Background: Invasive fungal diseases (IFDs) are an increasing global threat and associated with ≥1.5 million deaths annually1. Infections such as candidemia (the presence of Candida cells in blood) require timely antifungal administration to avoid death (currently 40% - 70% mortality rate)2. Rapid detection of IFDs is critical for patients, however conventional detection techniques are culture based and can take > 4 days to return actionable results. We investigated the feasibility of using the speed and precision of flow cytometry to detect Candida spp. from positive blood culture (BC).
Methods: 29 Candida isolates from 6 species were spiked into BC bottles (with healthy donor blood) at 1 × 105 cells/mL and incubated in the BD Bactec™ FX automated BC incubator. Once positive, human cells were lysed using a combination of 0.5% Triton-X solution and water. Samples were stained using a combination of Calcofluor white (CFW), Anti-Candida FITC (ACF), and/or Concanavalin-A (ConA), and analysed using the Attune NxT flow cytometer. The optimised method was applied to 2 clinical BC samples with concurrent plate counts performed. Fluorescence enhancement (FE) was used to measure the binding specificity of each of the stains.
Results: CFW and ACF fluorescent stains were duplexed into a single tube and displayed the best binding specificity. FE Mean (SD) values of 470.9 (308.2) and 248.2 (238.4) were observed for fungal cells compared to 5.7 (1.9) and 12.5 (6.5) for human cells when stained with CFW and ACF respectively (P < 0.0001). ConA showed statistically significant increases in fluorescence when bound to fungal cells (P = 0.0075), however there was non-specific staining of human cell components. Using CFW/ACF, we tested 2 clinical BC and were able to specifically enumerate yeast cells (lower limit of detection at 3.61 × 103 cells/mL, count agreement within 0.5 log10 CFU/mL).
Conclusions: Our novel flow cytometric assay can accurately detect and enumerate Candida cells in positive BC samples in just 1 hour.