10.22376/ijpbs.2019.10.1.p1-12 Volume 7 Issue 4 2016 (October - December) Standard culture-based drug susceptibility testing (DST) of lessThan i greaterThan Mycobacterium tuberculosis lessThan /i greaterThan (MTB) to pyrazinamide (PZA) is difficult to perform. PZA is a prodrug that has to be converted to its active form pyrazinoic acid (POA) by pyrazinamidase (PZase) enzyme, encoded by lessThan i greaterThan pncA lessThan /i greaterThan gene and loss of PZase activity is associated with PZA resistance. To further define genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having lessThan i greaterThan pncA lessThan /i greaterThan mutations. A 100 MTB isolates were examined for PZA susceptibility and analyzed. MTB were identified by phenotypic & genotypic methods. lessThan i greaterThan pncA lessThan /i greaterThan genes from these strains were amplified. Mutations in the sequences of lessThan i greaterThan pncA lessThan /i greaterThan genes from PZA-resistant strains were identified by comparison with wild-type MTB gene sequence using CLC protein workbench version 5.0 software. The frequency of mutations and amino acid changes in the target lessThan i greaterThan pncA lessThan /i greaterThan gene were analyzed. Phylogenetic analysis was performed to know the strain variation among the drug resistant population. 34 among 100 had lessThan i greaterThan pncA lessThan /i greaterThan mutations including nucleotide substitutions (missense mutations), insertions (64.7%) and small deletions (nonsense mutations) (29.4%), causing amino acid substitutions in most cases; frame shifts leading to nonsense polypeptides (5.9%). Identified mutations found dispersed along lessThan i greaterThan pncA lessThan /i greaterThan gene, but some degree of clustering of mutations observed in Pro38Leu, Ile16Ser, Val150Ala and Asp97Asx regions. PCR based sequencing assay targeting lessThan i greaterThan pncA lessThan /i greaterThan mutations, can reliably detect PZA resistance in Multi Drug Resistant (MDR) and rules out PZA resistance in non-MDR MTB isolates. Molecular assays are probably the way forward for detecting PZA resistance. VAISHALI R WABALE, AMEETA A JOSHI, MUTHURAJ MUTHAIAH AND ABHAY S CHOWDHARY Pyrazinamide resistance, pncA gene sequence analysis, pncA gene mutations, Mycobacterium tuberculosis. 648-654