Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi

Background: The poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries. Results: We established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i. e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i. e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera. Identification effectiveness was improved by increasing the number of both RMS per strain (p<10(-4)) and strains for a given species (p<10(-4)) in a multivariate analysis. Conclusion: Addressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi.