| Autor: |
Carvalho-Pereira J; Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal., Fernandes F; Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal., Araújo R; Department Medical Biotechnology, Health Sciences, Flinders University, Adelaide 5042, Australia., Springer J; Department of Internal Medicine II, University Hospital of Würzburg, 97080 Würzburg, Germany., Loeffler J; Department of Internal Medicine II, University Hospital of Würzburg, 97080 Würzburg, Germany., Buitrago MJ; Mycology Reference Laboratory, National Centre of Microbiology, Instituto de Salud Carlos III, 28220 Madrid, Spain., Pais C; Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal., Sampaio P; Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho, 4710 Braga, Portugal. |
| Abstrakt: |
A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopus arrhizus . The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes. |
