Open AccessUse of Quantitative Real-Time PCR To Study the Kinetics of Extracellular DNA Released from Candida albicans , with Implications for Diagnosis of Invasive Candidiasis
Author(s) -
Miki Kasai,
Andrea Francesconi,
Rūta Petraitienė,
Vidmantas Petraitis,
Amy M. Kelaher,
Hee Sup Kim,
Joseph Meletiadis,
Tin Sein,
John Bacher,
Thomas J. Walsh
Publication year - 2006
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.44.1.143-150.2006
Subject(s) - candida albicans , corpus albicans , biology , microbiology and biotechnology , dna , bovine serum albumin , systemic candidiasis , in vitro , in vivo , real time polymerase chain reaction , gene , immunology , biochemistry , genetics
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 fromCandida albicans in vitro and in vivo. We developed aC. albicans -specific assay targeting the rRNA gene complex and studied the kinetics of DNA released fromC. 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 ofC. albicans incubated in Hanks balanced salt solution (±10% bovine serum albumin [BSA]) or RPMI (±10% BSA) showed a significant release of DNA atT equal to 24 h compared toT equal to 0 h (P ≤ 0.01).C. albicans incubated with H-MNCs exhibited a greater release of DNA thanC. 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 ofC. albicans , demonstrating that DNA extracted from plasma reflected fungal cell-free DNA. In summary, these studies of the kinetics of DNA release byC. 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 ofC. albicans DNA.