Dos interesantes contaminantes fúngicos filamentosos en muestras clínicas positivas a Trychophyton rubrum: una situación de curiosidad morfológica / Two interesting filamentous fungal contaminants in clinical samples positive to Trychophyton rubrum: a situation of morphological curiosity

Las dermatofitosis corresponden a un grupo de enfermedades micóticas comunes en piel y fanéreas, donde Trichophyton rubrum es el agente causante más frecuente a nivel mundial y presente en nuestros 2 casos de pacientes masculinos con estas micosis, una en uñas y la otra en piel. Sin embargo, el enfoque de esta publicación se basa principalmente en la presencia de 2 interesantes contaminantes (uno en cada caso clínico) presentes solo en los cultivos de las primeras siembras como saprófitos y por ende como propágulos de dispersión, asociados al ambiente y sin intervención clínica demostrada en ambas micosis. La descripción morfofisiológica de estos 2 contaminantes Metarhizium purpureo-genum(similis) y Monascus ruber fue más bien una curiosidad esencial que el micólogo clínico adquiere en su contínua formación y ante la posibilidad de infecciones mixtas, pudiendo conjugar sus hallazgos junto al análisis taxonómico y los factores geográficos y edáficos asociados a su distribución. (AU)

Dermatophytoses belongs to a group of common mycotic diseases in skin and pharynals, where Trichophyton rubrum is the most frequent causative agent worldwide and present in our 2 cases of male patients with these mycoses, one in nails and the other in skin. However, the focus of this publication is mainly about the presence of 2 interesting contaminants (one in each clinical case) present only in the crops of the first sowings as saprophytes and therefore as dispersal propagules, associated with the environment and without clinical intervention demonstrated in both mycoses. The morphophysiological description of these 2 contaminants, Metarhizium purpureogenum (similis) and Monascus ruber was rather an essential curiosity that the clinical mycologist acquires in his continuous training and in the face of the possibility of mixed infections, being able to combine his findings together with the taxonomic analysis and the geographic and edaphic factors associated with its distribution. (AU)

Visualising virulence factors: Trichophyton benhamiaes subtilisins demonstrated in a guinea pig skin ex vivo model.

BACKGROUND: Dermatophytoses rank among the most frequent communicable diseases in humans, and the zoonotic transmission is increasing. The zoophilic dermatophyte Trichophyton (T.) benhamiae is nowadays one of the main causes of tinea faciei et corporis in children. However, scientific data on molecular pathomechanisms and specific virulence factors enabling this ubiquitous occurrence are scarce. OBJECTIVES: To study tissue invasion and the expression of important virulence factors of T. benhamiae, isolates that were recovered from two groups of hosts (humans vs. guinea pigs (GP)) using an ex vivo skin model. METHODS: After confirmation of species identity by ITS sequencing, CFU suspensions of dermatophyte isolates (n = 20) were applied to the skin infection model and cultured. Employing specific immunofluorescence staining techniques, the expression of subtilisin 3 and 6 and metallocarboxypeptidase A was analysed. The general mode of invasion was explored. Results were compared with biopsies of naturally infected GP. RESULTS: All isolates were successfully recovered and proliferated well after application to the infection model. Progressive invasion of hyphae through all skin structures and destruction of explants were observed with early events being comparable to natural infection. An increasing expression of the examined virulence factors towards the end of culture was noticed but no difference between the two groups of isolates. CONCLUSIONS: For the first time, important in vivo markers of dermatophytosis were visualised immunohistochemically in an ex vivo skin infection model and in skin biopsies of GP naturally infected with T. benhamiae. More research on the underlying pathomechanisms of dermatophyte infection is urgently needed.

Comparison of in vitro antifungal activity of novel triazoles with available antifungal agents against dermatophyte species caused tinea pedis.

OBJECTIVE: Dermatophytes are a group of keratinophilic fungi that invade and infect the keratinized tissues and cause dermatophytosis. We investigated effectiveness of novel triazole (luliconazole and lanaconazole) in comparison with available antifungal agents against dermatophyte species isolated from patients with tinea pedis. MATERIAL AND METHODS: A total of 60 dermatophytes species were isolated from the patients with tinea pedis. Identification of species was done by DNA sequencing of the ITS1-5.8S rDNA-ITS2 rDNA region. In vitro antifungal susceptibility testing with luliconazole and lanaconazole and available antifungal agent was done in accordance with the Clinical and Laboratory Standards Institute, M38-A2 document. RESULTS: In all investigated isolates, luliconazole had the lowest minimum inhibitory concentration (MIC) (MIC range=0.0005-0.004µg/mL), while fluconazole (MIC range=0.4-64µg/mL) had the highest MICs. Geometric mean MIC was the lowest for luliconazole (0.0008µg/mL), followed by lanoconazole (0.003µg/mL), terbinafine (0.019µg/mL), itraconazole (0.085 µg/mL), ketoconazole (0.089µg/mL), econazole (0.097µg/mL), griseofulvin (0.351 µg/mL), voriconazole (0.583µg/mL) and fluconazole (11.58µg/mL). CONCLUSION: The novel triazoles showed potent activity against dermatophytes and promising candidates for the treatment of tinea pedis caused by Trichophyton and Epidermophyton species. However, further studies are warranted to determine the clinical implications of these investigations.

Ex vivo nail infection as an effective preclinical method for screening of new topical antifungals.

Onychomycosis are fungal nail infections comprising of about 50% of onychopathies and are commonly caused by dermatophytes. The treatment of this dermatomycosis requires a long period of time and is associated with high rates of recurrence. In view of the need to evaluate the antifungal performance of promising preclinical compounds, we developed, in this study, a practical and accessibleex vivo model for establishing a Trichophyton rubrum onychomycosis framework using porcine hooves. This model has as its main advantage the similar structural and three-dimensional characteristics that the porcine hooves have with the human nail. The proposed model allowed to evaluate the antifungal activity of a new antifungal compound and a reference drug (terbinafine), both already incorporated into a nail lacquer for topical use. Treatments with compound 3-selenocyanate-indole (Se4a) and with terbinafine incorporated into this nail lacquer completely inhibited fungal growth, corresponding to the profile of in vitro activity observed against T. rubrum. This study concludes that the ex vivo porcine hoof model is an effective alternative method for preclinical screening of drugs or new topical compounds developed to combat onychomycosis. Further studies are needed to compare the permeability of porcine hooves with human nails permeability.

The Role of Reactive Oxygen Species and Nitric Oxide in the Inhibition of Trichophyton rubrum Growth by HaCaT Cells.

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.

Evaluation of an Explanted Porcine Skin Model to Investigate Infection with the Dermatophyte Trichophyton rubrum.

Dermatophytosis is a fungal infection of skin, hair and nails, and the most frequently found causative agent is Trichophyton rubrum. The disease is very common and often recurring, and it is therefore difficult to eradicate. To develop and test novel treatments, infection models that are representative of the infection process are desirable. Several infection models have been developed, including the use of cultured cells, isolated corneocytes, explanted human skin or reconstituted human epidermis. However, these have various disadvantages, ranging from not being an accurate reflection of the site of infection, as is the case with, for example, cultured cells, to being difficult to scale up or having ethical issues (e.g., explanted human skin). We therefore sought to develop an infection model using explanted porcine skin, which is low cost and ethically neutral. We show that in our model, fungal growth is dependent on the presence of skin, and adherence of conidia is time-dependent with maximum adherence observed after ~ 2 h. Scanning electron microscopy suggested the production of fibril-like material that links conidia to each other and to skin. Prolonged incubation of infected skin leads to luxurious growth and invasion of the dermis, which is not surprising as the skin is not maintained in conditions to keep the tissue alive, and therefore is likely to lack an active immune system that would limit fungal growth. Therefore, the model developed seems useful to study the early stages of infection. Furthermore, we demonstrate that the model can be used to test novel treatment regimens for tinea infections.

Preparation, characterization and antidermatophytic activity of free- and microencapsulated cinnamon essential oil.

Essential oils (EO) are effective natural antimicrobials but are susceptible to oxidation. Microencapsulation improves EO stability, reduces toxicity, and controls release. The aim of this study was preparation, characterization and antidermatophytic activity of free and microencapsulated cinnamon essential oil (MP). MP were prepared by the spray drying method and the success of MP encapsulation was confirmed by UV-vis spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. The antifungal effect of EO and MP was evaluated by the broth microdilution method against Microsporum gypseum and Trichophyton mentagrophytes. The checkerboard method was used to assess synergistic interactions. Fluorescence microscopy and scanning electron microscopy were used to investigate the inhibition of hyphal growth by EO and MP. A cytotoxic assay was performed using the VERO cell line. Microencapsulated cinnamon essential oil was found to be micrometric, with a round, regular structure. The minimum inhibitory concentration of EO was found to be between 125-250µg/mL, while that of MP was 220.5-440.5µg/mL. EO was synergistic with fluconazole while microencapsulated oil was less cytotoxic than EO.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein.

BACKGROUND: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80-90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. RESULTS: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. CONCLUSIONS: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Antioxidant and antifungal activities of marrubiin, extracts and essential oil from Marrubium vulgare L. against pathogenic dermatophyte strains.

OBJECTIVE: Medicinal plants extracts and plant-derived compounds are one of the natural sources for discovering new antifungal agents, the objectives of this work were to investigate for the first time the antidermatophytic, antipathogenic activities of methanol, acetone extracts, and essential oil of Marrubium vulgare L. grown in Tunisia and its active compound marrubiin on pathogenic for animals and humans, such as some dermatophytes and pathogenic for plants, and to evaluate antioxidant activities of different extracts with consideration to their chemical compositions. MATERIAL AND METHODS: Acetone and methanol extracts were evaluated by HPLC, the essential oil was also analyzed by GC/MS. PCL assay was used to determine the antioxidant activity. RESULTS: Results showed that methanol and acetone extracts exhibited a significant antioxidant activity (261.41 and 272.90µmol TE/g respectively), while the lowest one was observed in the case of marrubiin and essential oil. The antifungal activity of different extracts, marrubiin and essential oil at two concentrations (20 and 100µg/mL) were screened against the dermatophytes fungi Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum and against two fungi strains (Botrytis cinerea, Pythium ultimum). Among tested extracts, marrubiin at 100µg/mL showed about 50% inhibition for T. mentagrophytes and E. floccosum. The anti-phytopathogenic activity was also carried out, only marrubiin had in activity against B. cinerea at the highest dose (32.40%), while methanol extract of M.vulgare and marrubiin are able to increase the mycelial growth of P. ultimum at the highest concentration (45.15 and 40.30% respectively). CONCLUSION: In our study, we conclude that M.vulgare and marrubiin can be used as natural antioxidants and antifungal agent for treatment of skin dermatophyte infections.

Linalool modulates dermatophyte susceptibility to azole drugs.

This study investigated the monoterpene linalool and its resistance modulating activity involving ergosterol biosynthesis inhibitors (ketoconazole, fluconazole, and itraconazole) in strains of Microsporum spp. and Trichophyton spp. The minimum inhibitory concentration (MIC) of test-drugs were determined by microdilution. The modulating effect of linalool was evaluated by determining the MIC of the antifungals in the presence of subinhibitory concentrations of linalool. We also investigated the association effect (checkerboard) of linalool together with ketoconazole and itraconazole. The fungi became more sensitive to ketoconazole and itraconazole in the presence of linalool. The linalool and azole drug associations presented synergism.

Genes coding for LysM domains in the dermatophyte Trichophyton rubrum: A transcription analysis.

The filamentous fungus Trichophyton rubrum is a pathogen that causes superficial mycoses in humans, predominantly in keratinized tissues. The occurrence of dermatophytoses has increased in the last decades, mainly in immunocompromised patients, warranting research on the mechanisms involved in dermatophyte virulence. The genomes of dermatophytes are known to be enriched in genes coding for proteins containing the LysM domain, a carbohydrate-binding module, indicating the possible involvement of these genes in virulence. Although the LysM domains have already been described in other fungi, their biological functions in dermatophytes are unknown. Here we assessed the transcription of genes encoding proteins containing the LysM domains in T. rubrum grown on different substrates using quantitative real-time polymerase chain reaction. Some of these genes showed changes in transcription levels when T. rubrum was grown on keratin. In silico analyses suggest that some of these proteins share features, namely, they are anchored in the plasma membrane and contain the catalytic domain chitinase II and signal peptide domains. Here we show a detailed study of genes encoding the proteins with LysM-containing domains in T. rubrum, aiming to contribute to the understanding of their functions in dermatophytes.

Clinical retrospective analysis of long-pulsed 1064-nm Nd:YAG laser in the treatment of onychomycosis and its effect on the ultrastructure of fungus pathogen.

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/cm2. When irradiated with cumulative laser energy ≥ 25600 J/cm2, 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.

Simultaneous dermatophytosis and keratomycosis caused by Trichophyton interdigitale infection: a case report and literature review.

BACKGROUND: Dermatophytosis is a fungal infectious disease caused by dermatophytes, which produce protease and keratinase to digest keratin, leading to the colonization, invasion, and infection of the stratum corneum of the skin, hair shafts, and nails. Trichophyton interdigitale belongs to Trichophyton mentagrophytes complex, which is the common pathogen causing dermatophytosis. Fungal keratitis, also called keratomycosis, is an infectious disease of cornea. CASE PRESENTATION: Here, we report a case of simultaneous dermatophytosis and keratomycosis caused by Trichophyton interdigitale. A 67-year-old man presented with extensive erythema all over the body since 4 years ago, fungal infection of left eye for 2 years, and loss of vision in the eye. These symptoms had become aggravated in the last month. Dermatological examinations showed extensive erythematous plaques with clear borders and scales, scattered red papules with ulceration, and scabs throughout the body. Onychomycosis was observed on the nails of left hand, conjunctival infection with secretion and loss of vision were noted in left eye. Hyaline septate hyphae were observed under direct microscopic examination, fungal culture and internal transcribed spacer sequencing revealed T. interdigitale. Histopathological examination suggested infectious granuloma. A diagnosis of dermatophytosis and keratomycosis caused by T. interdigitale with loss of vision in left eye was made. The patient was treated with luliconazole cream (two applications per day) and itraconazole (100 mg, BID, PO). Complete clinical remission was achieved after 1 month. Subsequently, the patient underwent left eye enucleation in the ophthalmology department. CONCLUSIONS: In the present study, we reported a case of simultaneous dermatophytosis and keratomycosis caused by T. interdigitale, and reviewed the literature on corneal infection caused by Trichophyton. A total of 10 articles with 45 patients were published between 1973 and 2018. The pathogen of 27 patient were identified to species level. There were T. schoenleinii (17), T. mentagrophytes (4), T. verrucosum (3), T. rubrum (1), T. erinacei (1), and T. interdigitale (1). Five patients had corneal trauma, one had contact lens use history. Direct microscopic examination, fungal culture, and analysis of physiological characteristics were the main methods of identification. Early diagnosis and prompt treatment may help improve the management and outcomes.

A microtiter plate-based quantitative method to monitor the growth rate of dermatophytes and test antifungal activity.

Dermatophytosis is one of the most common superficial fungal infections, which is mainly caused by filamentous fungi such as Trichophyton species. A challenging aspect in dermatophyte research is the lack of a straightforward method to measure the rate of growth, in particular when growing dermatophytes in small volumes such as in microtitre plates. However, one characteristic of dermatophytes is their ability to produce compounds such as ammonia that make the growth medium more alkaline. The objective of this study was to test whether the change in pH in a liquid medium, colourimetrically established using the indicator phenol red, was linearly and directly proportional to the growth rate for Trichophyton rubrum and Trichophyton interdigitale. The changes in the colour determined by the phenol-red based assay showed a good correlation with the amount of fungal biomass over an incubation period of 24-120 h. The functionality of the phenol red assay was also validated in experiments on the growth of T. rubrum in the presence of antifungals. The changes in colour showed a clear dose-response relationship compounds and enabled determination of the minimum inhibitory concentration. The phenol red assay is thus a simple and straightforward assay to monitor the rate of growth of Trichophyton spp. and test antifungal activity.

Biomass-Derived Dialdehyde Cellulose Cross-linked Chitosan-Based Nanocomposite Hydrogel with Phytosynthesized Zinc Oxide Nanoparticles for Enhanced Curcumin Delivery and Bioactivity.

A sustainable biomass-based nanocomposite hydrogel was formulated, characterized, and applied for curcumin delivery. Phytosynthesized zinc oxide nanoparticles (ZnO NPs) employing musk melon (Cucumis melo) seed extract was embedded in the hydrogel matrices and cross-linked using Dialdehyde cellulose prepared from sugarcane (Saccharum officinarum) bagasse (SCB). Nanoparticle incorporation enhanced the hydrogel's swelling degree to 4048% at pH 4.0. Also, an improved tensile strength of 14.1 ± 0.32 MPa was exhibited by the nanocomposite hydrogel compared to 9.79 ± 0.76 MPa for the pure chitosan cellulose hydrogel. A curcumin loading efficiency of 89.68% with around 30% increased loading was exhibited for the nanocomposite hydrogel. A Fickian diffusion-controlled curcumin release mechanism with maximum release at pH 7.4 was obtained. The synergistic effect on the antimicrobial activity was exhibited against Staphylococcus aureus and Trichophyton rubrum. The in vitro cytotoxicity studies employing L929 cells and A431 cells demonstrated good biocompatibility and enhanced anticancer activity of the curcumin-loaded green nanocomposite hydrogel compared to pure curcumin.

Influence of the cultivation medium and pH on the pigmentation of Trichophyton rubrum.

Trichophyton rubrum is a human pathogenic fungus. As a dermatophyte it causes athlete's foot, fungal infection of nails, jock itch and ringworm. The pigmentation of T. rubrum is variable and can range from white or yellow to wine-red. We demonstrate that the pigmentation is strongly influenced by pH. Under alkaline conditions, T. rubrum has a red pigmentation, whereas at acid conditions, T. rubrum has a yellow pigmentation. Moreover, the color change immediately from yellow to red by adding NaOH and reverse immediately from red to yellow by adding HCl. We suggest that the chemical compound Xanthomegnin is responsible for red as well for yellow pigmentation in T. rubrum. To figure out, why T. rubrum has red pigmentation on Trichophyton medium, adjust to alkaline, but not on Synthetic-Complete medium, also adjusted to alkaline, we measure the pH of liquid media, adjusted to pH 3.5, 6 and 8, over a period of four weeks. The pH of both cultivation media changes significantly, with a maximum of five pH levels. Whereas the Trichophyton medium, initially adjusted to pH 8, stays alkaline, the pH of the Synthetic-Complete medium drops to acid conditions. The acidification of the SC medium and the alkalization of the Trichophyton medium explains the different pigment color of the T. rubrum colonies.

Cold Atmospheric Pressure Plasma Jet Reduces Trichophyton rubrum Adherence and Infection Capacity.

This study aimed to evaluate the effects of cold atmospheric pressure plasma (CAPP) jet on Trichophyton rubrum growth, germination and adherence to nail. The effects of plasma jet on T. rubrum conidia germination and on mycelial growth were evaluated by in vitro assays. An ex vivo nail infection model was used to evaluate the effects on conidia adherence and infection. Biochemical analyses of nail fragments exposed or not to CAPP were performed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Plasma jet exposure for 10 and 15 min completely inhibited mycelial growth after only one exposure. Fifteen minutes of exposure could reduce conidia germination in suspension. Fungal suspensions exposed to plasma jet for 10 and 15 min were not able to infect nail specimens. These results were corroborated by ATR-FTIR analyses of nail fragments. In conclusion, single exposure to CAPP for 15 min was able to inhibit fungal growth, adherence and infection capacity. The results suggest that cold atmospheric plasma jet can be a promising alternative for the treatment of onychomycoses caused by T. rubrum.

In Vitro Activity of Antifungal Drugs Against Trichophyton rubrum and Trichophyton mentagrophytes spp. by E-Test Method and Non-supplemented Mueller-Hinton Agar Plates.

Trichophyton rubrum and Trichophyton mentagrophytes spp. are two of the most frequently isolated dermatophytes causing dermatophytosis worldwide. Since the incidence of resistance to antifungal agents is increasing, antifungal susceptibility tests are needed to successfully treat dermatophytoses. Most of the methods currently available are complicated, time-consuming and lack of reference procedures. The aim of this work was to establish a simple protocol to test the susceptibility of dermatophytes isolated from clinical samples against five antifungal drugs using E-test and disk diffusion methods. We used the E-test on non-supplemented Mueller-Hinton agar plates to determine the minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole and amphotericin B, and disk diffusion method to determine the interpretive MIC of terbinafine. Fifty dermatophytes-10 T. rubrum and 40 T. mentagrophytes spp.-were assessed after only 96 h of colony growth. Terbinafine was the most active antifungal agent with an inhibition diameter greater than 70 mm (sensitivity > 20 mm), followed by voriconazole, itraconazole and amphotericin B with MICs ranging from 0.032 to 0.38 µg/mL, from 0.006 to 0.125 µg/mL and from 0.5 to 1.5 µg/mL, respectively. All isolates were resistant to fluconazole. Collectively, the less laborious E-test and disk diffusion method were shown to be suitable and reliable to determine antifungal sensitivity of dermatophytes. This simple standard protocol could be employed in the routine of clinical laboratories.

A new acridone with antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans.

AIM: The increase in the number of fungal infections worldwide, coupled with the limitations of current antifungal chemotherapy, demand the development of safe and effective new antifungals. Here, we presented the synthesis of a novel acridone (M14) and its antifungal properties against Candida and dermatophytes species. METHODS AND RESULTS: A series of 17 acridones was designed, synthesized and tested for its antifungal activity. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Only the acridone M14 showed growth-inhibitory activity against reference strains and clinical isolates of Candida and dermatophytes, with MIC range of 7·81-31·25 µg ml-1 . Moreover, M14 exhibited fungicidal activity and prevented biofilm formation by C. albicans as well as reduced the viability of preformed biofilms, even at sub-MICs. The confocal laser scanning microscopy analysis revealed that C. albicans hyphal growth was completely inhibited in the presence of M14. Similarly, there was a severe inhibition on hyphal growth of Trichophyton rubrum. We also found that M14 has relatively low toxicity to human fibroblasts. CONCLUSIONS: The new acridone M14 has antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans. In addition, M14 is relatively selective to fungal cells compared to human normal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its in vitro antifungal activity, anti-Candida biofilm effect and moderate cytotoxicity towards normal human cell, M14 may serve as a valuable lead compound to develop a new antifungal agent.

Ciclopirox and Efinaconazole Transungual Permeation, Antifungal Activity, and Proficiency To Induce Resistance in Trichophyton rubrum.

Onychomycosis is a nail fungal infection, mostly caused by dermatophytes. The treatment efficacy is impaired by difficulties of reaching effective drug levels at the site of infection; frequent relapses occur after cessation of antifungal therapy. The aim of the study was to compare two commercial products containing ciclopirox or efinaconazole for antimycotic activity and antifungal drug resistance. A study of permeation and penetration through bovine hoof membranes, as a nail model, was performed to evaluate the antimycotic activity of permeates against clinical isolates of selected fungi, and the frequency of spontaneous in vitroTrichophyton rubrum-resistant strains was assessed by broth microdilution assays. The results suggest that ciclopirox creates a depot in the nail, leading to a gradual release of the drug over time with action on both the nail plate and bed. Conversely, efinaconazole, mildly interacting with nail keratin, mainly exerts its antifungal activity in the nail bed. However, in the case of T. rubrum, the antifungal activities of the drugs in the nail plate seem comparable. Finally, efinaconazole showed a potential for induction of resistance in T. rubrum, which may limit its efficacy over time. Ciclopirox did not show any potential to induce resistance in T. rubrum and appears endowed with a more complete activity than efinaconazole in the management of onychomycosis as the nail keratin is a substrate for the growth of fungal cells, and the availability of drug in large concentration just in the nail bed may not be sufficient to guarantee the complete eradication of pathogens.