高级检索
中华结核和呼吸杂志
期刊首页
过刊列表
高级检索
稿件发表
• 论著 •
ENGLISH ABSTRACT
扩增子测序技术检测石蜡包埋组织标本耐药结核基因的研究
宋婧
张治国
董宇杰
杜伟丽
王宇轩
刘子臣
李琨
张倩
孙倩
车南颖
作者及单位信息
·
DOI： 10.3760/cma.j.issn.1001-0939.2020.03.019
Detection of tuberculosis genes associated with drug-resistance in paraffin-embedded tissue specimens using next generation sequencing technology
Song Jing
Zhang Zhiguo
Dong Yujie
Du Weili
Wang Yuxuan
Liu Zichen
Li Kun
Zhang Qian
Sun Qian
Che Nanying
Authors Info & Affiliations
Song Jing
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Zhang Zhiguo
Changping District Center for Tuberculosis Prevention and Treatment, Beijing 102200, China
Dong Yujie
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Du Weili
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Wang Yuxuan
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Liu Zichen
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Li Kun
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
Zhang Qian
Changping District Center for Tuberculosis Prevention and Treatment, Beijing 102200, China
Sun Qian
Changping District Center for Tuberculosis Prevention and Treatment, Beijing 102200, China
Che Nanying
Department of Pathology, Beijing Key Laboratory for Drug Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China
·
DOI： 10.3760/cma.j.issn.1001-0939.2020.03.019
915
251
0
0
1
1
PDF下载
APP内阅读
摘要

目的用基于多重PCR（multiplex PCR，mPCR）扩增子的二代测序（next-generation sequencing，NGS）技术（mPCR-NGS）检测MTB的耐药基因突变，并评估其检测石蜡包埋组织标本诊断耐药结核病的临床应用价值。

方法选取北京市昌平区结核病防治所2013年4月至2015年10月常规进行了利福平、异烟肼、乙胺丁醇、链霉素、卷曲霉素、卡那霉素、阿米卡星和氧氟沙星比例法检测且具有药敏试验结果的50株MTB临床分离株，包括42株耐药MTB和8株敏感MTB菌株。参考耐药基因数据库及相关文献，针对以上8种抗结核药物主要耐药基因及常见位点设计耐药基因组合，建立有效的检测方案。收集首都医科大学附属北京胸科医院病理科2017年11月至2018年9月耐药结核病患者55例的石蜡包埋组织标本，探针熔解曲线法检测结果为利福平、异烟肼、乙胺丁醇和氟喹诺酮类至少存在一种耐药相关基因突变。首先对已知表型药敏试验结果的MTB临床分离株进行mPCR-NGS检测，验证该方法检测纯菌样本的有效性，再进一步检测结核病患者石蜡包埋组织标本进行临床验证。主要评价指标为敏感度和特异度以及一致性。

结果以比例法为金标准，mPCR-NGS检测利福平、异烟肼、链霉素和乙胺丁醇的敏感度分别为95%（38/40）、93%（27/29）、93%（27/29）和72%（13/18），特异度分别为100%（10/10）、95%（20/21）、100%（21/21）和94%（30/32）；二线注射类药物卷曲霉素、卡那霉素和阿米卡星敏感度均为100%（2/2，3/3，3/3），特异度分别为98%（47/48）、100%（33/33）和100%（47/47）；氧氟沙星的敏感度为70%（7/10），特异度为100%（40/40）。总符合率为94%，一致性检验 Kappa值为0.87。纳入的55例石蜡包埋组织标本经探针熔解曲线法检测，28例利福平耐药，37例异烟肼耐药，13例乙胺丁醇耐药，17例氟喹诺酮类耐药。mPCR-NGS与探针熔解曲线法比较，利福平、异烟肼、乙胺丁醇和氟喹诺酮类的敏感度分别为100%（28/28）、95%（35/37）、100%（13/13）和100%（17/17）；特异度均为100%（27/27，18/18，42/42，38/38）。两种方法总符合率为99%，一致性检验 Kappa值为0.98。

结论mPCR-NGS检测MTB临床分离株及结核病患者石蜡包埋组织标本耐药基因突变的敏感度、特异度和符合率较高，有望成为耐药结核病准确、快速的分子病理诊断新技术。

分枝杆菌，结核;石蜡包埋组织;抗药性;基因测定
ABSTRACT

ObjectiveTo evaluate the use of multiplex PCR amplicon sequencing (mPCR-NGS) technology in detecting gene mutations related to drug resistance of Mycobacterium tuberculosis (MTB) in formalin-fixed paraffin-embedded tissue specimens, and to explore its clinical value in the diagnosis of drug-resistant tuberculosis.

MethodsFifty clinical MTB strains isolated in the Changping District Tuberculosis Control Institute of Beijing from April 2013 to October 2015 with drug susceptibility test (DST) results of rifampicin, isoniazid, ethambutol, streptomycin, ofloxacin, capreomycin, kanamycin and amikacin available were recovered, including 42 drug-resistant strains and 8 drug-sensitive strains. The mPCR-NGS test was established to detect genes related to the 8 anti-tuberculosis drugs according to the previously published studies and databases. Fifty-five paraffin-embedded tissue specimens from drug-resistant tuberculosis patients were collected in the Department of Pathology, Beijing Chest Hospital, Capital Medical University during November 2017 to September 2018. All the specimens showed no less than one mutation in the gene regions related to drug resistance of any of the 4 drugs (rifampicin, isoniazid, ethambutol or fluoroquinolones) by probe melting curve assay. The effectiveness of mPCR-NGS test was evaluated on clinical MTB isolates using phenotypic DST as the reference. Clinical evaluation of mPCR-NGS test on formalin-fixed paraffin-embedded specimens from TB patients was performed using probe melting curve assay as the reference. The sensitivity, specificity and coincidence of mPCR-NGS were analyzed.

ResultsUsing phenotypic DST as the reference, the sensitivities of the mPCR-NGS for detecting drug-resistance of rifampicin, isoniazid, streptomycin, and ethambutol were 95% (38/40), 93% (27/29), 93% (27/29), and 72% (13/18), respectively; and the specificities were 100% (10/10), 95% (20/21), 100% (21/21), and 94% (30/32), respectively. The sensitivities for capreomycin, kanamycin and amikacin were all 100% (2/2, 3/3, 3/3), and the specificities were 98% (47/48), 100% (33/33) and 100% (47/47), respectively. The sensitivity and specificity of ofloxacin were 70% (7/10) and 100% (40/40), respectively. The total coincidence rate for the 8vdrugs was 94%, and the Kappa value was 0.87. The 55 paraffin-embedded tissue specimens included in this study were all tested by probe melting curve assays. Among them 28 were resistant to rifampicin, 37 resistant to isoniazid, 13 resistant to ethambutol, and 17 resistant to fluoroquinolones. Using the probe melting curve assay as the reference, the sensitivities of the mPCR-NGS for detecting resistant to rifampicin, isoniazid, ethambutol, and fluoroquinolones were 100% (28/28), 95% (35/37), 100%, and 100%, respectively; and the specificities were all 100% (42/42, 38/38). The total coincidence rate of the two methods was 99%, and the Kappa value was 0.98.

ConclusionsmPCR-NGS showed good sensitivities and specificities in detecting drug-resistant gene mutations both in clinical MTB isolates and paraffin-embedded tissue specimens. mPCR-NGS has the potential to be an accurate and rapid molecular pathological technology for diagnosis of drug-resistant tuberculosis.

Mycobacterium tuberculosis ;Tissue,paraffin-embedded;Drug resistance;Genetic testing
Che Nanying, Email: mocdef.3ab618440ynehc

These authors contributed equally: Song Jing, Zhang Zhiguo

Copyright by Chinese Medical Association
No content published by the journals of Chinese Medical Association may be reproduced or abridged without authorization. Please do not use or copy the layout and design of the journals without permission.
All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
引用本文

宋婧,张治国,董宇杰,等. 扩增子测序技术检测石蜡包埋组织标本耐药结核基因的研究[J]. 中华结核和呼吸杂志,2020,43(03)：234-241.

DOI:10.3760/cma.j.issn.1001-0939.2020.03.019

PERMISSIONS

Request permissions for this article from CCC.

评价本文
*以上评分为匿名评价

版权归中华医学会所有。

未经授权，不得转载、摘编本刊文章，不得使用本刊的版式设计。

除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。

我国是全球结核病高负担国家之一，2017年新发结核病患者88.9万例，仅次于排名第1位的印度 [ 1 ]。耐药结核病是结核病防控的难题之一。目前，诊断耐药结核病的金标准是基于培养的表型药敏试验（drug sensitivity test，DST），但该方法受生物安全、培养时间长等因素影响，不能满足临床快速诊断的需求。据估算，我国菌阴肺结核患者约占所有肺结核患者的70% [ 2 ]，病理学的诊断具有重要作用 [ 3 ]。随着分子病理学的发展，通过检测组织病灶中MTB耐药基因突变成为快速诊断耐药结核病的新途径。文献报道探针熔解曲线法成功用于检测石蜡包埋组织标本诊断耐药结核病 [ 4 ]，但目前的分子检测试剂盒覆盖的抗结核药物种类有限。
二代测序（next-generation sequencing，NGS）的发展为MTB的耐药、进化、传播及基因组变异等研究提供了准确、高效的方法 [ 5 ]。全基因组测序成本相对较高，数据量较大，一般只能对纯菌样本进行检测，目前主要用于科学研究。基于多重PCR（multiplex PCR，mPCR）扩增子的NGS技术（mPCR-NGS）可以对靶基因测序，覆盖度更深，更加高效、经济、简便 [ 6 , 7 , 8 ]。经文献检索，mPCR-NGS可直接检测痰标本，而不需要分离纯菌 [ 9 ]，但未见该技术检测结核患者石蜡包埋组织标本的报道。本研究通过对文献中耐药相关基因功能位点分析 [ 10 , 11 ]，选取8种常用抗结核药物的10个耐药相关基因特定区域，建立mPCR-NGS方法，检测已知药敏试验结果的MTB临床分离株和结核病患者石蜡包埋组织标本进行初步临床诊断价值的评估。
参考文献
[1]
World Health Organization. Global tuberculosis report 2018[M]. Geneva:World Health Organization, 2018.
返回引文位置
[2]
安军,逄宇. 重视新型实验室技术,助力菌阴肺结核诊断[J]. 中国防痨杂志, 2018,40(4):345-347. DOI: 10.3969/j.issn.1000-6621.2018.04.001 .
返回引文位置Google Scholar
百度学术
万方数据
[3]
中华医学会结核病学分会,结核病病理学诊断专家共识编写组. 中国结核病病理学诊断专家共识[J]. 中华结核和呼吸杂志, 2017,40(6):419-425. DOI: 10.3760/cma.j.issn.1001-0939.2017.06.005 .
返回引文位置Google Scholar
百度学术
万方数据
[4]
Che NY , Huang SJ , Ma Y ,et al. Comparison of histological, microbiological, and molecular methods in diagnosis of patients with TBLN having different anti-TB treatment background[J]. Biomed Environ Sci, 2017,30(6):418-425. DOI: 10.3967/bes2017.055 .
返回引文位置Google Scholar
百度学术
万方数据
[5]
徐鹏,甘明宇,高谦. 二代测序技术在结核分枝杆菌研究中的应用进展[J]. 微生物与感染, 2015,10(1):54-60.
返回引文位置Google Scholar
百度学术
万方数据
[6]
Walker TM , Kohl TA , Omar SV ,et al. Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study[J]. Lancet Infect Dis, 2015,15(10):1193-1202. DOI: 10.1016/S1473-3099(15)00062-6 .
返回引文位置Google Scholar
百度学术
万方数据
[7]
Zignol M , Cabibbe AM , Dean AS ,et al. Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study[J]. Lancet Infect Dis, 2018,18(6):675-683. DOI: 10.1016/S1473-3099(18)30073-2 .
返回引文位置Google Scholar
百度学术
万方数据
[8]
Park J , Jang W , Kim M ,et al. Molecular drug resistance profiles of Mycobacterium tuberculosis from sputum specimens using ion semiconductor sequencing[J]. J Microbiol Methods, 2017,145:1-6. DOI: 10.1016/j.mimet.2017.12.003 .
返回引文位置Google Scholar
百度学术
万方数据
[9]
Doyle RM , Burgess C , Williams R ,et al. Direct whole-genome sequencing of sputum accurately identifies drug-resistant Mycobacterium tuberculosis faster than MGIT culture sequencing[J]. J Clin Microbiol, 2018,56(8):e00666-18. DOI: 10.1128/JCM.00666-18 .
返回引文位置Google Scholar
百度学术
万方数据
[10]
Miotto P , Tessema B , Tagliani E ,et al. A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis[J]. Eur Respir J, 2017,50(6):1701354. DOI: 10.1183/13993003.01354-2017 .
返回引文位置Google Scholar
百度学术
万方数据
[11]
CRyPTIC Consortium and the 100,000 Genomes Project Allix-Beguec C,Arandjeloric I,et al. Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing[J]. N Engl J Med, 2018,379(15):1403-1415. DOI: 10.1056/NEJMoa1800474 .
返回引文位置Google Scholar
百度学术
万方数据
[12]
Sowajassatakul A , Prammananan T , Chaiprasert A ,et al. Molecular characterization of amikacin, kanamycin and capreomycin resistance in M/XDR-TB strains isolated in Thailand[J]. BMC Microbiol, 2014,14(1):165. DOI: 10.1186/1471-2180-14-165 .
返回引文位置Google Scholar
百度学术
万方数据
[13]
Torres JN , Paul LV , Rodwell TC ,et al. Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates[J]. Emerg Microbes Infect, 2015,4(7):e42. DOI: 10.1038/emi.2015.42 .
返回引文位置Google Scholar
百度学术
万方数据
[14]
Otchere ID , Asante-Poku A , Osei-Wusu S ,et al. Detection and characterization of drug-resistant conferring genes in Mycobacterium tuberculosis complex strains: a prospective study in two distant regions of Ghana[J]. Tuberculosis, 2016,99:147-154. DOI: 10.1016/j.tube.2016.05.014 .
返回引文位置Google Scholar
百度学术
万方数据
[15]
Rinder H , Mieskes KT , Loscher T ,Heteroresistance in Mycobacterium tuberculosis[J]. Int J Tuberc Lung Dis, 2001,5(4):339-345. DOI: 10.1258/0956462011922931 .
返回引文位置Google Scholar
百度学术
万方数据
[16]
刘宇红,王甦民,那希宽. 比例法测试结核分枝杆菌药物敏感度的探讨[J]. 中华结核和呼吸杂志, 2000,23(2):89-92. DOI: 10.3760/j:issn:1001-0939.2000.02.009 .
返回引文位置Google Scholar
百度学术
万方数据
[17]
吴慧娜,孙付胜,刘清文,等. 荧光PCR探针熔解曲线法检测结核分枝杆菌复合群对利福平和异烟肼耐药性的价值[J]. 中国防痨杂志, 2019,41(1):74-79. DOI: 10.3969/j.issn.1000-6621.2019.01.016 .
返回引文位置Google Scholar
百度学术
万方数据
[18]
André E , Goeminne L , Colmant A ,et al. Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays[J]. Clin Microbiol Infect, 2017,23(4):267.e5-267.e7. DOI: 10.1016/j.cmi.2016.12.009 .
返回引文位置Google Scholar
百度学术
万方数据
[19]
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.
返回引文位置
备注信息
A
车南颖，Email： mocdef.3ab618440ynehc
B

宋婧和张治国对本研究具有同等贡献

C
所有作者均声明不存在利益冲突
D
北京市科技计划医药协同科技创新研究 (Z181100001918027)
北京市科学技术委员会重点项目 (D181100000418003)
北京市医院管理局登峰计划 (DFL20151501，DFL20181601)
北京市卫生与健康科技成果和适宜技术推广项目 (2018-TG-41)
通州区高层次人才发展支持计划 (YHLD2018006)
评论 （0条）
注册
登录
时间排序
暂无评论，发表第一条评论抢沙发
最新推荐
更多
宏基因组学二代测序对结核性脑膜炎的诊断价值
孔娇 等
中华传染病杂志 2023,41(07)
下呼吸道感染宏基因组二代测序报告临床解读路径专家共识
中华医学会呼吸病学分会
中华结核和呼吸杂志 2023,46(04)
高通量宏基因组测序技术检测病原微生物的临床应用规范化专家共识
中华医学会检验医学分会
中华检验医学杂志 2020,43(12)
中国宏基因组学第二代测序技术检测感染病原体的临床应用专家共识
《中华传染病杂志》编辑委员会
中华传染病杂志 2020,38(11)
MedAI助手（体验版）
文档即答
智问智答
机器翻译
回答内容由人工智能生成，我社无法保证其准确性和完整性，该生成内容不代表我们的态度或观点，仅供参考。
生成快照
文献快照

你好，我可以帮助您更好的了解本文，请向我提问您关注的问题。

0/2000

《中华医学会杂志社用户协议》 | 《隐私政策》

《SparkDesk 用户协议》 | 《SparkDesk 隐私政策》

网信算备340104764864601230055号 | 网信算备340104726288401230013号

技术支持：

历史对话
本文全部
还没有聊天记录
设置
模式
纯净模式沉浸模式
字号
小中大