Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance.