BackgroundHand, foot and mouth disease (HFMD) has become a major public health problem across the Asia-Pacific region, and is commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A6 (CV-A6), CV-A10 and CV-A16. Generating pathogen whole-genome sequences is essential for understanding their evolutionary biology. The frequent replacements among EV serotypes and a limited numbers of available whole-genome sequences hinder the development of overlapping PCRs for whole-genome sequencing.We developed and evaluated a non-ribosomal random PCR (rPCR) and next-generation sequencing based assay for sequence-independent whole-genome amplification and sequencing of HFMD pathogens. A total of 16 EV-A71/CV-A6/CV-A10/CV-A16 PCR positive rectal/throat swabs (Cp values: 20.9–33.3) were used for assay evaluation.ResultsOur assay evidently outperformed the conventional rPCR in terms of the total number of EV-A71 reads and the percentage of EV-A71 reads: 2.6 % (1275/50,000 reads) vs. 0.1 % (31/50,000) and 6 % (3008/50,000) vs. 0.9 % (433/50,000) for two samples with Cp values of 30 and 26, respectively. Additionally the assay could generate genome sequences with the percentages of coverage of 94–100 % of 4 different enterovirus serotypes in 73 % of the tested samples, representing the first whole-genome sequences of CV-A6/10/16 from Vietnam, and could assign correctly serotyping results in 100 % of 24 tested specimens. In all but three the obtained consensuses of two replicates from the same sample were 100 % identical, suggesting that our assay is highly reproducible.ConclusionsIn conclusion, we have successfully developed a non-ribosomal rPCR and next-generation sequencing based assay for sensitive detection and direct whole-genome sequencing of HFMD pathogens from clinical samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0580-9) contains supplementary material, which is available to authorized users.

Background Brainstem encephalitis is a serious complication of hand foot and mouth disease (HFMD) in children. Autonomic nervous system (ANS) dysregulation and hypertension may occur, sometimes progressing to cardiopulmonary failure and death. Vietnamese national guidelines recommend use of milrinone if ANS dysregulation with Stage 2 hypertension develops. We wished to investigate whether magnesium sulfate (MgSO 4 ) improved outcomes in children with HFMD if used earlier in the evolution of the ANS dysregulation (Stage 1 hypertension). Methods During a regional epidemic we conducted a randomized, double-blind, placebo-controlled trial of MgSO 4 in children with HFMD, ANS dysregulation and Stage 1 hypertension, at the Hospital for Tropical Diseases in Ho Chi Minh city. Study participants received an infusion of MgSO 4 or matched placebo for 72 h. We also reviewed data from non-trial HFMD patients in whom milrinone failed to control hypertension, some of whom received MgSO 4 as second line therapy. The primary outcome for both analyses was a composite of disease progression within 72 h - addition of milrinone (trial participants only), need for ventilation, shock, or death. Results Between June 2014 and September 2016, 14 and 12 participants received MgSO 4 or placebo respectively, before the trial was stopped due to futility. Among 45 non-trial cases with poorly controlled hypertension despite high-dose milrinone, 33 received MgSO 4 while 12 did not. There were no statistically significant differences in the composite outcome between the MgSO 4 and the placebo/control groups in either study (adjusted relative risk (95%CI) of [6/14 (43%) vs. 6/12 (50%)], 0.84 (0.37, 1.92), p = 0.682 in the trial and [1/33 (3%) vs. 2/12 (17%)], 0.16 (0.01, 1.79), p = 0.132 in the observational cohort). The incidence of adverse events was similar between the groups. Potentially toxic magnesium levels occurred very rarely with the infusion regime used. Conclusion Although we could not demonstrate efficacy in these studies, there were no safety signals associated with use of 30-50 mg/kg/hr. MgSO 4 in severe HFMD. Intermittent outbreaks of HFMD are likely to continue across the region, and an adequately powered trial is still needed to evaluate use of MgSO 4 in controlling hypertension in severe HFMD, potentially involving a higher dose regimen. Trial registration ClinicalTrials.gov Identifier: NCT01940250 (Registered 22 AUG 2013). Trial sponsor : University of Oxford Electronic supplementary material The online version of this artic...

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