Urinary tract infection (UTI) is the most common bacterial infection amongst adult women and has a high rate of recurrence, particularly in postmenopausal women [1]. Recurrent UTI (rUTI) is defined as ≥3 symptomatic, clinically diagnosed UTIs in 12 months or ≥2 symptomatic, clinically diagnosed UTIs in 6 months [1]. Despite its high prevalence, no robust detection method is available for the rapid diagnosis of rUTI. Urine dipstick, which is a widely used method for the diagnosis of UTI, offers limited specificity and sensitivity. Leukocyte esterase, which is the urine dipstick marker for pyuria, may yield misleading results due to factors such as urine contamination with vaginal fluid, elevated specific gravity, protein, and glucose [2]. Urine culture is more accurate, but the average time to diagnosis is 72 hours [2]. Therefore, the development of simple, reliable, and rapid point-of-care diagnostic tests for UTI is crucial. 
rUTI is, in part, an inflammatory disease. Evidence of inflammation, termed cystitis, has been reported in bladders of rUTI patients during cystoscopy [1]. Studies in mouse models suggest that mice with chronic cystitis show increased urinary levels of specific cytokines and chemokines early during infection that may affect susceptibility to recurrent infection [3]. Based on mouse model data, we hypothesized that the levels of proinflammatory cytokines and chemokines are elevated in the urine of women with rUTI. We also hypothesized that these urinary cytokines may serve as diagnostic and/or prognostic biomarkers of rUTI. Thus, the goal of this study is to identify urinary cytokines and chemokines associated with rUTI and define their utility as diagnostic or prognostic biomarkers for rUTI in postmenopausal women.

Upon IRB approval, a cross-sectional cohort of postmenopausal women passing inclusion criteria for uncomplicated rUTI were stratified into two cohorts based on their history of rUTI and current UTI status: Never (no UTI history, n=26) and Relapse (rUTI history, current symptomatic rUTI, n=31). Clean catch urine was collected and immediately stored in liquid nitrogen. We performed a priori power analysis to determine the appropriate sample size and found that a minimum of 17 patients per group was necessary to detect an effect size of 0.5 between Never and Relapse groups with a power >0.8. Urinary concentrations of 13 inflammatory cytokines IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 were then measured from patients in the Never and Relapse groups (Total n=46) using LEGENDplex™ Human Inflammation Panel 1 (BioLegend). Data was acquired using the BD Fortessa flow cytometer located in the UT Dallas core facility. LEGENDplex™ Data Analysis Software was used to calculate the concentration. Statistical analysis was performed using GraphPad Prism software. Urinary concentrations of cytokines were then normalized to urinary creatinine (uCr) (pg/µg), using a colorimetric assay kit (Thermo Fisher), to account for variations in water reabsorption in the patient urine samples.

Quantification of urinary cytokines in our cohort of postmenopausal women revealed that the urinary levels of cytokines IL-8, MCP-1, IL-1β, and IL-18 were elevated in the Relapse patients compared to the Never group (Fig 1). This increase was statistically significant in both the raw and normalized data. The median fold change in the raw concentration values for these cytokines were 94.6, 7.73, 45.6, and 4.8, respectively.

The data indicates that the urinary levels of IL-8, MCP-1, IL-1β, and IL-18 are significantly elevated in postmenopausal women with active rUTI as compared to Never UTI controls. These cytokines may represent candidate urinary biomarkers for rUTI in postmenopausal women. IL-8 and MCP-1 are involved in neutrophil and monocyte recruitment, respectively. IL-1β and IL-18 are produced due to inflammasome genesis. Inflammasome formation often induces pyroptosis, which is a highly inflammatory form of cell death.

Our data indicates that IL-8, MCP-1, IL-1β, and IL-18 may be candidate diagnostic or prognostic urinary biomarkers for rUTI in postmenopausal women. As part of our future directions, we will employ linear regression models to determine their sensitivity and selectivity as diagnostic biomarkers and perform Kaplan-Meir analysis of collected time to relapse data to assess their potential as prognostic biomarkers of rUTI in postmenopausal women. Identification of urinary biomarkers of rUTI with improved sensitivity and selectivity over existing rapid diagnostic methods will be an essential step in the development of improved point-of-care diagnostic tools for UTI and rUTI.

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