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...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.展开更多
目的通过鉴定耐多药(multidrug-resistant,MDR)结核分枝杆菌中吡嗪酰胺(pyrazinamide,PZA)耐药的表型和基因型特性,为制定合适的治疗方案提供实验室依据。方法采用液体培养对耐多药结核分枝杆菌进行PZA药物敏感性测定。通过测序鉴定pnc...目的通过鉴定耐多药(multidrug-resistant,MDR)结核分枝杆菌中吡嗪酰胺(pyrazinamide,PZA)耐药的表型和基因型特性,为制定合适的治疗方案提供实验室依据。方法采用液体培养对耐多药结核分枝杆菌进行PZA药物敏感性测定。通过测序鉴定pncA的突变情况,并与标准菌株H37Rv的序列进行比较。结果75株耐多药结核菌中,52株(69.33%)为PZA表型耐药菌株。80.76%的准广泛耐药(pre-extensive drug resistant,pre-XDR)菌株、92.31%的广泛耐药(extensive drug resistant,XDR)菌株以及84.61%的对四种一线药物(异烟肼、利福平、乙胺丁醇、链霉素)耐药的菌株发生了PZA耐药。在PZA耐药菌株中45株(45/52,86.53%)的pncA基因发生了突变。在这些MDR菌株共存在40种不同类型的pncA的突变,包括33个点突变和7个插入缺失突变。在这些突变类型中,有17个为新发现位点。结论对四种一线药物全部耐药的菌株,准广泛耐药菌株和广泛耐药菌株的PZA具有较高耐药率。大多数PZA耐药与pncA相关,该突变散在分布且具有多样性。展开更多
文摘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.
文摘目的通过鉴定耐多药(multidrug-resistant,MDR)结核分枝杆菌中吡嗪酰胺(pyrazinamide,PZA)耐药的表型和基因型特性,为制定合适的治疗方案提供实验室依据。方法采用液体培养对耐多药结核分枝杆菌进行PZA药物敏感性测定。通过测序鉴定pncA的突变情况,并与标准菌株H37Rv的序列进行比较。结果75株耐多药结核菌中,52株(69.33%)为PZA表型耐药菌株。80.76%的准广泛耐药(pre-extensive drug resistant,pre-XDR)菌株、92.31%的广泛耐药(extensive drug resistant,XDR)菌株以及84.61%的对四种一线药物(异烟肼、利福平、乙胺丁醇、链霉素)耐药的菌株发生了PZA耐药。在PZA耐药菌株中45株(45/52,86.53%)的pncA基因发生了突变。在这些MDR菌株共存在40种不同类型的pncA的突变,包括33个点突变和7个插入缺失突变。在这些突变类型中,有17个为新发现位点。结论对四种一线药物全部耐药的菌株,准广泛耐药菌株和广泛耐药菌株的PZA具有较高耐药率。大多数PZA耐药与pncA相关,该突变散在分布且具有多样性。
