飞行时间质谱在耐药结核病检测中的应用价值

doi:10.6040/j.issn.1671-7554.0.2024.0303

摘要/Abstract

摘要： 目的旨在建立一种基于基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF-MS)平台鉴定结核分枝杆菌及检测结核耐药突变位点的方法,并对其用于临床耐药结核病检测的应用价值进行评估。方法采用MALDI-TOF-MS检测3株国家参考品菌株,测定其对于结核鉴定位点和耐药位点的最低检出限。以非随机抽样法,收集昆明市第三人民医院结核科2023年1月至9月疑似结核病患者呼吸道样本,包括痰液样本120例,肺泡灌洗液样本100例,每例样本含量3~5 mL。供试样本均提取核酸,先用结核荧光定量PCR(quantitative real-time PCR, qPCR)试剂盒筛选出Ct值≤32的结核阳性样本,然后用MALDI-TOF-MS检测与利福平(rifampicin, RIF)、异烟肼(isoniazide, INH)、乙胺丁醇(ethambutol, EMB)、莫西沙星(moxifloxacin, MXF)、链霉素(streptomycin, SM)和吡嗪酰胺(pyrazinamide, PZA)相关的耐药基因位点。同时采用表型药敏法和三代测序(Nanopore平台)对结核阳性样本进行检测并与MALDI-TOF-MS结果进行对比分析,验证其检测耐药结核的准确性。结果对于结核鉴定位点IS6110和ext_RD9,MALDI-TOF-MS的最低检出限为60~100 CFU/mL;对于结核耐药位点,MALDI-TOF-MS的最低检出限为100~1 000 CFU/mL。经qPCR法鉴定Ct值≤32的结核阳性标本共132例。对比表型药敏结果,MALDI-TOF-MS检测耐药的敏感度、特异度和kappa值分别是:RIF为93.75%、100%和0.96;INH为98.33%、90.28%和0.88;EMB为83.33%、97.50%和0.78;MXF为95.45%、100%和0.97。与三代测序结果相比,MALDI-TOF-MS检测耐药的敏感度、特异度和kappa值分别是:RIF为90.91%、100%和0.94;INH为92.96%、100%和0.92;EMB为92.86%、100%和0.96;MXF为95.45%、100%和0.97;SM为95.74%、100%和0.97;PZA为66.67%、100%和0.80。结论 MALDI-TOF-MS针对抗结核药物的耐药检测结果与表型药敏和三代测序检测结果对比高度一致,可作为快速检测结核耐药的一种有效方法。

关键词: 飞行时间质谱, 结核分枝杆菌, 耐药, 表型药敏, 三代测序

Abstract: Objective To explore the matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)platform for identifying Mycobacterium tuberculosis and detecting drug-resistant mutations and evaluate its application value. Methods MALDI-TOF-MS was used to detect three national reference strains, and the minimum detection limits for tuberculosis-specific identifying genes and drug-resistant mutations were determined. Respiratory tract clinical specimens of suspected tuberculosis patients were collected from January to September 2023 in the Tuberculosis Department of Kunming Third Peoples Hospital by non-random sampling method, including 120 sputum samples and 100 bronchoalveolar lavage fluid samples, with the content of 3-5 mL in each sample. Nucleic acid was extracted from all the samples, and the tuberculosis positive samples with Ct value≤32 were identified by qPCR kit for tuberculosis identification. The drug-resistant mutations of rifampicin(RIF), isoniazid(INH), ethambutol(EMB), moxifloxacin(MXF), streptomycin(SM)and pyrazinamide(PZA)were detected by MALDI-TOF-MS. The phenotypic drug susceptibility test(pDST)and third-generation sequencing(nanopore platfom)were used to detect the TB-positive samples, and the results were compared with those of MALDI-TOF-MS to verify its accuracy. Results The lowest detection limit of MALDI-TOF-MS was 60-100 CFU/mL for MTB identification genes IS6110 and ext_RD9, and 100-1 000 CFU/mL for drug-resistance mutations. A tolal of 132 tuberculosis positive samples with Ct value ≤ 32 were screened by qPCR. Compared with pDST results, the sensitivity, specificity and kappa value of MALDI-TOF-MS for detection of drug-resistant mutations were 93.75%, 100% and 0.96 for RIF, 98.33%, 90.28% and 0.88 for INH, 83.33%, 97.50% and 0.78 for EMB and 95.45%, 100% and 0.97 for MXF, respectively. Compared with nanopore sequencing, the sensitivity, specificity and kappa values of MALDI-TOF-MS for detection of drug-resistant mutations were 90.91%, 100% and 0.94 for RIF, 92.96%, 100% and 0.92 for INH, 92.86%, 100% and 0.96 for EMB, 95.45%, 100% and 0.97 for MXF, 95.74%, 100% and 0.97 for SM, 66.67%, 100% and 0.80 for PZA, respectively. Conclusion MALDI-TOF-MS can be used as an effective method for rapid detection of Mycobacterium tuberculosis drug-resistant mutations because its results were highly consistent with those of pDST and nanopore sequencing.

Key words: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Mycobacterium tuberculosis, Drug resistance, Phentypic drug susceptibility test, Nanopore sequencing

中图分类号:

R446.9

刘超,刁婷婷,张红吉,刘红伟,杨永锐,李晓非. 飞行时间质谱在耐药结核病检测中的应用价值[J]. 山东大学学报 (医学版), 2024, 62(4): 61-67.

LIU Chao, DIAO Tingting, ZHANG Hongji, LIU Hongwei, YANG Yongrui, LI Xiaofei. Application value of matrix-assisted laser desorption ionization time-of-flight mass spectrometry in the detection of drug resistance of Mycobacterium tuberculosis[J]. Journal of Shandong University (Health Sciences), 2024, 62(4): 61-67.

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