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)

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.

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.

Antifungal effects of a 1,3,4-thiadiazole derivative determined by cytochemical and vibrational spectroscopic studies.

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 ß(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 ß(1→3) glucans and ß(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.

Inhibitory Effects of Berberine Hydrochloride on Trichophyton mentagrophytes and the Underlying Mechanisms.

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.

Ultrastructural changes of Trichophyton rubrum in tinea unguium after itraconazole therapy in vivo observed using scanning electron microscopy.

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.

A 61-year-old man with erythematous forearm papules three months after liver transplantation.

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.

Clinical laser treatment of toenail onychomycoses.

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.

Morphological Effect of the New Antifungal Agent ME1111 on Hyphal Growth of Trichophyton mentagrophytes, Determined by Scanning and Transmission Electron Microscopy.

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.

Epizootic and epidemic dermatophytose outbreaks caused by Trichophyton mentagrophytes from rabbits in Portugal, 2015.

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.

Detection of dermatophytes in human nail and skin dust produced during podiatric treatments in people without typical clinical signs of mycoses.

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.

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes.

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.

Chemical composition and antifungal activity of essential oil from Eucalyptus smithii against dermatophytes

ABSTRACT 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.

An ultrastructural study of Trichophyton rubrum induced onychomycosis.

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.

Scanning electron microscopy of superficial white onychomycosis.

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.

Scanning electron microscopy of superficial white onychomycosis

AbstractSuperficial 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.

Synergistic combination of violacein and azoles that leads to enhanced killing of major human pathogenic dermatophytic fungi Trichophyton rubrum.

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.

Novel in vivo observations on double acting points of luliconazole on Trichophyton rubrum: an ultrastructural study.

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.

Case of dermatophyte abscess caused by Trichophyton rubrum: a case report and review of the literature.

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.

In vitro antifungal activity and mechanism of essential oil from fennel (Foeniculum vulgare L.) on dermatophyte species.

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.