Reliable and rapid identification of terbinafine resistance in dermatophytic nail and skin infections.

BACKGROUND: Fungal infections are the most frequent dermatoses. The gold standard treatment for dermatophytosis is the squalene epoxidase (SQLE) inhibitor terbinafine. Pathogenic dermatophytes resistant to terbinafine are an emerging global threat. Here, we determine the proportion of resistant fungal skin infections, analyse the molecular mechanisms of terbinafine resistance, and validate a method for its reliable rapid identification. METHODS: Between 2013 and 2021, we screened 5634 consecutively isolated Trichophyton for antifungal resistance determined by hyphal growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine. All Trichophyton isolates with preserved growth capacity in the presence of terbinafine underwent SQLE sequencing. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. RESULTS: Over an 8-year period, the proportion of fungal skin infections resistant to terbinafine increased from 0.63% in 2013 to 1.3% in 2021. Our routine phenotypic in vitro screening analysis identified 0.83% (n = 47/5634) of Trichophyton strains with in vitro terbinafine resistance. Molecular screening detected a mutation in the SQLE in all cases. Mutations L393F, L393S, F397L, F397I, F397V, Q408K, F415I, F415S, F415V, H440Y, or A398 A399 G400 deletion were detected in Trichophyton rubrum. Mutations L393F and F397L were the most frequent. In contrast, all mutations detected in T. mentagrophytes/T. interdigitale complex strains were F397L, except for one strain with L393S. All 47 strains featured significantly higher MICs than terbinafine-sensitive controls. The mutation-related range of MICs varied between 0.004 and 16.0 µg/mL, with MIC as low as 0.015 µg/mL conferring clinical resistance to standard terbinafine dosing. CONCLUSIONS: Based on our data, we propose MIC of 0.015 µg/mL as a minimum breakpoint for predicting clinically relevant terbinafine treatment failure to standard oral dosing for dermatophyte infections. We further propose growth on Sabouraud dextrose agar medium containing 0.2 µg/mL terbinafine and SQLE sequencing as fungal sporulation-independent methods for rapid and reliable detection of terbinafine resistance.

Trichophyton rubrum Azole Resistance Mediated by a New ABC Transporter, TruMDR3.

The mechanisms of terbinafine resistance in a set of clinical isolates of Trichophyton rubrum have been studied recently. Of these isolates, TIMM20092 also showed reduced sensitivity to azoles. The azole resistance of TIMM20092 could be inhibited by milbemycin oxime, prompting us to examine the potential of T. rubrum to develop resistance through multidrug efflux transporters. The introduction of a T. rubrum cDNA library into Saccharomyces cerevisiae allowed the isolation of one transporter of the major facilitator superfamily (MFS) conferring resistance to azoles (TruMFS1). To identify more azole efflux pumps among 39 ABC and 170 MFS transporters present within the T. rubrum genome, we performed a BLASTp analysis of Aspergillus fumigatus, Candida albicans, and Candida glabrata on transporters that were previously shown to confer azole resistance. The identified candidates were further tested by heterologous gene expression in S. cerevisiae Four ABC transporters (TruMDR1, TruMDR2, TruMDR3, and TruMDR5) and a second MFS transporter (TruMFS2) proved to be able to operate as azole efflux pumps. Milbemycin oxime inhibited only TruMDR3. Expression analysis showed that both TruMDR3 and TruMDR2 were significantly upregulated in TIMM20092. TruMDR3 transports voriconazole (VRC) and itraconazole (ITC), while TruMDR2 transports only ITC. Disruption of TruMDR3 in TIMM20092 abolished its resistance to VRC and reduced its resistance to ITC. Our study highlights TruMDR3, a newly identified transporter of the ABC family in T. rubrum, which can confer azole resistance if overexpressed. Finally, inhibition of TruMDR3 by milbemycin suggests that milbemycin analogs could be interesting compounds to treat dermatophyte infections in cases of azole resistance.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Epizootic and epidemic dermatophytose outbreaks caused by Trichophyton mentagrophytes from rabbits in Portugal, 2015.

We report an outbreak of dermatophytoses in rabbits, which was the origin of a dermatophytose epidemic in an agricultural school in central Portugal, affecting 15 people. Both the phenotypic characteristics and internal transcribed spacer (ITS) sequence of the dermatophytes isolated from the rabbits and patients were identical, suggesting that a single strain was responsible for both the epizootic and epidemic dermatophytoses and confirming that these two outbreaks were linked. The ITS sequences were also 100% identical to the ITS sequence of five strains isolated from rabbits in Greece and Italy, but different from that of Trichophyton mentagrophytes commonly isolated from dogs and cats. These results suggest that a particular T. mentagrophytes genotype could be prevalent in rabbits in southern Europe.

Which Fungus Originally was Trichophyton mentagrophytes? Historical Review and Illustration by a Clinical Case.

Several dermatophytes producing numerous pyriform or round microconidia were called Trichophyton mentagrophytes. Among these dermatophytes are the teleomorph species Arthroderma benhamiae, Arthroderma vanbreuseghemii and Arthroderma simii, and other species such as Trichophyton interdigitale, Trichophyton erinacei and Trichophyton quinckeanum for which only the anamorph is known. Confusion exists about which fungus should be really called T. mentagrophytes and about the rational use of this name in practice. We report a case of beard ringworm (tinea barbae) with A. vanbreuseghemii. According to both clinical signs and the type of hair parasitism, this case was exactly compatible to the first description of a non-favic dermatophytosis by Gruby under the name of "mentagrophyte" from which was derived the dermatophyte epithet mentagrophytes. In addition, the phenotypic characters of the isolated fungus in cultures perfectly matched with those of the first description of a dermatophyte under T. mentagrophytes by Blanchard (Parasites animaux et parasites végétaux à l'exclusion des Bactéries, Masson, Paris, 1896). In conclusion, T. mentagrophytes corresponds to the fungus later named A. vanbreuseghemii. However, because the neotype of T. mentagrophytes was not adequately designated in regard to the ancient literature, we would privilege the use of A. vanbreuseghemii and abandon the name of T. mentagrophytes.

Survey on Dermatophytes Isolated from Animals in Switzerland in the Context of the Prevention of Zoonotic Dermatophytosis.

Most inflammatory dermatophytoses in humans are caused by zoophilic and geophilic dermatophytes. Knowledge of the epidemiology of these fungi in animals facilitates the prevention of dermatophytosis of animal origin in humans. We studied the prevalence of dermatophyte species in domestic animals in Switzerland and examined the effectiveness of direct mycological examination (DME) for their detection compared to mycological cultures. In total, 3515 hair and skin samples, collected between 2008 and 2022 by practicing veterinarians, were subjected to direct fluorescence microscopy and fungal culture. Overall, 611 dermatophytes were isolated, of which 547 (89.5%) were from DME-positive samples. Cats and dogs were the main reservoirs of Trichophyton mentagrophytes and Microsporum canis, whereas Trichophyton benhamiae was predominantly found in guinea pigs. Cultures with M. canis significantly (p < 0.001) outnumbered those with T. mentagrophytes in DME-negative samples (19.3% versus 6.8%), possibly because M. canis can be asymptomatic in cats and dogs, unlike T. mentagrophytes, which is always infectious. Our data confirm DME as a reliable, quick, and easy method to identify the presence of dermatophytes in animals. A positive DME in an animal hair or skin sample should alert people in contact with the animal to the risk of contracting dermatophytosis.

Identification of infectious agents in onychomycoses by PCR-terminal restriction fragment length polymorphism.

A fast and reliable assay for the identification of dermatophyte fungi and nondermatophyte fungi (NDF) in onychomycosis is essential, since NDF are especially difficult to cure using standard treatment. Diagnosis is usually based on both direct microscopic examination of nail scrapings and macroscopic and microscopic identification of the infectious fungus in culture assays. In the last decade, PCR assays have been developed for the direct detection of fungi in nail samples. In this study, we describe a PCR-terminal restriction fragment length polymorphism (TRFLP) assay to directly and routinely identify the infecting fungi in nails. Fungal DNA was easily extracted using a commercial kit after dissolving nail fragments in an Na(2)S solution. Trichophyton spp., as well as 12 NDF, could be unambiguously identified by the specific restriction fragment size of 5'-end-labeled amplified 28S DNA. This assay enables the distinction of different fungal infectious agents and their identification in mixed infections. Infectious agents could be identified in 74% (162/219) of cases in which the culture results were negative. The PCR-TRFLP assay described here is simple and reliable. Furthermore, it has the possibility to be automated and thus routinely applied to the rapid diagnosis of a large number of clinical specimens in dermatology laboratories.

Identification of Dermatophyte and Non-Dermatophyte Agents in Onychomycosis by PCR and DNA Sequencing-A Retrospective Comparison of Diagnostic Tools.

Rapid and reliable fungal identification is crucial to delineate infectious diseases, and to establish appropriate treatment for onychomycosis. Compared to conventional diagnostic methods, molecular techniques are faster and feature higher accuracy in fungal identification. However, in current clinical practice, molecular mycology is not widely available, and its practical applicability is still under discussion. This study summarizes the results of 16,094 consecutive nail specimens with clinical suspicion of onychomycosis. We performed PCR/sequencing on all primary nail specimens for which conventional mycological diagnostics remained inconclusive. In specimens with a positive direct microscopy but negative or contaminated culture, molecular mycology proved superior and specified a fungal agent in 65% (587/898). In 75% (443/587), the identified pathogen was a dermatophyte. Positive cultures for dermatophytes, yeasts and non-dermatophyte molds (NDMs) were concordant with primary-specimen-DNA PCR/sequencing in 83% (10/12), 34% (22/65) and 45% (76/169), respectively. Among NDMs, agreement was high for Fusarium spp. (32/40; 80%), but low for Penicillium spp. (5/25; 20%) and Alternaria spp. (1/20; 5%). This study underlines the improvement in diagnostic yield by fungal primary-specimen-DNA PCR/sequencing in the event of a negative or contaminated culture, as well as its significance for the diagnosis of dermatophyte and non-dermatophyte onychomycosis. Molecular mycology methods like PCR and DNA sequencing should complement conventional diagnostics in cases of equivocal findings, suspected NDM onychomycosis or treatment-resistant nail pathologies.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex.

Isolates of the Trichophyton mentagrophytes complex vary phenotypically. Whether the closely related zoophilic and anthropophilic anamorphs currently associated with Arthroderma vanbreuseghemii have to be considered as members of the same biological species remains an open question. In order to better delineate species in the T. mentagrophytes complex, we performed a mating analysis of freshly collected isolates from humans and animals with A. benhamiae and A. vanbreuseghemii reference strains, in comparison to internal transcribed spacer (ITS) and 28S rDNA sequencing. Mating experiments as well as ITS and 28S sequencing unambiguously allowed the distinction of A. benhamiae and A. vanbreuseghemii. We have also shown that all the isolates from tinea pedis and tinea unguium identified as T. interdigitale based on ITS sequences mated with A. vanbreuseghemii tester strains, but had lost their ability to give fertile cleistothecia. Therefore, T. interdigitale has to be considered as a humanized species derived from the sexual relative A. vanbreuseghemii.

Efficacious treatment of non-dermatophyte mould onychomycosis with topical amphotericin B.

BACKGROUND: Dermatophytes are the main cause of onychomycosis, but various non-dermatophyte moulds (NDMs) are often the infectious agents in abnormal nails. In particular, Fusarium spp. and other NDMs are mostly insensitive to standard onychomycosis treatment with topical agents as well as with oral terbinafine and itraconazole. OBJECTIVE: The aim of this work is to report the efficacy of a topical amphotericin B solution on NDM onychomycosis in a series of 8 patients resistant to multiple conventional topical and systemic treatments. METHODS: Treatment consisted in the application of an optimized amphotericin B solution once daily to the affected nails and surrounding tissue. No mechanical debridement or medications were allowed except for trimming excessively long nails or in some cases occasionally applying urea-based cream to soften thickened nail plates. RESULTS: Onychomycosis was clinically cured in all patients after a 12-month treatment. Mycological cure was obtained in all but 1 patient. CONCLUSIONS: Topical amphotericin B is an efficacious, safe, cheap and easy-to-apply treatment which should be considered as first-line therapy for NDM onychomycosis.

Epidemiology of Dermatophytoses in Switzerland According to a Survey of Dermatophytes Isolated in Lausanne between 2001 and 2018.

Dermatophytes are the most common pathogenic agents of superficial mycoses in humans and animals. Knowledge of their epidemiology can facilitate the prevention of dermatophytosis and improve prophylactic measures. We sought to determine the incidence of the different dermatophyte species diagnosed in Lausanne (Switzerland) from 2001 to 2018. In total, 10,958 dermatophytes were isolated from patients and 459 from pets. Overall, 99% of tinea unguium and tinea pedis were caused by Trichophyton rubrum and Trichophyton interdigitale with a prevalence ratio of 3:1. Trichophyton violaceum and Trichophyton soudanense were mainly found in tinea capitis in patients of African and Mediterranean origin. Interestingly, while Epidermophyton floccosum and Trichophyton verrucosum were prevalent 50 years ago in an epidemiological analysis carried out in the same laboratory from 1967 to 1970, these two species were rarely detected from 2001 to 2018. Trichophyton mentagrophytes, Trichophyton benhamiae and Microsporum canis were the prevalent zoophilic pathogenic species in children and young adults. Our investigation of animal samples revealed the main reservoirs of these zoophilic species to be cats and dogs for T. mentagrophytes and M. canis, and Guinea pigs for T. benhamiae. This study provides an epidemiological overview of dermatophytoses in Switzerland to improve their surveillance.

Pets as the main source of two zoonotic species of the Trichophyton mentagrophytes complex in Switzerland, Arthroderma vanbreuseghemii and Arthroderma benhamiae.

In cases of highly inflammatory dermatophytosis in humans, it is important to identify the possible source of animal transmission in order to prevent recurrence, family outbreaks or rapidly progressing epidemics. A survey of dermatophytes in pets during a 14-month period in Switzerland revealed, in addition to Microsporum canis, two different species of the Trichophyton mentagrophytes complex, Arthroderma benhamiae and Arthroderma vanbreuseghemii, all causing inflammatory dermatophytoses. Arthroderma benhamiae was only and frequently isolated from guinea pigs. Arthroderma vanbreuseghemii was isolated mainly from European short hair cats, but also from dogs and in one case from a pure-bred cat. Ninety-three percent of the cats carrying A. vanbreuseghemii were hunters and all had skin lesions. In contrast, cats with skin lesions that were strictly indoors were found to be almost exclusively infected by M. canis. Therefore, it can be suspected that infection with A. vanbreuseghemii occurred during hunting and that the natural source of this dermatophyte is either soil or an animal other than the cat, most probably a rodent.

Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses.

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10% of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1-3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

The dermatophyte species Arthroderma benhamiae: intraspecies variability and mating behaviour.

Arthroderma benhamiae is a zoophilic dermatophyte belonging to the Trichophyton mentagrophytes species complex. Here, a population of A. benhamiae wild strains from the same geographical area (Switzerland) was studied by comparing their morphology, assessing their molecular variability using internal transcribed spacer (ITS) and 28S rRNA gene sequencing, and evaluating their interfertility. Sequencing of the ITS region and of part of the 28S rRNA gene revealed the existence of two infraspecific groups with markedly different colony phenotypes: white (group I) and yellow (group II), respectively. For all strains, the results of mating type identification by PCR, using HMG (high-mobility group) and α-box genes in the mating type locus as targets, were in total accordance with the results of mating type identification by strain confrontation experiments. White-phenotype strains were of mating type + (mt+) or mating type - (mt-), whilst yellow-phenotype strains were all mt-. White and yellow strains were found to produce fertile cleistothecia after mating with A. benhamiae reference tester strains, which belonged to a third group intermediate between groups I and II. However, no interfertility was observed between yellow strains and white strains of mt+. A significant result was that white strains of mt- were able to mate and produce fertile cleistothecia with the white A. benhamiae strain CBS 112371 (mt+), the genome of which has recently been sequenced and annotated. This finding should offer new tools for investigating the biology and genetics of dermatophytes using wild-type strains.

Sulphite efflux pumps in Aspergillus fumigatus and dermatophytes.

Dermatophytes and other filamentous fungi excrete sulphite as a reducing agent during keratin degradation. In the presence of sulphite, cystine in keratin is directly cleaved to cysteine and S-sulphocysteine, and thereby, reduced proteins become accessible to hydrolysis by a variety of secreted endo- and exoproteases. A gene encoding a sulphite transporter in Aspergillus fumigatus (AfuSSU1), and orthologues in the dermatophytes Trichophyton rubrum and Arthroderma benhamiae (TruSSU1 and AbeSSU1, respectively), were identified by functional expression in Saccharomyces cerevisiae. Like the S. cerevisiae sulphite efflux pump Ssu1p, AfuSsu1p, TruSsu1p and AbeSsu1p belong to the tellurite-resistance/dicarboxylate transporter (TDT) family which includes the Escherichia coli tellurite transporter TehAp and the Schizosaccharomyces pombe malate transporter Mae1p. Seven genes in the A. fumigatus genome encode transporters of the TDT family. However, gene disruption of AfuSSU1 and of the two more closely related paralogues revealed that only AfuSSU1 encodes a sulphite efflux pump. TruSsulp and AbeSsulp are believed to be the first members of the TDT family identified in dermatophytes. The relatively high expression of TruSSU1 and AbeSSU1 in dermatophytes compared to that of AfuSSU1 in A. fumigatus likely reflects a property of dermatophytes which renders these fungi pathogenic. Sulphite transporters could be a new target for antifungal drugs in dermatology, since proteolytic digestion of hard keratin would not be possible without prior reduction of disulphide bridges.