A rapid, highly sensitive and culture-free detection of pathogens from blood by positive enrichment

- Human genomic DNA background affects detection of microbes at < 10 CFU/mL when present in blood
- We demonstrate that positive enrichment using ApoH beads allows the detection of these microbes at 1 CFU/mL in blood
- An analytical sensitivity of 1 CFU/mL was demonstrated with E. coli, Enterococcus gallinarum and Candida albicans
- This method also decreases the sample volume significantly thus simplifying the subsequent nucleic acid extraction process

Molecular diagnostics is a promising alternative to culture based methods for the detection of bloodstream infections, notably due to its overall lower turnaround time when starting directly from patient samples. Whole blood is usually the starting diagnostic sample in suspected bloodstream infections. The detection of low concentrations of pathogens in blood using a molecular assay necessitates a fairly high starting volume of blood sample in the range of 5-10 mL. This large volume of blood sample has a substantial accompanying human genomic content that interferes with pathogen detection. In this study, we have established a workflow using magnetic beads coated with Apolipoprotein H that makes it possible to concentrate pathogens from a 5.0 mL whole blood sample, thereby enriching pathogens from whole blood background and also reducing the sample volume to ~ 200 μL or less. We have also demonstrated that this method of enrichment allows detection of 1 CFU/mL of Escherichia coli, Enterococcus gallinarum and Candida tropicalis from 5 mL blood using quantitative PCR; a detection limit that is not possible in unenriched samples. The enrichment method demonstrated here took 30 min to complete and can be easily integrated with various downstream molecular and microbiological techniques.