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)

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.

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.

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.

The role of formulation and follicular pathway in voriconazole cutaneous delivery from liposomes and nanostructured lipid carriers.

In general, colloids provide increased cutaneous permeation of drugs. Still, skin interaction and main pathways for drug diffusion may vary depending on system and formulation characteristics. The knowledge of how different colloidal systems interact with biological membranes and the formulation impact on delivery is especially relevant for drugs that can be encapsulated in multiple nanosystems, as voriconazole (VOR). In here, we compared VOR release and permeation profile from liposomes (LP) and nanostructured lipid carriers (NLC) in aqueous colloidal dispersions and in gel formulations. Despite the controlled drug release provided by gel formulations, formulation only had a significant impact on drug skin accumulation from LP. The reduced mobility in gel formulations compromised follicle deposition and drug retention in the skin. Such a hypothesis was confirmed by permeation experiments evaluating follicle pathway influence. Follicular route also had an influence on delivery from NLC, which was only significant for total drug that reached the acceptor medium. These differences could be attributed to the mechanisms of colloid interaction with the skin and subsequent drug release. Follicle LP deposition and slow drug release leads to higher cutaneous amounts whilst NLC interaction with skin and fast drug release leads to fast drug diffusion and deeper penetration. By the low MIC50 values encountered against Trichophyton rubrum (∼ 0.001 µg/mL), permeated amounts could inhibit fungal growth, regardless the system. In conclusion, both LP and NLC seem to be valuable systems for cutaneous VOR delivery. Fluidic formulations could provide better efficiency for cutaneous drug delivery from LP.

Quantitative and structural analyses of the in vitro and ex vivo biofilm-forming ability of dermatophytes.

PURPOSE: The aim of this study was to evaluate the in vitro and ex vivo biofilm-forming ability of dermatophytes on a nail fragment. METHODOLOGY: Initially, four isolates of Trichophyton rubrum, six of Trichophyton tonsurans, three of Trichophyton mentagrophytes, ten of Microsporum canis and three of Microsporum gypseum were tested for production biomass by crystal violet assay. Then, one strain per species presenting the best biofilm production was chosen for further studies by optical microscopy (Congo red staining), confocal laser scanning (LIVE/DEAD staining) and scanning electron (secondary electron) microscopy. RESULTS: Biomass quantification by crystal violet assay, optical microscope images of Congo red staining, confocal microscope and scanning electron microscope images revealed that all species studied are able to form biofilms both in vitro and ex vivo, with variable density and architecture. M. gypseum, T. rubrum and T. tonsurans produced robust biofilms, with abundant matrix and biomass, while M. canis produced the weakest biofilms compared to other species. CONCLUSION: This study sheds light on biofilms of different dermatophyte species, which will contribute to a better understanding of the pathophysiology of dermatophytosis. Further studies of this type are necessary to investigate the processes involved in the formation and composition of dermatophyte biofilms.

Investigation of the antifungal potential of linalool against clinical isolates of fluconazole resistant Trichophyton rubrum.

The aim of this study was to investigate the activity of the monoterpene linalool against clinical isolates of Trichophyton rubrum. Initially, a sensitivity assay for commercial antifungals with solid disks in diffusion medium was performed. Minimum inhibitory concentration (MIC) of linalool and ketoconazole (positive control) were determined by microdilution in RPMI 1640 medium (CLSI M38-A2). We then evaluated the action of linalool and ketoconazole at different concentrations (1/2MIC, MIC and 2×MIC) on mycelial growth (radial mycelial growth), conidia production and conidia germination using a hemacytometer. The effects on cell membrane (release of intracellular material) were also investigated. Finally, changes in fungal morphology as induced by the test drugs were analyzed. Based on the sensitivity tests, the fungal strains showed resistance to 5-fluorocytosine and fluconazole. The linalool MIC values ranged from 256µg/mL to 512µg/mL, whereas ketoconazole showed values of 4µg/mL to 8µg/mL. For the LM 305 strain, the test drugs showed the following MIC values: linalool 256µg/mL and ketoconazole 8µg/mL. The mycelial growth of T. rubrum LM 305 was inhibited by linalool (2×MIC) and ketoconazole (1/2MIC, MIC, 2×MIC), in 7 days of treatment (P<0.05). The test-drugs also inhibited conidial germination and conidiogenesis (P<0.05). Linalool also caused leakage of intracellular material (P<0.05). Finally, we verified the effectiveness of linalool and ketoconazole to induce micro-morphological changes, forming abnormal, wide, short and crooked hyphae. Based on these results, we conclude that linalool presents as an antifungal agent with anti-Trichophyton rubrum potential, an important dermatophytosis agent.

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms.

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 µg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 µg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.

Dermatoscopic findings as a complementary tool in the differential diagnosis of the etiological agent of tinea capitis.

Tinea capitis is a scalp infection caused by fungi. In Brazil, the main causative agents are Microsporum canis and the Trichophyton tonsurans. Etiological diagnosis is based on suggestive clinical findings and confirmation depends on the fungus growth in culture. However, it is not always possible to perform this test due to lack of availability. We reveal the dermoscopic findings that enable distinction between the main causative agents of Tinea capitis, M. canis and T. tonsurans. The association of clinical and dermatoscopic findings in suspected Tinea capitis cases may help with the differential diagnosis of the etiological agent, making feasible the precocious, specific treatment.

Dermatoscopic fi ndings as a complementary tool in the differential diagnosis of the etiological agent of tinea capitis

Tinea capitis is a scalp infection caused by fungi. In Brazil, the main causative agents are Microsporum canis and the Trichophyton tonsurans. Etiological diagnosis is based on suggestive clinical findings and confirmation depends on the fungus growth in culture. However, it is not always possible to perform this test due to lack of availability. We reveal the dermoscopic findings that enable distinction between the main causative agents of Tinea capitis, M. canis and T. tonsurans. The association of clinical and dermatoscopic findings in suspected Tinea capitis cases may help with the differential diagnosis of the etiological agent, making feasible the precocious, specific treatment.

[Evaluation of the in vitro susceptibility pattern of clinical isolates of Trichophyton mentagrophytes and Trichophyton rubrum in Santiago, Chile]. / Evaluación del perfil de sensibilidad in vitro de aislamientos clínicos de Trichophyton mentagrophytes y Trichophyton rubrum en Santiago, Chile.

BACKGROUND: Dermatophytes are a group of keratinophilic fungi able to produce dermatophytosis or tinea infections. In Chile, Trichophyton rubrum and Trichophyton mentagrophytes are the ones most commonly isolated in adults, while Microsporum canis is found among children. Treatment of these infections is usually with topical or oral antifungals, such as griseofulvin or azole derivatives (clotrimazole, itraconazole, fluconazole), allylamines (terbinafine) or new drugs that are available. AIMS: Evaluation of the in vitro susceptibility of dermatophytes to five antifungal agents and the comparison of the susceptibility pattern with that of previous years. METHODS: Sixty-two clinical isolates of dermatophyte fungi were studied (March-June 2010). The CLSI M38-A2 micromethod was used. RESULTS: Fluconazole MIC values for T. rubrum and T. mentagrophytes varied between 0.25 and 1 µg/ml; MIC range to clotrimazole, terbinafine and itraconazole was 0.03-0.06 µg/ml, and MIC values for griseofulvin were 0.015-0.03 µg/ml. No statistically significant differences were found between susceptibility patterns, except for fluconazole. CONCLUSIONS: Fluconazole was less active in comparison with other drugs tested (0.25-1 µg/ml). None of the isolates were resistant to any of the drugs, and no changes in the susceptibility pattern were observed when comparing the results with data previously reported concerning dermatophytes in Chile.

Detection of synergistic combinations of Baccharis extracts with terbinafine against Trichophyton rubrum with high throughput screening synergy assay (HTSS) followed by 3D graphs. Behavior of some of their components.

Forty four extracts from nine Baccharis spp. from the Caulopterae section were tested in combination with terbinafine against Trichophyton rubrum with the HTSS assay at six different ratios with the aim of detecting those mixtures that produced a ≥50% statistically significant enhancement of growth inhibition. Since an enhanced effect of a combination respective of its components, does not necessarily indicate synergism, three-dimensional (3D) dose-response surfaces were constructed for each selected pair of extract/antifungal drug with the aid of CombiTool software. Ten extracts showed synergistic or additive combinations which constitutes a 22% hit rate of the extracts submitted to evaluation. Four flavonoids and three ent-clerodanes were detected in the active Baccharis extracts with HPLC/UV/ESI-MS methodology, all of which were tested in combination with terbinafine. Results showed that ent-clerodanes but not flavonoids showed synergistic or additive effects. Among them, bacchotricuneatin A followed by bacrispine showed synergistic effects while hawtriwaic acid showed additive effects.

Aislamiento de Sporothrix pallida y Trichophyton rubrum en onicomicosis de mano / Isolation of Sporothrix pallida and Trichophyton rubrum in hand onychomycosis

Se presenta un caso de onicomicosis de mano, de la cual se aisló en repetidas ocasiones Sporothrix pallida y Trichophyton rubrum. Se discute sobre los principales agentes de onicomicosis, el rol de los hon- gos ambientales y del aislamiento de S.pallida en este y en otro tipo de muestras.

It reports a case of hand onychomycosis, which was isolated repeatedly Sporothrix pallida and Trichophyton rubrum. We discuss the main agents of onychomycosis, the role of the environmental fungi and S.pallida isolation in this and other samples.

Comparison of direct microscopy, culture and calcofluor white for the diagnosis of onychomycosis.

BACKGROUND: Mycological diagnosis of onychomycosis can be performed by direct microcopy (KOH), cultures and calcofluor white. AIMS: To compare the percentage of positivity and the degree of correlation of KOH, cultures and calcofluor white for the diagnosis of onychomycosis. METHODS: Descriptive, transversal and comparative study. Samples of toenails with onychomycosis were used for KOH, cultures and calcofluor white under fluorescence. The percentage of positivity of the different techniques was calculated and the degree of correlation between them was determined (Epi Info v 3.4.3(©)). RESULTS: KOH was positive in 66.67% of the cases, cultures in 33.33% and calcofluor white in 57.58%. KOH and calcofluor white had a higher percentage of positivity than culture (p<0.01 and p<0.05 respectively). The degree of correlation between KOH and calcofluor white was excellent (κ=0.8085; p<0.0001); however, the degree of correlation between KOH and culture and between calcofluor white and culture was poor. CONCLUSIONS: The use of calcofluor white is not recommended in routine laboratories because it does not seem to bring any additional benefits when comparing with KOH. This is especially important when funding is a great problem.