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  Table of Contents  
BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 3  |  Page : 429-431
 

Serotyping of dengue viruses circulating during 2014–2015 in Assam, India


1 Department of Microbiology, Dr. Damanis Nursing Home, Dibrugarh, Assam, India
2 Department of Microbiology, Gauhati Medical College, Guwahati, Assam, India
3 Regional Medical Research Centre, Dibrugarh, Assam, India
4 Department of Microbiology, Assam Medical College, Dibrugarh, Assam, India

Date of Web Publication14-Nov-2018

Correspondence Address:
Dr. Ajanta Sharma
Department of Microbiology, Gauhati Medical College, Guwahati - 781 032, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_121

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 ~ Abstract 

Dengue has become endemic in India with outbreaks caused by all the four serotypes occurring almost every year. Dengue cases have been increasing alarmingly in Assam also. This study aimed to identify the dengue serotypes circulating in Assam. Clinically suspected dengue fever cases were included in the study. Viral RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Nested reverse transcriptase polymerase chain reaction was done for serotyping. The frequency of dengue was 25.23% with a peak during the period from September (22.56%) to October (26.22%). Two serotypes were detected, DEN-1 (72.7%) and DEN-2 (12.1%) and dual infection in 15.1%.


Keywords: Assam, dengue fever, dengue serotype, dengue virus, epidemiological feature


How to cite this article:
Rahman M, Sharma A, Patgiri S, Hussain E, Borah AK, Saikia L. Serotyping of dengue viruses circulating during 2014–2015 in Assam, India. Indian J Med Microbiol 2018;36:429-31

How to cite this URL:
Rahman M, Sharma A, Patgiri S, Hussain E, Borah AK, Saikia L. Serotyping of dengue viruses circulating during 2014–2015 in Assam, India. Indian J Med Microbiol [serial online] 2018 [cited 2019 Nov 14];36:429-31. Available from: http://www.ijmm.org/text.asp?2018/36/3/429/245374



 ~ Introduction Top


Dengue is a mosquito-borne viral disease of global public health concern and is a major cause of morbidity in most of the endemic regions of the world.[1] Dengue is caused by dengue virus (DENV) that comprises four serotypes (DENV 1–4) and transmitted in humans by Aedes mosquito species.[2] Infection may range from mild, self-limiting febrile illness (dengue fever) to a more severe form of dengue haemorrhagic fever and dengue shock syndrome.[3]

In India, DENV-2 and DENV-3 were the most predominant serotypes during 2003–2007, however, DENV-1 replaced these strains in the year 2008.[4] Dengue cases have been increasing alarmingly in Assam. DENV-2 has been reported from Assam and other neighbouring states earlier.[5],[6] Due to scanty information on dengue serotype distribution in Assam, the present study was undertaken to identify the serotypes of DENV circulating in Assam.


 ~ Materials and Methods Top


The study was carried out in the Department of Microbiology, Assam Medical College, Dibrugarh, Assam, for a period of 1 year (2014–2015) after taking the Institutional Ethics Committee approval. NS1 antigen detection was done by ELISA (Panbio Diagnostic) using Manufacturer's protocol.[7] The study was hospital based as well as samples were collected also from the community where suspected dengue fever cases occurred.

Extraction of Viral RNA was done using QIAamp Viral RNA Mini Kit manufactured by Qiagen as per standard protocol. Initially, a dengue pan-specific polymerase chain reaction (PCR) was done for confirmation of dengue infection. A nested reverse transcription (RT)-PCR assay was done comprising of two-step PCR reaction involving an initial RT and amplification using universal dengue primers targeting a region of the virus genome (C-pr M) followed by a serotype-specific amplification.[3] The sequences of the oligonucleotide primers (Gibco-BRL, USA) used to amplify and serotyping of DENVs are presented in [Table 1].
Table 1: Sequences of the oligonucleotide primers used to amplify and type dengue viruses

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Reverse transcription and amplification of RNA

The target sequence of the viral RNA was converted to a complementary DNA copy (cDNA) using RT and the DENV downstream consensus primer (D2), homologous to the genomic RNA of the four serotypes, before enzymatic DNA amplification. Subsequently, Taq polymerase amplification of resulting cDNA was performed using the upstream DENV consensus primer (D1).[3]

The cDNA was synthesised by incubating RT-PCR mixture consisting of buffer (250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2), 1.25 mM of each dNTPs (Gibco BRL, Maryland, USA), 25 pmol D2 primer, 200 units of Superscript II and 20 units of RNase inhibitor (Promega, Madison, USA) at 37°C for 60 min, followed by enzyme inactivation at 100°C for 10 min.

In the first round of PCR, 12 μl of cDNA was added to 38 μl of the PCR mixture which consisted of PCR buffer (Promega, Madison, USA), 2.5 mM of each dNTPs (Gibco BRL, Maryland, USA), 12 pmol D1 primer and 1 unit Taq DNA polymerase (Promega, Madison, USA). The reaction was carried out in a thermal cycler at 94°C for 5 min of initial denaturation followed by 35 cycles of denaturation (94°C for 1 min), primer annealing (55°C for 1.5 min) and primer extension (72°C for 2.5 min) followed by final extension at 72°C for 7 min.

The amplicon was subjected to ethidium bromide stained 2.0% agarose gel electrophoresis. The size of different bands was compared with a standard marker for the identification of dengue serotypes (DENV type, 482 bp [Den-1], 119 bp [Den-2], 290 bp [Den-3] and 389 bp [Den-4]) [Figure 1].
Figure 1: Gel image (Lane A - 100 bp DNA ladder, Lane B - negative control, Lane C - positive control, Lane D to H and K - DENV 1 [482 bp], Lane I and J - DENV 2 [119 bp])

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Statistical analysis of data was done using Epi info version 7. Chi-square test was done to calculate P value to show the statistical significance.


 ~ Results Top


Out of 650 samples, 164 (25.2%) were found to be laboratory confirmed dengue cases with symptoms of classical dengue fever, of which 57 (34.7%) were positive for NS1 antigen-positive cases. The overall frequency of dengue infection was more during the period from September (22.56%) to October (26.22%). Chi-square test showed a significant difference in month-wise distribution of the dengue cases.

Of the 57 cases, 4 (7%) cases were seen in <15 years of age, 42 (73.6%) cases were seen between 16 and 40 years and 11 (19.3%) cases in the age group of >40 years. Children were found to be least affected. Chi-square test showed this finding to be statistically significant (P < 0.0001). Males (73.6%) were found to be more affected than the females (26.3%) with a male-to-female ratio of 2.8:1, which was statistically significant (P < 0.0001).

DENV 1 (72.7%) was the predominant serotype in Assam followed by DENV 2 (12.1%). Mixed infection with DENV 1 and DENV 2 was found in 15.1% of cases. In the age group of <15 years, only DENV 1 was found, and DENV 1 was the most prevalent serotype in both the sexes.


 ~ Discussion Top


In the present study, the frequency of dengue fever was found to be 25.3% which correlates with the reports of Bandyopadhyay et al., 2013 (25.6%), Patankar et al., 2014 (21%) and Chakravarti et al., 2012 (31.1%).[4],[8],[9] However, Parida et al. and Raja et al. have reported a higher frequency of dengue fever (64.10% and 46.84%, respectively).[10],[11] The complex epidemiology of dengue fever in the Indian subcontinent has substantially changed over the past six decades regarding prevalent strains, affected geographical locations and severity of disease. The upward trend is due to increases in long-distance travel, population growth and urbanisation, lack of sanitation, ineffective mosquito control and increases in the surveillance and official reporting of dengue cases.

The present study also observed the occurrence of dengue mostly during post-monsoon period (September–November) which corresponds with the previous report by Bandyopadhyay et al.[8] Probably during monsoon period vector multiply and their number reaches maximum in post-monsoon period, causing more transmission of dengue during that period.


 ~ Conclusion Top


Dengue is endemic in Assam with the circulating serotypes DENV-1 and DENV-2. Dengue cases appear mostly in the post-monsoon period. Hence, the appropriate preventive measure should be initiated during the monsoon season only. In the absence of a licensed vaccine or specific drugs, the containment of spread of the vector and the disease is still important.

Acknowledgement

The authors would like to acknowledge the Department of Biotechnology, Ministry of Science and Technology, Government of India, for funding this study vide sanction order no. BT/Med/15/Vision-NER/2011, dated 2nd November 2011. The authors are also thankful to Dr. Biswa Barkakoti, Scientist D, Regional Medical Research Centre, Dibrugarh, Assam, for his technical help in the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
World Health Organization and the Special Programme for Research and Training in Tropical Diseases (TDR). Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva, Switzerland: WHO Press; 2009. p. 3-147. Available from: http://www.whqlibdoc.who.int-publications-2009-9789241547871_eng.pdf. [Last accessed on 2015 Oct 21].  Back to cited text no. 1
    
2.
Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. Indian J Med Res 2012;136:373-90.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992;30:545-51.  Back to cited text no. 3
    
4.
Chakravarti A, Matlani M, Kashyap B, Kumar A. Awareness of changing trends in epidemiology of dengue fever is essential for epidemiological surveillance. Indian J Med Microbiol 2012;30:222-6.  Back to cited text no. 4
  [Full text]  
5.
Dutta P, Mahanta J. Potential vectors of dengue and the profile of dengue in the North-Eastern region of India: An epidemiological perspective. Dengue Bull 2006;30:234-42.  Back to cited text no. 5
    
6.
Sankari T, Hoti SL, Singh TB, Shanmugavel J. Outbreak of dengue virus serotype-2 (DENV-2) of Cambodian origin in Manipur, India – Association with meteorological factors. Indian J Med Res 2012;136:649-55.  Back to cited text no. 6
[PUBMED]  [Full text]  
7.
World Health Organization. Dengue guidelines for diagnosis, treatment, prevention and control: New edition. Geneva: World Health Organization; 2009.  Back to cited text no. 7
    
8.
Bandyopadhyay B, Bhattacharyya I, Adhikary S, Konar J, Dawar N, Sarkar J, et al. Comprehensive study on the 2012 dengue fever outbreak in Kolkata, India. ISRN Virol 2013;2013:1-5  Back to cited text no. 8
    
9.
Patankar MC, Patel BV, Gandhi VP, Shah PD, Vegad MM. Seroprevalence of dengue in Gujarat, Western India: A study at a tertiary care hospital. Int J Med Sci Public Health 2014;3:16-8.  Back to cited text no. 9
    
10.
Parida MM, Dash PK, Upadhyay C, Saxena P, Jana AM. Serological & virological investigation of an outbreak of dengue fever in Gwalior, India. Indian J Med Res 2002;116:248-54.  Back to cited text no. 10
    
11.
Raja D, Phukan C, Hazarika NK. Seroprevalence and epidemiological trends of dengue in Gauhati Medical College & Hospital. Assam J Int Med 2014;4:30-4.  Back to cited text no. 11
    


    Figures

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    Tables

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