|Year : 2017 | Volume
| Issue : 1 | Page : 33-36
Prevalence and characterization of rotaviruses in children hospitalized for diarrheal disease in a tertiary care hospital, Pune
Sae Satish Pol1, Ashwini Kacharu Dedwal1, Sujata Sudhir Ranshing2, Shobha Dattatray Chitambar2, Sushma Narayan Pednekar1, Renu Satish Bharadwaj1
1 Department of Microbiology, B.J. Government Medical College, Sassoon General Hospital, Pune, Maharashtra, India
2 Rotavirus Division, National Institute of Virology, Pune, Maharashtra, India
|Date of Web Publication||16-Mar-2017|
Sae Satish Pol
Department of Microbiology, B.J. Government Medical College, Sassoon Hospital Campus, Station Road, Pune - 411 001, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Diarrhoea remains the second most common cause of death among children below 5 years globally. Among various enteric pathogens, rotavirus appears to be the most important aetiological agent of acute gastroenteritis in infants and young children. Increased understanding of epidemiology of rotavirus infections is needed to improve the vaccine efficacy. Aim: This study aims to determine prevalence rotavirus infection and prevalent circulating strains of rotavirus in and around Pune. Setting and Design: Prospective hospital-based study. The study was approved by Institutional Ethical Committee. Materials and Methods: Stool samples (n = 100) were collected from children aged <5 years, hospitalised for acute diarrhoea in paediatric ward at a tertiary care hospital. Samples were subjected for rotavirus antigen capture ELISA. The viral RNA was subjected to multiplex reverse transcription polymerase chain reaction to amplify VP7 genotypes G1–G4, G8–G10 and G12 and VP4 genotypes P, P, P, P, P and P. Nontypable rotavirus strains were sequenced. Results: About 35% stool samples were positive for rotavirus antigen by ELISA. G9P (28.6%) was found to be the most prevalent rotavirus strain. The detection of emerging strain G12P (14.3%) and rare reassortant strain G9P was the significant finding. Conclusion: Genotypes found in circulation are not present in the currently used vaccine. Thus, an emergence of newer genotypes over a period calls for the continued surveillance and genomic characterisation of rotaviruses to improve the vaccine efficacy.
Keywords: Characterisation, diarrhoea, efficacy, genotype, polymerase chain reaction, rotavirus
|How to cite this article:|
Pol SS, Dedwal AK, Ranshing SS, Chitambar SD, Pednekar SN, Bharadwaj RS. Prevalence and characterization of rotaviruses in children hospitalized for diarrheal disease in a tertiary care hospital, Pune. Indian J Med Microbiol 2017;35:33-6
|How to cite this URL:|
Pol SS, Dedwal AK, Ranshing SS, Chitambar SD, Pednekar SN, Bharadwaj RS. Prevalence and characterization of rotaviruses in children hospitalized for diarrheal disease in a tertiary care hospital, Pune. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 29];35:33-6. Available from: http://www.ijmm.org/text.asp?2017/35/1/33/202348
| ~ Introduction|| |
Diarrhoeal disease is a major public health concern for both developed and developing countries. Diarrhoea remains the second most common cause of death among children below 5 years globally. Among various enteric pathogens, rotavirus appears to be the most important aetiological agent of acute gastroenteritis in infants and young children. Rotavirus causes deaths of approximately 453,000 children annually, and most of them occur in developing countries in the Asian subcontinent, Africa and Latin America.,, It is estimated that in India, rotavirus accounts for 122,000–153,000 deaths, 454,000–884,000 hospitalisations and 2 million outpatient visits in children <5 years of age.
Two rotavirus vaccines, Rotarix™ and RotaTeq ® introduced in several countries for the use against rotavirus diarrhoea have been reported to have low efficacies (39%–49%) in low-income countries., In India, immunogenicity of these vaccines along with seroconversion rates has been reported to be 58.3% and 83% among healthy infants.,
However, limited data are available on the vaccine efficacy. In view of this, increased understanding of epidemiology of rotavirus infections is needed. This study was aimed to determine the prevalence of rotavirus infections and genotypes in the stool specimens from children suffering from the acute diarrhoeal disease in a tertiary care hospital from Pune, Western India. The hospital has 1200 beds which include 77 beds for paediatric patients. Patients visiting this hospital belong to lower socioeconomic class and are residents of rural or urban region of Pune district.
| ~ Materials and Methods|| |
Stool specimens were collected from children aged <5 years, hospitalised for acute diarrhoea in paediatrics ward admitted to Sassoon General Hospital located centrally in Pune city.
A case of acute gastroenteritis enrolled in the present study was defined as the passage of ≥3 loose or watery stools a day with or without associated symptoms such as vomiting, fever and abdominal pain. All the patients were examined for fever, number of episodes and duration of vomiting and diarrhoea, extent of dehydration and treatment for the assessment of severity of disease., Epidemiologic data were recorded for all patients. The study was approved by hospital Ethics Committee.
Testing for rotavirus antigen
Samples were subjected for rotavirus antigen capture ELISA by IVD ELISA kit (Carlsbad, CA, USA). Ten per cent (w/v) suspensions of all rotavirus positive stool specimens prepared in 0.01 M phosphate buffered saline (pH 7.2) were transported to National Institute of Virology, Pune.
G- and P-typing
The viral nucleic acids were extracted from 10% (w/v) suspensions of all ELISA positive stool specimens using Trizol (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. The VP7 (G) andVP4 (P) genes were genotyped by multiplex reverse transcription-polymerase chain reaction (RT-PCR) according to the method described earlier. The viral RNA was subjected to one-step RT-PCR (Qiagen, Hilden, Germany) using the sets of outer primers: 9Con1-L/VP7-R; Con 3/Con 2 and oligonucleotide primers that could amplify VP7 genotypes G1–G4, G8–G10 and G12 and VP4 genotypes P[ 4 ], P[ 6 ], P[ 8 ], P[ 9 ]; P[ 10] and P[ 11 ]. All PCR products were analysed by electrophoresis using Tris-acetate-EDTA buffer, pH 8.3 on 2% agarose gels, containing ethidium bromide (0.5 µg/ml) and visualised under UV illumination. To determine the VP7 and VP4 genotypes of nontypeable rotavirus strains in multiplex PCR, first round PCR products obtained in agarose gel electrophoresis were sequenced using ABI-PRISM Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster, CA, USA) and a ABI-PRISM 310 Genetic analyser (Applied Biosystems, Foster, CA, USA) after purification on minicolumns (QIAquick: Qiagen, Valencia, CA, USA). The nucleotide sequences obtained were subjected to Basic Local Alignment Search Tool analysis in NCBI database to determine the genotype.
| ~ Results|| |
A total of 100 stool specimens were collected from children hospitalised for acute gastroenteritis during September 2012–August 2013. Of these, 35 (35%) were positive for rotavirus antigen by ELISA. The highest incidence for rotavirus positivity occurred in the age group of 7–12 months (51.4%) followed by 13–18 months (25.7%) and 19–24 months (11.4%). Although admissions of males outnumbered females, the detection rates of rotavirus did not differ significantly by gender (51.4% among males vs. 48.6% in females). Clinical severity score in rotavirus-infected patients was very severe 4%, severe 55%, moderate 39% and mild 2% and in rotavirus noninfected patients was very severe 3%, severe 46%, moderate 40% and mild 11%. Analysis of the clinical severity scores indicated that patients infected with rotavirus had severe disease as compared to rotavirus noninfected patients. Among the rotavirus-positive patients, watery diarrhoea (100%) was the predominant clinical feature, followed by dehydration (85.7%), fever (80%) and vomiting (74.3%). Rotavirus infections were detected in all months of the year. However, maximum positivity was observed during November–February (74.3%).
All the rotavirus-positive specimens were subjected to multiplex PCR for G andP typing. However, out of 35 ELISA-positive samples, 14 samples (40%) could be genotyped for both the genes. G9 and P[ 4 ] were the common genotypes detected in the present study [Table 1].
Among the strains typed for both VP7 and VP4 genes, G9P[ 4 ] strains attained the highest score (28.5%) [Figure 1]. This was followed by G1P and G2P[ 4 ] (21.4%); G12P[ 6 ] (14.3%) and G9P (7.1%). Mixed rotavirus strain G2P P[ 6] was detected in 7.1% of the specimens [Table 2].
| ~ Discussion|| |
In the present study, the prevalence of rotavirus in acute diarrhoeal patients has been found to be 35%. This data reaffirms earlier reports documenting large rotavirus disease burden among children in Pune and other cities of India.,
In the present study, the rotavirus strains G1P[ 8 ], G2P[ 4 ] and G9P[ 8 ] often detected in human infections have been found to contribute to diarrhoeal infections at 7.1%–21.4% level. Infection with rare reassortant strain G9P[ 4 ] (28.6%) and emerging strain G12P[ 6 ] (14.3%) was the significant outcome. Mixed infection was detected in one case with G2P[ 4 ], P[ 6 ].
| ~ Conclusion|| |
In a study published by Ramani and Kang  and Kang et al., dominant strain in South India was G2P[ 4 ] and in north India G1P[ 8 ]. On the contrary, Gladstone et al., Mathew et al. and Babji et al. documented G1P to be the dominant strain in Vellore, Kerala and Tamil Nadu (South India). G9P[ 4 ] was the dominant strain in the present study at Pune. During 2009–2012, G9P[ 4 ] rotavirus strain showed ~ 10% prevalence in Pune, western India.
From 2006 to 2009, there was a notable shift in the strains causing Rotavitus diarrhoea in specific regions of India. In particular, there was an increase in G12 strains, especially G12P strain in both Western (8%–24%) and northern region (8%–39%). G12, detected in the present study, was first detected in 1987–1988 in Philippines  and was then seen to be emerging all over the world. In 2003, G12 was first identified in India and since then been identified in Indian infants., The emergence of G12 strain may parallel the earlier emergence of G9 strain, which may have developed from a genetic reassortment event.
Melody et al. in 2012, reviewed 33 peer-reviewed manuscripts from the Indian subcontinent and found that the most common G-types (G1–4) and P-types (P and P) accounted for three-fourths of all strains in the subcontinent. However, strains varied by region, and temporal analysis showed the decline of G3 and G4 in recent years and the emergence of G9 and G12.
With the objective of reducing severe diarrhoea and hospitalisations caused commonly by rotavirus, the use of two oral rotavirus vaccines, Rotarix, a monovalent type containing an attenuated G1P [ 8 ] human strain (89–12) and RotaTeq, a pentavalent type containing G1, G2, G3, G4 and P1A[ 8 ]) human-bovine (WC3) reassortant has been permitted in several countries of world by the World Health Organization (WHO). Well-documented evidence shows that immunogenicity and efficacy of these vaccines in low- and middle-income countries is much lower than in high-income countries.,,, Among various factors, genotype diversity of rotavirus strains in developing countries has been reported to be one of the reasons., These data reinforce the necessity of monitoring of circulating strains.
Genotypes G9, G12 and P[ 6 ] obtained in the present study are not included in currently available vaccines recommended by the WHO. Although the importance of type-specific immunologic protection against rotavirus disease is still under discussion, many investigators suggested that genomic characterisation of rotaviruses is needed to assess whether vaccine efficacy might be altered by the changing pattern in the distribution of different G andPgenotypes.
Thus, an emergence of newer genotypes over a period calls for the continued surveillance and genomic characterisation of rotaviruses to improve the vaccine efficacy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]