Urinary extracellular vesicles: Assessment of pre-analytical variables and development of a quality control with focus on transcriptomic biomarker research.

Urinary extracellular vesicles (uEV) are a topical source of non-invasive biomarkers for health and diseases of the urogenital system. However, several challenges have become evident in the standardization of uEV pipelines from collection of urine to biomarker analysis. Here, we studied the effect of pre-analytical variables and developed means of quality control for uEV isolates to be used in transcriptomic biomarker research. We included urine samples from healthy controls and individuals with type 1 or type 2 diabetes and normo-, micro- or macroalbuminuria and isolated uEV by ultracentrifugation. We studied the effect of storage temperature (-20°C vs. -80°C), time (up to 4 years) and storage format (urine or isolated uEV) on quality of uEV by nanoparticle tracking analysis, electron microscopy, Western blotting and qPCR. Urinary EV RNA was compared in terms of quantity, quality, and by mRNA or miRNA sequencing. To study the stability of miRNA levels in samples isolated by different methods, we created and tested a list of miRNAs commonly enriched in uEV isolates. uEV and their transcriptome were preserved in urine or as isolated uEV even after long-term storage at -80°C. However, storage at -20°C degraded particularly the GC-rich part of the transcriptome and EV protein markers. Transcriptome was preserved in RNA samples extracted with and without DNAse, but read distributions still showed some differences in e.g. intergenic and intronic reads. MiRNAs commonly enriched in uEV isolates were stable and concordant between different EV isolation methods. Analysis of never frozen uEV helped to identify surface characteristics of particles by EM. In addition to uEV, qPCR assays demonstrated that uEV isolates commonly contained polyoma viruses. Based on our results, we present recommendations how to store and handle uEV isolates for transcriptomics studies that may help to expedite standardization of the EV biomarker field.

Can a semi-quantitative method replace the current quantitative method for the annual screening of microalbuminuria in patients with diabetes? Diagnostic accuracy and cost-saving analysis considering the potential health burden.

OBJECTIVES: Diabetes is a global epidemic, and the high cost of annually and quantitatively measuring urine albumin excretion using the turbidimetric immunoassay is challenging. We aimed to determine whether a semi-quantitative urinary albumin-creatinine ratio test could be used as a screening tool for microalbuminuria in diabetic patients. METHODS: We assessed the diagnostic accuracy of the semi-quantitative method. The costs of false results in the semi-quantitative method were calculated based on the annual probability of disease progression analyzed through a systematic literature review and meta-analysis. The pooled long-term cost-saving effect of the semi-quantitative method compared with the quantitative test was assessed using a Markov model simulating a long-term clinical setting. Diagnostic accuracy and the cost-saving effect were also validated in an independent external cohort. RESULTS: Compared with the quantitative test, the semi-quantitative method had sensitivities of 93.5% and 81.3% and specificities of 61.4% and 63.1% in the overall sample of diabetic patients (n = 1,881) and in diabetic patients with eGFR ≥60 ml/min/1.73 m2 and a negative dipstick test (n = 1,110), respectively. After adjusting for direct and indirect medical costs, including the risk of disease progression, which was adjusted by the meta-analyzed hazard ratio for clinical outcomes, it was determined that using the semi-quantitative method could save 439.4 USD per person for 10 years. Even after adjusting the result to the external validation cohort, 339.6 USD could be saved for one diabetic patient for 10 years. CONCLUSIONS: The semi-quantitative method could be an appropriate screening tool for albuminuria in diabetic patients. Moreover, it can minimize the testing time and inconvenience and significantly reduce national health costs.

The compliance and cost-effectiveness of smartphone urinalysis albumin screening for people with diabetes in England.

BACKGROUND: People with diabetes are at increased risk of developing chronic kidney disease (CKD) and should undergo annual screening, but adherence is poor. A home urinalysis self-test has been developed to improve compliance with screening. The objective of this paper is to report on a clinical evaluation and economic analysis of home urinalysis self-testing. RESEARCH DESIGN AND METHODS: People with diabetes who had not undergone screening within the previous 18 months were recruited to a single-arm clinical evaluation to assess the uptake and compliance of home urinalysis self-testing. An economic evaluation assessed the likely cost-consequences of the use of home urinalysis self-testing over a lifetime time horizon. RESULTS: A total of 2,196 people with diabetes were contacted as part of the clinical evaluation. Of these, 695 people agreed to be sent a home urinalysis self-testing kit and 499 people completed and returned the test. Cost savings of £2,008 per person were estimated over a lifetime due to increased CKD diagnosis and reduced progression to end stage renal disease. CONCLUSIONS: Home urinalysis self-testing of ACR in people with diabetes is estimated to be a cost-effective use of NHS resources in England in people who would otherwise not comply with standard care.

Diagnostic accuracy of self-administered urine glucose test strips as a diabetes screening tool in a low-resource setting in Cambodia.

OBJECTIVE: Screening for diabetes in low-resource countries is a growing challenge, necessitating tests that are resource and context appropriate. The aim of this study was to determine the diagnostic accuracy of a self-administered urine glucose test strip compared with alternative diabetes screening tools in a low-resource setting of Cambodia. DESIGN: Prospective cross-sectional study. SETTING: Members of the Borey Santepheap Community in Cambodia (Phnom Penh Municipality, District Dangkao, Commune Chom Chao). PARTICIPANTS: All households on randomly selected streets were invited to participate, and adults at least 18 years of age living in the study area were eligible for inclusion. OUTCOMES: The accuracy of self-administered urine glucose test strip positivity, Hemoglobin A1c (HbA1c)>6.5% and capillary fasting blood glucose (cFBG) measurement ≥126 mg/dL were assessed against a composite reference standard of cFBGmeasurement ≥200 mg/dL or venous blood glucose 2 hours after oral glucose tolerance test (OGTT) ≥200 mg/dL. RESULTS: Of the 1289 participants, 234 (18%) had diabetes based on either cFBG measurement (74, 32%) or the OGTT (160, 68%). The urine glucose test strip was 14% sensitive and 99% specific and failed to identify 201 individuals with diabetes while falsely identifying 7 without diabetes. Those missed by the urine glucose test strip had lower venous fasting blood glucose, lower venous blood glucose 2 hours after OGTT and lower HbA1c compared with those correctly diagnosed. CONCLUSIONS: Low cost, easy to use diabetes tools are essential for low-resource communities with minimal infrastructure. While the urine glucose test strip may identify persons with diabetes that might otherwise go undiagnosed in these settings, its poor sensitivity cannot be ignored. The massive burden of diabetes in low-resource settings demands improvements in test technologies.

Bayesian hierarchical joint modeling of repeatedly measured continuous and ordinal markers of disease severity: Application to Ugandan diabetes data.

Modeling of correlated biomarkers jointly has been shown to improve the efficiency of parameter estimates, leading to better clinical decisions. In this paper, we employ a joint modeling approach to a unique diabetes dataset, where blood glucose (continuous) and urine glucose (ordinal) measures of disease severity for diabetes are known to be correlated. The postulated joint model assumes that the outcomes are from distributions that are in the exponential family and hence modeled as multivariate generalized linear mixed effects model associated through correlated and/or shared random effects. The Markov chain Monte Carlo Bayesian approach is used to approximate posterior distribution and draw inference on the parameters. This proposed methodology provides a flexible framework to account for the hierarchical structure of the highly unbalanced data as well as the association between the 2 outcomes. The results indicate improved efficiency of parameter estimates when blood glucose and urine glucose are modeled jointly. Moreover, the simulation studies show that estimates obtained from the joint model are consistently less biased and more efficient than those in the separate models.

Urine protein/creatinine ratio as a mortality risk predictor in non-diabetics with normal renal function.

OBJECTIVE: Determine the relative mortality in apparently healthy adults with various levels of urinary protein measured by urine protein/creatinine (p/c) ratio. METHOD: By use of the Social Security Death Master File, mortality in 2010 was determined for 7.5 million life insurance applicants age 20 to 89 providing urine samples between 1992 and 2006. Relative mortality by Cox regression for bands of p/c ratios was determined using age and sex as covariates and with an age split at 60 after excluding those with hematuria (> 3 red cells/hpf), diabetes, evidence of blood sugar elevation, or eGFR < 60 mL/min. RESULTS: After the exclusions noted above, relative mortality increased to 160% beginning at a p/c ratio of 0.11 mg/mg and rose steadily above that value regardless of sex and age. Most of this risk was not explained by a history of hypertension or elevated systolic blood pressure. Albumin testing identified roughly a third of urine samples with elevated p/c ratios as not containing albumin; those cases appeared to be associated with much lower risk as long as the p/c ratio was < or = 1.0 mg/mg. CONCLUSION: Low levels of proteinuria identified as urine protein/creatinine ratios of 0.11 mg/mg or higher (much lower than the usual lower cut-off value of 0.21) are associated with substantial excess mortality risk, even after excluding diabetics and those with reduced kidney function or hematuria.

The assessment of pulmonary diffusing capacity in diabetes mellitus with regard to microalbuminuria.

OBJECTIVE: The current study was conducted to determine whether or not the lung is one of the target organs in the development of vascular complications in diabetic patients. We also investigated the relationship between pulmonary diffusing capacity and microalbuminuria. MATERIALS AND METHODS: A total of 68 patients with type 2 diabetes and 44 with type 1 diabetes (male/female [M/F] n=19/49 and 15/29; age: 52.4+/-8.8 and 32.5+/-11 years;) and 80 healthy controls (M/F=22/58; age=40.1+/-12.4 years) were recruited for the study. All of the participants were evaluated with simple spirometric tests and a simple breath carbon monoxide (CO) diffusion test. CO lung diffusion capacity (DLCO) and the ratio of DLCO value to the alveolar volume (VA) were used to assess alveolar membrane permeability (DLCO/VA). Urinary albumin excretion (UAE) per day of the participants was also evaluated. RESULTS: DLCO, DLCO% and DLCO/VA% values were significantly lower in diabetics than in the control group (p=0.006; p=0.039; p=0.003, respectively). UAE was inversely correlated with DLCO, DLCO/VA, DLCO/VA% and DLCO% (p=0.050; p=<0.001; p=0.001; p=0.004, respectively). CONCLUSION: This study demonstrated that alveolar gas exchange capacity is significantly decreased in diabetic patients. Microalbuminuria may be one of the predictors of this decline.

Design of a portable urine glucose monitoring system for health care.

This paper describes the design of a monitoring system that can be used to measure urine glucose during daily life. It consists of a bio-chemical sensor, hardware with PIC microcontroller and control circuits, and signal analyzing part. To evaluate the performance, we compared the analyzed glucose levels of the developed system to a standard instrument, YSI glucose analyzer, based on regression analysis using standard glucose solutions mixed with urine. Also, standard deviation and coefficient of variation were computed. In conclusion, the developed system showed it could be used for the measurement of urine glucose.

[Useful and useless tests for diabetes diagnosis and care]. / Examens utiles et inutiles dans le diabète de type 2.

The investigations that can be performed in diabetes are not all of equal interest. Some are necessary and sufficient, others useless as their results are non-reproducible, and others still are of no therapeutic value. In order to complete the clinical assessment, the doctor must have at his disposal several simple tests, the results of which should lead to a modification of treatment. The diagnosis of diabetes rests on the level of the fasting glucose. The level of the glycosylated haemoglobin is the best investigation for an appreciation of the glycaemic control. At diagnosis and once a year thereafter, the measurement of the level of the total cholesterol and HDL, triglycerides, microalbuminuria, and creatinine, along with fundoscopy and a resting electrocardiogram are the necessary and sufficient investigations. Combined with the measurement of the arterial blood pressure and an assessment of tobacco use, these permit a good appreciation of the level of cardiovascular risk and the existence or not of complications.

Social disadvantage: its impact on the use of Medicare services related to diabetes in NSW.

OBJECTIVE: To use Medicare data to examine the impact of social disadvantage on the use of health services related to diabetes. METHOD: Information on number of diabetic individuals and number of services for select Medicare item codes were retrieved by New South Wales postcodes using a Health Insurance Commission data file. The postcodes were graded into quintiles of social disadvantage. RESULTS: People at most social disadvantage were significantly less likely to be under the care of a general practitioner (adjusted OR 0.41; 95% CI 0.40-0.41) or consultant physician (adjusted OR 0.50; 95% CI 0.48-0.53), despite this group having the highest prevalence of diabetes. The difference in attendance to other specialists was less marked but nevertheless significant (adjusted OR 0.71; 95% CI 0.68-0.75). Once under a doctor's care, patients at most disadvantage were slightly more likely to undergo HbA1c or microalbuminuria estimation (adjusted OR 1.04; 95% CI 1.00-1.10 and adjusted OR 1.22; 95% CI 1.12-1.33, respectively) but were less likely to undergo lipid or HDL cholesterol estimation (adjusted OR 0.81; 95% CI 0.48-0.53 and adjusted OR 0.85; 95% CI 0.79-0.90, respectively). CONCLUSION: While access to medical care is decreased for people at most social disadvantage, once under a doctor's care they receive a level of monitoring that is relatively equal to that provided to people less disadvantaged. IMPLICATION: Strategies are required to ensure equal access to medical services for all persons with diabetes, especially for persons who are at most social and medical disadvantage.

Assessment of coagulation system activation using spot urine measurements.

Coagulation system activation is most commonly assessed by measuring levels of one or more proteins in peripheral blood. Because faulty blood-drawing can cause activation of the coagulation system, artifactual elevations of such markers have been reported. We have therefore investigated the possibility of using randomly collected ('spot') urine samples as a non-invasive means of assessing the state of coagulation system activation. Using a commercially available enzyme-linked immunosorbent assay kit designed to measure plasma levels of fragment 1 + 2, we found immunoreactive fragment 2 in healthy control subjects, and significantly increased levels in diabetic and non-diabetic pregnant subjects, and patients with venous thromboembolism, prostate cancer, and diabetes. Measurements of excretion of immunoreactive fragment 2 are worth further study as an adjunct or alternative to plasma-based assays designed to detect or quantify coagulation system activation.

Renal assessment practices and the effect of nurse case management of health maintenance organization patients with diabetes.

OBJECTIVE: To examine baseline renal screening practices and the effect of nurse case management of patients with diabetes in a group model health maintenance organization (HMO). RESEARCH DESIGN AND METHODS: We performed both 1-year retrospective and 1-year prospective studies of renal assessment practices and ACE inhibitor usage in a cohort of 133 diabetic patients enrolled in a randomized controlled trial of a diabetes nurse case management program in a group model HMO. In accordance with American Diabetes Association recommendations, urine dipstick and quantitative protein and microalbuminuria testing rates were calculated. RESULTS: At baseline, 77% of patients were screened for proteinuria with dipsticks or had quantitative urine testing. Of patients with negative dipstick findings, 30% had appropriate quantitative protein or microalbumin follow-up at baseline. Baseline ACE inhibitor usage was associated with decreased follow-up testing (relative risk = 0.47). Nurse case management was associated with increased quantitative protein or or microalbumin testing and increased follow-up testing (relative risk = 1.65 and 1.60, respectively). CONCLUSIONS: We found a higher degree of adherence to recommendations for renal testing than has been reported previously. Nurse case management intervention further increased renal screening rates. The inverse association between ACE inhibitor usage and microalbumin testing highlights a potentially ambiguous area of current clinical pathways.

Assessment of simple colorimetric procedures to determine smoking status of diabetic subjects.

The performance of a simple colorimetric assay for urinary nicotine metabolites to assess smoking status in diabetic subjects (n = 251) was investigated. Several variations of the colorimetric assay and a qualitative extraction procedure were evaluated in comparison with a cotinine immunoassay as the "gold standard." Among these, the best overall performance was achieved with the qualitative test (sensitivity 95%; specificity 100%). The quantitative measurement of total nicotine metabolites performed less well (sensitivity 92%; specificity 97%) but could be improved by incorporating a blank extraction (sensitivity 98%; specificity 98%). Allowance for diuresis appeared to offer no advantage over the other methods. These results support previous findings regarding the use of these colorimetric procedures in nondiabetic subjects and, contrary to other recent observations, their performance was not impaired in diabetic patients.

Monitoring glycemia in diabetes. Short-term assessment.

The monitoring of glycemic status is considered a cornerstone of diabetes care. This article reviews current recommendations for routine glycemia monitoring, with emphasis on practical applications. A description of the newly developed National Glycohemoglobin Standardization Program also is provided.

Finding diabetics--a method of screening in general practice.

BACKGROUND: It is well known that many diabetic patients go undiagnosed until complications have started to develop. Screening can be expensive in time and money, and ineffective, and is therefore unpopular with general practitioners (GPs). AIM: This study aimed to develop a screening method that was cost-effective and practical within the setting of an ordinary general practice. METHOD: Urine-testing sticks for glucose were sent with an explanatory letter to all (1736) non-diabetic subjects over 50 years old in a general practice. At the same time, diabetic recall and care were audited and improved in the practice. RESULTS: Reply slips were received from 1204 patients (69.4%). Of these, 2.6% were positive and 97.4% were negative. Eight new diabetic patients were therefore found at a cost of 78.25 pounds each. CONCLUSION: This method of screening a selected part of a general practice population is practical and effective. Apart from the human cost, the financial cost of finding a new diabetic patient is small compared with that of caring for a diabetic patient who is blind or an amputee.

Microalbuminuria screening in the UK: are we meeting European standards?

European guidelines recommend annual screening for microalbuminuria in patients with Type 1 (insulin-dependent) diabetes mellitus (IDDM) of greater than 5 years' duration and in those with Type 2 (non-insulin-dependent) diabetes mellitus (NIDDM) from diagnosis. To determine the current provision of screening for microalbuminuria we performed a postal survey of all diabetologists in the United Kingdom. Of 556 questionnaires sent, 326 (59%) were returned (246 adult, 57 paediatric, 3 adolescent clinics) and of these 306 (55%) were suitable for analysis. Screening programmes have been established by 210 (69%) diabetologists: 70 of these in the last 2 years. 46 more plan to screen patients with IDDM within 2 years. 155 (92%) of 169 adult programmes perform annual screening in IDDM, 74% according to European guidelines (39% in NIDDM). Other clinics use age, type of diabetes or criteria such as blood pressure to target screening. An albumin/creatinine ratio (52%) on an early morning urine (56%) or random (29%) urine sample is most commonly requested. Financial constraint was the principal reason given in 32 (33%) of 96 clinics that do not currently screen. Other reasons included implementation of other developments with a higher priority (24%) and doubts about the medical value of screening (46%). Assuming respondents are representative of current UK practice, we conclude that microalbuminuria screening is available to patients in many clinics, but is neither universal nor always performed according to European guidelines.