Analysis of the dermatophyte species isolated in the British Isles between 1980 and 2005 and review of worldwide dermatophyte trends over the last three decades.

Infections of the skin, hair and nails by dermatophyte fungi are common in developed and developing countries alike. However, the species involved and the resulting clinical entities vary both geographically and with time. We have surveyed 15,333 dermatophytes obtained from primary isolations at the Mycology Reference Laboratory, Bristol, UK from 1980 through 2005. Several striking trends in dermatophyte prevalence were apparent over this period. The relative frequencies of isolations of Microsporum canis (cat and dog ringworm), Trichophyton verrucosum (cattle ringworm), T. mentagrophytes var. mentagrophytes (rodent ringworm) and Epidermophyton floccosum (a cause of human groin and foot infections) all decreased by 90%. Conversely, the contributions of T. tonsurans and T. violaceum (two anthropophilic scalp-infecting species) to total dermatophyte isolations increased by 1000% over the same period. Finally, T. rubrum and T. mentagrophytes var. interdigitale, the two common causes of foot infection comprised 80% of all dermatophytes isolated in 1980 and 90% of isolations in 2005. Similar trends in dermatophyte prevalence were evidenced throughout the British Isles, based on the voluntary reporting of isolations from a large number of British laboratories at 5-yearly intervals over the same period. The implications of these changing patterns of dermatophyte species, and the clinical entities they produce are discussed in the context of a review of worldwide dermatophyte isolations over the last three decades, with emphasis on the causal agents of tinea capitis.     

Suppression of In Vitro Lymphocyte Transformation During an Experimental Dermatophyte Infection

During primary Trichophyton mentagrophytes infection of strain 2 guinea pigs, the colony-forming units (CFU) of fungi present within the lesion peaked between days 7 and 14, whereas the severity of the lesion itself peaked between days 11 and 16. Concomitant with the latter peak, a pronounced depression in the in vitro mitogenic activity of spleen cells (SPC) and lymph node cells (LNC) was observed. Only after resolution of the primary infection (day 21) did LNC show increased deoxyribonucleic acid (DNA) synthesis in the presence of fungal antigens. During cutaneous reinfection, there was no distinct peak fungal load and CFU appeared to decrease steadily during the accelerated course of a reinfection disease. LNC from guinea pigs with severe, ulcerated reinfection lesions generally exhibited a heightened response to fungal antigen in vitro. LNC from guinea pigs with mild reinfection dermatophytosis had depressed in vitro reactivity to mitogens and dermatophyte antigen. The suppression of blastogenic activity during dermatophyte infection appeared to be associated with autologous serum components, since increased DNA synthesis resulted when SPC or LNC were cultured with fetal calf serum. The depressed in vitro DNA synthesis of lymphocytes (cultured with dermatophyte antigens) that were harvested during reinfection was not accompanied by an impaired ability of infected guinea pigs to respond with a delayed-type hypersensitivity skin test in vivo. These results support the hypothesis that experimental T. mentagrophytes dermatophytosis is a cell-mediated hypersensitivity disease that can be modified by immunosuppressive control mechanisms elaborated or induced by the fungus.     

Infection stages of the dermatophyte pathogen Trichophyton: microscopic characterization and proteolytic enzymes.

Dermatophytes are pathogenic fungi that infect human skin, nails and hair and cause dermatophytosis. Trichophyton mentagrophytes is one of the most widespread species that belong to this group. Infection of the skin tissues include several stages, i.e., adhesion to the surface of the skin, invasion into the sublayers by the penetration of fungal elements and secretion of enzymes that degrade the skin components. In this study we have followed the morphology of the fungal elements, such as arthroconidia and hyphae, during the adhesion and invasion stages. Skin explants were inoculated with the dermatophyte and observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Skin explants were also inoculated with a transgenic isolate of T. mentagrophytes expressing the green fluorescent protein (GFP). The infected sublayers were investigated by confocal scanning laser microscopy (CSLM). As an adaptation to the tissue environment, the dermatophyte produced long fibrils when it is on the open surface of the stratum corneum, while short and thin fibrils are produced inside the dense sublayers. The short and long projections might have a role in adhesion. Invasion may be produced by mechanical and biochemical means. Invasion of the tissue showed hyphal branching and growth in multiple directions. The proteolytic profile was assayed by substrate gel and proteolytic activity. Two serine proteases of similar molecular weight were secreted during growth on the epidermal matrix components keratin and elastin. The dermatophyte may use the proteolytic enzymes to invade the surface and also the deep layer of the skin in immunocompromised patients. Dermatophytes, which are well adapted infectious agents, seem to use their mechanical and biochemical capabilities to invade the skin tissue effectively.     

Single-step PCR using (GACA)4 primer: utility for rapid identification of dermatophyte species and strains

Dermatophytes are fungi that belong to three genera: Epidermophyton, Microsporum, and Trichophyton. Identification of dermatophyte species is essential for appropriate diagnosis and treatment of dermatophytosis. Routine identification depends on macroscopic and microscopic morphology, which is time-consuming and does not identify dermatophyte strains. In this study, two PCR-based methods were compared for their abilities to identify 21 dermatophyte isolates obtained from Egyptian patients to the species and strain levels. The first method employed a two-step method: PCR amplification, using ITS1 and ITS4 as primers, followed by restriction enzyme digestion using the endonuclease MvaI. The second method employed a one-step approach employing the repetitive oligonucleotide (GACA)(4) as a primer. Dermatophyte strains were also identified using a conventional culture method. Our results showed that the conventional culture method identified four species: Microsporum canis, Trichophyton mentagrophytes, Trichophyton rubrum, and Trichophyton violaceum. Moreover, both PCR methods agreed with the diagnosis made using the conventional approach. Furthermore, ITS1/ITS4-based PCR provided no strain differentiation, while (GACA)(4)-based PCR identified different varieties among the T. mentagrophytes isolates. Taken together, our results suggest that (GACA)(4)-based PCR has utility as a simple and rapid method for identification of dermatophyte species as well as utility for differentiation of T. mentagrophytes variants.     

Isolation of glycopeptides with skin test activity from dermatophytes.

By using ethylene glycol extraction of whole submerged cultures followed by Sephadex G-200 and diethylaminoethyl-Sephadex chromatography, we isolated four distinct glycopeptides from Trichophyton mentagrophytes, T. rubrum, and Microsporum canis. Chemical analyses revealed that these glycopeptides contained mostly carbohydrate (42.5 to 81.6%) and protein (4.3 to 11.3%), with lesser amounts of phosphorus (0.4 to 6.0%) and hexosamines (0.3 to 0.6%). Based upon total carbohydrate and monosaccharide content, these dermatophyte glycopeptides could be divided into two chemical groups: glucopeptides (DSI1) and mannopeptides (DSI2, DSII1, and DSII2). The mannopeptides and glucopeptides of each species of dermatophyte were not significantly different chemically from those derived from the other two dermatophyte species studied. Skin testing of DSI1-glycopeptides or DSI2-mannopeptides in immunized guinea pigs indicated that only the DSI2-mannopeptides elicited a delayed hypersensitivity reaction. Skin testing T. mentagrophytes 62-infected guinea pigs with the four purified DS-glycopeptides, as well as earlier fractions from the purification scheme, derived from T. mentagrophytes, T. rubrum, and M. canis, again indicated that only the DSI2-mannopeptides of the two Trichophyton species elicited a delayed hypersensitivity reaction. The number of infections or duration of infection had no effect on the size of the skin test response. DSI2-mannopeptides were non-cross-reactive between genera when tested in Trichophyton-immunized or -infected guinea pigs and Microsporum-immunized guinea pigs.     

Trichophyton sensitivity in allergic and nonallergic asthma

BACKGROUND: Although the role of inhaled fungi in inducing asthma has been repeatedly confirmed, there are few reports about the association of asthma with dermatophyte sensitivity and the causal role of Trichophyton allergy in asthma. The objective was to investigate the presence of Trichophyton sensitivity among patients with allergic and nonallergic asthma in combination with tinea, and to compare the situation with several control groups in order to evaluate the factors determining Trichophyton sensitivity. METHODS: A total of 86 subjects (55 female, 31 male) with a mean age of 38.6 +/- 11.1 years were included in the study. The patients were divided into five groups: 1) nonallergic asthma plus tinea (n = 19) 2) allergic asthma plus tinea (n = 15) 3) asthma without tinea (n = 22) 4) tinea without asthma (n = 17) 5) healthy controls (n = 13). Skin tests with standardized extracts of T. rubrum and specific IgE measurements were performed in all subjects. All patients were also subjected to microscopic evaluation and fungal culture for dermatophyte infection. RESULTS: The skin test positivity rate to Trichophyton extract of groups 1 (63.1%), 2 (46.7%), and 4 (47.1%) was higher than that in groups 3 (4.4%) and 5 (7.7%) (P < 0.05). Although not significant, the rates of sensitivity to T. rubrum (63.1%) and of severe asthma (31.6%) were higher in the group with nonallergic asthma with tinea (group 1) than in other groups. Among 51 patients in whom direct microscopic evaluation revealed dermatophyte infection, 60.8% had positive fungal cultures for T. rubrum (58.1%), T. mentagrophytes (35.5%), and Candida (6.4%). CONCLUSION: According to our data, the presence of fungal infection seems to be an important determinant in hypersensitivity to Trichophyton whether or not the subject is asthmatic and/or allergic. Since a greater proportion of patients with nonallergic asthma--in whom the rate of severe asthma was also higher - showed positive skin tests to Trichophyton extracts in this study, we believe that patients with severe, intrinsic asthma should be examined for signs of fungal infection and tested to determine immediate hypersensitivity to dermatophyte antigens.     

Chitin synthase (CHS) gene analysis of dermatophytes]

About 620-bp genomic DNA fragments of CHS1 genes were amplified from 13 species of dermatophytes by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of CHS1 gene fragments of these dermatophyte species revealed that 3 genera of Epidermophyton, Microsporum and Trichophyton were genetically different from each other. The molecular analysis of CHS1 genes will provide useful information for the identification of dermatophytes. The species-specific primers were designed from the nucleotide sequences of CHS1 gene in 3 teleomorphs of T. mentagrophytes. Using these primers the PCR analysis identified the clinical isolates of T. mentagrophytes from rabbit as A. benhamiae. By PCR analysis with the dermatophyte specific primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples. The full length of CHS1 and CHS2 genes of Arthroderma benhamiae (one of the teleomorphs of T. mentagrophytes) was sequenced by 5'-RACE and 3'-RACE methods using cDNA as a template. The full length cDNA sequences of CHS1 gene (3158bp) and CHS2 gene (3392bp) were proved to encode 890 and 419 amino acids, respectively. The amino acid sequences of A. benhamiae CHS1 and CHS2 in the conserved regions shared, respectively, about 70% and 80% sequence similarity with those of the other filamentous ascomycetes registered in the data base of the GeneBank. RT-PCR analysis suggested that chitin synthase inhibitors (nikkomycin Z and polyoxin D) might stimulate the expression of CHS1 mRNA in A. benhamiae, and not the expression of CHS2 mRNA.     

Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.     

Inhibition of dermatophytes by the antimicrobial peptides human β-defensin-2, ribonuclease 7 and psoriasin

Previous studies have described some antibacterial effects of antimicrobial peptides (AMPs) expressed in human skin, but little is known about their possible activity against dermatophytes. Therefore we have tested the effects of human β-defensin 2 (hBD-2), ribonuclease 7 (RNase 7) and psoriasin on the in vitro growth of four dermatophyte species. Germinating conidia of Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Epidermophyton floccosum were exposed in vitro to hBD-2, RNase 7, psoriasin and fluconazole. Subsequent fungal growth was measured photometrically over 168 hours. All AMPs significantly inhibited fungal growth, with the degree of inhibition dependent on the dermatophyte species and the specific AMP. E. floccosum was found to be the most susceptible species in that it was markedly suppressed by all AMPs, whereas M. canis was inhibited only by psoriasin. Overall, psoriasin was the most effective AMP and had even stronger inhibitory effects on some dermatophytes than fluconazole. Our findings show that AMPs expressed in human skin can, in principal, inhibit the growth of dermatophytes in vitro. Therefore the question whether AMPs are relevant for human protection against tineas is justified and should be addressed by investigating their role in vivo.     

In vitro and in vivo photosensitized inactivation of dermatophyte fungi by heterotricyclic dyes.

The ability of three heterotricyclic dyes to photosensitize dermatophyte fungi was studied with Trichophyton mentagrophytes and Microsporum gypseum. In vitro studies showed that methylene blue, neutral red, and proflavine were capable of killing these fungi when used in conjunction with broad-spectrum light. Proflavine, however, killed both fungi most rapidly and was used for further studies. Fungal killing by proflavine plus light was dependent on dye concentration, pH, light wavelength, and light intensity. Based on the in vitro studies, a treatment regimen was developed for in vivo use on experimentally infected animals. When treatment of guinea pigs inoculated with T. mentagrophytes was begun during fungal invasion, lesion formation at inoculated sites was either prevented or substantially reduced. When treatment was begun after lesion formation, however, light-plus-dyed treated sites showed only slightly faster curing than untreated sites.     

Epidemiology of dermatophytic infections in Rome, Italy: a retrospective study from 2002 to 2004.

In the present study, we determined the incidence of dermatophyte species causing superficial mycoses among outpatients referred to the Department of Dermatology of the "La Sapienza" University of Rome between 2002 and 2004. Of the 3160 subjects studied, 1275 (40.3%) were positive for fungal infection, but only 252 (19.7%) of these had infections caused by dermatophytes. The dermatophyte most frequently isolated was Microsporum canis. Our epidemiological data were compared with those obtained previously by other authors in the same geographic area. For the first time we described an inversion of the T. rubrum/T. mentagrophytes ratio, the latter being more frequently encountered. We also observed the emergence of M. audouinii.     

Cell-mediated immunity in experimental murine dermatophytosis. I. Temporal aspects of T-suppressor activity caused by Trichophyton quinckeanum.

Cutaneous dermatophyte infections caused by Trichophyton quinckeanum were established in various strains of mice. All congenic BALB/B (H-2b), BALB/c (H-2d) and BALB/K (H-2k) strains showed high susceptibility to dermatophyte infection. Susceptibility is independent of MHC phenotype, since other strains with corresponding H-2 haplotypes such as C57BL/6 (H-2b), DBA/2 (H-2d) and CBA (H-2k) were resistant to the disease. During the acute phase of infection in BALB/c mice, the in vitro blastogenic responses of regional lymph node cells was suppressed. Suppression was observed for both the T cell mitogens concanavalin A and phytohaemagglutinin, and for the B cell mitogen lipopolysaccharide. A series of cell-mixing experiments revealed that lymph node cells from infected mice were able to suppress the T cell and B cell mitogenic responses of lymph node cells from normal mice. Suppression was mediated by T cells and abrogated by treatment of lymph node cells with either monoclonal anti-thy-1.2 or anti-Ly-2.2 and complement. In this report, we discuss the possibility that T-suppressor mechanism mediated by cells bearing the Ly-2+ phenotype and activated during dermatophyte infection may determine the course of the disease.     

Epidemiological investigation of tinea pedis in groups of healthy students, research workers and females wearing boots]

The infection rate, causative dermatophytes, and dermatophyte dissemination of tinea pedis in young healthy Japanese were studied by direct microscopic examination, slant cultures, and foot-press culture method. Questionnaires on subjective symptoms and treatments were also distributed. Among fifty-eight medical students with a mean age of 23.9 years, thirteen (22.4%) showed positive by direct microscopic examination and T. mentagrophytes was more dominant than T. rubrum by slant cultures. In one hundred and sixteen student feet, twelve were infected and disseminating dermatophytes, four were infected but not disseminating, three were not infected but adhering dermatophytes. The infection rate of tinea pedis was thus 24.1%. Among thirty-seven research workers (mean age: 34.8), twenty-one (56.8%) showed positive by direct microscopic examination. All the dermatophytes isolated by slant cultures were T. mentagrophytes. In seventy-four feet, twenty-nine were infected and disseminating, ten were infected but not disseminating, and three were adhering dermatophytes. The infection rate was 64.9%. Among thirty-one females wearing boots (mean age: 21.0), seven (22.6%) were infected and T. mentagrophytes was more dominant. In sixty-two feet, eight were infected and disseminating, one was infected but not disseminating, and five were adhering dermatophytes. The infection rate of tinea pedis was quite high and T.r/T.m rate was low in the three groups. Most of the patients had had no treatment and were disseminating dermatophytes.     

Identification of rare macroconidia-producing dermatophytic fungi by real-time PCR

To understand the pathogenicity and clinical significance of dermatophytes (also known as ringworms), the correct identification of these molds is essential. However, in routine practice they are notoriously difficult to classify and identify. The morphology of macroconidia, which are abundantly produced under suitable in vitro conditions, have provided useful criteria for the identification of many of the dermatophytes. However, several of them, including Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely produce macroconidia and cannot be easily identified. The objective of this study was to design, optimize, and evaluate real-time PCR as a tool for identifying dermatophytic fungi in a laboratory setting. The performance of the assay was evaluated using 64 dermatophyte isolates, i.e., 35 rare macroconidia-producing reference strains, including the six species mentioned above, and 29 clinical isolates from our laboratory, including M. canis (4), T. mentagrophytes (2), T. rubrum (20), T. rubrum with the 'raubitschekii' morphotype (2), and T. tonsurans (1). Real-time PCR correctly identified 10 taxonomically distinct dermatophytes, particularly rare macroconidia-producing species, with excellent sensitivity (100%). The advantages of the assay include the provision of accurate and reliable diagnoses of dermatophytic fungi.     

PCR identification of dermatophyte fungi Trichophyton rubrum, T. soudanense and T. gourvilii

Diagnosis of dermatophytosis employing conventional laboratory procedures has been complicated by the slow growth and varied morphological features shown by dermatophytes. After analysis of the nucleotide base sequences of a 1.2-kb fragment amplified from a dermatophyte fungus Trichophyton rubrum by arbitrarily primed PCR with random primer OPD18, a pair of primers (TRIF and TR1R) was designed and evaluated for specific identification of T. rubrum. The sensitivity of the primers TR1F and TR1R was high, as a specific PCR band of c. 600 bp was detected from as little as 7 pg of T. rubrum DNA. By examining 92 dermatophyte strains and clinical isolates, it was found that this pair of primers reacted in PCR with T. rubrum, T. soudanense and T. gourvilii through formation of the specific fragment of 600 bp, but not with any other of the dermatophyte species or varieties, fungi, yeasts or bacteria tested. As T rubrum is one of the most frequently isolated dermatophyte fungi, and T. soudanense and T. gourvilii are relatively uncommon in many parts of the world, these primers can be used for rapid, sensitive and specific identification and differentiation of T. rubrum from other fungi and micro-organisms.     

Relationship between Candida albicans producing proteinase (CAPP) and its environmental pH--comparison with a case of trichophyton mentagrophytes

Candida albicans produced a karatinolytic proteinase (KPase) or C. albicans producing proteinase (CAPP), a proposed new term for this enzyme, and Trichophyton mentagrophytes also produced KPase when cultivated in liquid medium containing human stratum corneum (HSC) as the nitrogen source, but were unable to do so when cultivated in sabouraud dextrose broth. Purified KPase from the culture supernatants of C. albicans had a molecular weight of 42,000 and an optimum pH at 4.0. The KPase was found to belong to the carboxyl proteinases group and its activity was strongly inhibited by pepstatin. Both fungi were able to grow by secreting KPase which digested HSC for nutrients. KPase from both fungi had high activity in each optimum pH, such as weakly acidic pH on C. albicans and neutral pH on T. mentagrophytes to adapt their surrounding environment by changing the environmental pH into their own optimum pH.     

Scanning electron microscopy of experimental Trichophyton mentagrophytes infections in guinea pig skin.

Trichophyton mentagrophytes invasion of guinea pig skin was examined by scanning electron microscopy. Biopsies were obtained daily for 12 days from experimental infection sites. Dermatophyte invasion, examined in detail by scanning electron microscopy of cross-sectioned, prefixed skin was evidenced by: the appearance of hyphae within the stratum corneum; follicular invasion by hyphae, which remained initially within the follicle wall; emergence of the hyphae from the wall into the follicular canal; proliferation of the fungus down the follicle, with furrowing of the follicle wall and hair shaft cuticle; penetration of hyphae into the hair shaft by subcuticular and transcuticular routes; and massive peripilar hyphal proliferation with arthrosporogenesis. A three-dimensional perception of the invasion sequence of a dermatophyte in guinea pig skin was obtained by scanning electron microscopy.     

rRNA gene internal transcribed spacer 1 and 2 sequences of asexual, anthropophilic dermatophytes related to Trichophyton rubrum

The ribosomal region spanning the two internal transcribed spacer (ITS) regions and the 5.8S ribosomal DNA region was sequenced for asexual, anthropophilic dermatophyte species with morphological similarity to Trichophyton rubrum, as well as for members of the three previously delineated, related major clades in the T. mentagrophytes complex. Representative isolates of T. raubitschekii, T. fischeri, and T. kanei were found to have ITS sequences identical to that of T. rubrum. The ITS sequences of T. soudanense and T. megninii differed from that of T. rubrum by only a small number of base pairs. Their continued status as species, however, appears to meet criteria outlined in the population genetics-based cohesion species concept of A. R. Templeton. The ITS sequence of T. tonsurans differed from that of the biologically distinct T. equinum by only 1 bp, while the ITS sequence of the recently described species T. krajdenii had a sequence identical to that of T. mentagrophytes isolates related to the teleomorph Arthroderma vanbreuseghemii.