Study on efficiency of Mycobacterium tuberculosis detection by real-time quantitative PCR with different clinical sample types and the improvement by grinding specimen with glass beads

doi:10.19982/j.issn.1000-6621.20210584

Abstract

Abstract:

Objective: To explore a method with improved efficiency for quantitatively detecting Mycobacterium tuberculosis (MTB) in different type of clinical samples by combining glass beads grinding with real-time quantitative PCR (qRT-PCR). Methods: A retrospective study was performed in Beijing Chest Hospital. Data of qRT-PCR examination performed between Jan. 1st, 2008 and Dec. 31th 2016 were retrieved. The outcomes of 11570 clinical specimens collected from tuberculosis suspects of inpatients and outpatients were acquired, including 1487 sputa, 273 purulent specimens, 1391 cerebrospinal fluids (CSF), 41 bronchoalveolar lavage fluids (BALF), and 4650 blood samples. Furthermore, 82 specimens collected from pulmonary tuberculosis patients were chosen by pseudorandom function, the quantitative outcomes were compared between the procedures with or without grinding with glass beads (diameter 0.3 mm). Results: The overall positive rate of the different clinical specimen types was 12.16% (1407/11570). Among them, BALF acquired the highest positive rate (36.59% (15/41)), and the outpatients had higher positive rate (43.48% (10/23)). The second highest positive rate was among purulent specimens (35.53%(97/273)), of which the inpatients yielded higher positive rate (36.26% (95/262)). The positive rate of sputa ranked the third (30.33% (451/1487)). The positive rate of the other leftover samples ranged over 10.43% (485/4650)-15.03% (209/1391). In contrast to the method without grinding, the nucleic acid quantity of the specimen grinded by class beads increased evidently (44.45 (9.05,71.62)×10⁷ copy/ml vs. 4.03 (1.87,5.92)×10⁷ copy/ml; Z=2.120, P=0.016). Additionally, 28 (34.15%) samples produced one order of magnitude increase outcomes, 66.67% (14/21) samples from 10² copy/ml to 10³ copy/ml by using glass bead grinding. Hence, the positive rate of the selected 82 specimens increased from 60.98% (50/82) to 78.05% (64/82). Conclusion: qRT-PCR had higher detection rates in BALF, pus and sputa than other sample types. Grinding specimen by glass bead could improve the positive detection rate.

Key words: Mycobacterium tuberculosis, Molecular diagnostic techniques, Laboratory techniques and procedures, Evaluation studies

CLC Number:

R52
R446

DAI Guang-ming, WANG fen, CAO Ting-ming, CHU Hong-qian, HUANG Hai-rong, SUN Zhao-gang. Study on efficiency of Mycobacterium tuberculosis detection by real-time quantitative PCR with different clinical sample types and the improvement by grinding specimen with glass beads[J]. Chinese Journal of Antituberculosis, 2022, 44(5): 450-454. doi: 10.19982/j.issn.1000-6621.20210584

Figures/Tables 4

检测菌株	0min	1min	2min	3min	4min	5min	6min
H37Rv标准株(×10⁷ 拷贝/ml, $x ¯$ ±s)	4.55±1.02	4.88±1.11	4.68±0.97	4.77±1.42	5.09±1.44	5.22±1.32	4.93±1.68
涂片(+)痰标本[×10³拷贝/ml,M(Q₁,Q₃)]	1.51(1.68, 2.86)	1.42(1.21, 2.98)	2.06(1.23, 3.46)	2.53(1.86, 3.91)	3.21(2.12, 3.95)	3.87(2.94, 5.09)	3.43(2.35, 4.76)

References 13

[1]	焦伟伟. 儿童结核病分子生物学诊断方法研究进展. 结核病与肺部健康杂志, 2021, 2(1): 69-72. doi: 10.3969/j.issn.2096-8493.2021.01.015. doi: 10.3969/j.issn.2096-8493.2021.01.015
[2]	吕纯芳, 吴健虹, 卢留珠, 等. 国产实时荧光定量PCR试剂与GeneXpert MTB/RIF检测结核分枝杆菌的对比分析. 中国防痨杂志, 2020, 42(1): 60-65. doi: 10.3969/j.issn.1000-6621.2020.01.014. doi: 10.3969/j.issn.1000-6621.2020.01.014
[3]	柴国祥, 李兴芳, 韩斌孝, 等. 实时荧光核酸恒温扩增检测技术对痰标本中结核分枝杆菌的诊断价值. 中国现代医学杂志, 2020, 30(11):39-42. doi: 10.3969/j.issn.1005-8982.2020.11.008. doi: 10.3969/j.issn.1005-8982.2020.11.008
[4]	中国疾病预防控制中心. 中国结核病防治规划痰涂片镜检质量保证手册. 北京: 中国协和医科大学出版社, 2004.
[5]	赵雁林, 逄宇. 结核病实验室检查规程. 北京: 人民卫生出版社, 2015.
[6]	World Health Organization. Update on the use of nucleic acid amplification tests to detect TB and drug-resistant TB: rapid communication. Geneva: World Health Organization, 2021.
[7]	黄雪欢, 沈惠兰, 曾华志, 等. 质量控制小组对提高结核科患者痰标本采集质量的作用. 中国防痨杂志, 2016, 38(12):1078-1081. doi: 10.3969/j.issn.1000-6621.2016.12.014. doi: 10.3969/j.issn.1000-6621.2016.12.014
[8]	刘权贤, 李正付, 陈玲, 等. 利福平耐药实时荧光定量核酸扩增技术检测结核病临床标本时异常结果的分析及处理. 中华结核和呼吸杂志, 2018, 41 (5): 386-387. doi: 10.3760/cma.j.issn.1001-0939.2018.05.021. doi: 10.3760/cma.j.issn.1001-0939.2018.05.021
[9]	唐婧, 王庆, 徐东芳, 等. 肺泡灌洗液不同检测方法在肺结核诊断中的临床价值. 临床肺科杂志, 2019, 24(1):133-134,182. doi: 10.3969/j.issn.1009-6663.2019.01.033. doi: 10.3969/j.issn.1009-6663.2019.01.033
[10]	邹自英, 朱冰, 汤雪晴, 等. 结核杆菌的细菌学、分子生物学和免疫学检测方法评价. 四川医学, 2011, 32(5):756-758. doi: 10.3969/j.issn.1004-0501.2011.05.060. doi: 10.3969/j.issn.1004-0501.2011.05.060
[11]	梁建琴, 高华方, 李洪敏, 等. PCR-荧光探针法检测临床痰标本结核分枝杆菌的可靠性研究. 中国防痨杂志, 2012, 34(5):271-274.
[12]	张军, 王晓飞, 王瑞东, 等. 通过增加痰液量和超声裂菌提高结核分枝杆菌荧光定量PCR检出率. 检验医学, 2014, 29(3):215-218. doi: 10.3969/j.issn.1673-8640.2014.03.004. doi: 10.3969/j.issn.1673-8640.2014.03.004
[13]	文岚, 张兵, 郭彦昌, 等. 5种前处理方法对痰中结核分枝杆菌DNA提取的影响. 实用预防医学, 2013, 20(9):1056-1059. doi: 10.3969/j.issn.1006-3110.2013.09.010. doi: 10.3969/j.issn.1006-3110.2013.09.010

样本类型	门诊患者			住院患者			合计
样本类型	总份数	阳性份数	阳性率 (%)	总份数	阳性份数	阳性率 (%)	总份数	阳性份数	阳性率 (%)
痰液	472	128	27.12	1015	323	31.82	1487	451	30.33
脓液	11	2	18.18	262	95	36.26	273	97	35.53
脑脊液	514	82	15.95	877	127	14.48	1391	209	15.03
腹腔积液	142	10	7.04	163	24	14.72	305	34	11.15
灌洗液	23	10	43.48	18	5	27.78	41	15	36.59
尿液	71	5	7.04	76	14	18.42	147	19	12.93
心包积液	29	4	13.79	59	8	13.56	88	12	13.64
胸腔积液	1307	126	9.64	1881	316	16.80	3188	442	13.86
血液	3653	379	10.38	997	106	10.63	4650	485	10.43
合计	6222	746	11.99	5348	1018	19.04	11570	1407	12.16

样本类型	菌株含量数量级(拷贝/ml)
样本类型	≤10¹	10²	10³	10⁴	10⁵	10⁶	≥10⁷
痰液(1487份)	1007(67.72)	29(1.95)	57(3.83)	123(8.27)	118(7.94)	114(7.67)	39(2.62)
脓液(273份)	175(64.10)	1(0.37)	8(2.93)	21(7.69)	30(10.99)	31(11.36)	7(2.56)
灌洗液(41份)	26(63.41)	0(0.00)	2(4.88)	3(7.32)	5(12.19)	1(2.44)	4(9.76)
脑脊液(1391份)	1161(83.47)	21(1.51)	51(3.67)	65(4.67)	58(4.17)	19(1.36)	16(1.15)
血液(4650份)	4508(96.95)	14(0.30)	28(0.60)	40(0.86)	32(0.69)	19(0.41)	9(0.19)
胸腔积液(3188份)	2681(84.10)	65(2.04)	128(4.02)	136(4.26)	81(2.54)	55(1.72)	42(1.32)
腹腔积液(305份)	264(86.56)	7(2.29)	12(3.93)	10(3.28)	7(2.30)	2(0.66)	3(0.98)
尿液(147份)	125(85.04)	3(2.04)	3(2.04)	7(4.76)	3(2.04)	5(3.40)	1(0.68)
心包积液(88份)	74(84.09)	2(2.27)	6(6.82)	3(3.41)	0(0.00)	2(2.27)	1(1.14)

检测结果	菌株含量数量级(拷贝/ml)
检测结果	<10¹	10²	10³	10⁴	10⁵	10⁶	10⁷
磨菌前浓度[×10⁷拷贝/ml,M(Q₁,Q₃)]	0	4.61(2.14, 7.21)	4.03(1.87, 5.92)	3.35(1.45, 6.43)	4.28(2.52, 6.72)	2.99(1.43, 4.88)	3.09(2.50, 4.66)
磨菌后浓度[×10⁷拷贝/ml,M(Q₁,Q₃)]	0	16.91(8.46, 20.12)	44.45(9.05, 71.62)	12.89(7.80, 36.81)	9.07(4.69, 9.83)	7.11(4.18, 9.75)	6.95(4.66, 8.34)
磨菌前份数	11	21	12	15	9	8	6
磨菌后份数	11	7	19	18	11	9	7
改善份数(改善率,%)]	0(0.00)	14(66.67)	7(58.33)	3(20.00)	2(2/9)	1(1/8)	1(1/6)

[1]	HE Yi-jun, CAO Xue-fang, GAO Lei. Interpretation of the Procedure of tuberculin skin test-interferon-gamma release assay two-step testing [J]. Chinese Journal of Antituberculosis, 2022, 44(5): 438-441.
[2]	JIN Xiao-wei, LI Ya-ru, DUAN Hai-xia, WEI Xiao-hui, PEI Yi-yi, LI Ying, JIN Hong-jian. Study on active screening of pulmonary tuberculosis among key population in key areas of Xinmi, He’nan [J]. Chinese Journal of Antituberculosis, 2022, 44(5): 467-472.
[3]	SUN Qing, LIAO Xin-lei, WANG Chen-qian, JIANG Guang-lu, DONG Ling-ling, WANG Fen, ZHAO Li-ping, HUANG Hai-rong, WANG Gui-rong. Characterization of rifampin resistance determining region mutations in tuberculosis patients with GeneXpert MTB/RIF positive RNA polymerase β subunit gene mutation [J]. Chinese Journal of Antituberculosis, 2022, 44(4): 349-353.
[4]	TIAN Li, ZHOU Wei, HUANG Xing, WU Xian-wei, ZHANG Hui-yong, LU Zhen-hui, ZHANG Shao-yan. Analysis of gene mutation characteristics of isoniazid-resistant Mycobacterium tuberculosis in China [J]. Chinese Journal of Antituberculosis, 2022, 44(4): 354-361.
[5]	WANG Yan, FANG Hong-xia, ZHENG Kai-qiao, TAN Xiao-ping, HUANG Dong-sheng, SU Lian-hui, LIU Chang-wei. Analysis of the effect of strengthening tuberculosis etiological examination on hospitalized patients from respiratory department of general hospital in Longhua of Shenzhen between 2018 and 2020 [J]. Chinese Journal of Antituberculosis, 2022, 44(4): 368-374.
[6]	MA Yan, LU Wei, GAO Lei, CHU Nai-hui, ZHOU Lin, CHENG Shi-ming. To end tuberculosis epidemic needs strengthen the management of screening and preventive treatment of latent tuberculosis infection in high-risk groups [J]. Chinese Journal of Antituberculosis, 2022, 44(3): 209-214.
[7]	LI Dong-shuo, WANG Bin, LU Yu, XU Jian. Study of expression and function of Mycobacterium tuberculosis membrane protein MmpL5-MmpS5 [J]. Chinese Journal of Antituberculosis, 2022, 44(3): 227-233.
[8]	FAN Ru, LI Xiao-fei. Research progress of molecular biology detection technology for tuberculosis [J]. Chinese Journal of Antituberculosis, 2022, 44(3): 294-298.
[9]	SUN Zhao-gang. Attention should be paid to the research and development of Mycobacterium tuberculosis antigen detection technology [J]. Chinese Journal of Antituberculosis, 2022, 44(2): 120-124.
[10]	LEI Jing, WU Xia, TAN Xiao-wen, LI Ai-fang, CUI Xiao-li, KANG Lei, PANG Jian-jian, REN Fei, WU Shou-zhen, YANG Han. Diagnostic value of GeneXpert MTB/RIF Ultra in testing surgical tissue specimens for diagnosing tuberculosis [J]. Chinese Journal of Antituberculosis, 2022, 44(2): 141-146.
[11]	JIN Long, TIAN Qi, ZHANG Bao-qing, XING Hai-dong, GAO Ming-xia, ZHANG Xiu-ying, WANG Li-hua, ZHANG Xiao-lei. Research of effectiveness of fluorescence PCR melting curve assay in detecting drug resistance of levofloxacin and moxifloxacin in patients with multidrug-resistant pulmonary tuberculosis [J]. Chinese Journal of Antituberculosis, 2022, 44(2): 159-163.
[12]	WANG Shao-hua, ZHAO Guo-lian, WANG Pei, TAN Xiao-wen, CUI Xiao-li, KANG Lei, DANG Li-yun. Analysis of inconsistency between genotypic and phenotypic results of Mycobacterium tuberculosis rifampicin susceptibility test [J]. Chinese Journal of Antituberculosis, 2022, 44(2): 169-173.
[13]	ZHANG Can-you, CHEN Hui, FA Li-feng, ZHANG Hui, CHENG Jun. Evaluation of the effects of tuberculosis infection control training courses in China, 2016-2019 [J]. Chinese Journal of Antituberculosis, 2022, 44(2): 174-180.
[14]	ZHOU Ting-ting, ZHENG Xiao-man, OUYANG Jing, LU Yan-qiu, CHEN Yao-kai. Research progress on genes and mechanism of Mycobacterium tuberculosis resistance to pyrazinamide [J]. Chinese Journal of Antituberculosis, 2022, 44(1): 102-105.
[15]	Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College , Chinese Center for Disease Control and Prevention , Union Medical Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences . Expert consensus on the estimation of the national burden on latent tuberculosis infection [J]. Chinese Journal of Antituberculosis, 2022, 44(1): 4-8.