Identification of dermatophytes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this study we evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of dermatophytes in diagnostic laboratories. First, a spectral database was built with 108 reference strains belonging to 18 species of the anamorphic genera Epidermophyton, Microsporum and Trichophyton. All strains were well characterized by morphological criteria and ITS sequencing (gold standard). The dendrogram resulting from MALDI-TOF mass spectra was almost identical with the phylogenetic tree based on ITS sequencing. Subsequently, MALDI-TOF MS SuperSpectra were created for the identification of Epidermophyton floccosum, Microsporium audouinii, M. canis, M. gypseum (teleomorph: Arthroderma gypseum), M. gypseum (teleomorph: A. incurvatum), M. persicolor, A. benhamiae (Tax. Entity 3 and Am-Eur. race), T. erinacei, T. interdigitale (anthropophilic and zoophilic populations), T. rubrum/T. violaceum, T. tonsurans and T. terrestre. Because T. rubrum and T. violaceum did not present enough mismatches, a SuperSpectrum covering both species was created, and differentiation between them was done by comparison of eight specific peptide masses. In the second part of this study, MALDI-TOF MS with the newly created SuperSpectra was tested using 141 clinical isolates representing nine species. Analyses were done with 3-day-old cultures. Results were compared to morphological identification and ITS sequencing; 135/141 (95.8%) strains were correctly identified by MALDI-TOF MS compared to 128/141 (90.8%) by morphology. Therefore, MALDI-TOF MS has proven to be a useful and rapid identification method for dermatophyte

Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Protein fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spec-trometry (MALDI-TOF MS) is a rapid, reliable, and economical method to characterize isolates of terrestrial fungi and other microorganisms. The objective of our study was to evaluate the suitability of MALDI-TOF MS for the identification of aquatic hyphomycetes, a polyphyletic group of fungi that play crucial roles in stream ecosystems. To this end, we used 34 isolates of 21 aquatic hyphomycete species whose identity was confirmed by spore morphology and internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) nuc rDNA sequencing. We tested the efficiency of three protein extraction methods, including chemical and mechanical treatments using 13 different protocols, with the objective of producing high-quality MALDI-TOF mass spectra. In addition to extraction protocols, mycelium age was identified as a key parameter affecting protein extraction efficiency. The dendrogram based on mass-spectrum similarity indicated good and relevant taxonomic discrimination; the tree structure was comparable to that of the phylogram based on ITS sequences. Consequently, MALDI-TOF MS could reliably identify the isolates studied and provided greater taxonomic accuracy than classical morphological methods. MALDI-TOF MS seems suited for rapid characterization and identification of aquatic hyphomycete species

Identification of dermatophytes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

In this study we evaluated the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of dermatophytes in diagnostic laboratories. First, a spectral database was built with 108 reference strains belonging to 18 species of the anamorphic genera Epidermophyton, Microsporum and Trichophyton. All strains were well characterized by morphological criteria and ITS sequencing (gold standard). The dendrogram resulting from MALDI-TOF mass spectra was almost identical with the phylogenetic tree based on ITS sequencing. Subsequently, MALDI-TOF MS SuperSpectra were created for the identification of Epidermophyton floccosum, Microsporium audouinii, M. canis, M. gypseum (teleomorph: Arthroderma gypseum), M. gypseum (teleomorph: A. incurvatum), M. persicolor, A. benhamiae (Tax. Entity 3 and Am-Eur. race), T. erinacei, T. interdigitale (anthropophilic and zoophilic populations), T. rubrum/T. violaceum, T. tonsurans and T. terrestre. Because T. rubrum and T. violaceum did not present enough mismatches, a SuperSpectrum covering both species was created, and differentiation between them was done by comparison of eight specific peptide masses. In the second part of this study, MALDI-TOF MS with the newly created SuperSpectra was tested using 141 clinical isolates representing nine species. Analyses were done with 3-day-old cultures. Results were compared to morphological identification and ITS sequencing; 135/141 (95.8%) strains were correctly identified by MALDI-TOF MS compared to 128/141 (90.8%) by morphology. Therefore, MALDI-TOF MS has proven to be a useful and rapid identification method for dermatophytes

Biomineralization Capacities of Chlorodendrophyceae: Correlation Between Chloroplast Morphology and the Distribution of Micropearls in the Cell

Several species of the genus Tetraselmis (Chlorodendrophyceae, Chlorophyta) were recently discovered to possess unsuspected biomineralization capacities: they produce multiple intracellular inclusions of amorphous calcium carbonate (ACC), called micropearls. Early light-microscopists had spotted rows of refractive granules in some species, although without identifying their mineral nature. Scanning electron microscope (SEM) observations showed that the distribution of the micropearls in the cell forms a pattern, which appears to be characteristic for a given species. The present study shows that this pattern correlates with the shape of the chloroplast, which differs between Tetraselmis species, because micropearls align themselves along the incisions between chloroplast lobes. This was observed both by SEM and in live cells by light microscopy (LM) using Nomarski differential interference contrast. Additionally, molecular phylogenetic analyses, of rbcL and ITS2 gene sequences from diverse strains of Chlorodendrophyceae, corroborated the morphological observations by identifying two groups among nominal Tetraselmis spp. that differ in chloroplast morphology, micropearl arrangement, and ITS2 RNA secondary structure. (C) 2020 The Authors. Published by Elsevier GmbH

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures

The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory