Development and evaluation of CD45-conjugated magnetic particles-based host cell depletion for enhanced metagenomic next-generation sequencing in bloodstream infection.

Metagenomic next-generation sequencing (mNGS) enables unbiased detection of human pathogens without prior assumptions. However, the direct detection of bloodstream infection pathogens is limited by host DNA interference, leading to the clinical adoption of microbial cell-free DNA mNGS (plasma cfDNA mNGS). This study developed a host cell depletion method based on immunomagnetic separation using CD45-conjugated magnetic particles (CD45-MPs, termed the IP method) to reduce host DNA interference and enhance mNGS performance for bloodstream pathogen detection. In simulated samples, known concentrations of pathogens (intact Escherichia coli and Candida albicans cells, fragmented Staphylococcus aureus genomic DNA) were spiked into whole blood samples, serially diluted 10-fold, and divided into whole blood, plasma, and IP-treated groups. These groups were analyzed using hematological analysis, microscopic smear, DNA concentration measurement, relative quantification of GAPDH using qPCR, and mNGS. Results showed that CD45-conjugated magnetic particles effectively removed host cells from whole blood (reducing cell count by 99.9%). The results of nucleic acid measurement and qPCR indicated that the supernatant from IP-treated samples contained significantly lower host DNA compared to whole blood and plasma groups. mNGS detection of simulated samples demonstrated that the IP method enabled detection of pathogens at concentrations as low as 100 CFU/mL for E. coli and S. aureus, and 50 CFU/mL for C. albicans. In clinical testing of 77 samples from patients with suspected bloodstream infections, mNGS combined with the IP method showed significantly higher positivity rates than plasma cfDNA mNGS. In conclusion, CD45-conjugated magnetic particles effectively deplete human cells and enhance the clinical performance of mNGS on the detection of bloodstream infections.