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.

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.

Batrachochytrium dendrobatidis in amphibians of Cameroon, including first records for caecilians.

Amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been hypothesised to be an indigenous parasite of African amphibians. In Cameroon, however, previous surveys in one region (in the northwest) failed to detect this pathogen, despite the earliest African Bd having been recorded from a frog in eastern Cameroon, plus one recent record in the far southeast. To reconcile these contrasting results, we present survey data from 12 localities across 6 regions of Cameroon from anurans (n = 1052) and caecilians (n = 85) of ca. 108 species. Bd was detected in 124 amphibian hosts at 7 localities, including Mt. Oku, Mt. Cameroon, Mt. Manengouba and lowland localities in the centre and west of the country. None of the hosts were observed dead or dying. Infected amphibian hosts were not detected in other localities in the south and eastern rainforest belt. Infection occurred in both anurans and caecilians, making this the first reported case of infection in the latter order (Gymnophiona) of amphibians. There was no significant difference between prevalence and infection intensity in frogs and caecilians. We highlight the importance of taking into account the inhibition of diagnostic qPCR in studies on Bd, based on all Bd-positive hosts being undetected when screened without bovine serum albumin in the qPCR mix. The status of Bd as an indigenous, cosmopolitan amphibian parasite in Africa, including Cameroon, is supported by this work. Isolating and sequencing strains of Bd from Cameroon should now be a priority. Longitudinal host population monitoring will be required to determine the effects, if any, of the infection on amphibians in Cameroon.

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.

Batrachochytrium dendrobatidis in Darwin's frog Rhinoderma spp. in Chile.

The presence of the chytrid fungus Batrachochytrium dendrobatidis in Chile was evaluated in 2 endangered frog species of the genus Rhinoderma. Specimens from a captive rearing facility, wild populations and preserved collection material were analyzed using histological and molecular techniques. The fungus was identified in the rearing facility and in wild populations, but not in the archived frogs. This study confirms, for first time, the presence of chytridiomycosis in Rhinoderma darwinii in Chile.

Widespread unidirectional transfer of mitochondrial DNA: a case in western Palaearctic water frogs.

Interspecies transfer of mitochondrial (mt) DNA is a common phenomenon in plants, invertebrates and vertebrates, normally linked with hybridization of closely related species in zones of sympatry or parapatry. In central Europe, in an area north of 48 degrees N latitude and between 8 degrees and 22 degrees E longitude, western Palaearctic water frogs show massive unidirectional introgression of mtDNA: 33.7% of 407 Rana ridibunda possessed mtDNA specific for Rana lessonae. By contrast, no R. lessonae with R. ridibunda mtDNA was observed. That R. ridibunda with introgressed mitochondrial genomes were found exclusively within the range of the hybrid Rana esculenta and that most hybrids had lessonae mtDNA (90.4% of 335 individuals investigated) is evidence that R. esculenta serves as a vehicle for transfer of lessonae mtDNA into R. ridibunda. Such introgression has occurred several times independently. The abundance and wide distribution of individuals with introgressed mitochondrial genomes show that R. lessonae mt genomes work successfully in a R. ridibunda chromosomal background despite their high sequence divergence from R. ridibunda mtDNAs (14.2-15.2% in the ND2/ND3 genes). Greater effectiveness of enzymes encoded by R. lessonae mtDNA may be advantageous to individuals of R. ridibunda and probably R. esculenta in the northern parts of their ranges.

Origins of microsatellite diversity in the Trichophyton rubrum-T. violaceum clade (Dermatophytes).

We analyzed the population structure of the anthropophilic dermatophyte species Trichophyton violaceum, which mainly causes tinea capitis, and T. rubrum, the most frequently isolated agent of dermatophytosis worldwide. A microsatellite marker (T1) was developed by using the enrichment technique for microsatellites. The T1 marker containing a (GT)(8-10) repeat was proven to specifically amplify both species, underlining their close kinship. Four polymorphic alleles were detected within a set of about 130 strains by using polyacrylamide gel electrophoresis with this marker. An association with geographic origin of the isolates was apparent. Given the close relatedness of both species, these data suggest an African origin of the entire T. rubrum complex, followed by the emergence of a new genotype (B) in Asia with subsequent spread of this genotype over Europe and the United States.