Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in...

Urinary tract infections (UTIs) affect 405 million people worldwide. Current diagnostics rely on cultures, which can take 2 to 4 days. This study evaluates eleven culture-independent methods for sample preparation from 78 complicated UTI patients, followed by real-time nanopore sequencing and data analysis. The metagenomic results are highly consistent with culture-based clinical routines (MALDI-TOF/VITEK-2). The optimized method demonstrated an accuracy score of 99% (100/101) for pathogen identification and 90% (589/653) for antimicrobial susceptibility profiling with 95% specificity. The method's robustness is highlighted by its ability to accurately identify pathogens with as few as 32 bacterial cells/µL and a low bacterial-to-host cell ratio limit of 0.5. Additionally, mNGS identified 13 pathogens that routine diagnostics missed, which were subsequently confirmed by Vivalytic or PCR. This method is up to 30% more economical than published studies and commercial kits. DNA yield and flow cytometry can be used for pre-screening to reduce costs, which is crucial for clinical adoption. This research highlights the rapid diagnosis of clinical UTIs using a cost-effective and scalable method that requires around four hours from sample collection to informed decision-making. Furthermore, it aims to improve antimicrobial and diagnostic stewardship by reducing empirical treatment and ensuring more judicious antibiotic use.