[Genome detection of dermatophytes : Current findings from the external quality assessment test]. / Genomnachweis von Dermatophyten : Aktuelle Erkenntnisse aus dem Ringversuch.

In addition to morphological diagnostics, molecular methods have become available for the diagnosis of suspected dermatophytoses. Since March 2016, INSTAND e. V., in cooperation with the National Reference Laboratory for Dermatophytes, has provided an external quality assessment (EQA) test for the genome detection of dermatophytes twice a year. More than half of the participants used commercial kits for the analysis of the samples. All kits with a high level of accuracy correctly determined the presence of Trichophyton rubrum or no dermatophytes. In species diagnostics beyond Trichophyton rubrum there are large differences between the kits. These are examined in more detail based on clinical studies and the results of the EQA test.

[Dermophytes and guinea pigs : An underestimated danger?] / Dermatophyten und Meerschweinchen : Eine unterschätzte Gefahr?

BACKGROUND: For several years, an increasing number of human infections, mainly affecting children, with the zoophilic dermatophyte Trichophyton benhamiae has been observed. It is predominantly transmitted by pet guinea pigs. The prevalence of the dermatophyte on guinea pigs which are for sale in pet shops is unknown. OBJECTIVE: Therefore, the aim of this study was to analyze the frequency of T. benhamiae on symptomatic and asymptomatic guinea pigs from pet shops in Berlin. METHODS: We sampled 59 guinea pigs from 15 pet shops using toothbrushes (MacKenzie brush technique) and FLOQswabs™ and analyzed the material for the presence of T. benhamiae with polymerase chain reaction (PCR) and culture. RESULTS: We detected T. benhamiae on more than 90% of the guinea pigs; 9% of which showed visible tinea symptoms. The majority was identified as asymptomatic carriers of the dermatophyte. CONCLUSION: Pet shop guinea pigs have a high risk of being carriers of T. benhamiae, which can be transmitted to humans via physical contact, even though there is no visible infection in most cases. It is therefore recommended to have newly purchased animals examined by a veterinarian.

[Tinea corporis due to the rare geophilic dermatophyte Microsporum praecox]. / Tinea corporis durch den seltenen geophilen Dermatophyten Microsporum praecox.

A 10-year-old girl suffered from tinea corporis with erythematosquamous and centrifugal growing, sparse itching lesions of her right lower arm. Fluorescence optical Blankophor® preparation from skin scrapings revealed fungal hyphae. On Sabouraud's dextrose agar, the fast growing dermatophyte formed flat, peripheral radiating and convolved colonies with white, slightly yellowish to beige brown stained granular and powdery surface. The reverse side of the colonies was smooth with luminous yellow colour. Microscopically, an attitude of thin-walled spindle-shaped and echinulate (with small spins) and lanceolate macroconidia appeared. The small based macroconidia are raised in the middle and end part, however, pointy at the end ("spearhead"). Three to six or seven across septae are formed. The small piriform microconidia had an orthotropic arrangement. Chlamydospores were also formed. Urease activity was positive. Macromorphologically, Trichophyton (T.) interdigitale (formerly T. mentagrophytes) was suspected. Due to the shape of macroconidia, Microsporum (M.) gypseum and M. fulvum were also considered as possible species identification. Direct uniplex-PCR-EIA of the strains revealed negative results for T. rubrum, T. interdigitale, T. anamorph of Arthroderma benhamiae and M. canis. Sequencing analysis of the ribosomal ITS-region (18 S rRNA, ITS1, 5.8 S rRNA, ITS2, 28 S rRNA) and of the translation elongation factor 1­alpha (tef-1-alpha) gene revealed the dermatophyte species M. praecox. Topical treatment was done using ciclopiroxolamine cream. M. praecox represents a geophilic dermatophyte, morphologically resembling M. gypseum. Horses are often the source of infection. In humans, M. praecox causes tinea corporis and tinea capitis. For oral treatment of dermatomycosis due to M. praecox, griseofulvin and terbinafine can be used.

The agony of choice in dermatophyte diagnostics-performance of different molecular tests and culture in the detection of Trichophyton rubrum and Trichophyton interdigitale.

Dermatophytosis caused by dermatophytes of the genera Trichophyton and Microsporum belong to the most frequent mycoses worldwide. Molecular detection methods proved to be highly sensitive and enable rapid and accurate detection of dermatophyte species from clinical specimens. For the first time, we compare the performance of different molecular methods with each other and with conventional diagnostics in the detection of dermatophytoses caused by Trichophyton rubrum and Trichophyton interdigitale in clinical specimens (nail, skin and hair). The compared molecular methods comprise two already published PCR-ELISAs, a published quantitative RT-PCR as well as a newly developed PCR-ELISA targeting the internal transcribed spacer region. We investigated the sensitivity of the assays by analysing 375 clinical samples. In 148 specimens (39.5%) a positive result was gained in at least one of the four molecular tests or by culture, but the number of detected agents differed significantly between some of the assays. The most sensitive assay, a PCR-ELISA targeting a microsatellite region, detected 81 T. rubrum infections followed by an internal transcribed spacer PCR-ELISA (60), quantitative RT-PCR (52) and a topoisomerase II PCR-ELISA (51), whereas cultivation resulted in T. rubrum identification in 37 samples. The pros and cons of all four tests in routine diagnostics are discussed.

[Trichophyton violaceum : Main cause of tinea capitis in children at Mbarara Regional Referral Hospital in Uganda]. / Trichophyton violaceum : Haupterreger der Tinea capitis bei Kindern im Mbarara Regional Referral Hospital in Uganda.

BACKGROUND: Tinea capitis is caused by anthropophilic, zoophilic or geophilic dermatophytes of the genera Microsporum or Trichophyton. OBJECTIVE: The aim of this study was to analyze the clinical presentation of tinea capitis among children in western Uganda. PATIENTS AND METHODS: From February to June 2012, skin and hair samples were obtained from 115 patients aged from 1 to 16 years presenting at Mbarara Regional Referral Hospital (MUSC) with clinically suspected tinea capitis. Conventional mycological diagnostics comprised Blancophor preparation and cultivation of fungi for species identification. RESULTS: Tinea capitis among the children included in the MUSC study was mainly noninflammatory showing mostly a seborrhoeic pattern or "black dot" and "gray patch" form and highly inflammatory kerion celsi. Blancophor preparation identified 82.6 % positive and 17.4 % negative samples. Cultural species differentiation showed Trichophyton (T.) violaceum as the causative agent for tinea capitis in 56.6 % of the patients. In 13 %, Microsporum (M.) audouinii was isolated followed by T. soudanense (2.6 %), and T. rubrum (1.7 %). In addition, moulds (contamination?) such as Scopulariopsis brevicaulis, Aspergillus niger, and Fusarium oxysporum were found as well as mixed infections. CONCLUSION: The anthropophilic dermatophyte T. violaceum represents the most frequent cause of tinea capitis in western Uganda. For successful management oral antifungal therapy is necessary together with supportive topical treatment.

Detection of common dermatophytes in clinical specimens using a simple quantitative real-time TaqMan polymerase chain reaction assay.

BACKGROUND: Dermatophytes are common fungal pathogens causing mostly superficial infections in humans with a high prevalence worldwide. Traditional detection techniques are time-consuming and insensitive, whereas molecular detection methods have proved to be much more rapid and sensitive. OBJECTIVES: To develop a modular singleplex quantitative real-time polymerase chain reaction (qRT-PCR) assay for the detection of the most common dermatophytes in clinical specimens. METHODS: The qRT-PCR assay is based on single-tube reactions with TaqMan probes. We validated the test with 311 clinical samples of human and animal origin submitted for routine diagnosis and compared the qRT-PCR results with microscopy and culture. RESULTS: qRT-PCR proved to be significantly more sensitive than microscopy and culture, with 21·2% more positive samples. Among the 201 dermatophytes identified 152 were Trichophyton rubrum (75·6%) and 34 were Trichophyton interdigitale (16·9%). Only 15 samples were determined as less common dermatophytes (Microsporum canis, Epidermophyton floccosum, Trichophyton verrucosum and Arthroderma benhamiae). In the present study, pathogen identification was achieved for 95·2% of all samples (including negatives) by applying only three detection tests (pan-dermatophyte, T. rubrum and T. interdigitale). CONCLUSIONS: The qRT-PCR assay developed in this study allows the specific and sensitive detection of relevant dermatophytes at low cost in a short time.

Unusual strains of Microsporum audouinii causing tinea in Europe.

We comment on an unusual strain of Microsporum (M.) audouinii. It was isolated from tinea corporis of a boy who lived in Germany and most likely had acquired his infection during a stay on a farm with animal husbandry in Poland. The strain showed features of M. canis (plenty of markedly rough-walled macroconidia, growth on rice, positive hair perforation) as well as of M. audouinii (white thallus, long macroconidia with central constriction) and in vitro it degraded hair of various mammals. Because its ribosomal internal transcribed spacer region showed 99.9% homology to a M. audouinii reference strain it was finally identified as M. audouinii. We relate these findings with recent observations of M. audouinii causing tinea in Europe. This appraisal suggests that irrespective of an identical ribosomal ITS region distinct M. audouinii strains can display a spectrum of morphological and physiological features that is broader than currently outlined in mycological textbooks. Certain unusual characteristics like an enhanced capacity to utilise keratins may even be associated with unexpected transmission routes. Above all sporadic M. audouinii infections in Europe that bear no relation to an endemic area should be analysed from this perspective.

[Trichophyton thuringiense H.A. Koch 1969. A rare geophilic dermatophyte--now isolated for the first time from man]. / Trichophyton thuringiense H.A. Koch 1969. Ein seltener geophiler Dermatophyt, erstmals vom Menschen isoliert.

BACKGROUND: In 1969, Kolipp and Hoffmann isolated Trichophyton (T.) thuringiense spec. nov. Koch when they performed their thesis dealing with the distribution and epidemiology of dermatophytes and keratinophilic fungi in mice and other small mammals. At that time, T. thuringiense was detected as saprophytic fungus of the skin of different mice species (e.g. Mus musculus) both in rural and urban settings in the area of Thuringia in Germany. There were no further reports on this dermatophyte species until now, neither in animals, nor in man. PATIENT, METHODS AND RESULTS: Currently, we were able to isolate this geophilic fungus for the first time from a human being. A 58 year old patient baker by trade and living in a rural setting (village) suffered from nail changes like hyperkeratosis and thickening of the nail plate of his big toe. From his nail samples grew a dermatophyte with peripheral radiating and flat colonies which were a bit cottony in the centre. On Sabouraud's 4 % dextrose agar the thallus of the fungus was white to purple stained, the reverse side showed a dark red to brown color. In a typical manner, macroconidia were cylindrical to clavate, microconidia obovoidal to short-clavate with broad base. The species identification of T. thuringiense was done and confirmed by sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA. Antifungal treatment has been refused from the patient. CONCLUSION: In conclusion, this is the second description of the geophilic dermatophyte T. thuringiense, which could be isolated for the first time from a human being, in particular from nail sample of the big toe under the suspicion of onychomycosis. However, it is still uncertain if this fungus should be considered either as secondary colonization of the nail plate, or as causative agent of tinea unguium or onychomycosis.

[Molecular biological detection of dermatophytes in clinical samples when onychomycosis or tinea pedis is suspected. A prospective study comparing conventional dermatomycological diagnostics and polymerase chain reaction]. / Molekularbiologischer Direktnachweis von Dermatophyten im klinischen Material bei Verdacht auf Onychomykose und Tinea pedis. Eine prospektive Studie zum Vergleich konventioneller dermatomykologischer Diagnostik und der Polymerasekettenreaktion.

BACKGROUND: The prevalence of onychomycosis is rising worldwide. Before starting antifungal treatment, an exact mycological diagnosis should be obtained. The current laboratory diagnosis of dermatomycoses is based on the detection of the causative agent by microscopy and culture. These conventional diagnostic methods for fungal infections often are not the best solution because they are time-consuming, cultures are false-negative and direct examination identifies non-vital structures which cannot be used for speciation. PATIENTS AND METHODS: A total of 218 patients presenting in a surgical practice over 3 months with clinical signs of tinea pedis and/or onychomycosis were involved in the prospective study. All patients had predisposing factors for tinea pedis and tinea unguium, such as vascular insufficiency, diabetes mellitus, and leg ulcers. Nail specimens and skin scrapings were investigated for fungi using Blancophor® preparation, and cultured. In addition to conventional diagnostics, PCR (polymerase chain reaction) for detection of dermatophyte DNA was employed. This PCR-Elisa assay is based on the use of specific primers which target the topoisomerase II gene. This allows the highly specific molecular identification of Trichophyton (T.) rubrum, T. interdigitale, and Epidermophyton floccosum directly in clinical samples. RESULTS: 23.9 % of patients were culture-positive for dermatophytes (either T. rubrum, or T. interdigitale). With PCR, dermatophyte DNA either of T. rubrum or T. interdigitale could be detected in nail samples and skin scrapings from at least 29.9 % of all patients. Epidermophyton floccosum was not found in this study, neither by cultivation nor by PCR. The diagnostic sensitivity of the PCR-Elisa assay was calculated as 79.0% ; the diagnostic specificity as 85.5 %. CONCLUSION: PCR-Elisa evaluation makes possible a rapid, specific and sensitive diagnosis of dermatophytosis of the nails and skin within 24 (maximal 48) hours with identification of the involved species.

Development of a novel polymerase chain reaction-enzyme-linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis.

BACKGROUND: The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost-effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. OBJECTIVES: To develop a microsatellite-based polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (MS-ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. METHODS: An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS-ELISA with an earlier published topoisomerase PCR-ELISA (TI-ELISA) using template DNA extracted by another method. RESULTS: The MS-ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI-ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS-ELISA proved to be twice as sensitive as the TI-ELISA. CONCLUSIONS: We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis.

[Dermatomycoses due to pets and farm animals : neglected infections?]. / Dermatomykosen durch Haus- und Nutztiere : Vernachlässigte Infektionen?

Dermatomycoses due to contact with pets and livestock frequently affect children and young adults. Zoophilic dermatophytes are the main important causative agents. It has long been known that the often high inflammatory dermatophytoses of the skin and the scalp are caused mostly by Microsporum canis. Due to an absence of an obligation for reporting fungal infections of the skin to the Public Health Office in Germany, an unnoticed but significant change in responsible pathogens has occurred. Today an increasing number of infections due to zoophilic strains of Trichophyton interdigitale (formerly Trichophyton mentagrophytes) and Trichophyton species of Arthroderma benhamiae are found. The latter mentioned dermatophyte is the anamorph species of the teleomorph Arthroderma benhamiae, which originally was isolated in the Far East (Japan). Source of infection of these dermatophytes are small rodents, in particular guinea pigs. These animals are bought in pet shops by the parents of those children who later are affected by the fungal infection. The coincidental purchase of the relevant fungal pathogen is not obvious to the parents. As a consequence, highly contagious dermatophytoses occur, often tinea capitis sometimes with kerion formation. Further dermatophytes should be considered as cause of a zoophilic dermatomycosis. Both Trichophyton verrucosum, the cause of the ringworm in cattle, and Trichophyton erinacei following contact to hedgehogs are worthy of note. Yeasts cannot be ignored as cause of dermatomycosis, especially Malassezia pachydermatis, the only non-lipophilic species within the genus Malassezia, which can be transferred from dog to men. Cryptococcus neoformans also comes from animal sources. The mucous yeast occurs in bird's dropping, and it causes both pulmonary and central nervous system infections, but also primary and secondary cutaneous cryptococcosis in immunocompromised patients (HIV/AIDS) as possible consequence after contact to these animals.

[Tinea capitis profunda due to Trichophyton verrucosum with cMRSA superinfection in an infant]. / Tinea capitis profunda durch Trichophyton verrucosum mit abszedierender Superinfektion durch cMRSA bei einem Kleinkind.

A 28-month-old boy developed a cutaneous and subcutaneous lesion of the scalp together with alopecia. Treatment with sulfadiazine silver ointment and oral administration of cefaclor failed. The boy lived on a farm where cows and calves were present. He presented with a 5 cm erythematous, erosive, edematous, and sharply defined lesion with yellow crusts and circumscribed alopecia on the temporoparietal scalp. Peripheral hairs were easily epilated. Swabs from the wound revealed cMRSA (community acquired methicillin-resistant Staphylococcus aureus, Panton Valentine Leukocidin [PVL] toxin negative). There was no improvement after treatment with cefuroxime intravenously over 3 days. Therapy was changed to vancomycin and fosfomycin. Because of the purulent abscess, surgical incision was performed. PCR (polymerase chain reaction)-Elisa assay detected Trichophyton (T.) interdigitale-DNA from wound secretion and skin biopsy. Because of the clinical and molecular diagnosis of tinea capitis, oral antifungal therapy with fluconazole 5 mg kg(-1) body weight was started, along with cotrimoxazole and fosfomycin for the cMRSA. After 4 weeks incubation, the causative agent T. verrucosum was grown on culture and its identity confirmed by sequencing of the "internal transcribed spacer" (ITS) region of the ribosomal DNA. After 4 weeks of fluconazole, the lesion was nearly healed.

New records of Batrachochytrium dendrobatidis in Chilean frogs.

We used molecular techniques to examine 11 species of frogs in 6 localities in southern Chile to ascertain the incidence of the chytrid fungus Batrachochytrium dendrobatidis (Bd). We detected the fungus in 2 localities (Coñaripe and Raúl Marín Balmaceda) in 3 species: Batrachyla leptopus, Pleurodema thaul and Rhinoderma darwinii. Our findings expand the list of Bd hosts to include B. leptopus and P. thaul and extend the spatial distribution in Chile to include the southernmost Bd record at Raúl Marín Balmaceda.

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato.

BACKGROUND: Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. OBJECTIVES: The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. METHODS: One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction-amplified internal transcribed spacer (ITS) region of the rDNA. RESULTS: Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. CONCLUSIONS: Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.

Tinea corporis caused by an unusual strain of Microsporum audouinii that perforates hair in vitro.

We report on a dermatophyte infection acquired by a young woman from Germany who had worked in Ghana. The strain isolated from her skin lesions showed morphological and physiological features compatible with Microsporum audouinii but a clearly positive hair perforation test made its definite identification by conventional methods equivocal. A genetic analysis finally unambiguously revealed Microsporum audouinii. This is the first observation of a Microsporum audouinii strain with a positive hair perforation test. The ability to perforate hair may be related to attributes favouring an inflammatory host response.

Changes in frequency of agents of tinea capitis in school children from Western China suggest slow migration rates in dermatophytes.

Tinea capitis is a common dermatophyte infection of the scalp of children in Western China, with the gray-patch from being the most prevalent. Twenty years ago, the most widespread etiologic agent was reported to be Trichophyton violaceum, which was later succeeded by Microsporum ferrugineum and Trichophyton schoenleinii. In the framework of our recent study, 97 isolates were collected from patients with clinically suspected tinea capitis. Identification was performed by conventional methods and by sequencing the ribosomal DNA internal transcribed spacer region. In the case of T. violaceum an additional microsatellite primer set (T1) was used. Five species (in order of frequency, Trichophyton violaceum, T. schoenleinii, Microsporum ferrugineum, zoophilic strains of Arthroderma vanbreuseghemii, and Trichophyton tonsurans) were identified. Results of molecular and phenotypic ID of the same strains showed good correspondence. Comparison with earlier data showed that dermatophytes species in former rural societies must have migrated extremely slowly. Preponderance of local transmission from domesticated animals was proven by the occurrence of zoophilic strains of Arthroderma vanbreuseghemii. Etiologic agents in the rural communities of Western China tend to be different from those of the other regions in the country, despite modern communication and traffic.

Evolution of CDC42, a putative virulence factor triggering meristematic growth in black yeasts.

The cell division cycle gene (CDC42) controlling cellular polarization was studied in members of Chaetothyriales. Based on ribosomal genes, ancestral members of the order exhibit meristematic growth in view of their colonization of inert surfaces such as rock, whereas in derived members of the order the gene is a putative virulence factor involved in expression of the muriform cell, the invasive phase in human chromoblastomycosis. Specific primers were developed to amplify a portion of the gene of 32 members of the order with known position according to ribosomal phylogeny. Phylogeny of CDC42 proved to be very different. In all members of Chaetohyriales the protein sequence is highly conserved. In most species, distributed all over the phylogenetic tree, introns and 3(rd) codon positions are also invariant. However, a number of species had paralogues with considerable deviation in non-coding exon positions, and synchronous variation in introns, although non-synonomous variation had remained very limited. In some strains both orthologues and paralogues were present. It is concluded that CDC42 does not show any orthologous evolution, and that its paralogues haves the same function but are structurally relaxed. The variation or absence thereof could not be linked to ecological changes, from rock-inhabiting to pathogenic life style. It is concluded that eventual pathogenicity in Chaetothyriales is not expressed at the DNA level in CDC42 evolution.

Intraspecific diversity of species of the Pseudallescheria boydii complex.

In order to establish intraspecific diversity of Pseudallescheria boydii and Scedosporium apiospermum, and to develop tools for their identification, variability within P. boydii and related species was investigated at different levels of diversity. Sequences of the D1/D2 region of large subunit (LSU) and of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) gene were analyzed for a set of 57 strains, as well as partial sequences of the elongation factor 1-alpha (EF 1-alpha). Incongruence among 3 locus lineages was detected by partition homogeneity test (PHT). The maximum parsimony (MP) tree of the combined sequence data set, with the exception of strain CBS 499.90, formed 3 clades with high bootstrap support, corresponding to previously described nuclear DNA (nDNA)/DNA reassociation groups. These groups are known to differ slightly in predilection and temperature relations. Using Structure software, population genetic analysis revealed 3 clusters within the complex on the basis of multi-locus genotype data. Strain distribution in the clusters was concordant with that in the 3 clades of combined multi-locus MP tree. Recombination among individuals of a clade in evolutional history was found in 2 of the 3 clades. There was population differentiation among the 3 clades. Restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer (IGS) region of rDNA gene was added to further characterize subspecific entities. When the IGS regions of 22 strains were digested with the restriction endonucleases Hae III and Mbo I, seven and five distinct patterns were detected, respectively. This subtyping did not reveal any correspondence with geographic origin or clinical appearance. Though we need more evidence to locate the 3 clades of the P. boydii complex at species or population level, the sequence of the D1/D2 region is sufficiently variable for identification of taxa belonging to the P. boydii complex.