Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

背景：结核病（TB）的早期诊断，以及鉴定结核分枝杆菌（Mycobacterium tuberculosis）对各类抗结核药物的耐药菌株，被认为是结核病防控的核心要素。目的：标准化实时聚合酶链反应（real-time polymerase chain reaction，qPCR）检测技术，并将其应用于直接在齐-尼染色玻片（Ziehl-Neelsen stained slides，ZN）上鉴定与结核分枝杆菌对异烟肼（Isoniazid，INH）耐药相关的基因突变。方法：本研究对55份从结核分枝杆菌临床分离株中提取的独立DNA样本进行测序分析。为将该技术应用于结核病耐药诊断，本研究共纳入59份齐-尼染色玻片。本研究测定了该检测方法的灵敏度、特异度及Kappa值，并给出其95%置信区间（CI95%）。结果：以katG基因为靶点时，两种检测方法的一致性Kappa值为0.89（CI95%：0.7~1.0），对应的灵敏度与特异度分别为97.6%（CI95%：87.7~99.9）与91.7%（CI95%：61.5~99.5）。针对inhA靶点，Kappa值为0.92（CI95%：0.8~1.0），灵敏度与特异度分别为94.4%（CI95%：72.7~99.8）与97.3%（CI95%：85.8~99.9）。与其他耐药结核病标准检测方法相比，采用齐-尼染色玻片进行耐药结核病检测可获得显著性检测结果。主要结论：实时聚合酶链反应（qPCR）基因分型技术可有效检测赋予结核分枝杆菌对异烟肼（INH）耐药性的相关基因。因此，qPCR基因分型可作为测序技术的替代检测方案。