Severe Hand Foot and Mouth Disease in Vietnamese Children: Clinical Features and Management Strategies

Hand, Foot and Mouth Disease (HFMD) plays a major role in childhood morbidity and mortality in Vietnam and the Asia-Pacific region. The spectrum of severe disease and predictors for poor outcome are not well understood, and the evidence-base for management of hypertension in severe cases is lacking. In this thesis I describe the clinical features, their evolution over time, and the viral serotype/genotype findings, among 1272 HFMD cases admitted to PICU at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, during the the 2011-2012 Vietnam outbreak. Fever, skin rash, and myoclonic jerks happened in the early stage while the more severe features, including hypertension, tachypnea, irregular breathing, pulmonary edema and shock occurred after day 3. Most severe features were associated with EV-A71 infection rather than with Coxsackieviruses (CV). However having mouth ulcers without skin lesions was associated with CV infection. The C4 genogroup of EV-A71 was associated with more severe neurological involvement. Risk factors present within 24 hours of PICU admission that were associated with subsequent deterioration to severe outcome were skin lesions and tachypnea, while presence of mouth ulcers alone was protective. I also describe the development and execution of a randomized controlled trial of a novel treatment, MgSO4, for severe HFMD with hypertension secondary to ANS dysregulation. However, only 26 of the planned 190 patients were enrolled before the outbreak was controlled, and with this small number we found no evidence of benefit in this trial. In another approach, I assessed the efficacy of MgOS4 on severe hypertension among 33 patients treated with open-label MgSO4 compared to 12 patients of similar clinical severity who did not receive this intervention, and found a significant reduction in mean arterial pressure among the MgSO4 recipients. However, It is clear that with the small sample size, these data are insufficient to address the important question originally posed. Using our pre-prepared protocol as a basis, a much larger trial could be developed at short notice in the event of another outbreak, to properly evaluate whether MgSO4 has a role in controlling the blood pressure in severe HFMD

Assessing the efficacy and safety of magnesium sulfate for management of autonomic nervous system dysregulation in Vietnamese children with severe hand foot and mouth disease.

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 (MgSO4) 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 MgSO4 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 MgSO4 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 MgSO4 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 MgSO4 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 MgSO4 while 12 did not. There were no statistically significant differences in the composite outcome between the MgSO4 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. MgSO4 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 MgSO4 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

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

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. Keywords: Hand foot and mouth disease, Enteroviruses, Enterovirus A71, Real-time RT-PCR, Diagnosi

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples

Viral Etiology of Encephalitis in Children in Southern Vietnam: Results of a One-Year Prospective Descriptive Study

Viral encephalitis is associated with high morbidity and mortality in Vietnam. However little is known about the causes of the disease due to a lack of diagnostic facilities in this relatively resource-poor setting. Knowledge about the etiologies and clinical outcome of viral encephalitis is necessary for future design of intervention studies targeted at improvement of clinical management, treatment and prevention of the disease. We report the viral agents, clinical outcome and prognostic factors of mortality of encephalitis in children admitted to a referral hospital for children in southern Vietnam. We show that about one third of the enrolled patients die acutely, and that mortality is independently associated with patient age and Glasgow Coma Scale on admission. Japanese encephalitis, dengue virus and enterovirus (including enterovirus 71) are the major viruses detected in our patients. However, more than half of the patients remain undiagnosed, while mortality in this group is as high as in the diagnosed group. This study will benefit clinicians and public health in terms of clinical management and prevention of childhood encephalitis in Vietnam

Electrochemical Determination of Uric Acid in Urine by Using Zeolite Imidazolate Framework-11 Modified Electrode

In the present article, the synthesis of zeolite imidazole framework-11 (ZIF-11) by ultrasonic-assisted hydrothermal process and its application as an electrode modifier for electrochemical determination of uric acid in urine are demonstrated. The obtained materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and nitrogen adsorption/desorption isotherms. It was found that the ZIF-11 with rhombic dodecahedron topology and high surface area (1066 m2.g-1) was synthesized in a certain temperature and found in around 25–40°C, and other crystalline phases of zinc benzimidazolate deferring from ZIF-11 phase were found in less 25°C or higher than 40°C. The ZIF-11 is stable in the pH range 6-10. The modification of glassy carbon electrode was performed with ZIF-67 using the drop-casting procedure. The present ZIF-11 modified electrode was employed to study the electrochemical behavior of uric acid (UA). UA oxidation is catalyzed by this electrode in aqueous buffer solution (pH 7) with a decrease of 70 mV in overpotential compared to glassy carbon electrode. With the differential pulse–anodic stripping voltammetry (DP-ASV) method, the oxidation current of UA versus its concentration shows good linearity in the range 20–540μM (R=0.998) with a detection limit of 0.48 μM (S/N=3). The obtained ZIF-11 modified electrode was applied in the detection of UA content in urine samples, and satisfied results were obtained

Skin dendritic cell and T cell activation associated with dengue shock syndrome

The pathogenesis of severe dengue remains unclear, particularly the mechanisms underlying the plasma leakage that results in hypovolaemic shock in a small proportion of individuals. Maximal leakage occurs several days after peak viraemia implicating immunological pathways. Skin is a highly vascular organ and also an important site of immune reactions with a high density of dendritic cells (DCs), macrophages and T cells. We obtained skin biopsies and contemporaneous blood samples from patients within 24 hours of onset of dengue shock syndrome (DSS), and from healthy controls. We analyzed cell subsets by flow cytometry, and soluble mediators and antibodies by ELISA; the percentage of migratory CD1a+ dermal DCs was significantly decreased in the DSS patients, and skin CD8+ T cells were activated, but there was no accumulation of dengue-specific antibodies. Inflammatory monocytic cells were not observed infiltrating the skin of DSS cases on whole-mount histology, although CD14dim cells disappeared from blood.Published versio