Shih Keng Loong

Chikungunya virus of Asian and Central/East African genotypes in Malaysia

BACKGROUND Chikungunya virus (CHIKV) of the Central/East African genotype has caused large outbreaks worldwide in recent years. In Malaysia, limited CHIKV outbreaks of the endemic Asian and imported Central/East African genotypes were reported in 1998 and 2006. Since April 2008, an unprecedented nationwide outbreak has affected Malaysia. OBJECTIVE To study the molecular epidemiology of the current Malaysian CHIKV outbreak, and to evaluate cross-neutralisation activity of serum from infected patients against isolates of Asian and Central/East African genotypes. STUDY DESIGN Serum samples were collected from 83 patients presenting in 2008, and tested with PCR for the E1 gene, virus isolation, and for IgM. Phylogenetic analysis was performed on partial E1 gene sequences of 837bp length. Convalescent serum from the current outbreak and Bagan Panchor outbreak (Asian genotype, 2006) were tested for cross-neutralising activity against representative strains from each outbreak. RESULTS CHIKV was confirmed in 34 patients (41.0%). The current outbreak strain has the A226V mutation in the E1 structural protein, and grouped with Central/East African isolates from recent global outbreaks. Serum cross-neutralisation activity against both Central/East African and Asian genotypes was observed at titres from 40 to 1280. CONCLUSIONS The CHIKV strain causing the largest Malaysian outbreak is of the Central/East African genotype. The presence of the A226V mutation, which enhances transmissibility of CHIKV by Aedes albopictus, may explain the extensive spread especially in rural areas. Serum cross-neutralisation of different genotypes may aid potential vaccines and limit the effect of future outbreaks.

Genotypic and Phenotypic Characterization of Chikungunya Virus of Different Genotypes from Malaysia

Background Mosquito-borne Chikungunya virus (CHIKV) has recently re-emerged globally. The epidemic East/Central/South African (ECSA) strains have spread for the first time to Asia, which previously only had endemic Asian strains. In Malaysia, the ECSA strain caused an extensive nationwide outbreak in 2008, while the Asian strains only caused limited outbreaks prior to this. To gain insight into these observed epidemiological differences, we compared genotypic and phenotypic characteristics of CHIKV of Asian and ECSA genotypes isolated in Malaysia. Methods and Findings CHIKV of Asian and ECSA genotypes were isolated from patients during outbreaks in Bagan Panchor in 2006, and Johor in 2008. Sequencing of the CHIKV strains revealed 96.8% amino acid similarity, including an unusual 7 residue deletion in the nsP3 protein of the Asian strain. CHIKV replication in cells and Aedes mosquitoes was measured by virus titration. There were no differences in mammalian cell lines. The ECSA strain reached significantly higher titres in Ae. albopictus cells (C6/36). Both CHIKV strains infected Ae. albopictus mosquitoes at a higher rate than Ae. aegypti, but when compared to each other, the ECSA strain had much higher midgut infection and replication, and salivary gland dissemination, while the Asian strain infected Ae. aegypti at higher rates. Conclusions The greater ability of the ECSA strain to replicate in Ae. albopictus may explain why it spread far more quickly and extensively in humans in Malaysia than the Asian strain ever did, particularly in rural areas where Ae. albopictus predominates. Intergenotypic genetic differences were found at E1, E2, and nsP3 sites previously reported to be determinants of host adaptability in alphaviruses. Transmission of CHIKV in humans is influenced by virus strain and vector species, which has implications for regions with more than one circulating CHIKV genotype and Aedes species.

Multisensitive community-acquired methicillin-resistant Staphylococcus aureus infections in Malaysia

The 1st 9 clinical isolates of multisensitive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) from Malaysia carry SCCmec type IV and predominantly cause skin and soft-tissue infections. Seven were classified as nosocomially acquired. There was considerable clonal diversity, with both pandemic and novel multilocus sequence types detected. CA-MRSA rates appear to be increasing in our hospital, warranting close surveillance.

Real-time polymerase chain reaction for diagnosis and quantitation of negative strand of chikungunya virus☆

Quantitative real-time polymerase chain reaction (qRT-PCR) is useful for diagnosis and studying virus replication. We developed positive- and negative-strand qRT-PCR assays to detect nsP3 of chikungunya virus (CHIKV), a positive-strand RNA alphavirus that causes epidemic fever, rash, and arthritis. The positive- and negative-strand qRT-PCR assays had limits of quantification of 1 and 3 log10 RNA copies/reaction, respectively. Compared to a published E1 diagnostic assay using 30 laboratory-confirmed clinical samples, the positive-strand nsP3 qRT-PCR assay had higher R(2) and efficiency and detected more positive samples. Peak viral load of 12.9 log(10) RNA copies/mL was reached on day 2 of illness, and RNA was detectable up to day 9, even in the presence of anti-CHIKV IgM. There was no correlation between viral load and persistent arthralgia. The positive-strand nsP3 assay is suitable for diagnosis, while the negative-strand nsP3 assay, which uses tagged primers to increase specificity, is useful for study of active viral replication kinetics.

Utility of 16S rDNA Sequencing for Identification of Rare Pathogenic Bacteria.

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.

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.

Draft genome of Bordetella pseudohinzii BH370 isolated from trachea and lung tissues of a laboratory mouse

In this study, we present the draft genome sequence of B. pseudohinzii BH370 recovered from the trachea and lung tissues of an ICR mouse in Malaysia. The genome consists of 4,474,040 bp with a GC content of 66.4%. Annotation using RAST algorithm displayed 5119 protein encoding and 52 RNA genes. The CRISPR-cas genomic sequences previously reported in B. pseudohinzii were identified. The nucleotide sequences of BH370 was deposited into the European Nucleotide Archive under the genome assembly accession number FPJN01000000.

A Handy Field Portable ELISA-system for Rapid Onsite Diagnosis of Infectious Diseases

Enzyme-linked immunosorbent assays (ELISAs) are considered the gold standard for the detection of various immunological reactions and can be used for the detection of infectious diseases during outbreaks or in the care of individual patients. To be useful in the timely implementation of prevention and control measures against infectious diseases, a diagnostic modality should be rapid, accurate, and affordable. In the current study, we demonstrate the efficiency (90% less time and volume consumption compared with those of a standard 96-well ELISA), detection capability, and ease of operation of a field-portable, battery-operated ELISA system, approximately the size of a cellular phone (12 × 6 × 5.5 cm), in the serological diagnosis of measles and rubella viruses that has the potential for onsite testing such as during disease outbreaks.

Synovial Tissue Infection with Burkholderia fungorum

To the Editor: The genus Burkholderia, first proposed in 1992, has now expanded to consist of many species (1). Bacteria of this genus exhibit extensive ubiquity; they have been isolated from human, animal, and environmental sources (1,2). Although rare, Burkholderia fungorum has been implicated in several human infections (2–4). We isolated B. fungorum from the synovial tissue of a patient’s knee.

Draft genome of the emerging pathogen, Kocuria marina, isolated from a wild urban rat

Kocuria marina has recently emerged as a cause for catheter-related bloodstream infections in patients with underlying health complications. One K. marina strain was recently isolated from the lung tissues of a wild urban rat (Rattus rattus diardii) caught during rodent surveillance. Here, we present the draft genome of the first K. marina animal isolate, K. marina TRE150902.