Abstract
BackgroundHand 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.MethodsWe 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.ResultsThe 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0316-2) contains supplementary material, which is available to authorized users.
Highlights
Hand foot and mouth disease (HFMD) is a commonly benign and self-limiting viral infection of infants and young children worldwide
The disease is caused by acute infection with Enterovirus A of the family Picornaviridae including coxsackievirus A6 (CV-A6), CV-A10, CV-A16, and enterovirus A71 (EV-A71) [1,2,3,4,5]
In contrast to the common mild patterns of previous sporadic epidemics, since 1997 fulminant outbreaks of HFMD involving millions of children with neurological involvement and sometimes fatal cardiopulmonary complications have occurred across the Asia-Pacific region with EV-A71 being the most frequent pathogen isolated from patients with clinical complications and fatal cases [6,7,8,9]
Summary
Hand foot and mouth disease (HFMD) is a commonly benign and self-limiting viral infection of infants and young children worldwide. The availability of a rapid, high-throughput and accurate diagnostic assay that can simultaneously detect and distinguish between EVs and EV-A71 is an ideal aid to patient management and may help outbreak response with regards to predicting the possible level of severity of the outbreak and thereby initiating appropriate public health interventions. Molecular assays based on specific amplification viral nucleic acids, in particular real-time PCR, are sensitive, specific and high-throughput and are methods of choice [11, 12]. Hand foot and mouth disease (HFMD) is a disease of public health importance across the Asia-Pacific region. The availability of a robust diagnostic assay to distinguish EV-A71 from other EVs is important for patient management and outbreak response
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