Targeted next-generation sequencing: a promising approach for pathogen and antimicrobial resistance gene detection in...

Urinary tract infections (UTIs) are common and frequently recurrent. As traditional diagnostic methods fail to meet clinical needs, this study aimed to construct and evaluate the performance of targeted next-generation sequencing (tNGS) for detecting pathogens and antimicrobial resistance genes (ARGs) in urine specimens.

A total of 446 urine samples from patients with suspected UTIs were tested by tNGS, routine culture, and antimicrobial susceptibility testing (AST), with discordant results resolved by metagenomic NGS (mNGS) and whole-genome sequencing (WGS). The performance of tNGS was evaluated against culture and a composite reference standard (CRS) for pathogen detection, and against AST and WGS for resistance prediction.

tNGS achieved limit of detections of 500-1,000 CFU/mL, with high precision. Compared to culture, tNGS achieved a higher positive rate (62.78%), along with an overall sensitivity of 90.05% and specificity of 56.28%, whereas against the CRS the sensitivity and specificity reached 99.54% and 97.49%, respectively. For the seven common UTI bacteria, both metrics exceeded 90% regardless of the reference standard. Additionally, tNGS outperformed culture in detecting hard-to-culture microbes and polymicrobial infections. Among 132 samples with available AST results, ARG-based prediction showed strong concordance with resistance phenotypes for carbapenems (100.00%) and third generation cephalosporins + β-lactamase inhibitors (91.67%). Factors including sex and urine leukocyte count > 10 cells/μL were found associated with positive tNGS results.

tNGS enables rapid, sensitive and comprehensive detection of UTI pathogens and ARGs, covering hard-to-culture pathogens and mixed infections, thereby showing promise in improving diagnosis. However, it complements rather than replaces phenotypic AST, particularly for penicillin-class antibiotics.