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 < 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.

Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients

“…However, these findings cannot readily be generalized to candidaemic patients, and the data presented here suggest that this pattern may not hold true for candidaemia. Furthermore, studies of the kinetics of DNA release by Candida species demonstrate that cell-free fungal DNA is released into the bloodstream of hosts with disseminated candidiasis, and it seems that phagocytic cells play an active role in increasing this release over time (Bougnoux et al, 1999;Kasai et al, 2006). Consequently, the apparently better performance of serum samples in this study could perhaps be related to the fact that the patients were non-neutropenic.…”

“…It has been postulated by Bougnoux et al (1999) and Kasai et al (2006) that cell-free fungal DNA is present in the bloodstream of patients with invasive candidiasis and that this may readily be extracted from serum; however, it is noteworthy that this is based on an experimental rabbit model. Notwithstanding this, our previously reported data provide clinical support for the applicability of serum in this context to humans .…”

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

“…Separate PCR-fluorescence resonance energy transfer reactions were performed on all samples to test for any inhibitors of PCR. A master mix with Candida albicans primers-probes was used as previously described (17). Additionally, 5 l of water was added to 15 l of the master mix as a reference for results showing no inhibition.…”

Gastrointestinal tract distribution of Salmonella enteritidis in orally infected mice with a species-specific fluorescent quantitative polymerase chain reaction

“…Based on previous studies, the standard cur ve was generated as follows [7][8][9] : Standard DNA from S. enteritidis was used to establish a standard curve. The standard DNA contained amplified target DNA in different quantities which was included in each LightCycler run.…”