Rapid and direct quantification of viable Candida species in whole blood by use of immunomagnetic separation and solid-phase cytometry

Abstract

Candida species are a common source of nosocomial bloodstream infections in critically ill patients. The sensitivity of the traditional diagnostic procedure based on blood culture is variable, and it usually takes 2 to 4 days before growth of Candida species is detected. We developed a 4-h method for the quantification of Candida species in blood, combining immunomagnetic separation (IMS) with solid-phase cytometry (SPC) using viability labeling. Additionally, Candida albicans cells could be identified in real time by using fluorescent in situ hybridization. By analysis of spiked blood samples, our method was shown to be sensitive and specific, with a low detection limit (1 cell/ml of blood). In a proof-of-concept study, we applied the IMS/SPC method to 16 clinical samples and compared it to traditional blood culture. Our method proved more sensitive than culture (seven samples were positive with IMS/SPC but negative with blood culture), and identification results were in agreement. The IMS/SPC data also suggest that mixed infections might occur frequently, as C. albicans and at least one other Candida species were found in five samples. Additionally, in two cases, high numbers of cells (175 to 480 cells/ml of blood) were associated with an endovascular source of infection.

FIG. 1.

Numbers of Candida cells in spiking solutions (white bars) compared to the numbers obtained after application of the IMS/SPC procedure to spiked blood samples from 10 healthy volunteers (gray bars).

FIG. 2.

Numbers of C. albicans cells obtained with the IMS/SPC procedure for two spiked blood samples from one healthy volunteer (triangles and squares), determined at different time points during storage of the samples at 4°C.