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A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination

A revalidation and critique of assumptions about urinary sample collection methods, specimen... Introduction and hypothesis Midstream urine (MSU) is key in assessing lower urinary tract syndrome (LUTS), but contingent on some assumptions. The aim of this study was to compare the occurrence of contamination and the quality of substrates obtained from four different collections: MSU, catheter specimen urine (CSU), a commercial MSU collecting device (Peezy) and a natural void. Contamination was quantified by differential, uroplakin-positive, urothelial cell counts. Methods This was a single blind, crossover study conducted in two phases. First, we compared the MSU with CSU using urine culture, pyuria counts and differential counting of epithelial cells after immunofluorescence staining for uroplakin III (UP3). Second, we compared the three non-invasive (MSU, Peezy MSU™, natural void) methods using UP3 antibody staining only. Results The natural void was best at collecting bladder urinary sediment, with the majority of epithelial cells present derived from the urinary tract. CSU sampling missed much of the urinary sediment and showed sparse culture results. Finally, the MSU collection methods did not capture much of the bladder sediment. Conclusion We found little evidence for contamination with the four methods. Natural void was the best method for harvesting shed urothelial cells and white blood cells. It provides a richer sample of the inflammatory exudate, including parasitised urothelial cells and the microbial substrate. However, if the midstream sample is believed to be important, the MSU collection device is advantageous. . . . . . Keywords Midstream Urine Sample Catheter Peezy UTI Introduction related to hospitalisations in the USA [4]. A UTI is a debili- tating condition causing the onset of painful urination (dys- A urinary tract infection (UTI) is the leading reason why pa- uria), increased urinary frequency, the inability to start urinat- tients seek treatment in primary care [1]. One in three women ing (hesitancy) and the sensation of a sudden need to urinate will be treated with antibiotics for UTI by the age of 24 and (urgency) [5], all of which are classified as lower urinary tract 40% to 50% of women will experience one or more UTIs in symptoms (LUTS) [6]. their lifetime, with 10–15% experiencing recurrent infections Many clinical guidelines advocate urinary dipstick testing [2]. A UTI is commonly identified among young adolescents for leucocyte esterase and nitrite as a means of detecting UTI, attending sexual health centres and accounts for 17% of treat- but evidence suggests that their utility might be limited [7]. ment cases [3]. In the adult population of 65 years and older, a Dipstick analyses are surrogate tests, referenced not to micro- UTI is the second most common cause of infectious disease scopic pyuria but to a gold standard urine culture threshold for 3 −1 UTI, which guidelines accept as being between 10 cfu ml 6 −1 and 10 cfu ml of the pure growth of a single urinary path- ogen [8]. This test has attracted criticism too [9–11]. * Linda Collins Furthermore, the literature shows that the methods of sample [email protected] collection for urine culture have never been validated in ap- propriate clinical trials. Justification for the use of such methods is based on plausible assumptions without supporting School of Nursing, Kingston University, Frank Lampl Building, Kingston Hill Campus, London, UK evidence. It is now recognised that routine urinalyses, includ- Department of Renal Medicine, Division of Medicine, University ing dipstick and culture, are insensitive, thus missing genuine College London, London, UK infection in many symptomatic patients [12–14]. Int Urogynecol J There are three commonly used urine collection methods: voided urine, in which we measured microscopic epithelial the midstream clean catch technique (MSU) [15], the catheter cell counts using UP3 staining. specimen of urine (CSU) [16], and suprapubic aspiration [17]. Sample contamination is a key concern, particularly with regard to MSU. Our understanding is hampered by the variable defi- Materials and methods nitions of “contamination” described in the literature. Some have claimed that contamination is indicated by isolation of This study was presented to the National Research Ethics microbes typical of skin flora, such as Corynebacterium and Service Committee (NRES) in Harrow, London, UK, to obtain Staphylococcus [18–20]. According to Collier et al. [21], con- approval to conduct the investigation. Ethical approval was taminated urine samples are revealed by finding squamous ep- granted, which signified that this study complied with condi- ithelial cells on urine microscopy. Wilson and Gaido [22]de- tions that were favourable and worthy of safe research prac- fined contamination as ≥2 different types of organisms at tice. Ethical approval was given subject to all clinicians in the 5 −1 4 −1 >10 ml or 1 organism at <10 ml . Others have reported study having undergone training in good clinical practice urine samples as contaminated, without describing the criteria (GCP). The two-part comparative studies began in January used [23, 24]. These disparities add to the confusion that affects 2013 and lasted until March 2015. Patients with LUTS attend- these diagnostic methods and there are no data available to aid ing the Community LUTS Clinic, Hornsey Central Health clarification. An important consideration is the substrate that Centre, London, UK, made up the participant population. these collection methods should seek to obtain for culture. The patient sample was drawn from women diagnosed with Historically, the emphasis has been on uncontaminated urine chronic UTI, painful bladder syndrome (PBS), overactive samples being cell-free [22]. However, contemporary studies bladder (OAB) and general LUTS. have shown that UTI is associated with microbial parasitisation Patients were given an information sheet about the study of urothelial cells, via surface attachment and/or intracellular and were offered the opportunity to ask questions and address invasion [25–28]. This parasitisation stimulates urothelial cell any concerns. The first part of the study was designed to shedding as part of an innate immune response [27]. It has been ascertain whether there were outcome signals that merited a found that the urinary urothelial cell counts are elevated in focused analysis of urinary epithelial cells in relation to sam- association with other markers of infection and that the propor- ple collection. On the day of recruitment, patients were invited tion of parasitised cells among the cell sediment increases dur- to the centre, and written informed consent was obtained. ing UTIs, along with inflammatory markers and changes in the Each participant was given a unique non-identifiable number. urinary microbiome [13, 27, 29]. If the primary pathology is the All patients completed a validated female LUTS questionnaire microbial invasion of urothelial cells, stimulating the concom- (FLUTS and FLUTSqol) [31] and provided MSU and CSU itant innate immune response of increased urothelial cell shed- samples. The MSU method was accomplished by the patient ding, it is plausible that these cells would make a better sub- spreading her labia apart with one hand and then wiping the strate for urine culture than would supernatants of planktonic urethra area with moistened wipes using the other hand [20]; bacteria. This was demonstrated in our previous work [12]. the CSU was achieved by placing the patient in the lithotomy Shed urothelial cells are an attractive option for study when position, inserting the urethral catheter along the urethra and seeking to examine contamination with different sampling into the bladder to collect part of the urine outflow [32]. methods. Uroplakin III (UP3), a transmembrane protein found The substrate of interest in urinalysis is the inflammatory exclusively in the urinary tract [30], is a useful biomarker for exudate, which contains microbes, white cells, shed urothelial discerning the origin of epithelial cells in a urine sample. cells and other debris. This may become contaminated with Thus, it is possible to discriminate contaminating cutaneous external debris during collection. The collection methods sam- and vaginal squamous cells from urothelial umbrella cells ple different parts of the bladder urine (Fig. 1). The presence using a specific antibody against this protein with immunoflu- of sediment and contaminants may be influenced by the dif- orescence staining protocols. ferent collection methods. Thus, we should seek to examine This study consisted of two parts. The first was a compar- the markers that reflect these different elements. ative study of the urinalysis results obtained from samples The urine sampling methods were randomly sequenced collected by MSU and CSU. It was a random allocation, and obtained with a 1-h interval between specimen collec- cross-over design and the urine sampling performances were tions. The specimen volume requirement was small and well compared using microscopic pyuria counts, epithelial cell within the capacity of an hour. Urine aliquots were obtained counts, microbial growth from spun cell sediment culture from the unspun specimens and these were introduced, un- and UP3-positive cell counts to measure contamination. The stained, into a Neubauer counting chamber; a microscope second part of the study was a random allocation, cross-over was then used to count the white blood cells (WBCs), red comparison study of contamination obtained with MSU, a blood cells (RBCs) and epithelial cells. A sediment culture novel MSU sampling device called a “Peezy” and naturally [12] was carried out. All urine samples were collected in a Int Urogynecol J Fig. 1 a Illustration of the fact that the cellular sediment collects at the bladder base under the influence of gravity. A catheter passes through this sediment to obtain a specimen from the urine that is above this substrate. The sequence in b–e illustrates the phases of the voiding processes. The inflammatory sediment is a Catheter Specimen shown accumulating at the bladder neck under the influence of gravity. The first part of the stream contains the largest quantity from the sediment at the bladder neck and this reduces over the course of the voiding process b Pre-void with sediment c Initial stream rich in d Midstream e Terminal stream accumulating at the sediment of urine bladder neck 30-ml container and spun down in a Denley refrigerated cen- against the perineum whilst passing urine. As the patient be- trifuge at 800 rpm (~75 relative centrifugal force [RCF]). The gan to pass urine, the first part of the stream entered the funnel remaining cell pellet was re-suspended in phosphate-buffered and caused a sponge valve to swell and block flow through the saline (PBS) and three ten-fold serial dilutions were made in main funnel exit. Once the blockage was established, a mid- PBS. A volume of 50 μl of the re-suspended pellet and dilu- stream specimen accumulated in the funnel and was passed tions was plated onto chromogenic CPS3 (now renamed CPS- out of an overflow side drain into the universal container. The Elite) agar (bioMérieux, France) [33] and dispersed evenly Peezy MSU™ device was then discarded into the clinical using a spreader. The culture plates were incubated aerobically waste bin. All urine samples were obtained using each meth- at 37 °C for 24 h, followed by colony enumeration and iden- od, through random sequencing, with one method each day tification using the Chrom ID™ colour chart provided. over 3 consecutive days. An aliquot of spun sediment cells was collected and stained This second study commenced in August 2014. Patients for UP3 as described previously [27]. Briefly, epithelial cells recruited from the first study were asked to participate in the were adhered to a glass slide using a Shandon Cytospin second part, but fresh consent was obtained. A random code Cytocentrifuge (Thermo Scientific) with 80 μl of a urine sam- dictated the order of sampling, which occurred on the first day ple for 5 min at 75 RCF. The cells were fixed with 4% form- of the 3 consecutive days. For experiment 2, samples were aldehyde in PBS for 15 min, blocked with 10% normal goat used to examine the epithelial cell content using the same serum for 30 min and stained for 1 h with anti-UP3 antibody. methods as in experiment 1. After three washes with PBS, the secondary antibody (goat anti-mouse IgG conjugated to Alexa-flour 488) was applied Sample size for 40 min, washed three times and the DNA stain 4′,6- diamidino-2-phenylindole (DAPI) was later applied for The sample was calculated using the G Power software pack- 20 min before mounting. age. The effect size was set as Cohen’s d = 0.55; α = 0.05; The second part of the study was conducted in the wake of power (1 – β err probability) = 0.8; non-centrality parameter the first experiment and was designed to compare the quanti- δ = 2.9; critical t = 2; df = 27; sample size = 28; actual power = tative and qualitative properties of epithelial cells captured in 0.8 or 80%. specimens obtained from an MSU, an MSU collection device (Peezy) and a naturally voided specimen, which was a straightforward urine sample collected by the patient in a con- Statistical analysis tainer without a technique. The Peezy MSU™ is a urine col- lection method achieved by urinating into an engineered de- The data were not normally distributed and exhibited wide vice [21]. The patients were instructed to wash their hands, variance; thus, to analyse quantitative measures we used the clean the genital area with wet wipes, attach the collection non-parametric Friedman test, which achieves a one-way bottle to the Peezy and position the Peezy MSU™ device analysis of variance by ranks. This is an alternative to the Int Urogynecol J Kruskal–Wallis when analysing repeated measures. We used culture [12] on the samples. Table 6 describes the occurrences the Chi-squared test to analyse the differences in proportions. of the top 90% of isolates across all patients cultured from the three different specimen collection methods. The data show that the spun sediment culture performed on MSU samples Results was the most productive method and the technique performed with CSU samples had significantly more negative results. Demography and symptoms The standard NHS culture method was far less productive whether it was from MSU or CSU; taken together, these re- In the first study, 60 female adult patients were enrolled. The sults emphasise the insensitivities of both collection methods. mean age of the patient group was 60 years (SD = 12). The The CSU seems to trade purity for sensitivity. patients suffered from chronic LUTS and were being treated for chronic UTI. The demographic data are shown in Table 1 Evaluation of UP3-positive cells, comparing MSU, and demonstrate that the patients were suffering from signif- Peezy MSU™ and natural void icant symptoms. Thirty-one patients were enrolled in this study, which used the Evaluation of pyuria, epithelial cells and UP3-positive UP3-positive properties of urothelial cells to identify the ori- cells gin of epithelial cells, given that skin and vaginal cells are UP3-negative. The mean age of the patients was 62 (SD = To evaluate the presence of pyuria, epithelial cells and UP3- 10). In total, 93 non-invasive urine samples were collected positive cells, a total of 118 urine samples were examined by MSU, Peezy MSU™ and natural voided urine. Tables 7, 8,and 9 reports the analysis of the UP3-positive cells. The (MSU = 60, CSU = 58). The patients were at various stages of disease. The data analysis results are shown in Tables 2, 3, mean count of UP3-positive cells from the natural void meth- −1 od was greatest compared with the MSU and Peezy MSU™. 4,and 5. Microscopic pyuria counts (log WBC μl )were significantly lower in CSU compared with MSU. Similarly, Thus, natural void achieved the greatest abundance of the there were greater numbers of epithelial cells in MSU, the target substrate. The lowest UP3-positive cell count was ob- majority of which were UP3-positive, indicating urothelial served with the Peezy technique; thus, this method captured origin. There was no significant difference in the proportion the substrate the least. The UP3-negative cells were sparse, of UP3-positive cells between the MSU (0.81) and CSU (0.9) with no difference between specimen collection techniques methods. If the CSU method collected a significantly less (Table 7, 8,and 9). Thus, the proportion of all cells that were contaminated sample, the proportion would be expected to UP3-positive did not differ between the sampling methods, be significantly higher with the CSU method. These results which implies that, contrary to assumptions, contamination by extra-urinary tract cells is not influenced by the sampling suggest that MSU might enrich a urine sample for urinary tract cell sediment compared with the CSU, without increased or technique and was low overall. appreciable contamination. Evaluation of culture results Discussion All 118 urine samples were sent to the National Health Service The purpose of this study was to measure contamination and (NHS) laboratory for routine culture (plating of 1 μlof urinary substrate content for four different methods of obtaining urine supernatant) and of these, 110 specimens were reported as samples. It also used the fact that urothelial cells can be dis- “negative” (defined as below the 10 cfu/ml threshold) or tinguished from squamous epithelial cells by UP3 staining. “mixed growth”, which is commonly reported for chronic This enabled measurement of contamination by the extra- UTI patients [12, 13, 37]. Thus, in addition to routine culture, urinary milieu compromising these various methods. The pro- we enriched infected cells by performing a spun sediment portion of UP3-positive cells found in MSU samples was Table 1 The average symptom scores and quality of life scores for patients who attended the Community Lower Urinary Tract Symptom (LUTS) Clinic with LUTS Participants Mean age Mean urgency [34]score Mean pain score [34] FLUTS [34] (without FLUTSqol [36] (maximum = 64) (maximum = 12) urgency and pain) (maximum = 288) Normal = 0 [35] Normal = 0 [35] (maximum = 149) Normal = N/A Normal = 0 [35] Patients 60 (SD = 12) 16 (SD = 11) 10 (SD = 7) 64 (SD = 40) 151 (SD = 75) Int Urogynecol J Table 2 Evaluation of microscopic pyuria for the midstream urine Table 4 Evaluation of uroplakin-positive cells (UP3) for the midstream (MSU) and catheter specimen urine (CSU) samples urine (MSU) and catheter specimen urine (CSU) samples Analysis Pyuria count MSU Pyuria count CSU p Analysis UP3 cells UP3 cells p −1 −1 (WBCs μl ) (WBCs μl ) (cells/80 μl) MSU (cells/80 μl) CSU Mean 65 24 0.001 Mean 17 5 0.006 Median 0 0 Median 3 1 Standard deviation 321 148 Standard deviation 30 12 WBCs white blood cells given that healthy urine is known to harbour many species of previously reported as 75% (Q1 = 68, Q3 = 78.5) by Horsley bacteria, including known uropathogens [39], it is not known et al [27]. This study had a similar outcome, and has extended whether these microbes are pathogenic or harmless commen- this to three other sample collection methods. Taken together sals, a problem affecting all current urinary microbial detec- with a wide body of literature demonstrating that urothelial tion methods. cell shedding is a common innate immune response to infec- The symptomatic measurements pain, urgency, FLUTS tion, this study suggests that the widely held assumption that and LUTSqol were concordant with the pyuria and culture epithelial cells in a urine sample indicate contamination might data [13]. It should be appreciated that CSU sampling missed need to be revised [26]. The sample collection method did a pathological signal in a significant proportion of patients influence the absolute epithelial cell counts, the lowest num- with appropriate symptoms, pyuria and urothelial cell shed- ber with CSU samples (Fig. 1a) and the greatest number with ding. Compared with CSU, the MSU technique provided a natural void (Fig. 1c), during which the bladder contracts and more substantial sample of the inflammatory sediment. UTI collapses to eliminate bladder urinary substrate. The Peezy involves colonisation of the urothelial cells by pathogenic device seemed to do what it claimed, achieving a sample of microbes [12, 27, 40]. The innate immune response to this fewer cells and very few uroplakin-negative cells. However, microbial colonisation is increased urothelial cell shedding, the sediment trapped in the sponge valve of the Peezy device which may be promoted by mast cells [41]. The shed cells may, counter-intuitively, prove to be a richer substrate for may be colonised or unaffected, but the proportion of microbiological investigation, as bacteria are known to adhere parasitised cells in the face of infection would be expected to and colonise the urothelial cells. to increase [42]. This sediment, a mixture of white cells, epi- The rationale of midstream sampling has always been the thelial cells and debris, is likely to collect under the influence avoidance of contamination. This was never properly tested of gravity at the bladder base, forming a sampling target that and relied on assumptions. In this experiment we were able to should not be influenced by dilution effects. This is made analyse contamination directly by using UP3 staining of the evident by the characteristic milky quality of the terminal flow epithelial cells as a proxy, and we found that contrary to what when a catheter is used to drain an infected bladder. A cathe- has been assumed, the majority of epithelial cells in all sam- ter, as well as a suprapubic stab, samples urine above this ples originated from the urinary tract. collection at the bladder base (Fig. 1a), and therefore contains The standard NHS MSU culture achieved a very small fewer epithelial cells, white cells and microbes, a point number of positive cultures, in contrast to the spun sediment reflected in the data reported here. Thus, a CSU sample may cultures, despite the patients exhibiting significantly fewer be an inferior option because it may miss a substantial amount urinary tract symptoms; this discrepancy has been reported of the pathology. previously by our team and others [38]. Urinary microscopic With the advent of more sensitive genomic technology, a detection of pyuria and increased urothelial cell shedding pro- number of groups have reported that the healthy bladder is not vided strong evidence for the presence of UTI. The sediment sterile, and that polymicrobial colonisation is the norm in both culture generated a greater abundance of isolates. However, healthy and infected bladders [12, 14, 43]. Polymicrobial Table 3 Evaluation of microscopic urinary epithelial cells for the Table 5 Evaluation of the proportion of uroplakin-positive cells (UP3) midstream urine (MSU) and catheter specimen urine (CSU) samples for the MSU and CSU sample Analysis Epithelial cells Epithelial cells p Analysis Proportion of cells Proportion of cells p −1 −1 (μl )MSU CSU (μl ) UP3-positive MSU UP3-positive CSU Mean 21 2 0.002 Mean 0.81 0.9 0.98 Median 2 0 Median 1 1 Standard deviation 103 4 Standard deviation 0.3 0.2 Int Urogynecol J Table 6 Results of the spun urinary sediment culture carried out at the community LUTS clinic laboratory and NHS laboratory Number (%) Number Number Number Number Number Number with Total culturing Ecoli culturing culturing culturing culturing culturing no growth Enterococcus Streptococcus Staphylococcus Proteus yeast MSU spun sediment 18 (30) 13 (22) 12 (20) 5 (8) 8 (13) 4 (7) 0 (0) 60 culture, LUTS laboratory CSU spun sediment 4 (7) 15 (26) 1 (2) 0 2 (3) 5 (9) 31 (53) 58 culture, LUTS laboratory NHS MSU 1 (1) 2(2) 1(1) 0 0 1(1) 100(95) 105 laboratory results NHS CSU 1 (8) 2 (15) 0 0 0 0 10 (77) 13 laboratory results growth in urine specimens has been associated with LUTS in long-held assumptions about urine sampling. The natural void a recent study of similar patients [21]. Indeed, the sediment captures the first part of the urinary stream; the influence of cultures carried out on urine samples collected by MSU and gravity would encourage such samples to contain a larger CSU in this study also demonstrated polymicrobial growth. In proportion of the sediment because of settlement at the blad- patients with symptoms of UTI, the species dispersion is much der base (Fig. 1b). By avoiding the initial stream, the MSU wider than in asymptomatic controls [12, 18, 43–45]. At this and Peezy MSU™ methods collected samples containing time it is not known what species of these mixes are respon- fewer cells, the Peezy MSU™ providing the clearest speci- sible for the disease. In this study, the data on the UP3-positive mens (Fig. 1d). The remaining urine in the bladder proceeds cells argued against the common assumption that with a terminal flow, with very few bladder sediments (Fig. polymicrobial results and/or epithelial cells are pointers to 1e). contamination. A recent study has shown the isolation of a The Peezy MSU™ device works with a sponge in the fun- different microbial population using the Peezy compared with nel exit, which inflates gradually on contact with the first part one isolated from peri-urethral swabs, based on the assump- of the urinary stream, eventually blocking the outflow so that tion of peri-urethral contamination of MSU samples [46]. the remainder of the stream escapes by a side channel into a The natural void provided the greatest number of urothelial collecting tube. Thus, the bulk of the sediment deposit from cells and even in this situation, UP3 analysis did not show the bladder neck would be expected to be trapped in the evidence of increased contamination, which contradicts sponge. If the bladder base sediment is indeed, as recent work suggests, the best specific target for diagnostic analysis, so is Table 7 Evaluation of UP3-positive cells from midstream urine (MSU), the content of the sponge. As matters stand, the Peezy MSU™ Peezy MSU™ and natural void, which are all non-invasive urine collec- achieves what it sets out to do and provides an MSU sample tion methods UP3- UP3- UP3-positive p Table 8 Evaluation of UP3-negative cells from midstream urine positive positive cells Natural (MSU), Peezy MSU™ and natural void, which are all non-invasive urine cells cells void (cells/ collection methods MSU Peezy 80 μl) (cells/ MSU™ UP3- UP3-negative UP3- p 80 μl) (cells/ negative cells Peezy negative 80 μl) cells MSU MSU™ cells natural (cells/ (cells/80 μl) void (cells/ 80 μl) 80 μl) Analysis Mean 8 3 16 0.001 Analysis Median 2 2 3 Mean 1 0.87 3 0.325 Standard 17 8 27 Median 0 0 0 deviation Test statistics Standard 22 9 deviation N 31 Test statistics Chi-squared N 31 14.- 041 Chi-squared 2.250 df 2 df 2 Int Urogynecol J Table 9 Evaluation of the proportion of UP3-positive cells from mid- 3. Huppert JS, Biro F, Lan D, Mortensen JE, Reed J, Slap GB. 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Intracellular lifestyles and immune eva- tion in urological outpatients with lower urinary tract symptoms sion strategies of uropathogenic Escherichia coli. Annu Rev without acute frequency and dysuria. J Urol. 2010;183(5):1843–7. Microbiol. 2010;64:203–21. 39. Tamadonfar KO, Omattage NS, Spaulding CN, Hultgren SJ. 27. Horsley H, Malone-Lee J, Holland D, et al. Enterococcus faecalis Reaching the end of the line: urinary tract infections. Microbiol subverts and invades the host urothelium in patients with chronic Spectr. 2019; https://doi.org/10.1128/microbiolspec.BAI-0014- urinary tract infection. PloS One. 2013;8(12):e83637. 28. Scott VC, Haake DA, Churchill BM, Justice SS, Kim JH. 40. Horsley H, Dharmasena D, Malone-Lee J, Rohn JL. A urine- Intracellular bacterial communities: a potential etiology for chronic dependent human urothelial organoid offers a potential alternative lower urinary tract symptoms. Urology. 2015;86(3):425–31. to rodent models of infection. 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Qual Life Res. 2005;14(8):1901–13. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Urogynecology Journal Springer Journals

A revalidation and critique of assumptions about urinary sample collection methods, specimen quality and contamination

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Abstract

Introduction and hypothesis Midstream urine (MSU) is key in assessing lower urinary tract syndrome (LUTS), but contingent on some assumptions. The aim of this study was to compare the occurrence of contamination and the quality of substrates obtained from four different collections: MSU, catheter specimen urine (CSU), a commercial MSU collecting device (Peezy) and a natural void. Contamination was quantified by differential, uroplakin-positive, urothelial cell counts. Methods This was a single blind, crossover study conducted in two phases. First, we compared the MSU with CSU using urine culture, pyuria counts and differential counting of epithelial cells after immunofluorescence staining for uroplakin III (UP3). Second, we compared the three non-invasive (MSU, Peezy MSU™, natural void) methods using UP3 antibody staining only. Results The natural void was best at collecting bladder urinary sediment, with the majority of epithelial cells present derived from the urinary tract. CSU sampling missed much of the urinary sediment and showed sparse culture results. Finally, the MSU collection methods did not capture much of the bladder sediment. Conclusion We found little evidence for contamination with the four methods. Natural void was the best method for harvesting shed urothelial cells and white blood cells. It provides a richer sample of the inflammatory exudate, including parasitised urothelial cells and the microbial substrate. However, if the midstream sample is believed to be important, the MSU collection device is advantageous. . . . . . Keywords Midstream Urine Sample Catheter Peezy UTI Introduction related to hospitalisations in the USA [4]. A UTI is a debili- tating condition causing the onset of painful urination (dys- A urinary tract infection (UTI) is the leading reason why pa- uria), increased urinary frequency, the inability to start urinat- tients seek treatment in primary care [1]. One in three women ing (hesitancy) and the sensation of a sudden need to urinate will be treated with antibiotics for UTI by the age of 24 and (urgency) [5], all of which are classified as lower urinary tract 40% to 50% of women will experience one or more UTIs in symptoms (LUTS) [6]. their lifetime, with 10–15% experiencing recurrent infections Many clinical guidelines advocate urinary dipstick testing [2]. A UTI is commonly identified among young adolescents for leucocyte esterase and nitrite as a means of detecting UTI, attending sexual health centres and accounts for 17% of treat- but evidence suggests that their utility might be limited [7]. ment cases [3]. In the adult population of 65 years and older, a Dipstick analyses are surrogate tests, referenced not to micro- UTI is the second most common cause of infectious disease scopic pyuria but to a gold standard urine culture threshold for 3 −1 UTI, which guidelines accept as being between 10 cfu ml 6 −1 and 10 cfu ml of the pure growth of a single urinary path- ogen [8]. This test has attracted criticism too [9–11]. * Linda Collins Furthermore, the literature shows that the methods of sample [email protected] collection for urine culture have never been validated in ap- propriate clinical trials. Justification for the use of such methods is based on plausible assumptions without supporting School of Nursing, Kingston University, Frank Lampl Building, Kingston Hill Campus, London, UK evidence. It is now recognised that routine urinalyses, includ- Department of Renal Medicine, Division of Medicine, University ing dipstick and culture, are insensitive, thus missing genuine College London, London, UK infection in many symptomatic patients [12–14]. Int Urogynecol J There are three commonly used urine collection methods: voided urine, in which we measured microscopic epithelial the midstream clean catch technique (MSU) [15], the catheter cell counts using UP3 staining. specimen of urine (CSU) [16], and suprapubic aspiration [17]. Sample contamination is a key concern, particularly with regard to MSU. Our understanding is hampered by the variable defi- Materials and methods nitions of “contamination” described in the literature. Some have claimed that contamination is indicated by isolation of This study was presented to the National Research Ethics microbes typical of skin flora, such as Corynebacterium and Service Committee (NRES) in Harrow, London, UK, to obtain Staphylococcus [18–20]. According to Collier et al. [21], con- approval to conduct the investigation. Ethical approval was taminated urine samples are revealed by finding squamous ep- granted, which signified that this study complied with condi- ithelial cells on urine microscopy. Wilson and Gaido [22]de- tions that were favourable and worthy of safe research prac- fined contamination as ≥2 different types of organisms at tice. Ethical approval was given subject to all clinicians in the 5 −1 4 −1 >10 ml or 1 organism at <10 ml . Others have reported study having undergone training in good clinical practice urine samples as contaminated, without describing the criteria (GCP). The two-part comparative studies began in January used [23, 24]. These disparities add to the confusion that affects 2013 and lasted until March 2015. Patients with LUTS attend- these diagnostic methods and there are no data available to aid ing the Community LUTS Clinic, Hornsey Central Health clarification. An important consideration is the substrate that Centre, London, UK, made up the participant population. these collection methods should seek to obtain for culture. The patient sample was drawn from women diagnosed with Historically, the emphasis has been on uncontaminated urine chronic UTI, painful bladder syndrome (PBS), overactive samples being cell-free [22]. However, contemporary studies bladder (OAB) and general LUTS. have shown that UTI is associated with microbial parasitisation Patients were given an information sheet about the study of urothelial cells, via surface attachment and/or intracellular and were offered the opportunity to ask questions and address invasion [25–28]. This parasitisation stimulates urothelial cell any concerns. The first part of the study was designed to shedding as part of an innate immune response [27]. It has been ascertain whether there were outcome signals that merited a found that the urinary urothelial cell counts are elevated in focused analysis of urinary epithelial cells in relation to sam- association with other markers of infection and that the propor- ple collection. On the day of recruitment, patients were invited tion of parasitised cells among the cell sediment increases dur- to the centre, and written informed consent was obtained. ing UTIs, along with inflammatory markers and changes in the Each participant was given a unique non-identifiable number. urinary microbiome [13, 27, 29]. If the primary pathology is the All patients completed a validated female LUTS questionnaire microbial invasion of urothelial cells, stimulating the concom- (FLUTS and FLUTSqol) [31] and provided MSU and CSU itant innate immune response of increased urothelial cell shed- samples. The MSU method was accomplished by the patient ding, it is plausible that these cells would make a better sub- spreading her labia apart with one hand and then wiping the strate for urine culture than would supernatants of planktonic urethra area with moistened wipes using the other hand [20]; bacteria. This was demonstrated in our previous work [12]. the CSU was achieved by placing the patient in the lithotomy Shed urothelial cells are an attractive option for study when position, inserting the urethral catheter along the urethra and seeking to examine contamination with different sampling into the bladder to collect part of the urine outflow [32]. methods. Uroplakin III (UP3), a transmembrane protein found The substrate of interest in urinalysis is the inflammatory exclusively in the urinary tract [30], is a useful biomarker for exudate, which contains microbes, white cells, shed urothelial discerning the origin of epithelial cells in a urine sample. cells and other debris. This may become contaminated with Thus, it is possible to discriminate contaminating cutaneous external debris during collection. The collection methods sam- and vaginal squamous cells from urothelial umbrella cells ple different parts of the bladder urine (Fig. 1). The presence using a specific antibody against this protein with immunoflu- of sediment and contaminants may be influenced by the dif- orescence staining protocols. ferent collection methods. Thus, we should seek to examine This study consisted of two parts. The first was a compar- the markers that reflect these different elements. ative study of the urinalysis results obtained from samples The urine sampling methods were randomly sequenced collected by MSU and CSU. It was a random allocation, and obtained with a 1-h interval between specimen collec- cross-over design and the urine sampling performances were tions. The specimen volume requirement was small and well compared using microscopic pyuria counts, epithelial cell within the capacity of an hour. Urine aliquots were obtained counts, microbial growth from spun cell sediment culture from the unspun specimens and these were introduced, un- and UP3-positive cell counts to measure contamination. The stained, into a Neubauer counting chamber; a microscope second part of the study was a random allocation, cross-over was then used to count the white blood cells (WBCs), red comparison study of contamination obtained with MSU, a blood cells (RBCs) and epithelial cells. A sediment culture novel MSU sampling device called a “Peezy” and naturally [12] was carried out. All urine samples were collected in a Int Urogynecol J Fig. 1 a Illustration of the fact that the cellular sediment collects at the bladder base under the influence of gravity. A catheter passes through this sediment to obtain a specimen from the urine that is above this substrate. The sequence in b–e illustrates the phases of the voiding processes. The inflammatory sediment is a Catheter Specimen shown accumulating at the bladder neck under the influence of gravity. The first part of the stream contains the largest quantity from the sediment at the bladder neck and this reduces over the course of the voiding process b Pre-void with sediment c Initial stream rich in d Midstream e Terminal stream accumulating at the sediment of urine bladder neck 30-ml container and spun down in a Denley refrigerated cen- against the perineum whilst passing urine. As the patient be- trifuge at 800 rpm (~75 relative centrifugal force [RCF]). The gan to pass urine, the first part of the stream entered the funnel remaining cell pellet was re-suspended in phosphate-buffered and caused a sponge valve to swell and block flow through the saline (PBS) and three ten-fold serial dilutions were made in main funnel exit. Once the blockage was established, a mid- PBS. A volume of 50 μl of the re-suspended pellet and dilu- stream specimen accumulated in the funnel and was passed tions was plated onto chromogenic CPS3 (now renamed CPS- out of an overflow side drain into the universal container. The Elite) agar (bioMérieux, France) [33] and dispersed evenly Peezy MSU™ device was then discarded into the clinical using a spreader. The culture plates were incubated aerobically waste bin. All urine samples were obtained using each meth- at 37 °C for 24 h, followed by colony enumeration and iden- od, through random sequencing, with one method each day tification using the Chrom ID™ colour chart provided. over 3 consecutive days. An aliquot of spun sediment cells was collected and stained This second study commenced in August 2014. Patients for UP3 as described previously [27]. Briefly, epithelial cells recruited from the first study were asked to participate in the were adhered to a glass slide using a Shandon Cytospin second part, but fresh consent was obtained. A random code Cytocentrifuge (Thermo Scientific) with 80 μl of a urine sam- dictated the order of sampling, which occurred on the first day ple for 5 min at 75 RCF. The cells were fixed with 4% form- of the 3 consecutive days. For experiment 2, samples were aldehyde in PBS for 15 min, blocked with 10% normal goat used to examine the epithelial cell content using the same serum for 30 min and stained for 1 h with anti-UP3 antibody. methods as in experiment 1. After three washes with PBS, the secondary antibody (goat anti-mouse IgG conjugated to Alexa-flour 488) was applied Sample size for 40 min, washed three times and the DNA stain 4′,6- diamidino-2-phenylindole (DAPI) was later applied for The sample was calculated using the G Power software pack- 20 min before mounting. age. The effect size was set as Cohen’s d = 0.55; α = 0.05; The second part of the study was conducted in the wake of power (1 – β err probability) = 0.8; non-centrality parameter the first experiment and was designed to compare the quanti- δ = 2.9; critical t = 2; df = 27; sample size = 28; actual power = tative and qualitative properties of epithelial cells captured in 0.8 or 80%. specimens obtained from an MSU, an MSU collection device (Peezy) and a naturally voided specimen, which was a straightforward urine sample collected by the patient in a con- Statistical analysis tainer without a technique. The Peezy MSU™ is a urine col- lection method achieved by urinating into an engineered de- The data were not normally distributed and exhibited wide vice [21]. The patients were instructed to wash their hands, variance; thus, to analyse quantitative measures we used the clean the genital area with wet wipes, attach the collection non-parametric Friedman test, which achieves a one-way bottle to the Peezy and position the Peezy MSU™ device analysis of variance by ranks. This is an alternative to the Int Urogynecol J Kruskal–Wallis when analysing repeated measures. We used culture [12] on the samples. Table 6 describes the occurrences the Chi-squared test to analyse the differences in proportions. of the top 90% of isolates across all patients cultured from the three different specimen collection methods. The data show that the spun sediment culture performed on MSU samples Results was the most productive method and the technique performed with CSU samples had significantly more negative results. Demography and symptoms The standard NHS culture method was far less productive whether it was from MSU or CSU; taken together, these re- In the first study, 60 female adult patients were enrolled. The sults emphasise the insensitivities of both collection methods. mean age of the patient group was 60 years (SD = 12). The The CSU seems to trade purity for sensitivity. patients suffered from chronic LUTS and were being treated for chronic UTI. The demographic data are shown in Table 1 Evaluation of UP3-positive cells, comparing MSU, and demonstrate that the patients were suffering from signif- Peezy MSU™ and natural void icant symptoms. Thirty-one patients were enrolled in this study, which used the Evaluation of pyuria, epithelial cells and UP3-positive UP3-positive properties of urothelial cells to identify the ori- cells gin of epithelial cells, given that skin and vaginal cells are UP3-negative. The mean age of the patients was 62 (SD = To evaluate the presence of pyuria, epithelial cells and UP3- 10). In total, 93 non-invasive urine samples were collected positive cells, a total of 118 urine samples were examined by MSU, Peezy MSU™ and natural voided urine. Tables 7, 8,and 9 reports the analysis of the UP3-positive cells. The (MSU = 60, CSU = 58). The patients were at various stages of disease. The data analysis results are shown in Tables 2, 3, mean count of UP3-positive cells from the natural void meth- −1 od was greatest compared with the MSU and Peezy MSU™. 4,and 5. Microscopic pyuria counts (log WBC μl )were significantly lower in CSU compared with MSU. Similarly, Thus, natural void achieved the greatest abundance of the there were greater numbers of epithelial cells in MSU, the target substrate. The lowest UP3-positive cell count was ob- majority of which were UP3-positive, indicating urothelial served with the Peezy technique; thus, this method captured origin. There was no significant difference in the proportion the substrate the least. The UP3-negative cells were sparse, of UP3-positive cells between the MSU (0.81) and CSU (0.9) with no difference between specimen collection techniques methods. If the CSU method collected a significantly less (Table 7, 8,and 9). Thus, the proportion of all cells that were contaminated sample, the proportion would be expected to UP3-positive did not differ between the sampling methods, be significantly higher with the CSU method. These results which implies that, contrary to assumptions, contamination by extra-urinary tract cells is not influenced by the sampling suggest that MSU might enrich a urine sample for urinary tract cell sediment compared with the CSU, without increased or technique and was low overall. appreciable contamination. Evaluation of culture results Discussion All 118 urine samples were sent to the National Health Service The purpose of this study was to measure contamination and (NHS) laboratory for routine culture (plating of 1 μlof urinary substrate content for four different methods of obtaining urine supernatant) and of these, 110 specimens were reported as samples. It also used the fact that urothelial cells can be dis- “negative” (defined as below the 10 cfu/ml threshold) or tinguished from squamous epithelial cells by UP3 staining. “mixed growth”, which is commonly reported for chronic This enabled measurement of contamination by the extra- UTI patients [12, 13, 37]. Thus, in addition to routine culture, urinary milieu compromising these various methods. The pro- we enriched infected cells by performing a spun sediment portion of UP3-positive cells found in MSU samples was Table 1 The average symptom scores and quality of life scores for patients who attended the Community Lower Urinary Tract Symptom (LUTS) Clinic with LUTS Participants Mean age Mean urgency [34]score Mean pain score [34] FLUTS [34] (without FLUTSqol [36] (maximum = 64) (maximum = 12) urgency and pain) (maximum = 288) Normal = 0 [35] Normal = 0 [35] (maximum = 149) Normal = N/A Normal = 0 [35] Patients 60 (SD = 12) 16 (SD = 11) 10 (SD = 7) 64 (SD = 40) 151 (SD = 75) Int Urogynecol J Table 2 Evaluation of microscopic pyuria for the midstream urine Table 4 Evaluation of uroplakin-positive cells (UP3) for the midstream (MSU) and catheter specimen urine (CSU) samples urine (MSU) and catheter specimen urine (CSU) samples Analysis Pyuria count MSU Pyuria count CSU p Analysis UP3 cells UP3 cells p −1 −1 (WBCs μl ) (WBCs μl ) (cells/80 μl) MSU (cells/80 μl) CSU Mean 65 24 0.001 Mean 17 5 0.006 Median 0 0 Median 3 1 Standard deviation 321 148 Standard deviation 30 12 WBCs white blood cells given that healthy urine is known to harbour many species of previously reported as 75% (Q1 = 68, Q3 = 78.5) by Horsley bacteria, including known uropathogens [39], it is not known et al [27]. This study had a similar outcome, and has extended whether these microbes are pathogenic or harmless commen- this to three other sample collection methods. Taken together sals, a problem affecting all current urinary microbial detec- with a wide body of literature demonstrating that urothelial tion methods. cell shedding is a common innate immune response to infec- The symptomatic measurements pain, urgency, FLUTS tion, this study suggests that the widely held assumption that and LUTSqol were concordant with the pyuria and culture epithelial cells in a urine sample indicate contamination might data [13]. It should be appreciated that CSU sampling missed need to be revised [26]. The sample collection method did a pathological signal in a significant proportion of patients influence the absolute epithelial cell counts, the lowest num- with appropriate symptoms, pyuria and urothelial cell shed- ber with CSU samples (Fig. 1a) and the greatest number with ding. Compared with CSU, the MSU technique provided a natural void (Fig. 1c), during which the bladder contracts and more substantial sample of the inflammatory sediment. UTI collapses to eliminate bladder urinary substrate. The Peezy involves colonisation of the urothelial cells by pathogenic device seemed to do what it claimed, achieving a sample of microbes [12, 27, 40]. The innate immune response to this fewer cells and very few uroplakin-negative cells. However, microbial colonisation is increased urothelial cell shedding, the sediment trapped in the sponge valve of the Peezy device which may be promoted by mast cells [41]. The shed cells may, counter-intuitively, prove to be a richer substrate for may be colonised or unaffected, but the proportion of microbiological investigation, as bacteria are known to adhere parasitised cells in the face of infection would be expected to and colonise the urothelial cells. to increase [42]. This sediment, a mixture of white cells, epi- The rationale of midstream sampling has always been the thelial cells and debris, is likely to collect under the influence avoidance of contamination. This was never properly tested of gravity at the bladder base, forming a sampling target that and relied on assumptions. In this experiment we were able to should not be influenced by dilution effects. This is made analyse contamination directly by using UP3 staining of the evident by the characteristic milky quality of the terminal flow epithelial cells as a proxy, and we found that contrary to what when a catheter is used to drain an infected bladder. A cathe- has been assumed, the majority of epithelial cells in all sam- ter, as well as a suprapubic stab, samples urine above this ples originated from the urinary tract. collection at the bladder base (Fig. 1a), and therefore contains The standard NHS MSU culture achieved a very small fewer epithelial cells, white cells and microbes, a point number of positive cultures, in contrast to the spun sediment reflected in the data reported here. Thus, a CSU sample may cultures, despite the patients exhibiting significantly fewer be an inferior option because it may miss a substantial amount urinary tract symptoms; this discrepancy has been reported of the pathology. previously by our team and others [38]. Urinary microscopic With the advent of more sensitive genomic technology, a detection of pyuria and increased urothelial cell shedding pro- number of groups have reported that the healthy bladder is not vided strong evidence for the presence of UTI. The sediment sterile, and that polymicrobial colonisation is the norm in both culture generated a greater abundance of isolates. However, healthy and infected bladders [12, 14, 43]. Polymicrobial Table 3 Evaluation of microscopic urinary epithelial cells for the Table 5 Evaluation of the proportion of uroplakin-positive cells (UP3) midstream urine (MSU) and catheter specimen urine (CSU) samples for the MSU and CSU sample Analysis Epithelial cells Epithelial cells p Analysis Proportion of cells Proportion of cells p −1 −1 (μl )MSU CSU (μl ) UP3-positive MSU UP3-positive CSU Mean 21 2 0.002 Mean 0.81 0.9 0.98 Median 2 0 Median 1 1 Standard deviation 103 4 Standard deviation 0.3 0.2 Int Urogynecol J Table 6 Results of the spun urinary sediment culture carried out at the community LUTS clinic laboratory and NHS laboratory Number (%) Number Number Number Number Number Number with Total culturing Ecoli culturing culturing culturing culturing culturing no growth Enterococcus Streptococcus Staphylococcus Proteus yeast MSU spun sediment 18 (30) 13 (22) 12 (20) 5 (8) 8 (13) 4 (7) 0 (0) 60 culture, LUTS laboratory CSU spun sediment 4 (7) 15 (26) 1 (2) 0 2 (3) 5 (9) 31 (53) 58 culture, LUTS laboratory NHS MSU 1 (1) 2(2) 1(1) 0 0 1(1) 100(95) 105 laboratory results NHS CSU 1 (8) 2 (15) 0 0 0 0 10 (77) 13 laboratory results growth in urine specimens has been associated with LUTS in long-held assumptions about urine sampling. The natural void a recent study of similar patients [21]. Indeed, the sediment captures the first part of the urinary stream; the influence of cultures carried out on urine samples collected by MSU and gravity would encourage such samples to contain a larger CSU in this study also demonstrated polymicrobial growth. In proportion of the sediment because of settlement at the blad- patients with symptoms of UTI, the species dispersion is much der base (Fig. 1b). By avoiding the initial stream, the MSU wider than in asymptomatic controls [12, 18, 43–45]. At this and Peezy MSU™ methods collected samples containing time it is not known what species of these mixes are respon- fewer cells, the Peezy MSU™ providing the clearest speci- sible for the disease. In this study, the data on the UP3-positive mens (Fig. 1d). The remaining urine in the bladder proceeds cells argued against the common assumption that with a terminal flow, with very few bladder sediments (Fig. polymicrobial results and/or epithelial cells are pointers to 1e). contamination. A recent study has shown the isolation of a The Peezy MSU™ device works with a sponge in the fun- different microbial population using the Peezy compared with nel exit, which inflates gradually on contact with the first part one isolated from peri-urethral swabs, based on the assump- of the urinary stream, eventually blocking the outflow so that tion of peri-urethral contamination of MSU samples [46]. the remainder of the stream escapes by a side channel into a The natural void provided the greatest number of urothelial collecting tube. Thus, the bulk of the sediment deposit from cells and even in this situation, UP3 analysis did not show the bladder neck would be expected to be trapped in the evidence of increased contamination, which contradicts sponge. If the bladder base sediment is indeed, as recent work suggests, the best specific target for diagnostic analysis, so is Table 7 Evaluation of UP3-positive cells from midstream urine (MSU), the content of the sponge. As matters stand, the Peezy MSU™ Peezy MSU™ and natural void, which are all non-invasive urine collec- achieves what it sets out to do and provides an MSU sample tion methods UP3- UP3- UP3-positive p Table 8 Evaluation of UP3-negative cells from midstream urine positive positive cells Natural (MSU), Peezy MSU™ and natural void, which are all non-invasive urine cells cells void (cells/ collection methods MSU Peezy 80 μl) (cells/ MSU™ UP3- UP3-negative UP3- p 80 μl) (cells/ negative cells Peezy negative 80 μl) cells MSU MSU™ cells natural (cells/ (cells/80 μl) void (cells/ 80 μl) 80 μl) Analysis Mean 8 3 16 0.001 Analysis Median 2 2 3 Mean 1 0.87 3 0.325 Standard 17 8 27 Median 0 0 0 deviation Test statistics Standard 22 9 deviation N 31 Test statistics Chi-squared N 31 14.- 041 Chi-squared 2.250 df 2 df 2 Int Urogynecol J Table 9 Evaluation of the proportion of UP3-positive cells from mid- 3. Huppert JS, Biro F, Lan D, Mortensen JE, Reed J, Slap GB. Urinary stream urine (MSU), Peezy MSU™ and natural void, which are all non- symptoms in adolescent females: STI or UTI? J Adolesc Health. invasive urine collection methods 2007;40(5):418–24. 4. Curns AT, Holman RC, Sejvar JJ, Owings MF, Schonberger LB. Proportion of Proportion Proportion p Infectious disease hospitalizations among older adults in the United UP3-positive UP3-positive UP3-positive States from 1990 through 2002. Arch Intern Med. 2005;165(21): cells MSU cells Peezy cells natural 2514–20. (cells/80 μl) MSU™ (cells/ void (cells/ 5. Bono MJ, Reygaert WC. Urinary tract infection. In StatPearls. 80 μl) 80 μl) 2018. Treasure Island: StatPearls Publishing. 6. Rosen JM, Klumpp DJ. Mechanisms of pain from urinary tract Analysis infection. Int J Urol. 2014;21(Suppl 1):26–32. Mean 0.91 0.88 0.91 0.5 7. Kupelian AS, Horsley H, Khasriya R. 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Published: Mar 5, 2020

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