Urine Pellet Research Articles

Development of sensitive single-round pol or env RT-PCR assays to screen for XMRV in multiple sample types

The potential association between xenotropic murine leukemia virus-related virus (XMRV) and prostate cancer and chronic fatigue syndrome (CFS) has been much debated. To help resolve the potential role of XMRV in human disease, it is critical to develop sensitive and accurate reverse transcriptase (RT)-PCR assays to screen for the virus. Single-round RT-PCR assays were developed on the automated m2000™ system for detection of the pol or env regions of XMRV in whole blood, plasma, urine cell pellets and urogenital swab samples. Assay performance was assessed by testing two blinded panels, one comprised of whole blood and the other of plasma spiked with serial dilutions of XMRV-infected tissue culture cells and supernatant, respectively, prepared by the Blood XMRV Scientific Research Working Group (SRWG). For both whole blood and plasma panel testing, the assays showed excellent specificity and sensitivity as compared to the other tests included in the SRWG phase I study. Analytical specificity of the assays was also evaluated. Neither pol nor env PCR assays detected a panel of potential cross-reactive microorganisms, although some cross-reaction was observed with mouse genomic DNA. Screening of 196 normal human blood donor plasma, 214 HIV-1 seropositive plasma, 20 formalin-fixed paraffin-embedded (FFPE) prostate cancer specimens, 4 FFPE benign prostate specimens, 400 urine pellets from prostate cancer patients, 166 urine pellets from non-prostate cancer patients, and 135 cervical swab specimens, detected no samples as unequivocally XMRV positive.

Comparative Proteomic Analysis of Differentially Expressed Proteins in the Urine of Reservoir Hosts of Leptospirosis

Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection.

English

The aim of the present study was to evaluate the diagnostic value of nested PCR in genitourinary tuberculosis (GUTB) compared with acid fast staining and culture method. In total 200 urine samples from suspected cases of GUTB were collected during the period of study. Urine pellets were used for smear preparation, culture and DNA extraction by ether-chloroform method. Nested PCR was performed according to standard protocol using primers based on IS6110 gene fragment. The results obtained by PCR were compared with those obtained by standard acid-fast bacilli stain and culture method. Based on obtained results, the positivity rate of urine samples in this study was 5.0% by using culture and PCR methods and 2.5% for acid fast staining. Four out of total samples showed positive results in all three methods (2%). The sensitivity of PCR in this study was estimated as high as culture equal to 100%, while the sensitivity for direct smear staining was 41.6%. In conclusion, the obtained rate of GUTB in our study was 5.0%. Since the detection rate of culture and nested PCR was identical, we could suggest PCR as a rapid alternative to culture especially for confirmed cases of GUTB.

HMG–CoA Reductase Activation and Urinary Pellet Cholesterol Elevations in Acute Kidney Injury

Experimental acute kidney injury (AKI) activates the HMG-CoA reductase (HMGCR) gene, producing proximal tubule cholesterol loading. AKI also causes sloughing of proximal tubular cell debris into tubular lumina. This study tested whether these two processes culminate in increased urinary pellet cholesterol content, and whether the latter has potential AKI biomarker utility. Urine samples were collected from 29 critically ill patients with (n = 14) or without (n= 15) AKI, 15 patients with chronic kidney disease, and 15 healthy volunteers. Centrifuged urinary pellets underwent lipid extraction, and the extracts were assayed for cholesterol content (factored by membrane phospholipid phosphate content). In vivo HMGCR activation was sought by measuring levels of RNA polymerase II (Pol II), and of a gene activating histone mark (H3K4m3) at exon 1 of the HMGCR gene (chromatin immunoprecipitation assay of urine chromatin samples). AKI+ patients had an approximate doubling of urinary pellet cholesterol content compared with control urine samples (versus normal; P < 0.001). The values significantly correlated (r, 0.5; P < 0.01) with serum, but not urine, creatinine concentrations. Conversely, neither critical illness without AKI nor chronic kidney disease raised pellet cholesterol levels. Increased HMGCR activity in the AKI+ patients was supported by three- to fourfold increased levels of Pol II, and of H3K4m3, at the HMGCR gene (versus controls or AKI- patients). (1) Clinical AKI, like experimental AKI, induces HMGCR gene activation; (2) increased urinary pellet cholesterol levels result; and (3) urine pellet cholesterol levels may have potential AKI biomarker utility. The latter will require future testing in a large prospective trial.

P3-S1.31 Comparison of Copan UriSwab with BD ProbeTec urine preservative transport kit for preservation and detection of CT and NG in the ProbeTec assay

BackgroundUrine specimen collection is better accepted by the patients than invasive collection techniques for detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). Self-collection is useful for Sexually Transmitted Infection...

A prototype RT-PCR assay for detection of XMRV in multiple human sample types

Results For the SRWG whole blood panel, all six XMRV negative samples were assay negative, while all the 22Rv1-spiked samples from 0.5 cells/mL to 9,900 cells/mL were detected (3/3 for each panel member). For the SRWG plasma panel, all six XMRV negative samples were assay negative, while the 22Rv1 supernatant-spiked plasma samples were detected as positive, 1/3 for the 3.2 XMRV copies/mL panel member, 2/3 for the 16 copies/mL panel member, and 3/3 for panel members containing > 80 copies/mL. For screening of clinical specimen, assay positive results were obtained for 10% (2/20) of prostate cancer FFPE specimens, 0.5% (2/400) of prostate cancer urine pellets, and 0% (0/135) of the cervical swab specimens.

Diagnostic, Staging, and Grading of Urothelial Carcinomas from Urine: Performance of BCA-1, a Mini-Array Comparative Genomic Hybridisation–Based Test

BackgroundCytogenetic abnormalities occur at an early stage of bladder urothelial carcinomas (BUC), and their frequency increases as the cancer becomes more advanced. ObjectiveTo assess the diagnostic performance of a test based on cytogenetic abnormalities to diagnose, stage, and grade BUC from the urine. Design, setting, and participantsWe used a 341 bacterial artificial chromosome (BAC) comparative genomic hybridisation (CGH)–array chip (BCA-1) designed to include loci affected in BUC. The chip was first used on 32 frozen BUC biopsies to design staging (BN0) and grading (BN1 and BN2) prediction models based on Bayesian networks analysis. The models were then validated on external data obtained from 98 tumour samples using a 2464 BAC CGH-array chip. The performance of the test was finally assessed on 44 urine pellets collected, including 22 patients who had BUC and 22 controls. MeasurementsWe measured sensitivity and specificity to diagnose BUC stage and grade from urine pellets. Results and limitationsIn the urine, BCA-1 test sensitivity was 95%, specificity was 86%, and accuracy was 91%. The BN0 staging model identified T1–4 tumours in the urine with a sensitivity of 90%, a specificity of 83%, and an accuracy of 87%. The BN1 and BN2 grading models detected high-grade disease with a sensitivity, specificity, and accuracy of 86%, 88%, and 87%, respectively, using BN1 and 100%, 63%, and 82%, respectively, using BN2. BN models performed with similar sensitivity but reduced specificity using the external data. BCA-1 failed to produce results for eight additional samples (failure rate: 9%). The test needed high quantities and quality of DNA, and external validation in larger, prospective, and better-designed studies is necessary to confirm feasibility and performance. ConclusionsThe BCA-1 mini–CGH-array chip detected BUC in urine with a high diagnostic performance. It could also accurately discriminate low-grade from high-grade tumours and, to a lesser extent, lamina propria–invasive tumours from pTa tumours.

Potential molecular marker for detecting transitional cell carcinoma

Objectives. To assess the expression rate of a cancer-related gene, hepatoma-up-regulated protein (HURP), in the tumor tissue of transitional cell carcinoma (TCC), and to assess the potential suitability of using this gene as a novel molecular marker for detecting TCC in voided urine. Methods. Total RNA was extracted from 80 TCC tissue samples of the urinary tract. Forty-five of the 80 tumor-adjacent tissue samples were from the same patients and 15 were from control subjects (patients with benign prostatic hyperplasia). The expression levels of HURP mRNA in these specimens were examined using reverse transcriptase-polymerase chain reaction. The HURP mRNA transcripts in voided urine pellets from 14 additional patients were determined using the same method. The messages were normalized to beta-actin mRNA. Results. The detection of HURP expression in the TCC tissue samples had a sensitivity of 88.8% (71 of 80) and a specificity of 100% (15 of 15). Ten of the 45 grossly tumor-adjacent tissue samples expressed HURP mRNA, which may indicate subtle genetic changes in tissue adjacent to tumor. All seven urine specimens from the patients with TCC revealed HURP expression; however, no specimens from patients with nonmalignant diseases did so. Conclusions. A potential molecular marker for detecting TCC with tissue specimens and voided urine samples has been found. Although the real clinical application of this marker requires additional evaluation, the high sensitivity and specificity of the HURP gene amplification method warrants more investigation in the future.

Detection of urinary tract infections by reduction of nitroblue tetrazolium: Technical Note

Nitroblue tetrazolium (NBT) reduction to formazan has been used as a marker for nitric oxide synthase (NOS). Since inducible NOS activity is elevated in urine from patients with urinary tract infections (UTIs), we investigated the accuracy of NBT reduction as an early predictor of UTIs and quantified the relationship between inducible NOS and NBT. Urine samples from 434 patients were screened for the presence of UTIs with leukocyte-esterase and nitrite dipsticks and with NBT reduction. The rapid screening results from each test were compared to urine culture results. In addition, NBT reduction parameters were measured in urine pellet at 595 nm after incubation with one of four factors: NOS cofactors, NOS inhibitors, NADH, or superoxide dismutase/catalase. As a urine screening test for UTIs, NBT reduction was more sensitive with a higher negative predictive accuracy than the nitrite dipstick. NBT reduction also was more specific with a higher positive predictive accuracy and negative predictive accuracy than the leukocyte-esterase dipstick. In infected urine pellet, both NADPH, a NOS cofactor, and NADH increased NBT reduction. Superoxide dismutase/catalase decreased NBT reduction. Although NOS may not be the only NBT reducing enzyme, rapid, visible reduction of NBT is induced in urine from patients with UTIs.

Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients

We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable. Int. J. Cancer 74:625–629, 1997.© 1997 Wiley-Liss, Inc.

Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients

We performed microsatellite analysis and tested telomerase activity in paired tissue and urine of bladder cancer patients from frozen archived samples. DNA obtained from microdissected tumor and urine sediment was analyzed and compared to peripheral lymphocytes for microsatellite alterations (loss of heterozygosity [LOH] or instability) using a panel of 20 microsatellite markers in 15 patients with transitional or squamous cell carcinoma of the urinary tract. Additionally, telomerase activity was determined in 12 microdissected tumor specimens and corresponding frozen urine pellets. Tumor cell DNA was detected by microsatellite analysis (LOH or shift) in at least one marker in 14/15 microdissected tumor specimens and in 13/15 DNA samples obtained from urine sediments. Telomerase activity was present in 11/12 tumor samples but could not be detected in any of the corresponding urine sediments. Frozen archived urine samples are useful for retrospective studies utilizing microsatellite analysis or other PCR-based approaches after DNA extraction. However, the evaluation of telomerase protein activity in stored urine samples appears to be unsuitable.

Urinary Nitric Oxide Synthase Activity and Cyclic GMP Levels are Decreased with Interstitial Cystitis and Increased with Urinary Tract Infections

Purpose Since urinary nitric oxide synthase (NOS) activity correlates with certain disease processes affecting the urinary tract and since nitric oxide increases cyclic GMP levels by activating guanylyl cyclase, urinary particulate NOS activity and cyclic GMP levels are evaluated in female patients with interstitial cystitis (IC) and compared with those from female controls and female patients with urinary tract infections (UTIs). Materials and Methods Urinary NOS activity is measured as the formation of [sup 14 C]-L-citrulline from [sup 14 C]-L-arginine, and urinary cyclic GMP levels are measured by an [sup 125 I]-radioimmunoassay. Results Female patients with IC have significantly less NOS activity in their urine pellet particulate fractions than female controls or females with UTIs, 2.3 plus/minus 1.0, 14 plus/minus 3.0, and 120 plus/minus 10 pmol. citrulline formed/min./mg. protein. Urinary cyclic GMP levels also are significantly lower in IC patients than in female controls or females with UTIs: 0.50 plus/minus 0.06, 0.82 plus/minus 0.14, and 3.72 plus/minus 0.81 micromole cyclic GMP/g. creatinine. Conclusion Regulation of urinary NOS activity with subsequent changes in nitric oxide and cyclic GMP may be an important determinant of symptoms and immunologic responses to UTIs and IC.

Detection ofChlamydia trachomatis in the urine of young Norwegian males by enzyme immunoassay

First-void urine samples from 392 Norwegian military conscripts were investigated for the presence of Chlamydia trachomatis by enzyme immunoassay (EIA) on day 1 and day 5 after collection. Positive samples were subsequently investigated by direct immunofluorescence (IF) microscopy for the presence of chlamydial elementary bodies (EBs) in the urine pellet, and urethral swab material taken from the EIA-positive individuals was cultured. 4.8% (19/392) of the urine samples were EIA-positive on day 1, and 5.4% (21/392) were positive on day 5, with a combined total of 6.6% (26/392). Twenty-four of the 26 urine samples were confirmed as positive on IF microscopy. Urethral swabs were taken from 21 EIA-positive individuals. Six of the swabs were positive on cell culture, whereas nine were positive on IF microscopy of swab material, suggesting that these techniques perform better in symptomatic cases than in male Chlamydia trachomatis carriers. In the urine samples a notable discrepancy in EIA results was seen when the same refrigerated samples were retested on day 5 compared to day 1. This discrepancy was probably due to storage-related factors.

Detection of human immunodeficiency virus type 1 (HIV-1) in urine cell pellets from HIV-1-seropositive individuals.

Fresh urine pellets from human immunodeficiency virus type 1 (HIV-1)-seropositive individuals were examined for the presence of the HIV-1 genomic sequence and gene products. By using the polymerase chain reaction technique, HIV-1 DNA proviral sequences were detected in 53 of 80 (66.25%) fresh urine pellets from HIV-1-seropositive individuals, while urine pellets from all 24 healthy heterosexual controls were negative. HIV-1 RNA in urine pellets was detected by reverse transcriptase polymerase chain reaction in 2 of 43 (4.7%) HIV-1-seropositive individuals. In addition, HIV-1 p24 core antigen was demonstrated in 3 of 80 urine pellets from HIV-1-seropositive individuals by enzyme-linked immunosorbent assay. Moreover, HIV-1 p24 core antigen and HIV-1 RNA were shown in the cellular component of urine pellets from HIV-1-seropositive individuals by immunohistochemical staining and in situ hybridization. These results indicate that HIV-1 can be present in urine pellets from HIV-1-infected individuals.

Dot immunoperoxidase assay using monoclonal antibody for direct detection of cytomegalovirus in urine samples.

A rapid, simple dot immunoperoxidase assay for the direct detection of cytomegalovirus in clinical urine samples was developed. The assay was performed on nitrocellulose paper dotted with urine pellets free of cellular debris. Cytomegalovirus was detected with a monoclonal antibody to the capsid antigen, and the complex was visualized by immunoperoxidase staining. Positive reactions appeared as well-defined dark blue spots. Of the 87 urine samples examined, 10 proved positive in the dot immunoperoxidase assay, and 77 proved negative. The results agreed completely with the detection of cytomegalovirus-induced antigens in cell cultures inoculated with clinical specimens.

Odor response characteristics of thalamic mediodorsal nucleus neurons in the rabbit.

Extracellular responses were recorded from neurons in the thalamic mediodorsal nucleus (MD) of the lightly anesthetized rabbit. Eighty-seven neurons responded to electrical stimulation of the lateral olfactory tract (LOT shocks). They were located in the medial portion of the MD. In the same portion, negative field potentials with a short latency were evoked by the electrical stimulation of the olfactory projection area in the neocortex (OPA shocks). Fifty-nine MD neurons responded both to LOT and to OPA shocks. Among them, 17 thalamocortical relay neurons (which responded antidromically to OPA shocks) were found to respond transsynaptically to LOT shocks. Of the 87 LOT-responsive MD neurons, 48 responded to the odors applied. Eight odor-sensitive neurons were found to be the thalamocortical relay neurons. Thus, it was proven for the first time that a portion of the olfactory input to the OPA is mediated via relay neurons in the MD. Characteristics of response of MD neurons to odor stimulation were compared with those of OPA neurons. MD neurons did not show a selectivity of response to odors of urine, feces, or dry food pellets, to which OPA neurons responded exclusively. These results were discussed in relation to the functional role of the MD-OPA projection system in the discrimination of specific odors.

Isolation of virus-like particles in urine from a psoriatic patient.

Virus-like particles have been isolated from urine of a psoriatic patient. A 70,000-dalton glycoprotein, gp70, located on the surface of the particles was purified by concanavalin A fractionation and immunosorbent chromatography with antibodies against virus-like particles produced by a cell culture established from a psoriatic lesion. Rabbit antiserum against purified gp70 seemed to be highly specific when anti-gp70 immunobeads reacted with detergent-disrupted urine pellet. When the immunobeads were added to urine in the absence of detergent, the proteins p27, p15, and p12 were extracted in addition to gp70. The three former proteins were radioactive labelled only after detergent-disruption of the adsorbed material in contrast to gp70. The results demonstrate that p27, p15, and p12 are located within the same particle which contains gp70 on the surface. An approximate quantitation of isolated core particles suggested that about 20 micrograms virus-like particles can be extracted from one litre urine by anti-gp70 immunobeads.

Influence of parathyroid extract on blood and urine mineral levels in iguanid lizards

The influence of parathyroid extract on calcium and phosphorus levels in the blood and urine of two species of iguanid lizards has been investigated. The Desert Iguana, Dipsosaurus dorsalis, and the Mesquite lizard, Sceloporus grammicus, were injected daily with parathyroid extract for 1 week. Urine pellets were excreted within 5–7 days. Urinary mineral levels were determined as concentration and total clearance per pellet. After PTE treatment, hypercalcemia was observed in D. dorsalis, while bone citric acid was slightly, but not significantly, elevated. Both D. dorsalis and S. grammicus responded to repetitive doses of PTE by elevated urine calcium and phosphorus titers. Plasma phosphorus, studied only in PTE-treated D. dorsalis, showed no change from normal. A marked decrease in urine pellet weight was noted in PTE-treated lizards, while control animals kept in the laboratory a comparable length of time showed little fluctuation in quantity of excreted urine. Neither the duration nor conditions of short-term captivity influenced urine mineral levels of normal iguanid lizards.

The unexplained high erythrocyte sedimentation rate.

Model systems demonstrate that progression to ESRD is driven by progressive podocyte depletion (the podocyte depletion hypothesis) and can be noninvasively monitored through measurement of urine pellet podocyte mRNAs. To test these concepts in humans, we analyzed urine pellet mRNAs from 358 adult and pediatric kidney clinic patients and 291 controls (n=1143 samples). Compared with controls, urine podocyte mRNAs increased 79-fold (P