Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Fails To Identify Nontuberculous Mycobacteria from Primary Cultures of Respiratory Samples

Abstract

We have assessed matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification (Bruker) of nontuberculous mycobacteria from newly positive liquid cultures of respiratory samples. Twelve (22%) of 54 isolates were identified directly from liquid medium. After subculture and with manual laser operation, this rose to 49/54 isolates (91%). MALDI-TOF MS is less promising than previously suggested. N ontuberculous mycobacteria (NTM) are increasingly recognized as mostly opportunistic pathogens of humans. The most frequent disease manifestation is a chronic pulmonary infection (1). Since NTM are environmental microorganisms, their presence in pulmonary samples need not indicate disease per se. The clinical relevance of NTM isolation differs strongly by species (2). Hence, correct identification is of paramount importance (3).Identification of NTM is done mostly by using molecular tools. While accurate, these require a good laboratory infrastructure and trained personnel and are costly. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has revolutionized identification in general bacteriology and has also been tried for NTM (3). Most studies of MALDI-TOF MS identification of NTM have applied it to pure cultures from strain collections (4) rather than new positive cultures from clinical samples from nonsterile sites.We assessed MALDI-TOF MS identification of NTM directly from new positive liquid cultures of respiratory samples in a mycobacterial disease reference clinic.Primary cultures of respiratory samples were performed with the Mycobacterium Growth Indicator Tube (MGIT) system (BD Bioscience, Erembodegem, Belgium), as well as on Löwenstein-Jensen slants, both at 37°C, after decontamination by the 1% Nacetyl-L-cysteine-sodium hydroxide method (3).All of the positive cultures that we obtained during the MarchApril 2014 and January-February 2015 periods that tested negative in the TBc-ID immunochromatography assay (BD Bioscience) were studied.We performed MALDI-TOF MS identification with the MALDI Biotyper (Bruker Daltonics, Bremen, Germany) platform. Protein extraction from liquid MGIT medium for MALDI-TOF MS analysis was performed by the manufacturer's MycoEx protocol. In short, we collected biomass by aspirating 1.2 ml of liquid medium from the bottom of the MGIT tube and transferring it to a reaction tube, which was then centrifuged for 2 min at 13,000 rpm (8,124 ϫ g; 43-mm rotor radius); the supernatant was pipetted off, the pellet was resuspended in 300 l of high-performance liquid chromatography grade water, and cells were then inactivated by boiling for 30 min. A 900-l volume of pure cold ethanol was then added, and the tube was vortexed and centrifuged for 2 min at 13,000 rpm. The supernatant was pipetted off. The last two steps were repeated once. The pellet was dried at room temperature before the addition of zirconium beads and 10 to 50 l of pure acetonitrile, depending on the pellet size. After 1 min of vortexing, we adde...

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

“…So we suggest performing each extraction in duplicate or in triplicate. As observed by van Eck et al (31) in a real-life setting from primary cultures of respiratory samples, MALDI-TOF MS identification yields insufficient results when protein extraction is performed from low-AFB-wealth liquid medium.…”

“…As recently shown by van Eck et al with MALDI Biotyper (Bruker Daltonics), identification rates drastically dropped, from 91% to 22%, between colonies subcultured and primary culture of respiratory samples. Although two distinct media were compared (Middlebrook 7H10 plates for subcultures and MGIT medium for primary cultures), these data highlighted the problem of mixed-protein patterns and possible interference with proteins from patients (31). A similar prospective study on Vitek MS would be helpful to define real performances of IVD v3.0 from primary cultures of respiratory samples.…”

“…Identification of mycobacteria has not reached an optimal level of routine testing yet and published studies showed discrepant performances, depending on spectrometer, extraction method, algorithm, library, media, and biomass used for the assays (15)(16)(17)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). In this study, we compared two distinct databases of Vitek-MS, the Saramis v4.12 open database and the new IVD v3.0 closed database.…”

“…It was initially evaluated with mycobacterial intact cells (9)(10)(11)(12), but several investigators worked on improving inactivation and protein extraction (13)(14)(15)(16)(17)(18)(19)(20). Most published studies assessed the performance of MALDI-TOF BioTyper (Bruker Daltonics, Bremen, Germany) (15,17,(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Few of them evaluated Vitek MS (bioMérieux, Marcy l'Etoile, France) (16,17,20,24,25,32).…”

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria

“…The same set of MBT-ASTRA parameters (RG, 0.5; AUC, 0.015; calculation window, m/z 5,000 to 13,000) could be applied to all tested combinations of mycobacterial species with either bactericidal (e.g., isoniazid) or bacteriostatic (e.g., linezolid) antibiotics, indicative of the robustness of the technique. Finally, its application indirectly resolved some outstanding technical issues with automated MALDI-TOF MS-based mycobacterial identifications, i.e., flawed direct analysis of newly positive flagged MGIT cultures, and the superior quality of solid over liquid cultures (32,34,37). Indeed, both the increase in bacterial biomass during the required incubation step and the presence of the internal standard proteins facilitated automated spectrum acquisition.…”