Voided Urine Samples Research Articles

Comparison of urine samples and penile swabs for detection of human papillomavirus in HIV-negative Dutch men

ObjectivesPenile swab sampling is the method of choice when testing for human papillomavirus (HPV) in men. Urine sampling is already used in routine sexually transmitted infections (STI) diagnostics and could...

A multiplex immunoassay for the non-invasive detection of bladder cancer.

BackgroundUrine based assays that can non-invasively detect bladder cancer (BCa) have the potential to reduce unnecessary and invasive procedures. The purpose of this study was to develop a multiplex immunoassay that can accurately and simultaneously monitor ten diagnostic urinary protein biomarkers for application as a non-invasive test for BCa detection.MethodsA custom electrochemiluminescent multiplex assay was constructed (Meso Scale Diagnostics, LLC, Rockville, MD, USA) to detect the following urinary proteins; IL8, MMP9, MMP10, ANG, APOE, SDC1, A1AT, PAI1, CA9 and VEGFA. Voided urine samples from two cohorts were collected prior to cystoscopy and samples were analyzed blinded to the clinical status of the participants. Means (±SD) and receiver operating characteristic (ROC) curve analysis were used to compare assay performance and to assess the diagnostic accuracy of the diagnostic signature.ResultsComparative diagnostic performance analyses revealed an AUROC value of 0.9258 for the multiplex assay and 0.9467 for the combination of the single-target ELISA assays (p = 0.625), so there was no loss of diagnostic utility for the MSD multiplex assay. Analysis of the independent 200-sample cohort using the multiplex assay achieved an overall diagnostic sensitivity of 0.85, specificity of 0.81, positive predictive value 0.82 and negative predictive value 0.84.ConclusionsIt is technically feasible to simultaneously monitor complex urinary diagnostic signatures in a single assay without loss of performance. The described protein-based assay has the potential to be developed for the non-invasive detection of BCa.

A multiplex immunoassay for the non-invasive detection of bladder cancer.

471 Background: Urine based assays that can non-invasively detect bladder cancer (BCa) have the potential to reduce unnecessary and invasive procedures. The purpose of this study was to develop a multiplex immunoassay that can accurately and simultaneously monitor 10 diagnostic urinary protein biomarkers for application as a non-invasive test for BCa detection Methods: A custom electrochemiluminescent (ECL) multiplex assay was constructed (Meso Scale Diagnostics, LLC, Rockville, MD) to detect the following urinary proteins; IL8, MMP9, MMP10, ANG, APOE, SDC1, A1AT, PAI1, CA9 and VEGFA. Voided urine samples from two cohorts (cohort #1 n = 62 and cohort #2 n = 200) were collected prior to cystoscopy and samples were analyzed blinded to the clinical status of the participants. Means (±SD) and receiver operating characteristic (ROC) curve analysis were used to compare assay performance and to assess the diagnostic accuracy of the diagnostic signature. Results: Comparative diagnostic performance analyses revealed an AUROC value of 0.9258 for the multiplex assay and 0.9467 for the combination of the single-target ELISA assays (p = 0.625), so there was no loss of diagnostic utility for the MSD multiplex assay. Analysis of the independent 200-sample cohort using the multiplex assay achieved an overall diagnostic sensitivity of 0.85, specificity of 0.81, positive predictive value 0.82 and negative predictive value 0.84. Conclusions: It is technically feasible to simultaneously monitor complex urinary diagnostic signatures in a single assay without loss of performance. The described protein-based assay has the potential to be developed for the non-invasive detection of BCa.

Gene expression test for the non-invasive diagnosis of bladder cancer: A prospective, blinded, international and multicenter validation study

ObjectiveThis study aimed to validate, in a prospective, blinded, international and multicenter cohort, our previously reported four non-invasive tests for bladder cancer (BC) diagnosis based on the gene expression patterns of urine. MethodsConsecutive voided urine samples from BC patients and controls were prospectively collected in five European centres (n=789). Finally, 525 samples were successfully analysed. Gene expression values were quantified using TaqMan Arrays and previously reported diagnostic algorithms were applied to gene expression data. Results from the most accurate gene signature for BC diagnosis were associated with clinical parameters using analysis of variance test. ResultsHigh diagnostic accuracy for the four gene signatures was found in the independent validation set (area under curve [AUC]=0.903–0.918), with the signature composed of two genes (GS_D2) having the best performance (sensitivity: 81.48%; specificity: 91.26%; AUC: 0.918). The diagnostic accuracy of GS_D2 was not affected by the number of tumours (p=0.58) but was statistically associated with tumour size (p=0.008). Also, GS_D2 diagnostic accuracy increases with increasing BC tumour risk. We found no differences in the performance of the GS_D2 test among the populations and centres in detecting tumours (p=0.7) and controls (p=0.2). ConclusionsOur GS_D2 test is non-invasive, non-observer dependent and non-labour-intensive, and has demonstrated diagnostic accuracy in an independent, international and multicenter study, equal or superior to the current gold standard (cystoscopy combined with cytology). Additionally, it has higher sensitivity than cytology while maintaining its specificity. Consequently, it meets the requirements for consideration as a molecular test applicable to clinical practice in the management of BC.

Role of Urinary and Serum Carbohydrate Antigen 19-9 as a Biomarker in Diagnosis of Adult Giant Hydronephrosis.

The most common cause of adult Giant Hydronephrosis (GH) is congenital Uretero-Pelvic Junction (UPJ) obstruction. Conventional imaging modalities, like Intravenous Urography (IVU) and Computed Tomography Urography (CTU) and radionuclide renal scan can be fallacious. Serum carbohydrate antigen 19-9 (CA19-9) is a useful tumour marker for gastrointestinal and pancreatic cancer. Only a few studies and case reports have shown raised serum levels due to benign hydronephrosis and GH. To investigate the prognostic role of urine and serum CA19-9 in the diagnosis and follow-up of adult GH due to UPJ obstruction. The present hospital based observational study was conducted on 24 adult patients (Group 1) with unilateral GH due to UPJ obstruction. Twenty four healthy adults were included as control (Group 2). Serum and voided urine samples were collected to evaluate Carbohydrate Antigen (CA) 19-9 in each group. During surgery, urine from the affected pelvis was collected to determine CA19-9 level. Patients were followed up after surgery at 3 and 9 months with serum and voided urine samples for CA19-9 level. Preoperative Serum and voided urine CA19-9 were significantly greater in Group1 than in controls, which significantly correlated inversely with preoperative percentage renal function and glomerular filtration rate. Postoperative improvement in renal function significantly correlated inversely with serum and voided urine CA19-9 at 3 and 9 months. Voided urine CA19-9 can be a non-invasive clinical marker in adult GH due to UPJ obstruction. The clinical implications of these data for diagnosis and follow-up of these patients are significant. Our findings suggest, significant decrease in urinary Ca19-9 level during follow-up is predictive of excellent surgical outcome and resolution of renal damage.

Genital Mycoplasma and Chlamydia trachomatis infections in patients with genital tract infections attending a tertiary care hospital of North India.

Limited data are available on the prevalence of genital mycoplasmas and Chlamydia trachomatis (CT) among Indian patients with genital tract infections. The objectives of the study were to determine the prevalence of Ureaplasma urealyticum (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), and CT in patients with genital tract infections. The antimicrobial susceptibilities of UU and MH were also assessed. Endocervical swabs/urethral swabs and first void urine samples of patients (n = 164) were collected. UU and MH were detected by culture and multiplex polymerase chain reaction (PCR). MG and CT were identified by PCR. Ureaplasma isolates were further biotyped and serotyped. Antimicrobial susceptibility was done by microbroth dilution method. UU, MH, MG, and CT were detected in 15.2%, 5.4%, 1.2%, and 6% patients, respectively. Ureaplasma parvum serovar 3/14 was the most prevalent. All isolates of UU and MH were uniformly susceptible to doxycycline and josamycin. Routine screening for these pathogens and antimicrobial susceptibility testing is warranted to prevent sequel of infections and formulate treatment guidelines.

Optimizing Urine Processing Protocols for Protein and Metabolite Detection

In urine, factors such as timing of voids, and duration at room temperature (RT) may affect the quality of recovered protein and metabolite data. Additives may aid with detection, but can add more complexity in sample collection or analysis. We aimed to identify the optimal urine processing protocol for clinically-obtained urine samples that allows for the highest protein and metabolite yields with minimal degradation. Healthy women provided multiple urine samples during the same day. Women collected their first morning (1st AM) void and another "random void". Random voids were aliquotted with: 1) no additive; 2) boric acid (BA); 3) protease inhibitor (PI); or 4) both BA + PI. Of these aliquots, some were immediately stored at 4°C, and some were left at RT for 4 hours. Proteins and individual metabolites were quantified, normalized to creatinine concentrations, and compared across processing conditions. Sample pools corresponding to each processing condition were analyzed using mass spectrometry to assess protein degradation. Ten Caucasian women between 35-65 years of age provided paired 1st morning and random voided urine samples. Normalized protein concentrations were slightly higher in 1st AM compared to random "spot" voids. The addition of BA did not significantly change proteins, while PI significantly improved normalized protein concentrations, regardless of whether samples were immediately cooled or left at RT for 4 hours. In pooled samples, there were minimal differences in protein degradation under the various conditions we tested. In metabolite analyses, there were significant differences in individual amino acids based on the timing of the void. For comparative translational research using urine, information about void timing should be collected and standardized. For urine samples processed in the same day, BA does not appear to be necessary while the addition of PI enhances protein yields, regardless of 4°C or RT storage temperature.

Clinical value of digital image analysis in the diagnosis of urinary bladder cancer, particularly in aggressive tumors: a preliminary report.

The aim of the project was to evaluate the clinical value of a computer analysis of cytological specimen images obtained from urine and bladder washing samples. Three sample types (voided urine, catheterized urine and bladder washing) from 59 patients with primary or recurrent tumor were analyzed. All patients underwent cystoscopy and biopsy or resection. The histological results were compared with the results of the image analyzing computer system of collected urine samples. The consistency between the computer diagnosis and the clinical or histological diagnosis both in the presence and absence of cancer was as follows: 77% for voided urine samples, 72.5% for catheterized urine samples and 78% for bladder washing samples. The specificity of the method at the standard pathology level was 71%, and the sensitivity was 83%. The positive and negative predictive values (PPV and NPV) were 87.5% and 63% respectively. The sensitivity for G3 or CIS or T2 or T3 tumors reached nearly 100%. Computer analysis of urine provided correct diagnoses in cancer and control patients with the sensitivity of 83% and specificity of 71% and gave excellent results in aggressive tumors such as T2, T3, G3 and in CIS.

Editorial Comment

After resection of a primary non–muscle-invasive bladder cancer (NMIBC), about two-thirds of patients will develop recurrences. Kompier 1 Kompier L.C. van der Aa M.N. Lurkin I. et al. The development of multiple bladder tumour recurrences in relation to the FGFR3 mutation status of the primary tumour. J Pathol. 2009; 218: 104-112 Crossref PubMed Scopus (47) Google Scholar followed 118 patients with NMIBC. During a median follow-up of 8.8 years, these patients underwent 2133 cystoscopies and 80 patients developed 414 recurrences. The 38 patients who did not develop any recurrences underwent on average 14 cystoscopies. For these patients, surveillance by cystoscopy can be considered as overtreatment. Cystoscopy is an invasive technology that is associated with anxiety and pain. 2 van der Aa M.N. Steyerberg E.W. Sen E.F. et al. Patients' perceived burden of cystoscopic and urinary surveillance of bladder cancer: a randomized comparison. BJU Int. 2008; 101: 1106-1110 Crossref PubMed Scopus (88) Google Scholar Although the gold standard, its sensitivity is estimated to be between 46% and 86% and specificity between 31% and 93%. 3 Witjes J.A. Douglass J. The role of hexaminolevulinate fluorescence cystoscopy in bladder cancer. Nat Clin Pract Urol. 2007; 4: 542-549 Crossref PubMed Scopus (107) Google Scholar The limited sensitivity is often ignored, and this oversight is one of the reasons that urine tests have not been introduced in the clinic. We and others have demonstrated that molecular assays can detect recurrences in cells obtained from voided urine with a sensitivity that allows implementation of urine diagnostics especially for patients who are at low risk of progression and which is also cost-effective. 4 Zuiverloon T.C. Tjin S.S. Busstra M. et al. Optimization of nonmuscle invasive bladder cancer recurrence detection using a urine based FGFR3 mutation assay. J Urol. 2011; 186: 707-712 Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar , 5 Zuiverloon T.C. Beukers W. van der Keur K.A. et al. Combinations of urinary biomarkers for surveillance of patients with incident nonmuscle invasive bladder cancer: the European FP7 UROMOL project. J Urol. 2013; 189: 1945-1951 Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar , 6 van Kessel K.E. Kompier L.C. de Bekker-Grob E.W. et al. FGFR3 mutation analysis in voided urine samples to decrease cystoscopies and cost in nonmuscle invasive bladder cancer surveillance: a comparison of 3 strategies. J Urol. 2013; 189: 1676-1681 Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar The Diagnostic and Prognostic Performance of Urinary FGFR3 Mutation Analysis in Bladder Cancer Surveillance: A Prospective Multicenter StudyUrologyVol. 86Issue 6PreviewTo assess the diagnostic and prognostic performance of a noninvasive FGFR3 mutation analysis. After transurethral resection (TUR) of noninvasive bladder transitional cell carcinoma (B-TCC), recurrence occurs in 70% of patients, thus justifying cystoscopic surveillance. Full-Text PDF

Elevated plasma and urinary concentrations of green tea catechins associated with improved plasma lipid profile in healthy Japanese women

This study investigated green tea catechins in plasma and urine and chronic disease biomarkers. We hypothesized that plasma and urinary concentration of green tea catechins are associated with cardiovascular disease and diabetes biomarkers. First void urine and fasting plasma samples were collected from 57 generally healthy females aged 38 to 73 years (mean, 52 ± 8 years) recruited in Himeji, Japan. The concentrations of plasma and urinary green tea catechins were determined by liquid chromatography coupled with mass tandem spectrometer. Low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, glucose, insulin, glycated hemoglobin, and C-reactive protein in plasma/serum samples were analyzed by a commercial diagnostic laboratory. Statistical associations were assessed using Spearman correlation coefficients. The results showed weak associations between plasma total catechin and triglyceride (r = −0.30) and LDL cholesterol (r = −0.28), whereas plasma (−)-epigallocatechin-3-gallate, (−)-epigallocatechin, (−)-epicatechin-3-gallate, and (−)-epicatechin exhibited weak to moderate associations with triglyceride or LDL cholesterol, but little associations with HDL cholesterol, body fat, and body mass index were evident. Urinary total catechin was weakly associated with triglyceride (r = −0.19) and LDL cholesterol (r = −0.15), whereas urinary (−)-epigallocatechin-3-gallate (r = −0.33), (−)-epigallocatechin (r = −0.23), and (−)-epicatechin-3-gallate (r = −0.33) had weak to moderate correlations with triglyceride and similarly with body fat and body mass index. Both plasma (r = −0.24) and urinary (r = −0.24) total catechin, as well as individual catechins, were weakly associated with glycated hemoglobin. Plasma total and individual catechins were weakly to moderately associated with C-reactive protein, but not the case for urinary catechins. In conclusion, we found weak to moderate associations between plasma and urinary green tea catechin concentrations and plasma biomarkers of cardiovascular disease and diabetes.

Urine specimen collection following consensual intercourse – A forensic evidence collection method for Y-DNA and spermatozoa

The purpose of the prospective research was to evaluate the benefit of urine specimen as a collection technique for biological forensic evidence in adult volunteers following consensual intercourse. For detecting Y-chromosomal material Buccal Swab Spin Protocol® was used in DNA extraction and purification and samples were analysed with Quantifiler Y Human Male DNA Quantification Kit®. The time frame for positive Y-DNA was evaluated. Immediate microscopy for detection of spermatozoa was performed.Y-DNA was detected in 173/205 (84.4%) urine samples. Of the 86 first post-coital void urine samples available, Y-DNA was detected in 83 (96.5%) specimens. Of the 119 urine samples from volunteers with post-coital activities Y-DNA was still measurable in 70 (58.8%) urine specimens. The male DNA amount was below 0.023 ng/μl in 28/153 (18.3%) urine samples.Of the 22 urine samples obtained after 24 post-coital hours, 9 (40.9%) were still Y-DNA positive.No associations were found between coital durance, coital frequency during the past two weeks prior to the study intercourse, post-coital activities, and the urine sample Y-DNA positivity.Of the 111 urine samples where the immediate microscopy was performed, in 66 (59.5%) samples spermatozoa were verified and one sample even contained motile spermatozoa. Microscopy detected 66 (67.3%) and failed to detect spermatozoa in 32 (32.7%) of Y-DNA positive samples.In addition to conventional invasive swab techniques, urine samples seem to be an effective biological trace collection method for Y-DNA and spermatozoa within 24 h following penile-vaginal penetration. Furthermore, it may be considered as a non-invasive collection method in suspected acute child sexual abuse cases to diminish time delay in forensic evidence collection and to improve patients' positive attitudes towards evidence collection.

Potential biomarkers for the early detection of acute kidney injury after percutaneous nephrolithotripsy

This study aims to investigate the role of urinary biomarkers in the determination of the potential risks of renal parenchymal tubular damage in adult patients who underwent percutaneous nephrolithotomy (PNL) with the indication of renal stone. A randomized and prospective controlled study was performed between June and December 2013. We enrolled 29 consecutive patients with renal calculi > 2 cm and who underwent PNL, as well as 47 healthy control subjects. Urine samples, including 2 h before surgery, 2 and 24 h after surgery were collected from the patient group. Freshly voided urine samples were collected from the control group. Kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl-glucosaminidase (NAG), and liver-type fatty acid binding protein (LFABP) levels were measured from these urine samples. The mean KIM-1/Cr value that measured 24 h after the operation was statistically significant, higher than its preoperative (preop) level (p = 0.045). A significant difference was detected between the mean preop and postoperative (postop) 24 h NAG/Cr values (p < 0.001). Also, postop 24 h NGAL/Cr levels were statistically significant, higher than its preop levels (p = 0.013). According to the comparison of preop and postop levels, an increase in LFABP/Cr values secondary to surgical intervention was observed without any statistically significant difference. Besides the LFABP/Cr levels do not change after percutaneous kidney surgery, KIM-1/Cr, NAG/Cr, and NGAL/Cr levels increase postop period, especially at 24 h. Further studies with a larger series and repeated measurements should be performed to clarify if they can be used to demonstrate renal damage after percutaneous surgery or not.

Urinary biomarker concentrations of captan, chlormequat, chlorpyrifos and cypermethrin in UK adults and children living near agricultural land.

There is limited information on the exposure to pesticides experienced by UK residents living near agricultural land. This study aimed to investigate their pesticide exposure in relation to spray events. Farmers treating crops with captan, chlormequat, chlorpyrifos or cypermethrin provided spray event information. Adults and children residing ≤100 m from sprayed fields provided first-morning void urine samples during and outwith the spray season. Selected samples (1–2 days after a spray event and at other times (background samples)) were analysed and creatinine adjusted. Generalised Linear Mixed Models were used to investigate if urinary biomarkers of these pesticides were elevated after spray events. The final data set for statistical analysis contained 1518 urine samples from 140 participants, consisting of 523 spray event and 995 background samples which were analysed for pesticide urinary biomarkers. For captan and cypermethrin, the proportion of values below the limit of detection was greater than 80%, with no difference between spray event and background samples. For chlormequat and chlorpyrifos, the geometric mean urinary biomarker concentrations following spray events were 15.4 μg/g creatinine and 2.5 μg/g creatinine, respectively, compared with 16.5 μg/g creatinine and 3.0 μg/g creatinine for background samples within the spraying season. Outwith the spraying season, concentrations for chlorpyrifos were the same as those within spraying season backgrounds, but for chlormequat, lower concentrations were observed outwith the spraying season (12.3 μg/g creatinine). Overall, we observed no evidence indicative of additional urinary pesticide biomarker excretion as a result of spray events, suggesting that sources other than local spraying are responsible for the relatively low urinary pesticide biomarkers detected in the study population.

Comparison of residents' pesticide exposure with predictions obtained using the UK regulatory exposure assessment approach

The UK regulatory methods currently used for estimating residents' potential pesticide exposure were assessed to determine whether they provide sufficiently conservative estimates. A non-random sample of 149 residents living within 100 m of fields where pesticides were sprayed provided first morning void urine samples one and/or two days after spraying. Using farmers’ spray information, regulatory exposure assessment (REA) models were applied to estimate potential pesticide intake among residents, with a toxicokinetic (TK) model used to estimate urinary biomarker concentrations in the mornings of the two days following the spray. These were compared with actual measured urinary biomarker concentrations obtained following the spray applications. The study focused on five pesticides (cypermethrin, penconazole, captan, chlorpyrifos and chlormequat). All measured cypermethrin urinary biomarker levels were lower than the REA-predicted concentrations. Over 98% and 97% of the measured urinary biomarker concentrations for penconazole and captan respectively were lower than the REA-predicted exposures. Although a number of the chlorpyrifos and chlormequat spray-related urinary biomarker concentrations were greater than the predictions, investigation of the background urinary biomarker concentrations suggests these were not significantly different from the levels expected had no pesticide spraying occurred. The majority of measured concentrations being well below the REA-predicted concentrations indicate that, in these cases, the REA is sufficiently conservative.

Diagnostic and prognostic value of bladder cancer-related transcript markers in urine

Since cytology as the current "gold standard" for noninvasive detection of bladder cancer (BCa) is characterized by a relatively low sensitivity, urinary transcript levels of survivin (SVV), Ki-67 and cytokeratin 20 (CK20) were evaluated as alternative or complementary biomarkers. Furthermore, their prognostic value was investigated. Voided urine samples from 105 BCa patients and 156 controls were included. Total RNA was isolated from urine pellets and reverse-transcribed into cDNA. Expression levels of SVV, Ki-67 and CK20 were determined by quantitative PCR and normalized to the housekeeping gene TBP. Diagnostic performance of transcript markers and cytology was assessed by receiver operating characteristic (ROC) curve analyses. The prognostic value of the transcript markers was calculated by Cox proportional hazards models. ROC analyses resulted in AUC values between 0.71 (Ki-67) and 0.86 (CK20), indicating an appropriate diagnostic power. Using specifically defined cutoff values, the expression levels of the assessed biomarkers were significantly higher in urine specimens from BCa patients compared to control group (Mann-Whitney U test p < 0.001). Specificity ranged from 75% (SVV) to 84% (CK20) and sensitivity from 56% (Ki-67) to 87% (CK20). In combination with cytology, the sensitivity increased up to 97% (CK20). With regard to prognostic power, only SVV showed a significant, but not independent impact on the risk of recurrence (p = 0.008). Quantitative assessment of tumor-related transcript markers, particularly of CK20, may serve as a noninvasive method to identify patients with BCa. Moreover, SVV appears to be useful as a marker for a high risk of recurrence.

P07.05 Utility of pooled urine samples for detection ofchlamydia trachomatisinfection in asymptomatic pregnant women in northern india

Background Urogenital infections by Chlamydia trachomatis (CT) are the most prevalent sexually-transmitted bacterial diseases in women. Cost effective screening of women for C. trachomatis infection in developing countries is highly desirable for reducing morbidity and complications like pelvic inflammatory diseases, ectopic pregnancy and infertility. Results of noninvasive urine samples tests for C. trachomatis are nearly identical to samples collected directly from the cervix or urethra. The present study used the pooled urine samples to decrease the cost of screening for C. trachomatis . Methods First void urine samples were collected from 1000 asymptomatic pregnant women having gestational age less than 24 weeks attending the Antenatal Clinics at PGIMER, Chandigarh during July 2009 to June 2012. The pooled urine samples (5x pooled processed specimen) were tested for presence of C. trachomatis by Amplicor CT PCR kit (Roche Diagnostic) and positive results were further tested separately on each urine sample. Direct fluroscent antibody test (DFA) assay was used on urine specimen which were positive by PCR to confirm the positive results. Results Overall C. trachomatis infection tested by both PCR and DFA was present in 1.6% (16/1000) of asymptomatic pregnant women. A total of 200 pools of urine samples were tested and 20 pools were positive for C. trachomatis . When these pools were tested individually, 20 (10%) samples were positive for CT (In one pool, 2 samples were positive and one pool was false positive). Four samples were negative by DFA. Pooling of urine samples saved 70% of reagent costs in our study. Conclusions The study shows C. trachomatis infection was present in 1.6% of pregnant women which indicates low prevalence of infection in northern India. Pooling of urine samples saved labour, cost (70% reduction) and time in screening large number of samples in resource-limited settings. Disclosure of interest statement None (No conflict of interest). The study was funded by Indian Council of Medical Research, New Delhi. No pharmaceutical grants were received in the development of this study.

Processing of voided urine for prostate cancer RNA biomarker analysis.

Voided urine samples have been shown to contain cells released from prostate tumors. Could good quality RNA from cells in urine be obtained from every donor for multimarker analysis? In addition, could urine donation be as simple as possible, a practical consideration for a lab test, without involving a prostate massage (as indicated for PCA3 testing), which precludes frequent collection; needing it done at a specific time of day (e.g., first or second urine); and requiring prompt processing of samples in clinics with limited molecular biology capability? Collected urine samples were pelleted, and the RNA isolated was processed for cDNA synthesis and in vitro transcription to generate amplified sense aRNA. The resultant aRNA was rigorously analyzed for possible introduced changes. DMSO was used as a cell preservative for frozen storage of urine samples. Good quality aRNA was obtained for over 100 samples collected at two different institutions. The process of RNA amplification removed co-isolated DNA in some samples, which did not affect RNA amplification. Amplification did not amplify genes that were absent and produce other expression alterations. The sense aRNA could be used to generate urinary transcriptomes specific to individual patients. No chaotropic agents for RNA preservation were added to the urine samples so that the supernatant could be used for analysis of secreted protein biomarkers. The time of donation was not important since patients were seen during the entire day. DMSO was an effective cell preservative for freezing urine. Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.

Urine proteome analysis to evaluate protein biomarkers in children with autism

BackgroundAutism is a complex developmental disability for which no specific diagnostic markers have been identified so far. The present study aimed to evaluate whether there is any abnormal protein(s) excreted in the urine of autistic children by proteome analysis which may act as diagnostic marker. MethodsUrine proteome analysis was carried out in first void urine samples of autistic and normal children (n=30) in the age group of 4–12years by 2D-PAGE followed by MALDI–TOF–MS analysis. ResultsComparison of 2D-PAGE gels revealed that many urinary proteins are expressed differentially in autistic children. Total numbers of spots observed were 250 and 159 in autism and normal samples respectively, out of which 95 matches were observed. In addition, 3 spots of abnormally expressed peptides were selected, excised and analyzed. Peptide sequence with significant match score was for kininogen-1 (KNG-1)–50 (spot-1), IgG1 heavy chain variable region-35(spot-2) and mannan-binding lectin serine protease-2 isoform-2 precursor-45(spot-3). All the autistic children showed significant increase (p<0.001) in urinary kininogen level measured quantitatively by ELISA, when compared to normal children. ConclusionIncreased urinary kininogen-1 level in all the autistic children and the possibility of this protein as a diagnostic marker need further investigation.

Association of Urinary 6-Sulfatoxymelatonin (aMT6s) Levels and Objective and Subjective Sleep Measures in Older Men: The MrOS Sleep Study.

Sleep and melatonin have been associated with healthy aging. In this study, we examine the association between melatonin levels and sleep among older men. Cross-sectional study of a community-dwelling cohort of 2,821 men aged 65 years or older recruited from six U.S. centers. First morning void urine samples were collected to measure melatonin's major urinary metabolite, 6-sulfatoxymelatonin (aMT6s). We also assessed objective and subjective sleep parameters. We used logistic regression models to calculate multivariate (MV) odds ratios (ORs), and 95% confidence intervals (CIs) adjusted for important demographic variables and comorbidities. In the overall sample, the only significant finding in fully adjusted models was that aMT6s levels were inversely associated with subjectively measured daytime sleepiness (sleepiness mean score of 5.79 in the top aMT6s quartile, and 6.26 in the bottom aMT6s quartile, MV OR, 1.32; 95% CI, 0.95-1.84; p trend ≤ .02). When restricting to men without β-blocker use (a known melatonin suppressant), aMT6s levels were significantly associated with shorter sleep time, that is, less than 5 hours (MV OR, = 1.90; 95% CI, 1.21-2.99; p trend = .01), and worse sleep efficiency, that is, less than 70% (MV OR, 1.58; 95% CI, 1.28-2.65; p trend < .001). aMT6s were not associated with subjective sleep quality or respiratory disturbance in any of our analyses. Lower nocturnal melatonin levels were associated with worsened daytime sleepiness, sleep efficiency, and shorter sleep time in older men. The role of circadian interventions, and whether melatonin levels are a modifiable risk factor for poor sleep in older men, warrants further study.

Abstract 549: A urine based assay to select patients for initial cystoscopy

Abstract Objective Only 2.6 - 28% of the patients that present with haematuria will be diagnosed with bladder cancer. Unnecessary cystoscopies and upper tract imaging lead to high diagnostic costs and patient burden. In order to better select patients for diagnostic cystoscopy and thereby reduce cost and over-testing, we aimed to develop a diagnostic urine assay by combining mutation status of FGFR3, TERT and PIK3CA with methylation markers OTX1 and ONECUT2. Material and Methods A total of 258 patients undergoing cystoscopy for haematuria were included, 175 were diagnosed with bladder cancer and 83 had a non-malignant source of the haematuria. Voided urine samples were collected prior to cystoscopy. DNA was extracted and analyzed for mutations in FGFR3, TERT and PIK3CA. Methylation assays were done using a single-nucleotide probe extension assay with multiple probes for OTX1 (4 probes) and ONECUT2 (5 probes). Methylation percentages were calculated and cut-off values (sensitivity ≥ 85%) were determined for dichotomization. All results were combined and the optimal model was selected through logistic regression analysis. The Bootstrap procedure was used for internal validation. Results Combining the results of the dichotomized methylation markers OTX1 (probe 2), ONECUT2 (probe 1+4) with the mutation markers FGFR3, TERT, PIK3CA and the clinical parameters Age and Gender led to a prediction model with an AUC of 0.95 (95% CI 0.92-0.98) and an optimism-corrected AUC of 0.94. To further improve the model, analyses were repeated for continuous methylation marker variables. Now, adding ONECUT2 probe 2 enhanced the predictive capacity of the model even further (AUC 0.97, 95% CI 0.94 - 0.99 and an optimism-corrected AUC of 0.96). Overall material cost was estimated to be 20 euro per patient. A mutation-analysis first strategy would lead to a potentially larger reduction in cost. This strategy identified 120 patients with bladder cancer based on mutation assays alone. For the remaining 125 mutation-naive patients the prediction model containing the previously selected continuous methylation markers combined with Age and Gender was used. The overall combined strategy sensitivity was 92,1% and specificity was 89% (AUC of 0.96). Conclusion Analyzing haematuria patients for their risk of bladder cancer may lead to a reduction of unnecessary diagnostic cystoscopies and upper urinary tract imaging. This will present a major cost reduction in the urological practice. Combining continuous marker variables for OTX1 and three probes for ONECUT2 with mutation markers FGFR3, TERT, PIK3CA and Gender and Age results in an accurate prediction model with an AUC of 0.97 and an optimism-corrected AUC of 0.96. A mutation-analysis first strategy seems most practical and feasible (Sens 92%, Spec 89%). The assay must be further improved to reach a more satisfactory overall sensitivity (&amp;gt;95%), for instance by adding RAS mutation analysis. Citation Format: Kim E.M. van Kessel, Willemien Beukers, Irene Lurkin, Kirstin A. van der Keur, Lars Dyrskjot, Ulrika Segersten, Torben F. Ørntoft, Nuria Malats, Per-Uno Malmström, Francisco X. Real, Chris H. Bangma, Ellen C. Zwarthoff. A urine based assay to select patients for initial cystoscopy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 549. doi:10.1158/1538-7445.AM2015-549