Fast-tracking fungal detection: a rapid flow cytometric method for the detection of yeasts in blood cultures

Invasive fungal diseases (IFDs), such as candidemia are increasing in prevalence globally and impose greater risk of life-threatening infection for immunocompromised and hospitalized patients if not treated with appropriate antifungals. The "gold-standard" method for candidemia detection is culture based with low sensitivity, often taking days to obtain results. We have developed a novel flow-cytometric method that detects yeast cells in blood cultures using two fluorescent markers, providing results in just over 1 h. In our study, we investigated the performance of the markers in clinical isolates across 6 different yeast species in spiked blood cultures. Using flow-cytometry and confocal microscopy we investigated the staining specificity of the markers to non-yeast cells (i.e., human cells and bacteria) and validated our gating strategy using imaging cytometry. Our limit of detection using conventional flow cytometry was 2.871 × 10³ cells/mL in spiked cultures and we successfully applied our assay to yeast positive clinical blood cultures, with the ability to detect unusual species such as Saccharomyces cerevisae. Compared to conventional microscopy, our assay demonstrates a superior LOD and with further optimization, could detect candidemia earlier to potentially improve patient outcomes.