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Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease.

ABSTRACT: BACKGROUND:Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response. METHODS:We developed and validated an internally controlled one-step single-tube real-time RT-PCR in terms of sensitivity, linearity, precision, and specificity for simultaneous detection of EVs and EV-A71. Subsequently, the assay was then applied on throat and rectal swabs sampled from 434 HFMD patients. RESULTS:The assay was evaluated using both plasmid DNA and viral RNA and has shown to be reproducible with a maximum assay variation of 4.41 % and sensitive with a limit of detection less than 10 copies of target template per reaction, while cross-reactivity with other EV serotypes was not observed. When compared against a published VP1 nested RT-PCR using 112 diagnostic throat and rectal swabs from 112 children with a clinical diagnosis of HFMD during 2014, the multiplex assay had a higher sensitivity and 100 % concordance with sequencing results which showed EVs in 77/112 (68.8 %) and EV-A71 in 7/112 (6.3 %). When applied to clinical diagnostics for 322 children, the assay detected EVs in throat swabs of 257/322 (79.8 %) of which EV-A71 was detected in 36/322 (11.2 %) children. The detection rate increased to 93.5 % (301/322) and 13.4 % (43/322) for EVs and EV-A71, respectively, when rectal swabs from 65 throat-negative children were further analyzed. CONCLUSION:We have successfully developed and validated a sensitive internally controlled multiplex assay for rapid detection of EVs and EV-A71, which is useful for clinical management and outbreak control of HFMD.

SUBMITTER: Thanh TT

PROVIDER: S-EPMC4464700 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease.

Thanh Tran Tan TT Anh Nguyen To NT Tham Nguyen Thi NT Van Hoang Minh Tu HM Sabanathan Saraswathy S Qui Phan Tu PT Ngan Tran Thuy TT Van Tran Thi My TT Nguyet Lam Anh LA Ny Nguyen Thi Han NT Thanh Le Thi My le TM Chai Ong Kien OK Perera David D Viet Do Chau do C Khanh Truong Huu TH Ha Do Quang do Q Tuan Ha Manh HM Wong Kum Thong KT Hung Nguyen Thanh NT Chau Nguyen Van Vinh NV Thwaites Guy G van Doorn H Rogier HR Van Tan Le L

Virology journal 20150609

<h4>Background</h4>Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The disease is caused by enteroviruses (EVs), in particular enterovirus A71 (EV-A71). In EV-A71-associated HFMD, the infection is sometimes associated with severe manifestations including neurological involvement and fatal outcome. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response. ...[more]

PMID: 26050791

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Project description:UnlabelledEnterovirus A71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011 and 2013 and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time frame of sampling, whereas that of B5 increased and remained >1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated area of endemicity. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia.ImportanceEV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

Dataset Information

Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease.

Publications

Validation and utilization of an internally controlled multiplex Real-time RT-PCR assay for simultaneous detection of enteroviruses and enterovirus A71 associated with hand foot and mouth disease.

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