Urinary Assay Research Articles

TERT Promoter Mutations as Simple and Non-Invasive Urinary Biomarkers for the Detection of Urothelial Bladder Cancer in a High-Risk Region.

Bladder cancer (BC) is the 10th most common cancer in the world. While there are FDA-approved urinary assays to detect BC, none have demonstrated sufficient sensitivity and specificity to be integrated into clinical practice. Telomerase Reverse Transcriptase (TERT) gene mutations have been identified as the most common BC mutations that could potentially be used as non-invasive urinary biomarkers to detect BC. This study aims to evaluate the validity of these tests to detect BC in the Kerman province of Iran, where BC is the most common cancer in men. Urine samples of 31 patients with primary (n = 11) or recurrent (n = 20) bladder tumor and 50 controls were prospectively collected. Total urinary DNA was screened for the TERT promoter mutations (uTERTpm) by Droplet Digital PCR (ddPCR) assays. The performance characteristics of uTERTpm and the influence by disease stage and grade were compared to urine cytology results. The uTERTpm was 100% sensitive and 88% specific to detect primary BC, while it was 50% sensitive and 88% specific in detecting recurrent BC. The overall sensitivity and specificity of uTERTpm to detect bladder cancer were 67.7% and 88.0%, respectively, which were consistent across different tumor stages and grades. The most frequent uTERTpm mutations among BC cases were C228T (18/31), C250T (4/31), and C158A (1/31) with mutant allelic frequency (MAF) ranging from 0.2% to 63.3%. Urine cytology demonstrated a similar sensitivity (67.7%), but lower specificity (62.0%) than uTERTpm in detecting BC. Combined uTERTpm and urine cytology increased the sensitivity to 83.8%, but decreased the specificity to 52.0%. Our study demonstrated promising diagnostic accuracy for the uTERTpm as a non-invasive urinary biomarker to detect, in particular, primary BC in this population.

Clinically relevant urine creatinine underestimation in the low concentration range on the Siemens Dimension Vista®

While considerable efforts have been accomplished to standardize the measurement of plasma creatinine (PCr), urine creatinine (UCr) has not been subject to the same scrutiny. UCr is importantly used when measuring biomarkers in spot urines, to assess urine output and variable dilution of urine samples. Here, we report underestimation of Jaffe UCr measurements on the Siemens Dimension Vista® analyzer, critically affecting samples with UCr ≤2 mmol/L. We demonstrate that this error is caused by automatic urine pre-dilution by the Vista’s «urine mode», and that UCr measured in «plasma mode» without pre-dilution does not present this error. In the absence of a comprehensive solution proposed by Siemens, we propose simple formulae that can be easily implemented in a laboratory to correct these low UCr measurements. Importantly, the observed UCr underestimation can significantly influence reported results for biomarkers/UCr ratios measured in spot urine. Indeed, these results can be overestimated up to +84.4 % before correction using our formulae. This can sometimes lead to misclassification according to clinical thresholds, e.g. Kidney disease: improving global outcomes (KDIGO) guidelines for urine albumin/creatinine. This highlights the need for every clinical laboratory to assess the detection limits of their assays, including for lesser-discussed parameters such as UCr. Indeed, the error we reported here may affect other urine assays performing systematic urine pre-dilution and could have significant repercussions on the clinical management of patients.

Penicillin V prophylaxis uptake among children living with sickle cell disease in a specialist sickle cell clinic in Ghana: A cross-sectional study.

Penicillin V prophylaxis protects children living with sickle cell disease (SCD) from bacteria infections especially Streptococcus pneumonia. However, the uptake of penicillin V prophylaxis is difficult to assess and often poor among SCD patients. Therefore, this study sought to investigate oral penicillin V prophylaxis adherence among SCD children using urine assay and self-reported methods and the associated factors. The study employed an analytical cross-sectional design in the assessment of penicillin V prophylaxis adherence using both urine assay and self-reported methods. Multiple logistic regression analysis was used to determine the factors associated with penicillin V prophylaxis adherence. A pvalue < 0.05 was considered statistically significant. Among the 421 SCD patients recruited, penicillin V prophylaxis adherence was observed to be 30.0% and 68.0% for the objective and subjective methods of assessment, respectively. For the objective method of assessment, being cared for by grandparents increased the odds of penicillin V adherence (adjusted odds ratio[aOR] = 3.68, confidence interval[CI] = 1.03-13.15). However, SCD patients within the ages of 10-14 years (aOR = 0.36, CI = 0.17-0.80), >14 years (aOR = 0.17, CI = 0.05-0.61), SCD patient cared for by married caregivers/parents (aOR = 0.32, CI = 0.14-0.72), SCD patient cared for by divorced caregivers/parents (aOR = 0.23, CI = 0.07-0.75), SCD patients taking homemade (herbal) preparations for the treatment of SCD (aOR = 0.42, CI = 0.21-0.83), and inappropriate intake of penicillin V prophylaxis (aOR = 0.27, CI = 0.11-0.67) reduced the odds of penicillin V adherence. For the subjective method of assessment, taking homemade preparation (herbal) for the treatment of SCD (aOR = 0.52, CI = 0.30-0.89) and inappropriate intake of penicillin V (aOR = 0.32, CI = 0.17-0.60) reduced the odds of penicillin V adherence. This study reports a relatively low adherence rate of penicillin V prophylaxis among children living with SCD. Educating and counseling both SCD patients and/or caregivers on the need to be adherent to penicillin V prophylaxis could prevent complications that may arise from nonadherence.

Urinary PCA3 a Superior Diagnostic Biomarker for Prostate Cancer among Ghanaian Men

Introduction Prostate cancer is one of the most commonly diagnosed cancers in men. Prostate-specific antigen (PSA) has been the biomarker of choice for screening and diagnosis of prostate cancer. However, inefficiencies exist with its diagnostic capabilities. This study thus evaluated the diagnostic and prognostic potential of urinary PCA3 as an alternative biomarker for prostate cancer in the Ghanaian population. Methods A hospital-based cross-sectional study was conducted at the Urology Department of the 37 Military Hospital, Accra, Ghana. A total of 237 participants aged 40 years and above with any form of suspected prostate disorder were recruited into the study after written informed consent was obtained. Total serum PSA levels was measured using the electrochemiluminescence method and transrectal ultrasound-guided systematic core needle biopsies were obtained from each study participant. Receiver operating characteristic curve (ROC) analysis was used to evaluate the diagnostic accuracies of serum PSA, DRE, and PCA3 as diagnostic tools for prostate cancer. These three diagnostic tools were also evaluated in various combinations to ascertain the combinations with the best diagnostic accuracy. Results Prostate cancer was diagnosed in 26.6% of the participants. Benign prostate hyperplasia and prostatitis were diagnosed in 48.5% and 24.9% participants, respectively. DRE had a sensitivity of 93.7% and a specificity of 12.1%. PSA had a sensitivity of 92.1% and a specificity of 16.1%. PCA3 had a sensitivity of 57.1% and a specificity of 85.6% and showed a better accuracy (AUC = 83.0) compared to PSA (AUC = 60.0) and DRE (AUC = 65.0) as individual diagnostic tools. The combination of DRE+PCA3 score had the best diagnostic accuracy (AUC = 0.80) with a sensitivity and specificity of 60.3% and 80.5%, respectively. Conclusion The urinary PCA3 assay showed a better diagnostic performance compared to serum PSA and DRE. PCA3 as a stand-alone and in combination with DRE could be a suitable complimentary marker in diagnosis and management of prostate cancer.

THE CHEST TOBACCO TREATMENT TOOLKIT: CHRONIC DISEASE MANAGEMENT, POINT-OF-CARE BIOCHEMISTRY ASSAYS, AND TITRATION OF TOBACCO TREATMENT MEDICATIONS INCREASES QUIT RATES

THE CHEST TOBACCO TREATMENT TOOLKIT: CHRONIC DISEASE MANAGEMENT, POINT-OF-CARE BIOCHEMISTRY ASSAYS, AND TITRATION OF TOBACCO TREATMENT MEDICATIONS INCREASES QUIT RATES

A Urine-based Genomic Assay Improves Risk Stratification for Patients with High-risk Hematuria Stratified According to the American Urological Association Guidelines

BackgroundAccording to the recent American Urological Association (AUA) guideline on hematuria, patients are stratified into groups with low, intermediate, and high risk of urothelial carcinoma (UC). These risk groups are based on clinical factors and do not incorporate urine-based tumor markers. ObjectiveTo evaluate whether a urine-based genomic assay improves the redefined AUA risk stratification for hematuria. Design, setting, and participantsWe selected patients with complete biomarker status, as assessed on urinary DNA, from a previously collected prospective Dutch hematuria cohort (n = 838). Patients were stratified into the AUA risk categories on the basis of sex, age, and type of hematuria. Biomarker status included mutation status for the FGFR3, TERT, and HRAS genes, and methylation status for the OTX1, ONECUT2, and TWIST1 genes. Outcome measurements and statistical analysisThe primary endpoint was the diagnostic model performance for different hematuria risk groups. Further analyses assessed the pretest and post-test UC probability in the hematuria subgroups using a Fagan nomogram. Results and limitationsOverall, 65 patients (7.8%) were classified as low risk, 106 (12.6%) as intermediate risk, and 667 (79.6%) as high risk. The UC incidence differed significantly between the gross hematuria (21%, 98/457) and microscopic hematuria (4%, 14/381) groups (p < 0.001). All cancer cases were in the high-risk group, which had UC incidence of 16.8% (112/667). Application of the diagnostic model revealed robust performance among all risk groups (area under the receiver operating characteristic curve 0.929–0.971). Depending on the risk group evaluated, a negative urine assay was associated with post-test UC probability of 0.3–2%, whereas a positive urine assay was associated with post-test UC probability of 31-42%. ConclusionsThis study shows the value that a urine-based genomic assay adds to the AUA guideline stratification for patients with hematuria. It seems justified to safely withhold cystoscopy for patients with AUA low risk who have a negative urine assay. In addition, evaluation should be expedited for patients with AUA intermediate or high risk and a positive urine assay. Patient summaryPatients who have blood in their urine (hematuria) can be classified as having low, intermediate, or high risk of having cancer in their urinary tract. We found that use of a urine-based genetic test improves the accuracy of predicting which patients are most likely to have cancer. Patients with a negative test may be able to avoid invasive tests, while further tests could be prioritized for patients with a positive test.

RNA-based urinary assays for non-muscle invasive bladder cancer.

To provide an overview of the recent literature on RNA-based molecular urine assays for the diagnosis and surveillance of non-muscle invasive bladder cancer (NMIBC). Articles were eligible for inclusion if performance metrics sensitivity, specificity, and negative-predictive value (NPV) were reported or could be calculated. Only prospective studies published between 2020-2022 were included. Five out of fourteen studies addressed the primary diagnostic setting; the proportion of gross hematuria patients in all study populations was >50%. Only one study reported performance metrics within a microscopic hematuria subgroup. This study evaluated Xpert Bladder and reported a sensitivity: 73%, specificity: 84%, NPV: 99%, and PPV: 12%. Ten studies assessed test performance during surveillance for NMIBC. For the detection of high-grade (HG) and high-risk (HR) NMIBC, sensitivity, specificity, NPV, and PPV varied between 78-100%, 64-89%, 97.0-99.7%, and 9.2-39%. Multiple RNA-based urine assays have been investigated for the detection of urothelial cancer in the primary or surveillance setting. However, studies included within this review have important limitations, hampering the interpretation of study results. As such, performance metrics should be interpreted with caution and further research is required to evaluate the clinical impact of RNA-based urine assays in daily practice.

The NIMH intramural healthy volunteer dataset: A comprehensive MEG, MRI, and behavioral resource

The NIMH Healthy Research Volunteer Dataset is a collection of phenotypic data characterizing healthy research volunteers using clinical assessments such as assays of blood and urine, mental health assessments, diagnostic and dimensional measures of mental health, cognitive and neuropsychological functioning, structural and functional magnetic resonance imaging (MRI), along with diffusion tensor imaging (DTI), and a comprehensive magnetoencephalography battery (MEG). In addition, blood samples of healthy volunteers are banked for future analyses. All data collected in this protocol are broadly shared in the OpenNeuro repository, in the Brain Imaging Data Structure (BIDS) format. In addition, task paradigms and basic pre-processing scripts are shared on GitHub. There are currently few open access MEG datasets, and multimodal neuroimaging datasets are even more rare. Due to its depth of characterization of a healthy population in terms of brain health, this dataset may contribute to a wide array of secondary investigations of non-clinical and clinical research questions.

Good Adherence to the Mediterranean Diet Lowered Risk of Renal Glomerular Impairment in Children: A Longitudinal Study.

Healthy diet patterns have a positive effect on chronic non-communicable diseases in the pediatric population, but the evidence is limited on the association between kidney impairment and adherence to a Mediterranean diet. We aim to determine the associations between Mediterranean diet adherence and longitudinal tubular and glomerular impairment in children. Based on four waves of urine assays conducted from October 2018 to November 2019, we assayed urinary β2-microglobulin (β2-MG) and microalbumin (MA) excretion to determine transient renal tubular and glomerular impairment during the follow-up of the child cohort (PROC) study in Beijing, China. We assessed Mediterranean diet adherence using the 16-item Mediterranean Diet Quality Index in children and adolescents (KIDMED) among 1914 primary school children. Poor, intermediate, and good adherence rates for the Mediterranean diet were 9.0% (KIDMED index 0–3), 54.4% (KIDMED index 4–7) and 36.5% (KIDMED index 8–12), respectively. A short sleep duration was more prevalent in children with lower Mediterranean diet adherence, with no significant differences presenting in the other demographic and lifestyle covariates. The results of linear mixed-effects models showed that a higher urinary MA excretion was inversely associated with a higher KIDMED score (β = −0.216, 95%CI: −0.358, −0.074, p = 0.003), after adjusting for sex, age, BMI z-score, SBP z-score, screen time, sleep duration and physical activity. Furthermore, in generalized linear mixed-effects models, consistent results found that transient renal glomerular impairment was less likely to develop in children with intermediate Mediterranean diet adherence (aOR = 0.68, 95%CI: 0.47, 0.99, p = 0.044) and in children with good Mediterranean diet adherence (aOR = 0.60, 95%CI: 0.40, 0.90, p = 0.014), taking poor Mediterranean diet adherence as a reference. We visualized the longitudinal associations between each item of the KIDMED test and kidney impairment via a forest plot and identified the main protective eating behaviors. Children who adhere well to the Mediterranean diet have a lower risk of transient glomerular impairment, underscoring the necessity of the early childhood development of healthy eating patterns to protect kidney health.

Inter-method reliability of silicone exposome wristbands and urinary biomarker assays in a pregnancy cohort

Silicone wristbands act as passive environmental samplers capable of detecting and measuring concentrations of a variety of chemicals. They offer a noninvasive method to collect complex exposure data in large-scale epidemiological studies. We evaluated the inter-method reliability of silicone wristbands and urinary biomarkers in the New Hampshire Birth Cohort Study. A subset of study participants (n = 96) provided a urine sample and wore a silicone wristband for 7 days at approximately 12 gestational weeks. Women were instructed to wear the wristbands during all their normal activities. Concentrations of urinary compounds and metabolites in the urine and parent compounds in wristbands were compared. High detection rates were observed for triphenyl phosphate (76.0%) and benzophenone (78.1%) in wristbands, although the distribution of corresponding urinary concentrations of chemicals did not differ according to whether chemicals were detected or not detected in wristbands. While detected among only 8.3% of wristbands, median urinary triclosan concentrations were higher among those with triclosan detected in wristbands (9.04 ng/mL) than without (0.16 ng/mL). For most chemicals slight to fair agreement was observed across exposure assessment methods, potentially due to low rates of detection in the wristbands for chemicals where observed urinary concentrations were relatively low as compared to background concentrations in the general population. Our findings support the growing body of research in support of deploying silicone wristbands as an important exposure assessment tool.

Comparison of urinary telomerase, CD44, and NMP22 assays for detection of bladder squamous cell carcinoma.

BackgroundSquamous cell carcinoma (SCC) of the bladder is common in many regions around the world. Prognosis is very poor, as most cases are diagnosed at an advanced stage due to a lack of affordable and valid screening markers for this type of cancer. The diagnostic accuracy of urinary nuclear matrix protein-22 (NMP22), telomerase activity, and CD44 were evaluated in urine samples of patients with bladder SCC.Materials and methodsWe conducted a case-control study comprised of 60 consecutive newly diagnosed bladder SCC patients diagnosed by cystoscopy and histopathological examination, and controls were 60 outpatients with benign urologic conditions and healthy clinic visitors. Urine samples collected from each subject underwent testing for NMP22, telomerase activity, and CD44. Descriptive and correlational statistical analysis of cases and controls were carried out and receiver operating characteristic curve analysis was used to determine optimal cut-off points for the three assays.ResultsArea under the curve was calculated at 0.96, 0.93, and 0.62 for NMP22, telomerase, and CD44, respectively. Urine levels of NMP22 and telomerase activity were significantly higher in the SCC group compared to controls (p < 0.001). Urine CD44 levels were not significantly higher in the SCC group compared to controls (p = 0.111). The overall sensitivity of NMP22, telomerase, and CD44 was 96.7%, 87%, and 45%, respectively, while the specificity was 85%, 88.6%, and 86.7%, respectively.ConclusionsUrinary telomerase activity, followed by NMP22 urine levels, showed high diagnostic yield and could hold potential promise as urinary biomarkers for the diagnosis of bladder SCC.

Urine Cellular DNA Point Mutation and Methylation for Identifying Upper Tract Urinary Carcinoma.

Simple SummaryIt’s difficult to detect upper tract urothelial carcinoma at early stage. Invasive testing may increase risk of cancer recurrences in the bladder after radical nephroureterectomy. Thus, in the present study, we incorporated two-gene mutation and methylation biomarkers to conduct the diagnostic tool of upper tract urothelial carcinoma and performed external validation to investigate the utility and stability of the optimal panel. It showed a highly specific and robust performance. It may be used as a replaceable approach for early detection of upper tract urothelial carcinoma, resulting in less extensive examinations in patients at low risk. Background: To improve the selection of patients for ureteroscopy, avoid excessive testing and reduce costs, we aimed to develop and validate a diagnostic urine assay for upper tract urinary carcinoma (UTUC). Methods: In this cohort study we recruited 402 patients from six Hunan hospitals who underwent ureteroscopy for hematuria, including 95 patients with UTUC and 307 patients with non-UTUC findings. Midstream morning urine samples were collected before ureteroscopy and surgery. DNA was extracted and qPCR was used to analyze mutations in TERT and FGFR3 and the methylation of NRN1. In the training set, the random forest algorithm was used to build an optimal panel. Lastly, the Beijing cohort (n = 76) was used to validate the panel. Results: The panel combining the methylation with mutation markers led to an AUC of 0.958 (95% CI: 0.933–0.975) with a sensitivity of 91.58% and a specificity of 94.79%. The panel presented a favorable diagnostic value for UTUC vs. other malignant tumors (AUC = 0.920) and UTUC vs. benign disease (AUC = 0.975). Furthermore, combining the panel with age revealed satisfactory results, with 93.68% sensitivity, 94.44% specificity, AUC = 0.970 and NPV = 98.6%. In the external validation process, the model showed an AUC of 0.971, a sensitivity of 95.83% and a specificity of 92.31, respectively. Conclusions: A novel diagnostic model for analyzing hematuria patients for the risk of UTUC was developed, which could lead to a reduction in the need for invasive examinations. Combining NRN1 methylation and gene mutation (FGFR3 and TERT) with age resulted in a validated accurate prediction model.

Effects of day-to-day variation of Opisthorchis viverrini antigen in urine on the accuracy of diagnosing opisthorchiasis in Northeast Thailand.

Antigen detection in urine using an enzyme-linked immunosorbent assay (ELISA) is more sensitive than fecal examination for diagnosis of opisthorchiasis and for assessment of the effects of drug treatment. It is not known whether day-to-day variation of urine composition, including levels of Opisthorchis viverrini antigen, influences the urine assay. We investigated this topic with the cooperation of participants from two localities in Northeast Thailand. Project participants were screened for parasite infections for three consecutive days using the quantitative formalin-ethyl acetate concentration technique (FECT) to detect O. viverrini eggs and the urine ELISA for detection of O. viverrini antigen. A subset of participants (n = 801) with matched fecal and urine samples were analyzed for comparison of inter-day prevalence estimates and the performance of the urine assay compared against FECT for diagnosis of opisthorchiasis. The daily prevalence measured by the urine assay ranged between 29.0%-30.2% while those by FECT ranged between 11.9%-20.2%. The cumulative three-day prevalence estimate determined by the urine antigen assay was 30.3%, which was significantly higher than that by FECT (20.2%, p < 0.05). A significant positive correlation was found between the concentration of antigen in urine and fecal egg counts (p < 0.001). Overall, the urine assay had better diagnostic performance for opisthorchiasis than fecal examination by FECT. The high sensitivity plus negligible daily variation of O. viverrini antigen in urine indicates the utility of the urine assay for diagnosis, as well as population screening, of opisthorchiasis.

Diagnostic performance of urine analysis based on flow microimaging and artificial intelligence recognition technology in suspected urinary tract infection patients

To investigate the diagnostic performance and clinical value of flow microimaging and artificial intelligence recognition technology for rapid screening of suspected urinary tract infection (UTI). Standard strains of bacteria responsible for UTIs, Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were prepared and used to evaluate the accuracy of classifying and counting bacteria. A total of 146 specimens of clean, midstream urine were collected from adults with suspected UTI (excluding pregnant women, and patients with urethral catheterization) and analyzed by urinary culture and assay using a MUS-3600 analyzer. Fourfold tables were used to evaluate the diagnostic performance in measurements of nitrite and leukocyte esterase. ROC curves were generated to identify cutoff values for bacillus, coccus, leukocyte, and leukocyte cluster counts. Of the samples cultured, 85 (58%) were negative and 61 (42%) were positive. E. coli and S. aureus showed good linear relationships between measured and theoretical values in a series of standard strains samples (R 2>0.95). The sensitivity of the nitrite parameter for diagnosis of suspected UTI was 37.7% and the specificity was 100%. Cutoff values obtained from ROC analysis were 50 μl for bacillus (sensitivity: 69.5%; specificity: 96.5%) with positive predictive value of 93.2% and negative predictive value of 82%. WBC ≥ 23 μl or bacillus ≥50 μl gave the highest sensitivity of 81% and NPV of 86%. MUS-3600 demonstrated sufficient specificity for UTIs to have utility in reducing needless urinary culture. Accuracy in classifying bacillus shows great potential for the rapid identification of pathogens for clinical purposes.

Noninvasive monitoring of bilirubin photoisomer excretion during phototherapy

Lumirubin is the most prevalently excreted hydrophilic bilirubin photoisomer in phototherapy for neonatal jaundice caused by excess hydrophobic unconjugated bilirubin (ZZ-bilirubin). We developed a simple method to estimate the amount of lumirubin by monitoring the reverse photoisomerization of lumirubin to ZZ-bilirubin. Although lumirubin formation was long considered irreversible, exposure to blue light in the presence of the fluorescent protein UnaG, which binds specifically and tightly to ZZ-bilirubin, enables the reverse photoisomerization of lumirubin. This reaction was first detected using a fluorescence assay of neonatal urine sampled during phototherapy and purified lumirubin. The phenomenon of reverse photoisomerization of lumirubin was validated using liquid chromatography–mass spectrometry, which confirmed that lumirubin is reconverted to ZZ-bilirubin in the presence of UnaG. Analyses of 20 urine samples from 17 neonates revealed a significant correlation (correlation coefficient [r] = 0.978; 95% confidence interval 0.867–0.979; P < .001) between lumirubin and ZZ-bilirubin concentration before and after reverse photoisomerization. In general, the rate of photo-reconversion of lumirubin to ZZ-bilirubin is approximately 40%. In conclusion, we demonstrate here that lumirubin can be photo-reconverted to ZZ-bilirubin via exposure to blue light in the presence of UnaG. Utilizing this approach, urinary lumirubin levels can be estimated using an easy-to-perform fluorescence assay.

Validated single urinary assay designed for exposomic multi-class biomarkers of common environmental exposures.

Epidemiological studies often call for analytical methods that use a small biospecimen volume to quantify trace level exposures to environmental chemical mixtures. Currently, as many as 150 polar metabolites of environmental chemicals have been found in urine. Therefore, we developed a multi-class method for quantitation of biomarkers in urine. A single sample preparation followed by three LC injections was optimized in a proof-of-approach for a multi-class method. The assay was validated to quantify 50 biomarkers of exposure in urine, belonging to 7 chemical classes and 16 sub-classes. The classes represent metabolites of 12 personal care and consumer product chemicals (PCPs), 5 polycyclic aromatic hydrocarbons (PAHs), 5 organophosphate flame retardants (OPFRs), 18 pesticides, 5 volatile organic compounds (VOCs), 4 tobacco alkaloids, and 1 drug of abuse. Human urine (0.2 mL) was spiked with isotope-labeled internal standards, enzymatically deconjugated, extracted by solid-phase extraction, and analyzed using high-performance liquid chromatography-tandem mass spectrometry. The methanol eluate from the cleanup was split in half and the first half analyzed for PCPs, PAH, and OPFR on a Betasil C18 column; and pesticides and VOC on a Hypersil Gold AQ column. The second half was analyzed for tobacco smoke metabolites and a drug of abuse on a Synergi Polar RP column. Limits of detection ranged from 0.01 to 1.0 ng/mL of urine, with the majority ≤0.5 ng/mL (42/50). Analytical precision, estimated as relative standard deviation of intra- and inter-batch uncertainty, variabilities, was <20%. Extraction recoveries ranged from 83 to 109%. Results from the optimized multi-class method were qualified in formal international proficiency testing programs. Further method customization options were explored and method expansion was demonstrated by inclusion of up to 101 analytes of endo- and exogenous chemicals. This exposome-scale assay is being used for population studies with savings of assay costs and biospecimens, providing both quantitative results and the discovery of unexpected exposures.

Interaction of Hydration Status and Physical Activity Level on Early Renal Damage in Children: A Longitudinal Study.

BackgroundOptimal water intake positively affects various aspects of human physiology, especially renal function. Physical activity (PA) may have an impact on hydration status and renal health, but the interaction of hydration status and PA level on renal function is not well-studied in children.MethodsWe conducted four waves of urine assays in our child cohort (PROC) study from October 2018 to November 2019 in Beijing, China. We measured urinary specific gravity, β2-microglobulin (β2-MG), and microalbumin (MA) excretion to assess hydration status and renal damage in the context of PA level and other covariates among 1,914 primary school children. We determined the associations of renal damage with the interaction of hydration status and PA level using generalized linear mixed-effects models.ResultsThe prevalence of dehydration was 35.0%, 62.1%, 63.9%, and 63.3%, and the prevalence of insufficient PA was 86.2%, 44.9%, 90.4%, and 90.2% from wave 1 to wave 4 among 1,914 primary school children. From wave 1 to wave 4, the prevalence of renal tubular damage had a significant increasing trend of 8.8%, 15.9%, 25.7%, and 29.0% (Z = 16.9, P < 0.001), while the prevalence of glomerular damage revealed a declining trend of 5.6%, 5.5%, 4.4%, and 4.1% (Z = −2.4, P = 0.016). There were stable longitudinal associations of renal tubular and glomerular damage with hydration status (euhydration: OR = 0.50 and 0.33, respectively) but not with PA level. In multivariate analysis, significant interactions of hydration status and PA level were noted with renal tubular damage (β = 0.43, P = 0.014) and glomerular damage (β = 0.60, P = 0.047). Children with euhydration and insufficient PA were less likely to have renal tubular damage (OR = 0.46, 95% CI: 0.39, 0.53) or glomerular damage (OR = 0.28, 95% CI: 0.20, 0.39); children with euhydration and sufficient PA were also less likely to have renal tubular damage (OR = 0.57, 95% CI: 0.44, 0.75) or glomerular damage (OR = 0.47, 95% CI: 0.30, 0.74), adjusting for age, sex, BMI z-score, standardized SBP, sleep duration, computer/cell phone screen time, and fruit and vegetable intake.ConclusionChildren with euhydration and either sufficient or insufficient PA were less likely to have early renal damage. Adequate daily water intake for children is important, especially after PA.

Pneumococcal serotypes in adults hospitalized with community-acquired pneumonia in Greece using urinary antigen detection tests: the EGNATIA study, November 2017 – April 2019

ABSTRACT Greece introduced a 13-valent pneumococcal conjugate vaccine (PCV13) into the infant national immunization program in 2010 (3 + 1 schedule until June 2019). Since 2015, PCV13 has been recommended for adults aged 19–64 years with comorbidities and adults ≥65 years sequentially with 23-valent pneumococcal polysaccharide vaccine (PPSV23). We examined pneumococcal serotype distribution among Greek adults aged ≥19 years hospitalized with community-acquired pneumonia (CAP) during November 2017-April 2019. This was an interim analysis of EGNATIA, a prospective study of adult hospitalized CAP in the cities of Ioannina and Kavala. Pneumococcus was identified using cultures, BinaxNow®, serotype-specific urinary antigen detection assays (UAD-1/2). Our analysis included overall 482 hospitalized CAP patients (mean age: 70.5 years; 56.4% male). 53.53% of patients belonged to the highest pneumonia severity index (PSI) classes (IV-V). Pneumococcus was detected in 65 (13.5%) patients, with more than half (57%) of cases detected only by UAD. Approximately two-thirds of pneumococcal CAP occurred in those aged ≥65 years (n = 40, 8.3% of CAP). More than half of pneumococcal CAP (n = 35, 53.8%) was caused by PCV13 serotypes. Most frequently detected PCV13 serotypes were 3, 19A, 23F, collectively accounting for 83% of PCV13 vaccine-type (VT) CAP and 6% of all-cause CAP. Overall, 82.9% of PCV13 VT CAP occurred among persons with an indication (age/risk-based) for PCV13 vaccination. Even with a mature PCV13 childhood immunization program, a persistent burden of PCV13 VT CAP exists in Greek adults. Strategies to increase PCV13 (and higher-valency PCVs, when licensed) coverage in adults should be implemented to reduce the disease burden.

Utility of cell-free transrenal DNA for the diagnosis of Tuberculous Meningitis: A proof-of-concept study

Tuberculous Meningitis (TBM) diagnosis remains a grave challenge. We evaluated the utility of extracellular vesicles (EVs) as a source of cell-free transrenal-mycobacterial DNA (cf-Tr-MTB DNA) for TBM diagnosis from urine samples. We developed a qPCR-assay targeting a highly repetitive 36-bp sequence specific to Mycobacterium tuberculosis complex. EVs were isolated from urine samples of suspected TBM groups (n = 44) [categorized using composite reference standard as ‘Definite’ TBM (n = 8), ‘Probable’ TBM (n = 15), ‘Possible’ TBM (n = 21)] and ‘Non-TBM’ group (n = 26). cf-Tr-MTB DNA-based qPCR assay was applied to DNA isolated from EVs (EV-DNA) and EV-free-fraction (EV-free DNA). ROC-curves were generated using qPCR results of ‘Definite’ TBM and ‘Non-TBM’ category in both EV-DNA and EV-free DNA samples and cut-off values were selected to provide 100% (95%CI:69.1–100) specificity. The cf-Tr-MTB DNA assay gave a sensitivity of 54.5% (95%CI:38.8–69.6) for EV-DNA and 77.3% (95%CI:62.1–88.5) for EV-free DNA in the TBM group (n = 44). The combination of EV-DNA and EV-free DNA results (corresponding to performance cf-Tr MTB DNA assay in urine), gave an overall sensitivity of 81.8% (95%CI:67.2–91.8) in the TBM group. Our results confirmed EVs as one of the sources of cf-Tr-MTB DNA and we believe the cf-Tr-MTB DNA-based qPCR assay has a potential application for TBM diagnosis.

Urine ALCAM, PF4 and VCAM-1 Surpass Conventional Metrics in Identifying Nephritis Disease Activity in Childhood-Onset Systemic Lupus Erythematosus.

ObjectivesSerial kidney biopsy for repeat evaluation and monitoring of lupus nephritis (LN) in childhood-onset Systemic Lupus Erythematosus (cSLE) remains challenging, thus non-invasive biomarkers are needed. Here, we evaluate the performance of ten urine protein markers of diverse nature including cytokines, chemokines, and adhesion molecules in distinguishing disease activity in cSLE.MethodsEighty-four pediatric patients meeting ≥4 ACR criteria for SLE were prospectively enrolled for urine assay of 10 protein markers normalized to urine creatinine, namely ALCAM, cystatin-C, hemopexin, KIM-1, MCP-1, NGAL, PF-4, Timp-1, TWEAK, and VCAM-1 by ELISA. Samples from active renal (LN) and active non-renal SLE patients were obtained prior to onset/escalation of immunosuppression. SLE disease activity was evaluated using SLEDAI-2000. 59 patients had clinically-active SLE (SLEDAI score ≥4 or having a flare), of whom 29 patients (34.5%) were classified as active renal, and 30 patients (35.7%) were active non-renal. Twenty-five healthy subjects were recruited as controls.ResultsUrine concentrations of ALCAM, KIM-1, PF4 and VCAM-1 were significantly increased in active LN patients versus active non-renal SLE, inactive SLE and healthy controls. Five urine proteins differed significantly between 2 (hemopexin, NGAL, MCP1) or 3 (Cystatin-C, TWEAK) groups only, with the highest levels detected in active LN patients. Urine ALCAM, VCAM-1, PF4 and hemopexin correlated best with total SLEDAI as well as renal-SLEDAI scores (p < 0.05). Urine ALCAM, VCAM-1 and hemopexin outperformed conventional laboratory measures (anti-dsDNA, complement C3 and C4) in identifying concurrent SLE disease activity among patients (AUCs 0.75, 0.81, 0.81 respectively), while urine ALCAM, VCAM-1 and PF4 were the best discriminators of renal disease activity in cSLE (AUCs 0.83, 0.88, 0.78 respectively), surpassing conventional biomarkers, including proteinuria. Unsupervised Bayesian network analysis based on conditional probabilities re-affirmed urine ALCAM as being most predictive of active LN in cSLE patients.ConclusionUrinary ALCAM, PF4, and VCAM-1 are potential biomarkers for predicting kidney disease activity in cSLE and hold potential as surrogate markers of nephritis flares in these patients.