Parasitology-Mycology Laboratory, Farhat Hached Hospital, Sousse, Tunisia.
Mycoses. 2014 Jan;57(1):19-26. doi: 10.1111/myc.12096. Epub 2013 Jun 4.
Onychomycosis is one of the most prevalent dermatophytic diseases. Mycological methods used in the conventional diagnosis may not be optimal. Multiplex (MX) PCR was reported as a reliable alternative. Dermatophyte gene sequence records were used to design a MX PCR for detection and identification of dermatophytes in nail specimens. A MX PCR method based on the amplification of the chitin synthase 1 and internal transcribed spacer genes was developed. The study included 93 strains of dermatophytes and non-dermatophytic fungi, six dermatophytic reference strains and 201 nail specimens from patients with dermatophytic onyxis. DNA extraction directly from nail samples was carried out by using the QIAamp DNA extraction kit (Quiagen). A set of primers was designed and their specificity was assessed. MX PCR detected the causal agent in specimens from which Trichophyton rubrum and T. interdigitale grew in culture and also identified a dermatophyte species in an additional 32 specimens that were negative in microscopy and culture. None of the investigated non-dermatophytic strains was positive. Sensitivity of MX PCR was higher as compared to mycological examination (97% vs. 81.1%). MX PCR for direct detection of dermatophytes from nail samples yielded mixed flora in 32.8% of samples. MX PCR proved sensitive and adequate for the diagnosis of dermatophytic onychomycosis. It is much adapted to cases where culture is negative or contaminated by overgrowing moulds, which makes the identification of the causal agent problematic.
甲真菌病是最常见的皮肤真菌病之一。传统诊断中使用的真菌学方法可能并不理想。多重（MX）PCR 被报道为一种可靠的替代方法。根据角鲨烯合酶 1 和内部转录间隔区基因的扩增，设计了一种用于检测和鉴定指甲标本中皮肤真菌的 MX PCR。该研究包括 93 株皮肤真菌和非皮肤真菌、6 株皮肤真菌参考株和 201 例来自甲真菌病患者的指甲标本。直接从指甲样本中提取 DNA 是通过使用 QIAamp DNA 提取试剂盒（Quiagen）进行的。设计了一组引物并评估了它们的特异性。MX PCR 检测到培养物中生长的红色毛癣菌和 T. interdigitale 的标本中的病原体，并在另外 32 份显微镜和培养均为阴性的标本中鉴定出一种皮肤真菌。没有一种被调查的非皮肤真菌株呈阳性。与真菌学检查相比，MX PCR 的敏感性更高（97%比 81.1%）。MX PCR 直接从指甲样本中检测皮肤真菌，在 32.8%的样本中产生混合菌群。MX PCR 被证明对甲真菌病的诊断具有敏感性和适宜性。它非常适合培养物为阴性或被过度生长的霉菌污染的情况，这使得确定病原体变得困难。
