Non-classical Bordetella sp. (closely related to Bordetella hinzii and Bordetella pseudohinzii) lower respiratory tract infection in a patient with extensive bronchiectasis: a case report.

An increasing number of reports have described the pathogenic nature of several non-classical Bordetella spp. Among them, Bordetella hinzii and Bordetella pseudohinzii have been implicated in a myriad of respiratory-associated infections in humans and animals. We report the isolation of a genetically close relative of B. hinzii and B. pseudohinzii from the sputum of a woman in her early 60s with extensive bronchiectasis who presented with fever and brown colored sputum. The isolate had initially been identified as Bordetella avium by API 20NE, the identification system for non-enteric Gram-negative rod bacteria. Sequencing of the 16S rDNA, ompA, nrdA, and genes used in the Bordetella multilocus sequence typing scheme could not resolve the identity of this Bordetella isolate. Whole-genome single nucleotide polymorphism analysis positioned the isolate between B. hinzii and B. pseudohinzii in the phylogenetic tree, forming a distinct cluster. Whole-genome sequencing enabled the further identification of this rare organism, and should be considered for wider applications, especially the confirmation of organism identity in the clinical diagnostic microbiology laboratory.

Molecular Surveillance for Vector-Borne Bacteria in Rodents and Tree Shrews of Peninsular Malaysia Oil Palm Plantations.

Many human clinical cases attributed to vector-borne pathogens are underreported in Malaysia, especially in rural localities where healthcare infrastructures are lacking. Here, 217 small mammals, consisting of rodents and tree shrews, were trapped in oil palm plantations in the Peninsular Malaysia states of Johor and Perak. Species identification was performed using morphological and DNA barcoding analyses, and 203 small mammals were included in the detection of selected vector-borne bacteria. The DNA extracted from the spleens was examined for Orientia tsutsugamushi, Borrelia spp., Bartonella spp. and Rickettsia spp. using established PCR assays. The small mammals collected in this study included Rattus tanezumi R3 mitotype (n = 113), Rattus argentiventer (n = 24), Rattus tiomanicus (n = 22), Rattus exulans (n = 17), Rattus tanezumi sensu stricto (n = 1) and Tupaia glis (n = 40). Orientia tsutsugamushi, Borrelia spp. and Bartonella phoceensis were detected in the small mammals with the respective detection rates of 12.3%, 5.9% and 4.9%. Rickettsia spp., however, was not detected. This study encountered the presence of both Lyme disease and relapsing fever-related borreliae in small mammals collected from the oil palm plantation study sites. All three microorganisms (Orientia tsutsugamushi, Borrelia spp. and Bartonella phoceensis) were detected in the R. tanezumi R3 mitotype, suggesting that the species is a competent host for multiple microorganisms. Further investigations are warranted to elucidate the relationships between the ectoparasites, the small mammals and the respective pathogens.

Molecular evidence of rat bocavirus among rodents in Peninsular Malaysia.

Rat bocavirus (RBoV) and rodent bocavirus (RoBoV) have previously been detected in Rattus norvegicus; however, these viruses have not been reported in rodent populations in Malaysia. We investigated the presence of RBoV and RoBoV in archived rodent specimens. DNA barcoding of the rodent cytochrome c oxidase gene identified five different species: Rattus tanezumi R3 mitotype, Rattus tiomanicus, Rattus exulans, Rattus argentiventer, and Rattus tanezumi sensu stricto. Three spleens were positive for RBoV (1.84%; 3/163), but no RoBoV was detected. Phylogenetic analyzes of the partial non-structural protein 1 gene grouped Malaysian RBoV strains with RBoV strains from China. Further studies among rats from different geographical locations are warranted for this relatively new virus.

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.

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.

Disruption of predicted dengue virus type 3 major outbreak cycle coincided with switching of the dominant circulating virus genotype.

Dengue is hyperendemic in most of Southeast Asia. In this region, all four dengue virus serotypes are persistently present. Major dengue outbreak cycle occurs in a cyclical pattern involving the different dengue virus serotypes. In Malaysia, since the 1980s, the major outbreak cycles have involved dengue virus type 3 (DENV3), dengue virus type 1 (DENV1) and dengue virus type 2 (DENV2), occurring in that order (DENV3/DENV1/DENV2). Only limited information on the DENV3 cycles, however, have been described. In the current study, we examined the major outbreak cycle involving DENV3 using data from 1985 to 2016. We examined the genetic diversity of DENV3 isolates obtained during the period when DENV3 was the dominant serotype and during the inter-dominant transmission period. Results obtained suggest that the typical DENV3/DENV1/DENV2 cyclical outbreak cycle in Malaysia has recently been disrupted. The last recorded major outbreak cycle involving DENV3 occurred in 2002, and the expected major outbreak cycle involving DENV3 in 2006-2012 did not materialize. DENV genome analyses revealed that DENV3 genotype II (DENV3/II) was the predominant DENV3 genotype (67%-100%) recovered between 1987 and 2002. DENV3 genotype I (DENV3/I) emerged in 2002 followed by the introduction of DENV3 genotype III (DENV3/III) in 2008. These newly emerged DENV3 genotypes replaced DENV3/II, but there was no major upsurge of DENV3 cases that accompanied the emergence of these viruses. DENV3 remained in the background of DENV1 and DENV2 until now. Virus genome sequence analysis suggested that intrinsic differences within the different dengue virus genotypes could have influenced the transmission efficiency of DENV3. Further studies and continuous monitoring of the virus are needed for better understanding of the DENV transmission dynamics in hyperendemic regions.

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.

Genetic characterization of commensal Escherichia coli isolated from laboratory rodents.

BACKGROUND: Escherichia coli, a commensal in the intestines of vertebrates, is capable of colonizing many different hosts and the environment. Commensal E. coli strains are believed to be the precursor of pathogenic strains by means of acquisition of antimicrobial resistant and virulence genes. Laboratory rodents are inherently susceptible to numerous known infectious agents, which could transfer virulence determinants to commensal E. coli. Hence, in this study, the genetic structure of commensal E. coli found in laboratory rodents and their antimicrobial resistance profiles were investigated. RESULTS: E. coli strains belonging to phylogroup A were the predominant strain obtained from the animals used in the study. Four novel sequence types (ST746, ST747, ST748 and ST749) were discovered using the multi locus sequence typing, together with one common ST357 in the gastrointestinal tract, liver and, the trachea and lung. Serotyping demonstrated that these commensal E. coli strains were non-Shiga toxin-producers. Phenotypic and genotypic analyses of extended spectrum beta lactamases were also negative. CONCLUSIONS: These findings implied that the E. coli strains recovered from the laboratory rodents were truly commensal in nature. Further study is required to investigate the possible influence of gender on the susceptibility of hosts to E. coli colonization in laboratory rodents.

Molecular and antimicrobial analyses of non-classical Bordetella isolated from a laboratory mouse.

Accurate identification and separation of non-classical Bordetella species is very difficult. These species have been implicated in animal infections. B. hinzii, a non-classical Bordetella, has been isolated from mice in experimental facilities recently. We isolated and characterized one non-classical Bordetella isolate from the trachea and lung of an ICR mouse. Isolate BH370 was initially identified as B. hinzii by 16S ribosomal DNA and ompA sequencing. Additionally, isolate BH370 also displayed antimicrobial sensitivity profiles similar to B. hinzii. However, analyses of nrdA sequences determined its identity as Bordetella genogroup 16. The isolation of BH370 from a healthy mouse suggests the possibility of it being a commensal. The nrdA gene was demonstrated to possess greater phylogenetic resolution as compared with 16S ribosomal DNA and ompA for the discrimination of non-classical Bordetella species.