Four Hour UTI Test Outperforms Hospital Cultures on Speed and Accuracy

By Pooja Toshniwal PahariaReviewed by Frances BriggsDec 11 2025

A new study published in Nature Communications reports a step forward in urinary tract infection diagnosis. 

Study: Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in approximately four hours. Image Credit: Lothar Drechsel/Shutterstock.com

A research team has developed a culture-free nanopore metagenomic workflow that identifies pathogens and predicts antibiotic susceptibility in roughly four hours, compared with the two to four days required for standard urine cultures.

With faster and more accurate results, the new method could sharply reduce unnecessary empirical antibiotic treatment, possibly avoiding more than a billion doses of broad-spectrum drugs every year.

UTIs affect more than 400 million people annually and can escalate into severe conditions such as pyelonephritis and urosepsis. These complications account for almost a quarter of all sepsis cases. 

Traditional cultures are frequently affected by slow turnaround times, polymicrobial infections, and fastidious organisms. With antimicrobial resistance continuing to rise, clinicians need diagnostic tools that can guide targeted therapy from the beginning of treatment, rather than days later.

Get all the details: Grab your PDF here!

Testing Tests: Evaluating 11 UTI Kits

The researchers assessed eleven sample-processing strategies, including eight in-house methods and three commercial kits, using urine from 78 patients with complicated UTIs.

They aimed to overcome two persistent obstacles in metagenomic testing: low bacterial loads in urine and the overwhelming background of human DNA.

Their top-performing method used saponin to lyse host cells, followed by a medium salt-activated endonuclease to further remove human DNA. Additional lysozyme or bead-beating steps helped break open Gram-positive bacteria, improving DNA recovery.

The extracted material was sequenced using nanopore technology and benchmarked using MALDI-TOF for pathogen identification and VITEK-2 for antibiotic susceptibility profiling.

Results of the Investigation

The optimized workflow demonstrated a pathogen identification accuracy of 99 % and predicted antibiotic susceptibility with 90 % accuracy and 95 % specificity, an important safeguard against overestimating resistance.

The system detected pathogens at concentrations as low as 32 bacterial cells per microlitre and at bacteria-to-host ratios previously considered too low for reliable metagenomic testing.

Pathogens appeared within the first 25 minutes of sequencing, while resistance genes emerged in some instances in as little as three minutes.

A key achievement reported by the authors was a thousand-fold reduction in human DNA, far exceeding the performance of commercial kits and allowing clear detection of pathogens in mixed infections.

The method also uncovered thirteen pathogens that routine culture had missed, including Aerococcus urinae and Actinotignum schaalii. These identifications were later confirmed using Vivalytic or PCR-based assays.

The patient cohort reflected real-world complexity: 41 % of samples were polymicrobial, and 84 % of pathogens were resistant to at least one antibiotic. More than half were multidrug-resistant.

The metagenomic workflow provided high-resolution insights into resistance mechanisms, detecting features such as AcrAB-TolC efflux pumps and β-lactamase variants.

The authors noted, however, that the presence of chromosomal ampC variants or certain efflux pumps does not always translate into measurable resistance, a nuance essential for the clinical interpretation of metagenomic antibiotic predictions.

Cost and Practicality

The workflow costs approximately $36 per sample, around a third cheaper than comparable commercial kits.

The authors also demonstrated that DNA yield and flow cytometry bacterial cell counts could reliably predict culture positivity. Samples above roughly 273 nanograms of DNA or 451 bacterial cells per microlitre were highly likely to be true infections.

Using these thresholds as a pre-screening step could reduce the total cost to about six dollars per sample by avoiding the sequencing of negative cases.

However, because saponin disrupts sterol-containing membranes, the current workflow performs poorly with fungal pathogens. The team is refining the method to address this limitation and is validating it further in more than 200 additional clinical samples.

What This Could Mean for UTI Diagnosis

Reducing diagnostic time from days to hours could transform the management of complicated UTIs.

Earlier, more accurate treatment decisions would help prevent progression to severe disease, reduce hospital stays, and support global antimicrobial stewardship efforts by limiting unnecessary use of broad-spectrum antibiotics.

Journal reference

Bellankimath, A.B., Branders, S., Kegel, I. et al. (2025). Metagenomic sequencing enables accurate pathogen and antimicrobial susceptibility profiling in complicated UTIs in approximately four hours. Nat Commun, DOI: 10.1038/s41467-025-66865-8

https://www.nature.com/articles/s41467-025-66865-8