Purpose: To evaluate and compare the performance of three commercial culture media, two filamentous fungi libraries, and two different protein extraction procedures in MALDI-TOF MS fungal identification.

Methods: A total of 21 quality control samples were cultured on Sabouraud dextrose agar (SDA), ID fungi plate medium (IDFP), and Sabouraud gentamicin chloramphenicol 2 agar (SGC2). For four consecutive days, fungal growths were inoculated on a MALDI target plate both by using a direct transfer technique (DT) and by using a formic acid-ethanol protein extraction procedure (EEP). The MALDI-TOF MS-generated spectra were identified by the MBT Bruker library and the MSI database.

Results: Selective culture media (IDFP and SGC2) significantly outperformed the non-selective SDA medium. IDFP was superior to the SGC2 medium for dermatophyte identification. The EEP only demonstrated a benefit over DT in the underperforming SDA medium. The MBT Bruker library outperformed the MSI database in Aspergillus identification while the MSI database outperformed the MBT library in dermatophyte identification. For non-Aspergillus fungi, the libraries performed comparably.

Conclusion: The results of our study show the necessity of using selective culture media (IDFP and SGC2) for fungal identification with MALDI-TOF MS and demonstrate no significant benefit of the formic acid-ethanol protein extraction technique in these media. Given the relative strengths and weaknesses of the MBT library and the MSI database, it might currently be beneficial to consider these libraries as complementary and employ both databases to achieve optimal fungal identification.