Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex.

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide.

The microscopic observation drug susceptibility assay (MODS) was evaluated to determine susceptibility to pyrazinamide in Mycobacterium tuberculosis, and compared with the broth microdilution method (BMM), absolute concentration method (ACM), and pyrazinamidase (PZase) determination. We tested 34 M. tuberculosis clinical isolates (24 sensitive and eight resistant to pyrazinamide) and the control strains M. tuberculosis H37Rv (ATCC 27294) and Mycobacterium bovis AN5. The MODS, BMM, ACM and PZase determination provided results in average times of 6, 18, 28 and 7 days, respectively. All methods showed excellent sensitivity and specificity (p <0.05). Of the methods studied, the MODS proved to be faster, efficient, inexpensive, and easy to perform. However, additional studies evaluating the MODS in differentiating pyrazinamide-resistant and pyrazinamide-susceptible M. tuberculosis must be conducted with a larger number of clinical isolates.

pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates from the southeast region of Brazil.

OBJECTIVES: To investigate the presence of mutations in the pncA gene in 31 pyrazinamide-resistant Mycobacterium tuberculosis and 5 susceptible strains. MICs and pyrazinamidase (PZase) activity were also determined. METHODS: All 36 M. tuberculosis clinical isolates were genotyped by mycobacterial interspersed repetitive units (MIRUs) and most were also typed by spoligotyping. The MIC value necessary to inhibit 99% of the resistant mycobacterial isolates was determined by microplate Alamar Blue assay (MABA) and by Löwenstein-Jensen assay (LJA). The PZase activity was measured by pyrazinamide deamination to pyrazinoic acid and ammonia, and the entire pncA sequence including the 410 bp upstream from the start codon was determined by DNA sequencing of purified PCR products. RESULTS: Of the 31 isolates resistant to pyrazinamide, 26 (83.9%) showed at least one mutation in the pncA gene or in its putative regulatory region. Among the 22 different mutations detected in the pncA gene and in its regulatory region, 9 (40.9%) mutations (consisting of six substitutions, two insertions and one deletion) have not been described in previous studies. Three pyrazinamide-resistant isolates, confirmed by MIC varying from 800 to 1600 mg/L, carried the wild-type pncA sequence and retained PZase activity. CONCLUSIONS: These results contribute to the knowledge of the molecular mechanism of pyrazinamide resistance in Brazil and also expand the profile of pncA mutations worldwide. The MABA was successfully used to determine the MICs of pyrazinamide.