Detection of enteroviruses during a 2018 hand, foot and mouth disease outbreak in Malaysia

@#Hand, foot, and mouth disease (HFMD) is a common childhood disease caused by enteroviruses. In 2018, a HFMD outbreak in Malaysia affected over 76,000 children. In this study, we used RT-qPCR and CODEHOP PCR to detect the causative agents in 89 clinically diagnosed HFMD patients in Kuala Lumpur and Selangor. Most (62.9%) of the children were below 3 years old. PCR with either assay detected enteroviruses in 84.2% (75/89) and CODEHOP PCR successfully typed 66.7% (50/75) of the enteroviruses. Sequencing of CODEHOP amplicons showed co-circulation of multiple enteroviruses with coxsackievirus A6 (CV-A6) and A16 as the predominant serotypes, but not the neurovirulent enterovirus A71. CV-A6 infection was more common in children less than 12 months old (p=0.01) and was more likely to cause vesicles in the gluteal area (p=0.01) compared to other enteroviruses. Establishing a robust identification method during HFMD outbreaks is important for patient management and public health responses.

Causative agents of hand, foot and mouth disease in University Malaya Medical Centre, Kuala Lumpur, Malaysia in 2012-2013

Hand, foot and mouth disease (HFMD) is a childhood illness, commonly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). In recent years, unusual HFMD outbreaks caused by coxsackievirus A6 (CV-A6) have been reported. From May 2012 to September 2013, enteroviruses were detected in 25 HFMD patients in University Malaya Medical Centre, Kuala Lumpur, Malaysia. The predominant serotypes were EV-A71 (48%) and CV-A6 (48%), followed by CV-A16 (4%). CV-A6 patients (mean age, 2.1) were significantly younger than EV-A71 patients (mean age, 3.3). There were no significant differences observed in clinical features between EV-A71 and CV-A6 patients. Since enteroviruses are difficult to differentiate clinically, the conserved 5’ untranslated region (5’ UTR) was used to identify enterovirus serotypes. Phylogenetic analysis of 5’ UTR showed distinct clustering of viruses as EV-A71, CV-A16 and CV-A6. Further genotyping with capsid genes showed that all the EVA71 sequences belonged to subgenotype B5, while the CV-A16 sequence belonged to subgenotype B2b. CV-A6 sequences were clustered into genotypes D1 and D2, with recent isolates from Seri Kembangan, Malaysia and China. In summary, 59.5% of HFMD cases in our centre in 2012-2013 were caused by EV-A71, CV-A16 and the newly emerging CV-A6. This study also demonstrated that 5’ UTR is suitable for preliminary identification of enteroviruses during HFMD outbreaks, but specific capsid genes such as VP1 and VP4/VP2 are required for further genotyping. Apart from measures to control the spread of the virus during an outbreak of HFMD, identification of EV-A71 as the etiological agent is important as EV-A71 is a major cause of severe neurological complications and potentially fatal.

Comparison of Anyplex II RV16 assay with conventional methods for detection of respiratory viruses

Early detection of viral etiologies of acute respiratory tract infections of patients affects management and disease control in pediatric patients. In this study, the performance of Anyplex II RV16 assay (Seegene, Seoul, Korea) was evaluated by comparing with viral culture and direct immunofluorescence staining of clinical specimens for detection of respiratory viruses in patients. A total of 168 respiratory specimens were collected from 122 patients from November 2012 to May 2013 at the time of admission to the University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The Anyplex II RV16 assay, viral culture, and direct immunofluorescence staining were positive in 74.4%, 18.5% and 14.9% of the specimens, respectively. HRV was the predominant virus detected by the Anyplex II RV16 assay. In 47 cases, two or more respiratory viruses were detected by the Anyplex II RV16 assay, which were missed by conventional methods. The performance of the Anyplex II RV16 assay was better than viral culture and direct immunofluorescence staining of clinical specimens for the detection of respiratory viruses. The implementation of the Anyplex II RV16 assay in hospital laboratories will provide rapid diagnosis of major viral infections of the respiratory tract.

Diagnosis of human enterovirus A71 infection in Malaysia using a commercial IgM-capture enzyme-linked immunosorbent assay and an IgM-colloidal gold immunochromatographic assay

Hand, foot and mouth disease (HFMD) is a common childhood infection caused by many enteroviruses, including enterovirus A71 (EV-A71). As EV-A71 is associated with severe neurological disease, early diagnosis is critical for clinical and public health management. In developing countries such as Malaysia, laboratory capacity to carry out EV-A71 IgM detection is greater than that of the gold standard methods of virus culture or molecular detection. This study evaluated two diagnostic kits, EV-A71 IgM-capture enzyme-linked immunosorbent (ELISA) and EV-A71 IgM-colloidal gold immunochromatographic assay (GICA), which had previously only been assessed in China. The assays were tested with 89 serum samples from patients with suspected HFMD. The sensitivity, specificity, positive predictive value, and negative predictive value rates were 78.4%, 80.8%, 74.4%, and 84.0%, respectively, for the IgM-capture ELISA, and 75.7%, 76.9%, 70.0%, and 81.6% for the IgM GICA. These performance measures were similar between the two assays. Concordance between the two assays was 91.1%. The sensitivity rates were lower than those previously reported, likely because the multiple circulating EV-A71 genotypes in Malaysia differ from the C4 subgenotype found in China and used in the assays. Both assays had low false positive rates (12.5% and 16.7% for ELISA and GICA, respectively) when tested on sera from patients confirmed to have enteroviruses. Both diagnostic kits are suitable for early diagnosis of HFMD caused by EV- A71 in Malaysia, but confirmation with culture or PCR is still important.

Comparative genetic analysis of VP4, VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997-2008

Three genomic regions, VP4 capsid, VP1 capsid and 3D RNA polymerase of human enterovirus 71 (EV-71) and coxsackievirus A16 (CV-A16) were sequenced to understand the evolution of these viruses in Malaysia. A total of 42 EV-71 and 36 CV-A16 isolates from 1997-2008 were sequenced. Despite the presence of many EV-71 subgenotypes worldwide, only subgenotypes B3, B4, B5, C1 and C2 were present in Malaysia. Importation of other subgenotypes such as C3, C4/D and C5 from other countries was infrequent. For CV-A16, the earlier subgenotype B1 was replaced by subgenotypes B2a and the recent B2c. Subgenotype B2a was present throughout the study while B2c only emerged in 2005. No genetic signatures could be attributed to viral virulence suggesting that host factors have a major role in determining the outcome of infection. Only three EV-71 B3 isolates showed non-consistent phylogeny in the 3D RNA polymerase region which indicated occurrence of recombination in EV-71. High genetic diversity was observed in the Malaysian EV-71 but Malaysian CV-A16 showed low genetic diversity in the three genomic regions sequenced. EV-71 showed strong purifying selection, but that occurred to a lesser extent in CV-A16.