Early detection and identification of commonly encountered Candida species from simulated blood cultures by using a real-time PCR-based assay

J Mol Diagn. 2004 May;6(2):108-14. doi: 10.1016/S1525-1578(10)60498-9.

Abstract

In a recent study, Candida species in clinical blood samples were detected using a real-time PCR-based method (Maaroufi et al, J Clin Microbiol 2003, 41:3293-3298). For the present study, we evaluated the efficiency of this method as an adjunct to the BACTEC blood culture system to early detection of positivity and negativity of simulated low candidemias. We first established an in vitro correlation between the inoculum of the most frequently encountered Candida species and the time to positivity of these microorganisms. Then, aliquots from blood culture bottles infected with a final average candidal inoculum of 3.18 colony-forming units (CFU)/culture bottle (range, 1 to 6 CFU) were collected at increasing incubation times, and DNA was extracted and submitted to the TaqMan-based PCR assay. To optimize this assay, we evaluated the effect of adding 0.5% bovine serum albumin (BSA) to DNA extracts and found that it decreased the effects of inhibitors. Using specific probes for the tested Candida species, the PCR assay was positive on blood culture aliquots collected from the BACTEC system after a minimum culture turnaround time (TAT) of 3.11 +/- 1.24 hours. Addition of BSA to PCR reaction mixtures improves the TAT (1.84 +/- 0.41 hours). Hence, the combination of DNA "amplification" in the culture bottles by normal growth with an additional DNA amplification by PCR might be a reliable tool facilitating the early diagnosis of low candidemias.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Blood / microbiology*
  • Candida / classification*
  • Candida / genetics*
  • Candida / growth & development
  • Candida albicans / genetics
  • Candida albicans / growth & development
  • Candida albicans / isolation & purification
  • Candidiasis / diagnosis*
  • Candidiasis / microbiology
  • Cattle
  • Colony-Forming Units Assay
  • Culture Media
  • DNA, Fungal / blood
  • DNA, Fungal / isolation & purification*
  • Humans
  • Polymerase Chain Reaction / methods*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serum Albumin, Bovine / metabolism

Substances

  • Culture Media
  • DNA, Fungal
  • Serum Albumin, Bovine