Obručová Hana, Tihelková Radka, Kotásková Iva, Růžička Filip, Holá Veronika, Němcová Eva, Freiberger Tomáš
Centre for Cardiovascular Surgery and Transplantation Brno, Genetics Laboratory, Brno, Czech Republic Molecular Immunology and Microbiology, Central European Institute of Technology, Masaryk University Brno, Brno, Czech Republic.
Centre for Cardiovascular Surgery and Transplantation Brno, Genetics Laboratory, Brno, Czech Republic.
J Clin Microbiol. 2016 May;54(5):1295-303. doi: 10.1128/JCM.00118-16. Epub 2016 Mar 2.
Early diagnosis of fungal infection is critical for initiating antifungal therapy and reducing the high mortality rate in immunocompromised patients. In this study, we focused on rapid and sensitive identification of clinically important Candida species, utilizing the variability in the length of the ITS2 rRNA gene and fluorescent capillary electrophoresis (f-ITS2-PCR-CE). The method was developed and optimized on 29 various Candida reference strains from which 26 Candida species were clearly identified, while Candida guilliermondii, C. fermentati, and C. carpophila, which are closely related, could not be distinguished. The method was subsequently validated on 143 blinded monofungal clinical isolates (comprising 26 species) and was able to identify 88% of species unambiguously. This indicated a higher resolution power than the classical phenotypic approach which correctly identified 73%. Finally, the culture-independent potential of this technique was addressed by the analysis of 55 retrospective DNA samples extracted directly from clinical material. The method showed 100% sensitivity and specificity compared to those of the combined results of cultivation and panfungal PCR followed by sequencing used as a gold standard. In conclusion, this newly developed f-ITS2-PCR-CE analytical approach was shown to be a fast, sensitive, and highly reproducible tool for both culture-dependent and culture-independent identification of clinically important Candida strains, including species of the "psilosis" complex.
真菌感染的早期诊断对于启动抗真菌治疗以及降低免疫功能低下患者的高死亡率至关重要。在本研究中，我们利用ITS2 rRNA基因长度的变异性和荧光毛细管电泳（f-ITS2-PCR-CE），专注于快速、灵敏地鉴定临床上重要的念珠菌属菌种。该方法在29种不同的念珠菌参考菌株上进行了开发和优化，从中明确鉴定出了26种念珠菌属菌种，而密切相关的季也蒙念珠菌、发酵念珠菌和嗜鱼念珠菌无法区分。随后，该方法在143份盲法单真菌临床分离株（包含26个菌种）上进行了验证，能够明确鉴定出88%的菌种。这表明该方法的分辨能力高于传统的表型方法，传统方法的正确鉴定率为73%。最后，通过对直接从临床材料中提取的55份回顾性DNA样本进行分析，探讨了该技术不依赖培养的潜力。与作为金标准的培养及泛真菌PCR测序联合结果相比，该方法显示出100%的敏感性和特异性。总之，这种新开发的f-ITS2-PCR-CE分析方法被证明是一种快速、灵敏且高度可重复的工具，可用于依赖培养和不依赖培养的方式来鉴定临床上重要的念珠菌菌株，包括“滑囊炎”复合体的菌种。
