title: Identification of Mycobacterium tuberculosis clinical isolates in Bangladesh by a species distinguishable multiplex PCR.
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.
authors.affiliation: Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Kita-ku, Sapporo, Japan. cnakajim@czc.hokudai.ac.jp
authors.forename: Chie
authors.initials: C
authors.lastname: Nakajima
authors.forename: Zeaur
authors.initials: Z
authors.lastname: Rahim
authors.forename: Yukari
authors.initials: Y
authors.lastname: Fukushima
authors.forename: Isamu
authors.initials: I
authors.lastname: Sugawara
authors.forename: Adri G M
authors.initials: AG
authors.lastname: van der Zanden
authors.forename: Aki
authors.initials: A
authors.lastname: Tamaru
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D001459:Bangladesh; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D009163:Mycobacterium bovis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; C480221:Cfp32 protein, Mycobacterium tuberculosis; D004269:DNA, Bacterial
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