Data Sheet 1_Enhancing bloodstream infection diagnostics: a novel filtration and targeted next-generation sequencing approach for precise pathogen identification.docx

Bloodstream infections (BSIs) pose a significant diagnostic challenge, largely due to the limitations of traditional methods such as blood cultures. These methods often yield low positive rates, have lengthy processing times that delay treatment, and are limited in detecting only a narrow range of pathogens. Such delays and inaccuracies can critically impede timely clinical interventions, potentially compromising patient outcomes. Next-generation sequencing (NGS) is a powerful tool for rapid, precise pathogen identification. While metagenomic NGS (mNGS) offers broad pathogen coverage, it is often costly and complex. Targeted NGS (tNGS), however, focuses on key regions of clinically relevant pathogens, reducing costs and simplifying workflows while maintaining high sensitivity, making it more practical for routine diagnostics. In this study, we introduce a novel approach combining a human cell-specific filtration membrane with a multiplex tNGS panel to overcome these challenges. The filtration membrane, designed with surface charge properties to be electrostatically attractive to leukocytes for the selective capture of specific cells, demonstrated high efficiency in removing host cells and nucleic acids, achieving over a 98% reduction in host DNA and thereby minimizing background interference in pathogen detection. Additionally, we developed an effective multiplex tNGS panel targeting over 330 clinically relevant pathogens and verified its consistency with mNGS and blood culture results, demonstrating a significant improvement in detection sensitivity. By integrating these two methods, we achieved a synergistic enhancement in diagnostic capability, boosting pathogen reads by 6- to 8-fold, which enabled reliable identification even in cases of low-abundance pathogens. This approach provides faster, more accurate, and more sensitive detection of BSIs, enabling earlier identification of infections. This facilitates timely and targeted treatment, ultimately improving patient outcomes in critical care settings. Given the unique properties of the filtration membrane and the strengths of the tNGS panel, this approach shows promising applications in prenatal and genetic health support, as well as in advancing early cancer screening strategies.