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.

Spatiotemporal Evolution of SARS-CoV-2 Alpha and Delta Variants during Large Nationwide Outbreak of COVID-19, Vietnam, 2021.

We analyzed 1,303 SARS-CoV-2 whole-genome sequences from Vietnam, and found the Alpha and Delta variants were responsible for a large nationwide outbreak of COVID-19 in 2021. The Delta variant was confined to the AY.57 lineage and caused >1.7 million infections and >32,000 deaths. Viral transmission was strongly affected by nonpharmaceutical interventions.

Long-Term Humoral Immune Response in Persons with Asymptomatic or Mild SARS-CoV-2 Infection, Vietnam.

Antibody response against nucleocapsid and spike proteins of SARS-CoV-2 in 11 persons with mild or asymptomatic infection rapidly increased after infection. At weeks 18-30 after diagnosis, all remained seropositive but spike protein-targeting antibody titers declined. These data may be useful for vaccine development.

Superspreading Event of SARS-CoV-2 Infection at a Bar, Ho Chi Minh City, Vietnam.

We report a superspreading event of severe acute respiratory syndrome coronavirus 2 infection initiated at a bar in Vietnam with evidence of symptomatic and asymptomatic transmission, based on ministry of health reports, patient interviews, and whole-genome sequence analysis. Crowds in enclosed indoor settings with poor ventilation may be considered at high risk for transmission.

The Natural History and Transmission Potential of Asymptomatic Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: Little is known about the natural history of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We conducted a prospective study at a quarantine center for coronavirus disease 2019 in Ho Chi Minh City, Vietnam. We enrolled quarantined people with reverse-transcription polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection, collecting clinical data, travel and contact history, and saliva at enrollment and daily nasopharyngeal/throat swabs (NTSs) for RT-PCR testing. We compared the natural history and transmission potential of asymptomatic and symptomatic individuals. RESULTS: Between 10 March and 4 April 2020, 14 000 quarantined people were tested for SARS-CoV-2; 49 were positive. Of these, 30 participated in the study: 13 (43%) never had symptoms and 17 (57%) were symptomatic. Seventeen (57%) participants imported cases. Compared with symptomatic individuals, asymptomatic people were less likely to have detectable SARS-CoV-2 in NTS collected at enrollment (8/13 [62%] vs 17/17 [100%]; P = .02). SARS-CoV-2 RNA was detected in 20 of 27 (74%) available saliva samples (7 of 11 [64%] in the asymptomatic group and 13 of 16 [81%] in the symptomatic group; P = .56). Analysis of RT-PCR positivity probability showed that asymptomatic participants had faster viral clearance than symptomatic participants (P < .001 for difference over the first 19 days). This difference was most pronounced during the first week of follow-up. Two of the asymptomatic individuals appeared to transmit SARS-CoV-2 to 4 contacts. CONCLUSIONS: Asymptomatic SARS-CoV-2 infection is common and can be detected by analysis of saliva or NTSs. The NTS viral loads fall faster in asymptomatic individuals, but these individuals appear able to transmit the virus to others.

Neutralizing Antibodies against Enteroviruses in Patients with Hand, Foot and Mouth Disease.

Hand, foot and mouth disease (HFMD) is an emerging infection with pandemic potential. Knowledge of neutralizing antibody responses among its pathogens is essential to inform vaccine development and epidemiologic research. We used 120 paired-plasma samples collected at enrollment and >7 days after the onset of illness from HFMD patients infected with enterovirus A71 (EV-A71), coxsackievirus A (CVA) 6, CVA10, and CVA16 to study cross neutralization. For homotypic viruses, seropositivity increased from <60% at enrollment to 97%-100% at follow-up, corresponding to seroconversion rates of 57%-93%. Seroconversion for heterotypic viruses was recorded in only 3%-23% of patients. All plasma samples from patients infected with EV-A71 subgenogroup B5 could neutralize the emerging EV-A71 subgenogroup C4. Collectively, our results support previous reports about the potential benefit of EV-A71 vaccine but highlight the necessity of multivalent vaccines to control HFMD.

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.

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.

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.

Seroprotection against tetanus in southern Vietnam.

BACKGROUND: Ongoing tetanus cases and sporadic outbreaks of vaccine-preventable diseases associated with routine vaccination programmes remain problems in many low and middle-income countries, including Vietnam. With no human-to-human transmission or natural immunity, tetanus antibody levels indicate both individual risk of tetanus and gaps in vaccination programmes. METHODS: To investigate gaps in immunity to tetanus in Vietnam, a country with a historically high level of tetanus vaccination coverage, tetanus antibodies were measure by ELISA from samples selected from a long-term serum bank, established for the purposes of general-population seroepidemiological investigations in southern Vietnam. Samples were selected from 10 provinces, focussing on age-groups targeted by national vaccination programmes for infants and pregnant women (Expanded Programme on Immunization, EPI, and Maternal and Neonatal Tetanus, MNT). RESULTS: Antibodies were measured from a total of 3864 samples. Highest tetanus antibody concentrations occurred in children under 4 years old, over 90 % of whom had protective levels. Approximately 70 % of children aged 7-12 years had protective antibody concentrations although there was variation among provinces. For infants and children, there were no significant differences in tetanus protection between males and females, but for adults aged 20-35 years, in five of the ten provinces surveyed, protection against tetanus was higher in females (p < 0.05) who are eligible for booster doses under the MNT programme. In seven of ten provinces, antibody concentrations were inversely related to age (p < 0.01) and protection of older individuals was generally low. CONCLUSION: Widespread immunity to tetanus toxoid is seen in infants and young children consistent with the high coverage rates reported for diptheria tetanus toxoid and pertussis (DTP) in Vietnam. However, the lower antibody concentrations seen in older children and men suggest reduced immunity to tetanus in populations not targeted by EPI and MNT programmes.

Kinetics of Neutralizing Antibodies against Omicron Variant in Vietnamese Healthcare Workers after Primary Immunization with ChAdOx1-S and Booster Immunization with BNT162b2.

We studied the development and persistence of neutralizing antibodies against SARS-CoV-2 ancestral strain, and Delta and Omicron (BA.1 and BA.2) variants in Vietnamese healthcare workers (HCWs) up to 15 weeks after booster vaccination. We included 47 HCWs, including group 1 (G1, N = 21) and group 2 (G2; N = 26) without and with breakthrough Delta variant infection before booster immunization, respectively). The study participants had completed primary immunization with ChAdOx1-S and booster vaccination with BNT162b2. Neutralizing antibodies were measured using a surrogate virus neutralization assay. Of the 21 study participants in G1, neutralizing antibodies against ancestral strain, Delta variant, BA.1, and BA.2 were (almost) abolished at month 8 after the second dose, but all had detectable neutralizing antibodies to the study viruses at week 2 post booster dose. Of the 26 study participants in G2, neutralizing antibody levels to BA.1 and BA.2 were significantly higher than those to the corresponding viruses measured at week 2 post breakthrough infection and before the booster dose. At week 15 post booster vaccination, neutralizing antibodies to BA.1 and BA.2 dropped significantly, with more profound changes observed in those without breakthrough Delta variant infection. Booster vaccination enhanced neutralizing activities against ancestral strain and Delta variant compared with those induced by primary vaccination. These responses were maintained at high levels for at least 15 weeks. Our findings emphasize the importance of the first booster dose in producing cross-neutralizing antibodies against Omicron variant. A second booster to maintain long-term vaccine effectiveness against the currently circulating variants merits further research.

Immunogenicity of Oxford-AstraZeneca COVID-19 Vaccine in Vietnamese Health-Care Workers.

We studied the immunogenicity of the Oxford-AstraZeneca vaccine in health-care workers of a major infectious diseases hospital in Vietnam. We measured neutralizing antibodies before and 14 days after each dose, and at day 28 and month 3 after dose 1. A total of 554 workers (136 men and 418 women; age range, 22-71 years; median age, 36 years) participated with the study. Of the 144 participants selected for follow-up after dose 1, 104 and 94 gave blood for antibody measurement at weeks 6 and 8, and at month 3 after dose 1, respectively. The window time between the two doses was 6 weeks. At baseline, none had detectable neutralizing antibodies. After dose 1, the proportion of participants with detectable neutralizing antibodies increased from 27.3% (151 of 554) at day 14 to 78.0% (432 of 554) at day 28. Age correlated negatively with the development and the levels of neutralizing antibodies. However, at day 28, these differences were less profound, and women had a greater seroconversion rate and greater levels of neutralizing antibodies than men. After dose 2, these age and gender associations were not observable. In addition, the proportion of study participants with detectable neutralizing antibodies increased from 70.2% (73 of 104) before dose 2 (week 6, after dose 1) to 98.1% (102 of 104) 14 days later. At month 3, neutralizing antibodies decreased and 94.7% (89 of 94) of the study participants remained seropositive. The Oxford-AstraZeneca COVID-19 vaccine is immunogenic in Vietnamese health-care workers. These data are critical to informing the deployment of the COVID-19 vaccine in Vietnam and in Southeast Asia, where vaccination coverage remains inadequate.

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.

Absence of SARS-CoV-2 antibodies in pre-pandemic plasma from children and adults in Vietnam.

We tested pre-pandemic (2015--2019) plasma samples from 148 Vietnamese children and 100 Vietnamese adults at high risk of zoonotic infections for antibodies against SARS-CoV-2 nucleocapsid and spike proteins. None was positive. The data thus demonstrated no evidence of prior serological cross-reactivity with SARS-CoV-2 that might explain the low numbers of COVID-19 in Vietnam. No pre-existing cross-reactivity might explain Vietnam success of COVID-19 control.

An observational study of breakthrough SARS-CoV-2 Delta variant infections among vaccinated healthcare workers in Vietnam.

BACKGROUND: Data on breakthrough SARS-CoV-2 Delta variant infections in vaccinated individuals are limited. METHODS: We studied breakthrough infections among Oxford-AstraZeneca vaccinated healthcare workers in an infectious diseases hospital in Vietnam. We collected demographic and clinical data alongside serial PCR testing, measurement of SARS-CoV-2 antibodies, and viral whole-genome sequencing. FINDINGS: Between 11th-25th June 2021 (7-8 weeks after the second dose), 69 staff tested positive for SARS-CoV-2. 62 participated in the study. Most were asymptomatic or mildly symptomatic and all recovered. Twenty-two complete-genome sequences were obtained; all were Delta variant and were phylogenetically distinct from contemporary viruses obtained from the community or from hospital patients admitted prior to the outbreak. Viral loads inferred from Ct values were 251 times higher than in cases infected with the original strain in March/April 2020. Median time from diagnosis to negative PCR was 21 days (range 8-33). Neutralizing antibodies (expressed as percentage of inhibition) measured after the second vaccine dose, or at diagnosis, were lower in cases than in uninfected, fully vaccinated controls (median (IQR): 69.4 (50.7-89.1) vs. 91.3 (79.6-94.9), p=0.005 and 59.4 (32.5-73.1) vs. 91.1 (77.3-94.2), p=0.002). There was no correlation between vaccine-induced neutralizing antibody levels and peak viral loads or the development of symptoms. INTERPRETATION: Breakthrough Delta variant infections following Oxford-AstraZeneca vaccination may cause asymptomatic or mild disease, but are associated with high viral loads, prolonged PCR positivity and low levels of vaccine-induced neutralizing antibodies. Epidemiological and sequence data suggested ongoing transmission had occurred between fully vaccinated individuals. FUNDING: Wellcome and NIH/NIAID.

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.

Economic Burden Attributed to Children Presenting to Hospitals With Hand, Foot, and Mouth Disease in Vietnam.

BACKGROUND: Hand, foot, and mouth disease (HFMD) has become a major public health concern in the Asia-Pacific region. Knowledge of its economic burden is essential for policy makers in prioritizing the development and implementation of interventions. METHODS: A multi-hospital-based study was prospectively conducted at 3 major hospitals in Ho Chi Minh City, Vietnam, during 2016-2017. Data on direct and productivity costs were collected alongside clinical information and samples and demographic information from study participants. RESULTS: A total of 466 patients were enrolled. Two hundred three of 466 (43.6%) patients lived in Ho Chi Minh City, and 72/466 (15.5%) had severe HFMD. An enterovirus was identified in 74% of 466 patients, with EV-A71, CV-A6, CV-A10, and CV-A16 being the most common viruses identified (236/466, 50.6%). The mean economic burden per case was estimated at US$400.80 (95% confidence interval [CI], $353.80-$448.90), of which the total direct (medical) costs accounted for 69.7%. There were considerable differences in direct medical costs between groups of patients with different clinical severities and pathogens (ie, EV-A71 vs non-EV-A71). In Vietnam, during 2016-2017, the economic burden posed by HFMD was US$90 761 749 (95% CI, $79 033 973-$103 009 756). CONCLUSIONS: Our findings are of public health significance because for the first time we demonstrate that HFMD causes a substantial economic burden in Vietnam, and although multivalent vaccines are required to control HFMD, effective EV-A71 vaccine could substantially reduce the burden posed by severe HFMD. The results will be helpful for health policy makers in prioritizing resources for the development and implementation of intervention strategies to reduce the burden of HFMD.

Central Nervous System Infection Diagnosis by Next-Generation Sequencing: A Glimpse Into the Future?

Japanese encephalitis virus was detected by deep sequencing for the first time in urine of a 16-year-old boy with encephalitis. Seroconversion and polymerase chain reaction analysis confirmed the metagenomics finding. Urine is useful for diagnosis of flaviviral encephalitis, whereas deep sequencing can be a panpathogen assay for the diagnosis of life-threatening infectious diseases.