Method Description

DNA sequence analysis utilizes a 500-base pair region of the 16S rRNA gene as the target for identification of mycobacteria and is performed using the MicroSeq kit from Applied Biosystems. Sequence data generated is compared to several different databases of known mycobacterial and aerobic actinomycete sequences to obtain organism identification. These include MicroSeq, NCBI GenBank, and Mayo Clinic Mycobacteria database. A 100% agreement with a database strain is needed for an acceptable identification to the species level.(Hall L, Doerr KA, Wohlfiel SL, Roberts GD: Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol. 2003 Apr;41[4]:1447-1453)

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis is done using the Bruker BioTyper platform and the Bruker BDAL library, Bruker Mycobacterial Library, and the Mayo Clinic Library. A spectral score of 2.0 or greater is required for identification to the species level.

Rapid polymerase chain reaction (PCR) is performed following specimen digestion and decontamination using N-acetyl cysteine and sodium hydroxide. Genomic DNA is extracted using the MagNA Pure (Roche Applied Sciences) extraction platform. The purified genomic DNA is placed on the LightCycler instrument, which amplifies and monitors, by fluorescence, the development of target nucleotide sequences after each PCR cycle. A specific target sequence from a portion of the katG gene from Mycobacterium tuberculosis complex is amplified and the resulting segment is detected by melt-curve analysis using sequence-specific fluorescence resonance energy transfer hybridization probes. The LightCycler PCR assay is a closed PCR system that greatly reduces the potential for false-positive results due to specimen cross-contamination as compared with traditional open-system PCR or other amplification methods like transcription-mediated amplification.(Buckwalter SP, Connelly BJ, Louison LK, et al. Description, validation, and review of a decade of experience with a laboratory-developed PCR test for detection of M tuberculosis complex in pulmonary and extrapulmonary specimens. J Clin Tuberc Other Mycobact Dis. 2022;29:100340. doi:10.1016/j.jctube.2022.100340)

The method employed in this assay is broth microtiter dilution using a commercially available plate from Trek Diagnostics. Antimicrobials included in the assay are tested according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.(Clinical and Laboratory Standards Institute: Susceptibility testing of mycobacteria, nocardia spp., and other aerobic actinomycetes. 3rd ed. CLSI standard M24. CLSI; 2018)

This test method is based on growth or absence of growth of M tuberculosis complex isolates in broth cultures containing critical concentrations of the antimycobacterial agents isoniazid, rifampin, and ethambutol. One of two US Food and Drug Administration-cleared platforms, the BACTEC MGIT 960 (Becton Dickinson) or the VersaTREK (ThermoFisher) will be used.(Brown-Elliott BA, Cirillo DM, Musser KA, Rowlinson M-C. Susceptibility test methods: Mycobacteria, nocardia, and other actinomycetes. In: Carroll KC, Pfaller MA, eds. Manual of Clinical Microbiology. 13th ed. ASM Press; 2023)

This test utilizes the Sensititre MycoTB broth microtiter dilution plate (Trek/ThermoFisher). Antimicrobials are tested according to CLSI M24 guidelines.(Thermo Scientific Sensititre MIC Susceptibility Plates for Mycobacterium tuberculosis. Product Insert. 011-MYCOTB-CID9502. Revision Date: 09/07/2016; Brown-Elliott BA, Cirillo DM, Musser KA, Rowlinson M-C. Susceptibility test methods: Mycobacteria, nocardia, and other actinomycetes. In: Carroll KC, Pfaller MA, eds. Manual of Clinical Microbiology, 13th ed. ASM Press; 2023)