Application of isothermal amplification fluorescence method for rapid detection of Mycobacterium tuberculosis complex

doi:10.19982/j.issn.1000-6621.20220221

Abstract

Abstract:

Objective: To evaluate the clinical application value of isothermal amplification fluorescence method for detection of Mycobacterium tuberculosis complex (MTBC) in sputum samples. Methods: Between January and November 2020, a total of 1848 sputum samples from suspected pulmonary tuberculosis patients in Shanghai 6 designated tuberculosis hospitals were respectively tests by isothermal amplification fluorescence, sputum smear, BACTEC MGIT 960 liquid culture and GeneXpert MTB/RIF (GeneXpert). Test effectiveness of isothermal amplification fluorescence, sputum smear and GeneXpert were compared according to liquid culture method. Results: Of all the 1848 sputum samples, the positive detection rates of isothermal amplification fluorescence, sputum smear, liquid culture and GeneXpert were 16.7% (309/1848), 14.4% (266/1848), 25.4% (470/1848) and 28.7% (530/1848), respectively. Based on liquid culture, the sensitivity of isothermal amplification fluorescence, sputum smear and GeneXpert were 58.94% (277/470), 52.77% (248/470) and 80.43% (378/470), respectively. The specificity of isothermal amplification fluorescence, sputum smear and GeneXpert were 97.68% (1346/1378), 98.69% (1360/1378) and 88.97% (1226/1378), respectively. The concordance rate of the isothermal amplification fluorescence, sputum smear and GeneXpert were 87.82% (1623/1848), 87.01% (1608/1848) and 86.80% (1604/1848), respectively. The Kappa value were 0.638, 0.600 and 0.666, respectively. Among the four sputum samples with different characteristics (mucoid sputum, salivary sputum, caseous sputum and blood sputum), the highest detection rate was by GeneXpert with 51.5% (308/598), 66.7% (28/42) and 64.0% (16/25) respectively, and the highest detection rate in salivary sputum was liquid culture with 38.8% (59/152). Conclusion: The isothermal amplification fluorescence detection method showed good specificity for detecting MTBC in sputum samples, and the result was general consistent with the liquid culture for detecting MTBC. Multiple detection methods should be considered according to laboratory and patient’s condition to improve the pathogenic diagnosis rate of pulmonary tuberculosis.

Key words: Mycobacterium tuberculosis, Sputum, Nucleic acid amplification techniques, Diagnosis, differential

CLC Number:

R521
R446.5

Li Jing, Wu Zheyuan, Yang Jinghui, Yang Liyuan, Wang Lili, Yuan Feng, Shen Xin, Jiang Yuan. Application of isothermal amplification fluorescence method for rapid detection of Mycobacterium tuberculosis complex[J]. Chinese Journal of Antituberculosis, 2022, 44(11): 1167-1173. doi: 10.19982/j.issn.1000-6621.20220221

Figures/Tables 3

References 18

[1]	World Health Organization. Global tuberculosis report 2021. Geneva: World Health Organization, 2021.
[2]	World Health Organization. Updated on the use of nucleic acid amplification tests to detect TB and drug-resistant TB: rapid communication. Geneva: World Health Organization, 2021.
[3]	中华人民共和国国家卫生和计划生育委员会. WS 288—2017 肺结核诊断. 2017-11-09.
[4]	中华人民共和国卫生部疾病预防控制局, 中华人民共和国卫生部医政司, 中国疾病预防控制中心. 中国结核病防治规划实施工作指南(2008年版). 北京: 中国协和医科大学出版社, 2009:20.
[5]	赵雁林, 逄宇. 结核病实验室检验规程. 北京: 人民卫生出版社, 2015: 5-10, 39-44.
[6]	赵雁林, 姜广路. 痰涂片镜检标准化操作及质量保证手册. 北京: 中国协和医科大学出版社, 2009: 7-11.
[7]	夏辉, 赵雁林. 世界卫生组织《关于更新使用结核分枝杆菌及其耐药性核酸扩增检测技术的建议》的启示. 中国防痨杂志, 2021, 43(8):761-765. doi: 10.3969/j.issn.1000-6621.2021.08.002. doi: 10.3969/j.issn.1000-6621.2021.08.002
[8]	World Health Organization. WHO consolidated guidelines on tuberculosis. Module 3: diagnosis-rapid diagnostics for tuberculosis detection. Geneva: World Health Organization, 2020.
[9]	马晓光, 郑丹薇, 朱岩昆, 等. 恒温扩增荧光检测技术在基层结核病实验室的应用价值. 中国防痨杂志, 2018, 40(10):1071-1074. doi: 10.3969/j.issn.1000-6621.2018.10.008. doi: 10.3969/j.issn.1000-6621.2018.10.008
[10]	杨健, 张天华, 鲜小萍, 等. 环介导等温扩增技术在肺结核病诊断中的应用评价. 中国实验诊断学, 2018, 22(12):2095-2098. doi: 10.3969/j.issn.1007-4287.2018.12.016. doi: 10.3969/j.issn.1007-4287.2018.12.016
[11]	韩珍, 朱惠慧, 范远珍. 迪澳结核分枝杆菌恒温核酸检测法,Xpert MTB/RIF检测法与传统固体罗氏培养法的比较. 牡丹江医学院学报, 2019, 40(5):36-38.
[12]	徐银娟, 赵国连, 崔晓利, 等. 新型国产改良Middlebrook液体培养试剂对结核分枝杆菌培养效果的评价. 中国防痨杂志, 2022, 44(1):60-63. doi: 10.19982/j.issn.1000-6621.20210459. doi: 10.19982/j.issn.1000-6621.20210459
[13]	李金莉, 王峰, 彭毅, 等. 环介导等温扩增技术检测痰样结核分枝杆菌临床价值评价. 中国防痨杂志, 2015, 37(2):134-139. doi: 10.3969/j.issn.1000-6621.2015.02.004. doi: 10.3969/j.issn.1000-6621.2015.02.004
[14]	胡典琴, 张美芳. 痰诱导技术联合振动排痰仪在无痰或少痰肺结核患者痰标本留取中的应用. 养生保健指南, 2018(28): 296. doi: 10.3969/j.issn.1006-6845.2018.28.282. doi: 10.3969/j.issn.1006-6845.2018.28.282
[15]	丁卫忠, 陈巍, 石莲, 等. 环介导等温扩增法对痰标本中结核分枝杆菌检测效果的评估. 中国防痨杂志, 2016, 38(10):818-822. doi: 10.3969/j.issn.1000-6621.2016.10.007. doi: 10.3969/j.issn.1000-6621.2016.10.007
[16]	陈涛, 周琳, 周杰, 等. 环介导等温扩增法快速检测结核分枝杆菌的临床应用评估. 中国防痨杂志, 2012, 34(7):413-418.
[17]	宋红焕, 邵燕, 李燕, 等. 两种分子诊断技术对提高结核病病原学检测阳性率的评价研究. 结核病与肺部健康杂志, 2019, 8(3):168-171. doi: 10.3969/j.issn.2095-3755.2019.03.004. doi: 10.3969/j.issn.2095-3755.2019.03.004
[18]	田丽丽, 杨新宇, 代小伟, 等. 免疫学检测联合痰涂片和痰培养检测在活动性肺结核临床诊断中的价值. 中国防痨杂志, 2021, 43(10):1073-1078. doi: 10.3969/j.issn.1000-6621.2021.10.017. doi: 10.3969/j.issn.1000-6621.2021.10.017

检测方法	液体培养(例)		敏感度 [%(95%CI)]	特异度 [%(95%CI)]	阳性预测值 [%(95%CI)]	阴性预测值 [%(95%CI)]
检测方法	阳性^a	阴性	敏感度 [%(95%CI)]	特异度 [%(95%CI)]	阳性预测值 [%(95%CI)]	阴性预测值 [%(95%CI)]
恒温扩增荧光检测法			58.94 (54.33~63.40)	97.68 (96.70~98.38)	89.64 (85.56~92.71)	87.46 (85.68~89.50)
阳性	277	32
阴性	193	1346
痰涂片			52.77 (48.14~57.34)	98.69 (97.90~99.20)	93.23 (89.34~95.82)	85.97 (84.14~87.62)
阳性	248	18
阴性	222	1360
GeneXpert MTB/RIF			80.43 (76.49~83.86)	88.97 (87.17~90.55)	71.32 (67.23~75.10)	93.02 (91.47~94.31)
阳性	378	152
阴性	92	1226

痰样本	患者数 (例)	恒温扩增荧光检测法阳性 [例(检出率,%)]	痰涂片阳性 [例(检出率,%)]	液体培养阳性 [例(检出率,%)]	GeneXpert检测阳性 [例(检出率,%)]
黏液样痰	598	201(33.6)	188(31.4)	278(46.5)	308(51.5)
唾液样痰	152	29(19.1)	25(16.4)	59(38.8)	51(33.6)
干酪样痰	42	16(38.1)	14(33.3)	21(50.0)	28(66.7)
血样痰	25	9(36.0)	7(28.0)	15(60.0)	16(64.0)
合计	817	255(31.2)	234(28.6)	365(45.7)	403(49.3)

痰涂片级别	患者数 (例)	恒温扩增荧光检测法阳性 [例(检出率,%)]	GeneXpert 检测阳性 [例(检出率,%)]	χ²值	P值
阴性	583	60(10.3)	186(31.9)	81.790	0.001
涂阴培阳	163	41(25.2)	103(63.2)	47.830	0.001
涂阴培阴	420	19(4.5)	83(19.8)	45.710	0.001
低级别阳性	139	105(75.5)	128(92.1)	14.030	0.001
1~8条/300视野^a或1~9条/50视野^b	75	50(66.7)	70(93.3)	16.670	0.001
1+	64	55(85.9)	58(90.6)	0.680	0.410
高级别阳性	95	90(94.7)	89(93.7)	0.100	0.756
2+	44	41(93.2)	40(90.9)	0.160	0.694
3+	30	29(96.7)	29(96.7)	-	-
4+	21	20(95.2)	20(95.2)	-	-
合计	817	255(31.2)	403(49.3)	55.730	0.001