New approaches in fungal DNA preparation from whole blood and subsequent pathogen detection via multiplex PCR

Abstract

Sepsis is a life-threatening disease that results from excessive host responses to microbial infections. Fungal pathogens mainly contribute to lethal outcomes and high treatment budgets. Numerous trials revealed that the mortality rates of septic patients could be reduced if appropriate anti-infective approaches are promptly initiated. This demands a forthwith identification of the causative pathogen(s) and antibiotic resistances. However, standard procedures (e.g., blood cultures) deliver first results after 2-3 days. Facing the time-to-result for cultural pathogen detection, culture independent nucleic acid amplification techniques (NAT) are increasingly desirable to deliver a reliable basis for a targeted antibiotic regimen within the first decisive hours of the disease. Crucial steps in the detection of pathogens within whole blood concern cell lysis and the disproportion of pathogen and human background DNA Standard analytical methods applied and current developments in sepsis diagnostics are reviewed. New tools are introduced which accelerate the clinical investigation course and improve the sensitivity as well as the quality of NAT-based genus and species detections. © 2010 Springer-Verlag Berlin Heidelberg.