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NGS brings power and potential to molecular analysis of urine specimens


Next-generation DNA sequencing can identify more than 50,000 microorganisms.

Urine culture remains the standard of care for the diagnostic evaluation of patients with complicated and recurrent urinary tract infections (UTIs), but next-generation DNA sequencing (NGS) is its future, according to J. Curtis Nickel, MD.

J. Curtis Nickel, MD

J. Curtis Nickel, MD

“The development of molecular technology for diagnosing infections of the urinary tract has outstripped our knowledge of how to use it, but understanding of its clinical application is catching up, thanks to ongoing research,” said Nickel, who is a professor of urology at Queen’s University School of Medicine in Kingston, Ontario.

“When the studies are completed, I strongly believe that NGS will be ready for prime time, and the use of urine cultures will be relegated to the wastebasket of medical history,” Nickel said.

In interviews with Urology Times®, Jonathan Rubenstein, MD, and Jennifer Anger, MD, MPH, agreed that NGS holds great promise as a diagnostic test for patients with lower urinary tract symptoms, but they felt more work is needed before it is adopted into clinical care. In contrast, based on extensive experience using the technology, Timothy Hlavinka, MD, considers NGS a revolutionary development and invaluable for evaluating challenging patients with lower urinary tract symptoms.

Urine culture, which detects uropathogens based on visible inspection, is recognized to have limitations as a method for UTI diagnosis, including lack of clarity on the threshold for growth to define clinically significant bacteriuria and the difficulty of culturing most bacteria, including those living in biofilms.

Molecular diagnostic techniques for uropathogen detection, including NGS and polymerase chain reaction (PCR) methodology, are based on DNA detection. PCR assays the sample for unique gene sequences that are present in a defined and limited panel of bacteria representing the most common uropathogens. It is a rapid, semiquantitative test that reports bacteria load as low, medium, or high. It also can test for genes that mediate resistance to main antibiotic families.

In contrast, NGS can identify more than 50,000 microorganisms, including most fungi, and it provides a ranking of the quantity of the detected organisms based on their percentage of the total load.

“PCR may be good if you are looking for particular organisms. However, because we now understand that cystitis-like symptoms may be caused by an abnormal microbiome [called dysbiosis], not just a single pathogen, I prefer NGS, particularly as a research tool, since it shows whatever organisms are present,” Nickel said.

The 2019 American Urological Association (AUA)/Canadian Urological Association/Society of Urodynamics, Female Pelvic Medicine & Urogenital Reconstruction guideline on recurrent uncomplicated UTIs in women discussed sensitive molecular diagnostic techniques for UTIs, but cautioned against relying on their use, said Anger, lead author of the published guideline and associate professor of urology at Cedars-Sinai Medical Center in Los Angeles, California.1

Jennifer Anger, MD, MPH

Jennifer Anger, MD, MPH

“We know now that the lower urinary tract in healthy individuals is not sterile, but we still have a lot to learn about the urinary tract microbiome in health and disease,” Anger said. “We do not yet know if the DNA of bacteria and fungi identified by NGS represent DNA only, dead organisms, live commensal organisms, or organisms causing infection. The guideline committee concluded that the clinical utility of sensitive molecular diagnostic techniques for UTI remains unproven and may lead to overdiagnosis and associated overtreatment.”

Rubenstein said that he has used NGS in the work-up of patients who present with urinary symptoms that are worrisome for infection and that have not been diagnosed by routine culture. Whereas he has found the test useful if it is negative because it allows him to rule out infection, he remains uncertain about the value of a positive test.

Jonathan Rubenstein, MD

Jonathan Rubenstein, MD

“Although I have treated some patients based on the findings of the NGS test, I do not have enough outcome data to comment about its benefit,” said Rubenstein, clinical associate professor of surgery at the University of Maryland School of Medicine in Baltimore.

Nickel said that results from NGS were a source of confusion in some patients and likely led to overtreatment of others. However, he also had patients who were symptomatic and culture-negative respond to treatment based on NGS findings.

“Once we have more information on the urinary microbiome in health and disease, I believe we will be able to use NGS to consistently get more predictable responses,” he said.

Hlavinka, who has reviewed data from more than 800 assays that he ordered plus several hundred more from his practice partners, is convinced that NGS has enabled better patient

Timothy Hlavinka, MD

Timothy Hlavinka, MD

outcomes. He said he routinely sent specimens for parallel culture in the first 6 months after he began using NGS until he was confident there was no advantage to cross-checking the results.

“Repeating a negative or errant test for patients who have had multiple negative cultures or infections that are refractory to culture and sensitivity report-directed treatment only adds to cost,” said Hlavinka, who is in private practice in San Antonio, Texas.

“Cultures often detect an organism that is not clinically significant or fail to detect a significant organism. Even if it is just 2% of the bacterial load, a significant pathogen in a symptomatic patient with a refractory UTI must be covered by the antibiotic I prescribe. That may not happen with treatment decisions driven by culture and sensitivity testing, especially if the minor pathogen has significant resistance,” Hlavinka said.

Led by Nickel and Anger, an evaluation of urine specimens obtained from the Multidisciplinary Approach to the Study of Chronic Pelvic Pain (MAPP) Research Network will help describe the normal urinary microbiome and determine if Hunner’s lesions in interstitial cystitis/bladder pain syndrome may be caused by dysbiosis of the microbiome. Nickel also is studying the normal healthy microbiome in a study funded by MicroGenDx using the company’s NGS assay.

In addition, he is analyzing information contained in the more than 50,000 reports from symptomatic patients in the MicroGenDx database, aiming to define “community states” or urotypes of microorganism and then see whether patients with diagnoses of interstitial cystitis/bladder pain syndrome or chronic prostatitis fit into specific urotypes.

“The information from this study will help us figure out the relevance of NGS in the real world,” Nickel said.

Nickel also has obtained additional support from MicroGenDx for a study to examine the potential benefit of NGS-guided treatment for patients with cystitis and/or bladder pain who are culture-negative or refractory to culture-directed antibiotic treatment.

Hlavinka said that reimbursement for the NGS test is available through Medicare and most commercial insurance plans. Nevertheless, he cautions patients upfront about the potential for an out-of-pocket cost.

“In my experience, patients needing a work-up for perplexing urinary symptoms accept the cost of the test when it is discussed in terms of the alternative scenario that can involve multiple repeat cultures, copays for additional visits and antibiotic courses, and the potential for antibiotic-related side effects,” he said.

Rubenstein, who is chair of the AUA Coding and Reimbursement Committee, said that if he contemplates ordering an NGS test, he first explains to the patient how NGS differs from standard culture and that the test may not be covered by insurance.

Rubinstein emphasized that medical necessity is the overarching criterion for payment. He also stressed the importance of following local coverage determinations for appropriate ICD-10 codes and making sure that the ICD-10 code aligns with the laboratory billing code, which can be organism-specific.

Disclosures: Nickel has received research grants from MicroGenDx; Hlavinka is a consultant to MicroGenDx.


1. Anger J, Lee U, Ackerman AL, et al. Recurrent uncomplicated urinary tract infections in women: AUA/CUA/SUFU guideline. J Urol. 2019;202(2):282-289. doi:10.1097/JU.0000000000000296

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