基于BACTEC MGIT 960培养的结核分枝杆菌与非结核分枝杆菌混合感染检测方法的研究

doi:10.19982/j.issn.1000-6621.20240128

摘要/Abstract

摘要：

目的：探讨结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTBC)和非结核分枝杆菌(nontuberculous mycobacteria, NTM)混合感染的检测方法,为临床诊治混合感染提供参考依据。方法：使用脓肿分枝杆菌、鸟分枝杆菌、胞内分枝杆菌3种NTM和MTBC标准菌株(H37Rv)各制备3种不同初始浓度(10^-2 mg/ml、10^-4 mg/ml和10^-6 mg/ml)的菌液,将每个浓度的NTM与MTBC采用9种不同比例(NTM标准菌株与H37Rv按照1∶99、5∶95、10∶90、20∶80、50∶50、80∶20、90∶10、95∶5和99∶1)混合制成81种混合感染标本。3种感染模型分别为脓肿分枝杆菌和H37Rv、鸟分枝杆菌和H37Rv、胞内分枝杆菌和H37Rv。经BACTEC MGIT 960培养,采用MPB64抗原检测、对硝基苯甲酸(PNB)生长试验、荧光PCR熔解曲线法和恒温扩增实时荧光法4种方法对培养液进行检测。结果：3种混合感染模型的81份标本均在34d内报告阳性。其中,MPB64抗原检测阳性3份(3.7%,3/81);PNB生长试验阳性78份(96.3%,78/81);荧光PCR熔解曲线法仅检出相应的NTM 64份(79.0%,64/81),仅检出MTBC 3份(3.7%,3/81),同时检出相应的NTM和MTBC 14份(17.3%,14/81);恒温扩增实时荧光法检出MTBC 67份(82.7%,67/81)。结论：临床标本经BACTEC MGIT 960培养,仪器报告阳性后,用MPB64抗原检测和PNB生长试验初判,若有NTM生长,建议采用分子生物学方法鉴定NTM,同时用单一检测MTBC核酸的方法对培养液进行检测,以免MTBC 漏检。

关键词: 分枝杆菌, 结核, 非典型性细菌, 重叠感染, 培养技术, 研究技术

Abstract:

Objective: To investigate the detection methods for mixed infections with Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), and provide reference for clinical diagnosis and treatment of mixed infections. Methods: Bacterial suspensions with three different initial concentrations (10^-2 mg/ml, 10^-4 mg/ml, and 10^-6 mg/ml) were prepared respectively using MTBC standard strains (H37Rv) and three NTM species (M.abscessus, M.avium, and M.intracellulare). The bacterial suspensions at same initial concentrations of NTM and MTBC were mixed in nine different ratios (namely each NTM were mixed with H37Rv in a ratio of 1∶99, 5∶95, 10∶90, 20∶80, 50∶50, 80∶20, 90∶10, 95∶5, and 99∶1) to create a total of 81 simulated mixed infection specimens, which including three infection models: M.abscessus plus H37Rv, M.avium plus H37Rv, and M.intracellulare plus H37Rv. All mixed infection specimens were incubated in the MGIT 960 system and then tested using four methods: MPB64 antigen assay, p-nitrobenzoic acid (PNB) growth assay, fluorescent PCR melting curve method, and real-time fluorescence thermostatic amplification. Results: All 81 specimens from 3 mixed infection models reported positive within 34 days. Among them, 3 specimens (3.7%, 3/81) tested positive for the MPB64 antigen, while 78 specimens (96.3%, 78/81) tested positive for the PNB growth assay. By using the fluorescent PCR melting curve method, 64 specimens (79.0%, 64/81) were detected as corresponding NTM alone, 3 specimens (3.7%, 3/81) were MTBC alone, and 14 specimens (17.3%, 14/81) were both NTM and MTBC. MTBC was detected in 67 specimens (82.7%, 67/81) by using the thermostatic amplification real-time fluorescence method. Conclusion: The clinical specimens that were cultured and reported positive by BACTEC MGIT 960, MPB64 antigen test and PNB growth assay were recommended for preliminary assessment. If NTM growth in the MGIT tube is observed, a molecular biological method is recommended for identifying NTM. At the same time, another molecular method specific for MTBC nucleic acid can be used for the culture medium examination to avoid missed detection of MTBC.

Key words: Mycobacterium tuberculosis, Atypical bacterial forms, Superinfection, Culture techniques, Investigative techniques

中图分类号:

R446
R378.91

陈振华, 郭婧玮, 王珏, 胡培磊, 易松林, 刘怡, 谭云洪. 基于BACTEC MGIT 960培养的结核分枝杆菌与非结核分枝杆菌混合感染检测方法的研究[J]. 中国防痨杂志, 2024, 46(10): 1243-1249. doi: 10.19982/j.issn.1000-6621.20240128

Chen Zhenhua, Guo Jingwei, Wang Jue, Hu Peilei, Yi Songlin, Liu Yi, Tan Yunhong. Study on detection methods of mixed infection with Mycobacterium tuberculosis complex and nontuberculous mycobacteria based on BACTEC MGIT 960 culture[J]. Chinese Journal of Antituberculosis, 2024, 46(10): 1243-1249. doi: 10.19982/j.issn.1000-6621.20240128

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