Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
Institute of Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety, Graz, Austria.
Sci Rep. 2018 May 3;8(1):6964. doi: 10.1038/s41598-018-25129-w.
The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.
侵袭性真菌感染的发病率不断上升，真菌病原体的范围不断扩大，这使得早期准确鉴定致病病原体成为一项艰巨的任务。使用分子标记物进行诊断可以快速鉴定真菌，为传染病动态提供新的见解，并为传染病的控制和预防开辟新的可能性。我们对先前经过培养和/或内部转录间隔区（ITS）PCR 处理的疑似真菌感染患者的临床标本（N=233）进行了回顾性研究。我们使用这些标本评估了一种使用自动化 DNA 提取（QIAsymphony）、真菌核糖体小亚基（18S）rDNA RT-PCR 和扩增子测序进行真菌检测的高通量筛选方法。将真菌序列与经过精心整理、市售的 SmartGene IDNS 数据库中的序列进行比较，以进行病原体鉴定。18S rDNA RT-PCR 与培养结果的一致性为 91%，18S rDNA RT-PCR 与 ITS PCR 结果的一致性为 94%。此外，18S rDNA RT-PCR 和 Sanger 测序在临床确诊真菌感染患者的培养阴性（N=13）和 ITS PCR 阴性标本（N=12）中检测到真菌病原体。我们的结果支持使用 18S rDNA RT-PCR 诊断工作流程快速准确地鉴定临床标本中的真菌病原体。
