Previous studies have indicated that MS could be used to identify various filamentous fungi taxa of clinical interest, including Fusarium spp, dermatophytes, Aspergillus spp, and Pseudallescheria/ Scedosporium spp those of industrial interest, including Penicillium spp, Verticillium spp, and Trichoderma spp and various filamentous fungal contaminants frequently isolated in the clinical laboratory. When establishing a reference library for microbial identification purposes, many authors have used reference mass spectra, sometimes referred to as “metaspectra” or “superspectra”, which are generated by combining the results of a various number of individual spectra corresponding to technical replicates of a given sample. Using this technique, the identification of an unknown organism is performed by comparing the corresponding spectrum to a reference library of spectra. This technique is used to analyze microorganism content (primarily ribosomal proteins), thereby generating a spectrum that is considered the fingerprint of the microorganism. Recently, matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been applied to rapidly identify bacteria and yeasts in the clinical microbiology laboratory setting. Currently, only some clinical laboratories routinely use a molecular approach for microorganism identification, which is primarily due to the cost and application constraints. Nevertheless, the DNA sequence-based identification of filamentous fungi is primarily limited by the following: i) low DNA extraction yields because mold cells are difficult to lyse, ii) the presence of PCR inhibitors, iii) the presence of misidentified sequences in non-curated public DNA sequence databases, and iv) the cost and time required for sequencing. Therefore, multilocus DNA sequence analysis represents the recommended approach to accurately identify these microorganisms. Additionally, some distinct species, which are identified via DNA sequence analysis, are morphologically indistinguishable. It is a slow and complex process requiring highly skilled mycologists, and misidentifications may occur, even in experienced reference laboratories. The identification of mold in the clinical laboratory is classically based on macroscopic and microscopic examination of the colonies grown on mycological culture media. ConclusionĪddressing 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. 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. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera. 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. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries. 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.
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