Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Atypical chlamydoconidium-producing Trichophyton tonsurans strains from Ceará State, Northeast Brazil: investigation of taxonomy by phylogenetic analysis and biofilm susceptibility.

Chlamydoconidium-producing Trichophyton tonsurans strains isolated in Northeastern Brazil have morphological features different from the classic description of this dermatophyte species. This study investigated the phylogenetic relationship of chlamydoconidium-producing T. tonsurans strains isolated in Northeastern Brazil. Also, the effect of terbinafine and farnesol on mature biofilms of T. tonsurans strains was evaluated. The mass spectra of T. tonsurans strains were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The ITS and LSU loci regions of rDNA and the partial ß-tubulin gene were sequenced and the phylogenetic tree was analysed. The effects of terbinafine and farnesol on mature T. tonsurans biofilms were evaluated through the analysis of metabolic activity, quantification of biomass and observation by scanning electron microscopy. MALDI-TOF MS spectra of the chlamydoconidium-producing T. tonsurans strains differed from the spectrum of the control strain (ATCC 28942), presenting an intense ion peak at m/z 4155 Da. Phylogenetic tree analysis showed that the chlamydoconidium-producing strains isolated in Northeastern Brazil are allocated to a single cluster, differing from strains isolated from other countries. As for mature T. tonsurans biofilms, farnesol reduced biomass and metabolic activity by 64.4 and 65.9 %, respectively, while terbinafine reduced the biomass by 66.5 % and the metabolic activity by 69 %. Atypical morphological characteristics presented by chlamydoconidium-producing T. tonsurans strains result from phenotypic plasticity, possibly for adaptation to environmental stressors. Also, farnesol had inhibitory activity against T. tonsurans biofilms, demonstrating this substance can be explored for development of promising anti-biofilm drugs against dermatophytes.

Evaluation of a Method for Long-Term Cryopreservation of Fungal Strains.

The conservation of microorganisms is essential for their in-depth study. However, today's most widely used conservation methods, based on the use of distilled water, soil, oils, or silica, do not guarantee the stability of fungal cells, especially dermatophytes. This problem led us to evaluate the conservation capacity of a cryogenic vials system containing glass beads covered in a cryopreservant hypertonic solution as an alternative method of storage of fungal cells at -80°C. Up to 570 strains of fungi belonging to 27 different species, isolated from clinical samples, were inoculated into cryotubes containing 25 glass beads covered in a cryopreserving hypertonic solution. Suspensions were mixed vigorously and the cryopreserving solution was discarded. The tubes were frozen at -80°C for a period of 42 months and periodically, a glass bead was removed from each cryotube and inoculated onto Sabouraud dextrose agar, and incubated at 30°C for 7-14 days to evaluate the number of colonies recovered, their purity, and phenotypic characteristics. All yeast isolates were recovered, unlike 2 isolates (4.4%) of the mold group and 21 (10.7%) of the dermatophytes. Survival rates were close to 100% for yeasts and molds, with expiration times being estimated for almost indefinite stocks, and 62% for dermatophytes, with an average expiration date of 25.5 years. The phenotypic characteristics remained comparable to those of the strains before storage. Conservation at -80°C using cryogenic vials is a reliable and efficient system for the conservation of fungal collections, and although the behavior differs by groups, stratified survival data are obtained to avoid extinction.

Dermatomycoses Due to Nannizzia praecox (Formerly Microsporum praecox) in Germany: Case Reports and Review of the Literature.

Nannizzia praecox, formerly known as Microsporum praecox, is a geophilic dermatophyte. Up to now 31 cases of human tinea have been reported in the literature, most of them with an inflammatory course. Three recent cases diagnosed in Germany within 1 year suggest that the fungus might be a more common cause of human dermatophytosis than reported so far. This might be based on the fact that N. praecox is often found in an equine environment and that horse riding is becoming more popular recently.

[Arthroderma benhamiae strains in Germany : Morphological and physiological characteristics of the anamorphs]. / Arthroderma-benhamiae-Stämme aus Deutschland : Morphologische und physiologische Merkmale der anamorphen Formen.

BACKGROUND: Anamorphs of Arthroderma (A.) benhamiae, which can cause inflammatory tinea lesions in humans, have been progressively spreading in Germany. OBJECTIVES: Identification of A. benhamiae anamorphs by conventional methods. MATERIALS AND METHODS: Evaluation of our own results obtained with A. benhamiae anamorphs and from the relevant literature. RESULTS: Infections with A. benhamiae anamorphs are usually transferred by guinea pigs or other animals. A. benhamiae anamorphs can be cultured on growth media used for dermatophytes and can be characterized morphologically and physiologically. In Germany the yellow variant is seen most often but a white variant that equals Trichophyton (T.) interdigitale has also been observed. On Sabouraud agar the yellow variant develops markedly yellow thalli, whereas the white variant produces white aerial mycelium. Microconidia are formed by the yellow variant-if at all-only scarcely and delayed; they are small, roundish, and arranged in a grape-like order. The white variant produces peg-shaped microconidia alongside hyphae as well as roundish ones in grape-like clusters, and subsequently some macroconidia, chlamydospores, and spiral hyphae. In microcultures with both variants circuit-like hyphal structures can consistently be demonstrated. On Trichophyton agars only the yellow variant is clearly dependent on thiamine. The urease test is negative with the yellow variant and positive with the white variant. Most strains of both variants are negative in the hair perforation test. CONCLUSIONS: The characteristics described allow reliable identification of the yellow variant of the A. benhamiae anamorph by conventional methods; a distinction between the white variant and T. interdigitale can be more difficult. Dermatologists should be able to identify this agent in clinical routine.

Toma de muestras y examen directo en dermatomicosis / Sample Taking and Direct Examination in Dermatomycoses

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Morphological and physiological features of Arthroderma benhamiae anamorphs isolated in northern Germany.

The anamorph of Arthroderma benhamiae is an upcoming zoophilic dermatophyte that only in recent years has gained importance as a cause of tinea in humans. Its identification by conventional methods can cause problems. In this study we have subjected seven genetically confirmed strains of A. benhamiae anamorphs from northern Germany recently identified in our laboratory to a comprehensive assessment. Their macroscopic and microscopic morphology was checked on various agars and enzyme release stimulated by substrates with keratin, hair perforation and other physiological characteristics were tested. All strains were related to the previously described yellow phenotype of the A. benhamiae anamorph and showed a high resemblance among themselves. Coherent features were their uniform thallus morphology on Sabouraud glucose agar with yellow pigmentation, the formation of circuit-like hyphal structures and hyphal connections that had not been described previously, a lack of conidia, thiamine dependence, the spectrum of released enzymes and a good growth on human stratum corneum. With exception of the latter two these criteria are suggested for the identification of this anamorph phenotype that should be evaluated by future observations. Different phenotypes of the A. benhamiae anamorph may prevail in other geographic regions.

A comparative study on morphological versus molecular identification of dermatophyte isolates.

OBJECTIVE: Dermatophytes are taxonomically classified in the genera Trichophyton, Microsporum, and Epidermophyton. Pleomorphism, cultural variability, slow growth and sporulation, and the need for additional physiological tests make dermatophytes notoriously difficult to identify. The present study aimed to compare the results of morphological and molecular identification of certain groups of clinical isolates of dermatophytes with a view to evaluating the accuracy of molecular methods. PATIENTS AND METHODS: For each sample, the ITS1-5.8S-ITS2 rDNA region was amplified using the primers ITS1 and ITS4. PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using the enzyme MvaI and isolate identification was performed by comparing the electrophoretic RFLP patterns with reference profiles obtained previously. Finally, paired comparative analyses of molecular and conventional methods were performed. RESULTS: While morphology results from routine daily reports of the laboratories indicated that 18 (6.8%) and 136 (52.10%) of the isolates were T. rubrum and T. interdigitale, respectively, PCR-RFLP results suggested that T. rubrum was the most common etiological agent of ringworm accounting for 94 (36.01%), followed by T. interdigitale accounting for 71 (27.20%). Interestingly, 80.8% out of the 94 isolates identified as T. rubrum by molecular testing had been identified by morphological examination as belonging to different species, such as T. interdigitale (75.5%), E. floccosum (2.1%) and M. canis, T. verrucosum, and T. tonsurans (each 1.06%). Ten strains out of 261 (T. interdigitale, n=8; E. floccosum, n=2) had been defined as unknown species by morphological tests. CONCLUSION: An unexpected high percent of isolates identified as T. interdigitale by conventional methods were in effect T. rubrum shown by PCR-RFLP, and regarding the necessity of correct identification of dermatophytes recovered from different clinical forms of the infection, we highly recommend ITS-sequencing or ITS-RFLP of the isolates, particularly for epidemiological research studies.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including age-related macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 µM Pc 4 followed by 670 to 675 nm light at 2.0 J/cm(2) reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

Morphological and molecular identification of two strains of dermatophytes.

In this study, we used traditional morphological and molecular identification methods to preliminarily identify two strains of dermatophytes. The two strains were observed under the microscope. And then the dermatophytes were cultured on Sabouraud's dextrose agar (SDA). The 18S rRNA regions of the two dermatophyte strains were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced and compared with GenBank data. BLAST tools and DNAMAN software were used to analyze the sequences. To further determine highly homologous sequences, a phylogenetic tree was constructed using the Neighbor-Joining method. The two strains of dermatophytes were identified by traditional morphological identification as Epidermophyton floccosum and Microsporum ferrugineum. The 18S rRNA sequence analyses showed high similarities to Cladosporium cladosporioides isolate C115LM-UFPR and Ascomycete sp. LB68A1A2. Epidermophyton and Cladosporium belong to dermatophyte, while Microsporum ferrugineum and Ascomycete belong to microsporum. The two novel strains of dermatophytes were therefore identified as Cladosporium cladosporioides isolate C115LM-UFPR (JN650537, Cladosporium) and Ascomycete sp. LB68A1A2 (AY770409, Ascomycete sp).

Borelli's lactritmel agar induces conidiation in rare-macroconidia producing dermatophytic fungi.

Macroconidia are among the most important indicators used to identify dermatophytic fungi, but several do not usually sporulate and/or produce macroconidia on Sabouraud glucose agar. Specifically, Microsporum audouinii, M. ferrugineum, Trichophyton concentricum, T. schoenleinii, T. verrucosum, and T. violaceum (including T. soudanense and T. yaoundei) rarely form macroconidia and, therefore, cannot be easily identified. In this study, we investigated the production of macroconidia on nine common laboratory media, including Borelli's lactritmel agar (BLA), modified Borelli's lactritmel agar (MBLA), brain heart infusion agar (BHIA), Christensen's urease agar in Petri dishes (UPA), cornmeal dextrose agar (CMDA), Lowenstein-Jensen agar (LJA), malt extract agar (MEA), oatmeal agar (OA), and potato dextrose agar (PDA). The performance of these media was evaluated using 18 rare-macroconidia producing isolates, including representative of the six species mentioned above. All cultures in this study were incubated at 26°C on the bench, and conidia formation on each was investigated at 5, 10, 15, 20, 25, and 30 days of incubation. BLA apparently improved macroconidia production after 15 days and was the most useful nutrient agar medium to induce these phenotypic characters in daily practice, closely followed by OA, PDA, and MBLA.

Changes of dermatophytoses in southwestern Greece: an 18-year survey.

The isolation and distribution rate of dermatophytes as causative agents of superficial mycoses of skin, hair, and nails during an 18-year period (1991-2008) at a university hospital are presented. A comparative analysis of epidemiological differences within the first (1991-1999) and the second 9-year period (2000-2008) was performed. Skin scrapings, nail, and hair specimens were examined by a direct microscopic examination and culture. Identification of dermatophyte species was based on macroscopic and microscopic characteristics of colonies. During the complete period (18 years), 5,971 patients with suspected dermatophytosis were examined. Seven hundred and sixty-nine patients (12.8%) were found positive. Among them, 495 cases (64.3%) were of skin dermatophytoses, 91(11.8%) of hair, and 183 (23.7%) of nails. The most frequent etiological agents were Microsporum canis (54%), Trichophyton rubrum (38%), and T. mentagrophytes (6%). Epidermophyton floccosum, T. tonsurans, T. violaceum, and M. gypseum were responsible only for 16 cases (2%) of dermatophytoses. The prevalence of dermatophytoses seems to decrease significantly from 16.2% (1991-1999)-9.6% during the last 9-year period. The most frequent dermatophyte, M. canis, shows decreasing trends during the last period (from 58.5 to 45.7%), whereas T. rubrum shows an increasing isolation rate (from 35 to 43.6%), respectively. The most common form of dermatophytosis among children remains tinea capitis due to M. canis. The most frequent etiological agent of tinea unguium (81%) is T. rubrum.

Antifungal properties of crude extracts of five Egyptian medicinal plants against dermatophytes and emerging fungi.

Antifungal properties of the crude extracts of five medicinal plants (Artemisia judaica, Ballota undulate, Cleome amblyocarpa, Peganum harmala, and Teucrium polium) were tested against dermatophytes and emerging fungi. Ethanol extract of Ballota undulate was the most effective against all tested fungi. Paecilomyces lilacinus, P. variotii, and Candida albicans were the most sensitive organisms. The minimum inhibitory concentration (MIC) of Ballota undulate ethanol extract against C. albicans, P. lilacinus, and P. variotii was 25 mg/ml. GC-MS analysis revealed that Ballota undulate ethanol extract contains 35 aliphatic and aromatic hydrocarbons, sesquiterpene hydrocarbon along with some other essential oils, which could be involved in antifungal activity. Light microscopy and scanning electron microscopy (SEM) have proved that Ballota undulate ethanol extract exhibits fungicidal effect on P. lilacinus through alterations in hyphal structures including budding of hyphal tip, anomalous structure, such as swelling, decrease in cytoplasmic content, with clear separation of cytoplasm from cell wall in hyphae. SEM clearly showed distorted mycelium, squashed and flattened conidiophores bearing damaged metullae. Eventually, the mycelia became papillated, flattened, and empty. Puncturing and squashing of hyphae as well as complete cell wall disruption were clear signs of complete death of hyphae.

Evaluation of a modified microscopic direct diagnosis of dermatophytosis.

Here we present a modified protocol for dematophyte diagnosis, utilizing a simple centrifugation step to significantly decrease false-negative results of the original KOH direct microscopy-based technique. Although culture constitutes the gold-standard diagnosis, the time spent for results is a limit. Fast and low-cost techniques are important for infection screening in underdeveloped countries.

Dynamics of dermatophytosis frequency in Mexico: an analysis of 2084 cases.

We analysed 15,101 biological samples from patients presenting with superficial mycoses who attended outpatient services over a 10-year period. Scale samples were processed for direct microscopic examination with 15% KOH and cultured on Sabouraud glucose agar plus chloramphenicol and cycloheximide. Laboratory examination confirmed 4,709 cases of superficial mycosis (31.18%), of which 2,084 (44.26%) were dermatophytoses. The species most frequently encountered was Trichophyton rubrum (71.2%), followed by T. tonsurans (6.9%), T. mentagrophytes (5.5%), Microsporum canis (4.5%) and Epidermophyton floccosum (1.3%). The most frequent clinical form of dermatophytosis was tinea unguium (59.9%), followed by tinea pedis (24.5%). We demonstrate that the number of cases of T. rubrum is increasing in Mexico.

Epidemiological and mycological data of onychomycosis in Goiania, Brazil.

Onychomycosis defined as fungal infection of the nail represents more than 50% of all onychopathies. Epidemiological studies have shown that this mycosis is worldwide in occurrence, but with geographical variation in distribution. The direct microscopy and culture of the nail samples were performed to identify the causative agent. Out of 2273 patients with nail infection examined between January 2000 and December 2004 in Goiania, state of Goias, Brazil, diagnosis of onychomycosis was confirmed in 1282 cases, with dermatophytes and Candida species being the most common aetiological agents isolated. Dermatophyte onychomycosis was more common in toenails than in fingernails, while onychomycosis caused by yeast had a similar frequency in both toenails and fingernails. Among the species identified, Candida albicans was responsible for 492 cases (38.4%) of onychomycosis, Trichophyton rubrum was found in 327 cases (25.6%) and Trichophyton mentagrophytes in 258 cases (20.1%). Other fungi isolated from nail infections included Aspergillus sp., Trichosporon sp., Geotrichum sp. and Fusarium sp. In our study, yeast of the genus Candida were the dominant cause of onychomycosis in women and dermatophytes were the principal cause of this condition in men.

Diagnostic value of morphological, physiological and biochemical tests in distinguishing Trichophyton rubrum from Trichophyton mentagrophytes complex.

The two most frequently encountered dermatophyte etiologic agents of glabrous skin and nail dermatophytoses are Trichophyton rubrum and T. mentagrophytes. This study was aimed to discuss the efficacy of morphological, physiological and biochemical diagnostic tests commonly used in the identification of T. rubrum and members of the T. mentagrophytes complex. In this study, we evaluated; hydrolysis of urea in broth and on urea agar slants and Petri plates incubated at 22 degrees C, 28 degrees C and 37 degrees C, in vitro hair perforation (blond child, sheep and goat hair), pigment production on cornmeal dextrose agar (CMDA) and bromcresol purple-milk solids-glucose agar (BCP-MS-G), Tween opacity, sorbitol assimilation, and salt tolerance. Additionally, the production of micro- and macroconidia was investigated by using brain heart infusion agar (BHIA), Christensen's urea agar in Petri plates (UPA), CMDA, Lowenstein-Jensen agar (LJA), malt extract agar, oatmeal agar, Oxoid chromogenic Candida agar, and potato dextrose agar. All cultures were incubated at 28 degrees C, and conidial production was compared on days 5, 10 and 15. It was found that the urea hydrolysis test yielded more rapid and significant results when urea medium was prepared in Petri plates and incubated at 28 degrees C (P<0.01). LJA supported the highest production of microconidia after 15 days (P<0.001). Additionally, it was found that T. rubrum strains produced red pigment on CMDA (P<0.01) and BCP-MS-G, while strains of the T. mentagrophytes species complex did not. A special algorithm containing the various test procedures employed in these studies is presented which was found to be useful in the differentiation of T. rubrum strains from T. mentagrophytes complex. Our results revealed that UPA, CMDA, BCP-MS-G, LJA, and BHIA may be used as common mycological agars in routine practice.

Glucose improves the in vitro viability of Microsporum canis and Trichophyton mentagrophytes var. mentagrophytes.

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.