Application of rapid Nanopore metagenomic cell-free DNA sequencing to diagnose bloodstream infections: a prospective observational study

Abstract

Bloodstream infections are a major cause of mortality, often leading to sepsis or septic shock. Rapid initiation of effective antimicrobial therapy is essential for survival; however, the current gold standard for identifying pathogens in bloodstream infections, blood culturing, has limitations with long turnaround time and poor sensitivity. This delay in refining empirical broad-spectrum antimicrobial treatments contributes to increased mortality and the development of antimicrobial resistance. In this study, we developed a metagenomic next-generation sequencing assay utilizing the Oxford Nanopore Technologies platform to sequence microbial cell-free DNA from blood plasma. We demonstrated proof of concept in a prospective observational clinical study including patients (n = 40) admitted to the emergency ward on suspicion of bloodstream infection. Study samples were drawn from the same venipuncture as a blood culture sample from the included patients. Nanopore metagenomic sequencing confirmed all microbiological findings in patients with positive blood cultures (n = 11) and identified pathogens relevant to the acute infection in an additional 11 patients with negative blood cultures. This proof-of-concept study demonstrates that culture-independent Nanopore metagenomic sequencing directly on blood plasma could be a feasible supplementary test for infection diagnostics in patients admitted with severe infections or sepsis. These findings support further studies on Nanopore metagenomic sequencing for sepsis diagnostics in larger cohorts to validate and expand the results from this study.IMPORTANCEThis study demonstrates the potential of Nanopore metagenomic sequencing as a rapid, culture-independent diagnostic tool for bloodstream infections, identifying pathogens missed by conventional blood cultures. The study highlights the method's promise in improving pathogen detection and warrants further validation in larger clinical studies.

Keywords: Nanopore; bacteremia; bloodstream infections; cfDNA; metagenomics.

Fig 1

Flowchart of patient inclusion and selection for analysis.

Fig 2

(a) Flowchart of the sample handling workflow for blood cultures and mNGS samples (created with biorender.com). (b) A timeline for BSI diagnostics with Nanopore mNGS (15), Illumina mNGS (14), and blood culturing (8).

Fig 3

(a) Pie chart summarizing findings from blood culture and mNGS. (b) Total pathogen DNA burden for patients (measured in genome equivalents per microliter) positive by mNGS in BSI-confirmed and BSI-suspected and compared only for E. coli where identified. (c) Number of pathogens identified with mNGS per sample. (d) Plasma cfDNA concentrations across the different patient groups and blood donor samples.