实时聚合酶链反应检测 mpb64 基因对肺外结核的诊断价值。
Evaluation of real time polymerase chain reaction targeting mpb64 gene for diagnosis of extrapulmonary tuberculosis.
机构信息
Department of Microbiology, JIPMER, Puducherry, India.
Department of Pathology, JIPMER, Puducherry, India.
出版信息
Indian J Tuberc. 2021 Apr;68(2):242-248. doi: 10.1016/j.ijtb.2020.09.009. Epub 2020 Sep 12.
BACKGROUND
Paucibacillary nature of extrapulmonary tuberculosis (EPTB) has paved way for molecular methods increasingly being used for diagnosis. We undertook a study for evaluation of sensitivity and specificity of real-time polymerase chain reaction (RT-PCR) targeting mpb64 gene for diagnosis of EPTB.
METHODS
A total of 152 clinical samples from suspected cases of EPTB were included in this study. All samples were extracted using spin column based commercial DNA extraction kit and were subjected to RT-PCR targeting mpb64 and IS6110. Smear and culture was also done for samples whenever quantity was sufficient. Cytology report was noted from hospital information system. Receiver operating characteristic (ROC) curve analysis was done for determining cut-off Ct value for mpb64 RT-PCR. Melt curve analysis was done for samples whose cycle threshold (Ct) value was more than 37. The sensitivity and specificity of the mpb64 RT-PCR was calculated using a composite gold standard i.e., positive for one or more of the following: microscopy (including fine needle aspiration cytology (FNAC), acid-fast bacilli positivity), culture and IS6110 RT-PCR.
RESULTS
Out of the 152 samples, 72 (47.4%) were positive for tuberculosis by composite gold standard. Samples consisted of ascitic fluid (12), CSF (35), pus (23), lymph node aspirate (35), pleural fluid (37), synovial fluid (4), urine (1), pericardial fluid (1) and tissue bits (4). Microscopy (AFB smear including lymph node aspirate) was done for 124 samples of which 43 (34.7%) were positive. Culture results were available for 79 samples, 25 (31.6%) of which were positive and 42 (27.6%) of the 152 samples were positive by IS6110 PCR. Based on ROC and melt curve analysis, mpb64 RT-PCR was able to detect 38 (52.8%) of the 72 positive samples. In comparison to IS6110 RT PCR, 4 additional cases were detected by mpb64 RT-PCR. Compared to composite gold standard mpb64 showed overall sensitivity of 52.8%.
CONCLUSION
The mpb64 RT-PCR is highly specific or MTB and can be used as a supplemental test for diagnosis of EPTB along with other diagnostic tests. However the overall sensitivity of mpb64 RT-PCR is too low to be used as an independent test for diagnosis of EPTB. Combining the results of IS6110 RT PCR and mpb64 RT PCR improved the overall sensitivity and hence mpb64 can be used as an additional target for diagnosis of EPTB.
背景
肺外结核（EPTB）的少菌性特征为越来越多地使用分子方法进行诊断铺平了道路。我们进行了一项研究，以评估针对 mpb64 基因的实时聚合酶链反应（RT-PCR）检测 EPTB 的敏感性和特异性。
方法
本研究纳入了 152 例疑似 EPTB 临床样本。所有样本均采用基于离心柱的商业 DNA 提取试剂盒提取，并进行针对 mpb64 和 IS6110 的 RT-PCR。只要样本数量充足，就会进行涂片和培养。从医院信息系统中记录细胞学报告。进行受试者工作特征（ROC）曲线分析，以确定 mpb64 RT-PCR 的临界 Ct 值。对 Ct 值大于 37 的样本进行熔解曲线分析。使用复合金标准（即一种或多种以下检测结果阳性：显微镜检查（包括细针抽吸细胞学检查（FNAC）、抗酸杆菌阳性）、培养和 IS6110 RT-PCR）计算 mpb64 RT-PCR 的敏感性和特异性。
结果
在 152 例样本中，72 例（47.4%）经复合金标准检测为结核病阳性。样本包括腹水（12 例）、脑脊液（35 例）、脓液（23 例）、淋巴结抽吸物（35 例）、胸腔积液（37 例）、滑液（4 例）、尿液（1 例）、心包液（1 例）和组织块（4 例）。对 124 例样本进行了显微镜检查（包括淋巴结抽吸物的抗酸杆菌涂片），其中 43 例（34.7%）阳性。对 79 例样本进行了培养，其中 25 例（31.6%）阳性，152 例样本中 42 例（27.6%）经 IS6110 PCR 检测为阳性。基于 ROC 和熔解曲线分析，mpb64 RT-PCR 能够检测到 72 例阳性样本中的 38 例（52.8%）。与 IS6110 RT-PCR 相比，mpb64 RT-PCR 还检测到 4 例额外病例。与复合金标准相比，mpb64 显示出总体敏感性为 52.8%。
结论
mpb64 RT-PCR 对 MTB 具有高度特异性，可与其他诊断试验一起作为 EPTB 诊断的辅助试验。然而，mpb64 RT-PCR 的总体敏感性太低，不能作为 EPTB 诊断的独立试验。结合 IS6110 RT-PCR 和 mpb64 RT-PCR 的结果可提高总体敏感性，因此 mpb64 可作为 EPTB 诊断的附加靶标。
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