二代测序技术检测临床痰标本中结核分枝杆菌的初步评价

doi:10.19982/j.issn.1000-6621.20220097

摘要/Abstract

摘要： 目的评价二代测序(NGS)技术对临床痰标本中结核分枝杆菌的检测效能。方法采用前瞻性研究方法,选取北京市疾病预防控制中心(北京结核病控制研究与防治所)2021年8—10月接诊的49例疑似肺结核患者痰标本,同时进行抗酸杆菌涂片镜检(简称“涂片”)、分枝杆菌培养(包含罗氏培养和MGIT 960液体培养;简称“培养”)、GeneXpert MTB/RIF(简称“Xpert”)和NGS技术检测结核分枝杆菌,比较4种方法检测不同分类患者阳性率差异,并以最终临床诊断为参照标准,评价4种检测方法的检测效能。结果49例疑似肺结核患者中,最终肺结核诊断者40例(包括病原学确诊患者25例、病原学阴性临床诊断患者15例),非肺结核患者9例(包括肺炎6例,非结核分枝杆菌感染、慢性阻塞性肺疾病和哮喘各1例)。NGS技术检测49例疑似肺结核患者的阳性率[69.4%(34/49)]明显高于涂片[44.9%(22/49)]、培养[51.0%(25/49)]和Xpert[49.0%(24/49)],差异均有统计学意义(χ²=17.614、17.018、20.753,P值均=0.000);检测病原学阴性肺结核患者的阳性检出率为46.7%(7/15)。以临床诊断结果为参照标准,涂片、培养、Xpert、NGS技术对49例疑似患者痰标本的检测敏感度分别为55.0%(22/40)、60.0%(24/40)、60.0%(24/40)、80.0%(32/40),特异度分别为9/9、8/9、9/9、7/9,一致率分别为63.3%(31/49)、65.3%(32/49)、67.3%(33/49)、79.6%(39/49),Kappa值分别为0.310、0.297、0.355、0.459。结论以临床诊断为参考标准,NGS技术检测的敏感度最高,与临床诊断的一致性也最高,能够快速、高效检测痰标本中的结核分枝杆菌,可早期辅助诊断疑似肺结核患者。

关键词: 分枝杆菌,结核, 诊断技术和方法, 对比研究

Abstract:

Objective: To evaluate detection efficacy of next-generation sequencing (NGS) to Mycobacterium tuberculosis from clinical sputum samples. Methods: Sputum samples of 49 suspected pulmonary tuberculosis patients diagnosed in Tuberculosis Outpatient Department of Beijing Center for Disease Prevention and Control (Beijing Center for Tuberculosis Research and Control) were collected from August to November, 2021. Suspected pulmonary tuberculosis were detected from sputum samples with acid-fast staining (smear),L-J culture and MGIT 960 liquid culture (culture), GeneXpert MTB/RIF (Xpert) and NGS methods. The positive detection rates of 4 kinds of methods were compared in the selected patients. We also evaluated the detection efficacy of different methods based on the results of clinical diagnosis. Results: There were 40 pulmonary tuberculosis patients (25 confirmed pathogen-positive pulmonary tuberculosis patients and 15 clinical diagnosed cases) and 9 non-pulmonary tuberculosis patients (6 cases of pneumonia, 1 case of nontuberculous mycobacteria infection, 1 case of COPD and 1 case of asthma). The positive detection rates are statistically difference (χ²=17.614,17.018,20.753;Ps=0.000) among smear 44.9% (22/49), culture 51.0% (25/49), Xpert 49.0% (24/49) and NGS 69.4% (34/49). For etiology-negative pulmonary tuberculosis patients, the positive detection rate by the NGS was 46.7% (7/15). Sensitivity were 55.0% (22/40), 60.0% (24/40), 60.0% (24/40), 80.0% (32/40), and specificity were 9/9, 8/9, 9/9, 7/9, and concordance rate were 63.3% (31/49), 65.3% (32/49), 67.3% (33/49), 79.6% (39/49), Kappa values were 0.310,0.297,0.355,0.459 based on smear, culture, Xpert and NGS respectively. Conclusion: NGS has the highest sensitivity and consistency based on the national clinical diagnosis standard of pulmonary tuberculosis, which can diagnose suspected pulmonary tuberculosis earlier and detect Mycobacterium tuberculosis from sputum samples rapidly and effectively.

Key words: Mycobacterium tuberculosis, Diagnostic techniques and procedures, Comparative study

代小伟, 王嫩寒, 陈双双, 杨新宇, 田丽丽, 陈红, 张泓泰, 李传友. 二代测序技术检测临床痰标本中结核分枝杆菌的初步评价[J]. 中国防痨杂志, 2022, 44(7): 669-679. doi: 10.19982/j.issn.1000-6621.20220097

DAI Xiao-wei, WANG Nen-han, CHEN Shuang-shuang, YANG Xin-yu, TIAN Li-li, CHEN Hong, ZHANG Hong-tai, LI Chuan-you. Assessing next-generation sequencing for Mycobacterium tuberculosis diagnosis in clinical sputum samples[J]. Chinese Journal of Antituberculosis, 2022, 44(7): 669-679. doi: 10.19982/j.issn.1000-6621.20220097

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患者分类	总例数	涂片[例(阳性率,%)]	培养[例(阳性率,%)]	Xpert[例(阳性率,%)]	NGS[例(阳性率,%)]	χ²值	P值
肺结核	40	22(44.9)	24(49.0)	24(49.0)	32(65.3)	6.383	0.094
病原学阳性确诊	25	22(88.0)	24(96.0)	24(96.0)	25(100.0)	4.000	0.261
病原学阴性临床诊断	15	0(0.0)	0(0.0)	0(0.0)	7(46.7)	23.774	0.000
非肺结核	9	0(0.0)	1(11.1)	0(0.0)	2(22.2)	4.000	0.261
肺炎	6	0(0.0)	0(0.0)	0(0.0)	1(16.7)	-	-
非结核分枝杆菌病	1	0(0.0)	1(100.0)	0(0.0)	0(0.0)	-	-
慢性阻塞性肺疾病	1	0(0.0)	0(0.0)	0(0.0)	1(16.7)	-	-
哮喘	1	0(0.0)	0(0.0)	0(0.0)	0(0.0)	-	-