Quantitative multiplexed-tandem PCR for direct detection of bacteraemia in critically ill patients

Culture remains the gold standard for diagnosis of blood stream infections (BSI), but its clinical utility is limited by slow turnaround times. Here we describe a method for rapid quantitative detection of bacterial DNA directly extracted from whole blood using a multiplexed tandem real-time PCR (MT-PCR) assay targeting Staphylococcus, Streptococcus, Pseudomonas, Enterococcus and Enterobacteriaceae 16S rDNA genes. Results were available less than 3.5 hours after blood collection with all five bacterial targets having limits of detection between 101 and 103 CFU/mL. A small-scale clinical evaluation of the assay using blood samples collected from 15 patients admitted to the Intensive Care Unit at our institution demonstrated 93.3% (14/15) concordance between MT-PCR and blood culture when detection of persistent bacterial DNAemia by MT- PCR was considered a true result. Further evaluation with clinical samples is needed; however, this method has potential as an effective rule-in diagnostic tool for bacteraemic sepsis and septic shock.