[Conventional and molecular diagnostics in onychomycosis-part 2 : Molecular identification of causative dermatophytes by polymerase chain reaction and sequence analysis of the internal transcribed spacer region of ribosomal DNA]. / Konventionelle und molekulare Diagnostik bei Onychomykose ­ Teil 2 : Molekulare Identifizierung der ursächlichen Dermatophyten mit Polymerasekettenreaktion und Sequenzanalyse der ITS("internal transcribed spacer")-Region der ribosomalen Desoxyribonukleinsäure.

Dermatophyte identification using traditional methods such as optics-based direct fluorescence microscopy and culture is nowadays supplemented by molecular biological methods. The validity of dermatophyte DNA detection with direct uniplex-polymerase chain reaction-enzyme immunoassay (PCR-EIA) in nail samples was proven by sequence analysis of the ribosomal internal transcribed spacer (ITS) region. A total of 108 dermatophytes, isolated from patients with onychomycosis, were positive for Trichophyton rubrum (TR) and Trichophyton interdigitale (TI) in culture and/or uniplex-PCR-EIA. Conventional methods for dermatophyte identification were complemented by direct uniplex-PCR-EIA and sequence analysis of the ribosomal ITS region (18S rRNA, ITS1, 5.8S rRNA, ITS2, 28S rRNA). Of 108 patients (average age 62, median age 73), 56 showed cultural growth with 31 of them being identified as TR and 23 as TI. There was high agreement with the sequence analysis. Surprisingly, the pathogen of a single nail sample was identified as T. quinckeanum (formerly T. mentagrophytes sensu stricto), a rare zoophilic dermatophyte in Germany. A single TI strain turned out to be a misidentified T. tonsurans based on the sequence analysis. In all, 34 of the 52 specimens lacking cultural growth were detected by PCR as TR, and 18 specimens could be identified as TI. The results of dermatophyte identification of culture-negative nail samples were also in agreement with the results of sequence analysis. Molecular biological methods are well applicable, and they show high reliability for direct dermatophyte identification in nail samples without prior cultivation. Especially for nail samples without cultural growth, PCR-based dermatophyte identification was highly specific and sensitive.

[Conventional and molecular diagnostics in onychomycosis-part 1 : Conventional differentiation of dermatophytes-Trichophyton rubrum, Trichophyton interdigitale]. / Konventionelle und molekulare Diagnostik bei Onychomykose ­ Teil 1 : Konventionelle Differenzierung von Dermatophyten ­ Trichophyton rubrum, Trichophyton interdigitale.

Onychomycosis is a common infectious nail disease occurring worldwide. The mycological diagnosis of onychomycosis is primarily used for differential diagnostic differentiation from other, mostly inflammatory nail diseases, such as nail psoriasis or onychodystrophies of other causes. Conventional laboratory diagnostics when onychomycosis is suspected is based on microscopic detection of fungi in the nail material using fluorescence-optical potassium hydroxide preparations and culture of the pathogen. Molecular amplification methods allow a more sensitive and specific identification of the causative dermatophyte. Here, in 108 patients with onychomycosis, the dermatophytes were identified by culture and/or molecular biology using polymerase chain reaction (PCR) and the species identification was confirmed with subsequent sequencing. The dermatophytes were analyzed based on macromorphological and microscopic features. A dermatophyte was cultured in 56 of the 108 patients. Among them were 31 isolates of Trichophyton (T.) rubrum and 25 of T. interdigitale. All species identifications were subsequently confirmed by rDNA sequencing with concordant results in 54 of 56 patients. Two primarily as T. interdigitale identified specimens were revealed to be T. quinckeanum and T. tonsurans by molecular methods. T. quinckeanum, which is a zoophilic dermatophyte and a so-called emerging pathogen in dermatomycology, was isolated here for the first time as the causative agent of onychomycosis. The other dermatophyte, initially thought to be T. interdigitale, turned out to be T. tonsurans on molecular biology. This anthropophilic dermatophyte is also a rarity in onychomycosis. In addition, T. rubrum was identified by PCR in 34 of the 52 nail specimens that did not grow culture, and T. interdigitale in 18 nail specimens. However, the morphological identification of the four different dermatophytes species proved problematic. Neither the colony morphology nor the microscopic features of the dermatophytes allow clear differentiation of the pathogens. Microconidia, macroconidia, chlamydospores, and arthrospores are inconsistent in occurrence, number, microscopic distribution, and shape. The urease activity also did not allow an assignment of the dermatophyte species. These results indicate that the most sensitive detection and reliable identification of causative dermatophytes in onychomycosis is only possible by molecular methods.

Molecular epidemiology of Trichophyton quinckeanum - a zoophilic dermatophyte on the rise.

BACKGROUND: Formerly only referred to as a subspecies (T. mentagrophytes var. quinckeanum), T. quinckeanum once again constitutes a distinct species according to the updated taxonomy of dermatophytes. PATIENTS AND METHODS: During routine diagnostic tests conducted at the Mycology Laboratory, Mölbis, Germany, between 11/2013 to 1/2017 (three years and three months), all specimens sent in were examined for T. quinckeanum. Molecular biology methods employed included: 1) DNA hybridization (PCR ELISA), 2) gene sequencing of the ITS region and TEF-1α, and 3) in some cases, MALDI-TOF mass spectrometry. RESULTS: Overall, 62 strains of T. quinckeanum were found. Sixty-eight percent of patients were female; 43 % were children and adolescents (≤ 19 years of age). Cats were a frequent source of infection. Sequencing of all 62 strains revealed a concordance of 100 % with T. quinckeanum sequences contained in the NCBI database. Isolates analyzed by MALDI-TOF mass spectrometry showed specific spectra. CONCLUSIONS: In Germany, the zoophilic dermatophyte T. quinckeanum currently appears to be more prevalent than expected. T. quinckeanum strains were isolated from children and adults with dermatomycosis and tinea capitis. Sources of infection with T. quinckeanum include small rodents (mice), horses, and - remarkably commonly -  cats. Given that unequivocal morphological identification of this dermatophyte is not always possible, molecular methods have to be employed in the diagnosis.