BCS2.0: a capture sequencing platform for rapid differential diagnosis of bacterial infections and antimicrobial resistance

Introduction: The "Golden Hour" is the period immediately after trauma, stroke or a cardiac event when rapid intervention is critical to reducing morbidity and mortality. The same principle should also apply to infectious diseases. Rapid, sensitive detection of infectious agents, enabling targeted interventions, has the potential to reduce mortality, morbidity, and costs of infectious diseases, and to decrease the inappropriate use of antibiotics that drives the evolution of antimicrobial resistance (AMR). Methods: Probes were designed to represent the MetaPhlAn4 database covering 894 known or potential pathogenic bacterial species, 16S rRNA sequences from the SILVA database comprising 1,325 potentially pathogenic bacterial species, genes in the Virulence Factor Database and antimicrobial resistance determinants in the Comprehensive Antibiotic Resistance Database. Target sequences were tiled with 120 nucleotide probes distributed at 60 nt intervals and clustered at 99% sequence identity. Performance measures included limits of detection (LOD) and assay reproducibility in plasma and urine using contrived and clinical samples. Results: Analytical validation using 20 bacterial strains in contrived plasma and urine samples confirmed an LOD of 5 colony-forming units per milliliter and detection of mixed infections. Results obtained with urine and blood cultures were concordant with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI), and Blood Culture Identification (BCID) assays. Discussion: BCS2.0 enables sensitive detection of bacterial species and AMR genes and has the potential to expedite rapid, efficient infectious disease management.