Simple dual filter workflow for facilitating blood culture-free and sensitive detection of pathogenic bacteria from blood.

Bloodstream infections (BSI) caused by pathogenic bacteria are a global health threat, highlighting the need for improved diagnostic methods. Current methods, such as mass spectrometry, depend on time-consuming blood cultures, which hinder timely diagnosis. Nucleic acid amplification tests (NAATs) offer potential for sensitive bacteria detection but are bottlenecked by the inability to isolate bacteria at low concentrations from blood. This study introduces a facile dual filter workflow for isolating low concentrations of bacteria from blood without blood culture. The workflow, which involves blood dilution and osmolysis, blood cell filtration, chemical and enzymatic lysis, and filter-based bacteria capture, can be completed in ~ 30 min without special equipment. It readily combines with downstream broad-based bacteria detection via PCR and high-resolution melt (HRM), as well as pheno-molecular antimicrobial susceptibility testing (AST) via PCR. When coupled with species-specific PCR, ~ 10 CFU of bacteria in 0.5 mL of blood could be detected. The small sample volume is particularly attractive because it is comparable to those obtained from fingerstick collections or pediatric patients. With further optimization, testing with a wider range of bacteria, and integration with rapid point-of-care testing, this simple dual filter workflow can be a key driver for advancing blood culture-free bacterial BSI diagnosis, especially in resource-limited settings.