Performance of direct detection of Mycobacterium tuberculosis within Mycobacterium tuberculosis complex by routine...

Managing tuberculosis cases requires species and drug susceptibility identification, which are limited by the time taken by testing procedures due to slow bacterial growth. Lipid-based matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for identifying pathogenic mycobacterial species. This study aims to define and use species-specific lipid profiles of members of the Mycobacterium tuberculosis complex (MTBC) obtained by MALDI-TOF MS, to directly discriminate M. tuberculosis from other members of the MTBC, such as Mycobacterium africanum, Mycobacterium bovis, and M. bovis bacillus Calmette-Guerin (BCG). Reference strains (M. tuberculosis H37Rv, M. africanum, M. bovis, and M. bovis BCG) were grown in Middlebrook 7H11 media (supplemented with 10% oleic acid-albumin-dextrose-catalase growth supplement) and incubated for up to 6 weeks at 37°C to generate the large biomass (~10⁹ bacteria) required for optimization, to assess reproducibility of the assay, and to set the reference lipid database. In clinical use, standard shorter culture periods would be sufficient. A blinded study was then performed using a collection of 46 mycobacterial clinical isolate strains composed of 30 M. tuberculosis, 2 M. africanum, 9 M. bovis BCG, and 5 M. bovis and grown under the same conditions. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption target, followed by the addition of the matrix. Acquisition of the data was done using the negative ion mode. Using the species-specific glycolipid, sulfolipids, M. tuberculosis was discriminated within MTBC using the MALDI-TOF process with a sensitivity and specificity of 86.7% (95% confidence interval [CI] 69.3-96.2) and 93.7% (95% CI 69.8-99.8), respectively. Direct detection of M. tuberculosis within the MTBC based on mycobacterial lipid profiling provides a safe and accurate method, based on the detection of the sulfolipids, as a species-specific lipid biomarker of M. tuberculosis.IMPORTANCETuberculosis remains a major infectious disease in humans and mammals, but one of the major challenges is to accurately discriminate M. tuberculosis from the other mycobacterial species belonging to the MTBC. Here we report on a novel assay that can detect M. tuberculosis directly within the MTBC. This approach is simple and relies on the detection of species-specific lipids by routine MALDI-TOF MS.