Evaluation of TBMDR® and XDRA® for the detection of multidrug resistant and pre-extensively drug resistant tuberculosis.

This study evaluated the diagnostic performance of the AccuPower® TB&MDR Real-Time PCR (TBMDR®) and AccuPower® XDR-TB Real-Time PCR Kit-A (XDRA®) to detect multidrug-resistant (MDR-TB) and pre-extensively drug-resistant tuberculosis (pre-XDR-TB) in comparison with phenotypic drug susceptibility testing (DST) using MGIT 960 on 234 clinical Mycobacterium tuberculosis isolates. Discrepant results were confirmed by direct-sequencing. Sensitivity and specificity of TBMDR and XDRA for cultured isolates were 81.2% and 95.8% for isoniazid (INH) resistance, 95.7% and 95.7% for rifampicin (RIF) resistance, 84.1% and 99.1% for fluoroquinolone (FQ) resistance, and 67.4% and 100% for second-line injectables resistance. The sensitivities of each drug were equivalent to other molecular DST methods. High concordance was observed when compared to direct-sequencing. We also found that TBMDR and XDRA assays can detect INH, RIF and FQ resistance in isolates with low level resistance-associated mutations which were missed by phenotypic DST. Our study showed TBMDR and XDRA assays could be the useful tools to detect MDR-TB and pre-XDR-TB.

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BACKGROUND: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed. METHODS: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results. RESULTS: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST. CONCLUSION: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Geographic Differences in the Contribution of ubiA Mutations to High-Level Ethambutol Resistance in Mycobacterium tuberculosis.

Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA (Rv3806c) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis We evaluated the prevalence of ubiA and embB (Rv3795) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.

Genetic diversity of Mycobacterium tuberculosis isolates from a tertiary care tuberculosis hospital in South Korea.

Tuberculosis (TB) remains an immense public health problem in the Republic of Korea despite a more than fivefold decrease in the prevalence of the disease over the last 3 decades. The rise in drug-resistant TB has compounded the situation. We analyzed 208 clinical isolates of M. tuberculosis from the National Masan Tuberculosis Hospital by spoligotyping, IS6110 restriction fragment length polymorphism (RFLP), and 24-locus-based mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing to assess the diversity and transmission dynamics of the tubercle bacilli in the Republic of Korea. The majority of the isolates (97.1%) belonged to the Beijing genotype. Cluster analysis by MIRU-VNTR yielded a low clustering rate of 22.3%, with most of the clusters comprising isolates with diverse drug resistance patterns. The discriminatory capacity of the typing methods was high for RFLP and MIRU-VNTR (allelic diversity [h] = 0.99) but low for spoligotyping (h = 0.31). Although analysis of 19 MIRU-VNTR loci was needed to achieve maximum discrimination, an informative set of 8 loci (960, 1955, 2163b, 2165, 2996, 3192, 4052, and 4348) (h = 0.98) that was able to differentiate most of the closely related strains was identified. These findings suggest that 24-locus-based MIRU-VNTR typing is a likely suitable alternative to RFLP to differentiate clinical isolates in this setting, which is dominated by M. tuberculosis Beijing strains. Within the study limits, our results also suggest that the problem of drug-resistant TB in the Republic of Korea may be largely due to acquired resistance as opposed to transmission.