Clinical application and performance evaluation of metagenomic next-generation sequencing for pathogen detection in patients with suspected sepsis: a prospective study

Metagenomic next-generation sequencing (mNGS) enables robust pathogen identification, yet excess host-derived DNA interference limits its accuracy. This study sought to characterize and compare the performance of two mNGS testing strategies in patients with suspected sepsis, to inform optimized clinical mNGS utilization. We prospectively enrolled 121 patients with suspected sepsis and focal infections to systematically compared the diagnostic performance of peripheral blood (PB) host depletion (PB-HD) mNGS, plasma cell-free DNA (cfDNA) mNGS, their combined testing regimen, alongside conventional microbial culture and focal specimen mNGS. Mock samples containing quantitative microorganisms were employed to evaluate host depletion efficiency and pathogen detection capability of PB-HD mNGS. PB-HD mNGS exhibited relatively higher positive percent agreement (PPA) (73.33% vs. 66.67%), negative percent agreement (NPA) (62.63% vs. 41.76%), overall percent agreement (OPA) and species matching consistency for co-positive cases relative to cfDNA mNGS, noting they interrogate distinct blood fractions, indicating a tendency toward better consistency between PB-HD mNGS and blood culture. Stratified analysis based on inflammatory biomarkers revealed that cfDNA mNGS yielded higher positivity in patients with prominent systemic inflammatory activation (elevated neutrophil proportion, CRP and PCT), while logistic regression analyses identified CRP as the only independent inflammatory marker correlated with cfDNA mNGS and combined mNGS ​positivity. Additionally, PB-HD mNGS positive results were observationally linked to prolonged ICU stay and higher in-hospital mortality, whereas cfDNA mNGS outcomes showed no ​statistical correlation with these prognostic endpoints. PB-HD mNGS presented relatively higher concordance with blood culture in suspected sepsis and focal infections, while cfDNA mNGS tended to show higher detection positivity in severe inflammation and abdominal infection without lesion samples. The combination of PB-HD and cfDNA mNGS provides a robust alternative when invasive sampling is unavailable. These findings imply clinicians may select appropriate mNGS modalities tailored to patient inflammatory status and infection focus.