Characterization of pyrazinamide resistance among presumptive multidrug-resistant tuberculosis cases by targeted sequencing

Background and objectives Pyrazinamide (PZA) is a vital anti-tubercular drug used to treat multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). PZA resistance is a serious concern for the global tuberculosis (TB) control programme. In this study, we aimed to determine the prevalence of PZA resistance among presumptive MDR-TB patients and characterise it by targeted DNA sequencing to study the correlation between phenotypic and genotypic data. Methods In the present study, 2,631 smear-positive, presumptive MDR-TB patients were enrolled and tested using the line probe assay (LPA). Further, a subset (n=534/2631) of MDR/RR-TB or INH (isoniazid) -mono-resistant and RIF (rifampicin) - and INH-sensitive samples, as identified by LPA, was subjected to mycobacteria growth indicator tube (MGIT) culture followed by PZA drug susceptibility testing. Targeted DNA sequencing of genes associated with PZA resistance was performed in PZA-resistant isolates (n=83/534). Results The percentage of PZA resistance varied significantly among the RR-TB/MDR-TB isolates (45.6%) as compared to RIF and INH-sensitive isolates (5%). Correlation of PZA resistance was significant with RR-TB/MDR-TB ( P <0.001). In the DNA sequencing of the pncA gene, 72 isolates were found to be mutant (n=10 had S65S, a silent mutation), and n=3 were wild type (WT). Subsequently, pncA WT isolates and isolates with the S65S silent mutation were sequenced for panD and rpsA genes; however, all 13 samples were also found to be wild-type for both these genes. Interpretation and conclusions The high frequency of PZA resistance underscores the need for routine testing for PZA resistance, preferably by genotypic DST (targeted sequencing), which provides results with a short turnaround time. However, additional studies are also needed to determine the role of other genes that may be associated with PZA resistance.