Emerging Enterovirus A71 Subgenogroup B5 Causing Severe Hand, Foot, and Mouth Disease, Vietnam, 2023.

We report on a 2023 outbreak of severe hand, foot, and mouth disease in southern Vietnam caused by an emerging lineage of enterovirus A71 subgenogroup B5. Affected children were significantly older than those reported during previous outbreaks. The virus should be closely monitored to assess its potential for global dispersal.

Enterovirus A71 Phenotypes Causing Hand, Foot and Mouth Disease, Vietnam.

We investigated enterovirus A71-associated hand, foot and mouth disease in Vietnam and found that, after replacing subgenogroup C4 in 2013, B5 remained the leading cause of this disease. In contrast with previous observations, this switch did not result in an explosive outbreak, and B5 evolution was driven by negative selection.

Emerging Coxsackievirus A6 Causing Hand, Foot and Mouth Disease, Vietnam.

Hand, foot and mouth disease (HFMD) is a major public health issue in Asia and has global pandemic potential. Coxsackievirus A6 (CV-A6) was detected in 514/2,230 (23%) of HFMD patients admitted to 3 major hospitals in southern Vietnam during 2011-2015. Of these patients, 93 (18%) had severe HFMD. Phylogenetic analysis of 98 genome sequences revealed they belonged to cluster A and had been circulating in Vietnam for 2 years before emergence. CV-A6 movement among localities within Vietnam occurred frequently, whereas viral movement across international borders appeared rare. Skyline plots identified fluctuations in the relative genetic diversity of CV-A6 corresponding to large CV-A6-associated HFMD outbreaks worldwide. These data show that CV-A6 is an emerging pathogen and emphasize the necessity of active surveillance and understanding the mechanisms that shape the pathogen evolution and emergence, which is essential for development and implementation of intervention strategies.

Enterovirus serotypes in patients with central nervous system and respiratory infections in Viet Nam 1997-2010.

BACKGROUND: Enteroviruses are the most common causative agents of human illness. Enteroviruses have been associated with regional and global epidemics, recently, including with severe disease (Enterovirus A71 and D68), and are of interest as emerging viruses. Here, we typed Enterovirus A-D (EV) from central nervous system (CNS) and respiratory infections in Viet Nam. METHODS: Data and specimens from prospective observational clinical studies conducted between 1997 and 2010 were used. Species and serotypes were determined using type-specific RT-PCR and viral protein 1 or 4 (VP1, VP4) sequencing. RESULTS: Samples from patients with CNS infection (51 children - 10 CSF and 41 respiratory/rectal swabs) and 28 adults (28 CSF) and respiratory infection (124 children - 124 respiratory swabs) were analysed. Twenty-six different serotypes of the four Enterovirus species (A-D) were identified, including EV-A71 and EV-D68. Enterovirus B was associated with viral meningitis in children and adults. Hand, foot and mouth disease associated Enteroviruses A (EV-A71 and Coxsackievirus [CV] A10) were detected in children with encephalitis. Diverse serotypes of all four Enterovirus species were found in respiratory samples, including 2 polio-vaccine viruses, but also 8 CV-A24 and 8 EV-D68. With the exception of EV-D68, the relevance of these viruses in respiratory infection remains unknown. CONCLUSION: We describe the diverse spectrum of enteroviruses from patients with CNS and respiratory infections in Viet Nam between 1997 and 2010. These data confirm the global circulation of Enterovirus genera and their associations and are important for clinical diagnostics, patient management, and outbreak response.

Severe enterovirus A71 associated hand, foot and mouth disease, Vietnam, 2018: preliminary report of an impending outbreak.

Since January 2018, over 53,000 hospitalisations and six deaths due to hand, foot and mouth disease (HFMD) have occurred across Vietnam with most cases from September onward. In a large tertiary referral hospital, Ho Chi Minh City, enterovirus A71 subgenogroup C4 was predominant, while B5 was only sporadically detected. The re-emergence of C4 after causing a severe HFMD outbreak with > 200 deaths in 2011-12 among susceptible young children raises concern of another impending severe outbreak.

Phylodynamics of Enterovirus A71-Associated Hand, Foot, and Mouth Disease in Viet Nam.

UNLABELLED: Enterovirus A71 (EV-A71) is a major cause of hand, foot, and mouth disease (HFMD) and is particularly prevalent in parts of Southeast Asia, affecting thousands of children and infants each year. Revealing the evolutionary and epidemiological dynamics of EV-A71 through time and space is central to understanding its outbreak potential. We generated the full genome sequences of 200 EV-A71 strains sampled from various locations in Viet Nam between 2011 and 2013 and used these sequence data to determine the evolutionary history and phylodynamics of EV-A71 in Viet Nam, providing estimates of the effective reproduction number (Re) of the infection through time. In addition, we described the phylogeography of EV-A71 throughout Southeast Asia, documenting patterns of viral gene flow. Accordingly, our analysis reveals that a rapid genogroup switch from C4 to B5 likely took place during 2012 in Viet Nam. We show that the Re of subgenogroup C4 decreased during the time frame of sampling, whereas that of B5 increased and remained >1 at the end of 2013, corresponding to a rise in B5 prevalence. Our study reveals that the subgenogroup B5 virus that emerged into Viet Nam is closely related to variants that were responsible for large epidemics in Malaysia and Taiwan and therefore extends our knowledge regarding its associated area of endemicity. Subgenogroup B5 evidently has the potential to cause more widespread outbreaks across Southeast Asia. IMPORTANCE: EV-A71 is one of many viruses that cause HFMD, a common syndrome that largely affects infants and children. HFMD usually causes only mild illness with no long-term consequences. Occasionally, however, severe infection may arise, especially in very young children, causing neurological complications and even death. EV-A71 is highly contagious and is associated with the most severe HFMD cases, with large and frequent epidemics of the virus recorded worldwide. Although major advances have been made in the development of a potential EV-A71 vaccine, there is no current prevention and little is known about the patterns and dynamics of EV-A71 spread. In this study, we utilize full-length genome sequence data obtained from HFMD patients in Viet Nam, a geographical region where the disease has been endemic since 2003, to characterize the phylodynamics of this important emerging virus.

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.

Age-time-specific transmission of hand-foot-and-mouth disease enterovirus serotypes in Vietnam: A catalytic model with maternal immunity.

Hand, foot and mouth disease (HFMD) is highly prevalent in the Asia Pacific region, particularly in Vietnam. To develop effective interventions and efficient vaccination programs, we inferred the age-time-specific transmission patterns of HFMD serotypes enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), coxsackievirus A10 (CV-A10), coxsackievirus A16 (CV-A16) in Ho Chi Minh City, Vietnam from a case data collected during 2013-2018 and a serological survey data collected in 2015 and 2017. We proposed a catalytic model framework with good adaptability to incorporate maternal immunity using various mathematical functions. Our results indicate the high-level transmission of CV-A6 and CV-A10 which is not obvious in the case data, due to the variation of disease severity across serotypes. Our results provide statistical evidence supporting the strong association between severe illness and CV-A6 and EV-A71 infections. The HFMD dynamic pattern presents a cyclical pattern with large outbreaks followed by a decline in subsequent years. Additionally, we identify the age group with highest risk of infection as 1-2 years and emphasise the risk of future outbreaks as over 50% of children aged 6-7 years were estimated to be susceptible to CV-A16 and EV-A71. Our study highlights the importance of multivalent vaccines and active surveillance for different serotypes, supports early vaccination prior to 1 year old, and points out the potential utility for vaccinating children older than 5 years old in Vietnam.

Coxsackievirus A16 in Southern Vietnam.

Background: Hand, Foot and Mouth Disease (HFMD) is a major public health concern in the Asia-Pacific region. Most recent HFMD outbreaks have been caused by enterovirus A71 (EV-A71), coxsackievirus A16 (CVA16), CVA10, and CVA6. There has been no report regarding the epidemiology and genetic diversity of CVA16 in Vietnam. Such knowledge is critical to inform the development of intervention strategies. Materials and Methods: From 2011 to 2017, clinical samples were collected from in- and outpatients enrolled in a HFMD research program conducted at three referral hospitals in Ho Chi Minh City (HCMC), Vietnam. Throat or rectal swabs positive for CVA16 with sufficient viral load were selected for whole genome sequencing and evolutionary analysis. Results: Throughout the study period, 320 CVA16 positive samples were collected from 2808 HFMD patients (11.4%). 59.4% of patients were male. The median age was 20.8 months (IQR, 14.96-31.41). Patients resided in HCMC (55.3%), Mekong Delta (22.2%), and South East Vietnam (22.5%). 10% of CVA16 infected patients had moderately severe or severe HFMD. CVA16 positive samples from 153 patients were selected for whole genome sequencing, and 66 complete genomes were obtained. Phylogenetic analysis demonstrated that Vietnamese CVA16 strains belong to a single genogroup B1a that clusters together with isolates from China, Japan, Thailand, Malaysia, France and Australia. The CVA16 strains of the present study were circulating in Vietnam some 4 years prior to its detection in HFMD cases. Conclusion: We report for the first time on the molecular epidemiology of CVA16 in Vietnam. Unlike EV-A71, which showed frequent replacement between subgenogroups B5 and C4 every 2-3 years in Vietnam, CVA16 displays a less pronounced genetic alternation with only subgenogroup B1a circulating in Vietnam since 2011. Our collective findings emphasize the importance of active surveillance for viral circulation in HFMD endemic countries, critical to informing outbreak response and vaccine development.

Clinical, etiological and epidemiological investigations of hand, foot and mouth disease in southern Vietnam during 2015 - 2018.

Hand, foot and mouth disease (HFMD) continues to challenge Asia with pandemic potential. In Vietnam, there have been two major outbreaks occurring during 2011-2012 (>200,000 hospitalizations and >200 deaths) and more recently in 2018 (>130,000 hospitalizations and 17 deaths). Given the high burden and the complex epidemic dynamics of HFMD, synthesizing its clinical and epidemiological data remains essential to inform the development of appropriate interventions and design public health measures. We report the results of a hospital-based study conducted during 2015-2018, covering the severe HFMD outbreak recently documented in Vietnam in 2018. The study was conducted at three major hospitals responsible for receiving HFMD patients from southern Vietnam with a population of over 40 million. A total of 19 enterovirus serotypes were detected in 1196 HFMD patients enrolled in the clinical study during 2015-2018, with enterovirus A71 (EV-A71), coxsackievirus A6 (CV-A6), CV-A10 and CV-A16 being the major causes. Despite the emergence of coxsackieviruses, EV-A71 remains the leading cause of severe HFMD in Vietnam. EV-A71 was consistently detected at a higher frequency during the second half of the years. The emergence of EV-A71 subgenogroup C4 in late 2018 was preceded by its low activity during 2017-early 2018. Compared with EV-A71 subgenogroup B5, C4 was more likely to be associated with severe HFMD, representing the first report demonstrating the difference in clinical severity between subgenogroup C4 and B5, the two predominant EV-A71 subgenogroups causing HFMD worldwide. Our data have provided significant insights into important aspects of HFMD over four years (2015-2018) in Vietnam, and emphasize active surveillance for pathogen circulation remains essential to inform the local public health authorities in the development of appropriate intervention strategies to reduce the burden of this emerging infections. Multivalent vaccines are urgently needed to control HFMD.

Intravenous magnesium sulfate for the management of severe hand, foot, and mouth disease with autonomic nervous system dysregulation in Vietnamese children: study protocol for a randomized controlled trial.

BACKGROUND: Over the last 15 years, hand, foot, and mouth disease (HFMD) has emerged as a major public health burden across the Asia-Pacific region. A small proportion of HFMD patients, typically those infected with enterovirus 71 (EV71), develop brainstem encephalitis with autonomic nervous system (ANS) dysregulation and may progress rapidly to cardiopulmonary failure and death. Although milrinone has been reported to control hypertension and support myocardial function in two small studies, in practice, a number of children still deteriorate despite this treatment. Magnesium sulfate (MgSO4) is a cheap, safe, and readily available medication that is effective in managing tetanus-associated ANS dysregulation and has shown promise when used empirically in EV71-confirmed severe HFMD cases. METHODS/DESIGN: We describe the protocol for a randomized, placebo-controlled, double-blind trial of intravenous MgSO4 in Vietnamese children diagnosed clinically with HFMD plus ANS dysregulation with systemic hypertension. A loading dose of MgSO4 or identical placebo is given over 20 min followed by a maintenance infusion for 72 h according to response, aiming for Mg levels two to three times the normal level in the treatment arm. The primary endpoint is a composite of disease progression within 72 h defined as follows: development of pre-specified blood pressure criteria necessitating the addition of milrinone, the need for ventilation, shock, or death. Secondary endpoints comprise these parameters singly, plus other clinical endpoints including the following: requirement for other inotropic agents; duration of hospitalization; presence of neurological sequelae at discharge in survivors; and neurodevelopmental status assessed 6 months after discharge. The number and severity of adverse events observed in the two treatment arms will also be compared. Based on preliminary data from a case series, and allowing for some losses, 190 patients (95 in each arm) will allow detection of a 50 % reduction in disease progression with 90 % power at a two-sided 5 % significance level. DISCUSSION: Given the large numbers of HFMD cases currently being seen in hospitals in Asia, if MgSO4 is shown to be effective in controlling ANS dysregulation and preventing severe HFMD complications, this finding would be important to pediatric care throughout the region. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01940250 (Registered 22 August 2013).

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.