This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.
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