Objective: To evaluate the application of a high-resolution melting (HRM) curve analysis for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis (M. tuberculosis).
Methods: Clinical isolates of M. tuberculosis were collected from Shenzhen from 2007 - 2009 and pncA mutations were characterized by sequence analysis. HRM analysis method was established and used to determine pncA gene mutation in 49 pncA mutant isolates and 78 wild type isolates. BCATEC MGIT 960 was used as the standard method to evaluate application of the HRM curve.
Results: Among 49 pncA mutant isolates, 48 isolates were characterized as mutant and 1 isolate with double point mutations misclassified as wild type by using HRM detection. In 78 wild type isolates, HRM found 6 mutant isolates. Using the BCATEC MGIT 960 to detect pyrazinamide drug susceptibility, 62 isolates were found resistant and 65 were susceptible. Among the 62 resistant strains by HRM results 53 strains were resistant, and among the 65 susceptible stains by HRM results 64 were susceptible. Compared to the results of BACTEC MGIT 960, the sensitivity and specificity of HRM were 85.5% (53/62) and 98.5% (64/65), respectively. The Youden index was found to be 0.840.
Conclusions: The results showed a good correlation between HRM curve analysis and BACTEC MGIT 960. HRM curve analysis is a rapid and accurate method that can be used as a screening test for identification of resistance to pyrazinamide.