Viscoelastic Separation and Concentration of Fungi from Blood for Highly Sensitive Molecular Diagnostics

Abstract

Isolation and concentration of fungi in the blood improves sensitivity of the polymerase chain reaction (PCR) method to detect fungi in blood. This study demonstrates a sheathless, continuous separation and concentration method of candida cells using a viscoelastic fluid that enables rapid detection of rare candida cells by PCR analysis. To validate device performance using a viscoelastic fluid, flow characteristics of 2 μm particles were estimated at different flow rates. Additionally, a mixture of 2 μm and 13 μm particles was successfully separated based on size difference at 100 μl/min. Candida cells were successfully separated from the white blood cells (WBCs) with a separation efficiency of 99.1% and concentrated approximately 9.9-fold at the center outlet compared to the initial concentration (~2.5 × 10 7 cells/ml). Sequential 1st and 2nd concentration processes were used to increase the final number of candida cells to ~2.3 × 10 9 cells/ml, which was concentrated ~92-fold. Finally, despite the undetectable initial concentration of 10 1 CFU/ml, removal of WBCs and the additional buffer solution enabled the quantitative reverse transcription (RT)-PCR detection of candida cells after the 1st concentration (Ct = 31.43) and the 2nd concentration process (Ct = 29.30).