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

Miki Kasai, Andrea Francesconi, Ruta Petraitiene, Vidmantas Petraitis, Amy M. Kelaher, Hee Sup Kim, Joseph Meletiadis, Tin Sein, John Bacher, Thomas J. Walsh

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)143-150
Number of pages8
JournalJournal of Clinical Microbiology
Volume44
Issue number1
DOIs
StatePublished - Jan 2006
Externally publishedYes

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Invasive Candidiasis
Candida albicans
Real-Time Polymerase Chain Reaction
DNA
Candidiasis
Bovine Serum Albumin
Monocytes
Fungal DNA
Rabbits
Phagocytes
rRNA Genes

ASJC Scopus subject areas

  • Microbiology (medical)
  • Microbiology

Cite this

Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive candidiasis. / Kasai, Miki; Francesconi, Andrea; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Kim, Hee Sup; Meletiadis, Joseph; Sein, Tin; Bacher, John; Walsh, Thomas J.

In: Journal of Clinical Microbiology, Vol. 44, No. 1, 01.2006, p. 143-150.

Research output: Contribution to journalArticle

Kasai, M, Francesconi, A, Petraitiene, R, Petraitis, V, Kelaher, AM, Kim, HS, Meletiadis, J, Sein, T, Bacher, J & Walsh, TJ 2006, 'Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive candidiasis', Journal of Clinical Microbiology, vol. 44, no. 1, pp. 143-150. https://doi.org/10.1128/JCM.44.1.143-150.2006
Kasai, Miki ; Francesconi, Andrea ; Petraitiene, Ruta ; Petraitis, Vidmantas ; Kelaher, Amy M. ; Kim, Hee Sup ; Meletiadis, Joseph ; Sein, Tin ; Bacher, John ; Walsh, Thomas J. / Use of quantitative real-time PCR to study the kinetics of extracellular DNA released from Candida albicans, with implications for diagnosis of invasive candidiasis. In: Journal of Clinical Microbiology. 2006 ; Vol. 44, No. 1. pp. 143-150.
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