Major Dermatophytes Research Articles

Efficacy of common disinfection processes against infective spores (arthroconidia) and mycelia of Microsporum gallinae causing avian dermatophytosis.

Background and Aim:Microsporum gallinae is the major dermatophyte species that causes avian dermatophytosis. Disinfection plays an important role in controlling and preventing dermatophytosis; however, information about the effect of common disinfection processes on M. gallinae is limited. This study aimed to investigate the disinfection efficacy of ultraviolet (UV) irradiation, heat treatment, detergents, and germicides against infective spores (arthroconidia) and vegetative mycelia of M. gallinae.Materials and Methods:The minimum inhibitory and minimum fungicidal concentrations of benzalkonium chloride, chlorhexidine, ethanol, formaldehyde, glutaraldehyde, hydrogen peroxide, phenol, povidone-iodine, and sodium hypochlorite germicides against arthroconidia and mycelia of M. gallinae American type culture collection (ATCC) 90749 were determined by broth microdilution. Time-kill assays were used to determine the fungicidal efficacy of moist heat treatment, UV irradiation, commercially available detergents, and germicides.Results:There were no significant differences between the arthroconidia and mycelia growth stages of M. gallinae ATCC 90749 in the magnitude of the log10 cell reductions in the number of viable fungal cells induced by the disinfection treatments (all p > 0.05). Moist heat treatment at 40°C did not reduce the number of viable fungal cells at any time (1–60 min); however, treatment at 50°C for 25 min and either 60°C or 80°C for 5 min eliminated > 99.999% of viable fungal cells. Irradiation of fungal cultures with UVC and UVB at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, resulted in a 5-log10 reduction in the number of viable fungal cells, whereas UVA only reduced the number of viable fungal cells by < 2-log10 up to a dose of 1.6 J/cm2. All the tested detergents demonstrated minimal fungicidal effects with < 1-log10 reductions in the number of viable fungal cells at concentrations up to 8% w/v. All of the tested germicides eradicated the fungus after treatment for 1 min at 1–1000× minimum inhibitory concentration (MIC), except for hydrogen peroxide, which was not fungicidal after treatment for 20 min at 100× MIC.Conclusion:Moist heat treatment at temperatures greater than or equal to 50°C, UVC and UVB irradiation at doses higher than or equal to 0.4 and 0.8 J/cm2, respectively, and treatment with all tested germicides except hydrogen peroxide can be considered effective processes for disinfecting the fungus M. gallinae from the equipment employed in poultry farming. In contrast, commercially available detergents are not suitable for use as M. gallinae disinfectants.

Molecular Epidemiology, Genetic Diversity, and Antifungal Susceptibility of Major Pathogenic Dermatophytes Isolated From Human Dermatophytosis.

BackgroundDermatophytes are a homogeneous group of species with low genetic diversity, and there are still many uncertainties about the boundaries among species.ObjectivesAiming at clarifying the relationships among species in the genus and introducing suitable genes for multilocus sequence typing (MLST), a new MLST scheme approach was developed to characterize the major pathogenic dermatophytes.MethodsWe performed maximum parsimony (MP), MrBayes, RAxML, and eBURST analyses, based on the MLST scheme to scrutinize the evolution within 95 clinical isolates and four reference strains belonging to the four major dermatophytes species. Then, the discriminatory power, pairwise genetic distances, ratio dN/dS, and sequence types (STs) of these isolates were determined. Also, to study taxonomy, sequences of the internal transcribed spacer (ITS), Beta-tubulin (BT2), and translation elongation factor 1-α (TEF-1α) genes of other dermatophytes species available in the GenBank were analyzed.ResultsFindings of the present study indicated that three genes: BT2, ITS, and TEF−1α, which showed the greatest diversity among dermatophyte species, were suitable for MLST. The most prevalent STs were seen among the species of Trichophyton interdigitale. Also, two new genotypes, i.e., XXVII and XXVIII, were introduced for T. interdigitale and Trichophyton mentagrophytes. The least informative sites were found in Epidermophyton floccosum, Trichophyton rubrum, and T. mentagrophytes, while the most informative sites were observed in T. interdigitale. Furthermore, the most informative locus was TEF-1α. The phylogenetic tree, constructed by the combination of the three genes, shows a new topological pattern that confirms the derivation of the anthropophilic and zoophilic genera from the geophilic genus. Also, the phylogenetic analyses and pairwise distances of the combination of the three loci showed that Trichophyton tonsurans and Trichophyton equinum were a species complex, where T. equinum is derived from T. tonsurans.ConclusionsResults of this study showed that MLST is very effective in determining the boundaries between species and taxonomy. Considering that there is no database for MLST dermatophytes, further studies are needed to determine the suitable genes for MLST. Also, the determination of STs in epidemiological studies and raising epidemiological information are helpful. This study was a new starting point to determine the ST and a foundation for a dermatophyte MLST database.

Antifungal ME1111 in vitro human onychopharmacokinetics

This study determined ME1111 onychopharmacokinetics and possible topical antifungals’ clinical efficacy in human great toenails using an in vitro finite dose model. ME1111 topical formulations in 1, 5, 10 or 15% for 3 days observation and 1, 5 or 10% for 14 days observation, respectively, were used to determine ME1111 penetration rate and transungual kinetics. ME1111 concentrations in the deeper nail (ventral/intermediate layers) and a cotton pad/nail bed, were several orders of magnitude greater than MIC90 and MFC90 for three major dermatophytes. ME1111 concentrations 3 days after a single and 14 days after multiple dosing of 10% formulation were 253 and 7991 μg/g nail, respectively, and superior to those of 8% ciclopirox control. ME1111 concentration (μg equivalent/cm3) in the cotton pad following 10% ME1111 multiple applications increased linearly throughout the 336 h experiment and was significantly greater than that of 8% ciclopirox. Flux rate of ME1111 averaged as 50.9 μg/cm3/day, which was ca. two orders of magnitude greater than the MIC90 values. The novel antifungal ME1111 penetrated well into human nail plate and its concentrations in the deeper nail and cotton pad after application of 10% formulation were significantly greater than those of ciclopirox.

Econazole imprinted textiles with antifungal activity

In this work, we propose pharmaceutical textiles imprinted with lipid microparticles of Econazole nitrate (ECN) as a mean to improve patient compliance while maintaining drug activity. Lipid microparticles were prepared and characterized by laser diffraction (3.5±0.1μm). Using an optimized screen-printing method, microparticles were deposited on textiles, as observed by scanning electron microscopy. The drug content of textiles (97±3μg/cm2) was reproducible and stable up to 4months storage at 25°C/65% Relative Humidity. Imprinted textiles exhibited a thermosensitive behavior, as witnessed by a fusion temperature of 34.8°C, which enabled a larger drug release at 32°C (temperature of the skin) than at room temperature. In vitro antifungal activity of ECN textiles was compared to commercial 1% (wt/wt) ECN cream Pevaryl®. ECN textiles maintained their antifungal activity against a broad range of Candida species as well as major dermatophyte species. In vivo, ECN textiles also preserved the antifungal efficacy of ECN on cutaneous candidiasis infection in mice. Ex vivo percutaneous absorption studies demonstrated that ECN released from pharmaceutical textiles concentrated more in the upper skin layers, where the fungal infections develop, as compared to dermal absorption of Pevaryl®. Overall, these results showed that this technology is promising to develop pharmaceutical garments textiles for the treatment of superficial fungal infections.

Environmental and Genetic Factors on the Development of Onychomycosis.

Since the early 20th century, onychomycosis originated with the onset of war, the use of occlusive footwear, and the mass migration of people by transportation in the United States. Even though onychomycosis has a high prevalence in the US, other parts of the world including Canada, West Africa, Southeast Asia, Northern Australia, and Europe have been well documented with cases of fungal toenail infection in their environments. Trichophyton rubrum (T. rubrum) is the major dermatophyte responsible for toenail fungal infection and is typically diagnosed in conjunction with tinea pedis, especially in individuals older than 60 years. Gender roles, age, cultural habits, shoe gear, sports activities, and genetic predisposition all contribute to the different presentation of onychomycosis in these areas where organisms like dermatophytes, candida, and molds were isolated in a variety of cases. Despite the differences in isolated pathogens, treatment outcomes remained consistent. This literature review discusses the influence of tinea pedis, genetics, shoe gear, sports, and age on the development of onychomycosis.

Effects of &lt;i&gt;Ocimum Gratissimum&lt;/i&gt; Leaves on Common Dermatophytes and Causative Agent of &lt;i&gt;Pityriasis Versicolor&lt;/i&gt; in Rivers State, Nigeria

This antifungal study on Ocimum gratissimum is rare in Nigeria and hence this study seeks to justify the ethobotanical uses of the plant. The antifungal activity of Ocimum gratissimum used by traditional medicine practitioners against the three major Dermatophytes - Trichophyton, Microsporum, Epidermophyton together with Malassezia furfur (the cause of Pityriasis versicolor (Eczema)), were studied by well-in-agar diffusion technique using different concentrations of ethanolic extracts. Isolates from the scalp, skin, toes and feet of forty individuals (mainly children) were obtained in four locations namely Aluu, Choba, Rumuosi and Emohua areas of Rivers State, Nigeria. The results of the study revealed the significant inhibitory effect of Ocimum gratissimum at five different concentrations of 250mg/ml, 200mg/ml, 150mg/ml, 100mg/ml and 50mg/ml used. The diameter zones of inhibition exhibited by the extracts against the test fungal species ranged between 12.50 and 20mm. The minimum inhibitory concentrations (MICs) of the ethanol extract of O. gratissimum was 50.01, 52.40, 63.06 and 63.09 mg/ml for Malassezia furfur, Microsporum, Trichophyton and Epidermophyton, respectively. Assessment of the various MICs showed that Ocimum gratissimum has great potential for use as an antidermatophytic agent. The study showed that the extracts from the leaves of Ocimum gratissimum had pronounced antifungal activities on all the fungi tested. The study has shown that the leaves extracts of O. gratissimum are quite promising and have strongly indicated the antifungal activity spectra of leaves extract of the plant. In conclusion, the results from this study indicated potentials of leaves extract of Ocimum gratissimum as a source of antifungal compounds.

Direct detection of five common dermatophyte species in clinical samples using a rapid and sensitive 24-h PCR-ELISA technique open to protocol transfer

Identification of dermatophytes is usually based on morphological characteristics determined by time-consuming microscopic and cultural examinations. An effective PCR-ELISA method has been developed for rapid detection of dermatophyte species directly from clinical specimens within 24 h. Isolated genomic DNA of skin scrapings and nail samples from patients with suspected dermatophyte infections is amplified with species-specific digoxigenin-labelled primers targeting the topoisomerase II gene. The subsequent ELISA procedure with biotin-labelled probes allows a sensitive and specific identification of the five common dermatophytes -Trichophyton rubrum, T. interdigitale, T. violaceum, Microsporum canis and Epidermophyton floccosum. PCR-ELISA, based on the new polyphasic species concept, was assessed using 204 microscopy-positive samples in two university mycological laboratories in Munich and Tübingen, and 316 consecutive specimens - regardless of mycological findings - in a dermatological practice laboratory in Neu-Ulm. One of the five dermatophytes was confirmed by PCR-ELISA in 163 of 204 (79.9%) of the clinical samples from the university hospitals found positive using microscopy. Culture was positive for dermatophytes in 59.8% of the same cases. A significant difference between these two methods could be demonstrated using the McNemar test (P < 0.005). Analysis of specimens from Neu-Ulm confirmed the results in a dermatological practice laboratory as 25.0% of the specimens had positive PCR results, whereas only 7.3% were positive according to culture. Direct DNA isolation from clinical specimens and the PCR-ELISA method employed in this study provide a rapid, reproducible and sensitive tool for detection and discrimination of five major dermatophytes at species level, independent of morphological and biochemical characteristics.

NB-002, a Novel Nanoemulsion with Broad Antifungal Activity against Dermatophytes, Other Filamentous Fungi, and Candida albicans

NB-002 is an oil-in-water emulsion designed for use for the treatment of skin, hair, and nail infections. The activity of NB-002 was compared to the activities of the available antifungal drugs against the major dermatophytes responsible for cutaneous infections, Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Microsporum spp., as well as 12 other genera of filamentous fungi. NB-002 consistently displayed fungicidal activity against all dermatophytes. The comparator compounds were either fungistatic or fungicidal, and for some strain-drug combinations, tolerance was observed. Assessment of the development of spontaneous resistance to NB-002 in different dermatophyte species yielded few stably resistant mutants. For filamentous nondermatophyte fungi, the MIC range varied from 0.06 to 0.5 microg/ml for Alternaria spp. to 2 to 8 microg/ml for Paecilomyes spp. NB-002 had activity against both azole-susceptible and -resistant Candida albicans yeast isolates, with MIC(90)s of 2 microg/ml, respectively, and minimum fungicidal concentrations at which 90% of isolates are inhibited of 4 and 8 microg/ml, respectively. The kinetics of the fungicidal activity of NB-002 against T. rubrum isolates were compared to those of the other antifungal drugs. NB-002 killed both mycelia and microconidia even when the fungal forms were dormant or not actively growing. Electron micrographs of mycelia and spores treated with NB-002 showed the significant disruption of the fungal structure. The in vitro broad coverage of NB-002 against filamentous fungi, dermatophytes, and C. albicans, as well as its rapid fungicidal activity, warrants further investigations to ascertain if NB-002 would be useful for the treatment of cutaneous mycoses.

Specific IgE Response to Trichophyton and Asthma Severity

Sensitization to Trichophyton, a major dermatophyte, has been associated with asthma. Whether such sensitization is generally associated with the severity of asthma, like other molds such as Alternaria, is unknown. We compared 258 patients with asthma, which was classified by severity as mild (n = 123), moderate (101), or severe (34), and 114 healthy control subjects, with regard to specific IgE titers against Trichophyton rubrum and other common allergens such as mixed molds, house-dust mite, cat dander, dog dander, Japanese cedar pollen, mixed Graminea pollens and mixed weed pollens. Positive rate of Trichophyton-specific IgE was higher in the patients with moderate asthma (15.8%) than in the control subjects (7.0%, p = 0.04) and patients with mild asthma (4.9%, p < 0.006), and it was also higher in the patients with severe asthma (32.4%) than in control subjects (p = 0.0001), and patients with mild asthma (p < 0.0001) and moderate asthma (p = 0.04), but it did not differ between the control subjects and patients with mild asthma. The positive rates of mixed molds, cat dander, and dog dander were almost invariably higher in patients in all asthma subgroups than in the control subjects but did not differ among patients in the three asthma subgroups. The positive rates of other allergens were not different in all groups. Reanalysis of positive rate of Trichophyton-specific IgE after excluding 52 subjects with positive results for mixed molds showed a similar statistical trend to that of the original cohort. This may negate the potential effect of cross-reactivity to these molds. Multivariate analysis of asthma subgroups identified positive IgE results for Trichophyton as an independent determinant of asthma severity. Specific IgE response to Trichophyton may be associated with more severe asthma.

Molecular Approach to Pathology of and Immunity against Dermatophytes

Molecular biological studies of the host invasion mechanisms and possible virulence-related factors of dermatophytes have just begun. The identification of individual genes and large-scale investigations of transcripts expressed under different experimental culture conditions have provided useful information on the structure, expression, and regulation of the genes of major dermatophyte species such as Trichophyton rubrum. The next goal of dermatophytosis research will be to elucidate the functions and roles of the identified fungal genes during the infection process. It will also be necessary to investigate the host immune responses to fungal gene expression and regulation during infection. For such research, genetic manipulation techniques for dermatophytes, such as exogenous gene transfer into fungal cells and targeted gene disruption, are indispensable. However, such methods are not yet well established. We have developed an efficient dermatophyte genetic manipulation system using T. mentagrophytes. Here, we present our current research findings, mainly with regard to the system for exogenous gene transfer into T. mentagrophytes cells.

Reproducible genetic transformation system for two dermatophytes,Microsporum canisandTrichophyton mentagrophytes

A reproducible genetic transformation system was developed for two major dermatophytes, Microsporum canis and Trichophyton mentagrophytes. Two circular transformation vectors carrying either the bacterial hygromycin B phosphotransferase gene (hph) or both the hph and green fluorescent protein (eGFP) genes under the control of a promoter sequence from Cochlibolus heterostrophus were introduced independently into the protoplasts by a polyethylene glycol (PEG)-mediated method. Polymerase chain reaction (PCR) showed that the hph gene was integrated randomly into the chromosomal DNA of the transformants through non-homologous recombination. Southern blotting analysis also demonstrated a single or multiple integration of the hph gene into the chromosomal DNA. Fluorescence due to eGFP gene expression was observed in the T. mentagrophytes transformants, and the transformants retained mitotic stability through subculture. This reproducible transformation system provides a method for the genetic manipulation of these pathogens, which will facilitate detailed molecular analysis of dermatophytosis.

Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes

Background: We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. Objective: To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence. Methods: The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes ( Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed. Results: Of the eighteen species of dermatophytes, five species ( T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum. Conclusion: PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.

PCR-based identification of common dermatophyte species using primer sets specific for the DNA topoisomerase II genes

Background: We have determined nucleotide sequences of the DNA topoisomerase II genes of the dermatophyte species, and conducted a PCR-based identification system using species-specific primers for the nucleotide sequences. Objective: To identify the major dermatophytes, Trichophyton rubrum, T. mentagrophytes, T. violaceum, M. gypseum, M. canis and E. floccosum, by PCR amplifications at the species level, without determining the nucleotide sequence. Methods: For PCR-based identification of the major dermatophyte species, a common primer set (dPsD1) for these species and species-specific primer sets (PsT and PsME) for each species were designed based on the genomic sequences of the DNA topoisomerase II genes of the dermatophytes, and tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by the common primer set, followed by a second PCR with the primer sets consisting of species-specific primers for each dermatophyte species. Results: Using dPsD1, a DNA fragment of 3390 bp was amplified from the genomic DNA of all the dermatophyte species. In the subsequent nested PCR using species-specific primer sets (PsT and PsME), both sets amplified unique sizes of PCR products, all of which corresponded to a species of the dermatophytes even in the presence of other fungal DNA. Conclusion: We demonstrate that the PCR-based identification targeting the DNA topoisomerase II gene is rapid and simple, and is available as a tool for the identification of the major dermatophyte species.

Species identification system for dermatophytes based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1.

This describes a new and reliable species identification and classification system for dermatophytes based on the cluster analysis of nuclear ribosomal internal transcribed spacer 1 (ITS1) DNA sequences. In this system, some phenotypically similar species construct a compact monophyleic cluster which seems to be a species. This ITS1 sequence based species is called an ITS1-genospecies. The classification of genospecies is a practical concept for DNA sequence based species identification. It is possible to perform species identification and/or strain typing of 25 major dermatophytes (anamorphic genera Trichophyton, Microsporum, Epidermophyton, and the teleomorphic genus Arthroderma), some of which are hard to identify from their morphological features, by demonstrating their dendrogram using this system.

A new anti-mycotic drug tioxaprofen and its uncoupling effect on isolated mitochondria

An oxazole compound, tioxaprofen, exerted a strong anti-mycotic activity against Trichophyton mentagrophytes and T. rubrum, which were major dermatophytes from patients. It was found that tioxaprofen was a potent uncoupling agent of mitochondrial respiration.