GeneXpert MTB/RIF阳性RNA聚合酶β亚基基因突变的结核病患者中利福平耐药决定区突变特征研究

doi:10.19982/j.issn.1000-6621.20210635

中国防痨杂志 ›› 2022, Vol. 44 ›› Issue (4): 349-353.doi: 10.19982/j.issn.1000-6621.20210635

GeneXpert MTB/RIF阳性RNA聚合酶β亚基基因突变的结核病患者中利福平耐药决定区突变特征研究

孙晴, 廖鑫磊, 王晨倩, 姜广路, 董玲玲, 王芬, 赵立平, 黄海荣, 王桂荣()

首都医科大学附属北京胸科医院/北京市结核病胸部肿瘤研究所/国家结核病临床实验室/北京市耐药结核病研究重点实验室,北京 101149

收稿日期:2021-10-27 出版日期:2022-04-10 发布日期:2022-04-06
通信作者: 王桂荣 E-mail:wangguirong1230@ccmu.edu.cn
基金资助:
北京市通州区科技计划项目(KJ2021CX010);北京市优秀人才培养青年拔尖个人项目(2018000021223);北京市通州区“两高”人才工程 “领军人才”项目(YH201917)

Characterization of rifampin resistance determining region mutations in tuberculosis patients with GeneXpert MTB/RIF positive RNA polymerase β subunit gene mutation

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()

National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory for Drug Resistant Tuberculosis, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing 101149, China

Received:2021-10-27 Online:2022-04-10 Published:2022-04-06
Contact: WANG Gui-rong E-mail:wangguirong1230@ccmu.edu.cn
Supported by:
Science and Technology Project of Beijing Tongzhou District(KJ2021CX010);Beijing Talents Training Youth Top Individual Project(2018000021223);Leading Talents of Beijing Tongzhou District “Two High” Talent Project(YH201917)

摘要/Abstract

摘要：

目的: 通过分析GeneXpert MTB/RIF(简称“Xpert”)阳性RNA聚合酶β亚基(rpoB)基因突变的结核病患者中rpoB基因探针的突变分布与频率,阐述利福平耐药决定区的突变特征。方法: 连续纳入2019年6月至2020年6月就诊于首都医科大学附属北京胸科医院的Xpert检测阳性且rpoB基因突变的682例结核病患者,其中肺结核618例,肺外结核64例。收集患者临床检测标本,其中痰液465份,支气管肺泡灌洗液153份,脓液37份,胸腔积液19份,便标本7份,脑脊液1份。分析所有Xpert 阳性标本中结核分枝杆菌(MTB)的半定量结果及rpoB基因探针的突变分布情况。结果: 682份标本中,MTB含量半定量水平为高占6.45%(44/682),中占25.07%(171/682),低占25.51%(174/682),极低占42.96%(293/682)。肺外结核标本中MTB含量半定量极低水平高于肺结核标本[分别为64.06%(41/64)和40.78%(252/618)],差异有统计学意义(χ²=12.83,P<0.001)。标本涂阳率在MTB含量半定量水平为高、中、低和极低水平的标本中分别为84.62%(33/39),64.02%(105/164),27.27%(42/154)和7.84%(20/255)。rpoB基因单探针突变率为92.82%(633/682),双探针突变率为6.89%(47/682),三探针突变率为0.29%(2/682)。单探针突变的标本中,突变率最高的为探针E(74.34%,507/682),其次为探针D(8.80%,60/682);双探针突变的标本中,突变率最高的为探针D+E(4.25%,29/682),其次为探针A+B(1.76%,12/682);2例三探针突变均为探针A+D+E。结论: Xpert阳性rpoB基因突变MTB中以单探针突变为主,在肺结核和肺外结核中最常见的突变探针均为探针E和探针D。

关键词: 分枝杆菌,结核, 核酸扩增技术, 基因,细菌, 突变, 核酸探针

Abstract:

Objective: To analyze the mutation distribution and frequency of RNA polymerase β subunit (rpoB) gene probe in tuberculosis (TB) patients with GeneXpert MTB/RIF (Xpert) positive rpoB gene mutation, and to elucidate the mutation characteristics of rifampicin resistance determining region. Methods: A total of 682 consecutive TB patients with positive Xpert test and rpoB gene mutation who visited Beijing Chest Hospital, Capital Medical University from June 2019 to June 2020 were involved, including 618 pulmonary TB (PTB) cases and 64 extrapulmonary TB (EPTB) cases. Clinical examination samples, including 465 sputum, 153 bronchoalveolar lavage fluid, 37 pus, 19 pleural effusion, 7 stool and 1 cerebrospinal fluid, were collected from patients. All Xpert positive specimens were analyzed for semi-quantitative results of Mycobacterium tuberculosis (MTB) and mutation distribution of the rpoB gene probe. Results: Of the 682 samples, 6.45% (44/682) were high, 25.07% (171/682) were medium, 25.51% (174/682) were low, and 42.96% (293/682) were very low in MTB semi-quantitative. The proportion of very low levels of MTB semi-quantitative was significantly higher in EPTB samples than in PTB samples (64.06% (41/64) vs. 40.78% (252/618), χ ²=12.83, P<0.001). Smear positive rates were 84.62% (33/39), 64.02% (105/164), 27.27% (42/154), and 7.84% (20/255) in semiquantitatively high, medium, low, and very low levels of MTB specimens, respectively. The mutation rate of the rpoB gene was 92.82% (633/682) for single probe, 6.89% (47/682) for double probe, and 0.29% (2/682) for triple probe. The mutation rate of probe E was the highest in specimens with single probe mutation (74.34%, 507/682), followed by probe D (8.80%, 60/682); the mutation rate of probe D+E was the highest in specimens with double probe mutation (4.25%, 29/682), followed by probe A+B (1.76%, 12/682); and all three probe mutations were probe A+D+E (2 cases). Conclusion: Single probe mutation is predominant in Xpert positive rpoB gene mutation MTB, and the most common mutation probes in PTB and EPTB are probe E and probe D.

Key words: Mycobacterium tuberculosis, Nucleic acid amplification techniques, Genes,bacterial, Mutation, Nucleic acid probes

中图分类号:

孙晴, 廖鑫磊, 王晨倩, 姜广路, 董玲玲, 王芬, 赵立平, 黄海荣, 王桂荣. GeneXpert MTB/RIF阳性RNA聚合酶β亚基基因突变的结核病患者中利福平耐药决定区突变特征研究[J]. 中国防痨杂志, 2022, 44(4): 349-353. doi: 10.19982/j.issn.1000-6621.20210635

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. doi: 10.19982/j.issn.1000-6621.20210635

图/表 4

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参考文献 18

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患者特征	肺结核(618例)	肺外结核(64例)	合计(682例)
性别
男性	413(66.83)	38(59.37)	451(66.13)
女性	205(33.17)	26(40.63)	231(33.87)
年龄组(岁)
<18	17(2.75)	2(3.13)	19(2.79)
18~45	350(56.63)	40(62.50)	390(57.18)
46~65	176(28.48)	13(20.31)	189(27.71)
>65	75(12.14)	9(14.06)	84(12.32)

MTB半定量水平	合计(682例)	肺结核标本(618例)	肺外结核标本(64例)	χ²值	P值
高	44(6.45)	44(7.12)	0(0.00)	-^a	0.027
中	171(25.07)	169(27.35)	2(3.13)	18.11	<0.001
低	174(25.51)	153(24.76)	21(32.81)	1.98	0.159
极低	293(42.97)	252(40.77)	41(64.06)	12.83	<0.001

突变探针类型	氨基酸位点	合计 (682份)	肺结核标本 (618份)	肺外结核标本 (64份)	χ²值	P值
单探针
A	507~511	33(4.84)	27(4.37)	6(9.38)	-^a	0.114
B	512~518	31(4.55)	29(4.69)	2(3.13)	-^a	0.759
C	518~523	2(0.29)	1(0.16)	1(1.56)	-^a	0.179
D	523~529	60(8.80)	56(9.06)	4(6.25)	0.57	0.450
E	529~533	507(74.34)	464(75.08)	43(67.19)	1.89	0.169
双探针
A+B	507~518	12(1.76)	10(1.62)	2(3.13)	-^a	0.313
A+C	507~511、518~523	1(0.15)	1(0.16)	0(0.00)	-^a	1.000
A+D	507~511、523~529	2(0.29)	1(0.16)	1(1.56)	-^a	0.179
B+C	512~523	1(0.15)	0(0.00)	1(1. 56)	-^a	0.094
B+D	512~518、523~529	2(0.29)	2(0.32)	0(0.00)	-^a	1.000
D+E	523~533	29(4.25)	25(4.05)	4(6.25)	-^a	0.339
三探针
A+D+E	507~511、523~533	2(0.29)	2(0.32)	0(0.00)	-^a	1.000

GeneXpert MTB/RIF阳性RNA聚合酶β亚基基因突变的结核病患者中利福平耐药决定区突变特征研究

Characterization of rifampin resistance determining region mutations in tuberculosis patients with GeneXpert MTB/RIF positive RNA polymerase β subunit gene mutation

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MTB半定量水平	肺结核涂片结果(份)					肺外结核涂片结果(份)					合计 (份)	涂阳率 (%)
MTB半定量水平	阴性	+	++		+	阴性	+	++		+	合计 (份)	涂阳率 (%)
高	6	7	12	8	6	0	0	0	0	0	39	84.62
中	39	58	40	1	6	20	0	0	0	0	164	64.02
低	97	33	5	4	0	15	0	0	0	0	154	27.27
极低	210	12	5	2	0	25	0	1	0	0	255	7.84

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