Molecular characterization of pyrazinamide resistance in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is a highly infectious disease that causes the second highest mortality rate in human worldwide. The emergence of multi-drug resistance tuberculosis (MDR-TB) leads to a major public health problem in controlling TB-caused mortality. Pyrazinamide (PZA) is an important first-line drug in the treatment of MDR-TB. However, since the challenge in performing susceptibility test on PZA, World Health Organization has not published any data on the prevalence of PZA resistance in Mycobacterium tuberculosis (M. tuberculosis). Since the occurrence of PZA resistance makes MDR-TB more difficult to treat with poor prognosis, rapid detection method in PZA resistance is urgently needed. Since pncA mutation is highly associated with up to 98% PZA resistant M. tuberculosis strains, it is worthwhile to develop rapid molecular method for detecting PZA resistance. This study aims to identify the mutations in PZA resistant M. tuberculosis strains. The first part of this study aims to characterize the pattern of pncA mutation among PZA-resistant and PZA-susceptible M. tuberculosis using Sanger sequencing method. Among all clinical isolates, 12 out of 29 cases of M. tuberculosis were resistant to PZA. All PZA-resistant M. tuberculosis strains harbored pncA mutation, whereas no known mutations were found among those PZA-susceptible strains, giving the positive predictive value to be 100%. Eight mutation patterns were found among 12 resistant isolates. Four of these pncA mutations have not been described previously by other studies. Study also characterizes the pattern of pncA mutation in 19 sputum specimens, with 2 mutation patterns found. Overall 10 mutation patterns were found in this study. Results show that the mutation of pncA gene is highly associated with PZA-resistant M. tuberculosis. Results also suggest the scattered and more extensive mutations in pncA gene that confer PZA resistance to M. tuberculosis.

The second and the last part of this study aims to evaluate the possibility of using molecular method to detect PZA resistance in routine clinical laboratory. Results show that using molecular sequencing to detect PZA resistance can shorten the turnaround time to about 3-4 working days. Since mutation of pncA was scattered along the entire pncA gene, using DNA sequencing approach may be the best strategy for the rapid detection of PZA resistance in M. tuberculosis.