UTI detection by PCR: Improving patient outcomes

Urinary tract infections (UTIs) are among the most common infections in humans, with and estimated seven million patient visits and a cost of $1.6 billion USD per year [1]. UTIs can be classified as symptomatic or asymptomatic, and uncomplicated or complicated [2]. Uncomplicated UTIs traditionally occur in a patient who: is not immunocompromised, lacks urogenital tract anatomic or functional abnormalities, has no evidence of tissue involvement, and resolves upon treatment. UTIs in immunocompromised patients, involving urogenital abnormalities or in the context of catheter placement, or recurrent infections, may require additional care and management [2]. Treatment of UTI is based on a combination of symptoms and laboratory diagnosis, which historically has been urinalysis and urine culture [3].

Interestingly, urine culture was not developed to diagnose UTIs, but rather to identify patients at risk for pyelonephritis [4]. As early as 1963, physicians recognized the need for improved methods of diagnosing bacteriuria [5]. Nevertheless, urine culture is still considered the gold standard for diagnosing UTI [3].

The development of molecular diagnostic techniques has improved the speed of detection of infectious diseases, most notably during the coronavirus pandemic [6]. Polymerase chain reaction (PCR) based molecular diagnostic techniques are widely used in clinical laboratories and physician offices around the world resulting in improved medical treatment and patient outcomes for numerous ailments [7].

At Five Valleys Urology in Missoula, Montana, we use a PCR test to identify 21 microbes and 20 antibiotic resistance markers (Table 1) that may be present in human urine specimens from patients exhibiting symptoms of UTI. To do this, we developed our own laboratory developed test (LDT) since there are no Food and Drug Administration approved assays for this purpose. According to the Centers for Disease Control and Prevention, an LDT is “a type of in vitro diagnostic test that is designed, manufactured, and used within a single laboratory” [8]. Before using it on patient specimens for clinically actionable results, our laboratory test underwent a complex validation process [9] and was approved by a board-certified clinical chemist.

Table 1.

Molecular targets in the FVU PCR panel.

Microbiome Target Name	Antibiotic Resistance Marker Name
Escherichia coli	AmpC
Enterobacter cloacae	NDM
Candida tropicalis	KPC
Candida glabrata	OXA-48
Candida krusei	VIM
Candida albicans	IMP-7
Candida parapsilosis	SHV
Candida lusitaniae	TEM
Morganella morganii	CTX-M group 1
Proteus mirabilis	CTX-M group 2
Enterococcus faecalis	MefA
Klebsiella oxytoca	VanB
Klebsiella pneumoniae	ErmA
Providencia stuartii	ErmB
Serratia marcescens	MecA
Staphylococcus saprophyticus	FemA
Staphylococcus aureus	QnrB
Streptococcus agalactiae	QnrA
Ureaplasma urealyticum	VanA1
Pseudomonas aeruginosa	VanA2
Mycoplasma hominis

We report three individuals with recurrent culture-positive UTIs for whom prior treatment had failed to eradicate the infection. Unfortunately, patient data prior to their referral to Five Valleys Urology was unavailable. Thus, these cases demonstrate the benefit of our newly developed LDT PCR assay, but the treatment decisions leading up to referral are unavailable for review.

Case 1

An 87-year-old female was referred to Five Valleys Urology by her primary care provider (PCP) after having four culture-positive, uncomplicated urinary tract infections (UTIs) over five months. Despite prescribed antibiotics the patient’s symptoms: suprapubic pain, frequency, urgency, and dysuria, would recur once the course of antibiotics was completed. A complete history was taken at Five Valleys Urology and was largely unremarkable apart from the frequent, recurrent UTIs. In-house PCR testing identified both Escherichia coli and Pseudomonas aeruginosa (Table 2). However, it is unknown if Pseudomonas was a new organism or whether the previous cultures were unable to detect the presence of this microbial agent. Prior to PCR results, the provider had been treating with guideline appropriate nitrofurantoin. With PCR identification of P. aeruginosa infection, ciprofloxacin was prescribed to target both E. coli and P. aeruginosa, which broke the cycle of recurrent infection.

Case 2

An 80-year-old female with indolent chronic lymphocytic leukemia (CLL) diagnosed two years prior had battled recurrent UTIs and interstitial cystitis for over ten years but had never experienced pyelonephritis or nephrolithiasis. Despite frequent cystoscopy and imaging with an outside urologist, her infections were never adequately treated. Her PCP referred her to Five Valleys Urology for a second opinion after another attempt at treating six months of UTI symptoms.

Table 2.

(Case 1).

Date	Culture or PCR	Microbes	Antimicrobial Susceptibility or Antibiotic Resistance
January 2022	Culture	E. coli
February 2022	Culture	E. coli	Resistant to ampicillin and gentamicin
April 2022	Culture	E. coli	Resistant to ampicillin and gentamicin
May 2022	Culture	E. coli	Resistant to ampicillin and gentamicin
June 2022	In-house PCR	E. coli Pseudomonas aeruginosa	Resistant to ampicillin, cephalexin, cefazolin

Upon evaluation at our clinic, she had no fever, chills, costovertebral angle tenderness, hematuria, or dysuria. However, she still struggled with constant suprapubic pressure and discomfort, as well as increased urination frequency and urgency. While she had no overt nocturnal enuresis, she reported that she was up seven times per night due to nocturia; she also experienced leakage when transferring from bed to bathroom.

PCR detected Klebsiella pneumoniae and Streptococcus agalactiae. Antibiotic resistance markers to cephalexin, cefazolin, and ceftazidime were identified. A urine culture collected the same day took four days to result and missed the extended-spectrum beta-lactamase (ESBL) producing organism (Table 3). Based upon the PCR results the patient was prescribed nitrofurantoin and UTI symptoms resolved with no further UTIs reported.

Case 3

This case involves a 72-year-old male with Parkinson’s (or a variant thereof) who was referred for urinary retention, leaving him Foley catheter dependent after orthopedic repair of a traumatic humeral head fracture. Catheters are known to increase the risk of complicated UTIs [10], as in this patient’s case. Neither tamsulosin nor finasteride helped his voiding, which is common in neurogenic bladder syndromes. PCR detected Klebsiella oxytoca, Enterococcus faecalis, and Staphylococcus aureus allowing for appropriate antibiotic treatment (Table 4). Two months after PCR-guided UTI treatment, the patient was voiding normally with resolved dysuria, urgency, and frequency.

Table 3.

(Case 2).

Date	Culture or PCR	Microbes	Antimicrobial Susceptibility or Antibiotic Resistance
January 2020	Culture	E. coli	Resistant to ampicillin, tetracycline, and Bactrim
February 2021	Culture	E. coli	Resistant to ampicillin and Bactrim
March 2021	Culture	E. coli	Resistant to ampicillin and Bactrim
June 2021	Semi-quantitative PCR (outside facility)	Streptococcus enterococcus	50–99,000 Streptococcus <10,000 enterococcus
August 2021	In-house PCR	Klebsiella pneumoniae Streptococcus agalactiae	Resistant to (ESBL): cephalexin, cefazolin, ceftazidime No resistance detected
	Culture	Klebsiella pneumoniae	Resistant to ampicillin

Table 4.

(Case 3).

Date	Culture or PCR	Microbes	Antimicrobial Susceptibility or Antibiotic Resistance
December 2021	Culture	Klebsiella oxytoca	Resistant ampicillin and cefazolin
May 2022	In-house PCR	Klebsiella oxytoca, Enterococcus faecalis, Staphylococcus aureus	Staphylococcus aureus: Resistant to amoxicillin/clavulanate, azithromycin, cefazolin, cephalexin, clarithromycin, clindamycin, dicloxacillin, erythromycin

Discussion

Given the widespread prevalence of UTIs, diagnosis and treatment are essential for improved clinical outcomes. Urine culture is a reliable method, but it is slow, taking days rather than hours, and can miss relevant infections (especially difficult-to-grow organisms), erroneously reporting sterile urine in the setting of an infected, symptomatic patient [11], [12]. The assay used here was developed and validated using a QuantStudio™ 5 Real-Time PCR instrument with ThermoFisher UTI/ABR primers/probes. The validation process included extracting patient urine samples from a commercial reference laboratory and comparing the results to those of the reference laboratory. Validation steps included: 3-day inter- and 2-day intra-day precision, accuracy, limit of detection, specimen stability, DNA extraction control, and impact of interfering substances. The assay precision, calculated on cycle threshold (CT) values for each analyte, ranged between 0.87 % and 3.02 % CV for all analytes during the 3-day study. For analysis of accuracy during validation, twenty-three previous patient samples were extracted, amplified, analyzed, and compared back to the commercial reference laboratory’s results with between 87.5 % and 100 % concordance for all the analytes in the panel. One specimen showed a false negative for C. glabrata when compared to the reference laboratory’s results. Another specimen showed a false negative result for U. urealyticum, which then changed the associated antibiotic resistance marker, ErmB, to negative for the clinic sample. It is believed that both urine specimens had death of the pathogen during shipping and storage, highlighting the importance of in-house testing to avoid sample degradation prior to testing. The limit of detection (LOD) was determined to be 60 copies of target for all analytes except Enterobacter cloacae and AmpC, which had LODs of 300 copies. No significant interference was observed when urine was spiked with biological fluids (blood, semen, saliva) or a drug panel of common over the counter medications.

A retrospective cohort study of 2565 female patients with an uncomplicated UTI reported that 11.9 % were prescribed inappropriate antibiotics, and 29.9 % received suboptimal antibiotics [13]. According to the Infectious Diseases Society of America guidelines, inappropriate treatment includes not being prescribed a recommended antibiotic, while appropriate treatment includes being prescribed first-line therapy (e.g., Fosfomycin, nitrofurantoin, or trimethoprim-sulfamethoxazole monotherapy (SXT)) for the proper duration [14]. Suboptimal treatment was defined as a change in antibiotics within 28 days of first diagnosis/treatment due to initial antibiotic failure or receiving treatment to which the microbes were not susceptible. Antibiotic stewardship and antibiotic resistance are important aspects of medical care, and improved diagnostics (as illustrated in case 1) could make strides toward preventing iatrogenic antibiotic resistance at the reported rates of suboptimal or inappropriate antibiotic use in UTI management.

It is estimated that there are 250,000 cases of pyelonephritis in the United States each year with 7 % of those cases requiring hospitalization [1]. The use of molecular diagnostics to diagnose pyelonephritis and UTIs more quickly could save healthcare costs and improve patient outcomes. However, the high sensitivity of PCR is a valid concern, and its implementation should be limited to symptomatic or high-risk patients (e.g., pregnant) with reasonable cycle thresholds defining a positive result.

During a six-month period in 2022, 350 urology patients at Five Valleys Urology were tested, resulting in 196 positive specimens. Our assay enabled us to quickly identify the causative microbe(s) and resistance(s) and treat accordingly. Currently, there are no FDA-approved molecular diagnostic tests for UTIs, however, in-house LDT offers an opportunity to avoid delays, misdiagnosis, and inaccurate or inadequate treatment.

Conclusion

LDTs are essential for providing modern healthcare to patients. The Clinical Laboratory Improvement Amendments (CLIA) regulations, enacted by Congress, govern the tests performed on patient specimens. LDTs are diagnostic tests designed and used within a single laboratory, and they must be reviewed biennially by Centers for Medicare & Medicaid Services (CMS) representatives or staff members from deemed agencies, such as the College of American Pathologists (CAP) or COLA Inc. The passage of the Verifying Accurate Leading-edge IVCT Development (VALID) Act, or similar legislation, would impede important developments in patient care that come from LDTs like ours.

We have used molecular diagnostics in our urology clinic laboratory to identify and guide treatment of the causative agents of UTIs, which has improved patient outcomes and antimicrobial stewardship by quickly determining the appropriate antibiotic. This laboratory-developed PCR assay has demonstrated clinical utility and could benefit the greater medical community.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.