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Background: Enterovirus 71 subgenogroup C4 caused the largest outbreak of Hand, Foot and Mouth Disease (HFMD) in Vietnam during 2011-2012, resulting in over 200,000 hospitalisations and 207 fatalities., Methods: A total of 1917 samples with adequate volume for RT-PCR analysis were collected from patients hospitalised with HFMD throughout Vietnam and 637 were positive for EV71. VP1 gene (n=87) and complete genome (n=9) sequencing was performed. Maximum-likelihood phylogenetic analysis was performed to characterise the B5, C4 and C5 strains detected., Results: Sequence analyses revealed that the dominant subgenogroup associated with the 2012 outbreak was C4, with B5 and C5 strains representing a small proportion of these cases., Conclusions: Numerous countries in the region including Malaysia, Taiwan and China have a large influence on strain diversity in Vietnam and understanding the transmission of EV71 throughout Southeast Asia is vital to inform preventative public health measures and vaccine development efforts., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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.