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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Acknowledgments
 ~  References
 ~  Article Figures
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  Table of Contents  
BRIEF COMMUNICATION
Year : 2014  |  Volume : 32  |  Issue : 1  |  Page : 57-59
 

Group A rotavirus and bacterial agents associated with diarrhoea-induced hospitalisations in children below 5 years of age in Jammu


Division of Veterinary Microbiology and Immunology, Sher e Kashmir University of Agricultural Sciences and Technology, Ranbir Singh Pura, Jammu and Kashmir, India

Date of Submission07-Mar-2013
Date of Acceptance10-Sep-2013
Date of Web Publication4-Jan-2014

Correspondence Address:
S Gazal
Division of Veterinary Microbiology and Immunology, Sher e Kashmir University of Agricultural Sciences and Technology, Ranbir Singh Pura, Jammu and Kashmir
India
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Source of Support: SKUAST-Jammu and ICMR (by providing ICMR-Junior Research fellowship),, Conflict of Interest: None


DOI: 10.4103/0255-0857.124308

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 ~ Abstract 

Out of 210 faecal samples collected from children below 5 years attending different hospitals in Jammu and exhibiting clinical signs of diarrhoea, 41.9% samples were found positive for group A rotavirus by RNA-PAGE. Escherichia coli isolated in the study belonged to nine serogroups, out of which O69 was most frequent, being present in 12.38% samples. E. coli serogroups well recognised as enteropathogens viz. O69, O20 and O153 were present in 27.6% samples. Other bacterial pathogens associated with diarrhoea were present in 8.09% samples, out of which Shigella spp. was found in 4.76% samples followed by Salmonella spp. (2.38%) and Pseudomonas spp. (0.95%).


Keywords: Escherichia coli, Ribonucleic acid - Polyacrylamide gel electrophoresis, rotavirus, serogroups


How to cite this article:
Gazal S, Taku A, Bhat M A, Badroo G. Group A rotavirus and bacterial agents associated with diarrhoea-induced hospitalisations in children below 5 years of age in Jammu. Indian J Med Microbiol 2014;32:57-9

How to cite this URL:
Gazal S, Taku A, Bhat M A, Badroo G. Group A rotavirus and bacterial agents associated with diarrhoea-induced hospitalisations in children below 5 years of age in Jammu. Indian J Med Microbiol [serial online] 2014 [cited 2019 Dec 12];32:57-9. Available from: http://www.ijmm.org/text.asp?2014/32/1/57/124308



 ~ Introduction Top


Diarrhoea, especially acute diarrhoea, remains a major public health problem in the world. In India every year almost 10% infants and 14% in the age group of 0-4 years die due to diarrhoea. [1] Many different agents, including viruses, bacteria and parasites can cause acute diarrhoea. Rotavirus is a leading cause of infantile gastroenteritis worldwide and is responsible for approximately 20% of diarrhoea-associated deaths in children under 5 years of age. [2] Annually in India, rotavirus diarrhoea causes an estimated 122,000-153,000 deaths, 457,000-884,000 hospitalizations, and 2 million outpatient visits in children < 5 years of age. [3] India spends Rs 2.0-3.4 billion (US$ 41-72 million) annually in medical costs to treat rotavirus diarrhoea. [3] Among bacteria Escherichia coli and Salmonella spp. are major etiological agents of diarrhoea. Various E. coli serogroups have been associated with diarrhoea in humans and animals. The objective of the present study was to investigate the association of group A rotavirus and various bacterial agents with diarrhoea-induced hospitalizations in children in Jammu. None of the child under the study had been vaccinated for rotavirus. Since, India has a high under-5 mortality rate of 69 per 1000 live births [4] the age group selected for the investigation was 0-5 years.


 ~ Materials and Methods Top


A total of 210 diarrhoeic children (below 5 years) attending different hospitals in Jammu, J and K were included in the study. Diarrhoea was characterised by the occurrence of three or more loose, liquid or watery stools or at least one bloody loose stool in a 24-h period. Faecal samples (one from each subject) were collected within 24 h of hospital admission, kept at 4°C, and transferred to laboratory within 24 h. The remains of each sample after the first culture on the media were kept at -70°C for further work.

Rotavirus detection

Ribonucleic acid -Polyacrylamide gel electrophoresis (RNA-PAGE)

Faecal samples were suspended in phosphate buffered saline (PBS) and 10% suspensions were prepared followed by centrifugation at 10,000 g for 10 min Viral double-stranded RNA (ds RNA) was extracted from the clarified supernatant by sodium dodecyl sulphate (SDS): Phenol: Chloroform method as described previously. [5] RNA-PAGE was performed in minigel twin vertical electrophoresis apparatus (Biometra) using 5% stacking gel and 8% resolving gel. Electrophoresis was carried out using freshly prepared Tris glycine buffer at a current of 10 milli-amperes for stacking and 25 milli-amperes for resolving gel. The gel was stained by silver nitrate staining method [6] with minor modifications for minigel (fixation and staining, each for 15 min).

Bacterial isolation

Faecal samples were initially cultured on MacConkey Lactose agar (MLA, HiMedia) followed by 24 hrs of aerobic incubation at 37°C. From each plate only one colony exhibiting lactose fermentation (suspected for E. coli) was sub-cultured on Eosine Methylene Blue medium. Non-lactose fermenter colonies, one from each plate when present were sub-cultured on Salmonella-Shigella Agar (SS Agar) (HiMedia) for the isolation of Shigella and Salmonella spp. All the suspected isolates were confirmed by standard morphological and biochemical tests. [7]

Serogrouping

All the confirmed E. coli isolates were sent to National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (H.P) for serogrouping of E. coli 'O' antigen.


 ~ Results Top


Out of 210 diarrhoeic samples, 88 (41.9%) were positive for rotavirus. All of them showed the RNA migration pattern of group A rotavirus in PAGE [Figure 1]. A total of 210 E. coli isolates, one from each of the faecal samples were obtained, while 10 samples revealed the presence of Shigella spp., five Salmonella spp. and two samples showed the presence of Pseudomonas spp.
Figure 1: Typical 4-2-3-2 migration pattern of group A rotavirus in RNA-PAGE

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Out of 210 E. coli isolates, 140 belonged to nine different serogroups viz. O1, O11, O20, O39, O56, O59, O68, O69 and O153 while 32 (15.23%) and 38 (18.09%) isolates were rough and untypeable, respectively. O69 was the most prevalent serogroup of which there were 26 (12.38%) isolates, followed by O39 (11.42%), O20 (11.42%), O1 (10%), O59 (4.78%), O11 (4.28%), O56 (4.28%), O68 (4.28%) and 0153 (3.8%) as shown in [Table 1]. The details of rotavirus and bacterial agents (and their association, if any) are provided in [Table 2].
Table 1: Serogroups of E. coli isolated in the study

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Table 2: The details of rotavirus and bacterial agents
isolated in the study


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 ~ Discussion Top


Many studies have shown important role of rotavirus as a cause of diarrhoea in children in both developed and developing countries. [8] The present study showed that 41.9% of the children under study were positive for group A rotavirus by RNA-PAGE. The prevalence of rotavirus in our study was found to be higher than most parts of the country viz. Chandigarh (16-19%) [9] , Pune (28-30%) [10] and Kolkata (5-22%). [11] However, in Manipur the incidence has been reported to be as high as 41%. [12]

Among E. coli, O69 (12.38%), a shiga toxin producing E. coli serogroup was the most frequently obtained. The well-established human entropathogens viz. O20, O153 and O69 were isolated from 19.52% cases. Rotavirus or other bacterial agents were not associated with these samples. Moreover, 17 (8.09%) cases had mixed infection of rotavirus and any of these three serogroups. Thus in all 27.6% of the E. coli isolates obtained in the study were either cause of diarrhoea, or complicated rotavirus-induced diarrhoea. Other bacterial pathogens were isolated from 8.09% samples, out of which Shigella spp. was found in 4.76% samples followed by Salmonella spp. (2.38%) and Pseudomonas spp. (0.95%). Since, Salmonella, Shigella and Pseudomonas were isolated from rotavirus-negative samples; this indicates that these bacterial pathogens may be responsible for diarrhoea in 17 cases.

This study establishes rotavirus to be major pathogen responsible for causing hospitalisations due to diarrhoea in children of less than 5 years of age in Jammu. Since none of the child had been vaccinated, it can be said that rotavirus poses a threat in unvaccinated children. Two live, oral, attenuated vaccines against rotavirus infection (Rotarix® , manufactured by GlaxoSmithKline; and RotaTeq® , manufactured by Merck) were licensed by WHO-recognized regulatory authorities in 2006. Clinical trials in Europe, Latin America and the US demonstrated that they are safe and highly efficacious at preventing rotavirus-associated severe gastroenteritis. [13] Similar trials need to be carried out in India to determine the efficacy of the vaccines for rotavirus.


 ~ Acknowledgments Top


The authors are thankful to Department of Paediatrics SMGS Hospital, Government Medical College, Jammu for their help in sample collection.

 
 ~ References Top

1.Isalkar U. 50 pc medical staff dump ORS. Times of India. Pune: 2010 September 28.  Back to cited text no. 1
    
2.Lundgren O, Svensson L. Pathogenesis of rotavirus diarrhea. Microbes Infect 2001;3:1145-56.  Back to cited text no. 2
[PUBMED]    
3.Tate JE, Chitambar S, Esposito DH, Sarkar R, Gladstone B, Ramani S, et al. Disease and economic burden of rotavirus diarrhoea in India. Vaccine 2009;27:F18-24.  Back to cited text no. 3
[PUBMED]    
4.Nirmalan P, Shamanna BR and Saravanan S. Managing Diarrhea among children aged less than 5 years in India-Where do we stand? 2011. Diarrhoea-Community Management Perspectives. Available from: http://www. prashasa.co.in/documents/managing_diarrhea_in_under_5_children_fact_sheet.pdf [Last accessed on 2011 Dec].  Back to cited text no. 4
    
5.Herring AJ, Inglis NF, Ojeh CK, Snodgrass DR, Menzies JD. Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acids in silver stained polyacrylamide gels. J Clin Microbiol 1982;16:473-7.  Back to cited text no. 5
[PUBMED]    
6.Svensson L, Uhnoo I, Grandien M, Wadell G. Molecular epidemiology of rotavirus infections in Uppsala, Sweden in 1981: Disappearance of a predominant electropherotype. J Med Virol 1986;18:101-11.  Back to cited text no. 6
[PUBMED]    
7.Quinn PJ, Carter ME, Markey BK, Carter GR. Clinical Veterinary Microbiology. London: Wolfe Publishing; 1994. p. 209-42.  Back to cited text no. 7
    
8.Staat MA, Azimi PH, Berke T, Roberts N, Bernstein DI, Ward RL, et al. Clinical presentations of rotavirus infection among hospitalized children. Pediatr Infect Dis J 2002;21:221-7.  Back to cited text no. 8
[PUBMED]    
9.Singh V, Broor S, Mehta S, Mehta SK. Molecular epidemiology of human rotavirus infections in Chandigarh (India). Indian J Med Res 1990;91:9-14.  Back to cited text no. 9
    
10.Jain V, Das BK, Bhan MK, Glass RI, Gentsch JR. Indian Strain Surveillance Colleborating Laboratories. Great diversity of group A rotavirus strains and high prevalence of mixed rotavirus infections in India. J Clin Microbiol 2001;39:3524-9.  Back to cited text no. 10
    
11.Ghosh AR, Nair GB, Dutta P, Pal SC, Sen D. Acute diarrhoeal diseases in infants aged below six months in hospital in Calcutta, India: An aetiological study. Trans R Soc Trop Med Hyg 1991;85:796-8.  Back to cited text no. 11
[PUBMED]    
12.Jain V, Parashar UD, Glass RI, Bhan MK. Epidemiology of rotavirus in India. Indian J Pediatr 2001;68:855-62.  Back to cited text no. 12
[PUBMED]    
13.Ruiz-Palacios GM, Pérez-Schael I, Velázquez FR, Abate H, Breuer T, Clemens SA, et al. Human Rotavirus Vaccine Study Group. Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis. N Engl J Med 2006;354:11-22.  Back to cited text no. 13
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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