MALDI‐TOF MS identification of Exophiala species isolated in Japan: Library enrichment and faster sample preparation

Abstract

Exophiala species cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, which are occasionally fatally in immunocompromised patients. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) provides rapid and accurate examination of isolated bacteria and some fungal isolates, but the preparation method for filamentous fungi is complicated. In this study, 31 clinical isolates of Exophiala spp. in Japan were identified by MALDI‐TOF MS with a library enriched by adding data. To simplify the sample preparation method, two modified methods were compared with the standard method for filamentous fungi. The agar cultivation sample preparation method reduced the time required for liquid culture and was considered suitable for clinical use. In 30 of 31 clinical isolates of Exophiala spp., the species identified by MALDI‐TOF MS with the highest score matched the species identified by sequencing the internal transcribed spacer region. Exophiala dermatitidis, E. lecanii‐corni, and E. oligosperma were identified above the genus level, while E. jeanselmei and E. xenobiotica were often not identified at the species level. The identification scores tended to be lower for less‐registered strains in the in‐house library. It is suggested that library enrichment and the modified preparation method may facilitate early diagnosis of rare fungal infections by Exophiala spp. in clinical laboratories using MALDI‐TOF MS.

Formic acid sandwich method is well-suited for filamentous fungi identification and improves turn around time using Zybio EXS2600 mass spectrometry

“…In the present study, the pretreatment method of the protein extraction kit also was evaluated, and those results displayed that these two rapid methods had general applicability in most fungi species. However, the mis identification of E. jeanselmei and S. dehoogii suggests that challenges with protein extraction may be due to the inherent properties of fungal cell walls, impeding accurate fungal identification [ 8 , 19 ]. Future studies should be evaluated and optimized using different protein approaches for different species.…”

“…And the complexity protein fingerprints of filamentous fungi at different growth cycle periods could be another problem. Usually, filamentous fungi form a large number of hyphae on solid medium, and the conidia are difficult to lysis [ 8 ]. The most common pretreatment method for filamentous fungi involves the extraction of proteins with formic acid (EtOH-FA full extraction), a process that requires two rounds of centrifugation [ 9 ].…”