Urinary incontinence (UI) is prevalent in society, affecting women of all ages and with potential to seriously impact the physical, psychological, and social wellbeing of affected individuals. According to the International Continence Society (ICS), overactive bladder (OAB) syndrome is characterized by urinary urgency usually accompanied by frequency and nocturia, with or without urgency urinary incontinence, and in the absence of urinary tract infection or other obvious pathology (Abrams et al., 2003; Haylen et al., 2010). As a major cause of urinary incontinence, OAB affects 12.8% of women over the age of 18 years of age and the incidence rises with age (Irwin et al., 2006). The incidence of any OAB symptom is as high as 48% (de Boer et al., 2011), however, there is a paucity of evidence associating OAB symptoms with urinary infection. OAB patients that are refractory to treatment are a particular challenge with the lack of a treatment response often attributed to an undiagnosed infection, despite this having been ruled out by urinalysis and standard culture, or poor drug efficacy.

The impact of OAB on quality of life can be significant. With adverse effects ranging from poor sleep quality to impaired emotional well-being to social disruption and difficulties in education and employment, several studies have highlighted the negative impact that this syndrome can have on patients’ lives (Stewart et al., 2003; Bartoli et al., 2010). Added to this are significant healthcare costs associated with disease management, which although difficult to accurately determine, are estimated to be around €5bn for major economies across Europe and up to $82bn in the United States (Reeves et al., 2006; Reynolds et al., 2016).

Despite the option for non-pharmacological interventions, such as lifestyle changes, behavioral therapy, and pelvic floor muscle training; most women with OAB will need drug treatment, which can include antimuscarinic drugs, anticholinergics, beta 3-receptor antagonists or botulinum toxins (Andersson et al., 1999; NICE, 2016a). In cases where drug therapy fails, additional management strategies including percutaneous posterial tibial nerve stimulation or sacral nerve stimulation, augmentation cytoplasty or urinary diversion can be considered. Although these are only indicated following extensive multidisciplinary team review and the failure of other treatment options (NICE, 2016b).

A key facet of the diagnosis of OAB is the absence of urinary tract infection (UTI), which is defined by urinary pathogens within a clean-catch midstream urine (MSU) sample and the presence of clinical symptoms. Although OAB and UTI share similar symptoms such as urgency frequency and nocturia, the onset of symptoms is different; UTI exhibiting acute symptoms and OAB exhibiting chronic symptoms. The similarity of sensory bladder symptoms associated with OAB and UTI can complicate disease management if UTI is not first excluded. Although there is no officially accepted standard for the minimum level of bacteria in a MSU specimen defining an infection, many laboratories use 10⁵ colony forming units (CFU)/mL urine as the UTI threshold (Kass, 1957), whilst others have proposed a threshold of ≥10² CFU/ml in symptomatic women presenting with dysuria and cystitis-like symptoms (Stamm, 1983). Conventional MSU culture judged positive at ≥10⁵ CFU/ml can fail to detect 50% of all clinically significant, symptomatic urinary infections in OAB patients (Khasriya et al., 2008). As symptoms of OAB and cystitis overlap, a negative MSU is not entirely reliable.

The aim of the present study was to investigate whether urine from women with OAB symptoms (and no evidence of clinical infection by conventional MSU) have significant markers of sub-clinical infection when compared to individuals with no OAB symptoms.

The study was a prospective, blinded case control study approved by the Southwest Wales Research Ethics Committee (REC reference number 12/WA/0405), performed at Singleton Hospital, Swansea, and Swansea University Medical School from 2012 to 2015. The objectives of the study were to discover whether patients with OAB exhibit significant pyuria on fresh microscopy, harbor bacteria undetected by routine MSU cultures, and if so, to identify and quantify such bacteria. In parallel, we determined whether these patients displayed elevated levels of inflammatory markers.

147 subjects were recruited: 73 OAB patients and 74 controls. Inclusion criteria were female patients of at least 18 years of age presenting with OAB symptoms for more than 3 months. Symptoms were defined as greater than 1 episode/week of an uncontrolled urge to void causing incontinence. Exclusion criteria included hematuria, obstructive uropathy, vaginitis, urothelial carcinoma, urinary tract infection, pelvic radiation disease, neurogenic bladder, women with predominant stress urinary incontinence, liver disease, patients receiving antibiotics, an inability to give consent and understand English, and participation in other studies where the intervention is likely to affect their condition. The control group was recruited on a voluntary basis and comprised women aged at least 18 years of age, with no history of urinary infection, OAB symptoms or urge incontinence.

All participants completed a study specific Proforma and International Consultation on Incontinence Questionnaire-Overactive Bladder (ICIQ-OAB) (Avery et al., 2004). Information was obtained on patient’s age, BMI, past medical history, co-morbid conditions, urinary symptoms, prolapse symptoms and medication history. The ICIQ-OAB is a validated questionnaire (https://www.baus.org.uk/_userfiles/pages/files/Patients/Leaflets/ICIQ-OAB.pdf) used to screen for overactive bladder. It comprises 4 questions pertaining to frequency of micturition, nocturia, urgency and urge urinary incontinence. Each question is scored from 0 to 4 depending on the presence and severity of symptoms. The overall score of the ICIQ-OAB questionnaire ranges from 0-16 with greater values indicating increased symptom severity.

The significant impact of OAB on quality of life makes early diagnosis and effective treatment essential. Symptomatic similarity with other conditions and the unreliable nature of routine microbiological culture, complicates the diagnostic process and means there is a need for more optimized approaches. By exploiting enhanced culture techniques, we have demonstrated a significantly higher rate of bacterial infection in OAB (23.28%) patients compared to control subjects (10.8%), than that estimated via the routine testing typically used to exclude infection in urinary samples. Data that are consistent with earlier reports (Khasriya et al., 2008; Khasriya et al., 2009; Hilt et al., 2014). An increased prevalence, in the control group, of vaginal commensal bacteria, which are regarded as an important component of the vaginal mucosa defence against genital infections, was also noted. By acidifying the vaginal microenvironment, these bacteria inhibit the colonization of invading pathogens through competitive exclusion. Our data are consistent with such a role for commensal bacteria in providing genital track mucosa homeostasis in healthy women, and with the clinical picture (Thomas-White et al., 2018). Using routine culture methods, only 4% of OAB samples in the present study were positive for uropathogens, demonstrating its limitations, and the incidence of pyuria was similar between OAB patients and control subjects. Despite a lack of consistency in the literature regarding the role of pyuria as diagnostic in LUTS patients, with different studies recording white cell counts ranging from 58%-74% in patients with OAB (Malone-Lee et al., 2007) to 22%-42% in LUTS patients (Kim et al., 2006), pyuria is generally considered a weak marker of low-grade infections, which is consistent with our data.

In contrast to some other studies, and despite the detection of uropathogens by enhanced culture techniques. We did not find any significant increase in the inflammatory markers tested in this study, suggesting that the sub-clinical levels of bacteria detected had not triggered an inflammatory response. The presence of cytokines in the urine has been studied in various bladder and kidney disorders as it provides a non-invasive tool for inflammation (Ninan et al., 1999; Abdel-Mageed et al., 2003). However, there is inconsistency in the studies analyzing the role of inflammatory markers in OAB. Tyagi et al. analyzed the urine of 17 idiopathic OAB patients and eight asymptomatic controls for 12 different chemokines, cytokines, and growth factors in a prospective study finding elevated levels of nine of these markers in the urine of OAB patients (Tyagi et al., 2010). The small sample size, cross-sectional study design, lack of comorbidities matching, and age disparity between the OAB and control group, is a potential limitation of Tyagi’s study, however. Ghoniem et al. analyzed an array of cytokines in the urine of 20 OAB patients, 20 controls, and 16 patients with UTI in a prospective, single blind study. Consistent with our observations, most of the cytokines were not elevated in the urine of OAB patients compared to controls or UTI patients. In fact, certain cytokines were significantly downregulated in OAB (Ghoniem et al., 2011). Currently the induction of a cytokine response in OAB remains to be established, and their utility as biomarkers for OAB diagnosis does not appear useful.

Heterogeneous results are also apparent for urine prostaglandins. Significant elevation in PGE2 and PGF2α in OAB patients compared to controls has been reported by Kim et al. and Kang Jun Cho, although prostaglandin concentrations were not corrected for urine creatinine concentration (Kim et al., 2006; Cho et al., 2013). Corroborating our study, however, Hsin-Tzu Liu did not demonstrate any significant difference in urinary PGE2/Cr levels among patients with interstitial cystitis/bladder pain syndrome (IC/BPS), OAB or controls (Liu et al., 2010). Thus, the role of urinary prostaglandins in the diagnosis of OAB also appears limited. Whilst the literature supports a role for inflammation in the pathophysiology of the overactive bladder, with the presence of many inflammatory markers described in OAB, studies performed with the purpose of identifying specific inflammatory markers have not demonstrated consistency.

Together, our data and the observations of others (Khasriya et al., 2008; Tyagi et al., 2010; Hilt et al., 2014), demonstrate that bacterial infection cannot be reliably excluded by routine culture and that inflammatory markers are an unreliable indicator of infection. Enhanced techniques or other methods, such as the identification of bacterial DNA in urine appear to be needed in all patients presenting with symptoms of OAB. Approximately 40% of OAB patients either do not achieve an acceptable level of a therapeutic benefit or remain completely refractory to treatment with conservative measures and antimuscarinics (Giarenis et al., 2013). Based on our observations that OAB patients have a higher incidence of bacterial infection, as demonstrated by enhanced culture techniques, it is plausible that these patients have unidentified urinary infections. The eradication of these subclinical infections, identified through the enhanced culture method used in the current study, by treatment with antibiotics might lead to simultaneous improvements in OAB symptoms and should form the basis of a future clinical trial.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Southwest Wales Research Ethics Committee (REC reference number 12/WA/0405). The patients/participants provided their written informed consent to participate in this study.

ZK undertook the experimental work and prepared the draft manuscript. GH compiled the final manuscript and data tables. RP undertook part of the experimental work. NB, ER, and SE provided clinical input and study support. LM provided clinical input, conceived the study, and contributed to manuscript preparation. DG and RC conceived the study and contributed to manuscript preparation. All authors contributed to the article and approved the submitted version.

Swansea Bay University Health Board Research & Development Department. Medical Research Council UK Confidence in Concept grant (MC_PC_19053).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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