Characterization of pyrazinamide and ofloxacin resistance among drug resistant Mycobacterium tuberculosis isolates from Singapore.

OBJECTIVES: To evaluate rapid molecular approaches for the detection of pyrazinamide (PZA) and ofloxacin resistance, by screening 100 known drug-resistant Mycobacterium tuberculosis isolates. METHODS: Mycobacterium tuberculosis isolates were tested for phenotypic resistance to pyrazinamide and ofloxacin using the BACTEC 460 radiometric method and the E-test, respectively. Mutation screening was done by amplifying the pncA, gyrA, and gyrB genes by the polymerase chain reaction (PCR) and direct automated sequencing. RESULTS: Twelve isolates were PZA-resistant and 8 of 12 (66.7%) isolates had missense mutations or deletions at the pncA gene, suggesting that mutation or deletion at the pncA gene is the major molecular mechanism of PZA resistance among the Singaporean isolates. Using the E-test, 48 isolates were resistant to ofloxacin, with minimum inhibitory concentrations of 4 microg/mL or higher. No mutations were observed at the quinolone resistance-determining region (QRDR) of gyrA in all isolates. At the QRDR of gyrB, mutations were present in 1 of 48 ofloxacin-resistant isolates and 0 of 19 ofloxacin-susceptible isolates. CONCLUSIONS: In Singapore, genotypic analysis of resistance to PZA and ofloxacin is inadequate and should be complemented by conventional methods.