An update on the myriad antifungal resistance mechanisms in dermatophytes and the place of experimental and existential therapeutic agents for Trichophyton complex implicated in tinea corporis and cruris.

INTRODUCTION: There is an epidemic emergence of increased resistance in dermatophytes with to antifungal drugs with ergosterol1 (Erg1) and Erg11 mutations to terbinafine and azoles. Apart from mutations, mechanisms that predict clinical failure include efflux pumps, cellular kinases, heat shock proteins (Hsp), and biofilms. Apart from itraconazole and SUBATM (Super-Bioavailable) itraconazole, measures that can be used in terbinafine failure include efflux-pump inhibitors, Hsp inhibitors and judicious use of antifungal drugs (topical + systemic) combinations. AREAS COVERED: A PubMed search was done for the relevant studies and reviews published in the last 22 years using keywords dermatophytes OR Trichophyton, anti-fungal, resistance, mechanism and fungal AND resistance mechanisms. Our aim was to look for literature on prevalent species and we specifically researched studies on Trichophyton genus. We have analyzed varied antifungal drug mechanisms and detailed varied experimental and approved drugs to treat recalcitrant dermatophytosis. EXPERT OPINION: Apart from administering drugs with low minimum inhibitory concentration, combinations of oral and topical antifungals (based on synergy data) and new formulations of existing drugs are useful in recalcitrant cases. There is a need for research into resistance mechanism of the existent Trichophyton strains in therapeutic failures in tinea corporis & cruris instead of data derived from laboratory strains which may not mirror clinical failures.

Treatment recalcitrant cases of tinea corporis/cruris caused by T. mentagrophytes - interdigitale complex with mutations in ERG11 ERG 3, ERG4, MDR1 MFS genes & SQLE and their potential implications.

BACKGROUND: Recalcitrant dermatophyte infections are being reported from various parts of the world due to varied causes including strain variation, steroid misuse, SQLE mutations, and variable quality of itraconazole pellet formulations. The oral drug preferred in endemic areas is itraconazole, to which MIC levels remain low, and clinical failures to itraconazole reported defy a sound scientific explanation. OBJECTIVES: The objective of the study was to conduct a proteomic and genomic analysis on isolates from therapeutically recalcitrant case with isolation of gene mutations and enzymatic abnormalities to explain azole failures. METHODS: Trichophyton mentagrophyte interdigitale complex strains were isolated from seven clinically non-responding tinea corporis/cruris patients, who had failed a sequential course of 6 weeks of terbinafine 250 mg QD and itraconazole 100 mg BID. After AFST 1 strain, KA01 with high MIC to most drugs was characterized using whole genome sequencing, comparative proteomic profiling, and total sterol quantification. RESULTS: Sterol quantification showed that the standard strain of Trichophyton mentagrophytes (MTCC-7687) had half the ergosterol content than the resistant KA01 strain. Genomic analysis revealed mutations in SQLE, ERG4, ERG11, MDR1, MFS genes, and a novel ERG3 mutation. Proteomic analysis established the aberrant expression of acetyl Co-A transferase in the resistant strain and upregulation of thioredoxin reductase and peroxiredoxin. CONCLUSION: Our findings demonstrate possible reasons for multidrug resistance in the prevalent strain with mutations in genes that predict terbinafine (SQLE) and azole actions (ERG4, ERG11, ERG3) apart from efflux pumps (MDR1, MFS) that can explain multidrug clinical failures.

Metabolic phenotype analysis of Trichophyton rubrum after laser irradiation.

BACKGROUND: Biological phenotypes are important characteristics of microorganisms, and often reflect their genotype and genotype changes. Traditionally, Trichophyton rubrum (T. rubrum) phenotypes were detected using carbon source assimilation tests, during which the types of tested substances are limited. In addition, the operation is complicated, and only one substance can be tested at once. To observe the changes of the metabolic phenotype of T. rubrum after laser irradiation, a high-throughput phenotype microarray system was used to analyze the metabolism of different carbon, nitrogen, phosphorus and sulfur source substrates in a Biolog metabolic phenotyping system. RESULTS: The strain of T. rubrum used in this study can effectively utilize 33 carbon, 20 nitrogen, 16 phosphorus, and 13 sulfur source substrates prior to laser irradiation. After laser irradiation, the strain was able to utilize 10 carbon, 12 nitrogen, 12 phosphorus, and 8 sulfur source substrates. The degree of utilization was significantly decreased compared with the control. Both groups efficiently utilized saccharides and organic acids as carbon sources as well as some amino acids as nitrogen sources for growth. The number of substrates utilized by T. rubrum after laser irradiation were significantly reduced, especially carbon substrates. Some substrates utilization degree in the laser treated group was higher than control, such as D-glucosamine, L-glutamine, D-2-Phospho-Glyceric Acid, D-glucosamine-6-phosphate, and D-methionine. CONCLUSION: Laser irradiation of T. rubrum may lead to changes in the metabolic substrate and metabolic pathway, thus weakening the activity of the strain.

A chronic disseminated dermatophytosis due to Trichophyton violaceum.

A 48-year-old female had presented dandruff and breakable hair for more than 40 years, dry scaly erythema on bilateral palms and feet accompanying with nail destruction for 20 years, and scaling papules on the buttock for 5 years. Direct microscopic examination showed endothrix anthroconidia within broken hair and septate and branched hyphae within skin and nail lesion. Fungal cultures from all infected sites were examined by morphology, ITS sequencing, and random amplified polymorphic DNA fingerprinting, and were identified as Trichophyton violaceum from the same source. The patient was treated with oral terbinafine 0.25 g/day as well as with 1% terbinafine gel for external use and with 2% ketoconazole lotion for shampoo and bath. A follow-up after 4 weeks showed that the lesions decreased significantly.

[Action of Euphorbia humifusa effective fraction on membrane biosynthesis and the gene expression of proteases MEP and SUB of Trichophyton rubrum].

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.

Is Trichophyton simii endemic to the Indian subcontinent?

Trichophyton simii is considered to be prevalent only in the Indian subcontinent where it was isolated from soil, as well as from infections of humans and animals. We have investigated a case of onychomycosis caused by this exotic dermatophyte, not traceable to endemic areas. This case, as in others due to this fungus in man or animals, that have been previously and sporadically reported worldwide, suggests infections caused by T. simii might be underestimated, especially outside its primary geographic areas. Indeed, there are isolates that do not show species-specific morphology, as in our case isolate, and as a result may be misidentified by classical methods. By checking the identity of some strains preserved in the collection BCCM/IHEM, we found several that proved to be T. simii, originating from non-endemic areas (Belgium, France and Ivory Coast). Therefore, the natural distribution of T. simii is probably not as restricted as has previously been proposed.

Pothomorphe umbellata: antifungal activity against strains of Trichophyton rubrum.

Trichophyton rubrum is a dermatophyte, which can cause infections in human skin, hair and nail. Pothomorphe umbellata (L.) Miq. (Piperaceae) is a native Brazilian plant, in which phytochemical studies have demonstrated the presence of steroids, 4-nerolidylcatechol, sesquiterpenes and essential oils. The objective of this study was to analyze the in vitro activity of extracts and fractions of P. umbellata on resistant strains of T. rubrum. The microdilution plate method was utilized to test Tr1, H6 and ΔTruMDR2 strains of T. rubrum; ΔTruMDR2 strain was obtained from H6 by TruMDR2 gene rupture, which is involved in multiple drugs resistance. The highest antifungal activity to all strains was observed for dichloromethane and hexane fractions of the 70% ethanolic extract which showed minimal inhibitory concentration (MIC) and minimal fungicide concentration (MFC) of 78.13 µg/mL. This antifungal activity was also obtained by 70% ethanolic extract, which presented MIC and MFC of 78.13 µg/mL to ΔTruMDR2, whereas the MIC values for Tr1 and H6 were 78.13 and 156.25 µg/mL, respectively. Our results suggest the potential for future development of new antifungal drugs from P. umbellata, especially to strains presenting multiple resistance.

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source.

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts overexpressed in T. rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-ß-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes.

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyses the oxygenation of cysteine to cysteine sulphinic acid leading to the production of sulphite, sulphate and taurine as the final metabolites of cysteine catabolism. Keratinolytic fungi secrete sulphite and sulphate to reduce disulphide bridges in host tissue keratin proteins as the first step of keratinolysis. In the present study, we describe the identification of cDNA, as well as expression and characterisation of recombinant CDO protein from Trichophyton mentagrophytes. The cDNA was amplified using primers designed on the basis of high conservancy CDO regions identified in other fungi. PCR product was cloned and sequenced. Recombinant CDO was expressed in Escherichia coli, and affinity purified and identified by matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS). Enzyme activity was assayed by monitoring the production of cysteine sulphinate using mass spectrometry. The Cdo cDNA encodes for a protein consisting of 219 amino acids. Recombinant CDO protein C-terminally fused with a His tag was purified by affinity chromatography. The CDO purified under native condition was proved to be enzymatically active. Protein identity was confirmed by MALDI-TOF MS. Comparison of cDNA sequence with those identified in other fungi revealed significant homology. Identification of T. mentagrophytes CDO provides indispensable tools for future studies of dermatophyte pathogenicity and development of new approaches for prevention and therapy.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling.

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T. rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T. rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC(-) mutant suggests that, in T. rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH.

Preliminary study of activity of the thioredoxin inhibitor pleurotin against Trichophyton mentagrophytes: a novel anti-dermatophyte possibility.

Dermatophyte infections, while not life-threatening, are very common, and there is great interest in developing new antifungal agents. Transcriptional profiling of Trichophyton on keratin has identified some antioxidant genes as induced on this host substrate, including a thioredoxin gene TmTRX1. If thioredoxin is a virulence factor, or necessary for the growth on keratin, thioredoxin inhibitors should act as antifungals. As a first evaluation of this hypothesis, we have tested the activity of a thioredoxin-inhibitory natural product, pleurotin, against a clinical isolate of each of two fungal pathogens: the dermatophyte T. mentagrophytes and Candida albicans. Pleurotin inhibited the growth of the dermatophyte in vitro and in an ex vivo skin model, but had no effect on Candida. It may be possible to develop and optimise thioredoxin inhibitors, some of which are already under study in cancer chemotherapy, as antifungals.

Commonly affected body sites in 92 Japanese combat sports participants with Trichophyton tonsurans infection.

Outbreaks of Trichophyton tonsurans infection constitute one of the serious problems among combat sports practitioners in Japan. To facilitate the diagnosis of individuals at risk, we undertook a study to determine which body sites are most commonly infected. We reviewed medical data, hairbrush culture results and questionnaire information from patients with T. tonsurans infection who were admitted to the dermatology clinic of Juntendo University hospital from 2000 to 2004. The study included 92 patients (87 males), aged 6-38 years (mean age: 18.4 years old). Eighty-nine patients were judo practitioners and three were wrestlers. Twenty-eight patients (30.4%) were asymptomatic carriers. In 64 patients, 51 patients (55.4%) with tinea corporis, 27 patients (29.3%) with tinea capitis, and/or one patient (1.1%) with tinea manuum were seen. Tinea corporis was observed on the forehead, auricles, nape of the neck, bilateral shoulders, left side of the upper chest, both elbows, back of the left hand to the wrist and both knees. Tinea capitis was most common in the occipitonuchal region at the hairline and in the temporal and frontal regions, at both auricles. Initial screening of these sites might facilitate the identification of the infection especially in judo practitioners.

Diagnosis of Trichophyton rubrum from onychomycotic nail samples using polymerase chain reaction and calcofluor white microscopy.

BACKGROUND: A high rate of false-negative dermatophyte detection is observed when the most common laboratory methods are used. These methods include microscopic observation of potassium hydroxide-digested nail clippings and culture methods using agar-based media supplemented with cycloheximide, chloramphenicol, and gentamicin to isolate dermatophytes. Microscopic detection methods that use calcofluor white staining or periodic acid-Schiff staining may also be substituted for and have previously been reported to be more sensitive than potassium hydroxide-digested nail clippings. METHODS: Trichophyton rubrum infections were detected directly from nails in a double-round polymerase chain reaction assay that uses actin gene-based primers. This method was compared with detection of fungal hyphae by using calcofluor white fluorescence microscopy of nail samples collected from 83 patients with onychomycosis who were undergoing antifungal drug therapy. RESULTS: Twenty-six of 83 samples (31.3%) were found to be positive by calcofluor white fluorescence microscopy, and 21 of 83 samples (25.3%) yielded positive results for T rubrum when actin gene-based primers in a double-round polymerase chain reaction assay were used. When calcofluor white fluorescence microscopy and polymerase chain reaction assay were used, the combined detection was 46.9% compared with 31.3% when calcofluor microscopy and culture of nail samples on Sabouraud's dextrose agar supplemented with cycloheximide, chloramphenicol, and gentamicin were used. CONCLUSIONS: These results suggest that the use of a direct DNA protocol is an alternative method for detecting Trichophyton infections. When this protocol is used, the presence of T rubrum DNA is directly detected. However, the viability of the dermatophyte is not addressed, and further methods need to be developed for the detection of viable T rubrum directly from nail samples.

Analysis of Trichophyton rubrum gene expression in response to cytotoxic drugs.

Suppressive subtractive hybridization was used to isolate transcripts specifically upregulated during Trichophyton rubrum exposure to acriflavin, fluconazole, griseofulvin, terbinafine or undecanoic acid. Macro-array dot-blot and sequencing of 132 clones, which correspond to genes differentially expressed after exposition of T. rubrum to at least one of these cytotoxic drugs, revealed 39 unique genes. Of these, 32 have not been previously described in T. rubrum, representing an increase in the number of T. rubrum genes that have been identified. The upregulation of the novel genes encoding a retrotransposon element, a carboxylic ester hydrolase, a copper resistance-associated P-type ATPase, a DNA mismatch repair protein and a NIMA (never in mitosis A) interactive protein was confirmed by Northern blot.

Molecular typing and antifungal susceptibility of Trichophyton rubrum isolates from patients with onychomycosis pre- and post-treatment.

Forty sequential isolates of Trichophyton rubrum were obtained from patients suffering from onychomycosis at two time points, before and after antifungal oral therapy. Strain differentiation by specific amplification of the two tandemly repeated elements (TRS-1 and TRS-2) of the ribosomal DNA of T. rubrum was performed. In addition, susceptibility tests were executed by the microdilution method with nine antifungal drugs: ketoconazole, itraconazole, fluconazole, miconazole, clotrimazole, isoconazole, griseofulvin, cyclopiroxolamine and terbinafine. The combination of TRS-1 with TRS-2 PCR amplification patterns configured 11 T. rubrum genotypes and the three most prevalent (genotypes 1-I, 5-I and 2-I) accounted for 67.5% of the isolates. Seven isolates (35%) obtained before antifungal oral therapy exhibited genotype 1-I compared to the 11 (55%) obtained after the treatment. Twelve patients exhibited different strains before and after the antifungal therapy. With respect to in vitro susceptibility testing, terbinafine was the most potent agent, followed by itraconazole, clotrimazole, isoconazole, miconazole, cyclopiroxolamine, ketoconazole, griseofulvin and fluconazole. Furthermore, an increase in the minimum inhibitory concentrations (MIC) were observed for most of the azole agents when testing isolates obtained post-treatment from four patients. This increase in MIC occurred concomitantly with the major occurrence of genotype 1-I for isolates obtained after oral therapy. These data attempt to consider the relevance of in vivo drug resistance for onychomycosis caused by T. rubrum.

Genotype analysis of the variable internal repeat region in the rRNA gene of Trichophyton tonsurans isolated from Japanese Judo practitioners.

Tinea capitis due to Trichophyton tonsurans is currently epidemic among Japanese Judo practitioners. T. tonsurans has seven genotypes in a variable internal repeat (VIR) region of the rRNA gene. All 101 isolates obtained from Japanese Judo practitioners had the identical genotype. This suggests that a specific genotype strain occurs throughout Japan.