Urinary Microbiome and its Correlation with Disorders of the Genitourinary System.

PURPOSE: Until recently, the urine of healthy individuals was assumed to be sterile. However, improvement of bacterial detection methods has debunked this assumption. Recent studies have shown that the bladder contains microbiomes, which are not detectable under standard conditions. In this review, we aimed to present an overview of the published literature regarding the relationship between urinary microbiota and functional disorders of the genitourinary system. METHODS: We searched Medline, PubMed, Embase, The Cochrane library and Scopus to identify RCTs published, with MeSH and free keywords including microbiota, bladder pain syndrome, prostatitis, kidney stone disease, and bladder cancer until September 2020. Randomized controlled trials investigating microbiome and lower urinary tract symptoms were included. Non-randomized trials, cross-over trials and pooled studies were excluded. The articles were critically appraised by two reviewers. CONCLUSION: The urine microbiome is a newly introduced concept, which has attracted the attention of medical researchers. Since its recent introduction, researchers have conducted many fruitful studies on this phenomenon, changing our perspective toward the role of bacteria in the urinary tract and our perception of the genitourinary system health. RESULT: A deeper understanding of the urinary microbiome can help us to develop more efficient methods for restoring the microbiota to a healthy composition and providing symptom relief. Modification of the urinary microbiome without antibiotic use can be a possible venue for future research.

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.

Urine proteomics in kidney and urogenital diseases: Moving towards clinical applications.

To date, multiple biomarker discovery studies in urine have been conducted. Nevertheless, the rate of progression of these biomarkers to qualification and even more clinical application is extremely low. The scope of this article is to provide an overview of main clinically relevant proteomic findings from urine focusing on kidney diseases, bladder and prostate cancers. In addition, approaches for promoting the use of urine in clinical proteomics including potential means to facilitate the validation of existing promising findings (biomarker candidates identified from previous studies) and to increase the chances for success for the identification of new biomarkers are discussed.

Time-course of quantitative urinary leukocytes and bacteria counts during antibiotic therapy in women with symptoms of urinary tract infection.

BACKGROUND: Urinary tract infections are generally diagnosed by test strips and microscopic semi-quantitative sediment analyses. However, results are uncertain because of lacking standardisation and limited sensitivity in low-count-bacteriuria. Flow cytometry UF-100 was used to analyse particles quantitatively in urine in women with urinary tract infections during the period of antibiotic therapy. The aim was to follow the courses of leukocytes and bacteria during infections and to gain information about the reasons for successful or unsuccessful outcomes. METHOD: Quantitative leukocytes and bacterial counts in urine of 16 symptomatic women were performed at presentation and each day during the antibiotic treatment by flow cytometry UF-100. RESULTS: Leukocytes in urine were between 30 and 15,000 (x10(6)/L) at presentation (cut-off 20x10(6)/L). Bacteria counts from flow cytometry were mainly 5x10(9)/L-100x10(9)/L (cut-off of 3x10(9)/L). The deepest decreases in cell counts were noted during the first 24 h after initiation of therapy and gained normal values at the end of treatment in successful outcomes. A slower or no decrease was noted in unsuccessful treatments. CONCLUSION: The precise leukocyte and bacteria counting by flow cytometry and their follow-up during urinary tract infections gave early information about outcomes of therapy.

Use of quantitative real-time PCR to monitor the shedding and treatment of chlamydiae in the koala (Phascolarctos cinereus).

The aim of this study was to monitor chlamydial shedding patterns in clinically affected koalas before, during and following treatment using quantitative real-time PCR. Swab samples were obtained from 14 koalas presented for treatment at the Australian Wildlife Hospital. Four of these animals were followed over a period of 8-9 weeks. Primers were designed based on the consensus signature sequence of the 16S rRNA chlamydial gene. Additional primers were designed based on the sequence of the koala beta-actin gene and used to normalize chlamydial values when comparing results from different swab samples. Chlamydial 16S rRNA gene copy number was highest in swab samples from clinically affected sites. Daily injections of chloramphenicol resulted in a marked and rapid reduction in the numbers of chlamydiae being shed from all sites. In general, chlamydial copy number was no longer detectable by the end of the 2nd week of treatment. No evidence of relapse of infection was detected at 2 weeks after the cessation of treatment. In contrast, topical chloramphenicol treatment of the eyes required a longer treatment period and had little effect on the shedding of chlamydiae from other sites of the body. Further studies are required to confirm the efficacy of a shorter treatment period.

Prevalence of urogenital Chlamydia trachomatis infections in the Netherlands suggests selective screening approaches. Results from the PILOT CT Population Study.

Chlamydia trachomatis screening is being considered in the Netherlands, but policy recommendations are hampered by the lack of population-based data. We studied the prevalence of chlamydia infection in 15-29-year-old women and men in a national representative sample of 21,000 inhabitants of rural and urban areas in the Netherlands. Of this sample, 41% responded by sending in urine and an answered questionnaire, while 11% returned a refusal card. The overall prevalence of chlamydia infection was 2.0% (CI: 1.7-2.3); 2.5% (CI: 2.0-3.0) in women and 1.5% (1.1-1.8) in men. Chlamydia prevalence was significantly greater in very highly urbanized areas (3.2%, CI: 2.4-4.0) compared to rural areas (0.6%, CI: 0.1-1.1). In very highly urbanized areas the greatest prevalence was found among 15-19-year-old women (4.3%) and among 25-29-year-old men (4.2%). A risk profile could be determined and a prediction rule was developed. These data suggest that nationwide systematic screening is not indicated in the Netherlands and that targeted approaches are a better option. Roll-out of selective screening is recommended.

Quantitative evaluation of telomerase subunits in urine as biomarkers for noninvasive detection of bladder cancer.

The aim of our study was to prospectively evaluate the potential diagnostic value and clinical applicability of quantitative analysis of telomerase subunits gene expression in urine for noninvasive detection of bladder cancer. Expression levels of human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) were analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in urine samples from 163 subjects with bladder cancer and 237 controls (163 individuals with benign genitourinary diseases; 74 healthy subjects). The sensitivity, specificity and optimal cutoffs were determined and compared to the corresponding values obtained by voided urine cytology. Quantitative urinary hTR analysis detects bladder cancer with an overall sensitivity of 77.0%, whereas hTERT analysis reached a sensitivity of 55.2%. The majority of undetected tumors were small, low-grade pTa lesions. Both hTR and hTERT proved to be significantly more sensitive than cytology (34.5%; p < 0.001). Specificities for hTR, hTERT and cytology were 72.1%, 85.0% and 92.7%, respectively, in the total study population and 96.9%, 89.2% and 100%, respectively, in healthy subjects. Higher diagnostic accuracy was achieved by hTR than by hTERT analysis (p < 0.05). The specificity of hTR increased to 85.0% in the total population if urinary leukocyte contamination was excluded. These data suggest that quantitative hTR analysis is the most accurate telomerase-based test for bladder cancer detection and has the potential to replace cytology as a noninvasive biomarker for disease diagnosis and follow-up.

[Hygienic aspects of urinary system developing pathology in children (review)]. / Gigienicheskie aspekty formirovaniia patologii organov mochevoi sistemy u detei (obzor).

The paper deals with the impact of environmental factors of industrial cities and towns on children's health. Particular emphasis is laid on urinary tract pathology since the kidney is a xenobiotically exposed target organ. The main criteria for a risk of environment-induced diseases are ambient air pollution, low drinking water quality, soil heavy metal pollution, etc. The review covers the pathogenetic mechanisms and clinical features of the manifestation of renal diseases in children living in the regions with different heavy metal-loaded environment.

Evaluating adolescents in juvenile detention facilities for urogenital chlamydial infection: costs and effectiveness of alternative interventions.

CONTEXT: Adolescents in juvenile detention facilities present a unique opportunity to diagnose and treat sexually transmitted diseases. OBJECTIVE: To evaluate the effectiveness and costs of different strategies for the diagnosis and treatment of chlamydial infection in adolescents in juvenile detention.Design, Setting, and Subjects For a cohort of adolescents in a juvenile detention facility, sex-specific decision models were developed comparing strategies for diagnosing and treating chlamydial infection. These strategies included not screening, treating everyone, and testing (with leukocyte esterase [LE], ligase chain reaction [LCR], or history and symptoms) followed by treatment for those with positive test results. Two different time horizons were considered: immediate and extended. In the immediate time horizon, we performed a cost-effectiveness analysis looking only at the outcomes associated with treating current infections; in the extended time horizon, we performed a cost-minimization analysis comparing the estimated total costs of diagnosing and treating Chlamydia as well as those associated with complications occurring up to 20 years in the future. RESULTS: In males, the immediate-time-horizon evaluation revealed that treating on the basis of urine LE results produced the lowest incremental cost-effectiveness ratio ($80 per infection treated). In the extended-time-horizon cost-minimization analysis, treating males on the basis of urine LE results was again found to be the least expensive strategy ($10.11 per person). Two other strategies, confirming urine LE results with LCR ($10.96 per person) and screening with urine LCR ($14.04 per person), were found to be less expensive than not screening ($16.66 per person). In females, the immediate-time-horizon evaluation found that treating on the basis of symptoms and history resulted in treating about half the cases of chlamydial infection and produced the lowest incremental cost-effectiveness ratio ($74 per infection treated). More infections were treated when treatment was based on urine LCR results with only a small increase in the incremental cost per case treated ($95 per infection treated). In the extended-time-horizon cost-minimization analysis, treating all females empirically and treating based on results of urine LCR testing were the least expensive strategies ($18.81 and $18.98 per person, respectively). The results were sensitive to several variables, including prevalence of chlamydial infection, in both males and females. CONCLUSIONS: For adolescent males in juvenile detention facilities, screening with urine LE minimizes the costs associated with diagnosis, treatment, and sequelae of urogenital chlamydial infection. For adolescent females in juvenile detention, empiric treatment and that based on urine LCR test results are the optimal strategies for managing urogenital chlamydial infection.

Persistence of Chlamydia trachomatis infection detected by polymerase chain reaction in untreated patients.

BACKGROUND: Prior studies have used Chlamydia trachomatis culture methods to demonstrate both persistence and spontaneous clearance of genital C trachomatis infection. OBJECTIVE: To further assess the issue of persistence and spontaneous clearance of C trachomatis infection, untreated men and women were evaluated with repeated polymerase chain reaction (PCR) testing. METHODS: Ninety four untreated patients with a prior positive C trachomatis PCR test returning to the Denver Metro Sexually Transmitted Disease Clinic were retested by PCR. RESULTS: The median and range intervals from initial to follow-up testing were 9.0 (2-112) days for men and 10.0 (2-231) days for women. Repeated PCR tests were positive for 29 of 36 men (80.6%) and 45 of 58 women (77.6%). Persistent PCR positivity did not decrease with a longer testing interval. By multivariate analysis, independent predictors of a persistently positive PCR test included nonwhite ethnicity, an interval of more than 3 days since last sexual encounter before the initial test, and an initial PCR optical density value of greater than or equal to 3.0. CONCLUSIONS: In the absence of treatment, a large majority of patients testing positive for C trachomatis by PCR are likely to remain positive for variable periods of time, increasing the risk of transmission and immune-mediated damage. A low initial optical density value and recent sexual contact may be markers for exposure that does not establish infection.

Qualitative and quantitative aspects of the ligase chain reaction assay for Chlamydia trachomatis in genital tract samples and urines.

The performance of the ligase chain reaction (LCR) assay for Chlamydia trachomatis was evaluated in a genitourinary medicine (GUM) clinic population. Its sensitivity was 100%, 91% and 95%, respectively, for cervical, vaginal and urine samples from 417 women, when compared with direct fluorescent antibody (DFA) staining of cervical samples, and 100% and 91%, respectively, for urethral and urine samples from 317 men, when compared with DFA staining of urethral smears. An enzyme immunoassay (EIA) was only 65% sensitive for cervical samples. Urethral swabs from a number of treated men remained LCR-positive when antigen was no longer detectable by DFA staining. An association between quantitative data from the LCR assay (i.e. the optical density of samples, measured in relation to internal controls and calibrators) and the antigen load of the samples, measured by DFA staining, indicated a lack of significant inhibition in the LCR assay in this study. This was probably due to freezing of the samples before testing. Diluting 20 LCR-positive urines with a range of antigen loads resulted in loss of positivity in 3, and a reduction in the signal in 13. The implications of the antigen load on the performance of detection assays for chlamydia-positive patients are discussed.

Screening for Chlamydia trachomatis infection using the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine in a GUM clinic setting: a simple, fast and cost-effective alternative.

This study compared the BDProbeTec ET Chlamydia trachomatis amplified DNA assay on urine specimens with culture of genital swabs for the detection of C. trachomatis in patients attending the Department of Genitourinary Medicine (GUM), Cardiff Royal Infirmary. Almost twice as many patients tested positive by BDProbeTec ET than by culture. A similar difference was found for both males and females. The case notes of those patients positive by BDProbeTec ET alone were analysed and a significantly greater number were found to have risk indicators for C. trachomatis infection when compared with age and sex comparable controls, providing clinical validation of our findings. The BDProbeTec ET assay was easy to use, more importantly, the test format features an internal control integral with every sample. The cost per true positive was calculated as comparable with culture. We conclude that the BDProbeTec ET assay is a superior alternative to culture for identifying patients infected with C. trachomatis in the GUM clinic setting.

Sensitivity and specificity of antiproliferative factor, heparin-binding epidermal growth factor-like growth factor, and epidermal growth factor as urine markers for interstitial cystitis.

We previously determined that the urine of interstitial cystitis (IC) patients specifically contains a factor (antiproliferative factor [APF]) that inhibits primary bladder epithelial cell proliferation, and that it has significantly decreased levels of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and increased levels of epidermal growth factor (EGF) compared with urine from asymptomatic controls and patients with bacterial cystitis. We sought to confirm the specificity of these findings for IC using a larger patient population, including control patients with a variety of urogenital disorders. Clean catch urine specimens were collected from 219 symptomatic IC patients, 113 asymptomatic controls without bladder disease, and 211 patients with various urogenital diseases including acute bacterial cystitis, vulvovaginitis, chronic nonbacterial prostatitis, overactive bladder, hematuria, stress incontinence, neurogenic bladder, benign prostatic hyperplasia, bladder or pelvic pain without voiding symptoms, bladder cancer, prostate cancer, or miscellaneous diagnoses including anatomic disorders. APF activity was determined by (3)H-thymidine incorporation into primary normal adult human bladder epithelial cells. HB-EGF and EGF levels were determined by enzyme-linked immunosorbent assay. APF activity was present significantly more often in IC than control urine specimens (P <0.005 for IC vs any control group; sensitivity = 94%, specificity = 95%, P <10(-82) for IC vs all controls). HB-EGF levels were also significantly lower and EGF levels significantly higher in IC urine than in specimens from controls (P <10(-84) and P <10(-36), respectively). These findings confirm the utility of APF, HB-EGF, and EGF as markers for IC. Understanding the reasons for altered levels of these markers may lead to understanding the pathogenesis of this disorder.

Chlamydia trachomatis PCR (Cobas Amplicor) in women: endocervical specimen transported in a specimen of urine versus endocervical and urethral specimens in 2-SP medium versus urine specimen only.

The sensitivity of Roche Cobas Amplicor Chlamydia trachomatis polymerase chain reaction (PCR) including the internal control (IC) programme to identify inhibition, was investigated on 3 different samples from women: (1) swab samples from the urethra and the cervix pooled in 2-SP transport medium, (2) swab sample from the cervix transported in a urine sample from the same patient, and (3) urine sample alone. Out of the 2412 patients, 193 (8.0%) were chlamydia positive and in 14 of these the results showed discrepancies between sampling methods. The sensitivity of PCR on urethra/cervix, urine/cervix and urine was 98.4% (190/193), 97.9% (189/193) and 93.3% (180/193) respectively. The higher sensitivity of PCR on urethra/cervix and urine/cervix as compared with urine alone was statistically significant. Without the IC, the sensitivity of PCR on urethra/ cervix, urine/cervix and urine would have been 95.9% (185/193), 94.8% (183/193) and 90.7% (175/193) respectively. Factors influencing the rate of inhibition were also studied.

Recurrence of urogenital Chlamydia trachomatis infection evaluated by mailed samples obtained at home: 24 weeks' prospective follow up study.

OBJECTIVES: To evaluate the rate of recurrence of genital Chlamydia trachomatis infection after antibiotic therapy in a population of patients drawn from general practice, and to evaluate whether retesting after antibiotic therapy was advisable and, if so, whether it could be based on a strategy involving samples obtained at home and mailed to the laboratory for analysis. METHODS: Prospective follow up study of 42 patients with genital C trachomatis infection drawn from general practice. Patients at or above the age of 18, with a positive urogenital swab sample obtained by a general practitioner were invited to participate. Follow up testing was based on LCR testing (LCx, Abbott diagnostics) of first void urinary and vaginal flush samples taken by the patients at home and mailed to the laboratory at weeks 2, 4, 8, 12, and 24 after antibiotic therapy. RESULTS: Cumulated incidence of recurrent infection was calculated to 29% (95% CI: 12%-46%) during the 24 weeks of follow up. Previous or present sexually transmitted diseases other than C trachomatis were significantly associated with recurrence (OR 6.1, p = 0.03). 89% of patients tested negative at week 2, and all patients tested negative at some point during the first 4-8 weeks. 84% of the test kits mailed to the patients were returned to the laboratory for analysis. CONCLUSIONS: Recurrence of C trachomatis after antibiotic treatment is a substantial problem. Retesting should be carried out, but not sooner than 12-24 weeks after treatment. Requiring patients to take tests at home appears to be a promising method for retesting.

Creatinine at the evaluation of urinary 1-methyladenosine and pseudouridine excretion.

The elevation of urinary modified nucleosides levels in urine is found in patients with cancers. In the present study, we have tested 616 urine samples randomly collected from non-malignant cases. Thirty-two percent (194/616) and 11% (68/616) had elevated levels of 1-methyladenosine and pseudouridine, respectively (They are designated as false-positive cases). To elucidate the cause on non-specific elevation of the nucleosides, the correlation between creatinine excretion level and urinary nucleosides levels were determined. The result revealed that false-positive cases were frequently detected in patients with lower creatinine excretion levels. The mean creatinine levels of false-positive cases were significantly lower than those of negative cases. From these results, the false-positive of urinary 1-methyladenosine and pseudouridine might be due to the low creatinine excretion mainly caused by the renal dysfunction. Creatinine excretion in each individual should be taken into consideration in case of determining urinary modified nucleosides.

Comparison of performances of two commercially available tests, a PCR assay and a ligase chain reaction test, in detection of urogenital Chlamydia trachomatis infection.

The diagnostic performance of a PCR test (Roche Cobas Amplicor CT/NG Test) and that of a ligase chain reaction (LCR) test (Abbott LCx Chlamydia trachomatis assay) were compared by using endocervical and urethral swab specimen culture as a reference test. First-void urine (FVU) and endocervical and urethral swab specimens were collected from 1,015 unselected patients attending a sexually transmitted disease clinic and a clinic for adolescents in Helsinki, Finland. Chlamydia trachomatis was cultured from samples from the endocervix or urethra. PCR was performed with fresh and frozen urine and the culture transport medium. LCR was performed with fresh and frozen urine and LCx swab transport medium. Diagnostic consistency and diagnostic accuracy were statistically tested. The test results were identical for 984 patients (97%). Discrepant results were observed for 31 patients. Overall, LCR and PCR showed excellent kappa coefficients of consistency for both swab and FVU specimens (0.93 and 0.95, respectively). Sixty-one patients (6%) were culture positive. Testing of FVU by LCR or PCR increased the overall positivity rates to 7.0 and 7.7%, respectively. While PCR of FVU detected the greatest number of C. trachomatis infections (sensitivity, 96.1%), for some PCR-positive FVU specimens the results could not be confirmed (specificity, 99.6%). PCR and LCR were more sensitive than culture (sensitivities, 92 and 93% versus 79% for culture) in the diagnosis of genital C. trachomatis infection. In conclusion, both tests can be recommended for use in the clinical laboratory and for the screening of asymptomatic C. trachomatis infections.

Basic fetoprotein in normal and pathologic urine.

We developed a latex agglutination nephelometric immunoassay for urinary basic fetoprotein (BFP) that functioned well and had good specificity, precision, and recovery. Reference intervals started below 0.5 microgram/L, the lower limit of the range of sensitivity of the assay, and went up to 7.0 micrograms/L at the 97.5th percentile without age- or sex-related variation, in accordance with the NCCLS guidelines. BFP was unstable at pH 5.0 at 4 degrees C and 25 degrees C. The western blot method showed BFP found in the semen to be structurally identical to purified BFP from hepatoma ascites, in which concentration ranged from 94.2 to 145.2 micrograms/L and, further, to have the same molecular weight and reactivity with a monoclonal antibody. BFP levels were elevated in cases urinary BFP concentration included ureter stone, infection, and prostate and bladder cancer. Moreover, BFP concentration correlated closely with that of alpha 2-macroglobulin, indicating that BFP is probably secreted locally in close pathophysiologic association with post-renal hemorrhage. We thus conclude that BFP is a urinary nonspecific marker for inflammation or tumor. The best indication for BFP as a tumor marker may be follow-up when diagnosis of genitourinary cancer is definite.