Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology

Abstract

Within the past decade, clinical microbiology laboratories experienced revolutionary changes in the way in which microorganisms are identified, moving away from slow, traditional microbial identification algorithms toward rapid molecular methods and mass spectrometry (MS). Historically, MS was clinically utilized as a high-complexity method adapted for protein-centered analysis of samples in chemistry and hematology laboratories. Today, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS is adapted for use in microbiology laboratories, where it serves as a paradigm-shifting, rapid, and robust method for accurate microbial identification. Multiple instrument platforms, marketed by well-established manufacturers, are beginning to displace automated phenotypic identification instruments and in some cases genetic sequence-based identification practices. This review summarizes the current position of MALDI-TOF MS in clinical research and in diagnostic clinical microbiology laboratories and serves as a primer to examine the "nuts and bolts" of MALDI-TOF MS, highlighting research associated with sample preparation, spectral analysis, and accuracy. Currently available MALDI-TOF MS hardware and software platforms that support the use of MALDI-TOF with direct and precultured specimens and integration of the technology into the laboratory workflow are also discussed. Finally, this review closes with a prospective view of the future of MALDI-TOF MS in the clinical microbiology laboratory to accelerate diagnosis and microbial identification to improve patient care.

Fig 1

General schematic for the identification of bacteria and yeast by MALDI-TOF MS using the intact-cell method. Bacterial or fungal growth is isolated from plated culture media (or can be concentrated from broth culture by centrifugation in specific cases) and applied directly onto the MALDI test plate. Samples are then overlaid with matrix and dried. The plate is subsequently loaded into the MALDI-TOF MS instrument and analyzed by software associated with the respective system, allowing rapid identification of the organism.

Fig 2

General schematic for MS analysis of ionized microbiological isolates and clinical material. Once appropriately processed samples are added to the MALDI plate, overlaid with matrix, and dried, the sample is bombarded by the laser. This bombardment results in the sublimation and ionization of both the sample and matrix. These generated ions are separated based on their mass-to-charge ratio via a TOF tube, and a spectral representation of these ions is generated and analyzed by the MS software, generating an MS profile. This profile is subsequently compared to a database of reference MS spectra and matched to either identical or the most related spectra contained in the database, generating an identification for bacteria or yeast contained within the sample.

Fig 3

Additional suggestions for MALDI-TOF MS sample preparations for use with different classes of microbes. Different groups of microorganisms vary fundamentally in their cellular composition and architecture. These differences have been demonstrated to affect the quality of spectra generated during MS experiments and, thus, the accuracy of MALDI-TOF MS-derived identifications. As such, investigators from a number of independent studies have evaluated different methods for sample preparation of different groups of microorganisms, ranging directly from intact-cell to full-protein-extraction-based methodologies. Results from these studies are summarized here. Proper biological safety precautions should be followed with respect to dangerous members of these groups of organisms.

Fig 4

Current position of MALDI-TOF MS in the workflow of the clinical microbiology laboratory, including the current options for analysis of bacteria directly from patient specimens. The MALDI-TOF MS instrument fits easily into the clinical microbiology workflow, occupying the position once held by instruments for automated phenotypic-based identifications (blue arrows). Evaluated mechanisms for the processing of samples directly from patient specimens are included (hatched red arrows), as are options for the use of traditional (green arrows) and MALDI-TOF MS (hatched green arrows) mechanisms. Finally, results are imported into the laboratory information system from the MADLI-TOF MS instrument or other instruments and reported to physicians and pharmacists as indicated. BAL, bronchoalveolar lavage.