Abstract
Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.
Highlights
Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, its susceptibility is not routinely tested
Number of studies suggest that PZA resistance is not caused by a single mechanism, mutations in the pncA gene are considered as the main mechanism of PZA resistance in M. tuberculosis [4]
We describe an High-resolution melting (HRM) technique to detect pncA mutations in M. tuberculosis clinical isolates and compare its results to the phenotypic PZA susceptibility testing (The Bactec MGIT 960)
Summary
Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, its susceptibility is not routinely tested. HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, further modifications to improve its performance are required. The lack of reliable culture-based methods which require an acidic condition and high cost of the molecular techniques makes the testing of PZA resistance generally very difficult to perform [5]. It is very important to develop rapid and accurate drug susceptibility testing (DST) to prevent the spread of multi-drug resistant (MDR)-TB as well as extremely drug resistant (XDR)-TB
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