Direct-from-Blood Microbial Sequencing Assay for Pathogen and Antibiotic Resistance Detection in Bloodstream Infections
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Bloodstream infections (BSIs) are a leading cause of morbidity and mortality that require rapid identification of the infecting pathogen to guide treatment. However, nearly all available diagnostics for BSIs rely on blood culture and so are inherently slow, requiring 1-2 days for bacterial growth. Achieving whole genome recovery of bacterial pathogens directly from blood is challenging due to (1) low absolute abundance, with clinically relevant loads of 1 CFU/mL and only 10s of femtograms of bacterial DNA; (2) low relative abundance, as human DNA outnumbers DNA in clinical blood samples by 8-9 orders of magnitude; and (3) matrix challenges, as blood and blood collection containers contain molecular amplification inhibitors such as hemoglobin and SPS. We developed Blood2Bac™ , a novel method for species agnostic ultrahigh enrichment (UHE) of bacteria directly from whole blood that depletes human DNA by 8-10 orders of magnitude. Blood2Bac is followed by whole genome sequencing (WGS) on an Oxford Nanopore Technologies sequencing device for rapid data generation. WGS data is analyzed with Keynome® ID, our algorithm for species pathogen identification in mixed human/pathogen samples, and Keynome® gAST (genomic antibiotic susceptibility testing), our machine learning algorithm for determining AST from genomic sequences to accurately predict resistance. With an end-to-end turnaround time of 8 hours, we demonstrate comprehensive capabilities for whole genome recovery, species identification, and AST determination direct from blood at low input concentrations.