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Serological evidence has shown the presence of several mosquito-borne arbovirus infections among the inhabitants of the forest fringe areas of the tropics. Among these infections, Japanese encephalitis, dengue fever, chikungunya fever and Zika fever could be targeted for vaccination to overcome severe infection and limit the disease transmission. Seroprevalence data among this high-risk population are needed to provide an estimate of the potential cost-effectiveness of any vaccine programme targeting these infections. The present study was conducted at six indigenous people (Orang Asli) villages and FELDA (Federal Land Development Authority) settlements located at the forest fringes of Malaysia. All participants consented and provided blood samples and demographic data for the study. The blood samples were tested for the presence of antibodies against CHIKV, DENV, JEV and ZIKV individually using ELISA. Results obtained were also analysed to determine the predictors for CHIKV, DENV, JEV and ZIKV seropositivity. Among the 585 samples tested, 33.0% (N=193), 41.7% (N=244), 10.3% (N=60) and 21.0% (N=123) were positive for CHIKV IgG, DENV IgG, JEV IgG and ZIKV IgG, respectively. Approximately one-third (N=220, 37.6%) of the participants were tested negative for IgG antibodies against all four arboviruses. Age of participants and type of settlement were found to be a significant predictor for CHIKV, DENV, JEV and ZIKV seropositivity. Level of education was a significant predictor for CHIKV, DENV and ZIKV seropositivity. Gender, however, was not found to be a significant predictor for infection with any of these viruses. These findings reaffirmed the significant presence of infection involving these major arboviruses among the group of people living within the forest fringe areas of Peninsular Malaysia. Hence, any future consideration of vaccination for these infections must take into consideration the marginalized and underserved communities living at the forest fringe areas of the tropics where these infections are present.

The present study aimed at exploring whether sunlight exposure might account for the relative difference in COVID-19-related morbidity and mortality between tropical and non-tropical countries. A retrospective observational study was designed and data from the World Health Organization weekly COVID-19 epidemiological update was compiled. We examined the total number of confirmed COVID-19 cases per 100 000 population, as well as the total number of COVID-19-related mortalities per 100 000 population. Solar variables data were obtained from the Global Solar Atlas website (https://globalsolaratlas.info/). These data were analyzed to determine the association of sunlight exposure to COVID-19-related morbidity and mortality in tropical and non-tropical countries. Results revealed a statistically significant decrease in the number of confirmed COVID-19 cases per 100 000 population (P<0.001), as well as the number of COVID-19-related mortalities per 100 000 population (P<0.001) between tropical and non-tropical countries. Analyses of sunlight exposure data found that specific photovoltaic power output, global horizontal irradiation, diffuse horizontal irradiation and global tilted irradiation at optimum angle were significantly inversely correlated to COVID-19-related morbidity and mortality. This suggests that stronger sunlight exposure potentially leads to lower COVID-19-related morbidity and mortality. Findings from this study suggest that the relatively low COVID-19-related morbidity and mortality in tropical countries were possibly due to better sunlight exposure that translates into adequate vitamin D status.

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.

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 R 2 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.

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., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Chee-Sieng Khor, Boon-Teong Teoh, Sing-Sin Sam, Hooi-Yuen Khoo, Noor-Syahida Azizan, AsmaAnati CheMatSeri, Kim-Ling Chin, Zur-Raiha Hamim, Noor-Adila Mohamed-Romai-Noor, Che-Norainon Yaacob, Juraina Abd-Jamil, Hai-Yen Lee, Yih-Harng Soh, Sazaly AbuBakar.)

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.

Introduction: Recently, the rapid surge of reported COVID-19 cases attributed to the Omicron variant of severe acute respiratory syndrome coronavirus (SARS-CoV-2) created an immediate concern across nations. Local information pertaining to the new variant of concern (VOC) is lacking. We aimed to determine the clinical characteristics of COVID-19 during a period of Omicron prevalence among patients hospitalised from February 1 to 21, 2022 at Sungai Buloh Hospital and to estimate the risks of disease progression presumably caused by this variant in association with gender, age, comorbidity, and vaccination status., Materials and Methods: In this retrospective, singlecentered, retrospective cohort study, all hospitalised adults with laboratory-confirmed COVID-19, aged 18 and above, were recruited from February 1 to 21, 2022. Clinical characteristics, investigations, and outcomes were assessed., Results: A total of 2279 patients aged 18 years and above with laboratory-proven COVID-19 were recruited and analysed, excluding 32 patients owing to incomplete data. Majority of the study population had a mean age of 41.8 ± 17.7, was female-predominant (1329/2279, 58.6%), had completed a primary series of vaccination with a booster (1103/2279, 48.4%), and had no underlying medical conditions (1529/2279, 67.4%). The risk of COVID-19-related disease progression was significantly lower in hospitalised patients under the age of 50 who were female, had no comorbidity, and had completed two doses of the primary series with or without a booster. [respectively, OR 7.94 (95% CI 6.16, 10.23); 1.68 (1.34, 2,12); 2.44 (1.85, 3.22); 2.56 (1.65, 3.97), p< 0.001]., Conclusion: During the period of Omicron prevalence, a favourable outcome of COVID-19 was strongly associated with female gender, age below 50, a comorbidity-free condition, and having completed immunization. With this new observation, it could help improve public health planning and clinical management in response to the emergence of the latest VOC.

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.

In recent years, the number of leptospirosis cases, including the number of deaths, has exponentially increased in Malaysia. From June 2016 to February 2018, blood samples of 321 febrile patients with the presumptive diagnosis of dengue-like illness were examined for possible exposure to Leptospira. Two hundred fifty-five blood samples were tested as negative for dengue. Seminested polymerase chain reaction (PCR) and IgM ELISA for leptospirosis were performed. From the samples, an overall prevalence for leptospirosis based on PCR of 4.7% (12/255) was obtained. Eighteen percent (46/255) were positive for anti-Leptospira IgM antibodies. The genome sequences of six of 12 Leptospira PCR-positive samples showed > 97.0% similarity to Leptospira interrogans. One patient's sample consisted of Leptospira and chikungunya virus, suggesting a coinfection. Findings from the study suggest that leptospirosis is prevalent among dengue-negative febrile patients in Malaysia.

This dataset presents a cross-sectional survey and was conducted to assess the knowledge on Zika Virus infection among adults in Sabah. The data were collected from December 2019 to February 2021, 274 adults living in forest fringe communities were interviewed by trained personnel and have completed the distributed questionnaires. SPSS version 27.0 was used to analyzed the data. These data could serve as auxiliary information and/or research data for other researchers in Sabah. It could also serve as guide or reference data to other researchers outside Sabah who may be interested in carrying out similar research in other state., Competing Interests: The authors have no known competing financial interests or personal relationships that could have affected the work presented by the paper., (© 2022 The Authors.)

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).

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., Competing Interests: Conflict of interestThe analyses of HEV were performed using Wantai HEV IgG ELISA, of which all the HEV ELISA kits were sponsored by Beijing Wantai Biological Pharmacy Enterprise Co., Ltd, Beijing, China)., (© Indian Society of Hematology and Blood Transfusion 2021.)

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.

The recommended test guidelines for Zika virus (ZIKV) include using both molecular and serological tools. While the molecular tools are useful for detecting acute infection, the serological tools are useful for the detection of previous infections. Nevertheless, detection of ZIKV-specific antibodies remains a challenge due to the high cross-reactivity between ZIKV and other flaviviruses such as dengue virus (DENV) and Japanese encephalitis virus (JEV). The objective of this study is to evaluate the commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of ZIKV IgG. In this study, we evaluated 6 commercially available anti-ZIKV IgG ELISA kits. Pre-characterized serum panels consisting of 70 sera were selected for the evaluation. The diagnostic accuracy of each ELISA kits was determined and compared to the gold standard, Foci Reduction Neutralization Test (FRNT). The present study established that the performance of the NS1-based anti-ZIKV IgG ELISA kit was superior to that which uses of the E protein as antigen. Overall, commercial ZIKV IgG ELISA showed varying test performances, with some achieving moderate to high test sensitivities and specificities. When compared against the FRNT, the test sensitivities ranged from 7.1% to 78.6%, whereas, the test specificities ranged from 40.0% to 100%. Limitation to the study includes the cross reactivity between flavivirus and specificity of the kit in addressing the cross reactivity.

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., (© 2021. The Author(s).)

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.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 < 0.001)., Conclusion: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.

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.

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., (© 2019 Wiley Periodicals, Inc.)

Dengue is an endemic mosquito-borne viral disease prevalent in many urban areas of the tropic, especially the Southeast Asia. Its presence among the indigenous population of Peninsular Malaysia ( Orang Asli ), however, has not been well described. The present study was performed to investigate the seroprevalence of dengue among the Orang Asli (OA) residing at the forest fringe areas of Peninsular Malaysia and determine the factors that could affect the transmission of dengue among the OA. Eight OA communities consisting of 491 individuals were recruited. From the study, at least 17% of the recruited study participants were positive for dengue IgG, indicating past exposure to dengue. Analysis on the demographic and socioeconomic variables suggested that high seroprevalence of dengue was significantly associated with those above 13 years old and a low household income of less than MYR500 (USD150). It was also associated with the vast presence of residential areas and the presence of a lake. Remote sensing analysis showed that higher land surface temperatures and lower land elevations also contributed to higher dengue seroprevalence. The present study suggested that both demographic and geographical factors contributed to the increasing risk of contracting dengue among the OA living at the forest fringe areas of Peninsular Malaysia. The OA, hence, remained vulnerable to dengue., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2020 Juraina Abd-Jamil et al.)

Ticks are hematophagous vectors of arthropod-borne disease agents globally. In Malaysia, despite seroprevalence studies indicating the presence of tick-borne diseases among the indigenous people, the etiological agents of these diseases are still unclear. These indigenous people, also known as the Orang Asli, still live in forested areas with frequent contact with wildlife. Wild boar are ubiquitously found in the forested areas where the Orang Asli communities are located and are commonly hunted as a food supplement. In this study, we aim to determine the tick species parasitizing wild boar from an Orang Asli community, and explore the tick-associated bacterial communities using 16 s rRNA amplicon sequencing on the Ion Torrent PGM™ platform. A total of 72 ticks were collected from three wild boar and were morphologically identified as Haemaphysalis hystricis (n = 32), Dermacentor compactus (n = 15), Amblyomma testudinarium (n = 13), Dermacentor steini (n = 10) and Dermacentor atrosignatus (n = 2). Across all tick samples, 910 bacterial taxa were identified. Although the bacterial communities were not significantly distinct between tick species in beta-diversity analyses, Coxiella, Rickettsia and Francisella were detected at high relative abundance in H. hystricis, D. compactus and D. steini respectively. Many other bacterial genera, including those that have been described in many different tick species, were also identified, including Pseudomonas, Acinetobacter, Staphylococcus and Corynebacterium. Beta-diversity analyses also showed that the bacterial communities were separated based on the animal host from which the ticks were collected from, suggesting that the bacterial communities here may be influenced by the animal skin microflora, host blood or the environment. PCR screening confirmed the presence of Rickettsia sp. related to spotted fever group Rickettsia in some of the ticks. This study provides baseline knowledge of the microbiome of H. hystricis, D. atrosignatus, D. compactus, D. steini and A. testudinarium parasitizing wild boar in this region. The information gained in this study provides the basis to target our efforts in H. hystricis, D. compactus and D. steini for the future investigation of vector competence and the zoonotic potential for the Coxiella, Rickettsia and Francisella detected here, as well as their implications for the risks of tick-borne diseases among the Orang Asli communities., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

We identified dengue in ≈51% of patients given a clinical diagnosis of suspected dengue in Taiz, Yemen, during 2016. The cosmopolitan genotype of dengue virus type 2 was most common; viruses appeared to have originated in Saudi Arabia. Damage to public health infrastructure during the ongoing civil war might enable dengue to become endemic to Yemen.

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., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2019 Sazaly AbuBakar, Chee-Sieng Khor, Habibi Hassan, Nurul-Farhana Mohd-Rahim, Josephine-Rebecca Chandren, Siti-Sarah Nore, Jefree Johari, Shih-Keng Loong, Juraina Abd-Jamil, Jing-Jing Khoo, Hai-Yen Lee, Brian L Pike, Li-Ping Wong, Yvonne Ai-Lian Lim.)

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.

The Borrelia genus consists of spirochete bacteria known to cause Lyme disease (LD) and relapsing fever in humans. Borrelia pathogens are commonly transmitted via arthropod vectors such as ticks, mites, or lice. Here, we report the molecular screening of LD group Borrelia sp. from ticks (Acari: Ixodidae) collected from rodents trapped in recreational forests and a semiurban residential area in the Selangor state in Malaysia. Of 156 adult ticks surveyed, 72 ticks were determined as positive for Borrelia sp. by polymerase chain reaction (PCR). All Borrelia PCR-positive ticks belonged to the Ixodes granulatus Supino species. Borrelia sp. was not detected in other tick species examined, including Dermacentor sp. and Amblyomma sp. ticks. Thirteen Borrelia PCR-positive tick samples were selected for further sequence analyses. Phylogenetic analyses of partial flaB gene sequences revealed that the Borrelia sp. were closely related to the LD group borreliae, Borrelia yangtzensis; a novel Borrelia genospecies reported in East Asian countries including Japan, Taiwan, and China. To our knowledge, this is the first report of Borrelia sp. related to Borrelia yangtzensis detected in Malaysia and Southeast Asia. The zoonotic potential of the Borrelia sp. reported here merits further investigation, as it may explain the previously reported serological evidence for borrelial infections in Malaysia.

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.

The lifestyles of the indigenous people (Orang Asli) of Peninsular Malaysia who traditionally live close to the forest, put them at higher risk of exposure to zoonotic diseases. Leptospirosis has recently emerged as one of the most important diseases of public health concern. Here, we aimed to obtain a baseline data on the level of Leptospira exposure among the 107 Orang Asli volunteers using a recombinant antigen-based ELISA, previously shown to have sensitivity of ~90.0% in comparison to the microscopic agglutination test (MAT). Among the Orang Asli volunteers in this study, 60.7% had IgM against Leptospira and 57.9% were antiLeptospira IgG positive. Of these seropositive individuals, 29.9% had both anti-Leptospira IgM and IgG antibodies. Age was found to be a significant predictor for exposure to Leptospira (P < 0.05) with the younger Orang Asli population more likely to be tested positive for antiLeptospira IgM. The finding of high Leptospira exposure among the Orang Asli volunteers could be due to their socio-economic practices and dependency on the forest for their livelihood. The rapid and sensitive recombinant antigen-based ELISA used in the study, could possibly complement MAT for the epidemiological surveillance of leptospirosis, especially among the underserved populations.

Spirochetes from the Borrelia genus are known to cause diseases in humans, namely Lyme disease and relapsing fever. These organisms are commonly transmitted to humans by arthropod vectors including ticks, mite, and lice. Here, we report the molecular detection of a Borrelia sp. from a Haemaphysalis hystricis Supino tick collected from wildlife in an Orang Asli settlement in Selangor, Malaysia. Phylogenetic analyses of partial 16s rRNA and flaB gene sequences revealed that the Borrelia sp. is closely related to the relapsing fever group borreliae, Borrelia lonestari, Borrelia miyamotoi, and Borrelia theileri, as well as a number of uncharacterized Borrelia sp. from ticks in Portugal and Japan. To our knowledge, this is the first report of a Borrelia sp. detected in H. hystricis, and in Malaysia. The zoonotic potential of this Borrelia sp. merits further investigation., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: Author Disclosure Statement The authors declare that they have no competing interests. B.L.P. is a military service member of the U.S. Government. This work was prepared as part of his official duties. The opinions and assertions contained herein are those of the author(s) and are not to be construed as official or reflecting the views of the Department of the Navy, Department of Defense, or the U.S. Government. Title 17 U.S.C. §105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person's official duties.

Background: Phenotypic identification systems are established methods for laboratory identification of bacteria causing human infections. Here, the utility of phenotypic identification systems was compared against 16S rDNA identification method on clinical isolates obtained during a 5-year study period, with special emphasis on isolates that gave unsatisfactory identification., Methods: One hundred and eighty-seven clinical bacteria isolates were tested with commercial phenotypic identification systems and 16S rDNA sequencing. Isolate identities determined using phenotypic identification systems and 16S rDNA sequencing were compared for similarity at genus and species level, with 16S rDNA sequencing as the reference method., Results: Phenotypic identification systems identified ~46% (86/187) of the isolates with identity similar to that identified using 16S rDNA sequencing. Approximately 39% (73/187) and ~15% (28/187) of the isolates showed different genus identity and could not be identified using the phenotypic identification systems, respectively. Both methods succeeded in determining the species identities of 55 isolates; however, only ~69% (38/55) of the isolates matched at species level. 16S rDNA sequencing could not determine the species of ~20% (37/187) of the isolates., Conclusion: The 16S rDNA sequencing is a useful method over the phenotypic identification systems for the identification of rare and difficult to identify bacteria species. The 16S rDNA sequencing method, however, does have limitation for species-level identification of some bacteria highlighting the need for better bacterial pathogen identification tools., (© 2016 Wiley Periodicals, Inc.)

From 1989 to 2011 in Kuala Lumpur, Malaysia, multiple genotypes from both respiratory syncytial virus (RSV) subgroups were found co-circulating each year. RSV-A subgroup predominated in 12 out of 17years with the remaining years predominated by RSV-B subgroup. Local RSV strains exhibited temporal clustering with RSV strains reported in previous epidemiological studies. Every few years, the existing predominant genotype was replaced by a new genotype. The RSV-A genotypes GA2, GA5 and GA7 were replaced by NA1 and NA2, while BA became the predominant RSV-B genotype. A unique local cluster, BA12, was seen in 2009, and the recently-described ON1 genotype with 72-nt duplication emerged in 2011. Our findings will have important implications for future vaccine intervention., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Background: Viral respiratory tract infections (RTI) are relatively understudied in Southeast Asian tropical countries. In temperate countries, seasonal activity of respiratory viruses has been reported, particularly in association with temperature, while inconsistent correlation of respiratory viral activity with humidity and rain is found in tropical countries. A retrospective study was performed from 1982-2008 to investigate the viral etiology of children (≤ 5 years old) admitted with RTI in a tertiary hospital in Kuala Lumpur, Malaysia., Methods: A total of 10269 respiratory samples from all children ≤ 5 years old received at the hospital's diagnostic virology laboratory between 1982-2008 were included in the study. Immunofluorescence staining (for respiratory syncytial virus (RSV), influenza A and B, parainfluenza types 1-3, and adenovirus) and virus isolation were performed. The yearly hospitalization rates and annual patterns of laboratory-confirmed viral RTIs were determined. Univariate ANOVA was used to analyse the demographic parameters of cases. Multiple regression and Spearman's rank correlation were used to analyse the correlation between RSV cases and meteorological parameters., Results: A total of 2708 cases were laboratory-confirmed using immunofluorescence assays and viral cultures, with the most commonly detected being RSV (1913, 70.6%), parainfluenza viruses (357, 13.2%), influenza viruses (297, 11.0%), and adenovirus (141, 5.2%). Children infected with RSV were significantly younger, and children infected with influenza viruses were significantly older. The four main viruses caused disease throughout the year, with a seasonal peak observed for RSV in September-December. Monthly RSV cases were directly correlated with rain days, and inversely correlated with relative humidity and temperature., Conclusion: Viral RTIs, particularly due to RSV, are commonly detected in respiratory samples from hospitalized children in Kuala Lumpur, Malaysia. As in temperate countries, RSV infection in tropical Malaysia also caused seasonal yearly epidemics, and this has implications for prophylaxis and vaccination programmes.

The relationship between left ventricular mass (LVM) and the mean arterial blood pressure (MAP) was investigated, using M-Mode echocardiography. MAP was higher in hypertensive patients (p<0.05, n=9) compared to that of controlled subjects. The results showed that LVM index for hypertensive patients was significantly higher (p<0.05, n=9) than that for the normal group. LVM index correlates fairly (r=0.6) with MAP for hypertensive patients. The results also show that the increase of intraventricular septal wall thickness (IVST) was due to hypertension. The LVM (r =0.9) and IVST (r=0.75) of the normal subjects were linearly dependent on the body surface area (BSA). The hypertensive group revealed a non-linear relationship to the BSA.