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Open Access Highly Accessed Research article

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

Chie Nakajima1, Zeaur Rahim2*, Yukari Fukushima1, Isamu Sugawara3, Adri GM van der Zanden4, Aki Tamaru5 and Yasuhiko Suzuki1*

Author Affiliations

1 Department of Global epidemiology, Hokkaido University Research Center for Zoonosis Control, Kita20-Nishi10, Kita-ku, Sapporo 001-0020, Japan

2 Tuberculosis laboratory, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), GPO Box 128, Dhaka 1000, Bangladesh

3 Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose, Tokyo, Japan

4 Laboratory for Medical Microbiology and Public Health, P.O.Box 377, Burg. Edo Bergsmalaanl, 7512 AD Enschede, The Netherlands

5 Osaka Prefectural Institute of Public Health, 1-3-69, Nakamichi, Higashinari-ku, Osaka 537-0025, Japan

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BMC Infectious Diseases 2010, 10:118  doi:10.1186/1471-2334-10-118

Published: 15 May 2010

Abstract

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.