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