Detection and identification of dermatophytes based on currently available methods - a comparative study.

AIMS: Accurate identification of dermatophytes is essential for implementing appropriate antifungal treatment and epidemiological analysis. However, the limitations of conventional diagnostics are a frequently discussed topic, and new diagnostic techniques are constantly expanding. In this study, we assess the suitability of conventional diagnostic techniques in comparison to the real-time PCR assay and MALDI-TOF MS in detection and identification of dermatophytes. METHODS AND RESULTS: Strains included in this study were obtained from human and animals with symptomatic, and asymptomatic infection. A direct examination revealed that 31·7 and 60·9% of samples from symptomatic patients, and 25·7 and 60% from asymptomatic animals were positive, as shown by light and fluorescence microscopy respectively. In turn, dermatophytes were isolated from 90·2 and 71·4% of these samples. The pan-dermatophyte primers in real-time PCR assay facilitated detection in 85·3 and 82·9% of the symptomatic and asymptomatic dermatophytoses respectively. Additionally, species-specific PCR assays were positive in 70·7 and 37·1% of these samples. The MALDI-TOF MS analysis yielded positive results consistent with conventional techniques in 97·2 and 72% of symptomatic and asymptomatic infections respectively. CONCLUSIONS: Our study revealed that there is no universal diagnostic method that would be ideal in each of the cases considered. Nonetheless, conventional techniques are still the most effective and reliable tools for mycological diagnostics. SIGNIFICANCE AND IMPACT OF THE STUDY: Dermatologists and veterinarians have difficulties in making a diagnosis of dermatophytoses based only on observed symptoms of fungal infections, as they mimic symptoms of other dermatoses. In this context, a comparative analysis of the results of diagnostics performed using conventional methods and new technologies are crucial for implementing these pioneer methods into routine laboratory practice.

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry: Protocol standardisation, comparison and database expansion for faster and reliable identification of dermatophytes.

BACKGROUND: Accurate and early identification of dermatophytes enables prompt antifungal therapy. However, phenotypic and molecular identification methods are time-consuming. MALDI-TOF MS-based identification is rapid, but an optimum protocol is not available. OBJECTIVES: To develop and validate an optimum protein extraction protocol for the efficient and accurate identification of dermatophytes by MALDI-TOF MS. MATERIALS/METHODS: Trichophyton mentagrophytes complex (n = 4), T. rubrum (n = 4) and Microsporum gypseum (n = 4) were used for the optimisation of protein extraction protocols. Thirteen different methods were evaluated. A total of 125 DNA sequence confirmed clinical isolates of dermatophytes were used to create and expand the existing database. The accuracy of the created database was checked by visual inspection of MALDI spectra, MSP dendrogram and composite correlation index matrix analysis. The protocol was validated further using 234 isolates. RESULT: Among 13 protein extraction methods, six correctly identified dermatophytes but with a low log score (≤1.0). The modified extraction protocol developed provided an elevated log score of 1.6. Significant log score difference was observed between the modified protocol and other existing protocols (T. mentagrophytes complex: 1.6 vs. 0.2-1.0, p < .001; T. rubrum: 1.6 vs. 0.4-1.0, p < .001; M. gypseum:1.6 vs. 0.2-1.0, p < .001). Expansion of the database enabled the identification of all 234 isolates (73.5% with log score ≥2.0 and 26.4% with log scores range: 1.75-1.99). The results were comparable to DNA sequence-based identification. CONCLUSION: MALDI-TOF MS with an updated database and efficient protein extraction protocol developed in this study can identify dermatophytes accurately and also reduce the time for identifying them.

Towards a rapid identification and a novel proteomic analysis for dermatophytes from human and animal dermatophytosis.

BACKGROUND: Since accurate identification of dermatophyte species is essential for epidemiological studies and implementing antifungal treatment, overcoming limitations of conventional diagnostics is a fruitful subject. OBJECTIVES AND METHODS: In this study, we investigated real-time polymerase chain reaction(q-PCR), matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) and nano-electrospray ionisation mass spectrometry (nano-ESI-MS) to detect and identify the most frequently isolated dermatophytes from human and animal dermatophytosis in comparison with conventional methods. RESULTS: Among 200 samples, the identified species were Microsporum canis (78.22%), Trichophyton verrucosum (10.89%) and T. mentagrophytes (5.94%). Q-PCR assay displayed great execution attributes for dermatophytes detection and identification. Using MALDI-TOF MS, M. canis, but none of T. violacium, T. verrucosum or T. mentagrophytes, could be identified. Nano-ESI-MS accurately identified all species. The potential virulence attributes of secreted proteases were anticipated and compared between species. Secreted endoproteases belonging to families/subfamilies of metalloproteases, subtilisins and aspartic protease were detected. The analysed exoproteases are aminopeptidases, dipeptidyl peptidases and carboxypeptidases. Microsporum canis have three immunogenic proteins, siderophore iron transporter mirB, protease inhibitors, plasma membrane proteolipid 3 and annexin. CONCLUSION: In essence, q-PCR, MALDI-TOF MS and nano-ESI-MS assays are very nearly defeating difficulties of dermatophytes detection and identification, thereby, supplement or supplant conventional diagnosis of dermatophytosis.

Ultra-High-Resolution Mass Spectrometry for Identification of Closely Related Dermatophytes with Different Clinical Predilections.

In the present study, an innovative top-down liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification of clinically relevant fungi is tested using a model set of dermatophyte strains. The methodology characterizes intact proteins derived from Trichophyton species, which are used as parameters of differentiation. To test its resolving power compared to that of traditional Sanger sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), 24 strains of closely related dermatophytes, Trichophyton rubrum, T. violaceum, T. tonsurans, T. equinum, and T. interdigitale, were subjected to this new approach. Using MS/MS and different deconvolution algorithms, we identified hundreds of individual proteins, with a subpopulation of these used as strain- or species-specific markers. Three species, i.e., T. rubrum, T. violaceum, and T. interdigitale, were identified correctly down to the species level. Moreover, all isolates associated with these three species were identified correctly down to the strain level. In the T. tonsurans-equinum complex, eight out of 12 strains showed nearly identical proteomes, indicating an unresolved taxonomic conflict already apparent from previous phylogenetic data. In this case, it was determined with high probability that only a single species can be present. Our study successfully demonstrates applicability of the mass spectrometric approach to identify clinically relevant filamentous fungi. Here, we present the first proof-of-principle study employing the mentioned technology to differentiate microbial pathogens. The ability to differentiate fungi at the strain level sets the stage to improve patient outcomes, such as early detection of strains that carry resistance to antifungals.

Fast identification of dermatophytes by MALDI-TOF/MS using direct transfer of fungal cells on ground steel target plates.

Dermatophytes cause human infections limited to keratinised tissues. We showed that the direct transfer method allows reliable identification of non-dermatophytes mould and yeast by MALDI-TOF/MS. We aimed at assessing whether the direct transfer method can be used for dermatophytes and whether an own mass spectra library would be superior to the Bruker library. We used the Bruker Biotyper to build a dermatophyte mass spectra library and assessed its performance by 1/testing a panel of mass spectrum produced with strains genotypically identified and, 2/comparing MALDI-TOF/MS identification to morphology-based methods. Identification of dermatophytes using the Bruker library is poor. Our library provided 97% concordance between ITS sequencing and MALDI-TOF/MS analysis with a panel of 1104 spectra corresponding to 276 strains. Direct transfer method using unpolished target plates allowed proper identification of 85% of dermatophytes clinical isolates most of which were common dermatophytes. A homemade dermatophyte MSP library is a prerequisite for accurate identification of species absent in the Bruker library but it also improves identification of species already listed in the database. The direct deposit method can be used to identify the most commonly found dermatophytes such as T. rubrum and T. interdigitale/mentagrophytes by MALDI-TOF/MS.

MALDI-TOF-Based Dermatophyte Identification.

MALDI-TOF MS has become increasingly popular for microorganism identification in the routine laboratory. Compared with conventional morphology-based techniques, MALDI-TOF is relatively inexpensive (per-unit identification), involves a rapid result turnaround time and yields more accurate results without the need for highly qualified staff. However, this technology has been technically difficult to implement for filamentous fungi identification. Identification of dermatophytes, a type of filamentous fungi, remains particularly challenging, partly due to the lack of clear species definition for some taxa or within some species complexes. Review of the ten studies published between 2008 and 2015 shows that the accuracy of MALDI-TOF MS-based identification varied between 13.5 and 100 % for dermatophytes. This variability was partly due to inconsistencies concerning critical steps of the routine clinical laboratory process. Use of both a complete formic acid-acetonitrile protein extraction step and a manufacturer library supplemented with homemade reference spectra is essential for an accurate species identification. This technique is conversely unaffected by variations in other routine clinical laboratory conditions such as culture medium type, incubation time and type of mass spectrometry instrument. Provided that a reference spectra library is adequate for dermatophyte identification, MALDI-TOF MS identification is more economical and offers an accuracy comparable to that of DNA sequencing. The technique also represents an advantageous alternative to the protracted and labor-intensive dermatophyte identification via macroscopic and microscopic morphology in the routine clinical laboratory.

Evaluation of the Bruker Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) System for the Identification of Clinically Important Dermatophyte Species.

Dermatophytes can invade the stratum corneum of the skin and other keratinized tissues and are responsible for a broad diversity of diseases of skin, nails and hair. Although the standard identification of dermatophytoses depends on macroscopic and microscopic characterization of the colonies grown on special media, there are a number of limitations owing to intraspecies morphological variability, atypical morphology or interspecies morphological similarity which entails improvement in the identification methods. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel method which proved to be effective for rapid and reliable identification of dermatophytes grown in cultures when compared to conventional methods. We evaluated the performance of Bruker MALDI-TOF MS System (Bruker Daltonics, Germany) for identification of clinically relevant dermatophytes. In order to increase the identification capacity of the system, we created supplemental spectral database entries using ten reference dermatophyte strains (ten species in two genera). The utility of the generated database was then challenged using a total of 126 dermatophytes (115 clinical isolates and 11 additional reference strains). The results were evaluated by both manufacturer-recommended and lowered cutoff scores. MALDI-TOF MS provided correct identification in 122 (96.8 %) and 113 (89.7 %) of the isolates with the lowered scores and using the supplemented database, respectively, versus 65 (51.6 %) and 17 (13.5 %) correct identifications obtained by the unmodified database and recommended scores at the genus and species levels, respectively. Our results support the potential utility of MALDI-TOF MS as a routine tool for accurate and reliable identification of dermatophytes.

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.

Fast and accurate identification of dermatophytes by matrix-assisted laser desorption ionization-time of flight mass spectrometry: validation in the clinical laboratory.

The performance of a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) workflow using an extensive reference database for dermatophyte identification was evaluated on 176 clinical strains. Using a direct-deposit procedure after 3 incubation days yielded 40% correct identification. Both increasing incubation time and using an extraction procedure resulted in 100% correct identification.

Identification of Dermatophyte species after implementation of the in-house MALDI-TOF MS database.

Despite that matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool in the clinical microbiology setting, few studies have till now focused on MALDI-TOF MS-based identification of dermatophytes. In this study, we analyze dermatophytes strains isolated from clinical samples by MALDI-TOF MS to supplement the reference database available in our laboratory. Twenty four dermatophytes (13 reference strains and 11 field isolated strains), identified by both conventional and molecular standard procedures, were analyzed by MALDI-TOF MS, and the spectra obtained were used to supplement the available database, limited to a few species. To verify the robustness of the implemented database, 64 clinical isolates other than those used for the implementation were identified by MALDI-TOF MS. The implementation allowed the identification of the species not included in the original database, reinforced the identification of the species already present and correctly identified those within the Trichophyton mentagrophytes complex previously classified as Trichophyton. tonsurans by MALDI-TOF MS. The dendrogram obtained by analyzing the proteic profiles of the different species of dermatophytes reflected their taxonomy, showing moreover, in some cases, a different clusterization between the spectra already present in the database and those newly added. In this study, MALDI-TOF MS proved to be a useful tool suitable for the identification of dermatophytes for diagnostic purpose.

A MALDI-TOF MS procedure for clinical dermatophyte species identification in the routine laboratory.

The conventional identification of dermatophytes requires a long turnaround time and highly skilled mycologists. We have recently developed a tandardized matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay to routinely identify molds of potential clinical significance. This study objective was to determine if this same assay could also be employed to identify clinical dermatophytes in the routine laboratory setting. The effects of the inclusion of cycloheximide in the culture medium and incubation time were tested after building a reference spectra library that included 48 well-characterized isolates of 17 dermatophyte species. Then these same isolates were prospectively identified using this library. MALDI-TOF MS-based identification was effective regardless of the presence of cycloheximide or incubation time as 130/133 (97.8%) of the clinical isolates were appropriately identified. Two Microsporum canis isolates yielded uninformative spectra and one M. audouinii isolate was misidentified. Since one only requires a small colony for MALDI-TOF MS analysis, accurate identifications were obtained in 3-6 days and, specifically, before the appearance of their characteristic morphological features. Consequently, identification turnaround time was dramatically reduced as compared to that needed for conventional morphological identification. In conclusion, this standardized MALDI-TOF MS-based identification procedure for filamentous fungi effectively identifies clinical dermatophyte isolates and drastically reduces the response times in the routine clinical laboratory.

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.

The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

Evaluation of reflectance confocal microscopy in dermatophytosis.

Traditional diagnostic testing for dermatophyte infection currently requires skin scraping for light microscopy and/or fungal culture or skin biopsy. Immunofluorescent microscopy can also be used with calcofluor stain. All of these tests can be time-consuming to perform, require a waiting period for results and are invasive. This study aimed to define the in vivo reflectance confocal microscopy (RCM) features of superficial cutaneous fungal infections and to analyse concordance with microscopic examination. Totally, 45 patients, who were diagnosed with superficial cutaneous fungal infections according to the positive result of microscopic examination, were enrolled in this study. We selected three typical lesions examined by RCM, and then recorded the results. In the patients with the tinea manus and pedis, mycelium in stratum corneum was found by the RCM in 14 of 22 patients (14/22; 63.64%). In the patients with the tinea cruris, mycelium in stratum corneum was found by the RCM in 19 of 23 patients (19/23; 82.61%). RCM seems to be useful for microscopic evaluation of mycelium features and may have a scientific value in study of superficial cutaneous fungal infections.

Genome mining reveals the presence of a conserved gene cluster for the biosynthesis of ergot alkaloid precursors in the fungal family Arthrodermataceae.

Genome sequence analysis of different fungi of the family Arthrodermataceae revealed the presence of a gene cluster consisting of five genes with high sequence similarity to those involved in the early common steps of ergot alkaloid biosynthesis in Aspergillus fumigatus and Claviceps purpurea. To provide evidence that this cluster is involved in ergot alkaloid biosynthesis, the gene ARB_04646 of the fungus Arthroderma benhamiae was cloned into pQE60 and expressed in Escherichia coli. Enzyme assays with the soluble tetrameric His(6)-tagged protein proved unequivocally that the deduced gene product, here termed ChaDH, catalysed the oxidation of chanoclavine-I in the presence of NAD(+), resulting in the formation of chanoclavine-I aldehyde. The enzyme product was unequivocally proven by NMR and MS analyses. Therefore, ChaDH functions as a chanoclavine-I dehydrogenase. K(m) values for chanoclavine-I and NAD(+) were 0.09 and 0.36 mM, respectively. Turnover number was 0.76 s(-1).

Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Dermatophytes are keratinolytic fungi responsible for a wide variety of diseases of glabrous skin, nails, and hair. Their identification, currently based on morphological criteria, is hindered by intraspecies morphological variability and the atypical morphology of some clinical isolates. The aim of this study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a routine tool for identifying dermatophyte and Neoscytalidium species, both of which cause dermatomycoses. We first developed a spectral database of 12 different species of common and unusual dermatophytes and two molds responsible for dermatomycoses (Neoscytalidium dimidiatum and N. dimidiatum var. hyalinum). We then prospectively tested the performance of the database on 381 clinical dermatophyte and Neoscytalidium isolates. Correct identification of the species was obtained for 331/360 dermatophytes (91.9%) and 18/21 Neoscytalidium isolates (85.7%). The results of MALDI-TOF MS and standard identification disagreed for only 2 isolates. These results suggest that MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes and Neoscytalidium spp. in clinical mycology laboratories.

Dermatophyte identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The performance of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, ≥1.7; species, ≥2.0) and adjusted cutoff values established by this study (genus, ≥1.5; species, ≥1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P = 0.005)- and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.

Arthroderma benhamiae (the teleomorph of Trichophyton mentagrophytes) mating type-specific genes.

This study first report to identify the mating type (-)-specific gene of alpha-box and the mating type (+)-specific gene of the high-mobility-group (HMG) DNA-binding domain in zoophilic dermatophytes of Arthroderma benhamiae in an effort to understand the epidemiological characteristics of Trichophyton mentagrophytes. The sequence of the alpha-box gene (1,387 bp) was found to contain two exons, from 184 to 475 bp and from 525 to 1,387 bp, coding a protein of 384 amino acids, beginning with a putative initiating methionine (ATG). The sequence of the HMG gene (1,910 bp) contained two exons, from 234 to 415 bp and from 479 to 1,457 bp, coding a protein of 386 amino acids, beginning with a putative initiating methionine (ATG). PCR analysis detected the alpha-box gene in A. benhamiae (-) mating type strains but not in (+) mating type strains. On the other hand, the HMG gene was detected in A. benhamiae (+) mating type strains but not in (-) mating type strains. These findings suggest that the HMG and alpha-box genes could be specific to the (+) and (-) mating types, respectively.

Identification and characterization of parvalbumin-like protein in Trichophyton violaceum.

Parvalbumins play crucial physiological roles in neuromuscular systems of vertebrates, such as cell-cycle, development of neurons, contraction of muscles, and regulation of intracellular calcium. To perform these neuromuscular functions, parvalbumin may be in associated with other proteins including calbindin, carbonic anhydrase, and cytochrome oxidase. Humans may show an IgE-specific hypersensitivity to parvalbumins after consumption of some distinct fish species. While this protein is abundant in fish muscles, literature review of publications related to fish parvalbumins, do not point to the presence of parvalbumins in eukaryotic microbes. In this study, we propose that distantly related parvalbumins may be found in some non-fish species. Bioinformatics studies such as multiple sequence alignment (MSA), phylogenetic analysis as well as molecular-based experiments indicate that, at least two parvalbumins sequences (UniProt IDs: A0A178F775 and A0A178F7E4) with EF-hand domains and Ca2+-binding sites could be identified in Trichophyton violaceum, a pathogenic fungal species. It was determined that both genes consisted of a single exon and encoded for parvalbumin proteins possessing conserved amino acid motifs. Antigenicity prediction revealed antigenic sites located in both sides of the Ca2+-binding site of the first EF-hand domain. Our phylogenetic analysis revealed that one of parvalbumins (UniProt ID: 0A178F775) can be evolved to other parvalbumins in T. violaceum (UniProt ID: A0A178F7E4) and fish species through evolutionary phenomenon. To confirm our in-silico findings, we designed three primer pairs to detect one of the T. violaceum parvalbumins (UniProt ID: A0A178F7E4) by polymerase chain reaction (PCR); one primer pair showed a strong and specific band in agarose gel electrophoresis. To evaluate the specificity of the method, the primers were tested on extracted DNA from Trichophyton rubrum and T. mentagrophytes. The results demonstrated that the evaluated parvalbumin gene (UniProt ID: A0A178F7E4) was T. violaceum-specific and this pathogenic fungus can be differentiated from T. rubrum and T. mentagrophytes through identification of parvalbumin genes. Further studies are necessary to unravel the biochemical and physiological functions of parvalbumins in T. violaceum.

Multilocus phylogeny of the Trichophyton mentagrophytes species complex and the application of matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry for the rapid identification of dermatophytes.

Despite intensive studies of the Trichophyton mentagrophytes species complex, its taxonomy still causes confusion. In this study, more than 70 dermatophytes were analyzed based on nuc rDNA ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (D1D2), and ß-tubulin gene (TUBB) sequences to clarify phylogenetic relationships in the complex. This demonstrated that strains of the complex were divided into three major lineages with high statistical support: (i) T. benhamiae and related species; (ii) T. simii and two related species, T. quinckeanum and T. schoenleinii; and (iii) T. mentagrophytes, T. interdigitale, and related species. The major lineages could be further divided into 18 phylogroups, representing either individual species or phylogenetically distinct groups within species. Among strains of T. benhamiae, African isolates American Type Culture Collection (ATCC) 28064 and 28065 formed a phylogenetically distinct phylogroup from their type strain and were considered a distinct species. Strains of T. mentagrophytes were divided into at least four phylogroups based on combined sequence analysis, but some phylogroups showed closer relationships to T. interdigitale, T. equinum, and T. tonsurans when compared by individual genes. This indicates that identifying those species with one gene could lead to incorrect results. For rapid identification of those dermatophytes, each phylogroup was tested by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry using a database with customized reference spectra of each phylogroup. This system was able to identify all the tested strains to species level with higher than 91% accuracy, except for strains of T. interdigitale. The three phylogroups of T. benhamiae were well distinguished from one another with high identification accuracy, whereas phylogroups of T. mentagrophytes were often cross-identified to one another or to T. interdigitale. Further research should improve identification accuracy for some species, but the results suggested that MALDI-TOF MS could be a rapid and efficient identification tool for closely related dermatophytes in the T. mentagrophytes species complex.