Characterization of pyrazinamide and ofloxacin resistance among drug resistant Mycobacterium tuberculosis isolates from Singapore.
OBJECTIVE
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.
Lee
Ann S G
AS
Department of Clinical Research, Singapore General Hospital, Singapore. dmslsg@nccs.com.sg
Tang
Lynn L H
LL
Lim
Irene H K
IH
Wong
Sin Yew
SY
D000581:Amidohydrolases / Q000235:genetics; D000890:Anti-Infective Agents / Q000494:pharmacology*; D000995:Antitubercular Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D015242:Ofloxacin / Q000494:pharmacology*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D011718:Pyrazinamide / Q000494:pharmacology*; D012846:Singapore
D000890:Anti-Infective Agents; D000995:Antitubercular Agents; D004269:DNA, Bacterial; D011718:Pyrazinamide; D015242:Ofloxacin; D000581:Amidohydrolases; C022490:pyrazinamide deamidase; D027081:DNA Gyrase