A Practical Workflow for the Identification of Aspergillus, Fusarium, Mucorales by MALDI-TOF MS: Database, Medium, and Incubation Optimization

Abstract

There is an increasing body of literature on the utility of MALDI-TOF MS in the identification of filamentous fungi. However, the process still lacks standardization. In this study, we attempted to establish a practical workflow for the identification of three clinically important molds: Aspergillus, Fusarium, and Mucorales using MALDI-TOF MS. We evaluated the performance of Bruker Filamentous Fungi database v3.0 for the identification of these fungi, highlighting when there would be a benefit of using an additional database, the MSI-2 for further identification. We also examined two other variables, namely, medium effect and incubation time on the accuracy of fungal identification. The Bruker database achieved correct species level identification in 85.7% of Aspergillus and 90% of Mucorales, and correct species-complex level in 94.4% of Fusarium. Analysis of spectra using the MSI-2 database would also offer additional value for species identification of Aspergillus species, especially when suspecting species with known identification limits within the Bruker database. This issue would only be of importance in selected cases where species-level identification would impact therapeutic options. Id-Fungi plates (IDFP) had almost equivalent performance to Sabouraud dextrose agar (SDA) for species-level identification of isolates and enabled an easier harvest of the isolates with occasional faster identification. Our study showed accurate identification at 24 h for Fusarium and Mucorales species, but not for Aspergillus species, which generally required 48 h.

Keywords: Aspergillus; Fusarium; IDFP; MALDI-TOF; Mucorales; clinical mycology; cryptic species; filamentous fungi; immunocompromised; immunocompromised hosts; immunosuppressed; mass spectrometry; molds; mycology; proteomics.

FIG 1

Performance of Sabouraud dextrose agar (SDA) versus Id-fungi plates (IDFP). Aspergillus (A; n = 31), Fusarium (F; n = 18), and Mucorales (M; n = 20) isolates performance to species/clade, section/species complex, and genus level identification using the Bruker Filamentous Fungi v3 and Mass Spectral Identification-2 (MSI-2). The best score was taken from each replicate. The reference standard was β-tubulin, translation elongation factor, internal transcribed spacer 2 regions, and/or D1/D2 region of the 25 to 28S rRNA gene sequencing. Due to the limited number of Fusarium isolates that were successfully identified as species by reference sequencing (n = 16/18), only species complex, genus, and no ID was shown.

FIG 2

Performance of 24 h (24 HR) versus 48 h (48 HR) from Id-fungi plates (IDFP). Aspergillus (A; n = 31), Fusarium (F; n = 18), and Mucorales (M; n = 20) isolates performance to species/clade, section/species complex, and genus level identification using the Bruker Filamentous Fungi v3 and Mass Spectral Identification-2 (MSI-2). The best score was taken from each replicate. The reference standard was β-tubulin, translation elongation factor, internal transcribed spacer 2 regions, and/or D1/D2 region of the 25 to 28S rRNA gene sequencing. Due to the limited number of Fusarium isolates that were successfully identified as species by reference sequencing (n = 16/18), only species complex, genus, and no ID was shown.

FIG 3

Flow chart for the workup of Aspergillus species after MALDI-TOF MS and the identification using the Bruker Filamentous Fungi v3 database. ID = identification; MBT = MALDI biotyper; MSI-2 = mass spectrometry identification-2.

FIG 4

Flow chart for the workup of Fusarium species after MALDI-TOF MS and the identification using the Bruker Filamentous Fungi v3 database.

FIG 5

Flow chart for the workup of Mucorales after MALDI-TOF MS and the identification using the Bruker Filamentous Fungi v3 database. ID, identification; MSI-2, mass spectrometry identification-2.