Rapid detection of pathogens in blood culture bottles by real-time PCR in conjunction with the pre-analytic tool MolYsis

Abstract

Rapid detection of pathogens in blood from septic patients is essential for adequate antimicrobial therapy and prognosis of patients. Aim of this study is the acceleration of detection and identification of bacteria and fungi in blood cultures by molecular methods before positive signalling in an automated system. This would allow an earlier appropriate antimicrobial therapy and may improve the prognosis of septic patients. Samples were analysed with an eubacterial real-time PCR assay that enables detection of bacterial DNA and simultaneous differentiation of Gram-positive and Gram-negative bacteria. In addition, genus- and species-specific real-time PCR assays were used. DNA preparation was performed with the new tool MolYsis. With the Gram-differentiating PCR assay bacteria were detectable in concentrations of 10-20 CFU per PCR reaction. A positive PCR result was achieved in samples taken from spiked blood culture bottles between 5.0 and 8.7h prior to positive signalling of the BACTEC system. We were able to identify the causative organism in 11 out of 18 culture-positive blood cultures from patients with septicaemia with an average of 10.7h prior to positive signalling. Out of 83 culture-negative bottles six samples showed a positive PCR result. PCR analysis in conjunction with MolYsis DNA preparation allows rapid detection of pathogens in blood culture samples. Thus, the approach may be a valuable supplemental tool for blood cultures in patients with suspicion of infection with slow-growing pathogens or serious clinical condition.