Rapid and robust diagnosis of bloodstream infections by single-cell analysis

Sepsis, a common consequence of bloodstream infection (BSI), is a leading cause of global mortality and morbidity. Diagnosis is challenging and slow due to extremely low pathogen loads and reliance on time-consuming blood culture, necessitating empiric therapy that worsens outcomes and antimicrobial resistance. Here, we introduce sedimentation-assisted tandem rocking and enrichment for analysis and monitoring (STREAM), a platform that efficiently isolates and enriches pathogens near their natural doubling times directly from whole blood. By integrating STREAM with single-cell analysis, we achieved rapid and robust diagnosis of BSI from whole-blood samples. Using molecularly barcoded sequential fluorescence in situ hybridization, pathogen identification in 104 positive blood culture samples showed 96.15% concordance with clinical laboratory results. Gel-based single-cell antimicrobial susceptibility testing yields 97.96% essential agreement and 93.9% categorical agreement across 219 drug-dose combinations. The platform enabled comprehensive BSI diagnosis directly from whole blood, achieving a detection limit as low as 0.1 to 1 CFU/ml and delivering full diagnostic results within 6.75 to 17 hours.