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

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.

Epidemiological serosurvey of chikungunya fever post outbreak at Tanjung Sepat, Malaysia.

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.

Leptospirosis among Dengue-Negative Febrile Patients in Selangor, Malaysia.

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.

Genomic and In Vitro Phenotypic Comparisons of Epidemic and Non-Epidemic Getah Virus Strains.

Getah virus is an emerging mosquito-borne animal pathogen. Four phylogenetic groups of GETV, Group I (GI), GII, GIII and GIV, were identified. However, only the GETV GIII was associated with disease epidemics suggesting possible virulence difference in this virus group. Here, we compared the genetic and in vitro phenotypic characteristics between the epidemic and non-epidemic GETV. Our complete coding genome sequence analyses revealed several amino acid substitutions unique to the GETV GIII and GIV groups, which were found mainly in the hypervariable domain of nsP3 and E2 proteins. Replication kinetics of the epidemic (GIII MI-110 and GIII 14-I-605) and non-epidemic GETV strains (prototype GI MM2021 and GIV B254) were compared in mammalian Vero cells and mosquito C6/36 and U4.4 cells. In all cells used, both epidemic GETV GIII MI-110 and GIII 14-I-605 strains showed replication rates and mean maximum titers at least 2.7-fold and 2.3-fold higher than those of GIV B254, respectively (Bonferroni posttest, p < 0.01). In Vero cells, the epidemic GETV strains caused more pronounced cytopathic effects in comparison to the GIV B254. Our findings suggest that higher virus replication competency that produces higher virus titers during infection may be the main determinant of virulence and epidemic potential of GETV.

Group IV Getah Virus in Culex Mosquitoes, Malaysia.

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).

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

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.

Emergence of B.1.524(G) SARS-CoV-2 in Malaysia during the third COVID-19 epidemic wave.

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.

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

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.

Culturable bacteria in adults of a Southeast Asian black fly, Simulium tani (Diptera:Simuliidae).

Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed ß-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.

Detection and confirmation of dengue pre- and postintroduction of dengue NS1-antigen test at the University Malaya Medical Centre: An observational study.

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.

A reverse transcription loop-mediated isothermal amplification for broad coverage detection of Asian and African Zika virus lineages.

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.

Dengue fever among febrile patients in Taiz City, Yemen during the 2016 war: Clinical manifestations, risk factors, and patients knowledge, attitudes, and practices toward the disease.

The current war in Yemen has displaced millions of people from their homes into living in cramped shelters where the healthcare is limited. The breakdown of Yemen's healthcare and sanitation systems has facilitated the spread of infectious diseases including mosquito-borne diseases. The present study aimed to describe the prevalence of dengue virus (DENV) infection among the febrile patients of the Taiz governorate, Yemen as well as their knowledge, attitude and preventive practices (KAPs) regarding dengue fever (DF), and to investigate the factors associated with dengue preventive practices during the war. A total of 384 clinically dengue-suspected patients who sought health care in Taiz, Yemen during the period from July 2016 until October 2016 were recruited for the study. Serum samples were obtained and screened for the presence of DENV RNA and anti-DENV antibodies by reverse transcription-recombinase polymerase amplification (RT-RPA) and dengue IgM/IgG-capture ELISA, respectively. KAP questionnaires were obtained from all participants too. In the study, dengue was laboratory confirmed in approximately 49.3% (189/384) of the clinically suspected dengue patients. In general, 67.1% of the patients had low knowledge scores regarding DF. Low scores for knowledge about DF was significantly associated with those in the age groups of ≤20 years and 21-30 years, illiterates and patients with non-skilled jobs or jobless. The most common preventive practices reported by participants were covering stored water (78.6%) and putting a screen on the house's windows (65.3%). A low proportion of participants (6.7%) had 51-100% of good DF preventive practices. Low scores of positive attitudes toward DF was identified as a risk factor. The study participants showed poor knowledge about DF and their ways of dealing with the various aspects of DF prevention was quite limited, hence, preventive measures against the disease were less likely to be undertaken. Findings from the study highlight the peril of dengue in Taiz, Yemen, which is now comparable to that of endemic regions. The ongoing civil war with disruption in regular health services compounded by the low knowledge about DF as well as the limited DF preventive practices could result in entrenchment of dengue in Yemen.

Bacterial pneumonia and its associated factors in children from a developing country: A prospective cohort study.

INTRODUCTION: Pneumonia in children is a common disease yet determining its aetiology remains elusive. OBJECTIVES: To determine the a) aetiology, b) factors associated with bacterial pneumonia and c) association between co-infections (bacteria + virus) and severity of disease, in children admitted with severe pneumonia. METHODS: A prospective cohort study involving children aged 1-month to 5-years admitted with very severe pneumonia, as per the WHO definition, over 2 years. Induced sputum and blood obtained within 24 hrs of admission were examined via PCR, immunofluorescence and culture to detect 17 bacteria/viruses. A designated radiologist read the chest radiographs. RESULTS: Three hundred patients with a mean (SD) age of 14 (±15) months old were recruited. Significant pathogens were detected in 62% of patients (n = 186). Viruses alone were detected in 23.7% (n = 71) with rhinovirus (31%), human metapneumovirus (HMP) [22.5%] and respiratory syncytial virus (RSV) [16.9%] being the commonest. Bacteria alone was detected in 25% (n = 75) with Haemophilus influenzae (29.3%), Staphylococcus aureus (24%) and Streptococcus pneumoniae (22.7%) being the commonest. Co-infections were seen in 13.3% (n = 40) of patients. Male gender (AdjOR 1.84 [95% CI 1.10, 3.05]) and presence of crepitations (AdjOR 2.27 [95% CI 1.12, 4.60]) were associated with bacterial infection. C-reactive protein (CRP) [p = 0.007]) was significantly higher in patients with co-infections but duration of hospitalization (p = 0.77) and requirement for supplemental respiratory support (p = 0.26) were not associated with co-infection. CONCLUSIONS: Bacteria remain an important cause of very severe pneumonia in developing countries with one in four children admitted isolating bacteria alone. Male gender and presence of crepitations were significantly associated with bacterial aetiology. Co-infection was associated with a higher CRP but no other parameters of severe clinical illness.

Serological evidence of DENV, JEV, and ZIKV among the indigenous people (Orang Asli) of Peninsular Malaysia.

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.

Dengue Outbreak during Ongoing Civil War, Taiz, Yemen.

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.

Baicalein and baicalin as Zika virus inhibitors.

At present, there is no effective antiviral agent for Zika virus (ZIKV), an arbovirus that is known for its teratogenic effects on newborns. Baicalein and baicalin were found to be capable of downregulating ZIKV replication up to 10 hours postinfection, while prophylactic effects were evident in pre-treated cells. Baicalein exhibited its highest potency during intracellular ZIKV replication, whereas baicalin was most effective against virus entry. Our in silico interaction assays predicted that both compounds exhibited the strongest binding affinities towards ZIKV NS5, while the virus envelope glycoprotein was the least likely target protein. These findings serve as a crucial platform for further in-depth studies to decipher the underlying anti-ZIKV mechanism(s) of each compound.

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

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.

Operational utility of the reverse-transcription recombinase polymerase amplification for detection of dengue virus.

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.

Vector competence of Malaysian Aedes albopictus with and without Wolbachia to four dengue virus serotypes.

OBJECTIVE: To determine the susceptibility status of Aedes albopictus with and without Wolbachia to the four dengue virus serotypes. METHODS: Two newly colonised colonies of Ae. albopictus from the wild were used for the study. One colony was naturally infected with Wolbachia while in the other Wolbachia was removed by tetracycline treatment. Both colonies were orally infected with dengue virus-infected fresh blood meal. Dengue virus load was measured using quantitative RT-PCR at four-time intervals in the salivary glands, midguts and ovaries. RESULTS: Wolbachia did not significantly affect Malaysian Ae. albopictus dengue infection or the dissemination rate for all four dengue virus serotypes. Malaysian Ae. albopictus had the highest replication kinetics for DENV-1 and the highest salivary gland and midgut infection rate for DENV-4. CONCLUSION: Wolbachia, which naturally exists in Malaysian Ae. albopictus, does not significantly affect dengue virus replication. Malaysian Ae. albopictus is susceptible to dengue virus infections and capable of transmitting dengue virus, especially DENV-1 and DENV-4. Removal of Wolbachia from Malaysian Ae. albopictus would not reduce their susceptibility status.

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

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.