Abstract
BackgroundThe 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.ResultsWe 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.ConclusionAddressing 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.
Highlights
The poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF mass spectrometry (MS)-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories
174 isolates corresponded to 18 species, which were represented among those used to construct the eight libraries, whereas the 26 remaining isolates belonged to 20 species that were not represented in the libraries
Reference MS library validation All 104 spectra derived from the 26 clinical isolates for which the species was not included in the seven MS libraries (4 raw spectra per clinical isolate) yielded low Log Scores (LS) ranging from 0.45 to 1.79 regardless of the library utilized, which is markedly below the manufacturer recommended threshold of 2.00 for a valid identification
Summary
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. The identification of mold in the clinical laboratory is classically based on macroscopic and microscopic examination of the colonies grown on mycological culture media It is a slow and complex process requiring highly skilled mycologists, and misidentifications may occur, even in experienced reference laboratories [1]. Matrix-assisted desorption/ionization timeof-flight (MALDI-TOF) mass spectrometry (MS) has been applied to rapidly identify bacteria and yeasts in the clinical microbiology laboratory setting [7]. This technique is used to analyze microorganism content (primarily ribosomal proteins), thereby generating a spectrum that is considered the fingerprint of the microorganism [8]. Previous studies have indicated that MS could be used to identify various filamentous fungi taxa of clinical interest, including Fusarium spp [9,10,11], dermatophytes [12,13], Aspergillus spp [14,15], and Pseudallescheria/Scedosporium spp [16]; those of industrial interest, including Penicillium spp [17,18], Verticillium spp [19], and Trichoderma spp [20]; and various filamentous fungal contaminants frequently isolated in the clinical laboratory [21,22]
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