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Fulltext Multiyear prospective cohort study to evaluate the risk potential of MERS-CoV infection among Malaysian Hajj pilgrims (MERCURIAL): a study protocol

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.
Fulltext The Risk of Transfusion-Transmitted Hepatitis E Virus: Evidence from Seroprevalence Screening of Blood Donations

Wong LP, Lee HY, Khor CS, Abdul-Jamil J, Alias H, Abu-Amin N, et al.

Indian J Hematol Blood Transfus, 2021 Apr 16.
PMID: 33879981 DOI: 10.1007/s12288-021-01428-7

Throughout the world, there has been growing concern over the risk of hepatitis E virus (HEV) transmission via blood transfusion. The present study screened blood donor samples for anti-HEV immunoglobulin M (IgM) and immunoglobulin G (IgG). The prevalence of HEV infection was assessed on a total of 1,003 archived serum samples obtained from the National Blood Centre, Malaysia. The samples were collected from healthy blood donor from Klang Valley between 2017 and 2018. All samples were tested for IgM and IgG antibodies to HEV using enzyme-linked immunosorbent assays (ELISA). HEV-specific IgG antibodies were detected in 31/1003 (3.1%; 95% confidence interval [CI] 2.1%-4.4%) and IgM in 9/1003 (0.9%; 95% CI 0.4%-1.7%) samples. In bivariate analysis, there was no significant difference in the prevalence of anti-HEV IgG with respect to gender and district of origin. Although not statistically significant, males had higher odds of having anti-HEV IgG than females (odds ratio [OR] = 2.86; 95% CI 0.95-8.64). All anti-HEV IgG positive individuals were people of Chinese descent. Anti-HEV IgG increased significantly with age, from 0.6% (95% CI 0.1%-2.6%) of 18-30-year-old donors to 7.4% (95% CI 2.7%-17.0%) of donors older than 50 years and was highest among non-professional workers (5.3%; 95% CI 2.5%-10.5%). Increasing age and a non-professional occupation remained significant predictors for anti-HEV IgG in the multivariable analysis. Screening of blood donations for HEV in Malaysia is important to safeguard the health of transfusion recipients. The higher rates of HEV infection in blood from older donors and donors who are non-professional workers may provide insights into targeted groups for blood screening.
Seroprevalence of Q Fever Among the Indigenous People (Orang Asli) of Peninsular Malaysia

Khor CS, Mohd-Rahim NF, Hassan H, Chandren JR, Nore SS, Johari J, et al.

Vector Borne Zoonotic Dis, 2018 03;18(3):131-137.
PMID: 29336685 DOI: 10.1089/vbz.2017.2153

Q fever is a disease caused by Coxiella burnetii. It is a disease of public health concern in many parts of the world. In this study, we described the seroprevalence of Q fever among selected populations of Orang Asli (OA), indigenous people, many of whom live within the forest fringe areas of Peninsular Malaysia. Serum samples were obtained from 887 OA participants from selected villages. Samples were analyzed for the presence of IgG antibodies reactive against C. burnetii by enzyme-linked immunosorbent assay. Statistical methods were used to identify possible associations between seropositivity for C. burnetii and a number of demographic variables obtained from the questionnaires. In total, 9.6% (n = 85/887) of the serum samples were reactive to C. burnetii. Statistical results suggest that elderly male OA residing in OA village, Bukit Payung, were most likely to be tested seropositive for C. burnetii. This study suggests that OA are at a significant risk of contracting C. burnetii infection, and both demographic and geographic factors are important contributors to this risk. Further prospective studies are needed to establish the true burden of C. burnetii infection within the indigenous population as well as within Peninsular Malaysia as a whole.
Fulltext Seroprevalence of Borrelia burgdorferi among the indigenous people (Orang Asli) of Peninsular Malaysia

Khor CS, Hassan H, Mohd-Rahim NF, Chandren JR, Nore SS, Johari J, et al.

J Infect Dev Ctries, 2019 05 31;13(5):449-454.
PMID: 32053515 DOI: 10.3855/jidc.11001

INTRODUCTION: Lyme disease has been well-described in the North America and European countries. However, information is still very limited in the developing countries including Malaysia. The Orang Asli (OA), the indigenous people of Peninsular Malaysia reside mostly in the forest and forest fringe areas abundant with the vector for Lyme disease. Here, we described the seroprevalence of Borellia burgdorferi (B. burgdorferi) among the OA and demographic variables that could be associated with seroprevalence.
METHODOLOGY: A total of 16 OA villages distributed across 8 states in Peninsular Malaysia participated in this study. Sera obtained from 904 OA volunteers were screened for anti-B. burgdorferi IgG antibodies. ELISA results obtained and demographic information collected were analysed to identify possible variables associated with seroprevalence.
RESULTS: A total of 73 (8.1%) OA tested positive for anti-B. burgdorferi IgG antibodies. Among all the variables examined, village of residence (p = 0.045) was the only significant predictor for seropositivity. High (> 10.0%) prevalence was associated with three OA villages. Those living in one particular village were 1.65 times more likely to be seropositive as compared to other OA villages. Age, gender, marital status, household size, level of education, monthly household income and occupation were not significant predictors for seropositivity.
CONCLUSION: Results of the present study support earlier findings that B. burgdorferi infection among Malaysians is currently under-recognized. Further studies will be needed at these locations to confirm the presence of Lyme disease among these populations.
Fulltext Emergence of B.1.524(G) SARS-CoV-2 in Malaysia during the third COVID-19 epidemic wave

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.
Fulltext Lactococcus lactis Strain Plasma Intake Suppresses the Incidence of Dengue Fever-like Symptoms in Healthy Malaysians: A Randomized, Double-Blind, Placebo-Controlled Trial

Khor CS, Tsuji R, Lee HY, Nor'e SS, Sahimin N, Azman AS, et al.

Nutrients, 2021 Dec 16;13(12).
PMID: 34960061 DOI: 10.3390/nu13124507

Dengue fever (DF) is a mosquito-borne disease still with no effective treatment or vaccine available. A randomized, placebo-controlled, double-blinded, parallel-group trial was undertaken to evaluate the efficacy of oral intake of Lactococcus lactis strain plasma (LC-Plasma) on the presentation and severity of DF-like symptoms among healthy volunteers. Study participants (320) were assigned into two groups, and consumed either placebo or LC-Plasma tablets (approximately 100 billion cells/day) for 8 weeks. The clinical symptoms of DF were self-recorded through questionnaires, and exposure to DENV was determined by serum antibody and/or DENV antigen tests. No significant differences between groups were observed for exposure to DENV, or the symptomatic ratio. Results obtained showed that participants from the LC-Plasma group reported a significant reduction in the cumulative incidence days of DF-like symptoms, which include fever (p < 0.001), muscle pain (p < 0.005), joint pain (p < 0.001), and pain behind the eyes (p < 0.001), compared to that of the placebo group. Subgroup analysis revealed a significantly (p < 0.05) reduced severity score in the LC-Plasma group when study sites were separately analyzed. Overall, our findings suggest that LC-Plasma supplementation reduces the cumulative days with DF-like symptoms, and the severity of the symptoms. Daily oral intake of LC-Plasma, hence, is shown to mitigate the DF-like symptoms.
Fulltext Epidemiological serosurvey of chikungunya fever post outbreak at Tanjung Sepat, Malaysia

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.
Fulltext MERS-CoV seroconversion amongst Malaysian Hajj pilgrims returning from the Middle East, 2016-2018: results from the MERCURIAL multiyear prospective cohort study

Johari J, Hontz RD, Pike BL, Husain T, Rusli N, Mohd-Zain R, et al.

Emerg Microbes Infect, 2023 Dec;12(1):2208678.
PMID: 37101375 DOI: 10.1080/22221751.2023.2208678

Prospective cohort study to investigate the potential exposure to the Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) following Hajj pilgrims is still very limited. Here, we report the antibody seroconversion study results obtained from successive three years cohort studies (2016-2018) involving the Malaysian Hajj pilgrims returning from the Middle East. A cohort study of Hajj pilgrims from Malaysia enrolled 2,863 participants from 2016-2018, all of whom consented to provide paired blood samples for both pre- and post-Hajj travel to the Middle East. ELISAs and micro-neutralization assays were performed to detect the presence of MERS-CoV IgG antibodies. Sociodemographic data, symptoms experienced during Hajj, and history of exposure to camels or camel products were recorded using structured pre- and post-Hajj questionnaires. A 4-fold increase in anti-MERS-CoV IgG between paired pre-Hajj and post-Hajj serum samples in twelve participants was observed. None of the twelve ELISA-positive sera had detectable levels of virus-neutralizing antibodies. All reportedly had mild symptoms of respiratory symptoms at a certain point during the pilgrimage, implying mild or asymptomatic infections. No association between post-Hajj serum positivity and a history of exposure to camels or camel products was obtained. Findings from the study suggest that serologic conversion to MERS-CoV occurred in at least 0.6% of the Hajj pilgrims returning from the Middle East. Since all the seroconvertants had mild to no symptoms during the sampling period, it highlights the likelihood of occurrence of only low infectivity spillover infections among the Hajj pilgrims.
Fulltext Seroprevalence of Zika Virus among Forest Fringe Communities in Peninsular Malaysia and Sabah: General Population-Based Study

Khoo HY, Lee HY, Khor CS, Tan KK, Bin Hassan MR, Wong CM, et al.

Am J Trop Med Hyg, 2022 Jul 25;107(3):560-8.
PMID: 35895331 DOI: 10.4269/ajtmh.21-0988

Zika virus (ZIKV) has had a history in Malaysia since its first isolation in 1966. However, it is believed that the immunity status among forest fringe communities has been underreported. We conducted cross-sectional surveillance of forest fringe communities from 10 Orang Asli villages and their peripheral communities in Perak, Pahang, and Sabah in Malaysia. A total of 706 samples were collected from 2019 to 2020 and screened for ZIKV exposure using an anti-ZIKV IgG ELISA kit. A neutralization assay against ZIKV was used to confirm the reactive samples. The seroprevalence results reported from the study of this population in Malaysia were 21.0% (n = 148, 95% CI, 0.183-0.273) after confirmation with a foci reduction neutralization test. The presence of neutralizing antibodies provides evidence that the studied forest fringe communities in Malaysia have been exposed to ZIKV. Multivariate analysis showed that those older than 44 years and those with an education below the university level had been exposed significantly to ZIKV. In addition, higher seropositivity rates to ZIKV were also reported among secondary school students from Bentong (Pahang) and residents from Segaliud (Sabah). No associations were identified between Zika seropositivity and gender, household size, house radius to the jungle, and income level. The presence of neutralizing antibodies against ZIKV among the study population might indicate that the causative pathogen had already circulated widely in forest fringe regions. Intervention for vector control, protection from mosquito bites, and awareness improvement should be encouraged in this population.
Fulltext A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages

Teoh BT, Chin KL, Samsudin NI, Loong SK, Sam SS, Tan KK, et al.

BMC Infect Dis, 2020 Dec 11;20(1):947.
PMID: 33308203 DOI: 10.1186/s12879-020-05585-4

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required.
METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.
RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P
Serological evidence of DENV, JEV, and ZIKV among the indigenous people (Orang Asli) of Peninsular Malaysia

Khor CS, Mohd-Rahim NF, Hassan H, Tan KK, Zainal N, Teoh BT, et al.

J Med Virol, 2020 08;92(8):956-962.
PMID: 31814135 DOI: 10.1002/jmv.25649

Dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV) are mosquito-borne flavivirus of medical importance in tropical countries such as Malaysia. However, much remains unknown regarding their prevalence among the underserved indigenous people (Orang Asli) living in communities in the forest fringe areas of Peninsular Malaysia. Information on the prevalence of diseases is necessary to elevate the effectiveness of disease control and preventive measures. This study aimed to determine the seroprevalence of the three major flaviviruses among the Orang Asli and investigate the association between demographic factors and seropositivities. Sampling activities were conducted in the Orang Asli villages to obtain serum samples and demographic data from consenting volunteers. The presence of DENV, JEV, and ZIKV immunoglobulin G (IgG) antibodies in the sera were examined using commercial enzyme-linked immunosorbent assay kits. A focus reduction neutralization assay was performed to measure virus-specific neutralizing antibodies. A total of 872 serum samples were obtained from the Orang Asli volunteers. Serological assay results revealed that DENV IgG, JEV IgG, and ZIKV IgG seropositivities among the Orang Asli were at 4.9%, 48.4%, and 13.2%, respectively. Neutralizing antibodies (FRNT50 ≥ 1:40) against JEV and ZIKV were found in 86.7% and 100.0%, respectively, out of the samples tested. Positive serology to all three viruses corresponded significantly to the age of the volunteers with increasing seropositivity in older volunteers. Findings from the study suggest that Orang Asli are at significant risk of contracting JEV and ZIKV infections despite the lack of active transmission of the viruses in the country.
Molecular detection of pathogens from ectoparasites recovered from peri-domestic animals, and the first description of a Candidatus Midichloria sp. from Haemaphysalis wellingtoni from rural communities in Malaysia

Khoo JJ, Husin NA, Lim FS, Oslan SNH, Mohd Azami SNI, To SW, et al.

Parasitol Int, 2021 Feb;80:102202.
PMID: 33038482 DOI: 10.1016/j.parint.2020.102202

Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans. Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing. Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested. The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.
Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

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.
Fulltext Penicillin-Susceptible, Oxidase-Negative, Nonhemolytic, Nonmotile Bacillus megaterium in Disguise of Bacillus anthracis

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.
Fulltext A quantitative reverse transcription-polymerase chain reaction for detection of Getah virus

Sam SS, Teoh BT, Chee CM, Mohamed-Romai-Noor NA, Abd-Jamil J, Loong SK, et al.

Sci Rep, 2018 12 05;8(1):17632.
PMID: 30518924 DOI: 10.1038/s41598-018-36043-6

Getah virus (GETV), a mosquito-borne alphavirus, is an emerging animal pathogen causing outbreaks among racehorses and pigs. Early detection of the GETV infection is essential for timely implementation of disease prevention and control interventions. Thus, a rapid and accurate nucleic acid detection method for GETV is highly needed. Here, two TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were developed. The qRT-PCR primers and TaqMan MGB probe were designed based on the conserved region of nsP1 and nsP2 genes of 23 GETV genome sequences retrieved from GenBank. Only the qRT-PCR assay using nsP2-specific primers and probe detected all two Malaysia GETV strains (MM2021 and B254) without cross-reacting with other closely related arboviruses. The qRT-PCR assay detected as few as 10 copies of GETV RNA, but its detection limit at the 95% probability level was 63.25 GETV genome copies (probit analysis, P ≤ 0.05). Further validation of the qRT-PCR assay using 16 spiked simulated clinical specimens showed 100% for both sensitivity and specificity. In conclusion, the qRT-PCR assay developed in this study is useful for rapid, sensitive and specific detection and quantification of GETV.
Fulltext Group IV Getah Virus in Culex Mosquitoes, Malaysia

Sam SS, Mohamed-Romai-Noor NA, Teoh BT, Hamim ZR, Ng HY, Abd-Jamil J, et al.

Emerg Infect Dis, 2022 Feb;28(2):475-477.
PMID: 35076371 DOI: 10.3201/eid2802.204887

A new Getah virus (GETV) strain, B254, was isolated from Culex fuscocephalus mosquitoes captured at Mount Ophir, Malaysia, in 2012. Phylogenetic analyses revealed that GETV B254 is distinct from the old Malaysia GETV MM2021 strain but closely related to group IV GETV from Russia (LEIV16275Mag), China (YN12031), and Thailand (GETV/SW/Thailand/2017).
Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus

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.
Coxiella Detection in Ticks from Wildlife and Livestock in Malaysia

Khoo JJ, Lim FS, Chen F, Phoon WH, Khor CS, Pike BL, et al.

Vector Borne Zoonotic Dis, 2016 12;16(12):744-751.
PMID: 27763821

Recent studies have shown that ticks harbor Coxiella-like bacteria, which are potentially tick-specific endosymbionts. We recently described the detection of Coxiella-like bacteria and possibly Coxiella burnetii in ticks found from rural areas in Malaysia. In the present study, we collected ticks, including Haemaphysalis bispinosa, Haemaphysalis hystricis, Dermacentor compactus, Dermacentor steini, and Amblyomma sp. from wildlife and domesticated goats from four different locations in Malaysia. Coxiella 16s rRNA genomic sequences were detected by PCR in 89% of ticks tested. Similarity analysis and phylogenetic analyses of the 16s rRNA and rpoB partial sequences were performed for 10 representative samples selected based on the tick species, sex, and location. The findings here suggested the presence of C. burnetii in two samples, each from D. steini and H. hystricis. The sequences of both samples clustered with published C. burnetii sequences. The remaining eight tick samples were shown to harbor 16s rRNA sequences of Coxiella-like bacteria, which clustered phylogenetically according to the respective tick host species. The findings presented here added to the growing evidence of the association between Coxiella-like bacteria and ticks across species and geographical boundaries. The importance of C. burnetii found in ticks in Malaysia warrants further investigation.
Fulltext Comparative genetic analysis of VP4, VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997-2008

Chan YF, Wee KL, Chiam CW, Khor CS, Chan SY, Amalina W MZ, et al.

Trop Biomed, 2012 Sep;29(3):451-66.
PMID: 23018509 MyJurnal

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.
Bacterial profiling of head lice isolated from the Orang Asli: A first report in Malaysia

Abd Majid MA, Khoo JJ, Lim FS, Khor CS, Loong SK, Low VL, et al.

Trop Biomed, 2020 Dec 01;37(4):884-895.
PMID: 33612742 DOI: 10.47665/tb.37.4.884

This study was carried out to determine from bacterial profiling to the bacterial profiles of head lice among the Orang Asli communities. The head lice were collected from Orang Asli community volunteers. The surface sterilized head lice pools were subjected to genomic DNA extraction while next generation sequencing of the 16S rRNA gene was performed using the Illumina MiSeq platform. Six female and three male head lice identified as Pediculus humanus capitis were collected. A total of 111 368 number of NGS sequencing reads were recorded while another 223 bacterial taxa sequences were obtained. Symbiotic bacteria showed the highest number of reads, with Arsenophonus and Rhodococcus sequences being the most abundant genera in the female and male samples, respectively. The female head lice contained a more distinct microbial diversity. Amongst the pathogenic bacterial species sequences noted were the methicillin-resistant Staphylococcus aureus, Streptobacillus moniliformis, Haemophilus influenzae, Bordetella pertussis and Acinetobacter baumannii. The 16S rRNA genome sequencing revealed a number of rare and pathogenic bacterial species within the head lice of the Orang Asli. The socio-economic practices of the community which involved forest foraging and hunting, and their poor living conditions potentially facilitated the transmission of zoonotic bacterial pathogens, including those found within the head lice. Hence, there is the possibility that the head lice could serve as vectors for the transmission of pathogenic bacteria. This study highlighted the diverse microbial community found within the head lice's gut of the Orang Asli, with the detection of multiple rare and pathogenic bacteria capable of causing severe infections.

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