Optimizing and Evaluating Nanopore-Based Targeted and Metagenomic Sequencing Workflows for Rapid Diagnosis of Acute Invasive Infections from Normally Sterile Body Fluids

Rapid and accurate pathogen identification is critical for managing acute invasive infections. Conventional culture methods are time-consuming, delaying effective treatment. Nanopore sequencing offers real-time, long-read capabilities suitable for clinical diagnostics, yet standardized workflows remain lacking. This study developed and evaluated two optimized nanopore sequencing workflows: Nanopore Targeted Sequencing (NTS) and Nanopore Metagenomic Sequencing (NMgS), for pathogen and antimicrobial resistance (AMR) detection in 177 normally sterile body fluid samples. NTS used multiplex PCR to amplify 16S rRNA, ITS, and 21 AMR genes, while NMgS applied host DNA depletion followed by unbiased sequencing. Both workflows were benchmarked against culture-based diagnostics. Among the 304 species cultured from 177 body fluid samples, NTS identified 78.95%, with 77.30% meeting the threshold of relative abundance (TRA) of 0.058 and 71.38% having at least 10 classified reads. In comparison, NMgS identified 39.47% of cultured species at the species level and 9.54% at the genus level. Of the 28 samples containing AMR ESKAPE pathogens, NTS detected associated AMR genes in 24 samples (85.71%), while NMgS identified AMR genes linked to 9 of the 32 ESKAPE pathogens (28.13%). The turnaround times for NTS and NMgS workflows were 10.75 and 12.82 hours, respectively. In conclusion, this study demonstrated the clinical utility of Nanopore sequencing for rapid diagnosis in clinical microbiology. The heightened sensitivity of Nanopore targeted sequencing renders it ideal for routine clinical microbiology diagnoses, whereas unbiased Nanopore metagenomic sequencing is advantageous in identifying infections of unknown etiology.