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Spectra of biological replicates, prepared from two repeated cultivations of 15 different strains, matched the expected reference spectra resulting in log(score) values of at least 2.3 for all samples. PCAs with spectra of eight technical replicates of each of 15 strain preparations were also performed for the selected aspergilli (seven), fusaria (four) and Mucorales (four) strains, respectively (Fig.?1). Within each PCA, all technical replicates of a given reference spectrum form discrete clusters according to their strain, indicating that, in spite of little variation between technical replicates of spectra of a particular strain, spectra of the reference strains showed enough difference from each selleck kinase inhibitor other to allow discrimination between them. To validate the extended MALDI-TOF?MS reference database as a tool for fungal species identification, a panel of 103 challenge isolates of aspergilli, fusaria, Mucorales and other fungi (Table?2) that grew directly on diagnostic media, from clinical specimens collected during a 16-month microbiology laboratory routine, were matched against it. The challenge set was also composed of culture collection strains (see Table S1), in order to encompass all of the fungal species present in our reference database. In parallel with MALDI-TOF?MS identification, all strains were analysed by multilocus sequencing (see above for details), which was used as a reference method. As shown in Table?2, MALDI-TOF?MS identified, click here according to their designated species, 91 of 94 clinical isolates (96.8%) of Aspergillus, Fusarium and Mucorales, with no non-identification or misidentification with PFKM the other 3290 reference spectra contained in the database. The log(score) values of the 91 isolates with correct results were all higher than 2.0, whereas three isolates with a log(score) value of designations as compared with the results of the reference method. By contrast, the clinical isolates that belonged to species not included in our database had all log(score) values of