Displaying publications 21 - 40 of 40 in total

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  1. Young KI, Mundis S, Widen SG, Wood TG, Tesh RB, Cardosa J, et al.
    Parasit Vectors, 2017 Aug 31;10(1):406.
    PMID: 28859676 DOI: 10.1186/s13071-017-2341-z
    BACKGROUND: Mosquito-borne dengue virus (DENV) is maintained in a sylvatic, enzootic cycle of transmission between canopy-dwelling non-human primates and Aedes mosquitoes in Borneo. Sylvatic DENV can spill over into humans living in proximity to forest foci of transmission, in some cases resulting in severe dengue disease. The most likely vectors of such spillover (bridge vectors) in Borneo are Ae. albopictus and Ae. niveus. Borneo is currently experiencing extensive forest clearance. To gauge the effect of this change in forest cover on the likelihood of sylvatic DENV spillover, it is first necessary to characterize the distribution of bridge vectors in different land cover types. In the current study, we hypothesized that Ae. niveus and Ae. albopictus would show significantly different distributions in different land cover types; specifically, we predicted that Ae. niveus would be most abundant in forests whereas Ae. albopictus would have a more even distribution in the landscape.

    RESULTS: Mosquitoes were collected from a total of 15 sites using gravid traps and a backpack aspirator around Kampong Puruh Karu, Sarawak, Malaysian Borneo, where sylvatic DENV spillover has been documented. A total of 2447 mosquitoes comprising 10 genera and 4 species of Aedes, were collected over the three years, 2013, 2014 and 2016, in the three major land cover types in the area, homestead, agriculture and forest. Mosquitoes were identified morphologically, pooled by species and gender, homogenized, and subject to DNA barcoding of each Aedes species and to arbovirus screening. As predicted, Ae. niveus was found almost exclusively in forests whereas Ae. albopictus was collected in all land cover types. Aedes albopictus was significantly (P = 0.04) more abundant in agricultural fields than forests. Sylvatic DENV was not detected in any Aedes mosquito pools, however genomes of 14 viruses were detected using next generation sequencing.

    CONCLUSIONS: Land cover type affects the abundance and distribution of the most likely bridge vectors of sylvatic DENV in Malaysia Borneo. Conversion of forests to agriculture will likely decrease the range and abundance of Ae. niveus but enhance the abundance of Ae. albopictus.

  2. Chien YS, Chen FJ, Wu HC, Lin CH, Chang WC, Perera D, et al.
    Microbiol Spectr, 2023 Oct 13.
    PMID: 37831475 DOI: 10.1128/spectrum.02507-23
    Enteroviruses (EVs) are a group of viruses that cause various human illnesses. While the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) method can generate VP1 gene fragments for enterovirus genotyping, it is unable to detect recombinant strains. Recent advances in viral genome sequencing using next-generation sequencing technologies have enabled comprehensive analyses. However, the high cost poses a challenge for widespread adoption. To address this issue, this study proposes a cost-effective approach for generating complete enterovirus genome sequences using the Oxford Nanopore MinION sequencer. This protocol not only facilitates the generation of accurate genome sequences for various enterovirus strains but also allows for the differentiation of co-infections from viral isolates. In addition, the method can generate polyprotein sequences as well as peptide sequences of the upstream ORF (uORF) whose expression can impact virus infection. Through the analysis of complete enterovirus genomes, this study successfully identified seven enterovirus A71 isolates obtained during the 2018 enterovirus outbreak in Malaysia and Taiwan as recombinants between enterovirus A71 and coxsackievirus A2. Furthermore, our study has made a significant discovery by establishing a strong correlation between uORF trees and the epidemics of EVA71. This finding highlights the potential of uORF sequences as valuable indicators for monitoring and understanding the spread of EVA71 infections. We also identified notable amino acid changes in the transmembrane domain of the uORF protein within a newly identified lineage. These findings provide crucial insights into the molecular characteristics and evolutionary dynamics of EVA71, offering valuable information for future research and intervention strategies. IMPORTANCE By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.
  3. Tan XH, Chong WL, Lee VS, Abdullah S, Jasni K, Suarni SQ, et al.
    Vaccines (Basel), 2023 Aug 14;11(8).
    PMID: 37631931 DOI: 10.3390/vaccines11081363
    Hand, foot and mouth disease (HFMD) is a childhood disease caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Capsid loops are important epitopes for EV-A71 and CV-A16. Seven chimeric EV-A71 (ChiE71) involving VP1 BC (45.5% similarity), DE, EF, GH and HI loops, VP2 EF loop and VP3 GH loop (91.3% similarity) were substituted with corresponding CV-A16 loops. Only ChiE71-1-BC, ChiE71-1-EF, ChiE71-1-GH and ChiE71-3-GH were viable. EV-A71 and CV-A16 antiserum neutralized ChiE71-1-BC and ChiE71-1-EF. Mice immunized with inactivated ChiE71 elicited high IgG, IFN-γ, IL-2, IL-4 and IL-10. Neonatal mice receiving passive transfer of WT EV-A71, ChiE71-1-EF and ChiE71-1-BC immune sera had 100%, 80.0% and no survival, respectively, against lethal challenges with EV-A71, suggesting that the substituted CV-A16 loops disrupted EV-A71 immunogenicity. Passive transfer of CV-A16, ChiE71-1-EF and ChiE71-1-BC immune sera provided 40.0%, 20.0% and 42.9% survival, respectively, against CV-A16. One-day-old neonatal mice immunized with WT EV-A71, ChiE71-1-BC, ChiE71-1-EF and CV-A16 achieved 62.5%, 60.0%, 57.1%, and no survival, respectively, after the EV-A71 challenge. Active immunization using CV-A16 provided full protection while WT EV-A71, ChiE71-1-BC and ChiE71-1-EF immunization showed partial cross-protection in CV-A16 lethal challenge with survival rates of 50.0%, 20.0% and 40%, respectively. Disruption of a capsid loop could affect virus immunogenicity, and future vaccine design should include conservation of the enterovirus capsid loops.
  4. Ooi MH, Wong SC, Mohan A, Podin Y, Perera D, Clear D, et al.
    BMC Infect Dis, 2009 Jan 19;9:3.
    PMID: 19152683 DOI: 10.1186/1471-2334-9-3
    BACKGROUND: Human enterovirus 71 (HEV71) can cause Hand, foot, and mouth disease (HFMD) with neurological complications, which may rapidly progress to fulminant cardiorespiratory failure, and death. Early recognition of children at risk is the key to reduce acute mortality and morbidity.

    METHODS: We examined data collected through a prospective clinical study of HFMD conducted between 2000 and 2006 that included 3 distinct outbreaks of HEV71 to identify risk factors associated with neurological involvement in children with HFMD.

    RESULTS: Total duration of fever >or= 3 days, peak temperature >or= 38.5 degrees C and history of lethargy were identified as independent risk factors for neurological involvement (evident by CSF pleocytosis) in the analysis of 725 children admitted during the first phase of the study. When they were validated in the second phase of the study, two or more (>or= 2) risk factors were present in 162 (65%) of 250 children with CSF pleocytosis compared with 56 (30%) of 186 children with no CSF pleocytosis (OR 4.27, 95% CI2.79-6.56, p < 0.0001). The usefulness of the three risk factors in identifying children with CSF pleocytosis on hospital admission during the second phase of the study was also tested. Peak temperature >or= 38.5 degrees C and history of lethargy had the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 28%(48/174), 89%(125/140), 76%(48/63) and 50%(125/251), respectively in predicting CSF pleocytosis in children that were seen within the first 2 days of febrile illness. For those presented on the 3rd or later day of febrile illness, the sensitivity, specificity, PPV and NPV of >or= 2 risk factors predictive of CSF pleocytosis were 75%(57/76), 59%(27/46), 75%(57/76) and 59%(27/46), respectively.

    CONCLUSION: Three readily elicited clinical risk factors were identified to help detect children at risk of neurological involvement. These risk factors may serve as a guide to clinicians to decide the need for hospitalization and further investigation, including cerebrospinal fluid examination, and close monitoring for disease progression in children with HFMD.

  5. Ooi MH, Wong SC, Podin Y, Akin W, del Sel S, Mohan A, et al.
    Clin Infect Dis, 2007 Mar 01;44(5):646-56.
    PMID: 17278054
    BACKGROUND: Human enterovirus (HEV)-71 causes large outbreaks of hand-foot-and-mouth disease with central nervous system (CNS) complications, but the role of HEV-71 genogroups or dual infection with other viruses in causing severe disease is unclear.

    METHODS: We prospectively studied children with suspected HEV-71 (i.e., hand-foot-and-mouth disease, CNS disease, or both) over 3.5 years, using detailed virological investigation and genogroup analysis of all isolates.

    RESULTS: Seven hundred seventy-three children were recruited, 277 of whom were infected with HEV-71, including 28 who were coinfected with other viruses. Risk factors for CNS disease in HEV-71 included young age, fever, vomiting, mouth ulcers, breathlessness, cold limbs, and poor urine output. Genogroup analysis for the HEV-71-infected patients revealed that 168 were infected with genogroup B4, 68 with C1, and 41 with a newly emerged genogroup, B5. Children with HEV-71 genogroup B4 were less likely to have CNS complications than those with other genogroups (26 [15%] of 168 vs. 30 [28%] of 109; odds ratio [OR], 0.48; 95% confidence interval [CI], 0.26-0.91; P=.0223) and less likely to be part of a family cluster (12 [7%] of 168 vs. 29 [27%] of 109; OR, 0.21; 95% CI, 0.10-0.46; P

  6. Simarmata D, Ng DC, Kam YW, Lee B, Sum MS, Her Z, et al.
    Sci Rep, 2016 05 16;6:26097.
    PMID: 27180811 DOI: 10.1038/srep26097
    Chikungunya fever (CHIKF) is a global infectious disease which can affect a wide range of age groups. The pathological and immunological response upon Chikungunya virus (CHIKV) infection have been reported over the last few years. However, the clinical profile and immune response upon CHIKV infection in children remain largely unknown. In this study, we analyzed the clinical and immunological response, focusing on the cytokine/chemokine profile in a CHIKV-infected pediatric cohort from Sarawak, Malaysia. Unique immune mediators triggered upon CHIKV infection were identified through meta-analysis of the immune signatures between this pediatric group and cohorts from previous outbreaks. The data generated from this study revealed that a broad spectrum of cytokines/chemokines is up-regulated in a sub-group of virus-infected children stratified according to their viremic status during hospitalization. Furthermore, different immune mediator profiles (the levels of pro-inflammatory cytokines, chemokines and growth and other factors) were observed between children and adults. This study gives an important insight to understand the immune response of CHIKV infection in children and would aid in the development of better prognostics and clinical management for children.
  7. Ooi MH, Wong SC, Clear D, Perera D, Krishnan S, Preston T, et al.
    Clin Infect Dis, 2003 Mar 1;36(5):550-9.
    PMID: 12594634
    We report the virological and clinical features of 8 children who presented with adenovirus-associated acute flaccid paralysis (AFP) during an epidemic of enterovirus type 71 (EV71)-associated hand-foot-and-mouth disease (HFMD) in Sarawak, Malaysia, in 1997. Neutralization tests and phylogenetic analysis revealed adenovirus type 21 (Ad21), although DNA restriction digests suggested that this virus was different from the prototype Ad21. Four children had upper-limb monoparesis, 2 had lower-limb monoparesis (one of whom had changes in the anterior spinal cord noted on magnetic resonance imaging), and 2 had flaccid paraparesis. At follow-up, 4 children were noted to have made full recoveries and 3 had residual flaccid weakness and wasting. Neurophysiological investigation revealed a mixture of axonal and demyelinating features in motor and sensory nerves, with denervation. These findings suggest that Ad21 might cause AFP by anterior horn cell damage or neuropathy of the brachial or lumbosacral plexus. The occurrence of these unusual adenovirus infections during an outbreak of EV71-associated HFMD suggests that an interaction between the 2 viruses may have occurred.
  8. Amrun SN, Tan JJL, Rickett NY, Cox JA, Lee B, Griffiths MJ, et al.
    Sci Rep, 2020 03 02;10(1):3810.
    PMID: 32123257 DOI: 10.1038/s41598-020-60761-5
    Hand, foot and mouth disease (HFMD), caused by enterovirus A71 (EV-A71), presents mild to severe disease, and sometimes fatal neurological and respiratory manifestations. However, reasons for the severe pathogenesis remain undefined. To investigate this, infection and viral kinetics of EV-A71 isolates from clinical disease (mild, moderate and severe) from Sarawak, Malaysia, were characterised in human rhabdomyosarcoma (RD), neuroblastoma (SH-SY5Y) and peripheral blood mononuclear cells (PBMCs). High resolution transcriptomics was used to decipher EV-A71-host interactions in PBMCs. Ingenuity analyses revealed similar pathways triggered by all EV-A71 isolates, although the extent of activation varied. Importantly, several pathways were found to be specific to the severe isolate, including triggering receptor expressed on myeloid cells 1 (TREM-1) signalling. Depletion of TREM-1 in EV-A71-infected PBMCs with peptide LP17 resulted in decreased levels of pro-inflammatory genes for the moderate and severe isolates. Mechanistically, this is the first report describing the transcriptome profiles during EV-A71 infections in primary human cells, and the potential involvement of TREM-1 in the severe disease pathogenesis, thus providing new insights for future treatment targets.
  9. Chiu ML, Luo ST, Chen YY, Chung WY, Duong V, Dussart P, et al.
    Vaccine, 2020 01 03;38(1):1-9.
    PMID: 31679864 DOI: 10.1016/j.vaccine.2019.09.111
    Enteroviruses (EV), the major pathogens of hand, foot, and mouth disease (HFMD) and herpangina, affect millions of children each year. Most human enteroviruses cause self-limited infections except polioviruses, enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and several echoviruses (Echo) and coxsackieviruses (CV). Especially, EV-A71 has repeatedly caused large-scale outbreaks in the Asia-Pacific region since 1997. Some Asian countries have experienced cyclical outbreaks of severe EV-A71 infections and initiated development of EV-A71 vaccines. Five EV-A71 vaccine candidates have been clinically evaluated and three of them were approved for marketing in China. However, none of the China-approved products seek marketing approval in other countries. This situation supports a role for collaboration among Asian countries to facilitate clinical trials and licensure of EV-A71 vaccines. Additionally, enterovirus D68 outbreaks have been reported in the US and Taiwan currently and caused severe complications and deaths. Hence, an Asia-Pacific Network for Enterovirus Surveillance (APNES) has been established to estimate disease burden, understand virus evolution, and facilitate vaccine development through harmonizing laboratory diagnosis and data collection. Founded in 2017, the APNES is comprised of internationally recognized experts in the field of enterovirus in Asian countries working to raise awareness of this potentially fatal and debilitating disease. This article demonstrated the summaries of the first expert meeting, 2017 International Workshop on Enterovirus Surveillance and Vaccine Development, held by APNES in Taipei, Taiwan, March 2017.
  10. Piyasena TBH, Setoh YX, Hobson-Peters J, Prow NA, Bielefeldt-Ohmann H, Khromykh AA, et al.
    Vector Borne Zoonotic Dis, 2017 12;17(12):825-835.
    PMID: 29083957 DOI: 10.1089/vbz.2017.2172
    In Australia, infection of horses with the West Nile virus (WNV) or Murray Valley encephalitis virus (MVEV) occasionally results in severe neurological disease that cannot be clinically differentiated. Confirmatory serological tests to detect antibody specific for MVEV or WNV in horses are often hampered by cross-reactive antibodies induced to conserved epitopes on the envelope (E) protein. This study utilized bacterially expressed recombinant antigens derived from domain III of the E protein (rE-DIII) of MVEV and WNV, respectively, to determine whether these subunit antigens provided specific diagnostic markers of infection with these two viruses. When a panel of 130 serum samples, from horses with known flavivirus infection status, was tested in enzyme-linked immunosorbent assay (ELISA) using rE-DIII antigens, a differential diagnosis of MVEV or WNV was achieved for most samples. Time-point samples from horses exposed to flavivirus infection during the 2011 outbreak of equine encephalitis in south-eastern Australia also indicated that the rE-DIII antigens were capable of detecting and differentiating MVEV and WNV infection in convalescent sera with similar sensitivity and specificity to virus neutralization tests and blocking ELISAs. Overall, these results indicate that the rE-DIII is a suitable antigen for use in rapid immunoassays for confirming MVEV and WNV infections in horses in the Australian context and warrant further assessment on sensitive, high-throughput serological platforms such as multiplex immune assays.
  11. Wang X, Xiu L, Binder RA, Toh TH, Lee JS, Ting J, et al.
    One Health, 2021 Dec;13:100274.
    PMID: 34124332 DOI: 10.1016/j.onehlt.2021.100274
    We examined a collection of 386 animal, 451 human, and 109 archived bioaerosol samples with a new pan-species coronavirus molecular assay. Thirty-eight (4.02%) of 946 specimens yielded evidence of human or animal coronaviruses. Our findings demonstrate the utility of employing the pan-CoV RT-PCR assay in detecting varied coronavirus among human, animal, and environmental specimens. This RT-PCR assay might be employed as a screening diagnostic for early detection of coronaviruses incursions or prepandemic coronavirus emergence in animal or human populations.
  12. Van Tu P, Thao NTT, Perera D, Truong KH, Tien NTK, Thuong TC, et al.
    Emerg Infect Dis, 2007 Nov;13(11):1733-41.
    PMID: 18217559 DOI: 10.3201/eid1311.070632
    During 2005, 764 children were brought to a large children's hospital in Ho Chi Minh City, Vietnam, with a diagnosis of hand, foot, and mouth disease. All enrolled children had specimens (vesicle fluid, stool, throat swab) collected for enterovirus isolation by cell culture. An enterovirus was isolated from 411 (53.8%) of the specimens: 173 (42.1%) isolates were identified as human enterovirus 71 (HEV71) and 214 (52.1%) as coxsackievirus A16. Of the identified HEV71 infections, 51 (29.5%) were complicated by acute neurologic disease and 3 (1.7%) were fatal. HEV71 was isolated throughout the year, with a period of higher prevalence in October-November. Phylogenetic analysis of 23 HEV71 isolates showed that during the first half of 2005, viruses belonging to 3 subgenogroups, C1, C4, and a previously undescribed subgenogroup, C5, cocirculated in southern Vietnam. In the second half of the year, viruses belonging to subgenogroup C5 predominated during a period of higher HEV71 activity.
  13. Podin Y, Gias EL, Ong F, Leong YW, Yee SF, Yusof MA, et al.
    BMC Public Health, 2006 Jul 07;6:180.
    PMID: 16827926
    BACKGROUND: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region.

    METHODS: We established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs.

    RESULTS: During this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information.

    CONCLUSION: Knowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease.

  14. Sam IC, Chong YM, Abdullah A, Fu JYL, Hasan MS, Jamaluddin FH, et al.
    J Med Virol, 2021 Nov 10.
    PMID: 34757638 DOI: 10.1002/jmv.27441
    Malaysia has experienced three waves of coronavirus disease 2019 (COVID-19) as of March 31, 2021. We studied the associated molecular epidemiology and SARS-CoV-2 seroprevalence during the third wave. We obtained 60 whole-genome SARS-CoV-2 sequences between October 2020 and January 2021 in Kuala Lumpur/Selangor and analyzed 989 available Malaysian sequences. We tested 653 residual serum samples collected between December 2020 to April 2021 for anti-SARS-CoV-2 total antibodies, as a proxy for population immunity. The first wave (January 2020) comprised sporadic imported cases from China of early Pango lineages A and B. The second wave (March-June 2020) was associated with lineage B.6. The ongoing third wave (from September 2020) was propagated by a state election in Sabah. It is due to lineage B.1.524 viruses containing spike mutations D614G and A701V. Lineages B.1.459, B.1.470, and B.1.466.2 were likely imported from the region and confined to Sarawak state. Direct age-standardized seroprevalence in Kuala Lumpur/Selangor was 3.0%. The second and third waves were driven by super-spreading events and different circulating lineages. Malaysia is highly susceptible to further waves, especially as alpha (B.1.1.7) and beta (B.1.351) variants of concern were first detected in December 2020/January 2021. Increased genomic surveillance is critical.
  15. Guzman H, Contreras-Gutierrez MA, Travassos da Rosa APA, Nunes MRT, Cardoso JF, Popov VL, et al.
    Am J Trop Med Hyg, 2018 02;98(2):410-419.
    PMID: 29016330 DOI: 10.4269/ajtmh.17-0350
    Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.
  16. Labadin J, Hong BH, Tiong WK, Gill BS, Perera D, Rigit ARH, et al.
    Multimed Tools Appl, 2023;82(11):17415-17436.
    PMID: 36404933 DOI: 10.1007/s11042-022-14120-3
    Traditionally, dengue is controlled by fogging, and the prime location for the control measure is at the patient's residence. However, when Malaysia was hit by the first wave of the Coronavirus disease (COVID-19), and the government-imposed movement control order, dengue cases have decreased by more than 30% from the previous year. This implies that residential areas may not be the prime locations for dengue-infected mosquitoes. The existing early warning system was focused on temporal prediction wherein the lack of consideration for spatial component at the microlevel and human mobility were not considered. Thus, we developed MozzHub, which is a web-based application system based on the bipartite network-based dengue model that is focused on identifying the source of dengue infection at a small spatial level (400 m) by integrating human mobility and environmental predictors. The model was earlier developed and validated; therefore, this study presents the design and implementation of the MozzHub system and the results of a preliminary pilot test and user acceptance of MozzHub in six district health offices in Malaysia. It was found that the MozzHub system is well received by the sample of end-users as it was demonstrated as a useful (77.4%), easy-to-operate system (80.6%), and has achieved adequate client satisfaction for its use (74.2%).
  17. Ooi MH, Solomon T, Podin Y, Mohan A, Akin W, Yusuf MA, et al.
    J Clin Microbiol, 2007 Jun;45(6):1858-66.
    PMID: 17446325
    Human enterovirus 71 and coxsackievirus A16 are important causes of hand-foot-and-mouth disease (HFMD). Like other enteroviruses, they can be isolated from a range of sterile and nonsterile sites, but which clinical sample, or combination of samples, is the most useful for laboratory diagnosis of HFMD is not clear. We attempted virus culture for 2,916 samples from 628 of 725 children with HFMD studied over a 3 1/2-year period, which included two large outbreaks. Overall, throat swabs were the single most useful specimen, being positive for any enterovirus for 288 (49%) of 592 patients with a full set of samples. Vesicle swabs were positive for 169 (48%) of 333 patients with vesicles, the yield being greater if two or more vesicles were swabbed. The combination of throat plus vesicle swabs enabled the identification of virus for 224 (67%) of the 333 patients with vesicles; for this patient group, just 27 (8%) extra patients were diagnosed when rectal and ulcer swabs were added. Of 259 patients without vesicles, use of the combination of throat plus rectal swab identified virus for 138 (53%). For 60 patients, virus was isolated from both vesicle and rectal swabs, but for 12 (20%) of these, the isolates differed. Such discordance occurred for just 11 (10%) of 112 patients with virus isolated from vesicle and throat swabs. During large HFMD outbreaks, we suggest collecting swabs from the throat plus one other site: vesicles, if these are present (at least two should be swabbed), or the rectum if there are no vesicles. Vesicle swabs give a high diagnostic yield, with the added advantage of being from a sterile site.
  18. 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.

  19. 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.
  20. Azami NAM, Perera D, Thayan R, AbuBakar S, Sam IC, Salleh MZ, et al.
    Int J Infect Dis, 2022 Dec;125:216-226.
    PMID: 36336246 DOI: 10.1016/j.ijid.2022.10.044
    OBJECTIVES: This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program.

    METHODS: COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database.

    RESULTS: From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country.

    CONCLUSION: The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness.

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