Abstract
Norovirus, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus are all common causes of gastroenteritis. Conventional diagnoses of these causative agents are based on antigen detection and electron microscopy. To improve the diagnostic potential for viral gastroenteritis, internally controlled multiplex real-time polymerase chain reaction (PCR) methods have been recently developed. In this study, individual real-time PCRs were developed and optimized for specific detections of Norovirus genogroup I, Norovirus genogroup II, Rotavirus group A, the Hepatitis A virus, and Coxsackievirus group B1. Subsequently, individual PCRs were combined with multiplex PCR reactions. In general, multiplex real-time PCR assays showed comparable sensitivities and specificities with individual assays. A retrospective clinical evaluation showed increased pathogen detection in 29% of samples using conventional PCR methods. Prospective clinical evaluations were detected in 123 of the 227 (54%) total samples used in the multiplex real-time PCR analysis. The Norovirus genogroup II was found most frequently (23%), followed by Rotavirus (20%), the Hepatitis A virus (4.5%), Coxsackievirus (3.5%), and Norovirus genogroup I (2.6%). Internally controlled multiplex real-time PCR assays for the simultaneous detection of Rotavirus, Coxsackievirus group B, the Hepatitis A virus, and Norovirus genogroups I and II showed significant improvement in the diagnosis of viral gastroenteritis.
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Amar, C.F.L., East, C.L., Gray, J., Iturriza-Gomara, M., Maclure, E.A., and McLauchlin, J. 2007. 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. 26, 311–323.
De Wit, M.A.S., Koopmans, M.P.G., Kortbeek, L.M., Wannet, W.J.B., Vinjé, J., Van Leusden, F., Bartelds, A.I.M., and Van Duynhoven, Y.T.H.P. 2001. Sensor, a population-based cohort study on gastroenteritis in the Netherlands: Incidence and etiology. Am. J. Epidemiol. 154, 666–674.
Estes, M.K. 2001. Rotaviruses and their replication. In Knipe, D.M. and Howley, P.M. (eds.), Fields Virology, pp. 1747–1785. Lippincott Williams & Wilkins, Philadelphia, USA.
Fankhauser, R.L., Monroe, S.S., Noel, J.S., Humphrey, C.D., Bresee, J.S., Parashar, U.D., Ando, T., and Glass, R.I. 2002. Epidemiologic and molecular trends of “Norwalk-like viruses” associated with outbreaks of gastroenteritis in the United States. J. Infect. Dis. 186, 1–7.
Hoshino, Y., Jones, R.W., Ross, J., Honma, S., Santos, N., Gentsch, J.R., and Kapikian, A.Z. 2004. Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3. J. Virol. 78, 7795–7802.
Kang, G., Iturriza-Gomara, M., Wheeler, J.G., Crystal, P., Monica, B., Ramani, S., Primrose, B., Moses, P.D., Gallimore, C.I., Brown, D.W., and Gray, J. 2004. Quantitation of Group A rotavirus by real-time reverse-transcription-polymerase chain reaction: Correlation with clinical severity in children in south India. J. Med. Virol. 73, 118–122.
Koff, R.S. 1998. Hepatitis A. Lancet 30, 1643–1649.
Li, C.S.Y., Chan, P.K.S., and Tang, J.W. 2009. Prevalence of diarrhea viruses in hospitalized children in Hong Kong in 2008. J. Med. Virol. 81, 1903–1911.
Maarseveen, N.M.V., Wessels, E., Brouwer, C.S.D., Vossen, A.C.T.M., and Claas, E.C.J. 2010. 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. 49, 205–210.
Nguyen, T.A., Yagyu, F., Okame, M., Phan, T.G., Trinh, Q.D., Yan, H., Hoang, K.T., Cao, A.T.H., Hoang, P.L., Okitsu, S., and et al. 2007. Diversity of viruses associated with acute gastroenteritis in children hospitalized with diarrhea in Ho Chi Minh City, Vietnam. J. Med. Virol. 79, 582–590.
Pang, X.L., Joensuu, J., and Vesikari, T. 1999. Human calicivirus-associated sporadic gastroenteritis in Finnish children less than two years of age followed prospectively during a rotavirus vaccine trial. Pediatr. Infect. Dis. J. 18, 420–426.
Selinka, H.C., Huber, M., Pasch, A., Klingel, K., Aepinus, C., and Kandolf, R. 1998. Coxsackie B virus and its interaction with permissive host cells. Clin. Diagn. Virol. 10, 97–101.
Tanaka, J. 2000. Hepatitis A shifting epidemiology in Latin America. Vaccine 18, S57–S60.
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Kang, LH., Oh, Sh., Park, JW. et al. Simultaneous detection of waterborne viruses by multiplex real-time PCR. J Microbiol. 51, 671–675 (2013). https://doi.org/10.1007/s12275-013-3199-1
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DOI: https://doi.org/10.1007/s12275-013-3199-1