Urine diagnostic stewardship: the role of next-generation sequencing in recurrent, complicated, and culture-negative UTI

Urine diagnostic stewardship centers on using laboratory testing thoughtfully to improve patient outcomes while minimizing unnecessary antibiotics and misdiagnosis. Clinical guidance from organizations such as the Infectious Diseases Society of America emphasizes that urine testing should be driven by symptoms and clinical context, not performed routinely in asymptomatic individuals [1], and always interpreted with an understanding that not all detected microbes represent true infection. A key principle is distinguishing infection from colonization and contamination, particularly in a biological environment like the urinary tract where low levels of microbes may be present without causing disease.

Traditional urine culture remains the standard diagnostic method, but it has important limitations that affect patient care. It may fail to detect fastidious or slow-growing organisms, underestimate polymicrobial infections, and produce false negatives in patients who have already received antibiotics or who have recurrent or complicated infections. These gaps can lead to repeated empiric treatment, persistent symptoms, and frustration for both patients and clinicians.

Next-generation sequencing (NGS), also called clinical metagenomics in our field, introduces a complementary, culture-independent approach that can identify a broader spectrum of bacterial and fungal organisms, as well as antimicrobial resistance markers, directly from urine samples. When applied appropriately, NGS has particular value in complex clinical scenarios such as recurrent and complicated urinary tract infections, persistent symptoms despite treatment, or culture-negative cases where suspicion for infection remains high. However, antibiotic stewardship principles remain essential: detection alone does not equal disease, and results must be interpreted in the context of symptoms, organism burden (or presence/absence based on a clinical threshold), and clinical history. Advances such as machine learning–based classifiers can improve specificity by helping distinguish likely pathogens from background microbial signals.

BIOTIA-ID Urine is an NGS test designed to bring this advanced diagnostic capability into patient care by combining metagenomic sequencing with analytical methods that improve clinical relevance. The assay is analytically validated as a laboratory-developed test [2]; it enables detection of bacteria and fungi that may be missed by culture, supports identification of polymicrobial infections, and provides antimicrobial resistance gene insights that can guide therapy selection.

A key component of this approach is the integration of machine learning within the NGS workflow. By training on large clinical datasets, Biotia’s classifier (BIOTIA-DX) helps establish clinically meaningful thresholds that differentiate true infection from background microbiome, colonization, or contamination. Rather than relying solely on presence or absence of organisms, the model evaluates patterns such as relative abundance, co-detection signatures, and known pathogenicity to prioritize organisms most likely driving disease. This added layer of interpretation improves diagnostic accuracy, reduces false positives, and supports more confident clinical decision-making, particularly in complex or polymicrobial samples where conventional methods often fall short.

In practice, a stewardship-aligned approach begins with standard evaluation for patients presenting with typical, uncomplicated urinary symptoms. Initial management may still rely on urinalysis and culture where appropriate. However, when patients experience recurrent infections, fail to respond to treatment, have an infection that occurs alongside other health issues, physical blockages, or medical devices which indicates a potential complicated UTI, or have negative cultures despite ongoing symptoms, incorporating an NGS-based test such as BIOTIA-ID Urine can provide additional resolution. The results are then interpreted alongside the patient’s symptoms and history, using classifier-informed outputs to prioritize clinically meaningful organisms. This approach supports more targeted therapy, helps avoid unnecessary antibiotics directed at colonizing organisms, and can reduce the cycle of repeated empiric treatments.

Because a comprehensive, culture-independent assay detects organisms across the full spectrum from established urogenital pathogens to commensal and contaminating flora, its value depends on selecting the right patients. BIOTIA-ID Urine is intended as a problem-solving test for patients for whom standard culture is most likely to fail or to mislead, not as a screening test for every urinary complaint. Eligibility is established at the point of order through a structured intake that captures the patient’s current symptoms and their onset, risk factors, prior UTI history and previous response to treatments, culture and/or PCR results, comorbidities, and any urinary devices or procedures.

These inputs map to three groups of patients in whom NGS-based testing adds the most clinical value: those with recurrent or complicated UTI [3]; those who remain symptomatic despite negative or non-diagnostic cultures; and those at high risk for urosepsis or other poor outcomes because they are immunocompromised or medically complex.

The three indicated groups overlap in practice — a transplant recipient with recurrent, culture-negative symptoms may satisfy all three — and it is precisely these patients, who accumulate multiple risk factors, for whom conventional workup most often falls short. Conversely, the test is not indicated for asymptomatic bacteriuria or for routine, uncomplicated infections expected to respond to first-line therapy, where standard culture remains appropriate and additional testing would run counter to stewardship goals.

In a study conducted with SUNY Downstate Health Sciences University [4], BIOTIA-ID Urine was applied to 201 culture-negative urine specimens from symptomatic, high-risk adults — median age 64, with diabetes in 24%, immunosuppression in 12%, an indwelling catheter in 19%, and 35% requiring hospitalization — whose standard cultures showed no growth, mixed or contaminated flora, or growth below the diagnostic threshold. The assay identified a urogenital pathogen in 61.5% of these culture-negative specimens (including one-third of true no-growth cultures) and detected antimicrobial-resistance gene markers in 37.5%, with 100% agreement against confirmatory qPCR.

When the results were compared retrospectively with the antibiotics actually prescribed — blinded to the NGS result — empiric therapy was fully appropriate in only 15% of treated patients, while NGS findings indicated a potential change in management in 85%, including de-escalation of unnecessary broad-spectrum therapy in 56% of those on active treatment and detection of a treatable pathogen in 54% of patients who had received no antibiotics at all. As a single-center, retrospective analysis, these treatment implications reflect modeled concordance rather than prospectively measured outcomes; prospective, outcomes-linked studies are planned.

The culture-negative population is also resource-intensive. In hospitalized patients, inappropriate empiric therapy has been associated with roughly 5.2 additional hospital days and $10,312 in excess cost per patient [5], and a single complicated-UTI hospitalization averages about $15,000. By converting a culture-negative result into a specific pathogen-and-resistance profile within approximately 24–36 hours, BIOTIA-ID Urine can shorten time to appropriate therapy and support de-escalation — precisely where empiric management most often fails.