Alp Alpaslan, Sarıbaş Zeynep
Hacettepe University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey.
Mikrobiyol Bul. 2019 Oct;53(4):355-363. doi: 10.5578/mb.67783.
One of the most important steps for the control of tuberculosis is rapid and accurate detection of Mycobacterium tuberculosis in clinical samples. The early and accurate diagnosis of tuberculosis allows the initiation of the effective treatment regimen as early as possible. However the early diagnosis of tuberculosis can be achieved by the integration of molecular methods into the diagnostic algorithm of tuberculosis together with the gold standard culture methods. For this reason, molecular methods have become valuable diagnostic tools in routine diagnostic laboratories in recent years. The aim of this study was to determine the diagnostic efficacy of Anyplex MTB/NTM test (Seegene, South Korea) used for the molecular diagnosis of tuberculosis in routine molecular diagnostic laboratories. In addition to this aim, a preliminary evaluation of in-house polymerase chain reaction (PCR) primers that was designed to produce a kit as an alternative against imported commercial kits was performed. Ten thousand six hundred fifthy two clinical specimens that were collected from suspected tuberculosis cases in three years were included in the study. All samples were tested by microscopic examination after staining, culture and real-time PCR (Rt-PCR) methods. The smears were examined by microscope after staining with Kinyoun method for the existence of acid resistant bacilli. For culture, following the N-acetyl-L-sistein-sodium hydroxide homogenization and decontamination procedure, the samples were inoculated into the MGIT (Mycobacteria Growth Indicator Tube) tubes (Becton Dickinson, USA). Rt-PCR method was performed by using Anyplex MTB/NTM test. In the first stage of the study, the performance of the Anyplex MTB/NTM test was compared with the gold standard culture method. M.tuberculosis was isolated in 178 specimens out of 10.652 (1.7%). After the comparison with the gold standard culture method, the sensitivity and specificity of Anyplex MTB/NTM test was found to be 84% and 99% respectively in pulmonary samples, and 74% and 99% respectively in extrapulmonary samples. In the second stage of the study, PCR method with laboratory designed primers was applied to 100 culture positive samples. The PCR results of 98 samples were found to be in agreement with the culture, while M.tuberculosis DNA was not detected in two samples. As a result of the study it was concluded that Anyplex MTB/NTM test is a rapid, practical and reliable method that can be used in routine tuberculosis diagnosis. The high agreement between PCR method using the laboratory-designed primers and the PCR method used in routine practice will lighten the way for the development of national tuberculosis molecular diagnostic kits with a relevant cost. In this way, it will be possible to perform rapid diagnosis in a more cost-effective manner in routine diagnosis laboratories.
控制结核病最重要的步骤之一是在临床样本中快速准确地检测结核分枝杆菌。结核病的早期准确诊断能够尽早启动有效的治疗方案。然而，将分子方法与金标准培养方法相结合纳入结核病诊断算法，才能实现结核病的早期诊断。因此，近年来分子方法已成为常规诊断实验室中宝贵的诊断工具。本研究的目的是确定用于常规分子诊断实验室结核病分子诊断的Anyplex MTB/NTM检测（韩国Seegene公司）的诊断效能。除该目的外，还对设计用于生产试剂盒以替代进口商业试剂盒的内部聚合酶链反应（PCR）引物进行了初步评估。本研究纳入了从三年内疑似结核病病例中收集的10652份临床标本。所有样本均通过染色后的显微镜检查、培养和实时PCR（Rt-PCR）方法进行检测。涂片用金胺酚染色法染色后，在显微镜下检查是否存在抗酸杆菌。培养时，经N-乙酰-L-半胱氨酸-氢氧化钠匀浆和去污程序处理后，将样本接种到美国BD公司的分枝杆菌生长指示管（MGIT）管中。使用Anyplex MTB/NTM检测进行Rt-PCR检测。在研究的第一阶段，将Anyplex MTB/NTM检测的性能与金标准培养方法进行比较。在10652份标本中有178份（1.7%）分离出结核分枝杆菌。与金标准培养方法比较后发现，Anyplex MTB/NTM检测在肺部样本中的敏感性和特异性分别为84%和99%，在肺外样本中分别为74%和99%。在研究的第二阶段，将实验室设计引物的PCR方法应用于100份培养阳性样本。发现98份样本的PCR结果与培养结果一致，而两份样本未检测到结核分枝杆菌DNA。研究结果表明，Anyplex MTB/NTM检测是一种可用于常规结核病诊断的快速、实用且可靠的方法。使用实验室设计引物的PCR方法与常规实践中使用的PCR方法之间的高度一致性，将为开发具有相应成本效益的国家结核病分子诊断试剂盒铺平道路。这样，在常规诊断实验室中就可以以更具成本效益的方式进行快速诊断。
