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

Abstract

In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of >2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies.T o date, literature-based evidence has accumulated with respect to the reliability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of yeast isolates in diagnostic clinical microbiology laboratories (1-10). As already shown with bacteria, MALDI-TOF MS identifies yeasts with rapidity, accuracy, and superiority over conventional phenotypic methods (for review, see references 11-15).As an alternative to the ethanol/formic acid-based procedure, also referred to as complete tube extraction, recommended for use only with the Bruker MALDI Biotyper system (Bruker Daltonics, Bremen, Germany), the on-plate extraction or fast formic acid method (4, 16, 17), which consists of covering the smeared yeast colony with a formic acid solution, was proposed. Using the Bruker Biotyper 3.0 database and spectral scores of Ն1.7 as cutoffs for species-level identification, Theel et al. found that the formic acid-based direct on-plate method yielded identification percentages that were similar to those obtained with the more complex tube-based extraction method (18). 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 ...

Interlaboratory Comparison of Sample Preparation Methods, Database Expansions, and Cutoff Values for Identification of Yeasts by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Using a Yeast Test Panel

“…These studies showed 88.7% (27) and 96% correct identification rates with a score of Ͼ1.7 (21), compared to 97.4% (29) and 97.9% (30) with a score of Ͼ1.8. The Bruker MALDI Biotyper system did not generate erroneous fungal identifications (10,31).…”

“…In comparison with other identification methods, such as sequence analysis of the D1/D2 domains of the large subunit ribosomal DNA (rDNA) and the internal transcribed spacer (ITS) 1 and 2 regions of the rDNA, MALDI-TOF MS is able to provide accurate identifications of microorganisms with a short turnaround time (1,2). No major errors, such as genus-level misidentifications, have been reported in many MALDI-TOF MS-based studies on yeasts and filamentous fungi (3)(4)(5)(6)(7)(8)(9)(10)(11). Most yeasts can easily be processed and correctly identified; even sibling species that cannot be distinguished with common biochemical methods can be discriminated with MALDI-TOF MS (12,13).…”

Optimized Use of the MALDI BioTyper System and the FilmArray BCID Panel for Direct Identification of Microbial Pathogens from Positive Blood Cultures

“…Aliquots from each positive BC bottle/broth were subjected to routine Gram staining microscopy and solid-medium subcultures. After isolation from the cultures, bacteria and yeasts were identified by MALDI BioTyper analysis; in cases in which the isolate could not be identified (i.e., ID score was Ͻ2.0), conventional phenotypical tests and/or sequencing of the 16S rRNA gene or the rpoB gene (for bacterial isolates) and the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA gene region (for fungal isolates) were performed as previously described (11,15,27). Antimicrobial susceptibility testing of the bacterial isolates was performed as part of the routine BC analyses with the Vitek 2 (bioMérieux) and/or Etest (bioMérieux), and interpreted according to EUCAST guidelines.…”

MALDI-TOF MS for the Diagnosis of Infectious Diseases

“…The VITEK MS system identified 96% of the isolates; the Biotyper system identified 91% of isolates. De Carolis et al created an in-house library using spectra from 156 reference and clinical yeast isolates, generated with a fast sample preparation procedure involving suspending a single colony of yeast in 50 L of 10% formic acid, vortex mixing, and using 1 L of the lysate for analysis (39 ). Using their database and processing, they identified 96% of 4232 routinely isolated yeasts using a specieslevel cutoff of Ն2.000 and the Biotyper system (software v3.0).…”