Department of Dermatology, Rabin Medical Center - Beilinson Hospital, Petach Tikva, Israel.
DYN Diagnostics, Migdal HaEmeq, Israel.
Mycoses. 2018 Feb;61(2):119-126. doi: 10.1111/myc.12713. Epub 2017 Nov 15.
The gold-standard method for dermatophyte identification involves direct microscopy and culture, which have inherent shortcomings. Only few molecular methods have been standardised for routine clinical work. This study aimed to develop and test a platform for identifying the most common dermatophytes in Israel using multiplex real-time polymerase chain reaction (RT-PCR). Specific primers were designed for the multiplex system (LightCycler 480) according to known cultures and validated by reference isolates. The dermatophyte detection rate was compared to smear and culture in 223 clinical samples obtained from a tertiary medical centre. Inconsistencies between methods were evaluated by sequencing. The RT-PCR was further evaluated in 200 community-based samples obtained from a health maintenance organisation and 103 military-personnel-based samples analysed at a central laboratory. In hospital-based clinical samples, complete concordance between methods was observed in 190 samples (85%; Kappa = 0.69). In most cases of non-concordance, sequencing was consistent with RT-PCR results. RT-PCR correctly identified all smear- and culture-positive cases in community and military-personnel samples. The results were available within 4 hours. The multiplex RT-PCR platform is a rapid and efficient method for identifying dermatophyte species in clinical samples and may serve as a first step in the diagnostic algorithm of superficial fungal infections.
真菌鉴定的金标准方法包括直接显微镜检查和培养，但这两种方法都存在固有缺陷。仅有少数分子方法已标准化用于常规临床工作。本研究旨在开发和测试一种使用多重实时聚合酶链反应（RT-PCR）鉴定以色列最常见皮肤真菌的平台。根据已知培养物设计了多重系统（LightCycler 480）的特异性引物，并通过参考分离物进行了验证。将皮肤真菌的检测率与来自三级医疗中心的 223 份临床样本的涂片和培养进行了比较。通过测序评估了方法之间的不一致性。该 RT-PCR 进一步在来自健康维护组织的 200 份基于社区的样本和在中央实验室分析的 103 份军人样本中进行了评估。在基于医院的临床样本中，方法之间完全一致的有 190 个样本（85%；Kappa 值为 0.69）。在大多数不一致的情况下，测序与 RT-PCR 结果一致。RT-PCR 正确鉴定了社区和军人样本中所有涂片和培养阳性的病例。结果可在 4 小时内获得。多重 RT-PCR 平台是一种快速有效的方法，可用于鉴定临床样本中的皮肤真菌种类，并可作为浅表真菌感染诊断算法的第一步。
