Development and Evaluation of SYBR Green Real-Time PCR for Rapid and Specific Identification of Trichophyton...

Since 2017, dermatophytosis caused by the newly introduced species Trichophyton indotineae has gained new interest worldwide due to the rise in terbinafine resistance and difficulty in the treatment of recalcitrant infections. Distinguishing T. indotineae from other Trichophyton species based on morphological features is impossible and DNA sequencing is necessary for accurate identification. Though early identification of the species is not solely sufficient for the treatment of infected cases, it is important for clinicians to take the next appropriate modalities such as antifungal susceptibility testing especially when the patients have extensive skin lesions recalcitrant to therapy by terbinafine. Here, we developed a rapid diagnostic scheme using SYBR Green real-time PCR for the specific detection/identification of T. indotineae.

DNA was extracted from 397 dermatophyte isolates and two SYBR Green real-time PCR assays targeting the C120-287 and E054-58 intergenic loci were developed. Using a collection of 132 T. indotineae and 128 non-T. indotineae strains, all had already been identified by ITS-PCR-sequencing and 137 unknown dermatophyte isolates, the assays were evaluated.

In both real-time PCR assays, 130 out of 132 T. indotineae strains were positive while all non-T. indotineae species were negative. Among 137 unknown tested isolates, 72 were identified as T. indotineae based on two real-time PCR assays, while 65 showed no peak and were considered non-T. indotineae. Based on PCR-sequencing as the reference standard, the SYBR Green real-time PCR assays demonstrated a sensitivity of 98.48% and a specificity of 100%.

The developed diagnostic assays using SYBR Green real-time PCR provided a rapid and accurate method for the distinction of cultured T. indotineae isolates and can be considered to evaluate for the detection of T. indotineae directly from clinical samples.