Performance of direct detection of within complex by routine MALDI-TOF for diagnosis using species-specific lipid fingerprint.

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 complex (MTBC) obtained by MALDI-TOF MS, to directly discriminate from other members of the MTBC, such as , , and bacillus Calmette-Guerin (BCG). Reference strains ( H37Rv, , , and 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 (~10 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 . , 2 . , 9 . BCG, and 5 . 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, 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 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 .IMPORTANCETuberculosis remains a major infectious disease in humans and mammals, but one of the major challenges is to accurately discriminate from the other mycobacterial species belonging to the MTBC. Here we report on a novel assay that can detect directly within the MTBC. This approach is simple and relies on the detection of species-specific lipids by routine MALDI-TOF MS.