Real-time PCR: A rapid and sensitive method for diagnosis of dermatophyte induced onychomycosis, a comparative study

Abstract

Onychomycosis is the most common nail disorder, accounting for up to 50% of all nail problems and about 30% of cutaneous fungal infections. Treatment of onychomycosis is expensive. It requires long term therapy with an oral antifungal medication with potential side effects. Therefore, a proper diagnosis of infection is needed.

Aim of the study: This study aimed to compare real time PCR using novel primers targeting the pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) with nested PCR targeting the same gene, KOH microscopy, direct microscopy in relation to culture for diagnosis of clinically suspected onychomycosis.

Subjects and methods: This study was conducted during the period from April 2013 through May 2014. Eighty patients attending Outpatient Dermatology and Andrology clinic at Benha University Hospital were included in this study. They were 30 females and 50 males with suspected clinical diagnosis of onychomycosis. Their ages ranged from 22 to 77 years. Thirty eight of them were living in rural areas, while the other 42 came from urban areas. Nail scrapings were collected and examined by the following: direct KOH microscopic examination, culture, nested PCR using double sets of primers and finally real time PCR.

Results: As regards direct microscopy by KOH examination, 66 (82.50%) cases were positive, while 14 (17.5%) were negative. Culture was positive only in 38 (47.5%) of nail samples revealing different fungi. Dermatophytes were isolated from 30 (37.5%) cases; most of them were Trichophyton mentagrophytes, and in 8 cases the only isolated  non-dermatophytic organism was Aspergillus Niger spp. (10.00%). Nested PCR was positive in 52 (65.00%) of nail samples while real time PCR was positive in 58 (72.5%) of nail samples.

Conclusion: Real-time PCR followed by melting-point analysis, gives a diagnostic tool that has a higher sensitivity (93.3%) and is faster than nested PCR (73.3%) and other conventional methods.

Keywords: Onychomycosis; Dermatophytes; Nested PCR; Real time PCR