Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences.
BACKGROUND
Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture.


OBJECTIVE
This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method.


METHODS
The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum, T. mentagrophytes, Aspergillus spp., Scopulariopsis brevicaulis, Fusarium solani, F. oxysporum, F. verticillioides, Candida albicans and C. tropicalis were used after confirmation of their specificity.


RESULTS
Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83.0%): T. rubrum in 35 cases (74.5%) and T. mentagrophytes in eight cases (17.0%). Surprisingly, nondermatophytes were detected in 18 cases (38.3%), both dermatophytes and nondermatophytes in 10 cases (21.3%) and nondermatophytes alone in eight cases (17.0%). Aspergillus spp. alone was observed in five cases (10.6%).


CONCLUSIONS
This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.
Ebihara
M
M
Department of Dermatology, Tokyo Medical University, Nishishinjuku, 6-1-1 Shinjuku-ku, Tokyo 160-0023, Japan.
Makimura
K
K
Sato
K
K
Abe
S
S
Tsuboi
R
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001230:Aspergillus / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D014009:Onychomycosis / Q000175:diagnosis / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D014249:Trichophyton / Q000235:genetics / Q000302:isolation & purification*; D055815:Young Adult
D017931:DNA Primers; D004271:DNA, Fungal; D012339:RNA, Ribosomal, 28S