RNA恒温扩增实时检测技术与荧光定量PCR联合检测肺泡灌洗液对痰涂阴性肺结核的快速诊断价值

Value of RNA isothermal amplification real-time testing and fluorescence quantitative PCR combined with detection of bronchoalveolar lavage fluid in rapid diagnosis of sputum smear-negative pulmonary tuberculosis

目的通过采用RNA恒温扩增实时检测技术(FQ-PCR)两种不同类型靶标核酸检测方法联合检测支气管肺泡灌洗液(BALF)标本,评估联合应用对痰涂阴性肺结核的诊断价值。方法采集2014年11月-2016年5月医院收治的疑诊为肺结核并且痰涂片至少3次阴性的1 155例患者灌洗液标本,剔除295例未明确诊断病例,肺结核确诊患者672例纳入肺结核组,其他肺部疾病188例(包括43例非结核分枝杆菌疾病)纳入对照组,以临床诊断为金标准,计算SAT-TB与FQ-PCR联合检测的各类评价指标,并与单一SAT-TB、FQ-PCR、结核分枝杆菌快速培养(Bactec MGIT 960)、抗酸杆菌染色进行比较,绘制受试者工作特征曲线(ROC)进行分析比较。结果以临床诊断为"金标准",分别以AFS、MIGT960、SAT-TB、FQ-PCR、SAT-TB与FQ-PCR联合检测平行试验(单阳)、系列试验(双阳)为检验变量时,ROC曲线下面积(AUC)依次为0.507、0.501、0.670、0.660、0.734、0.658,准确性依次为29.45%、33.64%、58.67%、58.32%、67.40%、49.82%,SAT-TB和FQ-PCR单独检测或者联合检测其诊断价值均有统计学意义(P<0.001);对痰涂阴性肺结核病诊断价值比较,平行试验>SAT-TB>FQ-PCR>系列试验>涂片>MIGT960。结论与细菌学检测方法比较,SAT-TB与FQ-PCR联合检测痰涂阴性肺结核病诊断具有快速、敏感、鉴别结核分枝杆菌(MTB)与非结核分枝杆菌(NTM)等优势,缩短了确诊时间,提高准确率,给痰涂阴性肺结核患者提供一种有效辅助手段,值得临床推广应用。

OBJECTIVE To evaluate the value of detection of two types of target nucleic acids by RNA isothermal amplification real-time testing combined with detection of bronchoalveolar lavage fluid in diagnosis of sputum smear-negative pulmonary tuberculosis. METHODS From Nov 2014 to May 2016, the hronchoalveolar lavage fluid specimens were collected from 1 155 patients with suspected pulmonary tuberculosis who were tested negative for sputum smear for at least 3 times, 259 cases of undetermined diagnosis were excluded, 672 patients with confirmed pulmonary diagnosis were assigned as the pulmonary tuberculosis group, and 188 patients with other pul- monary diseases （including 43 cases of non-tuberculous mycobacteria diseases） were set as the control group. With the clinical diagnosis set as the golden standard, the evaluation indexes that were detected by SAT-TB combined with FQ-PCR were calculated and compared with single SAT-TB, FQ-PCR, rapid culture of Mycobacteriurn tuberculosis （Bactec MGIT 960）, and acid-fast bacilli staining; the receiver operating characteristic （ROC） curve was drawn for comparative analysis. RESULTS With the clinical diagnosis set as the golden standard, the areas under ROC curve of AFS, MIGT 960 culture, SAT-TB, FQ-PCR, SAT-TB combined with FQ-PCR parallel test （single positive）, and serail tests （double positive） were 0. 507,0. 501, 0. 670, 0. 660, 0. 734, and 0. 658, respectively; the accuracies were 29.45%, 33.64%, 58. 67%, 58.32%, 67. 40%, and 49.82%, respectively; there was significant difference in the value of diagnosis between the single detection of SAT-TB or FQ-PCR and the combined detection （P〈0.001） ; as compared with the value in diagnosis of sputum smear-negative pulmonary tuberculosis, the orders were as follows： parallel test, SAT-TB, FQ-PCR, serial tests, smear, MIGT960. CONCLUSION As compared with the bacteriological detection, SAT-TB Combined with FQ-PCR detection is rapid and sensitive in diagnosis of sputum-negative pulmonary tuberculosis and can discriminate from Mycobacterium tuberculosis （MTB） to non-tuberculous mycobacteria）, shorten the diagnosis time, raise the accuracy, and offer an effective auxiliary mean for diagnosis of the sputum smear-negative pulmonary tuberculosis, and it is worthy to be promoted in the hospital.