Performance of Vitek MS v3.0 for Identification of Mycobacterium Species from Patient Samples by Use of Automated Liquid Medium Systems.

The accuracy and robustness of the Vitek MS v3.0 matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) system was evaluated by identifying mycobacteria from automated liquid-medium systems using patient samples. This is the first report to demonstrate that proteins within the liquid medium, its supplements, and decontamination reagents for nonsterile patient samples do not generate misidentification or false-positive results by use of the Vitek MS v3.0 system. Prior to testing with patient samples, a seeded-culture study was conducted to challenge the accuracy of the Vitek MS system at identifying mycobacteria from liquid medium by mimicking a clinical workflow. Seventy-seven Mycobacterium strains representing 21 species, seeded in simulated sputum, were decontaminated, inoculated into BacT/Alert MP liquid culture medium, incubated until positivity, and identified using the Vitek MS system. A total of 383 liquid cultures were tested, of which 379 (99%) were identified correctly to the species/complex/group level, 4 (1%) gave a "no-identification" result, and no misidentifications were observed. Following the simulated-sputum study, a total of 73 smear-positive liquid-medium cultures detected using BD BBL MGIT and VersaTREK Myco liquid media were identified by the Vitek MS system. Sixty-four cultures (87.7%) were correctly identified to the species/complex/group level; 7 (9.6%) resulted in no identification; and 2 (2.7%) were misidentified at the species level. These results indicate that the Vitek MS v3.0 system is an accurate tool for routine diagnostics of Mycobacterium species isolated from liquid cultures.

Rapid inactivation of Mycobacterium and nocardia species before identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained.

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.