Validation of an expanded, in-house library and an optimized preparation method for the identification of fungal isolates using MALDI-TOF mass spectrometry.
Margarita Estreya ZvezdanovaManuel González de AledoJosé Israel López-MironesJesús OrtegaAndrés CanutCarmen CastroCarmen GomezSilvia HernáezMarina OviañoMaría ErcibengoaMiriam AlkortaPatricia MuñozDavid Rodríguez-TemporalBelén Rodríguez-SánchezPublished in: Medical mycology (2023)
The goal of this study was to validate an optimized sample preparation method for filamentous fungal isolates coupled with the use of an in-house library for the identification of molds using MALDI-TOF Mass Spectrometry in a multicenter context. For that purpose, 3 Spanish microbiology laboratories participated in the identification of 97 fungal isolates using MALDI-TOF MS coupled with the Filamentous Fungi library 3.0 (Bruker Daltonics) and an in-house library containing 314 unique fungal references. The isolates analyzed belonged to 25 species from the genus Aspergillus, Fusarium, Scedosporium/Lomentospora, the Mucorales order and the Dermatophytes group. MALDI-TOF MS identification was carried out from hyphae resuspended in water and ethanol. After a high-speed centrifugation step, the supernatant was discarded and the pellet submitted to a standard protein extraction step. The protein extract was analyzed with the MBT Smart MALDI Biotyper system (Bruker Daltonics). The rate of accurate, species-level identification obtained ranged between 84.5 and 94.8% and the score values were ≥ 1.8 for 72.2-94.9% of the cases. Two laboratories failed to identify only one isolate of Syncephalastrum sp. and Trichophyton rubrum, respectively and three isolates could not be identified in the third center (Fusarium proliferatum, n = 1; Trichophyton interdigitale, n = 2). In conclusion, the availability of an effective sample preparation method and an extended database allowed high rates of correct identification of fungal species using MALDI-TOF MS. Some species, such as Trichophyton spp. are still difficult to identify. Although further improvements are still required, the developed methodology allowed the reliable identification of most fungal species.
Keyphrases
- mass spectrometry
- liquid chromatography
- genetic diversity
- high performance liquid chromatography
- high resolution
- gas chromatography
- bioinformatics analysis
- capillary electrophoresis
- high speed
- oxidative stress
- emergency department
- clinical trial
- electronic health record
- molecularly imprinted
- atomic force microscopy
- drug induced