Unité de Biologie et Recherché Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Rabat, Morocco ; Laboratoire de Microbiologie, Hygiène et Virologie, Faculté des Sciences et Techniques, Mohammedia, Morocco.
Unité de Biologie et Recherché Médicale, Centre National de l'Energie, des Sciences et des Techniques Nucléaires (CNESTEN), Rabat, Morocco.
Infect Drug Resist. 2013 Nov 28;6:207-13. doi: 10.2147/IDR.S47724. eCollection 2013.
Tuberculosis (TB) is a major public health problem with high mortality and morbidity rates, especially in low-income countries. Disturbingly, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) TB cases has worsened the situation, raising concerns of a future epidemic of virtually untreatable TB. Indeed, the rapid diagnosis of MDR TB is a critical issue for TB management. This study is an attempt to establish a rapid diagnosis of MDR TB by sequencing the target fragments of the rpoB gene which linked to resistance against rifampicin and the katG gene and inhA promoter region, which are associated with resistance to isoniazid.
For this purpose, 133 sputum samples of TB patients from Morocco were enrolled in this study. One hundred samples were collected from new cases, and the remaining 33 were from previously treated patients (drug relapse or failure, chronic cases) and did not respond to anti-TB drugs after a sufficient duration of treatment. All samples were subjected to rpoB, katG and pinhA mutation analysis by polymerase chain reaction and DNA sequencing.
Molecular analysis showed that seven strains were isoniazid-monoresistant and 17 were rifampicin-monoresistant. MDR TB strains were identified in nine cases (6.8%). Among them, eight were traditionally diagnosed as critical cases, comprising four chronic and four drug-relapse cases. The last strain was isolated from a new case. The most recorded mutation in the rpoB gene was the substitution TCG > TTG at codon 531 (Ser531 Leu), accounting for 46.15%. Significantly, the only mutation found in the katG gene was at codon 315 (AGC to ACC) with a Ser315Thr amino acid change. Only one sample harbored mutation in the inhA promoter region and was a point mutation at the -15p position (C > T).
The polymerase chain reaction sequencing approach is an accurate and rapid method for detection of drug-resistant TB in clinical specimens, and could be of great interest in the management of TB in critical cases to adjust the treatment regimen and limit the emergence of MDR and XDR strains.
结核病（TB）是一个重大的公共卫生问题，具有高死亡率和发病率，特别是在低收入国家。令人担忧的是，耐多药（MDR）和广泛耐药（XDR）结核病病例的出现使情况恶化，引发了对未来几乎无法治疗的结核病流行的担忧。事实上，快速诊断 MDR-TB 是结核病管理的一个关键问题。本研究试图通过测序 rpoB 基因的靶片段来快速诊断 MDR-TB，该基因与利福平耐药相关，而 katG 基因和 inhA 启动子区域与异烟肼耐药相关。
为此，从摩洛哥招募了 133 例结核病患者的痰液样本。其中 100 例来自新病例，其余 33 例来自以前治疗过的患者（药物复发或失败、慢性病例），在充分的抗结核药物治疗后对药物没有反应。所有样本均通过聚合酶链反应和 DNA 测序进行 rpoB、katG 和 pinhA 突变分析。
分子分析显示，7 株为异烟肼单耐药，17 株为利福平单耐药。在 9 例（6.8%）中发现了 MDR-TB 菌株。其中，8 例被传统诊断为危重症，包括 4 例慢性病例和 4 例药物复发病例。最后一株分离自新病例。rpoB 基因中记录最多的突变是密码子 531 处 TCG > TTG 的取代（Ser531 Leu），占 46.15%。值得注意的是，在 katG 基因中发现的唯一突变是密码子 315（AGC 突变为 ACC），导致 Ser315Thr 氨基酸变化。只有一个样本在 inhA 启动子区域发生突变，是 -15p 位置的点突变（C > T）。
聚合酶链反应测序方法是一种准确、快速的方法，可用于检测临床标本中的耐药性结核病，对于管理危重症结核病患者调整治疗方案、限制 MDR 和 XDR 菌株的出现具有重要意义。
