Validation of a Quantitative Molecular Assay for Direct Detection of Invasive Aspergillosis
Recent increases in the number of immunocompromised individuals has led to concerns about invasive fungal infection (IFI) susceptibility, particularly among the general population in the United States. These individuals include those with neutropenia, HIV, recent major organ transplant, chemotherapy, and severe burns and injuries. These individuals are at increased risk for infection by Candida, Aspergillus, and Mucormycetes species, the most common fungal pathogens; and all associated with high mortality rates. Current diagnostic methods rely on culturing, microscopy, and histopathology to obtain a specieslevel diagnosis necessary to determine an effective treatment plan, but these methods are time-consuming and lack sensitivity. Culture, the “gold standard” for fungal identification, can take days to weeks for identification. Delayed or missed diagnosis is associated with significant increases in mortality, highlighting the need for a more rapid, accurate, and direct test. The goal of this project was to validate a quantitative molecular assay, designed and developed at the University of California Davis Medical Center, Clinical Microbiology Laboratory, for direct detection of invasive Aspergillus from clinical specimens using real-time polymerase chain reaction. The assay detects the four most common infectious Aspergillus species: A. fumigatus, A. niger, A.flavus, and A. terreus. Patient samples were tested for analytical sensitivity, analytical specificity, precision, and accuracy based on guidelines for the validation of quantitative molecular assays. Preliminary results show consistent identification of positive samples, lack of interference, good reproducibility, and comparable accuracy to culturing. This assay may decrease turnaround time for fungal diagnosis from 4-9 days to 3-4 hours, potentially leading to more rapid treatment and better health outcomes.