Diagnostic ability of Peptidase S8 gene in the Arthrodermataceae causing dermatophytoses: A metadata analysis.

An unambiguous identification of dermatophytes causing dermatophytoses is necessary for accurate clinical diagnosis and epidemiological implications. In the current taxonomy of the Arthrodermataceae, the etiological agents of dermatophytoses consist of seven genera and members of the genera Trichophyton are the most prevalent etiological agents at present. The genera Trichophyton consists of 16 species that are grouped as clades, but the species borderlines are not clearly delimited. The aim of the present study was to determine the discriminative power of subtilisin gene variants (SUB1-SUB12) in family Arthrodermataceae, particularly in Trichophyton. Partial and complete reads from 288 subtilisin gene sequences of 12 species were retrieved and a stringent filtering following two different approaches for analysis (probability of correct identification (PCI) and gene gap analysis) conducted to determine the uniqueness of the subtilisin gene subtypes. SUB1 with mean PCI value of 60% was the most suitable subtilisin subtype for specific detection of T.rubrum complex, however this subtype is not reported in members of T. mentagrophytes complex which is one of the most prevalent etiological agent at present. Hence, SUB7 with 40% PCI value was selected for testing its discriminative power in Trichophyton species. SUB7 specific PCR based detection of dermatophytes was tested for sensitivity and specificity. Sequences of SUB7 from 42 isolates and comparison with the ITS region showed that differences within the subtilisin gene can further be used to differentiate members of the T. mentagrophytes complex. Further, subtilisin cannot be used for the differentiation of T. benhamiae complex since all SUB subtypes show low PCI scores. Studies on the efficiency and limitations of the subtilisin gene as a diagnostic tool are currently limited. Our study provides information that will guide researchers in considering this gene for identifying dermatophytes causing dermatophytoses in human and animals.

Potential emergence of terbinafine resistance by squalene epoxidase gene mutations: An 18-month cohort study of onychomycosis patients in the United States.

BACKGROUND: There is a concerning rise in antifungal-resistant dermatophytosis globally, with resistance to terbinafine conferred by point mutations in the squalene epoxidase (SQLE) gene. OBJECTIVES: Report changes in the prevalence and profile of SQLE mutations in onychomycosis patients in the United States. METHODS: A longitudinal cohort study of toenail samples was collected from suspected onychomycosis patients over an 18-month period from 2022 to 2023. Samples were submitted from across the United States and subjected to multiplex real-time polymerase chain reactions for dermatophyte detection, with further screening of SQLE mutations at four known hotspots (393Leu, 397Phe, 415Phe and 440His). RESULTS: A total of 62,056 samples were submitted (mean age: 57.5 years; female: 60.4%). Dermatophytes were detected in 38.5% of samples, primarily Trichophyton rubrum complex (83.6%) and T. mentagrophytes complex (10.7%). A survey of SQLE mutations was carried out in 22,610 dermatophyte samples; there was a significant increase in the prevalence of SQLE mutations between the first quarter of 2022 and the second quarter of 2023 (29.0 to 61.9 per 1000 persons). The Phe397Leu substitution was the predominant mutation; Phe415Ser and His440Tyr have also emerged which were previously reported as minor mutations in skin samples. The temporal change in mutation rates can be primarily attributed to the Phe415Ser substitution. Samples from elderly patients (>70 years) are more likely to be infected with the T. mentagrophytes complex including strains harbouring the Phe415Ser substitution. CONCLUSION: The prevalence of SQLE mutations among onychomycosis patients with Trichophyton infections may be underestimated. Older individuals may have a higher risk.

Molecular Detection of Dermatophytes and Nondermatophytes in Onychomycosis in Antalya, Turkey.

BACKGROUND: Onychomycosis is a chronic nail infection, and dermatophytes, yeasts, and nondermatophytic molds may be the causative agents. This study aimed to determine the etiological agents of onychomycosis by using conventional and molecular methods. METHODS: Between June 2020 and July 2021, 37 patients with a presumptive diagnosis of onychomycosis and mycological evidence (culture and/or EUROArray Dermatomycosis assay) were included in the study. Organisms detected in cultured nail specimens were identified by combined phenotypic characteristics and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). An EUROarray Dermatomycosis assay was used for molecular detection of fungal pathogens. RESULTS: The EUROArray Dermatomycosis assay was positive for a single fungal target in 23 samples, and 14 samples were positive by culture. The most common pathogen was Trichophyton rubrum in both methods. Coinfection was detected in 14 samples by using molecular methods, and Trichophyton rubrum and Fusarium solani (9 samples) were the most common pathogens detected together. Trichophyton spp., nondermatophyte molds, and Candida spp. were detected in 33 (89.2%), 16 (43.2%), and 6 (16.2%) samples, respectively, when the two methods were evaluated together. CONCLUSIONS: Our results revealed that fungal culture allows the diagnosis of onychomycosis, but it is not as sensitive as the EUROArray Dermatomycosis test, especially in patients receiving antifungal therapy.

Epidemiological trends, antifungal drug susceptibility and SQLE point mutations in etiologic species of human dermatophytosis in Al-Diwaneyah, Iraq.

Dermatophytes show a wide geographic distribution and are the main causative agents of skin fungal infections in many regions of the world. Recently, their resistance to antifungal drugs has led to an obstacle to effective treatment. To address the lack of dermatophytosis data in Iraq, this study was designed to investigate the distribution and prevalence of dermatophytes in the human population and single point mutations in squalene epoxidase gene (SQLE) of terbinafine resistant isolates. The identification of 102 dermatophytes isolated from clinical human dermatophytosis was performed through morphological and microscopic characteristics followed by molecular analysis based on ITS and TEF-1α sequencing. Phylogeny was achieved through RAxML analysis. CLSI M38-A2 protocol was used to assess antifungal susceptibility of the isolates to four major antifungal drugs. Additionally, the presence of point mutations in SQLE gene, which are responsible for terbinafine resistance was investigated. Tinea corporis was the most prevalent clinical manifestation accounting for 37.24% of examined cases of dermatophytosis. Based on ITS, T. indotineae (50.98%), T. mentagrophytes (19.61%), and M. canis (29.41%) was identified as an etiologic species. T. indotineae and T. mentagrophytes strains were identified as T. interdigitale based on TEF-1α. Terbinafine showed the highest efficacy among the tested antifungal drugs. T. indotineae and T. mentagrophytes showed the highest resistance to antifungal drugs with MICs of 2-4 and 4 µg/mL, while M. canis was the most susceptible species. Three of T. indotineae isolates showed mutations in SQLE gene Phe397Leu substitution. A non-previously described point mutation, Phe311Leu was identified in T. indotineae and mutations Lys276Asn, Phe397Leu and Leu419Phe were diagnosed in T. mentagrophytes XVII. The results of mutation analysis showed that Phe397Leu was a destabilizing mutation; protein stability has decreased with variations in pH, and point mutations affected the interatomic interaction, resulting in bond disruption. These results could help to control the progression of disease effectively and make decisions regarding the selection of appropriate drugs for dermatophyte infections.

Comparative Transcriptome Analysis of T. rubrum, T. mentagrophytes, and M. gypseum Dermatophyte Biofilms in Response to Photodynamic Therapy.

Dermatophyte biofilms frequently count for inadequate responses and resistance to standard antifungal treatments, resulting in refractory chronic onychomycosis infection. Although antimicrobial photodynamic therapy (aPDT) has clinically proven to exert significant antifungal effects or even capable of eradicating dermatophyte biofilms, considerably less is known about the molecular mechanisms underlying aPDT and the potential dysregulation of signaling networks that could antagonize its action. The aim of this study is to elucidate the molecular mechanisms underlining aPDT combat against dermatophyte biofilm in recalcitrant onychomycosis and to decipher the potential detoxification processes elicited by aPDT, facilitating the development of more effective photodynamic interventions. We applied genome-wide comparative transcriptome analysis to investigate how aPDT disrupting onychomycosis biofilm formed by three distinct dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, and Microsporum gypseum, the most frequently occurring pathogenic species. In total, 352.13 Gb of clean data were obtained for the transcriptomes of dermatophyte biofilms with or without aPDT treatment, resulting in 2,422.42 million reads with GC content of 51.84%, covering 99.9%, 98.5% and 99.4% of annotated genes of T. rubrum, T. mentagrophytes, and M. gypseum, respectively. The genome-wide orthologous analysis identified 6624 transcribed single-copy orthologous genes in all three species, and 36.5%, 6.8% and 17.9% of which were differentially expressed following aPDT treatment. Integrative orthology analysis demonstrated the upregulation of oxidoreductase activities is a highly conserved detoxification signaling alteration in response to aPDT across all investigated dermatophyte biofilms. This study provided new insights into the molecular mechanisms underneath anti-dermatophyte biofilm effects of aPDT and successfully identified a conserved detoxification regulation upon the aPDT application.

High Prevalence of Terbinafine Resistance Among Trichophyton mentagrophytes/T. interdigitale Species Complex, a Cross-Sectional Study from 2021 to 2022 in Northern Parts of Iran.

Treatment-resistant dermatophytosis caused by the members of the Trichophyton mentagrophytes/Trichophyton interdigitale species group (TMTISG) is increasing worldwide. We aimed to determine the prevalence of TMTISG in patients with dermatophytosis in two centers from north of Iran and detect the possible mutations in the squalene epoxidase (SQLE) gene in relevant terbinafine (TRB) resistant pathogenic isolates. From November 2021 to December 2022, 1960 patients suspected to dermatophytosis and referred to two mycology referral laboratories in the north of Iran were included in the study. Identification of all dermatophyte isolates was confirmed by RFLP of rDNA internal transcribed spacer (ITS) regions. Antifungal susceptibility testing against five common antifungals using the CLSI-M38-A3 protocol was performed. The TMTISG isolates resistant to TRB, were further analyzed to determine the possible mutations in the SQLE gene. Totally, 647 cases (33%) were positive for dermatophytosis of which 280 cases (43.3%) were identified as members of TMTISG. These were more frequently isolated from tinea corporis 131 (44.56%) and tinea cruris 116 (39.46%). Of 280 TMTISG isolates, 40 (14.3%) were resistant to TRB (MIC ≥ 4 µg/mL), all found to be T. indotineae in ITS sequencing. In SQLE sequencing 34 (85%) of TRB-resistant isolates had coincident mutations of Phe397Leu and Ala448Thr whereas four and two isolates had single mutations of Phe397Leu and Leu393Ser, respectively. Overall, the resistance of Iranian TMTISG isolates to TRB greatly occurred by a mutation of Phe397Leu in the SQLE gene as alone or in combination with Ala448Thr. Nevertheless, for the occurrence of in vitro resistance, only the presence of Phe397Leu mutation seems to be decisive.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Dermatophytosis in domestic cats: Identification, and treatment in an Indian context.

The surge in domestic cat adoption across India, particularly the rising preference for high-pedigree cats, coupled with environmental factors, has resulted in increased incidence of dermatophytosis among feline companions. Despite this growing concern, there is a noticeable scarcity of studies in India delving into the etiological factors contributing to dermatophytosis in cats. This disease is a threat to animal health and carries public health significance, given that cats are recognized reservoir hosts for Microsporum canis, a common dermatophyte affecting humans and animals. This study endeavours to identify the dermatophytes affecting cats and establish a standardized therapeutic regimen while accounting for the local stigma surrounding the regular bathing of cats. The study involved the examination of 82 cats presenting dermatological lesions, when subjected to cultural examination in dermatophyte test medium revealed 36 afflicted with dermatophytes. Isolates were presumptively identified by staining using lactophenol cotton blue, Chicago sky blue 6B, and Calcofluor white stains. Molecular-level identification of the isolates was confirmed through PCR-RFLP, amplifying the Internal Transcribed Spacer Sequence of 16 s rDNA, followed by restriction digestion using the Mva1 enzyme. Among the thirty-six isolates, 29 were identified as M. canis, while the remaining 7 were M. gypseum. The cases were categorized into five groups and treated with Lime Sulphur dip, 4 % chlorhexidine shampoo, a shampoo containing 2 % miconazole and 4 % chlorhexidine, oral itraconazole alone, and a combination of oral itraconazole with lime-Sulphur dip. Statistical analysis revealed that the response was notably swifter with lime Sulphur dip when considering only topical therapy. Moreover, the mycological cure was most expeditious when combining Lime Sulphur dip with oral itraconazole. These findings underscore the pivotal role of topical biocides in feline dermatophytosis treatment, potentially reducing the reliance on specific antifungals and thereby contributing to the mitigation of antimicrobial resistance emergence.

An update on antifungal resistance in dermatophytosis.

INTRODUCTION: The reports of resistance to antifungal agents used for treating onychomycosis and other superficial fungal infections are increasing. This rise in antifungal resistance poses a public health challenge that requires attention. AREAS COVERED: This review explores the prevalence of dermatophytes and the current relationship between dermatophyte species, their minimum inhibitory concentrations (MICs) for terbinafine (an allylamine) and itraconazole (an azole), and various mutations prevalent in these species. The most frequently isolated dermatophyte associated with resistance in patients with onychomycosis and dermatophytosis was T. mentagrophytes. However, T. indotineae emerged as the most prevalent isolate with mutations in the SQLE gene, exhibiting the highest MIC of 8 µg/ml for terbinafine and MICs of 8 µg/ml and ≥ 32 µg/ml for itraconazole.Overall, the most prevalent SQLE mutations were Phe397Leu, Leu393Phe, Ala448Thr, Phe397Leu/Ala448Thr, and Lys276Asn/Leu415Phe (relatively recent). EXPERT OPINION: Managing dermatophyte infections requires a personalized approach. A detailed history should be obtained including details of travel, home and occupational exposure, and clinical examination of the skin, nails and other body systems. Relevant testing includes mycological examination (traditional and molecular). Additional testing, where available, includes MIC evaluation and detection of SQLE mutations. In case of suspected terbinafine resistance, itraconazole or voriconazole (less commonly) should be considered.

The Ptk2-Pma1 pathway enhances tolerance to terbinafine in Trichophyton rubrum.

The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum, we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2), and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae, Ptk2 knockout strains were more sensitive to terbinafine compared with the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. Pma1 is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in T. rubrum Ptk2 knockout strain (ΔTrPtk2) suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole, an inhibitor of plasma membrane proton pump Pma1, increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes.

New thymol-derived triazole exhibits promising activity against Trichophyton rubrum.

Fungal infections have emerged worldwide, and azole antifungals are widely used to control these infections. However, the emergence of antifungal resistance has been compromising the effectiveness of these drugs. Therefore, the objective of this study was to evaluate the antifungal and cytotoxic activities of the nine new 1,2,3 triazole compounds derived from thymol that were synthesized through Click chemistry. The binding mode prediction was carried out by docking studies using the crystallographic structure of Lanosterol 14α-demethylase G73E mutant from Saccharomyces cerevisiae. The new compounds showed potent antifungal activity against Trichophyton rubrum but did not show relevant action against Aspergillus fumigatus and Candida albicans. For T. rubrum, molecules nº 5 and 8 showed promising results, emphasizing nº 8, whose fungicidal and fungistatic effects were similar to fluconazole. In addition, molecule nº 8 showed low toxicity for keratinocytes and fibroblasts, concluding that this compound demonstrates promising characteristics for developing a new drug for dermatophytosis caused by T. rubrum, or serves as a structural basis for further research.

Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae.

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Terbinafine resistance in Trichophyton mentagrophytes and Trichophyton rubrum in the Czech Republic: A prospective multicentric study.

BACKGROUND: Terbinafine, an allylamine antifungal, is crucial for treating dermatophytosis by inhibiting squalene epoxidase (SQLE) in the ergosterol biosynthetic pathway. However, resistance is emerging, particularly in India and Southeast Asia, but reports of resistance spread worldwide. Despite this, comprehensive studies on terbinafine resistance in Trichophyton are still limited. OBJECTIVES: This research aimed to determine the prevalence of terbinafine resistance in the Czech Republic, with a focus on Trichophyton rubrum and Trichophyton mentagrophytes, and investigate the underlying molecular mechanisms. PATIENTS/METHODS: A total of 514 clinical strains of T. rubrum and 240 T. mentagrophytes collected from four Czech clinical institutions were screened for terbinafine resistance. Molecular investigations included DNA sequencing, specifically the ITS rDNA region and SQLE gene, as well as antifungal susceptibility testing following EUCAST guidelines. RESULTS: While no resistance was observed in T. rubrum, 2.5% of T. mentagrophytes strains exhibited resistance, marked by the F397L mutation in SQLE. Notably, resistance surged from 1.2% in 2019 to 9.3% in 2020 but reverted to 0% in 2021. All resistant strains were identified as T. mentagrophytes var. indotineae. Resistant strains exhibited high MICs for terbinafine (≥4 mg L-1 ) but low MICs to the other seven antifungals tested except for fluconazole. CONCLUSIONS: This study highlights the emergence of terbinafine-resistant T. mentagrophytes strains in the Czech Republic, with the F397L mutation being pivotal. Due to the relatively low resistance level, the current guidelines for dermatomycosis treatment in the Czech Republic remain effective, but ongoing surveillance is essential for timely adaptations if resistance patterns change.

Molecular Verification of Trichophyton in the Brazilian URM Culture Collection.

Trichophyton species cause dermatophytosis in humans, with a high, worldwide frequency of reports and important public health relevance. We evaluated 61 Trichophyton strains from different sources deposited in the University Recife Mycology (URM) culture collection of the Universidade Federal de Pernambuco, Brazil. Strains were phenotypically identified and confirmed by sequencing Internal Transcribed Spacers rDNA and partial beta-tubulin 2-exon. Additionally, we evaluated their susceptibility to terbinafine and itraconazole. Physiological analyses included urease activity and growth in casein medium. Phenotypic methods allowed the reliable identification of T. rubrum only, whereas, for other species, molecular methods were mandatory. All Trichophyton species exhibited susceptibility profiles to itraconazole (0.04-5.33 µg/mL) and terbinafine (0.17-3.33 µg/mL). Our analyses revealed a heterogeneous distribution of T. mentagrophytes, which does not support the current distribution within the species complex of T. mentagrophytes and its genotypes.

[Conventional and molecular diagnostics in onychomycosis-part 1 : Conventional differentiation of dermatophytes-Trichophyton rubrum, Trichophyton interdigitale]. / Konventionelle und molekulare Diagnostik bei Onychomykose ­ Teil 1 : Konventionelle Differenzierung von Dermatophyten ­ Trichophyton rubrum, Trichophyton interdigitale.

Onychomycosis is a common infectious nail disease occurring worldwide. The mycological diagnosis of onychomycosis is primarily used for differential diagnostic differentiation from other, mostly inflammatory nail diseases, such as nail psoriasis or onychodystrophies of other causes. Conventional laboratory diagnostics when onychomycosis is suspected is based on microscopic detection of fungi in the nail material using fluorescence-optical potassium hydroxide preparations and culture of the pathogen. Molecular amplification methods allow a more sensitive and specific identification of the causative dermatophyte. Here, in 108 patients with onychomycosis, the dermatophytes were identified by culture and/or molecular biology using polymerase chain reaction (PCR) and the species identification was confirmed with subsequent sequencing. The dermatophytes were analyzed based on macromorphological and microscopic features. A dermatophyte was cultured in 56 of the 108 patients. Among them were 31 isolates of Trichophyton (T.) rubrum and 25 of T. interdigitale. All species identifications were subsequently confirmed by rDNA sequencing with concordant results in 54 of 56 patients. Two primarily as T. interdigitale identified specimens were revealed to be T. quinckeanum and T. tonsurans by molecular methods. T. quinckeanum, which is a zoophilic dermatophyte and a so-called emerging pathogen in dermatomycology, was isolated here for the first time as the causative agent of onychomycosis. The other dermatophyte, initially thought to be T. interdigitale, turned out to be T. tonsurans on molecular biology. This anthropophilic dermatophyte is also a rarity in onychomycosis. In addition, T. rubrum was identified by PCR in 34 of the 52 nail specimens that did not grow culture, and T. interdigitale in 18 nail specimens. However, the morphological identification of the four different dermatophytes species proved problematic. Neither the colony morphology nor the microscopic features of the dermatophytes allow clear differentiation of the pathogens. Microconidia, macroconidia, chlamydospores, and arthrospores are inconsistent in occurrence, number, microscopic distribution, and shape. The urease activity also did not allow an assignment of the dermatophyte species. These results indicate that the most sensitive detection and reliable identification of causative dermatophytes in onychomycosis is only possible by molecular methods.

[Conventional and molecular diagnostics in onychomycosis-part 2 : Molecular identification of causative dermatophytes by polymerase chain reaction and sequence analysis of the internal transcribed spacer region of ribosomal DNA]. / Konventionelle und molekulare Diagnostik bei Onychomykose ­ Teil 2 : Molekulare Identifizierung der ursächlichen Dermatophyten mit Polymerasekettenreaktion und Sequenzanalyse der ITS("internal transcribed spacer")-Region der ribosomalen Desoxyribonukleinsäure.

Dermatophyte identification using traditional methods such as optics-based direct fluorescence microscopy and culture is nowadays supplemented by molecular biological methods. The validity of dermatophyte DNA detection with direct uniplex-polymerase chain reaction-enzyme immunoassay (PCR-EIA) in nail samples was proven by sequence analysis of the ribosomal internal transcribed spacer (ITS) region. A total of 108 dermatophytes, isolated from patients with onychomycosis, were positive for Trichophyton rubrum (TR) and Trichophyton interdigitale (TI) in culture and/or uniplex-PCR-EIA. Conventional methods for dermatophyte identification were complemented by direct uniplex-PCR-EIA and sequence analysis of the ribosomal ITS region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA). Of 108 patients (average age 62, median age 73), 56 showed cultural growth with 31 of them being identified as TR and 23 as TI. There was high agreement with the sequence analysis. Surprisingly, the pathogen of a single nail sample was identified as T. quinckeanum (formerly T. mentagrophytes sensu stricto), a rare zoophilic dermatophyte in Germany. A single TI strain turned out to be a misidentified T. tonsurans based on the sequence analysis. In all, 34 of the 52 specimens lacking cultural growth were detected by PCR as TR, and 18 specimens could be identified as TI. The results of dermatophyte identification of culture-negative nail samples were also in agreement with the results of sequence analysis. Molecular biological methods are well applicable, and they show high reliability for direct dermatophyte identification in nail samples without prior cultivation. Especially for nail samples without cultural growth, PCR-based dermatophyte identification was highly specific and sensitive.

Molecular typing of a collection of Iranian clinical Trichophyton tonsurans isolates based on the non-transcribed spacer region of rDNA and antifungal susceptibility testing of the species.

INTRODUCTION: Wrestling, considered the national sport of Iran, has gained immense popularity among Iranians. Wrestlers frequently encounter skin conditions, with dermatophyte fungal infections, particularly tinea gladiatorum (TG), being a common issue. TG, caused by the Trichophyton genus, has emerged as a major health concern for wrestlers and other contact sport athletes worldwide. This study aimed to assess the genotypic diversity and antifungal susceptibility of Trichophyton tonsurans isolates responsible for TG in Iranian wrestlers from Mazandaran province, northern Iran. MATERIALS AND METHODS: A total of 60 clinical T. tonsurans isolates collected from various cities in Mazandaran, were included in the study. The isolates were identified through PCR-restriction fragment length polymorphism and sequencing methods. Genomic DNA was extracted from these isolates, and the non-transcribed spacer (NTS) region of ribosomal RNA (rRNA) was targeted for genotyping using newly designed primers. Haplotype analysis was performed to explore genetic diversity, and antifungal susceptibility to terbinafine (TRB) and itraconazole (ITC) was assessed. RESULTS: The results revealed five distinct NTS types: NTS-I, NTS-II, NTS-III, NTS-IV and NTS-V, with NTS-IV being the most prevalent. The distribution of NTS types varied across different cities, suggesting potential transmission patterns among wrestlers. Antifungal susceptibility testing showed that all isolates were susceptible to TRB, while one isolate demonstrated resistance to ITC. Genotypic diversity was not correlated with antifungal susceptibility, emphasising the importance of monitoring susceptibility to ensure effective treatment. Haplotype analysis highlighted significant genetic diversity among the T. tonsurans isolates. This diversity may be attributed to factors such as human-to-human transmission, geographic location and lifestyle changes. The study's findings underscore the need for comprehensive genotypic analysis to understand the epidemiology and evolution of T. tonsurans infections in athletes. CONCLUSION: In conclusion, this study provides valuable insights into the genotypic diversity and antifungal susceptibility of T. tonsurans isolates causing TG in Iranian wrestlers. The presence of multiple NTS types and varying susceptibility patterns highlights the complexity of T. tonsurans infections in this population. Further research is warranted to track the transmission routes and genetic evolution of T. tonsurans strains among wrestlers and develop effective control measures.

Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum, 10 T. mentagrophytes, 5 T. interdigitale) and 12 TRB non-WT (7 T. rubrum, 5 T. indotineae) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum, 7 T. interdigitale, 6 T. tonsurans, 5 T. mentagrophytes) and 4 non-WT T. indotineae. Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories.

PCR-based methods in detection and identification of dermatophytes in dogs and cats with suspected dermatophytosis in 2021 in Poland.

Dermatophytes from Microsporum, Trichophyton and Epidermophyton genera are divided into geophilic, zoophilic and anthropophilic species which cause skin infection in humans and wide group of animals, mainly mammals. Main species causing dermatophytosis in dogs and cats are Microsporum and Trichophyton. Conventional mycological diagnostic technique includes Saburaud Dextrose Agar (SAD) and others medium cultures, 10% KOH mount and direct microscopy of hairs and scraping. Molecular diagnostic become more frequent in veterinary practice due to shortening of waiting time. In this study we based on two PCR methods. The nested PCR amplified CHS1 gene for dermatophytes detection, and multiplex PCR coding ITS1 and ITS2 fragments for species identification of detected derpatophytes. Most frequently detected species was Microsporum canis, mainly in young cats. Geophilic Microsporum gypseum and anthropophilic Trichophyton rubrum was found primarily in dogs. Molecular methods in dermatophytosis identification are rapid in contrast to routinely, long lasting culture.