Rapid and direct observation of fungal spores or hyphae in clinical liquid specimens poses a challenge for the diagnosis of invasive fungal infection. To allow rapid detection of fungal pathogens, we designed a new method of fungal cell detection involving double fluorescence staining with calcium fluorescent white (CFW) and SYTOX green combined with single-cell real-time imaging flow cytometry (IFC). IFC allowed quick detection and analysis of detailed morphology of the spores and pseudohyphae of Candida albicans, and small hyphae and typical truncated large mycelia of Aspergillus fumigatus. Further, cell sorting based on fluorescence, the width-to-height ratio and bright-field parameters preferentially identified spores or hyphae with a typical cell wall. The specificity and overall coincidence rate of IFC for fungi detection in common clinical samples were 100% and 98.18%, respectively. Moreover, the detection rate by IFC (102/105, 97.14%) was significantly higher (P=0.002) than that by wet mount method (89/105, 84.5%). Therefore, IFC is a reliable diagnostic method with a high potential for application for rapid diagnosis of fungal infection in the clinic.

在临床液体标本中快速直接观察真菌孢子或菌丝对侵袭性真菌感染的诊断提出了挑战。为了快速检测真菌病原体，我们设计了一种新的真菌细胞检测方法，包括钙荧光白 (CFW) 和 SYTOX 绿双荧光染色结合单细胞实时成像流式细胞术 (IFC)。IFC 可以快速检测和分析白色念珠菌的孢子和假菌丝的详细形态，以及烟曲霉的小菌丝和典型截短的大菌丝体。. 此外，基于荧光、宽高比和明场参数的细胞分选优先识别具有典型细胞壁的孢子或菌丝。IFC对常见临床样本真菌检测的特异性和总体符合率分别为100%和98.18%。此外，IFC的检出率（102/105，97.14%）显着高于湿贴法（89/105，84.5%）（P = 0.002）。因此，IFC 是一种可靠的诊断方法，在临床真菌感染的快速诊断方面具有很大的应用潜力。