Whole-Genome Sequencing of Streptomycin-Resistant Mycobacterium tuberculosis Strain SBH145 from Sabah, Malaysia.

This paper reports on the whole-genome sequencing of a streptomycin-resistant Mycobacterium tuberculosis strain that was isolated from a patient with pulmonary tuberculosis in Sabah state of Malaysian Borneo. The strain belongs to the EAI2-Manila family of lineage 1 and is clustered with M. tuberculosis strains from the Philippines, India, and Taiwan.

Identification of a Mycobacterium tuberculosis-specific gene marker for diagnosis of tuberculosis using semi-nested melt-MAMA qPCR (lprM-MAMA).

Tuberculosis (TB) is the deadliest of infectious diseases. TB diagnosis, based on sputum microscopy, culture, and nucleic acid amplification tests (NAATs) to identify its main causative agent, Mycobacterium tuberculosis (MTB), remains challenging. The current available NAATs, endorsed by World Health Organization (WHO), can differentiate MTB from some MTB complex (MTBC) members. Using bioinformatics, we identified a single nucleotide polymorphism (SNP) in lprM (Rv1970) gene that differentiate MTB from other MTBC members. A forward mismatch amplification mutation assay (MAMA) primer was designed for the targeted mutation and was used in a semi-nested melt-MAMA qPCR (lprM-MAMA). Using the optimized protocol, lprM-MAMA was positive with all MTB reference and clinical strains, and negative with other MTBC members, non-tuberculous mycobacteria (NTM) and other non-mycobacterial (NM) reference strains. The limit of detection (LOD) of lprM-MAMA was 76.29 fg. Xpert® MTB/RIF (Xpert)-positive sputum samples were also positive by lprM-MAMA, except for samples classified as having "very low" bacterial load by Xpert. Xpert-negative sputum samples were also negative by lprM-MAMA. In conclusion, lprM-MAMA demonstrated to be a useful tool for specific MTB diagnosis. Further evaluation with higher number of reference strains, including NTM and NM; and sputum samples are required to determine its potential for clinical application.

Whole genome sequencing data and analysis of a rifampicin-resistant Mycobacterium tuberculosis strain SBH162 from Sabah, Malaysia.

A Mycobacterium tuberculosis strain SBH162 was isolated from a 49-year-old male with pulmonary tuberculosis. GeneXpert MDR/RIF identified the strain as rifampicin-resistant M. tuberculosis. The whole genome sequencing was performed using Illumina HiSeq 4000 system to further investigate and verify the mutation sites of the strain through genetic analyses namely variant calling using bioinformatics tools. The de novo assembly of genome generated 100 contigs with N50 of 156,381bp. The whole genome size was 4,343,911 bp with G + C content of 65.58% and consisted of 4,306 predicted genes. The mutation site, S450L, for rifampicin resistance was detected in the rpoB gene. Based on the phylogenetic analysis using the Maximum Likelihood method, the strain was identified as belonging to the Europe America Africa lineage (Lineage 4). The genome dataset has been deposited at DDBJ/ENA/GenBank under the accession number SMOE00000000.

Evaluation of new multiplex PCR primers for the identification ofPlasmodium species found in Sabah, Malaysia.

BACKGROUND/AIM: Malaria is a major public health problem, especially in the Southeast Asia region, caused by 5 species of Plasmodium (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi). The aim of this study was to compare parasite species identification methods using the new multiplex polymerase chain reaction (PCR) against nested PCR and microscopy. MATERIALS AND METHODS: Blood samples on filter papers were subject to conventional PCR methods using primers designed by us in multiplex PCR and previously designed primers of nested PCR. Both sets of results were compared with microscopic identification. RESULTS: Of the 129 samples identified as malaria-positive by microscopy, 15 samples were positive for P. falciparum, 14 for P. vivax, 6 for P. knowlesi, 72 for P. malariae, and 2 for mixed infection of P. falciparum/P. malariae. Both multiplex and nested PCR identified 12 P. falciparum single infections. For P. vivax, 9 were identified by multiplex and 12 by nested PCR. For 72 P. malariae cases, multiplex PCR identified 58 as P. knowlesi and 10 as P. malariae compared to nested PCR, which identified 59 as P. knowlesi and 7 as P. malariae. CONCLUSION: Multiplex PCR could be used as alternative molecular diagnosis for the identification of all Plasmodium species as it requires a shorter time to screen a large number of samples.