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Fulltext 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

Thanh TT, Anh NT, Tham NT, Van HM, Sabanathan S, Qui PT, et al.

Virol J, 2015 Jun 09;12:85.
PMID: 26050791 DOI: 10.1186/s12985-015-0316-2

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.
Fulltext Coxsackievirus A16 in Southern Vietnam

Nhu LNT, Nhan LNT, Anh NT, Hong NTT, Van HMT, Thanh TT, et al.

Front Microbiol, 2021;12:689658.
PMID: 34248913 DOI: 10.3389/fmicb.2021.689658

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

Tan le V, Tuyen NT, Thanh TT, Ngan TT, Van HM, Sabanathan S, et al.

J Virol Methods, 2015 Apr;215-216:30-6.
PMID: 25704598 DOI: 10.1016/j.jviromet.2015.02.011

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.

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