School of Pharmaceutical Sciences, Biological Sciences Department, Paulista State University, Rodovia Araraquara-Jaú km 1, 14800-901 Araraquara, SP, Brazil.
Biomed Pharmacother. 2011 Sep;65(6):456-9. doi: 10.1016/j.biopha.2011.04.021. Epub 2011 Jun 12.
We determined the susceptibility profile of 80 Mycobacterium tuberculosis (MTB) clinical isolates from Brazil against isoniazid (INH) and rifampicin (RIF) drugs by two phenotypic methods (Resazurin Microtiter Assay - REMA and BACTEC™ MGIT™ Mycobacterial Detection System). DNA polymorphisms were also determined by PCR-SSCP in isolates resistant to INH and RIF. BACTEC™ MGIT™ 960 detected 22 susceptible isolates to INH and RIF, 48 MDR isolates (resistant at least to INH and RIF) and nine mono-resistant isolates (eight to INH and one to RIF). REMA performance was determined by Receiver Operating Characteristic curve, whose assay was validated utilizing as reference the BACTEC™ MGIT™ 960 system. ROC curve showed cut-off values of 0.0625μg/mL and 0.125μg/mL, for INH and RIF, respectively. REMA-INH demonstrated sensitivity and specificity of 100% while REMA-RIF showed sensitivity of 97.2% and specificity of 100%. PCR-SSCP detected DNA polymorphisms in 87.5% and 75.5% of isolates classified as INH-resistant and RIF-resistant, respectively. One discordant sample found to RIF (resistant by BACTEC™ MGIT™ 960 and susceptible by REMA) showed no mutation by PCR-SSCP. In conclusion, our studies demonstrated that the combination of phenotypic method REMA, which allowed rapid detection of MDR-MTB with higher levels of sensitivity and specificity, with the genotypic method PCR-SSCP, which demonstrated high accuracy in the search of polymorphisms in the resistance genes, proved to be a useful strategy to study MDR-MTB clinical isolates from national reference center located in São Paulo city.
我们通过两种表型方法（Resazurin 微量滴定法 - REMA 和 BACTEC™ MGIT™ 分枝杆菌检测系统）来确定 80 株巴西分枝杆菌（MTB）临床分离株对异烟肼（INH）和利福平（RIF）药物的敏感性。还通过 PCR-SSCP 确定了对 INH 和 RIF 耐药的分离株的 DNA 多态性。BACTEC™ MGIT™ 960 检测到 22 株 INH 和 RIF 敏感的分离株，48 株耐多药（至少对 INH 和 RIF 耐药）的分离株和 9 株单耐药（8 株对 INH，1 株对 RIF）的分离株。REMA 的性能通过接受者操作特征曲线确定，该曲线的测定是利用 BACTEC™ MGIT™ 960 系统作为参考进行验证的。ROC 曲线显示，INH 和 RIF 的临界值分别为 0.0625μg/mL 和 0.125μg/mL。REMA-INH 显示出 100%的敏感性和特异性，而 REMA-RIF 显示出 97.2%的敏感性和 100%的特异性。PCR-SSCP 检测到分别被归类为 INH 耐药和 RIF 耐药的分离株中 87.5%和 75.5%存在 DNA 多态性。一个被 BACTEC™ MGIT™ 960 检测为 RIF（耐药）而 REMA 检测为敏感的不一致样本未通过 PCR-SSCP 检测到突变。总之，我们的研究表明，表型方法 REMA（其可快速检测到具有更高敏感性和特异性的 MDR-MTB）与基因型方法 PCR-SSCP（其在耐药基因中检测到的多态性具有高度准确性）的组合，对于研究来自圣保罗国家参考中心的 MDR-MTB 临床分离株来说是一种有用的策略。
