1Medical Laboratory Technology Department, College of Applied Medical Sciences, Taibah University, Al-Madinah, Kingdom of Saudi Arabia.
2Biology Department, College of Science, Taibah University, Al-Madinah, Kingdom of Saudi Arabia.
Pol J Microbiol. 2023 Dec 16;72(4):421-431. doi: 10.33073/pjm-2023-040. eCollection 2023 Dec 1.
This study aimed to evaluate the accuracy of detecting drug-resistant complex (MTBC)-specific DNA in sputum specimens from 48 patients diagnosed with pulmonary tuberculosis. The presence of MTBC DNA in the specimens was validated using the GeneXpert MTB/RIF system and compared with a specific PCR assay targeting the IS and the 40 gene sequence fragments. Additionally, the results obtained by multiplex PCR assays to detect the most frequently encountered rifampin, isoniazid, and ethambutol resistance-conferring mutations were matched with those obtained by GeneXpert and phenotypic culture-based drug susceptibility tests. Of the 48 sputum samples, 25 were positive for MTBC using the GeneXpert MTB/RIF test. Nevertheless, the IS and 40 single-step PCR revealed the IS in 27 of the 48 sputum samples, while the 40 gene fragment was found in only 17 of them. Furthermore, multiplex PCR assays detected drug-resistant conferring mutations in 21 (77.8%) of the 27 samples with confirmed MTBC DNA, 10 of which contained single drug-resistant conferring mutations towards ethambutol and two towards rifampin, and the remaining nine contained double-resistant mutations for ethambutol and rifampin. In contrast, only five sputum specimens (18.5%) contained drug-resistant MTBC isolates, and two contained mono-drug-resistant MTBC species toward ethambutol and rifampin, respectively, and the remaining three were designated as multi-drug resistant toward both drugs using GeneXpert and phenotypic culture-based drug susceptibility tests. Such discrepancies in the results emphasize the need to develop novel molecular tests that associate with phenotypic non-DNA-based assays to improve the detection of drug-resistant isolates in clinical specimens in future studies.
本研究旨在评估 GeneXpert MTB/RIF 系统检测 48 例肺结核患者痰标本中耐药结核分枝杆菌（MTBC）特异性 DNA 的准确性，并与针对 IS 和 40 基因序列片段的特定 PCR 检测进行比较。此外，还将检测最常见的利福平、异烟肼和乙胺丁醇耐药相关突变的多重 PCR 检测结果与 GeneXpert 和表型基于培养的药敏试验结果相匹配。在 48 份痰样本中，25 份使用 GeneXpert MTB/RIF 试验检测到 MTBC 阳性。然而，IS 和 40 单步 PCR 显示 48 份痰样本中有 27 份存在 IS，而 40 个基因片段仅存在于其中 17 份。此外，多重 PCR 检测在 27 份确认含有 MTBC DNA 的样本中检测到 21 份（77.8%）耐药相关突变，其中 10 份样本含有单一的乙胺丁醇耐药相关突变，2 份样本含有单一的利福平耐药相关突变，其余 9 份样本含有乙胺丁醇和利福平的双重耐药相关突变。相比之下，只有 5 份痰标本（18.5%）含有耐药 MTBC 分离株，其中 2 份含有单一的乙胺丁醇和利福平耐药 MTBC 种，其余 3 份被 GeneXpert 和表型基于培养的药敏试验分别指定为对两种药物均耐药。这些结果的差异强调需要开发新的分子检测方法，与表型非 DNA 检测方法相结合，以提高未来研究中临床标本中耐药分离株的检测能力。
