Real-time, low-cost detection of individual fungal cells in blood using Fountain Flow™ cytometry

Background

As fungi are considerably more slow growing than bacteria and are not affected by broad-spectrum antibiotics, too often conventional diagnostics fail to identify fungal bloodstream infection and confirmation is made postmortem. We describe the initial tests of a Fountain Flow™ cytometer (FFC) to detect pathogenic fungi in human blood, rapidly and cost-effectively. This innovative technique is based on fluorescence in which a stream of solution containing the cells, labeled with a fluorescent stain, is illuminated with an LED (Figure 1). The resulting fluorescence is detected with a CMOS imager.

Figure 1

The Fountain Flow™ cytometer. Fluorescently stained cells flow through the flow cell toward the CMOS camera, illuminated by an LED or laser. Cell(s) in the CMOS camera focal plane are imaged by the camera through the flow cell window at the wavelength of fluorescence.

Materials and methods

The FFC used here was used by us in previous published research to detect individual bacteria and amoebae in river water at concentrations ≥200 bacteria/ml and 0.06 amoebae/ml. An optical stimulant of blood was made by mixing red blood cells (RBCs) in saline to obtain a RBC concentration comparable with that of human blood. This was further diluted 1:20 in distilled water to reduce the opacity. Then the solution was inoculated with Candida albicans from the American Type Culture Collection to obtain samples of varying concentration. To label the Candida, FUN1, a fluorescent, fungus-specific dye (Invitrogen), was used. The dye Trypan was used to suppress background, especially from RBCs, which absorb small amounts of FUN1.

Three 0.1 ml samples at each concentration were flowed into the FFC in 200 seconds (although a much greater rate was possible), while 500 images were taken continuously. The images were then analyzed (in minutes) with custom software to count the fluorescent spots in the images corresponding to Candida. Plate counts of Candida on YM agar (18 hours incubation) were used for comparison.

Results

Figure 2 shows the comparison of the FFC and plate counts.

Figure 2

Fountain Flow™ cytometer counts of C. albicans in 0.1 ml red blood cells. Comparison of Fountain Flow™ cytometer (FFC) counts and plate counts of C. albicans spiked into 1:20 diluted red blood cells (RBCs). The line of best fit gives a 52% counting efficiency.

Conclusion

The FFC has potential as a system for low-cost, real-time detection of low concentrations of fungi in human blood. Further work is required to lower the false detection rate, to increase the detection efficiency, and to perform tests on human blood with its additional complexity.