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
Objective To 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.Methods Fifty 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.Results Using 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/
作者
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
Song Jing;Zhang Zhiguo;Dong Yujie;Du Weili;Wang Yuxuan;Liu Zichen;Li Kun;Zhang Qian;Sun Qian;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;Changping District Center for Tuberculosis Prevention and Treatment,Beijing 102200,China)
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2020年第3期234-241,共8页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
北京市科技计划医药协同科技创新研究(Z18100001918027)
北京市科学技术委员会重点项目(D18110000418003)
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)
北京市通州区科技计划项目(KJ2019CX006)。
关键词
分枝杆菌
结核
石蜡包埋组织
抗药性
基因测定
Mycobacterium tuberculosis
Tissue
paraffin-embedded
Drug resistance
Genetic testing
