Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive Candidiasis

Abstract

Quantitative real-time PCR (qPCR) is considered one of the most sensitive methods to detect low levels of DNA from pathogens in clinical samples. To improve the design of qPCR for the detection of deeply invasive candidiasis, we sought to develop a more comprehensive understanding of the kinetics of DNA released from Candida albicans in vitro and in vivo. We developed a C. albicans-specific assay targeting the rRNA gene complex and studied the kinetics of DNA released from C. albicans alone, in the presence of human blood monocytes (H-MNCs), and in the bloodstream of rabbits with experimental disseminated candidiasis. The analytical qPCR assay was highly specific and sensitive (10 fg). Cells of C. albicans incubated in Hanks balanced salt solution (+/-10% bovine serum albumin [BSA]) or RPMI (+/-10% BSA) showed a significant release of DNA at T equal to 24 h compared to T equal to 0 h (P < or = 0.01). C. albicans incubated with H-MNCs exhibited a greater release of DNA than C. albicans cells alone over 24 h (P = 0.0001). Rabbits with disseminated candidiasis showed a steady increase of detectable DNA levels in plasma as disease progressed. Plasma cultures showed minimal growth of C. albicans, demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release by C. albicans collectively demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, that phagocytic cells may play an active role in increasing this release over time, and that plasma is a suitable blood fraction for the detection of C. albicans DNA.

FIG. 1.

Schematic of the rRNA gene showing the highly conserved regions of the 18S (partial), 5.8S, and 28S (partial) subunits with the less-conserved intervening ITS1 and ITS2 regions of C. albicans. Candida albicans-specific (CAS) (+) and CAS (−) primers for the qPCR assay anneal to ITS1 and ITS2, respectively. The FITC and RD640 fluorescent probes anneal to the ITS1 region. The amplicon generated by CAS (+) and CAS (−) was 218 bp in size.

FIG. 2.

(A) Serial dilutions of C. albicans genomic DNA, ranging from 1 × 10⁶ fg to 1 × 10⁰ fg. (B) The linear regression line shows the linearity of the assay (r = 1.00) with PCR efficiency equal to 1.95. F2/F1 is the ratio of the data from two of the channels of the LightCycler instrument which is applied for assays utilizing a LC-Red 640-fluorescein hybridization probe pair.

FIG. 3.

In vitro kinetics of extracellular DNA released from C. albicans cultured in HBSS alone and HBSS plus 10% BSA over a 24-h period. †, P = 0.001 in comparison to DNA levels at T = 0 h. The figure represents results from nine replicate experiments. The concentration of DNA is expressed in femtograms.

FIG. 4.

In vitro kinetics of extracellular DNA released from C. albicans cultured in RPMI alone and RPMI plus 10% BSA over a 24-h period. †, P = 0.001 in comparison to DNA levels at T = 0 h. The figure represents results from nine replicate experiments. The concentration of DNA is expressed in femtograms.

FIG. 5.

In vitro kinetics of extracellular DNA of C. albicans cultured in HBSS alone and in the presence of H-MNCs (effector-to-target ratio 1:1). Values are given as means ± SEM. Results were analyzed using a nonlinear regression analysis. Data were fit to a one-phase exponential association model (R² > 0.96, SEM < 0.5 log₂). P = 0.0001, comparing DNA levels of C. albicans alone versus C. albicans plus H-MNCs at T = 24 h. Concentration of DNA is expressed in femtograms.

FIG. 6.

Quantitative DNA levels in plasma and parallel quantitative cultures from plasma and blood pellet from rabbits with experimental candidiasis (days 1 through day 8 postinoculation). Quantitative cultures from blood pellet reflect the microbiological progression of disease in parallel with increasing plasma DNA concentrations. Low values of quantitative cultures of plasma indicate that DNA detected in plasma was predominantly fungal cell-free DNA. Concentration of DNA is expressed in femtograms.

FIG. 7.

Stability of cell-free C. albicans DNA suspended in (A) water, (B) rabbit plasma, and (C) human plasma over time (T = 0, 24, 48, and 72 h). Suspended DNA was incubated at room temperature, 4°C, and −30°C. Cell-free C. albicans DNA remains stable up to 72 h in water and rabbit and human plasma at all experimental temperatures.