Clinical Usefulness of PCR-REBA for Diagnosis of Onychomycosis / 대한의진균학회지

BACKGROUND: PCR-based reverse blot hybridization assay (PCR-REBA) has high sensitivity and specificity, can be performed directly on nail samples, is relatively cheaper than other molecular biologic methods, and is useful for diagnosing onychomycosis. OBJECTIVE: This study aims to compare the diagnostic efficacy of fungal culture and REBA Fungus-ID® which is a commercial PCR-REBA-based kit used for onychomycosis diagnosis. METHODS: Fifty nail samples were collected from 50 patients diagnosed with onychomycosis via direct microscopic examination using KOH preparation, and subjected to fungal culture and REBA Fungus-ID® test. RESULTS: The sensitivity of conventional fungal culture and REBA Fungus-ID® was 56% and 100%, respectively. In REBA Fungus-ID®, 43 of 50 samples were found to be infected with Trichophyton rubrum. Four of the remaining 7 samples were identified as infected with Trichophyton spp., one with Trichophyton mentagrophytes, and two revealed a panfungal DNA sequence. In fungal culture, 28 of 50 samples showed growth, of which 18 samples were identified as T. rubrum, 3 as Rhodotorula mucilaginosa, 3 as Cladosporium spp., 1 as Cyphellophora europaea, 1 as Penicillium cvjetkovicii, 1 as Lachnum soppittii, and 1 as non-dermatophytic mold. REBA Fungus-ID® and fungal culture were identical in 20 cases (40%). The non-dermatophytic fungi identified in fungal culture were considered contaminants. CONCLUSION: Nail specimens can be used directly for REBA Fungus-ID®, which has a high sensitivity for onychomycosis diagnosis. Therefore, it can be considered useful for diagnosis and identification of the causative organism in mixed infections like onychomycosis.

PCR-reverse Blot Hybridization Assay for Species Identification of Dermatophytes / 대한의진균학회지

BACKGROUND: Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) cause cutaneous mycoses called dermatophytosis. Forproper anti-dermatophytosis therapy, accurate and early diagnosis of dermatophytes is important. Laboratory diagnosis of dermatophytosis for dermatophytes still relies on microscopic and macroscopic examination of in vitro cultures and some physiological tests. These methods (conventional methods) are time-consuming (2~4 weeks) and yet, still have low sensitivity and specificity. Recently, in order to overcome such limitations of conventional methods, molecular-based methods have been developed to identify dermatophytes. The polymerase chain reaction-reverse blot hybridization assay (PCR-REBA) allows sensitive and specific identification of dermatophytes species. OBJECTIVE: This study was aimed to develop a new PCR-REBA with higher sensitivity using less amount of probe concentration, so the assay can be more practical in clinical settings. METHODS: For this, PCR primers and species-specific oligonucleotide probes were designed within the internal transcribed sequences 1 region between 5.8S and 18S rRNA. The species-specific probes designed in this study was to identify 6 species (T. rubrum, T. mentagrophytes, T. tonsurans, M. canis, M. gypseum, and E. floccosum) comprised 99% of dermatophytes isolatedin Korea. RESULTS: The detection efficiency of the PCR-REBA was compared with the microscopic method, and the results showed that the sensitivity of the PCR-REBA developed in this study is 100 times higher than previously developed one. Subsequently, the results of PCR-REBA were evaluated using clinical isolates. DNAs from a total of 68 clinical isolates were analyzed by PCR-REBA, and the inconsistent results between PCR-REBA and conventional microscopic identification results were confirmed by sequence analysis. CONCLUSION: In brief, the results showed that results of sequence analysis were identical with PCR-REBA implying newly developed PCR-REBA is very useful method for accurate and rapid identification of dermatophytes and would provide higher simplicity, specificity, sensitivity than conventional method.