Development and Clinical Detection of Rapid Molecular Diagnostic System for Pathogenic Dermatophytes of Tinea Capitis of Multiple Centres in China

Abstract

Objectives: Tinea capitis remains a common fungal infection in children worldwide. Species identification is critical for determining the source of infection and reducing transmission. In conventional methods, macro- and microscopic analysis is time-consuming and results in slow fungal growth or low specificity. We propose a rapid real-time diagnostic PCR method that allows species-specific identification of dermatophytes, including the Microsporum canis complex, Trichophyton mentagrophytes complex, Trichophyton rubrum complex and Trichophyton tonsurans, in patients with tinea capitis.

Methods: Hair and scrapings samples were collected from 231 patients with tinea capitis who were positive for fungal elements via direct microscopy with potassium hydroxide. Each sample was subjected to a two-step real-time PCR (RT-PCR) assay, which was designed on the basis of differences in the DNA fragments of the internal transcribed spacer (ITS) and β-tubulin covering the Microsporum canis complex, T. mentagrophyte complex, T. rubrum complex, T. tonsurans, T. verrucosum, T. schoenleinii and N. gypseum.

Results: In total, 186/231 samples (80.52%) were positive for fungal culture. The two-step RT-PCR was positive in 215/231 samples (93.07%), among which 179 were culture positive. The combined efficacy was 96.81%, which was significantly different when the RT-PCR assays were performed in parallel with fungal culture. A total of 126 samples (54.55%) were identified as Microsporum canis by fungal culture, among which the positive rate of M. canis complex RT-PCR was 97.62% (123/126). A total of 45 samples were negative for fungal culture, of which 80.0% (36/45) were positive by RT-PCR, and the percentage of M. canis complex-positive samples was 53.33% (24/45). The RT-PCR assays were negative for 16/231 samples, among which 7 were culture positive, including M. canis (n = 3), T. violaceum (n = 3) and N. gypseum (n = 1).

Conclusion: We developed a new diagnostic assay system using a rapid real-time TaqMan PCR assay with specific primers that can be applied in routine laboratory practice for hair and skin samples of tinea capitis to detect dermatophytes and increase diagnostic efficiency.

Keywords: RT–PCR; dermatophytosis; molecular diagnosis; tinea capitis.