Assessment of a pan-dermatophyte nested-PCR compared with conventional methods for direct detection and...

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture-positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies.