MALDI-TOF-MS-based species identification and typing approaches in medical mycology

Abstract

MALDI-TOF MS-based species identification has found its place in many clinical routine diagnostic laboratories over the past years. Several well-established commercial systems exist and these allow precise analyses not only among bacteria, but also among clinically important yeasts. This methodology shows higher precision than biochemical and microscopic methods at significantly reduced turnaround times. Furthermore, the differentiation of different filamentous fungi including most dermatophytes and zygomycetes has been established. The direct identification of yeasts from blood culture bottles will be possible in a routine fashion with new standardized procedures. In addition to species identification, the MALDI-TOF MS technology offers several further possibilities, like assays to detect or predict resistance phenotypes in fungi as well as subtyping approaches to detect clinically relevant subgroups. The differences between the commercial systems are discussed with respect to fungi and an overview of their performances provided. Factors influencing outcome of MALDI-TOF-based species identification are discussed.

Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure

“…As already shown with bacteria, MALDI-TOF MS identifies yeasts with rapidity, accuracy, and superiority over conventional phenotypic methods (for review, see references [11][12][13][14][15].…”

“…Nonetheless, Van Herendael et al showed that MALDI-TOF MS analysis using the short extraction method is suitable for the rapid identification of yeast isolates but that its use necessitates the identification threshold to be lower (Ͻ1.7) to account for the lower scores originating from the method (19). Therefore, it was speculated that the highest concordance between acquired spectra and those included in the reference library-in terms of high-confidence (percentage or score) identification-is achievable only if the sample preparation procedure employed for the MALDI-TOF MS system at hand does not differ from that employed to construct the system's reference library (11,12).…”

“…Nonetheless, Van Herendael et al showed that MALDI-TOF MS analysis using the short extraction method is suitable for the rapid identification of yeast isolates but that its use necessitates the identification threshold to be lower (Ͻ1.7) to account for the lower scores originating from the method (19). Therefore, it was speculated that the highest concordance between acquired spectra and those included in the reference library-in terms of high-confidence (percentage or score) identification-is achievable only if the sample preparation procedure employed for the MALDI-TOF MS system at hand does not differ from that employed to construct the system's reference library (11,12).To address this issue, we used a previously established protocol for fast fungal protein extraction (20)-a slight modification from the formic acid-based method-to develop a spectral database for the rapid and unambiguous identification of medically important yeasts by MALDI-TOF MS. First, we retrospectively evaluated the performance of this database by comparing it with the Bruker Biotyper library (V3.2.1.1) and using a clinical collection of wellcharacterized yeast isolates (n ϭ 100) that underwent the fast preparation method. Second, we evaluated our database prospectively by testing freshly collected yeast isolates which were recovered during 6 months of routine laboratory workflow (n ϭ 4,232) and prepared with the above method.…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…Most problems with identification are due to absence, mistakes or incomplete reference spectra as well as differences in sample handling procedures [169]. Hence, reliable identification of organisms by this technique is only possible if the strains used to construct the reference spectral database have been carefully selected and identified by a reliable method such as the sequencing of the ITS region [178].…”

“…It represents a fresh approach that has the potential to replace conventional fungal identification techniques for a majority of routine isolates encountered in clinical microbiology laboratories [169]. Biomolecules such as nucleic future science group Biomarkers for invasive aspergillosis: the challenges continue Review acids, metabolites, proteins or whole microorganisms are embedded in a crystalline matrix of α-cyano-4-hydroxy-cinnamic acid, vaporized and ionized in a vacuum by a short laser pulse.…”