Abstract
Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are two major aetiological agents of hand, foot and mouth disease (HFMD) in children. Recently there have been several large outbreaks of HFMD in Vietnam and the Asia-Pacific region. In this study, a multiplex RT-PCR assay was developed in order to detect simultaneously HEV71, CVA16 and other human enteroviruses. Enterovirus detection was performed with a mixture of three pairs of oligonucleotide primers: one pair of published primers for amplifying all known enterovirus genomes and two new primer pairs specific for detection of the VP1 genes of HEV71 and CVA16. Enterovirus isolates, CVA16 and HEV71 strains identified previously from patients with HFMD were examined to evaluate the sensitivity and specificity of the multiplex RT-PCR assay. The assay was then applied to the direct detection of these viruses in clinical specimens obtained from HFMD cases identified at Children's Hospital Number 2, Ho Chi Minh City, Vietnam. The multiplex RT-PCR assay showed 100% specificity in screening for enteroviruses and in identifying HEV71 and CVA16. Similar results were obtained when using the multiplex RT-PCR assay to screen for enteroviruses and to identify HEV71 and CVA16 in clinical specimens obtained from HFMD cases identified at the hospital. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of HEV71 or CVA16 infection in cases of HFMD and is also potentially useful for molecular epidemiological investigations.
Highlights
Hand, foot and mouth disease (HFMD) is a common childhood exanthem, characterised by a brief febrile illness and vesicular lesions on the hands, feet, mouth and buttocks (Grist et al, 1978)
HFMD caused by infection with these enteroviruses is clinically indistinguishable, infection with human enterovirus 71 (HEV71) is commonly associated with acute neurological disease, including aseptic meningitis, brainstem encephalitis, and acute flaccid paralysis (AFP), which may lead to permanent paralysis or death (McMinn, 2002)
Primer concentrations were titrated to optimise the amplification of all three cation of the 440 bp pan-enterovirus amplicon; all HEV71 strains generated the 264 bp HEV71-specific amplicon plus the 440 bp pan-enterovirus amplicon; all Coxsackievirus A16 (CVA16) strains generated the 550 bp CVA16-specific amplicon plus the 440 bp pan-enterovirus amplicon
Summary
Foot and mouth disease (HFMD) is a common childhood exanthem, characterised by a brief febrile illness and vesicular lesions on the hands, feet, mouth and buttocks (Grist et al, 1978). Coxsackievirus A16 (CVA16) and human enterovirus 71 (HEV71), members of the human enterovirus A (HEV-A) species, are the two major causative agents of HFMD (McMinn, 2002; Pallansch and Roos, 2007). There has been an increase in the prevalence of HFMD and acute neurological disease caused by HEV71 infection in the Asia-Pacific region (Fujimoto et al, 2002; Huang et al, 1999; Lin et al, 2003; McMinn, 2002; Podin et al, 2006; Tu et al, 2007). With the recent increase in prevalence of HEV71 infection in southern Vietnam, it has become necessary to develop a rapid, sensitive, N.T.T. Thao et al / Journal of Virological Methods 170 (2010) 134–139 specific and cost-effective assay to distinguish HFMD caused by HEV71 or CVA16
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