Development of a novel polymerase chain reaction-enzyme-linked immunosorbent assay for the diagnosis of Trichophyton rubrum onychomycosis

摘要

Background The prevalence of onychomycosis has increased steadily in the past decade. An accurate diagnosis at the outset is important for successful and cost-effective treatment of patients. However, current diagnostic tests for onychomycosis are not rapid, sensitive or specific. Objectives To develop a microsatellite-based polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (MS-ELISA) for the detection of Trichophyton rubrum, which is the most common aetiological agent of onychomycosis. Methods An archival set of 434 nail and skin specimens from 217 patients was included as the test sample in this study. We also compared MS-ELISA with an earlier published topoisomerase PCR-ELISA (TI-ELISA) using template DNA extracted by another method. Results The MS-ELISA detected the highest number of positive samples (69%) followed by direct microscopy (56%), TI-ELISA (44%) and fungal culture (30%). When an identical DNA extraction method was applied to 120 specimens, the MS-ELISA proved to be twice as sensitive as the TI-ELISA. Conclusions We have optimized a target gene and DNA extraction method for rapid detection of T. rubrum onychomycosis. What's already known about this topic? Conventional microscopy and culture methods rely on skilled staff, and are either nonspecific or take a long time to appear in culture. Since the introduction of molecular tools a few polymerase chain reaction assays to identify dermatophyte spp. directly from clinical specimens have been developed. What does this study add? The assay developed here is highly sensitive, very probably due to the special gene target used for the detection of Trichophyton rubrum, the most prevalent aetiological agent of onychomycosis. This is the first study directly comparing molecular identification methods for dermatophyte fungi. It shows that the target gene and DNA extraction method used have a large influence on the sensitivity of a molecular detection assay.