UNDERSTANDING THE VARIABILITY OF PNCA IN MYCOBACTERIUM TUBERCULOSIS CLINICAL ISOLATES BY MEANS OF VARIANT ANALYSIS, PHYLOGENETICS, AND MEASURING SELECTIVE PRESSURE

Includes bibliographical references (pages 47-56).Pyrazinamide (PZA) is an important first-line drug in combating pulmonary tuberculosis, caused by the agent Mycobacterium tuberculosis (Mtb). However, the frequency of PZA resistance is continuing to rise globally. Currently, the recommended platform to detect PZA susceptibility is a growth-based drug susceptibility test (DST). Unfortunately, Mtb is notoriously slow growing, requiring weeks to obtain results. An attractive alternative to DST are molecular assays, which analyze well-documented areas of the genome that are highly associated with drug resistance. The most widely used molecular assay is the Gene Xpert RIF/MTB test, which identifies TB disease and defines resistance to rifampicin. However, there is no such assay available to diagnose PZA resistance. Literature suggests mutations in pncA are associated with PZA resistance; therefore, a platform could be developed to analyze pncA to diagnose PZA resistance. A review was written as a part of this report to summarize and analyze mutations in pncA that were associated with PZA-resistance. The review demonstrated that mutations in 171 codons out of 187 were associated with PZA resistance. In addition, at least 14% of the global PZA-resistant TB population were not explained by mutations in pncA. Following the completion of the review, a set of 347 Mtb clinical strains from Global Consortium for Drug Resistant TB Diagnostics (GCDD) were analyzed for mutations in pncA. A total of 118 unique mutations were discovered in both resistant and susceptible isolates, 71 of which were amino acid subsitutions. The resulting sensitivity was 83%, close to the global statistic calculated in the review. Convergent evolution has been hypothesized to be concordant with drug resistance and was confirmed in this report in both gyrA and rrs, but was not confirmed in pncA. To better understand the importance of individual mutations in pncA, positive selection was calculated using a dN/dS method. Phylogenetic Analysis by Maximum Likelihood (PAML) calculated 48 positions to be under positive selection. Overall, these results warrant further research into other factors that may influence PZA resistance such as regulation, epigenetics, or missing steps in the mechanism of PZA resistance in Mtb