Identification of Candida albicans and Aspergillus fumigatus in spiked blood samples by real-time polymerase chain reaction (RT-PCR) method

Ref ID: 19244

Author:

A. Pietrzyk*, T. Gosiewski, M. Brzychczy-Wloch, P. Heczko, M. Bulanda

Author address:

Cracow, PL

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

Objectives: Increase in the incidence of invasive fungal infections (IFI) makes it necessary to search for effective and rapid methods of fungi detection in blood samples. The sensitivity of PCR methods is affected by techniques of DNA isolation and the number of fungal cells in samples.
The aim of this study was to evaluate three commercial DNA extraction kits for the recovery of fungal DNA and to detect the sensitivity of real-time PCR in the identification of Candida albicans (ALB) and Aspergillus fumigatus (FUM) in spiked blood samples.
Methods: Venous blood samples collected from healthy volunteers were contaminated with conidia of FUM (strain ATTC 14110) and with cells of ALB in the yeast form (strain ATTC10231). Blood samples were spiked with various concentrations of conidia or yeast cells and contained from 10^0 to 10^5 cells/ml. Isolation of DNA was performed using three kits (I) GeneMATRIX Quick Blood (EURx), (II) QIAamp DNA Blood (QIAGEN) and (III) Genomic Mini (A & A Biotechnology) and was preceded by a preliminary phase of cell lysis to remove PCR inhibitors: incubation with 0,17 M ammonium chloride, shaking of samples with glass beads (FastPrep, MP Biomedicals), incubation with 75mM NaOH and incubation with beta-mercaptoethanol and lyticase. Following pre-lysis step, DNA isolation was performed according to the manufacturers’ protocols. For each of the isolates purity and concentration of DNA were measured spectrophotometrically at a wavelength of 260 and 280 nm (NanoDrop, ThermoScientific). DNA was amplified in real-time thermal cycler CFX96 (BioRad) using primers, complementary to the 18S rRNA sequences and the TaqMan probes labeled with HEX and BHQ. The analysis was based on reading the C (t) value.
Results: The highest concentration of DNA was found for samples obtained with the III extraction kit. The highest purity was noted in samples obtained with the I set.
It was also found that the amplification signal was registered at the earliest in case of DNA isolates obtained using the I set. Sensitivity of the real-time PCR method was 3×10^2 cfu/ml for samples spiked with FUM as well as for those inoculated with ALB.
Conclusion: A real-time PCR assay is a useful tool for the rapid identification of ALB and FUM in spiked blood samples and may be useful in the early diagnosis of IFI. However, the analysis of blood samples derived from patients suspected of IFI is needed to validate this method for clinical applications.

Abstract Number: R2709

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=163643&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

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