RAPID DIAGNOSIS OF INVASIVE ASPERGILLOSIS WITH FOUNTAIN FLOW? CELL SORTING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目将证明一个低成本的，实时诊断侵袭性曲霉病的护理点系统的概念。侵袭性曲霉病发生在8-15%的骨髓移植患者和5-15%的实体器官移植患者。受感染的移植患者的死亡率从30%到70%不等，如果不治疗，死亡率几乎是100%。诊断是不可靠的；常规诊断往往无法识别真菌感染，只有在死后才能确认检测。 这项建议探索了一种新的方法，该方法有可能在几分钟内检测到支气管肺泡灌洗液(BAL)中单一微生物的水平，并使用荧光原位杂交(FISH)RNA探针和/或PCR在2-4小时内诊断出该物种。 这项工作的目的是建立和测试一种概念验证的细胞分选方法--喷泉流分选，用于检测BAL液中的真菌。含有廉价荧光真菌染料的BAL液流用LED照明，荧光真菌细胞用数码相机检测。每次检测后，真菌细胞被分类到较小的体积，然后可以用更昂贵的免疫标记或FISH探针进行曲霉确认。然后，可以使用快速PCR或FISH探针进行物种鉴定，从而能够早期诊断侵袭性曲霉病，特别是在免疫功能低下的患者中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project will prove the concept for a low-cost, point-of-care system for real-time diagnosis of invasive aspergillosis. Invasive aspergillosis occurs in 8-15% of bone marrow transplant patients and 5-15% of solid-organ transplant patients. Mortality of infected transplant patients ranges from 30% to 70% and is nearly 100% if left untreated. Diagnostics are unreliable; too often conventional diagnostics fail to identify fungal infection and a confirmed detection is made only post mortem. This proposal explores a novel method which has the potential to detect Aspergillus down to the level of a single microorganism in bronchoalveolar lavage (BAL) fluid in minutes and to diagnose the species in 2-4 hours, using fluorescent in-situ hybridization (FISH) RNA probes and/or PCR. The objective of this effort is to build and test a proof-of-concept cell-sorting method, Fountain Flow" Sorting, for the detection of fungi in BAL fluid. A stream of BAL fluid containing an inexpensive fluorescent, fungal dye is illuminated with an LED, and fluorescent fungal cells are detected with a digital camera. After each detection, a fungal cell is sorted into a smaller volume, which can be then stained with more-expensive, immunolabel or FISH probe for Aspergillus confirmation. Rapid PCR or FISH probes can then be used for species identification, allowing for an early diagnosis of invasive aspergillosis, particularly in immunocompromised patients.