Multiplex PCR for the detection of fungemia and bacteriemia in the same run

A. Kalkanci, M. Dizbay, A. Kalkanci, K. Hizel, K. Caglar, A. Fouad, D. Arman, S. Kustimur

Author address: 

Gazi University, ANKARA, Turkey

Abstract: 

Objectives: Fungi are emerging as important etiological agents of bloodstream infections as much as bacteria. We performed a multiplex Real Time PCR based methodology to determine an accurate diagnostic method for bloodstream infections. In the present study, detection of Aspergillus fumigatus , Candida albicans , Staphylococcus aureus and Escherichia coli were aimed. Methods: The specific multiplex PCR method used in this study targets one Gram-positive species Staphylococcus aureus , one Gram-negative species Escherichia coli , and two fungal species Aspergillus fumigatus and Candida albicans . The designed primers and probe sets were based on regions of identity within the 16S rRNA gene of bacteria. Fungal primers anneal within the ITS2 region. Primer ITS-R anneals to a highly conserved region of the 28S rRNA gene of fungi. For the detection and differentiation of Aspergillus species from Candida species, 2 different species-specific biprobes were designed. For in vitro testing, EDTA-anticoagulated blood was inoculated with S. aureus , E. coli , A. fumigatus and C. albicans . For in vivo testing, 4 rabbits were used as the animal model of sepsis. They were inoculated with the microorganismas and on the fifth day, the blood was collected. DNA was extracted from simulated blood samples as well as from blood samples of the rabbits. Results: The combination of a group-specific and a universal primers for bacteria and fungi in one reaction mixture facilitated rapid screening and species differentiation by the characteristic peak melting temperatures of the probes. Both assays performed either as single assays or simultaneously in the same LightCycler run. The analytical sensitivity using pure cultures and EDTA-anticoagulated blood was shown to be at least 10 CFU per PCR, corresponding to 10 to 100 CFU/ml blood. All of the simulated blood samples were found to be positive both for bacteria and fungi. A total of 3 rabbit samples were found to be positive. Conclusion: Our data suggest that our PCR method may be appropriate for use in clinical laboratories as simple and rapid screening tests for the most frequently encountered species causing bloodstream infections. Therefore, modern diagnostic tests for bacteremia and fungemia, including the multiplex PCR studied, deserve the attention of clinicians and laboratory specialists as well as further study in clinical settings.
2009

abstract No: 

P220

Full conference title: 

4th Trends in Medical Mycology
    • TIMM 4th (2012)