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  1. Lim YZ, Teoh BT, Sam SS, Azizan NS, Khor CS, Nor'e SS, et al.
    Trop Biomed, 2023 Sep 01;40(3):313-319.
    PMID: 37897164 DOI: 10.47665/tb.40.3.007
    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus with widespread distribution across the globe. Since 2016, CHIKV re-emerged in several countries including Indian subcontinent and Southeast Asia. A proper diagnostic tool for early diagnosis of CHIKV infection is crucial to facilitate patient management and control virus transmission at the earliest stage of outbreak. Therefore, a TaqMan minor groove binder (MGB) probe-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to detect and quantify the CHIKV. The primers and probe were designed based on a conserved genomic region of 730 global CHIKV sequences that is located between nsP1 and nsP2 genes. The nucleotide mismatches of primers and probe with 730 global CHIKV sequences and 13 alphaviruses were then analysed in silico. In this study, the last 5 nucleotides at 3' end of primers and 5' end of probe were considered to be the critical regions for priming. In silico analysis revealed that the critical regions of primers and probe were at least 99.6% matched with the 730 global CHIKV sequences. Besides, the primers and probe showed at least 5/20 (25.0%) and 4/17 (23.5%) nucleotide mismatches with 13 alphaviruses respectively. The amplification efficiency of qRT-PCR assay was 100.59% (95% CI= 93.06, 109.33) with a R2 score of 0.957. Its limit of detection (LOD) at 95% probability level was 16.6 CHIKV RNA copies (95% CI= 12.9, 28.9). The qRT-PCR assay was specific to CHIKV without cross-reacting with all dengue virus serotypes, Getah virus, Tembusu virus and Zika virus. The diagnostic results of qRT-PCR assay were perfectly agreed (k=1.000, p=0.003) with a commercial trioplex assay, with sensitivity of 100% (95% CI= 61, 100) and specificity of 100% (95% CI= 44, 100). Overall, the developed qRT-PCR assay is ideal for rapid, sensitive and specific detection as well as quantification of CHIKV.
  2. Tan KK, Azizan NS, Yaacob CN, Che Mat Seri NAA, Samsudin NI, Teoh BT, et al.
    BMC Infect Dis, 2018 04 11;18(1):169.
    PMID: 29642856 DOI: 10.1186/s12879-018-3065-1
    BACKGROUND: A method for rapid detection of dengue virus using the reverse-transcription recombinase polymerase amplification (RT-RPA) was recently developed, evaluated and made ready for deployment. However, reliance solely on the evaluation performed by experienced researchers in a well-structured and well-equipped reference laboratory may overlook the potential intrinsic problems that may arise during deployment of the assay into new application sites, especially for users unfamiliar with the test. Appropriate assessment of this newly developed assay by users who are unfamiliar with the assay is, therefore, vital.

    METHODS: An operational utility test to elucidate the efficiency and effectiveness of the dengue RT-RPA assay was conducted among a group of researchers new to the assay. Nineteen volunteer researchers with different research experience were recruited. The participants performed the RT-RPA assay and interpreted the test results according to the protocol provided. Deviation from the protocol was identified and tabulated by trained facilitators. Post-test questionnaires were conducted to determine the user satisfaction and acceptability of the dengue RT-RPA assay.

    RESULTS: All the participants completed the test and successfully interpreted the results according to the provided instructions, regardless of their research experience. Of the 19 participants, three (15.8%) performed the assay with no deviations and 16 (84.2%) performed the assay with only 1 to 5 deviations. The number of deviations from protocol, however, was not correlated with the user laboratory experience. The accuracy of the results was also not affected by user laboratory experience. The concordance of the assay results against that of the expected was at 89.3%. The user satisfaction towards the RT-RPA protocol and interpretation of results was 90% and 100%, respectively.

    CONCLUSIONS: The dengue RT-RPA assay can be successfully performed by simply following the provided written instructions. Deviations from the written protocols did not adversely affect the outcome of the assay. These suggest that the RT-RPA assay is indeed a simple, robust and efficient laboratory method for detection of dengue virus. Furthermore, high new user acceptance of the RT-RPA assay suggests that this assay could be successfully deployed into new laboratories where RT-RPA was not previously performed.

  3. Abd-Jamil J, Azizan NS, Che-Mat-Seri NA, Yaacob CN, Samsudin NI, Mahfodz NH, et al.
    J Med Virol, 2021 08;93(8):4714-4719.
    PMID: 33421159 DOI: 10.1002/jmv.26790
    Early diagnosis of dengue is important to ensure proper management of patients and effective implementation of control measures. The present study was undertaken to determine the outcome of the implementation of dengue NS1-antigen (Ag) rapid diagnostic test (RDT) in the confirmation of dengue at the first patient hospital visit at the University Malaya Medical Centre. A total of 1036 and 1097 sera from the year 2008 and 2015 were used, representing samples from before and after dengue NS1-Ag RDT was implemented as routine diagnostic at the hospital. Results showed that similar dengue confirmation percentage (56%) was made in 2008 and 2015, regardless of the main laboratory diagnostic method used. Confirmation of dengue, however, increased to 68% and 73% when dengue NS1-Ag test or dengue immunoglobulin M-capture enzyme-linked immunosorbent assay was used as the second test for the 2008 and 2015 samples, respectively. Detection of dengue virus (DENV) using multiplex reverse transcription-polymerase chain reaction (RT-PCR) showed that DENV-1 was the highest in circulation in 2008 and that both DENV-1 and DENV-2 were dominant in 2015. In summary, the present study demonstrated that the introduction and use of the dengue NS1-Ag RDT did not change or compromise confirmation of dengue, highlighting the advantage of using the method. With the reducing cost of molecular detection tools, DENV detection using RT-PCR remains a viable option for further confirmation of dengue in hospital settings.
  4. Mohd Ngesom AM, Ahmad Razi A, Azizan NS, Wasi Ahmad N, Md Lasim A, Liang Y, et al.
    Parasit Vectors, 2021 Aug 18;14(1):413.
    PMID: 34407881 DOI: 10.1186/s13071-021-04918-9
    BACKGROUND: Dengue is a significant public health issue that is caused by Aedes spp. mosquitoes. The current vector control methods are unable to effectively reduce Aedes populations and thus fail to decrease dengue transmission. Hence, there is an urgent need for new tools and strategies to reduce dengue transmission in a wide range of settings. In this study, the Mosquito Home System (MHS) and Mosquito Home Aqua (MHAQ) formulations were assessed as commercial autodissemination traps in laboratory and small-scale field trials.

    METHOD: Multiple series of laboratory and small-scale field trials were performed to assess the efficacy of MHS and MHAQ exposed to Ae. aegypti. In the laboratory trials, various parameters such as fecundity, fertility, wing size, oviposition preferences, residual effects, and MHAQ transference to other containers through controlled experiments were tested. For small-scale field trials, the efficacy of the MHS and MHAQ approaches was determined to ascertain whether wild mosquitoes could transfer the MHAQ formulation from MHS stations to ovitraps.

    RESULTS: The data revealed that Ae. aegypti was highly susceptible to low concentrations of MHAQ formulations and had a residual effect of up to 3 months, with MHAQ exposure affecting fecundity, fertility, and mosquito wing size. In the oviposition studies, gravid females strongly preferred the hay infusion compared to tap water and MHAQ during egg-laying in the laboratory. Nevertheless, the use of commercial MHAQ by MHS was highly attractive in field settings compared to conventional ovitraps among local Aedes spp. mosquitoes. In addition, MHAQ horizontal transfer activities in the laboratory and small-scale field trials were demonstrated through larval bioassays. These findings demonstrated the potential of MHAQ to be transferred to new containers in each study site.

    CONCLUSION: This study provided proof of principle for the autodissemination of MHAQ. Through further refinement, this technique and device could become an effective oviposition trap and offer an alternative preventive tool for vector control management.

  5. Tan KK, Tiong V, Tan JY, Wong JE, Teoh BT, Abd-Jamil J, et al.
    Trop Biomed, 2021 Sep 01;38(3):283-288.
    PMID: 34362871 DOI: 10.47665/tb.38.3.069
    Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.
  6. Loong SK, Teoh BT, Johari J, Khor CS, Abd-Jamil J, Nor'e SS, et al.
    Case Rep Infect Dis, 2017;2017:2578082.
    PMID: 28331641 DOI: 10.1155/2017/2578082
    Bacillus anthracis is a bacterial pathogen of major concern. The spores of this bacteria can survive harsh environmental conditions for extended periods and are well recognized as a potential bioterror weapon with significant implications. Accurate and timely identification of this Bacillus species in the diagnostic laboratory is essential for disease and public health management. Biosafety Level 3 measures and ciprofloxacin treatment were instituted when B. anthracis was suspected from a patient with gangrenous foot. 16S rDNA sequencing was performed to accurately identify the suspected bacterium, due to the superiority of this method to accurately identify clinically isolated bacteria. B. megaterium was identified as the causative agent and the organism was subsequently treated as a Biosafety Level 2 pathogen.
  7. Tan KK, Zulkifle NI, Abd-Jamil J, Sulaiman S, Yaacob CN, Azizan NS, et al.
    Infect Genet Evol, 2017 Oct;54:271-275.
    PMID: 28698156 DOI: 10.1016/j.meegid.2017.07.008
    Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006-2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%-100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions.
  8. Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.
    Trop Biomed, 2022 Dec 01;39(4):518-523.
    PMID: 36602210 DOI: 10.47665/tb.39.4.005
    Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.
  9. Tan KK, Tan JY, Wong JE, Teoh BT, Tiong V, Abd-Jamil J, et al.
    Sci Rep, 2021 11 11;11(1):22105.
    PMID: 34764315 DOI: 10.1038/s41598-021-01223-4
    The COVID-19 pandemic first emerged in Malaysia in Jan 2020. As of 12th Sept 2021, 1,979,698 COVID-19 cases that occurred over three major epidemic waves were confirmed. The virus contributing to the three epidemic waves has not been well-studied. We sequenced the genome of 22 SARS-CoV-2 strains detected in Malaysia during the second and the ongoing third wave of the COVID-19 epidemic. Detailed phylogenetic and genetic variation analyses of the SARS-CoV-2 isolate genomes were performed using these newly determined sequences and all other available sequences. Results from the analyses suggested multiple independent introductions of SARS-CoV-2 into Malaysia. A new B.1.524(G) lineage with S-D614G mutation was detected in Sabah, East Malaysia and Selangor, Peninsular Malaysia on 7th October 2020 and 14th October 2020, respectively. This new B.1.524(G) group was not the direct descendant of any of the previously detected lineages. The new B.1.524(G) carried a set of genetic variations, including A701V (position variant frequency = 0.0007) in Spike protein and a novel G114T mutation at the 5'UTR. The biological importance of the specific mutations remained unknown. The sequential appearance of the mutations, however, suggests that the spread of the new B.1.524(G) lineages likely begun in Sabah and then spread to Selangor. The findings presented here support the importance of SARS-CoV-2 full genome sequencing as a tool to establish an epidemiological link between cases or clusters of COVID-19 worldwide.
  10. Khor CS, Teoh BT, Sam SS, Khoo HY, Azizan NS, CheMatSeri A, et al.
    J Infect Dev Ctries, 2023 Jan 31;17(1):118-124.
    PMID: 36795935 DOI: 10.3855/jidc.16613
    INTRODUCTION: Chikungunya fever is a mosquito-borne viral disease that usually presents with prominent arthralgia. An outbreak of chikungunya fever was reported in Tanjung Sepat, Malaysia in 2019. The outbreak was limited in size with a low number of cases being reported. The present study sought to determine the possible variables that could have affected the transmission of the infection.

    METHODOLOGY: A cross-sectional study involving 149 healthy adult volunteers from Tanjung Sepat was performed soon after the outbreak had subsided. All the participants donated blood samples and completed the questionnaires. Laboratory detection of anti-CHIKV IgM and IgG antibodies was performed using enzyme-linked immunoassays (ELISA). Risk factors associated with chikungunya seropositivity were determined using logistic regression.

    RESULTS: The majority (72.5%, n = 108) of the study participants tested positive for CHIKV antibodies. Only 8.3% (n = 9) of the participants out of all the seropositive volunteers had an asymptomatic infection. Participants who resided with a febrile (p < 0.05, Exp(B) = 2.2, confidence interval [CI] 1.3-3.6) or a CHIKV-diagnosed person (p < 0.05, Exp(B) = 2.1, CI 1.2-3.6) in the same household were found likely to be tested positive for CHIKV antibodies.

    CONCLUSIONS: Findings from the study support that asymptomatic CHIKV infections and indoor transmission occurred during the outbreak. Hence, widespread community testing and indoor use of mosquito repellent are among the possible measures that can be implemented to reduce CHIKV transmission during an outbreak.

  11. Johari J, Hontz RD, Pike BL, Husain T, Chong CK, Rusli N, et al.
    BMJ Open, 2021 08 26;11(8):e050901.
    PMID: 34446498 DOI: 10.1136/bmjopen-2021-050901
    INTRODUCTION: Middle East respiratory syndrome (MERS) is a viral respiratory infection caused by the MERS-CoV. MERS was first reported in the Kingdom of Saudi Arabia in 2012. Every year, the Hajj pilgrimage to Mecca attracts more than two million pilgrims from 184 countries, making it one of the largest annual religious mass gatherings (MGs) worldwide. MGs in confined areas with a high number of pilgrims' movements worldwide continues to elicit significant global public health concerns. MERCURIAL was designed by adopting a seroconversion surveillance approach to provide multiyear evidence of MG-associated MERS-CoV seroconversion among the Malaysian Hajj pilgrims.

    METHODS AND ANALYSIS: MERCURIAL is an ongoing multiyear prospective cohort study. Every year, for the next 5 years, a cohort of 1000 Hajj pilgrims was enrolled beginning in the 2016 Hajj pilgrimage season. Pre-Hajj and post-Hajj serum samples were obtained and serologically analysed for evidence of MERS-CoV seroconversion. Sociodemographic data, underlying medical conditions, symptoms experienced during Hajj pilgrimage, and exposure to camel and untreated camel products were recorded using structured pre-Hajj and post-Hajj questionnaires. The possible risk factors associated with the seroconversion data were analysed using univariate and multivariate logistic regression. The primary outcome of this study is to better enhance our understanding of the potential threat of MERS-CoV spreading through MG beyond the Middle East.

    ETHICS AND DISSEMINATION: This study has obtained ethical approval from the Medical Research and Ethics Committee (MREC), Ministry of Health Malaysia. Results from the study will be submitted for publication in peer-reviewed journals and presented in conferences and scientific meetings.

    TRIAL REGISTRATION NUMBER: NMRR-15-1640-25391.

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