Chan Hsin-Hua, Wang Yu-Chen, Jou Ruwen
Tuberculosis Research Center, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Taiwan.
Reference Laboratory of Mycobacteriology, Centers for Disease Control, Ministry of Health and Welfare, Taipei, Taiwan.
J Clin Microbiol. 2024 Dec 11;62(12):e0122724. doi: 10.1128/jcm.01227-24. Epub 2024 Nov 18.
Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) treatment by eradicating the persisting complex (MTBC). Due to cost and technical challenges, end TB strategies are hampered by the lack of a simple and reliable culture-based PZA antimicrobial susceptibility testing (AST) for routine use. We initially developed a simplified chromogenic pyrazinamidase (PZase) test in the TB reference laboratory using a training set MTBC isolates with various drug-resistant profiles, and validated its performance using consecutive BACTEC MGIT 960 (MGIT)-culture-positive culture in 10 clinical laboratories. The gene Sanger sequencing results were used as the reference, and compared to the MGIT-PZA AST. Differential diagnosis of was conducted using patented in-house real-time PCR. Of the 106 training isolates, the PZase test and MGIT-PZA AST showed 100.0% and 99.1% concordance as compared to Sanger sequencing, respectively. We found 32.1% (34/106) isolates harbored mutations, including one isolate with silent mutation S65S. For validation, 1,793 clinical isolates were tested including 150 duplicate isolates from specimens of the same cases and 16 isolates with uncharacterized drug resistance (UDR)-associated mutations. Excluding duplicated and UDR isolates, we identified 2.6% (43/1,627) PZA-resistant isolates, including 1.3% (21/1,627) . isolates. The kappa values were 0.851-1.000. In addition, the accuracy of the PZase test conducted by 10 laboratories was 98.5%-100.0%. Our simplified PZase test demonstrated high concordance with Sanger sequencing and MGIT-PZA AST. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB.
We developed and validated a simple pyrazinamidase (PZase) test for pyrazinamide (PZA) antimicrobial susceptibility testing (AST). Our results demonstrated that the PZase test had high agreement with the gene sequencing and MGIT-PZA AST. Integrating PZase test into routine AST is effortless and represents an improvement in laboratory services for ending TB.
吡嗪酰胺（PZA）是治疗结核病（TB）的一种重要一线药物，可根除持留菌复杂菌群（MTBC）。由于成本和技术挑战，缺乏一种简单可靠的基于培养的PZA抗菌药物敏感性试验（AST）用于常规使用，这阻碍了终结结核病战略的实施。我们最初在结核病参考实验室开发了一种简化的显色吡嗪酰胺酶（PZase）试验，使用一组具有不同耐药谱的MTBC分离株进行训练，并在10个临床实验室使用连续的BACTEC MGIT 960（MGIT）培养阳性培养物对其性能进行验证。基因Sanger测序结果用作参考，并与MGIT-PZA AST进行比较。使用专利的内部实时PCR进行鉴别诊断。在106株训练分离株中，与Sanger测序相比，PZase试验和MGIT-PZA AST的一致性分别为100.0%和99.1%。我们发现32.1%（34/106）的分离株存在突变，包括1株沉默突变S65S的分离株。为了进行验证，对1793株临床分离株进行了检测，包括来自同一病例标本的150株重复分离株和16株具有未鉴定耐药性（UDR）相关突变的分离株。排除重复和UDR分离株后，我们鉴定出2.6%（43/1627）的PZA耐药分离株，包括1.3%（21/1627）的分离株。kappa值为0.851 - 1.000。此外，10个实验室进行的PZase试验的准确性为98.5% - 100.0%。我们简化的PZase试验与Sanger测序和MGIT-PZA AST显示出高度一致性。将PZase试验整合到常规一线AST中很容易，代表了终结结核病实验室服务的一项改进。
我们开发并验证了一种用于吡嗪酰胺（PZA）抗菌药物敏感性试验（AST）的简单吡嗪酰胺酶（PZase）试验。我们的结果表明，PZase试验与基因测序和MGIT-PZA AST具有高度一致性。将PZase试验整合到常规AST中很容易，代表了终结结核病实验室服务的一项改进。
