Adults had the highest number of clinically suspected (203, 51%) and laboratory-confirmed cases of dengue (79, 52.7%), with a mean age of 35.5 ± 11.6 years and found to be significant (P = 0.037). The next age group was paediatric with a mean age of 8.8 ± 5.2 years, tailed by geriatric group with 68.6 ± 6.8 years mean age. No gender difference (P = 0.569) with positivity was observed in the study. The male-to-female ratio in suspected dengue cases was 1.02:1 and in dengue positive cases was 0.95:1.

A total of 96 NS1-positive samples were serotyped by rRT PCR. The results are depicted in [Table 3] and gender-wise distribution of serotypes in [Table 4]. Of these, only 60 were positive by rRT PCR with a preponderance of DENV-2 serotype. Negative result for DENV does not preclude infection.[15] It may be because of low virus number or viral load in patient, being beyond detectable limit of the assay. The male-to-female ratio amongst dengue serotyped samples was 0.94:1. The distribution of serotypes is shown in [Figure 1]. The clinical manifestations of patients were given in [Table 5]. Nearly 51.67% (DENV-1 3 cases, DENV-2 16 cases, DENV-3 2 cases and DENV-4 ten cases) of cases were dengue fever without warning signs. Almost 43.33% were dengue fever with warning signs (DENV-1 3 cases, DENV-2 9 cases, DENV-3 3 cases and DENV-4 11 cases). Almost 5% (DENV-3 2 cases and DENV-4 one case) were severe dengue with warning signs.
Table 3: Distribution of dengue serotypes

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Table 4: Distribution of gender-wise dengue serotypes

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Figure 1: Pie chart showing distribution of dengue virus serotypes (n=60)

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Table 5: Clinical manifestations of patients (serotyped cases)

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 ~ Discussion Top


This prospective study was done to identify the circulating dengue serotypes in this region. Southern part of India is pivotal for several viral diseases, amongst which the DENV tops the list. Recurrent dengue outbreaks were experienced by South Indian states such as Telangana and Andhra Pradesh with increased disease severity, but regarding circulating serotypes, very few studies are available.[5] After 1965 outbreak report from Visakhapatnam,[16] limited data are available from Andhra Pradesh, though studies from Hyderabad [17] (now in Telangana) are available.

Different parts of the country showed different trends of subtypes from time to time. In this study, all four serotypes of dengue were reported. In South India, during 1970–2000, DENV-2 was the principal serotype, responsible for huge outbreak registered in 1996,[3],[17] which was slowly replaced by DENV-3. Later on outbreaks with DENV-1, DENV-2 and DENV-3 became common. After 1970, DENV-4 was seldom reported from India.[17] In 1963, DENV-1, DENV-2 and DENV-4 were isolated during an outbreak of dengue fever in Vellore, Tamil Nadu and all four serotypes were isolated in subsequent outbreaks.[3],[18] DENV-3 and DENV-4 were reported from Hyderabad during the outbreak of 2007; however, the circulation of all four serotypes was noted in the same place during 2014.[5],[19] The dominance of DENV-2 was reported from Karnataka, whereas three serotypes DENV-1, 2 and 3 and no cases due to DENV-4 were reported from Kerala.[20],[21] In Northern India, studies from Delhi have reported all circulating serotypes.[3],[22] During 2003 outbreak, DENV-3 was predominant and co-circulated with DENV-1 in 2006 in Delhi.[3] In 2013, DENV-2 subjugated the outbreak with few cases of DENV-1 and DENV-3.[22] A 4-year (2009–2012) study from Uttar Pradesh reported three DENV serotypes (DENV-1, 2 and 3), but the DENV-4 was not detected at all. The prevalence of each serotype has changed through the years. In 2009, DEN-3 was the dominant serotype, but in 2010 and 2011, DENV-2 became predominant which was later replaced by DENV-1.[23] In Eastern parts of India, studies from Odisha [2] and Kolkata reported DENV-2 and DENV-3 as the dominants circulating with no sign of DENV-4.[2],[24] A 9-year (2007–2010) study from Mumbai,[25] Western India, reported all serotypes with DENV-2 as the predominant serotype, whereas DENV-3 was more common during 2012–2015. Co-circulation of DENV-1, 2 and 3 was reported from Pune [26] during 2002–2008 with additional detection of DENV-4 serotype along others during 2009–2010.

Although circulation of all four DENV serotypes is reported, the dominant subtypes and epidemiology of DENV keep on fluctuating. In our study, all serotypes were detected with DENV-2 (41%) as the dominant serotype. This is in concordance with other studies from Delhi,[22] Uttar Pradesh [23] and Mumbai.[25] Although all dengue serotypes were circulating in different parts of the country, DENV-4 infections were very few as compared to other serotypes. Even absence of DENV-4 was observed in some of the outbreaks such as reported from Odisha,[2] Kolkata,[24] Uttar Pradesh [23] and Delhi.[22] In this study, DENV-4 serotype was the common serotype only preceded by DENV-2 with 37% positivity. Few cases of DENV-1 and DENV-3 were also detected. This was in contradiction to studies of Garg et al.[3] and Mishra et al.[23]

No concurrent infections were detected in our study which can be explained by small sample size. Studies with large sample size might be more helpful in finding the answers to the presence or absence of concurrent infections. Previously, a study from Delhi [9] reported first concurrent infections by different dengue serotypes. Study at Ernakulam, Kerala,[10] reported an approximate rate of 56.8% of concurrent infections with the association of DENV-1, 2 and 3. Another study from Kerala [27] reported a predominant combination of concurrent infections of DENV-1 and DENV-3 compared to other combinations.

Adult age group was most affected amongst all the age groups. This was in concordance with studies from Mumbai,[25] Delhi,[9] Odisha [2] and Uttar Pradesh.[23] This age group comprises working and student population. As dengue is mainly spread through Aedes mosquitoes which are day biters, the crowded areas such as workplaces and colleges may facilitate the spread of infection. Paediatric age group was next affected followed by geriatric age group. There was no significant association found in seropositivity between males and females. This was in opposition to studies from Mumbai,[25] Delhi,[22] Uttar Pradesh [23] and Northern Kerala,[27] wherein males comprised more number of cases as compared to females. Gender-based association of dengue serotypes was not observed in our study.

Fever was the most common presentation affecting all patients (100%). Other complaints included myalgia, arthralgia, diarrhoea and vomiting. Haemorrhagic fever was associated with all serotypes' infections with a high percentage in DENV-1 (50%) and DENV-4 (50%). Severe dengue was common in infections with DENV-3 and DENV-4, where jaundice, respiratory distress and elevated aspartate transaminase and alanine transaminase levels were observed. Similar observations were made in another study.[19] Impaired consciousness was also observed in DENV-4. Although DENV-2 was the predominant serotype in our study, the severity of infection was high in infections with DENV-3 and 4.

On testing with ELISA, 54 samples were found to be NS1 antigen negative but IgM antibody positive. NS1 antigen can be detected up to 6 days of onset of symptoms, whereas IgM can be detected from day 4 of onset of symptoms.[13] Low viral load or delay in reporting to the hospital may be one of the possible causes for undetected NS1 antigen in these samples. As we have not done dengue IgG ELISA, we cannot conclude whether the infection was primary or secondary.

Despite the study having limitations in the form of short study period and small data, the findings about the distribution of serotypes are invaluable. This is the first study on serotyping of DENV after the bifurcation of the state. Nevertheless, the trend of serotype dominance keeps on changing from time to time. Further continuous surveillance of dengue serotyping will provide the extent of distribution of different dengue serotypes. Further genotyping of serotypes should be done which can help in designing vaccines.


 ~ Conclusion Top


This study revealed the presence of four serotypes of dengue virus, with predominance of DENV-2, however severe infections were found in DENV-3 and 4. This information on predominant circulating serotypes helps us in forecasting dengue outbreaks, which will certainly strengthen our ability and preparedness to address recurrent epidemics in this part of India.

Acknowledgement

We are very grateful to Centers for Disease Control and Prevention (CDC), Atlanta for kindly providing CDC DENV-1-4 Real-Time RT-PCR Assay kit. We are thankful to ICMR-DHR and RCVDL, NIV, Pune, for supplying kits and reagents.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Cecilia D, Kakade MB, Bhagat AB, Vallentyne J, Singh A, Patil JA, et al. Detection of dengue-4 virus in Pune, Western India after an absence of 30 years – Its association with two severe cases. Virol J 2011;8:46.  Back to cited text no. 26
    
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Reddy MN, Dungdung R, Valliyott L, Pilankatta R. Occurrence of concurrent infections with multiple serotypes of dengue viruses during 2013-2015 in Northern Kerala, India. PeerJ 2017;5:e2970.  Back to cited text no. 27
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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