Rouhi Faezeh, Aboutalebian Shima, Rezaei-Matehkolaei Ali, Jahanshiri Zahra, Shidfar Mohammad-Reza, Chadeganipour Amir-Shayan, Shadzi Shahla, Kharazi Mahboobeh, Erami Mahzad, Mirhendi Hossein
Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Medical Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Mycoses. 2025 Jan;68(1):e70015. doi: 10.1111/myc.70015.
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.
自2017年以来，新引入的印氏毛癣菌引起的皮肤癣菌病在全球范围内引起了新的关注，这是由于特比萘芬耐药性的增加以及顽固性感染治疗困难所致。基于形态学特征无法将印氏毛癣菌与其他毛癣菌属物种区分开来，准确鉴定需要进行DNA测序。虽然早期鉴定该物种对于治疗感染病例并不完全足够，但对于临床医生来说，采取下一步适当的措施（如抗真菌药敏试验）非常重要，特别是当患者有广泛的皮肤病变且对特比萘芬治疗无效时。在此，我们开发了一种使用SYBR Green实时PCR的快速诊断方案，用于印氏毛癣菌的特异性检测/鉴定。
从397株皮肤癣菌分离株中提取DNA，并开发了两种针对C120 - 287和E054 - 58基因间隔位点的SYBR Green实时PCR检测方法。使用一组132株印氏毛癣菌和128株非印氏毛癣菌菌株（所有菌株均已通过ITS-PCR测序鉴定）以及137株未知皮肤癣菌分离株对检测方法进行评估。
在两种实时PCR检测中，132株印氏毛癣菌菌株中有130株呈阳性，而所有非印氏毛癣菌物种均为阴性。在137株未知检测分离株中，基于两种实时PCR检测，72株被鉴定为印氏毛癣菌，而65株未出现峰值，被认为是非印氏毛癣菌。以PCR测序作为参考标准，SYBR Green实时PCR检测方法的灵敏度为98.48%，特异性为100%。
所开发的使用SYBR Green实时PCR的诊断检测方法为区分培养的印氏毛癣菌分离株提供了一种快速准确的方法，可考虑用于直接从临床样本中检测印氏毛癣菌的评估。
