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Background  Species identification of isolates belonging to the  Mycobacterium tuberculosis  complex (MTC) seems to be important for the appropriate treatment of patients, since  M. bovis  is naturally resistant to a first line anti-tuberculosis (TB) drug, pyrazinamide, while most of the other MTC members are susceptible to this antimicrobial agent. A simple and low-cost differentiation method was needed in higher TB burden countries, such as Bangladesh, where the prevalence of  M. bovis  among people or cattle has not been investigated.    Methods  Genetic regions  cfp32 , RD9 and RD12 were chosen as targets for a species distinguishable multiplex PCR and the system was evaluated with twenty reference strains of mycobacterial species including non-tubercular mycobacteria (NTM). A total of 350 clinical MTC isolates obtained in Bangladesh were then analyzed with this multiplex PCR.    Results  All of the MTC reference strains gave expected banding patterns and no non-specific amplifications were observed in the NTM strains. Out of 350 clinical isolates examined by this method, 347 (99.1%) were positive for all of the  cfp32 , RD9 and RD12 and determined as  M. tuberculosis . Two isolates lacked  cfp32  PCR product and one lacked RD12, however, those three samples were further examined and identified as  M. tuberculosis  by the sequence analyses of  hsp65  and  gyrB .    Conclusions  The MTC-discrimination multiplex PCR (MTCD-MPCR) developed in this study showed high specificity and was thought to be very useful as a routine test because of its simplicity. In the current survey, all the 350 MTC isolates obtained from Bangladesh TB patients were determined as  M. tuberculosis  and no other MTC were detected. This result suggested the general TB treatment regimen including pyrazinamide to be the first choice in Bangladesh.

Identification of Mycobacterium tuberculosis clinical isolates in Bangladesh by a species distinguishable multiplex PCR