Performance of lipid fingerprint by routine matrix-assisted laser desorption/ionization time of flight for the diagnosis of Mycobacterium tuberculosis complex species

Objectives: Rapid detection of bacterial pathogens at species and sub-species levels is crucial for appropriate treatment, infection control, and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial lipid-based approach to identify MTBC cultures and sub-species.

Methods: A blinded study was conducted using 90 mycobacterial clinical isolate strains comprising MTBC strains sub-cultured in Middlebrook 7H11 medium supplemented with 10% oleic-acid, dextrose, catalase growth supplement and incubated for up to 6 weeks at 37°C and using the following seven reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, and M. bovis BCG) grown under the same conditions, to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption/ionization target followed by the addition of the matrix. Acquisition of the data was performed using the positive ion mode.

Results: Based on the identification of clear and defined lipid signatures from the seven reference strains, the method that we developed was fast (<10 minutes) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the matrix-assisted laser desorption/ionization time of flight were 94.4 (95% CI, 86.4-98.5) and 94.4 (95% CI, 72.7-99.9), respectively.

Conclusions: Mycobacterial lipid profiling provides a means of rapid, safe, and accurate discrimination of species within the MTBC.