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The objective of this study was to analyze retrospectively the clinical efficacy and fungal clearance of long-pulsed 1064-nm Nd:YAG laser for treating onychomycosis and explore the inhibitory effects of laser on the fungus pathogen-induced onychomycosis in vitro. We performed a systematic retrospective analysis of clinical patients (162 effected nails) of onychomycosis treatment applying laser with or without topical ketoconazole ointment and followed up 3 months after treatment. Trichophyton rubrum- and Trichophyton mentagrophytes-induced onychomycosis was irradiated with laser superimposed for different cumulative energy, respectively; then, the areas of fungus colonies and growth curve in different days were showed, and changes in ultrastructures were observed under SEM and TEM. The clinical effective rate and fungal clearance rate in the combined group were higher than those in the laser group; however, there was no significant difference between the two groups. In vitro, the areas of T. rubrum colonies were significantly reduced at days 1, 3, and 5 after irradiation with cumulative laser energy ≥ 6400 J/cm 2 . When irradiated with cumulative laser energy ≥ 25600 J/cm 2 , significant difference in the areas of T. mentagrophytes colonies was found at day 5. And ultrastructure of the two strains before and after laser irradiation was damaged at different degrees. This study confirmed that long-pulsed 1064-nm Nd:YAG laser is effective for treating onychomycosis, and the laser irradiation can inhibit the colony growth of T. rubrum and T. mentagrophytes and change their cellular ultrastructures. The mechanism of laser treatment of onychomycosis may be related to direct damage of fungus pathogen.

Trichophyton rubrum ( T. rubrum ) is one of the most important agents of dermatophyte infection in humans. The aim of this experiment was to evaluate the effect of HaCaT cells on T. rubrum , investigate the responsible mechanism of action, and explore the role of reactive oxygen species (ROS) and nitric oxide (NO) in the inhibition of T. rubrum growth by HaCaT cells. The viability of fungi treated with HaCaT cells alone and with HaCaT cells combined with pretreatment with the NADPH oxidase inhibitor (DPI) or the nitric oxide synthase (NOS) inhibitor L-NMMA was determined by enumerating the colony-forming units. NOS, ROS, and NO levels were quantified using fluorescent probes. The levels of the NOS inhibitor asymmetric dimethylarginine (ADMA) were determined by enzyme-linked immunosorbent assay (ELISA). Micromorphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, fungal keratinase activity was assessed by measuring dye release from keratin azure. In vitro fungal viability, keratinase activity, and ADMA content decreased after HaCaT cell intervention, whereas the levels of ROS, NO, and NOS increased. The micromorphology was abnormal. Fungi pretreated with DPI and L-NMMA exhibited opposite effects. HaCaT cells inhibited the growth and pathogenicity of T. rubrum in vitro. A suggested mechanism is that ROS and NO play an important role in the inhibition of T. rubrum growth by HaCaT cells., Competing Interests: The authors declare that they have no conflicts of interest to disclose., (Copyright © 2020 Meiling Huang et al.)

Compounds belonging to the group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diols exhibit a broad spectrum of biological activity, including antibacterial, antifungal, and anticancer properties. The mechanism of the antifungal activity of compounds from this group has not been described to date. Among the large group of 5-substituted 4-(1,3,4-thiadiazol-2-yl) benzene-1,3-diol derivatives, the compound 4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol, abbreviated as C1, was revealed to be one of the most active agents against pathogenic fungi, simultaneously with the lowest toxicity to human cells. The C1 compound is a potent antifungal agent against different Candida species, including isolates resistant to azoles, and molds, with MIC100 values ranging from 8 to 96 μg/ml. The antifungal activity of the C1 compound involves disruption of the cell wall biogenesis, as evidenced by the inability of cells treated with C1 to maintain their characteristic cell shape, increase in size, form giant cells and flocculate. C1-treated cells were also unable to withstand internal turgor pressure causing protoplast material to leak out, exhibited reduced osmotic resistance and formed buds that were not covered with chitin. Disturbances in the chitin septum in the neck region of budding cells was observed, as well as an uneven distribution of chitin and β(1→3) glucan, and increased sensitivity to substances interacting with wall polymerization. The ATR-FTIR spectral shifts in cell walls extracted from C. albicans cells treated with the C1 compound suggested weakened interactions between the molecules of β(1→3) glucans and β(1→6) glucans, which may be the cause of impaired cell wall integrity. Significant spectral changes in the C1-treated cells were also observed in bands characteristic for chitin. The C1 compound did not affect the ergosterol content in Candida cells. Given the low cytotoxicity of the C1 compound to normal human dermal fibroblasts (NHDF), it is possible to use this compound as a therapeutic agent in the treatment of surface and gastrointestinal tract mycoses., Competing Interests: The authors have declared that no competing interests exist.

Background: T. mentagrophytes can infect all mammals, including rabbits, causing serious infections with remarkable economic losses for rabbit farmers. Berberine is an alkaloid that is effective against a variety of microbial infections such as T. mentagrophytes . Growth curve by dry weight determination and in-vivo antifungal assay were carried out to clarify the inhibitory effect of berberine hydrochloride against T. mentagrophytes . Transcriptomics analyses were also carried out for better understanding of the underlying mechanisms., Results: The growth rate of T. mentagrophytes was significantly higher in control condition than under berberine hydrochloride or clotrimazole for 60 h. The growth rate of T. mentagrophytes was significantly slighter higher in berberine condition (1 mg) than under clotrimazole for 46 h. T. mentagrophytes seriously shrunk after berberine or clotrimazole treatment, as observed by TEM and in SEM. Significant recovery was evident in three berberine groups on day 6 compared with the DMSO group. Results from transcriptomics analyses showed 18,881 identified unigenes, including 18,754 and 12,127 in the NT and SwissProt databases. Among these, 12,011, 9174, and 11,679 unigenes belonged to 3 Gene Ontology (GO), 43 KEGG, and 25 KOG categories, respectively. Interestingly, we found that down-regulation of 14α-demethylase exposed to various medicines was slightly different, i.e., berberine hydrochloride (fold change -3.4956) and clotrimazole (fold change -2.1283) caused various degrees of alteration., Conclusions: Berberine hydrochloride could inhibit the growth of T. mentagrophytes . Berberine hydrochloride could also cure dermatosis induced by T. mentagrophytes . Down-regulation of 14α-demethylase exposed to various medicines was slightly different and might be one of the anti-resistance mechanisms of berberine hydrochloride in T. mentagrophytes . The present investigation provides considerable transcript sequence data that would help further assess the antifungal mechanisms against T. mentagrophytes , for antifungal medicine development.

Background: Tinea unguium is a dermatophyte infection of the nails. Itraconazole is a broad-spectrum antifungal drug used to treat this disease. It works by interfering with the synthesis of ergosterol in the cell membrane, thus causing changes that can be seen under scanning electron microscopy (SEM)., Aim: To observe under SEM the in vivo ultrastructural changes of tinea unguium caused by Trichophyton rubrum before and after itraconazole (ICZ) therapy, and to explore the in vivo antifungal effect of ICZ., Methods: Ten outpatients diagnosed by mycology and SEM as having with tinea unguium caused by T. rubrum were recruited. They received ICZ pulse therapy (200 mg twice/day for 1 week, stop for 3 weeks, repeated for 3-4 months). Clinical change, mycology and SEM results were obtained after therapy., Results: After ICZ therapy, 3 of the 10 cases were positive by both mycology and SEM; the latter showed completely dry, shrivelled, curved and folded hyphae with a relatively smooth surface or local destruction. Several hyphae remained intact with only some local bumps or crimps. The other cases were negative by SEM, but two were positive by mycology., Conclusion: In vivo ultrastructural changes of hyphae of T. rubrum in tinea unguium after ICZ treatment could be seen under SEM, demonstrating that ICZ fungistasis was effective and that SEM is a good tool for evaluating the effectiveness of ICZ in vivo. To our knowledge, this study is the first to show by SEM ICZ permeates the affected nail plate via the nail bed capillaries., (© 2018 British Association of Dermatologists.)

A 61-year-old Caucasian man presented with papules on his left forearm and hand three months after liver transplantation: images from physical exam, pathology, and microbiology are presented. Skin biopsy confirmed the presence of fungal elements within the hair shaft, which is consistent with Majocchi granuloma, also known as nodular granulomatous perifolliculitis. A combination of fungal culture, microscopic morphology, and gene sequencing was used to identify the causative organism. The patient recovered with appropriate systemic antifungal therapy., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Onychomycoses are fungal infections of the fingernails or toenails having a prevalence of 3% among adults and accounts for 50% of nail infections. It is caused by dermatophytes, non-dermatophyte filamentous fungi, and yeasts. Compressions and microtraumas significantly contribute to onychomycosis. Laser and photodynamic therapies are being proposed to treat onychomycosis. Laser light (1064 nm) was used to treat onychomycosis in 156 affected toenails. Patients were clinically followed up for 9 months after treatment. Microbiological detection of fungal presence in lesions was accomplished. A total of 116 samples allowed the isolation of at least a fungus. Most of nails were affected in more than two thirds surface (some of them in the full surface). In 85% of cases, after 18 months of the onset of treatment, culture turned negative. After 3 months months, only five patients were completely symptom-free with negative culture. In 25 patients, only after 6 months, the absence of symptoms was achieved and the cultures negativized; in 29 patients, 9 months were required. No noticeable adverse effects were reported. This study reinforces previous works suggesting the applicability of laser therapies to treat toenail onychomycosis.

The effects of ME1111, a novel antifungal agent, on the hyphal morphology and ultrastructure of Trichophyton mentagrophytes were investigated by using scanning and transmission electron microscopy. Structural changes, such as pit formation and/or depression of the cell surface, and degeneration of intracellular organelles and plasmolysis were observed after treatment with ME1111. Our results suggest that the inhibition of energy production by ME1111 affects the integrity and function of cellular membranes, leading to fungal cell death., (Copyright © 2016 American Society for Microbiology.)

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., (© 2016 Blackwell Verlag GmbH.)

Pedicures are the most common cosmetic foot treatment. Many pedicurists and podiatrists suffer from respiratory infections and diseases such as asthma, sinusitis, chronic cough and bronchitis. Skin and nail dust may play an important role in the development of occupational diseases and the transmission of mycosis to other clients. To examine the presence of dermatophytes in nail and skin dust produced during podiatric treatments of people without typical symptoms of mycosis and to assess the epidemiological hazards of tinea pedis for podiatrists as well as other clients. Seventy-seven samples underwent direct microscopy and culture. The results of direct microscopy were positive in 28/77 samples (36.36%) and doubtful in 3/77 (3.9%). Fungi were cultured from 36/77 samples (46.75%), including 8/77 (10.3%) positive for dermatophytes (Trichophyton rubrum-6 isolates and Trichophyton mentagrophytes-2). Material collected during podiatric treatments is potentially infected by pathogenic fungi; thus, there is a need to protect both workers who perform such treatments, as well as other clients, to prevent the transmission of pathogens in the Salon environment. Exposure to this occupational hazard may increase not only the risk of respiratory infections but also increase asthmatic or allergic reactions to Trichophyton., (© 2016 Blackwell Verlag GmbH.)

Background: Trichophyton rubrum (T.rubrum) caused onychomycosis is the most common nail fungal disease. The common diagnostic methods are direct microscopic examination and fungal culture. In this study we used scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to study the subungual ultrastructural changes in T. rubrum induced onychomycosis., Methods: Six outpatients with onychomycosis were recruited and T.rubrum infection was confirmed by fungal culture. Six toenail samples were collected and prepared for SEM characterization. The cultured fugal colonies were prepared for SEM and TEM characterization., Results: 1) SEM showed significant structural damages and the formation of a thin layer or a single layer of keratinocytes in all infected nail plates. Hyphae (piercing or penetrating keratinocytes layers), arthrospores and local bacterial aggregation were observed on the ventral surface of the nail plates. 2) SEM of the cultured fungal colony showed relatively straight, highly branched hyphae and microconidias; TEM showed branching hyphae that were composed of double-layer cell walls. Hyphae had nucleus, mitochondria, liposomes, lysosomes, scattered rough endoplasmic reticulum, myeloid bodies and aggregated ribosomes. There were high-density particles outside the hyphae., Conclusion: SEM showed a large number of hyphae penetrated the keratinocytes layer, suggesting that T. rubrum can cause severe damage to the stratum corneum. TEM showed the ultrastructural features of T. rubrum-induced infection before treatment.

Introduction: In this study, we evaluated the chemical composition of a commercial sample of essential oil from Eucalyptus smithii R.T. Baker and its antifungal activity against Microsporum canis ATCC 32903, Microsporum gypseum ATCC 14683, Trichophyton mentagrophytes ATCC 9533, T. mentagrophytes ATCC 11480, T. mentagrophytes ATCC 11481, and Trichophyton rubrum CCT 5507., Methods: Morphological changes in these fungi after treatment with the oil were determined by scanning electron microscopy (SEM). The antifungal activity of the oil was determined on the basis of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values., Results: The compound 1,8-cineole was found to be the predominant component (72.2%) of the essential oil. The MIC values of the oil ranged from 62.5μg·mL-1 to >1,000μg·mL-1, and the MFC values of the oil ranged from 125μg·mL-1 to >1,000μg·mL-1. SEM analysis showed physical damage and morphological alterations in the fungi exposed to this oil., Conclusions: We demonstrated the potential of Eucalyptus smithii essential oil as a natural therapeutic agent for the treatment of dermatophytosis.

Scales from lesional skin of 12 patients with tinea pedis were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to gain an insight into the spatial and morphological changes of dermatophytes after application of a clinical dosage of topical luliconazole 1% cream (Lulicon® cream 1%). In all cases, Trichophyton rubrum was identified. The scales from the lesions collected before and after topical luliconazole application were fixed with glutaraldehyde and subjected to SEM and TEM. For SEM, fixed specimens were first placed in 1N-KOH and then post-fixed and observed. SEM showed a swollen appearance of fungal hyphae as an early change, and then shrinkage of them showing a flattened and twisted appearance as a later change. TEM showed cell wall alterations with initial development of and accumulation of a granular structure in the outermost layer and subsequent amorphous and electron-lucent change of the thickened inner part of the cell wall. This is the first report of dramatic morphological changes of T. rubrum before and after topical luliconazole application in vivo demonstrated by SEM and TEM. We hypothesize that luliconazole has double acting points, on the plasma membrane and cell wall, of dermatophyte hyphae., (© The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis.

Superficial mycoses caused by dermatophytic fungi such as Trichophyton rubrum represent the most common type of worldwide human infection affecting various keratinized tissues in our body such as the skin, hair, and nails, etc. The dermatophytic infection is a significant public health threat due to its persistent nature and high recurrence rates, and thus alternative treatments to cure this fungal infection are urgently required. The present study mainly focused on the synergistic activity of violacein with four azole drugs (ketoconazole, fluconazole, clotrimazole, and itraconazole) against T. rubrum. The synergistic antifungal activities of violacein and azoles were measured by checkerboard microdilution and time-kill assays. In our study, combinations of violacein and azoles predominantly recorded synergistic effect (FIC index < 0.5). Significant synergistic value was recorded by the combination of violacein and clotrimazole. Time-kill assay by the combination of MIC concentration of violacein and azoles recorded that the growth of the T. rubrum was significantly arrested after 4-12 h of treatment. The combination of violacein and azoles leads to the enhanced inhibition of mycelial growth and conidial germination. Moreover combination enhanced the rate of release of intracellular materials. Morphological changes by SEM analysis were also prominent with the combination. A normal human cell line [Foreskin (FS) normal fibroblast] was used to check the cytotoxicity of violacein. Interestingly violacein recorded no cytotoxicity up to 100 μg/ml. The in vitro synergistic effect of violacein and azoles against clinically relevant fungi, T. rubrum, is reported here for the first time. Finally, our findings support the potential use of the violacein as an antifungal agent especially against dermatophytic fungi T. rubrum.

A 54-year-old Japanese man without apparent immunosuppression presented with nodules with purulent drainage on the right lower leg. He had ringworm of the right leg and tinea unguium. A biopsy specimen of the nodule showed intradermal abscesses with fungal elements, and Trichophyton rubrum was cultured from both the pus and the biopsy specimen. Treatment with oral terbinafine resolved the nodules. Dermatophyte abscess is a rare, deep and invasive dermatophytosis, which is often associated with immunocompromised conditions. We provide a review of the literature including Japanese cases., (© 2015 Blackwell Verlag GmbH.)

Fennel seed essential oil (FSEO) is a plant-derived natural therapeutic against dermatophytes. In this study, the antifungal effects of FSEO were investigated from varied aspects, such as MIC and minimum fungicidal concentration, mycelia growth, spore germination and biomass. The results indicated that FSEO had potent antifungal activities on Trichophyton rubrum ATCC 40051, Trichophyton tonsurans 10-0400, Microsporum gypseum 44693-1 and Trichophyton mentagrophytes 10-0060, which is better than the commonly used antifungal agents fluconazole and amphotericin B. Flow cytometry and transmission electron microscopy experiments suggested that the antifungal mechanism of FSEO was to damage the plasma membrane and intracellular organelles. Further study revealed that it could also inhibit the mitochondrial enzyme activities, such as succinate dehydrogenase, malate dehydrogenase and ATPase. With better antifungal activity than the commonly used antifungal agents and less possibility of inducing drug resistance, FSEO could be used as a potential antidermatophytic agent., (© 2015 The Authors.)

K101 Nail Solution (trademarks Emtrix(®), Nalox(™), Naloc(™)) is a combination of propylene glycol, urea and lactic acid in a topical formulation for the treatment of nails affected by onychomycosis. The aim of this study was to investigate the Minimal Cidal Concentration (MCC) of K101 Nail Solution against Trichophyton rubrum and Candida albicans as well as the effect of K101 Nail Solution on the micromorphology of these fungi. The MCC of K101 Nail Solution against T. rubrum and C. albicans was 50% after 60-min exposure time. A MCC of 50% for K101 Nail Solution means that K101 Nail Solution diluted with e.g. water to 50% will totally kill the fungi tested. In the scanning electron microscope C. albicans cells, treated with 50% K101 Nail Solution, showed a shrunken surface. T. rubrum cells were severely damaged shown as collapse and degradation of the cells. In the transmission electron microscope most C. albicans cells, treated with 50% K101 Nail Solution exhibited destroyed organelles and many necrotic cells were found. The cell wall was clearly degraded and the contact between the cell wall and the inner membrane was punctured. In T. rubrum most cells were necrotic. Some cells were clearly collapsed and the content in the cytoplasm was degraded shown as small membrane vesicles and many big vacuoles. The cell wall was clearly degraded and the membrane was punctured. In conclusion, this in vitro study documents the efficacy of K101 Nail Solution against T. rubrum and C. albicans., (© 2014 Blackwell Verlag GmbH.)

Trichophyton violaceum is a pathogen of tinea capitis and usually cause infection of scalp and hair in children. To investigate the parasitic form of T. violaceum in the human hair tissue, the infected hair strands were collected from a 9-year-old boy with tinea capitis due to T. violaceum and observed under both the scanning electron microscope (SEM) and transmission electron microscope (TEM). The SEM and TEM findings revealed that T. violaceum parasitically lives in the hair shaft in various forms and the morphological transformation of the fungus from hyphae into arthrospores was noted. The involved hair shaft was damaged to the great extent and its ultrastructural changes were evident. Those morphological characteristics of T. violaceum and the three-dimensional ultastructure changes of infected hairs give a better knowledge about the host-fungus relationship in tinea capitis., (© 2014 Wiley Periodicals, Inc.)

Bovine ringworm caused by Trichophyton verrucosum is a notifiable disease in Norway. New infected herds are reported to the Norwegian Food Safety Authority. To limit spread of the disease, restrictions are imposed on holdings including access to common pastures and sale of live animals. Bovine ringworm has been endemic in the Norwegian dairy population for decades. Since 1980 a vaccine (Bovilis Ringvac LTF-130, Merck Animal Health) has been available. The vaccine contains an attenuated strain of T. verrucosum and stimulates humoral and cellular immune responses conferring protection. Efficacy and safety of the vaccine have been evaluated in experimental and field studies. Vaccination campaigns in densely populated counties have contributed to a substantial decrease in number of ringworm outbreaks. The annual incidence of new infected herds decreased from 1.7% in 1980 to 0.043% in 2004. Few herds remained with restrictions and a "mopping up" project was established to offer assistance specifically to these holdings. A milestone was achieved in 2009; no new herds with cases of clinical ringworm caused by T. verrucosum were reported to the authorities. By end of 2012, there are only two herds with restrictions. Vaccination during the last 30 years has been a key control measure in the effort to prevent disease outbreaks and eradicate bovine ringworm in Norway., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trichophyton tonsurans is known to be the causative agent of a worldwide epidemic of dermatophytoses among contact sports practitioners, and is spreading among the general population of Japan. Prompt and simple identification of T. tonsurans in diagnostic laboratories is crucial to control infection. The present study evaluated the availability of observation of chlamydospore-like structures grown in culture media as a characteristic for identification of T. tonsurans. Twenty-five strains of T. tonsurans and five strains each of Trichophyton verrucosum, Trichophyton rubrum and Trichophyton mentagrophytes were inoculated on Mycosel agar plates and inoculated Petri dishes were observed by light microscopy from the reverse side. Twenty-three of 25 T. tonsurans strains showed chlamydospore-like structures within 5 days, and all strains at day 8. The numbers of chlamydospore-like structures were very abundant in most strains. The majority of strains of other species showed no chlamydospore-like structures, or very few when present. Positive for chlamydospore-like structures among 15 strains other than T. tonsurans was one strain at day 5 and six strains at day 8. As for the identification of T. tonsurans, presence of chlamydospore-like structures showed 92.0% sensitivity (23/25) and 93.3% specificity (14/15) at day 5, and 100% sensitivity (25/25) and 60.0% specificity (9/15) at day 8. Electron microscopic findings suggest chlamydospore-like structures are not true chlamydospores but are produced by inflation of actively growing hyphae by developing vacuoles in cells. In conclusion, observation of development of chlamydospore-like structures in culture media is the simplest method for identification of T. tonsurans., (© 2013 Japanese Dermatological Association.)

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans. Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

Background: The current trend of keeping "exotic" pets has led to the emergence of new types of fungal species that may be transmitted to humans [1]. We describe a form of dermatophytosis transmitted by a Guinea pig and caused by a new variety of dermatophyte., Case Report: A 13-year-old girl developed multiple erythematosquamous and vesicular lesions with a highly inflammatory edge several weeks after acquiring a Guinea pig of apparently healthy appearance. Direct examination and culture tests demonstrated the presence of a dermatophyte closely related to the erinacei variant of Trichophyton mentagrophytes, from which it differed in terms of microscopic and macroscopic characteristics. The condition resolved on therapy with topical imidazole., Discussion: This new type of dermatophyte has been identified in many patients coming into close contact with Guinea pigs in the region of Nancy. We would suggest the emergence of a novel variety of T. mentagrophytes, which has adapted to its new host following transmission to Guineas pigs from hedgehogs. We propose that it be named T. mentagrophytes var. porcellae., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Trichophyton rubrum (T. rubrum) is the most common causative agent of dermatophytosis worldwide. In this study, we examined the effect of laser irradiation on the growth and morphology of T. rubrum., Methods: Colonies of T. rubrum were isolated (one colony per plate), and randomly assigned to 5 treatment groups: Q-switched 694 nm ruby laser treatment, long-pulsed Nd:YAG 1064 nm laser treatment, intense pulsed light (IPL) treatment, 308 nm excimer laser treatment and the blank control group without treatment. Standardized photographs were obtained from grown-up fungal plates prior to treatment. Colonies were then exposed to various wavelengths and fluences of laser light. To compare the growth of colonies, they were re-photographed under identical conditions three and six days post-treatment. To investigate the morphology of T. rubrum, scanning electron microscope (SEM) and transmission electron microscope (TEM) images were obtained from specimens exposed to 24 hours of laser treatment., Results: Growth of T. rubrum colonies was significantly inhibited following irradiation by 694 nm Q-switched and 1064 nm long-pulsed Nd:YAG lasers. Other treatments exerted little or no effect. Q-switched laser irradiation exerted a stronger growth inhibitory effect than long-pulsed Nd:YAG laser irradiation. Following treatment by the Q-switched ruby laser system, T. rubrum hyphae became shrunken and deflated, and SEM images revealed rough, fractured hyphal surfaces, punctured with small destructive holes. TEM images showed that the hyphae were degenerating, as evidenced by the irregular shape of hyphae, rough and loose cell wall, and obscure cytoplasmic texture. Initially high electron density structure was visible in the cell; later, low-density structure appeared as a result of cytoplasmic dissolution. In contrast, the blank control group showed no obvious changes in morphology., Conclusion: The Q-switched 694 nm ruby laser treatment significantly inhibits the growth and changes the morphology of T. rubrum.

TDT 067 is a novel, carrier-based dosage form of terbinafine in Transfersome (1.5%) formulated for topical delivery of terbinafine to the nail, nail bed, and surrounding tissue. We examined the effects of TDT 067 and conventional terbinafine on the morphology of dermatophytes. Trichophyton rubrum hyphae were exposed to TDT 067 or terbinafine (15 mg/ml) and examined under white light, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Subungual debris from patients treated with TDT 067 in a clinical trial was also examined. Exposure of T. rubrum hyphae to TDT 067 led to rapid and extensive ultrastructural changes. Hyphal distortion was evident as early as 4 h after exposure to TDT 067. After 24 h, there was complete disruption of hyphal structure with few intact hyphae remaining. Exposure to terbinafine resulted in morphological alterations similar to those seen with TDT 067; however, the effects of TDT 067 were more extensive, whereas a portion of hyphae remained intact after 24 h of exposure to terbinafine. Lipid droplets were observed under TEM following 30 min of exposure to TDT 067, which after 24 h had filled the intracellular space. These effects were confirmed in vivo in subungual debris from patients with onychomycosis who received topical treatment with TDT 067. The Transfersome in TDT 067 may potentiate the action of terbinafine by delivering terbinafine more effectively to its site of action inside the fungus. Our in vivo data confirm that TDT 067 can enter fungus in the nail bed of patients with onychomycosis and exert its antifungal effects.

Electron microscopic techniques have been widely used to investigate the pathogenesis of dermatophytosis. In this article, morphological studies by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) concerned with the dermatophyte infection process are literary reviewed and we introduced our morphological study observing the parasitic form of dermatophytes in the lesional cornified layer. Various experimental models have been established to study fungal adhesion and invasion to the skin surface in the early stage of infection by several authors.In these studies, arthroconidial adherence to corneocytes and germination was demonstrated by SEM and TEM. To understand the host-fungus relationship in dermatophytosis, it is also important to identify parasitic forms of fungi and morphological changes of corneocytes in the lesion. The three-dimensional structure of growing or parasitic dermatophytes in the lesional cornified cell layer was observed using the adhesive strip method in tinea cruris and tinea glabrosa. In tinea unguium, however, it is difficult to apply this method because of subungual hyperkeratosis. We introduce the alkaline treatment method for SEM for use in observing fungal elements in a subungual hyperkeratotic lesion and scales in dermatophytosis. With this technique, small pieces of nails or scales were fixed with 2.5% glutaraldehyde, and were treated with low concentrations of alkaline and subjected to the conventional procedure for SEM observation. This method is applicable to various skin lesions, such as tinea unguium, tinea pedis and tinea capitis to investigate the parasitic forms of dermatophytes and the spatial relationship with corneocytes and is useful to understand the infection process of dermatophytosis.

Worldwide, dermatophytoses represent a high percentage of all superficial mycoses. The most frequently isolated dermatophyte is Trichophyton rubrum. Solanum chrysotrichum is a vegetal species widely used in Mexican traditional medicine to treat skin infections; its extract has been used to formulate an herbal medicinal product that is used successfully to treat Tinea pedis. Spirostanic saponin SC-2 from S. Chrysotrichum possesses high activity against dermatophytes. The present study reports the ultrastructural changes observed by means of transmission electron microscopy (TEM) in clinical isolates of T. rubrum, T. mentagrophytes, and Microsporum gypseum induced by saponin SC-2. Strains were grown in RPMI 1640 containing SC-2 (1600 microg/mL). Fungi were harvested at 6, 12, 24, and 48 h; controls without SC-2 were included. T. mentagrophytes was the most susceptible to the SC-2 saponin, followed by M. gypseum, while T. rubrum was the most resistant. The main alterations caused by the SC-2 saponin were as follows: i) loss of cytoplasmic membrane continuity; ii) organelle degradation; iii) to a lesser extent, irreversible damage to the fungal wall; and iv) cellular death., (Georg Thieme Verlag KG Stuttgart, New York.)

The antifungal effects of citral, eugenol, nerolidol and alpha-terpineol on Trichophyton mentagrophytes were investigated. Citral over 0.1 mg/ml strongly inhibited the hyphal growth of T. mentagrophytes, and the antifungal activity of alpha-terpineol was less effective. The morphological changes of the fungus exposed to the terpenes were observed by electron microscopy. The hyphae were distorted and collapsed at 0.2, 0.4 and 1 mg/ml of eugenol, nerolidol and alpha-terpineol respectively, and cell membrane and organelles were irreversibly damaged at 0.2 mg/ml citral. These suggested that four terpenes possess antifungal activity against T. mentagrophytes, and the activity might lead to irreversible cellular disruption.

Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.

We describe simple and cost-effective methods using carbohydrates to improve the in vitro viability of dermatophytes. Glucose and sucrose in different concentrations (3, 6, 9 and 12%) were used to maintain fifteen strains of M. canis and T. mentagrophytes var. mentagrophytes at 4 and -20 degrees C. The strains were phenotypically analyzed before storage and reevaluated at 1, 3, 6 and 9 months. At 1 and 3 months, any alterations in the viability or phenotype pattern of the stored strains were noted. At 6 months, both dermatophytes were 100% viable, when preserved in glucose (3, 6, 9 and 12%) at -20 degrees C. All T. mentagrophytes strains were also viable in sucrose (12%), at 4 degrees C and -20 degrees C. However, sucrose failed to improve the viability of M. canis at both temperatures. At 9 months, the higher viabilities without pleomorphism were seen for both dermatophytes preserved in glucose (9 and 12%) at -20 degrees C.

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.

The antifungal activity of onion (Allium cepa L.) on two important dermatophytes, Trichophyton rubrum and Trichophyton mentagrophytes, with special reference to morphological aspects was studied. Growth of both fungi was found to be strongly inhibited by aqueous onion extract (AOE) as a dose-dependent manner. The extract showed fungicidal effect for both fungi at concentrations >3.12% (v/v). The fungus T. mentagrophytes was more affected by the onion as compared to T. rubrum at all concentrations used. Morphological effects of onion exposure were examined in correlation with fungal growth. Corresponding to the growth inhibition, light and electron microscopy observations revealed morphological anomalies in hyphal compartments. The results demonstrated that AOE targets the cell membrane of the fungi as breaking down of both inner and outer membranes with consequent extrution of materials into the surrounding medium. Cytoplasmic membranes and other membranous structures of organelles, such as nuclei and mitochondria, were also disrupted. In correlation to the fungal growth, morphological alterations occurred to a less content for T. rubrum compared with T. mentagrophytes. The hyphae of T. rubrum were found to be mainly affected by converting to resistant forms, i.e., chlamidospores as a consequence of phenotype switching response to AOE. Plasmolysis accompanied by an almost complete depletion and disorganization of cytoplasmic structures were found to be the final event which led to cell death. Ultrastructural evidences obtained from this study strongly support that morphological changes of T. rubrum and T. mentagrophytes caused by AOE are associated with its fungistatic and fungicidal activities. With respect to the morphological results and the preliminary data on fungal biochemistry, a mechanism of action by interacting of AOE with thiol (-SH) groups present in essential compartments of the fungal cells was postulated.

Onychomycosis, or tinea unguium, is generically thought of as a fungus infection of the nails characterized by thickening, splitting, roughening, and discoloration. This article discusses the origins of this condition as well as methods of classification and evaluation.

Sequence analysis of the highly variable internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) of the nuclear DNA has been used to explore the phylogeny of the dermatophytes. Results have led some investigators to recommend that a number of dermatophyte species be reduced to synonymy with other established species. One such recommendation is that both varieties of the zoophilic dermatophyte Trichophyton equinum be reduced to synonymy with the anthropophilic species T. tonsurans. The morphologies of both species are reviewed, as are their roles in human infection, their physiological characteristics and their respective ecologies. Close examination of these attributes shows clear differences between the varieties of T. equinum and T. tonsurans. The significance of the homogeneity of the ITS sequences of these two dermatophytes is discussed in the context of more recent appraisals of the results of ITS sequencing in other fungi. It is concluded that the results of ITS analysis for both varieties of T. equinum and T. tonsurans are indicative only of common ancestry. Similar arguments could be put forward for rescinding other changes in dermatophyte nomenclature that have been published in recent years.

Trichophyton raubitschekii is currently regarded as a synonymous name betokening a variant form of Trichophyton rubrum. Nonetheless, isolates conforming to this morphotaxonomic concept have morphological, physiological and clinical features very different from those of typical T. rubrum. Isolates are mainly obtained from subjects originating from certain tropical and subtropical countries, and are mainly obtained from upper body skin infections, rarely from onychomycosis. In this paper the authors report the first known Italian case of onychomycosis caused by such an isolate. The patient, a male student from Cameroon, had a typical fingernail tinea unguium, without any other sign of skin or nail infection. 'T. raubitschekii' was identified on morphological and physiological grounds by the following features: velvety colony surface, brownish pigment, abundant macroconidia and microconidia, and positive urease activity. Such isolates may prove very difficult to identify correctly, especially in areas like Italy where T. rubrum is normally seen only as isolates presenting a strongly differing phenotype.

In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

Aim: A novel ex vivo model for the study of adherence and invasion of dermatophytes to the stratum corneum was developed., Materials and Methods: A skin of full epidermis thickness was infected by spores of Trichophyton mentagrophytes and examined after various periods of time by scanning and transmission electron microscopy., Results: After 12 h of inoculation a tenacious adherence between the spores and the stratum corneum was observed. There was a time dependent increase in the number of spores adhered to this surface. By 24 h, germination had commenced. The initial growth of germ tubes occurred extracellularly to the corneocytes. Three days after inoculation, the most prominent feature was proliferation of fungal hyphae and penetration of mycelium through the outer keratinocyte layer which is followed by invasion of the outer stratum corneum., Conclusion: The model introduced in the present study may contribute to a better understanding of the nature of the interaction between dermatophytes and skin cells in dermatophytosis process.

Background: The spectrum of organisms causing dermatophytoses is changing continuously., Patients/methods: Two strains of Trichophyton mentagrophytes var. nodulare are characterized mycologically; both were isolated for the first time in our laboratory within the past 2 years from patients with tinea pedis., Results: Distinctive features of Trichophyton mentagrophytes var. nodulare are a bright yellow-orange pigmentation of the thallus, a marked exudation of pigment, and the development of nodular bodies. Trichophyton mentagrophytes var. nodulare is an anthropophilic variety of Trichophyton mentagrophytes causing tinea and onychomycosis., Conclusions: Trichophyton mentagrophytes var. nodulare has been found only very rarely in Germany and is relatively unknown. An increased awareness of this dermatophyte is needed in order to track its possible spread.

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.

A microbiological and ultrastructural study of Trichophyton raubitschekii recovered from a patient with tinea faciei is presented. This is the second case of isolation of this fungus in Sao Paulo and the sixth case in Brazil. Upon culture, the morphological pattern and the physiological tests performed confirmed the identification of T. raubitschekii. The ultrastructural study of T. raubitschekii showed the presence of a membrane-like structure located in the outer portion of the hyphal walls. This structure was bi-stratified and very like the one observed in T. rubrum. Is T. raubitschekii a new fungus emerging in Brazil or is it a T. rubrum variant?

The antifungal activity of five new synthetic compounds was evaluated on two dermatophytes: Epidermophyton floccosum and Trichophyton rubrum. The data showed that the imidazo-pyrazole and pyrazolo-thiazoles were not particularly effective, while the two pyrazole-thiocyanates proved highly active on both fungi. The most active 5-amino-3-methyl-1-phenylpyrazolo-4-thiocyanate was chosen to perform SEM and TEM morphological studies on both fungi. Both SEM and TEM observations revealed interesting alterations on the two dermatophytes, particularly involving the endomembrane system.

The characteristic macro- and microconidia produced by dermatophytes in cultures and those living as saprobes are not produced in any dermatophyte lesions observed to date. However, arthroconidia, defined as a genetically programmed disarticulation of septate hyphae to produce a chain of conidia, are produced by dermatophytes, both in culture and in lesions, under certain predisposing conditions. Arthroconidia are seen, for example, within nail-bed infections caused by Trichophyton rubrum; within nail-plate infections caused by Trichophyton mentagrophytes interdigitale; and in the infections of scalp hair by soil or geophilic dermatophytes, where chains of arthroconidia form near or on the surface of the infected hairs. Dr. Rashid discusses his electron microscope studies of the adherence and germination of arthroconidia and the penetration of the germ tubes and initial hyphae into the stratum corneum.

Using terbinafine and clotrimazole as examples, Dr. Rashid's electron microscopy images illustrate the differences between fungicidal and fungistatic drugs, respectively.

A considerable volume of research has now been completed on the application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the analysis of bacteria; however, to date no definitive studies have been made using this technique on fungi. Preliminary studies on the application of the MALDI-MS methodology, previously developed for the analysis of bacteria, to the analysis of intact fungal spores are described here. MALDI-MS and electrospray mass spectrometry enable the high molecular weight analysis of proteins, glycoproteins, oligosaccharides and oligonucleotides. Using MALDI-MS with bacteria has demonstrated the ability to produce 'fingerprints' of the intact cells with the ions observed being associated with the proteinaceous components of the cell wall. This paper reports the adaptation of this technique to the direct analysis of fungal cells. The high percentage of carbohydrate in the fungal cell wall indicates that the ions observed in the mass spectrometric experiments may be of carbohydrate origin. Penicillium spp., Scytalidium dimidiatum and Trichophyton rubrum have been studied in this preliminary investigation and all show individually distinctive spectra which would appear to provide a profile of the cellular material with discrete peaks being observed over the mass range 2 to 13 kDa. The spectra obtained are reproducible within the method used but, as shown in our previous studies on bacteria, washing may selectively release components from the fungal cell wall., (Copyright 2000 John Wiley & Sons, Ltd.)

The effects of the newly synthesized triazole, 1-amino-6-methyl-4-phenylpyrazolo[3,4-d]-1,2,3-triazole (V5), on the dermatophyte Trichophyton rubrum were tested. Optical and electron microscopy showed that the treatment suppressed the various forms of saprophytic conidia, induced the formation of chlamydospores and accelerated the formation of arthroconidia. This new triazole did not reveal any fungistatic or fungicidal activity and could not be considered as an antifungal substance.

The antifungal activity of 4-amino-3-methyl-1-phenylpyrazolo-(3,4-c)isothiazole was studied on Trichophyton rubrum. The compound, at concentrations between 20 and 10 microg ml(-1), induces a remarkable reduction in the growth and causes deep morphogenetic anomalies. The ultrastructural modifications have demonstrated that the compound targets the cell membrane of the fungus, breaking down not only the endomembrane system, but also the 'outer' membrane, with consequent extrusion of materials in the medium. The results suggests a mechanism of action similar to other azoles clinically utilized.

There is no reference to the cell wall formation during the regeneration process in dermatophyte. Having succeeded in identifying the cell wall formation system of protoplast from Trichophyton mentagrophytes I introduce here the dynamics of the regeneration of this protoplast and the formation of cell wall using ultrahigh resolution low-voltage scanning electron microscopy and transmission electron microscopy (TEM), compare to the formation of yeast cell wall. Protoplasts were formed from mycelia of T. mentagrophytes; they regenerated the cell wall substances and were followed by a network of fibrils after 4-6 hr, which developed into mature mycelia after 21 hr. A network of twisted microfibrils and rope-shaped thick fiber was seen; it was covered with a sheath-shaped structure and was created on the surface of the developing mycelium. Intrafibrillar space and the surface of thick fibers were gradually filled with amorphous particles and a mature cell wall surface was formed. This protoplast regeneration was completely inhibited by the antifungal agent, lanoconazole (2 ng/ml ). The sectioned TEM image revealed that normal mycelium having a thin cell wall developed from the abortive bead-shaped mycelium which was visible in the lose fibrous structure. Based on this result and our previos work on the ultrastructure of the mycelial surface of T. rubrum with lanoconazole, a model of the cell wall composition of Trichophyton is proposed.

Onychomycosis is primarily caused by dermatophyte fungi but occasionally by yeasts and non-dermatophytic moulds. The aim of this study was to develop an in vitro model of nail invasion by dermatophytes, yeasts and non-dermatophytic moulds, and to provide an alternative system for studying the activity of different classes of antifungal drugs against fungi associated with onychomycosis. In the absence of extraneous nutrients, Trichophyton mentagrophytes was seen in electron microscopy to degrade completely healthy nail plate. Candida albicans germinated on nail fragments, but invasion of the nail plate was not seen. The mould Fusarium formed long channels through the matrix of the nail plate. Aspergillus versicolor appeared to penetrate the outer and intermediate surface of the nail plate only. Acremonium sp. and Scopulariopsis brevicaulis did not invade nail in this model. Exposure of nail fragments to terbinafine (0.25 mg/l for 3 h) inhibited invasion by T. mentagrophytes, C. albicans and the non-dermatophytic moulds. Itraconazole (0.25 mg/l for 3 h) prevented nail plate invasion by T. mentagrophytes, A. versicolor and Fusarium but did not totally inhibit the surface growth of Acremonium or S. brevicaulis. C. albicans grew in the presence of itraconazole. The results indicate that terbinafine is readily absorbed by the nail and that the drug is bio-available in nail keratin. A short exposure of nail to low concentrations of terbinafine acted as a barrier against fungal invasion. Itraconazole appeared to be effective against Trichophyton and some non-dermatophytic moulds.