Fast and sensitive detection of Trichophyton rubrum DNA from the nail samples of patients with onychomycosis by a double-round polymerase chain reaction-based assay.
BACKGROUND
Trichophyton rubrum is one of the most frequently isolated pathogens in onychomycosis. Isolation of T. rubrum from nail samples by traditional methods is time-consuming and has a high false-negative rate of detection.


OBJECTIVE
To investigate the detection of T. rubrum in nail samples using DNA detection methods.


METHODS
A total of 62 nail samples from onychomycosis patients with T. rubrum infection were evaluated by culture on Sabouraud's dextrose agar plus chloramphenicol, cycloheximide and gentamicin and compared with genotyping methods utilizing DNA extracted directly from nails. Trichophyton rubrum DNA isolated directly from nails was amplified using two different conserved regions [actin gene and internal transcribed spacer 1 (ITS)] in double-round polymerase chain reaction (PCR) assays.


RESULTS
Forty-eight of 62 (77.4%) samples were potassium hydroxide (KOH) positive, but T. rubrum culture was positive in only 14 of 62 (22.6%) samples. By contrast, direct T. rubrum DNA detection rate was 59.7% (37/62) by actin gene and 45.2% (28/62) by ITS1 region PCR assays corresponding to higher detection frequencies compared with culture with P < 0.001 and < 0.008, respectively. The combined detection of actin and ITS1 was 69.4% (43/62). Interestingly, T. rubrum DNA was detected in 9 out of 14 (64.3%) of KOH- and culture-negative samples. Importantly, 15 culture-negative samples collected from patients undergoing antifungal treatment tested PCR positive using the actin region.


CONCLUSIONS
These results suggest that a direct DNA detection protocol is more sensitive, accurate and faster than traditional culture-based methods. It can be useful to detect T. rubrum in patients undergoing antifungal therapy and who have been reported mycologically cured on the basis of a culture-based method.
Gupta
A K
AK
Division of Dermatology, Department of Medicine, Sunnybrook Health Sciences Center and the University of Toronto, Toronto, Ontario, Canada.
Zaman
M
M
Singh
J
J
D000199:Actins / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis*; D005260:Female; D005533:Foot Dermatoses / Q000382:microbiology*; D005656:Fungal Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D016533:Mycological Typing Techniques / Q000379:methods; D009262:Nails / Q000382:microbiology; D014009:Onychomycosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014249:Trichophyton / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D000199:Actins; D004271:DNA, Fungal; D005656:Fungal Proteins