Urinary tract infections can be diagnosed with the use of rapid molecular tests, however urine PCR tests do not yet preclude the use of gold-standard methods.

By Tyler Radke, MLS(ASCP)

Urinary tract infections (UTI) are an extremely common healthcare issue, being the most common infection seen in outpatients today. At least half of adult women1 will experience a UTI in their lifetime. For these reasons, accurate diagnosis and treatment of a UTI is of paramount importance.

How are UTIs Diagnosed Today?

Signs and symptoms of a UTI can be widely variable but include painful urination, flank or abdominal pain, and urinary frequency. Diagnosis of a UTI often involves evaluating a blend of patient clinical symptoms in combination with diagnostic testing. Laboratory tests include the urinalysis test, urine Gram stain, urine culture, and more recently the urine PCR test. 

Gold-standard laboratory tests vary in their ability to reliably detect a UTI, hence the approach to combine testing with patient signs and symptoms. Newer advancements to assess urine by multiplex PCR offer another diagnostic tool for laboratories to consider.

Traditional Laboratory Tests for UTI

Urinalysis Dipstick Test

Conventional dipstick test strips for sampling urine specimens contain multiple test pads. Each individual pad may undergo a color change in response to the presence or absence of select analytes such as red blood cells, bilirubin, protein, etc. For the assessment of a UTI, the leukocyte esterase and nitrite pads are the two most relevant dipstick measurements.  Leukocyte esterase is an enzyme released by white blood cells in response to bacteria but can also be present with certain inflammatory disorders. Detection of leukocyte esterase is associated with, but not indicative of, a UTI. Nitrite, the byproduct of nitrate, is found when E. coli and other Gram-negative bacteria in the urine reduce nitrate. A positive nitrite finding is highly specific for a Gram-negative UTI.

Urinalysis Microscopic Test

Urine is further screened by concentration and microscopic review for the presence of white blood cells, red blood cells, bacteria, and other microscopic findings. It should be noted that the presence of bacteria is not indicative of a UTI. Urine can become contaminated during the collection process, resulting in the presence of bacteria in the urine sample. Additionally, asymptomatic colonization2 can occur with foley catheter use, urinary stoma, and postmenopausal women. Findings of pyuria, the presence of white blood cells in urine, are common amongst patients with a UTI but are also found in patients with interstitial cystitis, urinary stones, kidney disease, and more.

Urine Gram Stain

Urine that has not been concentrated is placed onto a microscope slide for review. Urine may be stained or viewed with contrast. Findings of bacteria are reported as whole numbers per oil immersion field (OIF). In unspun urine, bacteria per OIF correlate to a culture result of >105 CFU/mL. The presence and staining pattern of bacteria can help influence early treatment decisions when a UTI is suspected.  However, this test can be influenced by contamination from normal urogenital flora with limited to no improvement in ability to detect UTI compared3 to urinalysis dipstick and microscopic exam.

Urine Culture

A small sample of urine is inoculated onto microbiology media and placed into an incubator to promote organism growth. The cultured bacteria (or yeast) is then identified and tested against a panel of antimicrobials. Antimicrobial susceptibility results guide provider prescribing practices as the susceptibly pattern confers which antibiotics should be effective. Further, susceptibility testing is helpful for patients with allergies and can guide inpatient isolation protocols. A major disadvantage of this test is the prolonged time it takes perform culture and antimicrobial susceptibility testing. 

Multiplex Urine PCR Panel

The application of polymerase chain reaction (PCR) on urinary samples is relatively new to UTI diagnostics. A small amount of urine is needed and the principles of nucleic acid amplification via PCR are applied to detect various bacterial and/or yeast DNA targets. This assay is performed in multiplex format with several commonly associated UTI pathogens being included in the panel. Further, these panels can include testing of antibiotic resistance genes simultaneous with pathogen detection. 

What are the benefits?

By its very nature, PCR is expected to be more sensitive than culture in the ability to detect microorganisms. As example, in one study4, PCR was positive for potential uropathogens while culture was negative 22% of the time (130/582). Positive agreement in this study between PCR and culture was 90%. This demonstrates ability to detect possible UTI organisms that culture may otherwise miss. Lack of culture growth may be due to fastidiousness of select organisms or low bacterial loads, traits less hindering to PCR as a methodology.

Another benefit is that testing can be completed same day, allowing for expedited identification, treatment decisions, and patient satisfaction.

With a multiplex design, urine PCR is able to detect a large number of microorganisms from a single sample being tested one time. By casting a wider net, PCR is not susceptible to bias that may arise from culture practices of identifying the same uropathogens repeatedly. Additionally, PCR is less likely to be confounded by bacterial overgrowth on culture that can effectively hide the true uropathogen. Along these lines, PCR multiplex testing allows for detection of co-infections which again may be harder to identify in a culture exhibiting bacterial overgrowth.

In addition, urine PCR multiplex testing includes detection of genetically encoded antibiotic resistance markers5.  Detection of resistance genes promotes rapid clinical decisions regarding appropriateness of antibiotic coverage.

What are the limitations?

The laboratory staff, patients, and provider attempting to interpret the results should be made well aware of the limitations of this test method. PCR, as a testing method, is unable to differentiate between live microorganisms and nonviable DNA6. Detection of nonviable DNA occurs post treatment, hence industry standard to avoid using PCR methods as a “test of cure7 for infectious diseases. The “test of cure” format leads to unexpected positive findings, additional treatment, unnecessary antibiotic exposure, and added healthcare waste.

Additionally, PCR cannot discriminate between asymptomatic colonization and symptomatic infection. With asymptomatic colonization occurring at rates of 5-50%2, molecular detection of organismal DNA alone cannot definitively be considered a UTI. Further complicating detection is the fact that some uropathogens are also found as normal urogenital flora8. Therefore, mere organism detection being interpreted as normal or abnormal is subject to provider interpretation, allowing for significant and impactful treatment variation from provider to provider.

Multiplex PCR testing is limited to panel design which restricts the number of pathogens being investigated. Urine PCR panels range from as low as 94 different organisms to as high as 279. By comparison, urine culture is not limited to available reaction wells, but rather the fastidious requirements of an organism; features taken into consideration when plating a urine specimen for culture.

Detection of genetic resistance markers in rapid format can be helpful in early decision-making processes. However, this, too, is limited by panel design and therefore contains limited resistance markers. Consideration then needs to be given to which resistance genes are utilized as there can be distinct differences in regional or national prevalence. That is to say, the likelihood of detecting an NDM10 or VIM producing organism may be so unlikely that there is no value by inclusion into a routine panel. 

Further, detection of resistance markers with PCR is based on the presence of an encoded gene. While many genetically encoded resistance mechanisms exist, more resistance is conferred through means not yet genetically defined. The phenotypic profile, rather than genotypic, is therefore required to assess the full spectrum of antibiotic susceptibility. 

In instances of organism co-detection, genetic resistance markers may not be easily linked to an organism of origin. As example, a urine specimen containing both Staphylococcus aureus and Staphylococcus epidermidis may produce detection of a mecA encoded gene, conferring resistance to methicillin. The mecA gene could originate from either organism. Staphylococcus epidermidis, is a common commensal organism that doesn’t require treatment unless present in a quantity minimally >103 CFU/mL11. Without means of indicating which organism is encoding the resistance gene, providers will have to use an alternative antibiotic instead of a preferred11 semi-synthetic penicillin or cephalosporin. Widespread use of alternative therapies such as fluoroquinolones lead to increased rates of antimicrobial resistance12.

Antibiotic resistance is also impacted by interactions occurring within a bacteria’s own sub-population. When setting up a broth microdilution susceptibility panel, laboratorians are taught to select multiple different colonies of a purely isolated organism as each colony may have slightly variable susceptibility. In an attempt to overcome this limitation of PCR, some vendors are offering a pooled susceptibility13 test, wherein the panel is directed against the pool of organisms in the patient’s urine. This, however, may drive toward more broadly treating and clearing all microorganisms in a site that shouldn’t be completely sterile. Unlike blood and other sterile sites, the urogenital tract should not be rendered sterile as this could lead to further dysbiosis and infection by other opportunistic organisms.

Urine PCR: Guidance from Regulatory Bodies

The Centers for Medicare and Medicaid Services (CMS) indicates there are currently no FDA cleared/approved7 uses for a urine PCR multiplexed panel as there is no peer-reviewed published literature that demonstrates improved patient outcomes. CMS requires urine PCR testing to include parallel testing using conventional culture-based detection for correlation of results. 

The College of American Pathologists (CAP) goes further with the creation of the new requirement MIC.21855 Antimicrobial Resistance Markers by Molecular Analysis. This new standard requires detection of genetic markers to be linked to a corresponding organism in the final laboratory report when molecular analysis is performed directly on patient specimens. CAP states that it wants labs to culture specimens to ensure susceptibility testing can be completed, associating resistance genes to the appropriate organisms.

Promising Future?

Urine PCR has found high praise amongst urology clinics by supplying possible uropathogens and generalized recommended treatments in a rapid format. That alone has proven valuable to those clinics and laboratories electing to use it. The ability to include fastidious organisms within the panel also provides some anecdotal benefits.

Although PCR is an undoubtedly more sensitive method than culture, multiplexed PCR is limited to detecting only those organisms included in panel design. Therefore, laboratories need to exercise considerable caution when selecting prefabricated panels. The same holds true for panels including genetic resistance markers. 

Without the use of a traditional susceptibility test or the more unique pooled susceptibility profile, treatment selection is limited to known inherent resistance profiles. Treating based on what is virtually an antibiogram, an aggregate report of generally approved antibiotics by organism, and site of infection, lacks a personalized approach.

It is clear from the CMS and CAP guidance that Urine PCR testing should not supplant parallel culture testing. The risks of doing so can be summarized to inappropriate therapy selection leading to therapy failure, propagation of antimicrobial resistance, and additional healthcare costs. 

It seems several methods will continue to exist for UTI diagnostics in a combination approach.  Individually, no test yet has adequate sensitivity and specificity for complete diagnosis and treatment management. Though there is promise for urine PCR to improve detection of UTI amongst hard-to-diagnose patients, literature supporting improved patient outcomes is still lacking.

About the Author

Tyler Radke, MLS(ASCP), has been both a generalist and technical lead of microbiology. Since 2017, he has been the laboratory manager at Bellin Memorial Hospital and Bellin Health Oconto Hospital in Wisconsin. He is also a member of the laboratory technical advisory group (LabTAG) for the Wisconsin Clinical Laboratories Network (WCLN).

References

1. Medina M, Castillo-Pino E. An introduction to the epidemiology and burden of urinary tract infections. Ther Adv Urol. 2019 May 2;11:1756287219832172. doi: 10.1177/1756287219832172. PMID: 31105774; PMCID: PMC6502976. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502976/#:~:text=Uncomplicated%20UTIs,-UTIs%20are%20the&text=The%20prevalence%20in%20women%20over,11%25%20in%20the%20overall%20population.&text=Between%2050%25%20and%2060%25%20of,UTI%20in%20the%20previous%20year.

2. Crader MF, Kharsa A, Leslie SW. Bacteriuria. [Updated 2022 Aug 7]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482276/

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4. https://dx.doi.org/10.1016/j.urology.2019.10.018

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6. Janssen KJH, Hoebe CJPA, Dukers-Muijrers NHTM, Eppings L, Lucchesi M, Wolffs PFG (2016) Viability-PCR Shows That NAAT Detects a High Proportion of DNA from Non-Viable Chlamydia trachomatis. PLoS ONE 11(11): e0165920. https://doi.org/10.1371/journal.pone.0165920

7. “MolDX: Molecular Syndromic Panels for Infectious Disease Pathogen Identification Testing.” CMS.gov. June 6, 2022. https://www.cms.gov/medicare-coverage-database/view/lcd.aspx?lcdid=39038&ver=6&bc=0

8. “Urine Good Hands: Diagnosing UTIs With Urine Cultures.” Brennan-Krohn, Thea. American Society for Microbiology. Feb. 8 2021. https://asm.org/Articles/2021/February/Urine-Good-Hands-Diagnosing-UTIs-With-Urine-Cultur

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11.  van der Zee A, Roorda L, Bosman G, Ossewaarde JM. Molecular Diagnosis of Urinary Tract Infections by Semi-Quantitative Detection of Uropathogens in a Routine Clinical Hospital Setting. PLoS One. 2016 Mar 8;11(3):e0150755. doi: 10.1371/journal.pone.0150755. PMID: 26954694; PMCID: PMC4783162. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783162/#!po=78.5714

12.  Shaikh, Nader and Hoberman, Alejandro. Urinary tract infections in infants older than one month and young children: Acute management, imaging, and prognosis. UpToDate.com. October 2022. https://www.uptodate.com/contents/urinary-tract-infections-in-infants-older-than-one-month-and-young-children-acute-management-imaging-and-prognosis?search=UTI%20treatment&sectionRank=1&usage_type=default&anchor=H8&source=machineLearning&selectedTitle=1~150&display_rank=1#H8

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