Diagnosis of Trichophyton rubrum from onychomycotic nail samples using polymerase chain reaction and calcofluor white microscopy

Background: A high rate of false-negative dermatophyte detection is observed when the most common laboratory methods are used. These methods include microscopic observation of potassium hydroxide-digested nail clippings and culture methods using agar-based media supplemented with cycloheximide, chloramphenicol, and gentamicin to isolate dermatophytes. Microscopic detection methods that use calcofluor white staining or periodic acid-Schiff staining may also be substituted for and have previously been reported to be more sensitive than potassium hydroxide-digested nail clippings. Methods: Trichophyton rubrum infections were detected directly from nails in a double-round polymerase chain reaction assay that uses actin gene-based primers. This method was compared with detection of fungal hyphae by using calcofluor white fluorescence microscopy of nail samples collected from 83 patients with onychomycosis who were undergoing antifungal drug therapy. Results: Twenty-six of 83 samples (31.3%) were found to be positive by calcofluor white fluorescence microscopy, and 21 of 83 samples (25.3%) yielded positive results for Trubrum when actin gene-based primers in a double-round polymerase chain reaction assay were used. When calcofluor white fluorescence microscopy and polymerase chain reaction assay were used, the combined detection was 46.9% compared with 31.3% when calcofluor microscopy and culture of nail samples on Sabouraud's dextrose agar supplemented with cycloheximide, chloramphenicol, and gentamicin were used. Conclusions: These results suggest that the use of a direct DNA protocol is an alternative method for detecting Trichophyton infections. When this protocol is used, the presence of T rubrum DNA is directly detected. However, the viability of the dermatophyte is not addressed, and further methods need to be developed for the detection of viable Trubrum directly from nail samples.