A novel liquid media mycobacteria extraction method for MALDI-TOF MS identification using VITEK® MS

A low-cost identification method that can be performed directly from a positive liquid medium culture is needed for the diagnosis of mycobacterial infections. Here, we describe a novel, cost-effective, and validated method that allows for direct and rapid identification of mycobacteria from a positive liquid culture using VITEK® MS with a total process duration under 45min. From a liquid mycobacteria culture a 3.0mL aliquot is removed 24-72h post positivity and centrifuged to create a pellet. After decanting, the tube is blotted dry, the pellet is re-suspended in 0.5mL of 70% ethanol and then transferred into a 2.0mL tube containing glass beads. Mycobacteria are disrupted mechanically followed by a 10min. incubation at room temperature to complete inactivation. Inactivated material is pelleted by centrifugation and then re-suspended in 10μL of 70% formic acid and 10μL of acetonitrile. After centrifugation, 1μL of supernatant (protein extract) is deposited onto target slide, allowed to dry, and then 1μL CHCA matrix is added. A seeded study was conducted to demonstrate the reliability of the method, a total of 251 culture samples obtained from automated culture systems (BacT/ALERT® MP bottles, BACTEC MGIT™ 960 tubes, and VersaTREK® Myco bottles), were tested and resulted in 98.8% correct identification. Reproducibility was shown by testing three organisms across three reagent lots, between four laboratory technicians, over the course of five days for three liquid media systems resulting in a total of 180 deposits with an overall correct identification of 98.9% with the remaining results giving no identification. Additional studies were performed including comparison of different mechanical disruption techniques, stability of frozen extracts, and stability of slide deposits to allow for flexibility in a routine clinical workflow. The described method proved to be safe while providing consistent and reproducible results for different species of mycobacteria and is compatible with the three most widely used liquid media medium detection systems.