Dept of Microbiology, The University of Hong Kong, Queen Mary Hospital, Hong Kong

Sixty two strains of Mycobacterium tuberculosis and 285 pulmonary and extra-pulmonary samples were collected for polymerase chain reaction (PCR) to amplify the katG, inhA and ahpC in Mycobacterium tuberculosis. PCR-RFLP of the katG amplicon was studied by restriction digestion using MspI. InhA and ahpC amplicons were studied by DNA sequencing. All the 62 strains of Mycobacterium tuberculosis were PCR positive for all three genes. Among the 285 clinical samples, 217 samples were culture positive for Mycobacterium tuberculosis among which 198 were PCR positive and 178 were positive for Acid Fast Bacilli (AFB). The other 68 samples exhibited negative PCR result among which 36 were culture negative for Mycobacterium and 32 were confirmed to be MOTT. PCR-RFLP of the katG amplicon identified 50% of all INH resistant strains whereas DNA sequencing for InhA and ahpC amplicons identified an additional 6% of INH resistance in Mycobacterium tuberculosis. For samples culture positive for Mycobacterium tuberculosis, PCR showed 100% and 68% sensitivity for AFB positive and negative samples respectively. Direct detection of INH resistant Mycobacterium tuberculosis in clinical samples provides rapid laboratory diagnosis and PCR-RFLP of the katG amplicon is especially suitable for routine laboratory with limited setup for molecular biology.