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Abstract

Enteric viruses play a major role in causing diarrhea in children. Early identification of the causative pathogen is still a challenge in the clinical laboratory. A multiplex PCR assay is a useful tool to screen a large number of clinical samples especially in an outbreak situation. In this study, a multiplex reverse transcription (RT)-PCR assay was developed to detect nine enteric viruses such as group A rotavirus, norovirus GGII, sapovirus, adenovirus, astrovirus, aichivirus, parechovirus, bocavirus and enterovirus in clinical samples of diarrheal cases. Stool samples (n=185) collected from infants and children with acute gastroenteritis cases in Pune, western India were analysed for nine different enteric viruses by currently developed multiplex RT- PCR. Predominance of group A rotavirus (76%) followed by enterovirus (11.5%), astrovirus (4.5%), adenovirus (2.7%) and norovirus GII (1.6%) was observed. A total of 44.8 % (82/185) samples analysed by this method showed high frequency of mixed infections. These results highlighted high prevalence and diversity of different enteric viruses in children. The multiplex PCR showed good concordance with monoplex RT-PCR for detection of these enteric viruses in clinical samples. This is the first report on the development of a multiplex RT-PCR assay for detection of multiple enteric viruses in diarrheal diseases from India.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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2019-03-29
2024-03-28
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References

  1. Lanata CF, Fischer-Walker CL, Olascoaga AC, Torres CX, Aryee MJ et al. Child Health Epidemiology Reference Group of the World Health Organization and UNICEF. Global causes of diarrheal disease mortality in children <5 years of age: a systematic review. PLoS One 2013; 4:e72788
    [Google Scholar]
  2. Platts-Mills JA, Operario DJ, Houpt ER. Molecular diagnosis of diarrhea: current status and future potential. Curr Infect Dis Rep 2012; 14:41–46
    [Google Scholar]
  3. van Maarseveen NM, Wessels E, de Brouwer CS, Vossen AC, Claas EC. Diagnosis of viral gastroenteritis by simultaneous detection of adenovirus group F, astrovirus, rotavirus group A, norovirus genogroups I and II, and sapovirus in two internally controlled multiplex real-time PCR assays. J Clin Virol 2010; 49:205–210 [View Article]
    [Google Scholar]
  4. Clark B, McKendrick M. A review of viral gastroenteritis. Curr Opin Infect Dis 2004; 17:461–469
    [Google Scholar]
  5. Chow BD, Ou Z, Esper FP. Newly recognized bocaviruses (HBoV, HBoV2) in children and adults with gastrointestinal illness in the United States. J Clin Virol 2010; 47:143–147
    [Google Scholar]
  6. Pham NT, Khamrin P, Nguyen TA, Kanti DS, Phan TG et al. Isolation and molecular characterization of Aichi viruses from fecal specimens collected in Japan, Bangladesh Thailand, and Vietnam. J Clin Microbiol 2007; 45:2287–2288
    [Google Scholar]
  7. Pham NT, Trinh QD, Chan-It W, Khamrin P, Shimizu H et al. A novel RT-multiplex PCR for detection of Aichi virus, human parechovirus, enteroviruses, and human bocavirus among infants and children with acute gastroenteritis. J Virol Methods 2010; 169:193–197
    [Google Scholar]
  8. Reuter G, Boldizsár A, Papp G, Pankovics P. Detection of Aichi virus shedding in a child with enteric and extraintestinal symptoms in Hungary. Arch Virol 2009; 154:1529–1532
    [Google Scholar]
  9. Akhter S, Turegun B, Kiyan M, Gerceker D, Guriz H et al. Investigation of seven different RNA viruses associated with gastroenteritis in children under five years old. Mikrobiyol Bul 2014; 48:233–241
    [Google Scholar]
  10. Amar CF, East CL, Gray J, Iturriza-Gomara M, Maclure EA et al. Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control infectious intestinal disease Study (1993–1996). Eur J Clin Microbiol Infect Dis 2007; 26:311–323
    [Google Scholar]
  11. Guan H, Zhang J, Xiao Y, Sha D, Ling X et al. Evaluation of PCR based assays for the improvement of proportion estimation of bacterial and viral pathogens in diarrheal surveillance. Front Microbiol 2016; 30:386
    [Google Scholar]
  12. Harvala H, Simmonds P. Human parechoviruses: biology, epidemiology and clinical significance. J Clin Virol 2009; 45:1–9
    [Google Scholar]
  13. Lu L, Jia R, Zhong H, Xu M, Su L et al. Molecular characterization and multiple infections of rotavirus, norovirus, sapovirus, astrovirus and adenovirus in outpatients with sporadic gastroenteritis in Shanghai, China, 2010–2011. Arch Virol 2015; 160:1229–1238
    [Google Scholar]
  14. Yan H, Nguyen TA, Phan TG, Okitsu S, Li Y et al. Development of RT-multiplex PCR assay for detection of adenovirus and group A and C rotaviruses in diarrheal fecal specimens from children in China. Kansenshogaku Zasshi 2004; 78:699–709 [View Article]
    [Google Scholar]
  15. Yan H, Yagyu F, Okitsu S, Nishio O, Ushijima H et al. Detection of norovirus (Gi, GII), sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR. J Virol Methods 2003; 114:37–44 [View Article]
    [Google Scholar]
  16. Rohayem J, Berger S, Juretzek T, Herchenröder O, Mogel M et al. A simple and rapid single-step multiplex RT-PCR to detect norovirus, astrovirus and adenovirus in clinical stool samples. J Virol Methods 2004; 118:49–59
    [Google Scholar]
  17. Khamrin P, Okame M, Thongprachum A, Nantachit N, Nishimura S et al. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhea. J Virol Methods 2011; 173:390–393 [View Article]
    [Google Scholar]
  18. Hamza IA, Jurzik L, Wilhelm M. Development of a Luminex assay for the simultaneous detection of human enteric viruses in sewage and river water. J Virol Methods 2014; 204:65–72
    [Google Scholar]
  19. Stockmann C, Pavia AT, Graham B, Vaughn M, Crisp R et al. Detection of 23 gastrointestinal pathogens among children who present with diarrhea. J Pediatric Infect Dis Soc 2017; 6:231–238
    [Google Scholar]
  20. Iturriza Gómara M, Wong C, Blome S, Desselberger U, Gray J. Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation. J Virol 2002; 76:6596–6601
    [Google Scholar]
  21. Allard A, Albinsson B, Wadell G. Rapid typing of human adenoviruses by a general PCR combined with restriction endonuclease analysis. J Clin Microbiol 2001; 39:498–505
    [Google Scholar]
  22. Belliot G, Laveran H, Monroe SS. Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region. Arch Virol 1997; 142:1323–1334
    [Google Scholar]
  23. Okada M, Yamashita Y, Oseto M, Shinozaki K. The detection of human sapoviruses with universal and genogroup-specific primers. Arch Virol 2006; 151:2503–2509
    [Google Scholar]
  24. Puig M, Jofre J, Lucena F, Allard A, Wadell G et al. Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Appl Environ Microbiol 1994; 60:2963–2970
    [Google Scholar]
  25. Yamashita T, Sakae K, Tsuzuki H, Suzuki Y, Ishikawa N et al. Complete nucleotide sequence and genetic organization of Aichi virus, a distinct member of the Picornaviridae associated with acute gastroenteritis in humans. J Virol 1998; 72:8408–8412
    [Google Scholar]
  26. van der Sanden S, de Bruin E, Vennema H, Swanink C et al. Prevalence of human parechovirus in the Netherlands in 2000 to 2007. J Clin Microbiol 2008; 46:2884–2889
    [Google Scholar]
  27. Kapoor A, Simmonds P, Slikas E, Li L, Bodhidatta L et al. Human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections. J Infect Dis 2010; 201:1633–1643
    [Google Scholar]
  28. Girish R, Broor S, Dar L, Ghosh D. Foodborne outbreak caused by a Norwalk-like virus in India. J Med Virol 2002; 67:603–607
    [Google Scholar]
  29. Lekana-Douki SE, Kombila-Koumavor C, Nkoghe D, Drosten C, Drexler JF et al. Molecular epidemiology of enteric viruses and genotyping of rotavirus A, adenovirus and astrovirus among children under 5 years old in Gabon. Int J Infect Dis 2015; 34:90–95 [View Article]
    [Google Scholar]
  30. Ouédraogo N, Kaplon J, Bonkoungou IJ, Traoré AS, Pothier P et al. Prevalence and genetic diversity of enteric viruses in children with diarrhea in Ouagadougou, Burkina Faso. PloS One 2016; 11:e0153652
    [Google Scholar]
  31. Ouyang Y, Ma H, Jin M, Wang X, Wang J et al. Etiology and epidemiology of viral diarrhea in children under the age of five hospitalized in Tianjin, China. Arch Virol 2012; 157:881–887
    [Google Scholar]
  32. Sdiri-Loulizi K, Gharbi-Khelifi H, De Rougemont A, Chouchane S, Sakly N et al. Acute infantile gastroenteritis associated with human enteric viruses in Tunisia. J Clin Microbiol 2008; 46:1349–1355
    [Google Scholar]
  33. Llor C, Bjerrum L. Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Ther Adv Drug Saf 2014; 5:229–241
    [Google Scholar]
  34. Stofanko M, Gonçalves-Dornelas H, Cunha PS, Pena HB, Vianna-Morgante AM et al. Simple, rapid and inexpensive quantitative fluorescent PCR method for detection of microdeletion and microduplication syndromes. PLoS One 2013; 8:e61328
    [Google Scholar]
  35. Wang J, Xu Z, Niu P, Zhang C, Zhang J et al. A two-tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea. Biomed Res Int 2014; 648520:
    [Google Scholar]
  36. Brault AC, Fang Y, Reisen WK. Multiplex qRT-PCR for the detection of western equine encephalomyelitis, St. Louis encephalitis, and West Nile viral RNA in mosquito pools (Diptera: Culicidae). J Med Entomol 2015; 52:491–499
    [Google Scholar]
  37. Zhang C, Niu P, Hong Y, Wang J, Zhang J et al. A probe-free four-tube real-time PCR assay for simultaneous detection of twelve enteric viruses and bacteria. J Microbiol Methods 2015; 118:93–98
    [Google Scholar]
  38. Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A et al. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA 2005; 102:12891–12896 [View Article]
    [Google Scholar]
  39. Benschop K, Thomas X, Serpenti C, Molenkamp R, Wolthers K. High prevalence of human Parechovirus (HPeV) genotypes in the Amsterdam region and identification of specific HPeV variants by direct genotyping of stool samples. J Clin Microbiol 2008; 46:3965–3970
    [Google Scholar]
  40. Cheng WX, Jin Y, Duan ZJ, Xu ZQ, Qi HM et al. Human bocavirus in children hospitalized for acute gastroenteritis: a case-control study. Clin Infect Dis 2008; 47:161–167
    [Google Scholar]
  41. Chhabra P, Payne DC, Szilagyi PG, Edwards KM, Staat MA et al. Etiology of viral gastroenteritis in children <5 Years of age in the United States, 2008–2009. J Infect Dis 2013; 208:790–800
    [Google Scholar]
  42. Román E, Wilhelmi I, Colomina J, Villar J, Cilleruelo ML et al. Acute viral gastroenteritis: proportion and clinical relevance of multiple infections in Spanish children. J Med Microbiol 2003; 52:435–440
    [Google Scholar]
  43. Taylor MB, Marx FE, Grabow WO. Rotavirus, astrovirus and adenovirus associated with an outbreak of gastroenteritis in a South African child care centre. Epidemiol Infect 1997; 119:227–230
    [Google Scholar]
  44. Wiemer D, Loderstaedt U, von Wulffen H, Priesnitz S, Fischer M et al. Real-time multiplex PCR for simultaneous detection of Campylobacter jejuni, Salmonella, Shigella and Yersinia species in fecal samples. Int J Med Microbiol 2011; 301:577–584
    [Google Scholar]
  45. Youssef M, Shurman A, Bougnoux M, Rawashdeh M, Bretagne S et al. Bacterial, viral and parasitic enteric pathogens associated with acute diarrhea in hospitalized children from northern Jordan. FEMS Immunol Med Microbiol 2000; 28:257–263
    [Google Scholar]
  46. Corcoran MS, van Well GT, van Loo IH. Diagnosis of viral gastroenteritis in children: interpretation of real-time PCR results and relation to clinical symptoms. Eur J Clin Microbiol Infect Dis 2014; 33:1663–1673
    [Google Scholar]
  47. Wolffs PF, Bruggeman CA, van Well GT, van Loo IH. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. J Clin Microbiol 2011; 49:1926–1931
    [Google Scholar]
  48. Lekana-Douki SE, Behillil S, Enouf V, Leroy EM, Berthet N. Detection of human bocavirus-1 in both nasal and stool specimens from children under 5 years old with influenza-like illnesses or diarrhea in Gabon. BMC Res Notes 2018; 11:495
    [Google Scholar]
  49. Sano K, Hamada H, Hirose S, Sugiura K, Harada S et al. Prevalence and characteristics of human parechovirus and enterovirus infection in febrile infants. Pediatr Int 2018; 60:142–147
    [Google Scholar]
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