High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity- and 4 NAAT-/Clarity+ samples, there were 26 CCNA- and 4 CCNA- samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin- and NAAT-/toxin- patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin- than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay.

Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA-) became CCCNA-, and 5/26 (19.2%) Clarity+/CCCNA- samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology.

Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

High-sensitivity cardiac troponin I is a biomarker for occult coronary plaque burden and cardiovascular events in patients with rheumatoid arthritis.

Objectives: Patients with RA display greater occult coronary atherosclerosis burden and experience higher cardiovascular morbidity and mortality compared with controls. We here explored whether pro-inflammatory cytokines and high-sensitivity cardiac troponin I (hs-cTnI), a biomarker of myocardial injury, correlated with plaque burden and cardiovascular events (CVEs) in RA. Methods: We evaluated 150 patients with 64-slice coronary CT angiography. Coronary artery calcium, number of segments with plaque (segment involvement score), stenotic severity and plaque burden were assessed. Lesions were described as non-calcified, mixed or fully calcified. Blood levels of hs-cTnI and pro-inflammatory cytokines were assessed during coronary CT angiography. Subjects were followed over 60 (s.d. 26) months for both ischaemic [cardiac death, non-fatal myocardial infarction (MI), stroke, peripheral arterial ischaemia] and non-ischaemic (new-onset heart failure hospitalization) CVEs. Results: Plasma hs-cTnI correlated with all coronary plaque outcomes (P < 0.01). Elevated hs-cTnI (⩾1.5 pg/ml) further associated with significant calcification, extensive atherosclerosis, obstructive plaque and any advanced mixed or calcified plaques after adjustments for cardiac risk factors or Framingham D'Agostino scores (all P < 0.05). Eleven patients suffered a CVE (1.54/100 patient-years), eight ischaemic and three non-ischaemic. Elevated hs-cTnI predicted all CVE risk independent of demographics, cardiac risk factors and prednisone use (P = 0.03). Conversely, low hs-cTnI presaged a lower risk for both extensive atherosclerosis (P < 0.05) and incident CVEs (P = 0.037). Conclusion: Plasma hs-cTnI independently associated with occult coronary plaque burden, composition and long-term incident CVEs in patients with RA. Low hs-cTnI forecasted a lower risk for both extensive atherosclerosis as well as CVEs. hs-cTnI may therefore optimize cardiovascular risk stratification in RA.

Fibroblast growth factor 23 and renal function among young and healthy individuals.

BACKGROUND: Fibroblast growth factor 23 (FGF-23), an osteocyte hormone involved in the regulation of phosphate metabolism, is associated with incident and progressive chronic kidney disease. We aimed to assess the association of FGF-23 with renal parameters, vascular function and phosphate metabolism in a large cohort of young and healthy individuals. METHODS: Healthy individuals aged 25-41 years were included in a prospective population-based study. Fasting venous blood and morning urinary samples were used to measure plasma creatinine, cystatin C, endothelin-1, phosphate and plasma FGF-23 as well as urinary creatinine and phosphate. Multivariable regression models were constructed to assess the relationship of FGF-23 with parameters of renal function, endothelin-1 and fractional phosphate excretion. RESULTS: The median age of 2077 participants was 37 years, 46% were males. The mean estimated glomerular filtration rate (eGFR - CKD-EPI creatinine-cystatin C equation) and fractional phosphate excretion were 110 mL/min/1.73 m2 and 8.7%, respectively. After multivariable adjustment, there was a significant inverse relationship of FGF-23 with eGFR (ß per 1 log-unit increase -3.81; 95% CI [-5.42; -2.20]; p<0.0001). Furthermore, we found a linear association between FGF-23 and endothelin-1 (ß per 1 log-unit increase 0.06; [0.01, 0.11]; p=0.01). In addition, we established a significant relationship of FGF-23 with fractional phosphate excretion (ß per 1 log-unit increase 0.62; [0.08, 1.16]; p=0.03). CONCLUSIONS: Increasing plasma FGF-23 levels are strongly associated with decreasing eGFR and increasing urinary phosphate excretion, suggesting an important role of FGF-23 in the regulation of kidney function in young and healthy adults.

Low Plasma Testosterone Is Associated With Elevated Cardiovascular Disease Biomarkers.

BACKGROUND: The relation between testosterone (T) plasma concentration and cardiovascular (CV) risk is unclear, with evidence supporting increased risk in men with low and high T levels. Few studies have assessed CV risk as a function of plasma T levels using objective biomarkers. AIM: To determine the relation between T levels and high-sensitivity CV risk biomarkers. METHODS: Ten thousand forty-one male patients were identified in the database of a commercial clinical laboratory performing biomarker testing. Patients were grouped by total T concentration and associations with the following biomarkers were determined: cardiac troponin I (cTnI), endothelin-1 (ET-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-17A, N-terminal pro-B-type natriuretic peptide (NTproBNP), high-density lipoprotein (HDL) cholesterol, high-sensitivity C-reactive protein (hs-CRP), hemoglobin A1c (HbA1c), and leptin. OUTCOMES: Association of CV risk markers with levels of T in men. RESULTS: The median age of the cohort was 58 years (interquartile range = 48-68), and the median plasma T level was 420 ng/dL (interquartile range = 304-565); T levels did not vary with patient age. An inverse relation between plasma T levels and CV risk was observed for 9 of 10 CV markers: cTnI, ET-1, IL-6, TNF-α, NTproBNP, HDL cholesterol, hs-CRP, HbA1c, and leptin. Even after adjusting for age, body mass index, HbA1c, hs-CRP, and HDL cholesterol levels, the CV markers IL-6, ET-1, NTproBNP, and leptin were significantly associated with a T level lower than 250 ng/dL. CLINICAL IMPLICATIONS: Men with low T levels could be at increased risk for increased CV disease as seen by increased CV risk markers. STRENGTH AND LIMITATIONS: This study was performed in a group of 10,041 men and is the first study to examine CV risk associated with circulating T levels using a large panel of 10 objective biomarkers. This study is limited by an absence of clinical data indicating whether men had pre-existing CV disease or other CV risk factors. CONCLUSION: Men with low plasma T levels exhibit increases in CV risk markers, consistent with a potential increased risk of CV disease. Pastuszak AW, Kohn TP, Estis J, Lipshultz LI. Low Plasma Testosterone Is Associated With Elevated Cardiovascular Disease Biomarkers. J Sex Med 2017;14:1095-1103.

Healthy lifestyle and glucagon-like peptide-1 in young and healthy adults: A population-based study.

A healthy lifestyle is associated with a lower risk of cardiovascular events and mortality, but underlying mechanisms are not fully understood. The aim of our study was to investigate the relationships between a healthy lifestyle and glucagon-like peptide-1 (GLP-1), an incretin hormone with both glycemic and cardiovascular properties. Healthy participants aged 25-41years without cardiovascular disease, diabetes or a body mass index (BMI) >35kg/m2 were enrolled in a population-based study. The following metrics were used to build a lifestyle score ranging from 0 to 7 (a higher score indicating a healthier lifestyle): blood pressure (BP) (<120/80mmHg), plasma levels of glycated hemoglobin (<5.7%), total cholesterol levels (<200mg/dl), BMI (<25kg/m2), not smoking cigarettes, moderate (≥150min/week) or vigorous (≥75min/week) physical activity and a healthy diet. Among 2133 participants median age was 36.7years and 53.3% were female. GLP-1 levels decreased significantly from 39.5 to 30.9ng/l (p<0.0001) across increasing lifestyle score categories. This linear relationship persisted in multivariable adjusted linear regression models (B for GLP-1 per 1-unit increase of the lifestyle score -0.06; 95% confidence intervals -0.07, -0.04; p<0.0001). Individual health metrics that were significantly associated with GLP-1 were a normal BMI (-0.07; -0.12, -0.03; p=0.001), low total cholesterol levels (-0.07; -0.12, -0.03; p=0.001), normal BP (-0.05; -0.10, -0.00; p=0.047) and not smoking (-0.06; -0.10, -0.01; p=0.01). A healthy lifestyle is strongly associated with lower GLP-1 levels in young and healthy adults.

Plasma levels of endothelin-1 and renal function among young and healthy adults.

BACKGROUND: Endothelin-1 (ET-1), a vasoconstrictive and pro-inflammatory peptide, is associated with several cardiovascular risk factors and outcomes. We aimed to investigate the association of plasma ET-1 levels and renal function among young and healthy adults. METHODS: Individuals aged 25-41 years were enrolled in a population-based cohort study. Main exclusion criteria were established kidney disease, cardiovascular diseases, diabetes mellitus and a body mass index>35 kg/m2. Fasting venous plasma samples were used to measure creatinine, cystatin C and ET-1. The estimated glomerular filtration rate (eGFR) was calculated using the creatinine based chronic kidney disease epidemiology collaboration (CKD-EPI) formula. Multivariable regression models were constructed to assess interrelationships of plasma ET-1 with parameters of renal function. RESULTS: Median age of the 2139 participants was 37 years, 47% males. Median creatinine and eGFR were 67 µmol/L and 112 mL/min/1.73 m2, respectively. Using quartile one as the reference group, the ß-coefficients (95% confidence intervals [CIs]) for eGFR were 0.06 (- 1.22 to 1.35),-0.66 (- 1.95 to 0.62) and-1.70 (- 3.01 to-0.39) for quartiles 2-4 (p-for-trend=0.0056), respectively and ß-coefficients (95% CIs) for cystatin C were 0.002 (- 0.01 to 0.02), 0.02 (0.003-0.03) and 0.03 (0.01-0.04) for quartiles 2-4 (p-for-trend<0.0001), respectively. Using ET-1 as a continuous variable, the ß-coefficient (95% CI) for eGFR per 1-unit increase was-1.82 (- 3.19 to-0.44, p=0.0095) and 0.02 (0.01-0.04, p=0.0003) for cystatin C. Similar results were found between creatinine and ET-1 levels. CONCLUSIONS: ET-1 levels are strongly associated with parameters of renal function among young and healthy adults, suggesting an important role of ET-1 and endothelial function in the regulation of kidney function.

Plasma levels of glucagon-like peptide 1 and markers of obesity among young and healthy adults.

OBJECTIVE: Glucagon-like peptide 1 (GLP-1)-related pathways may partially explain the strong relationship between obesity and type 2 diabetes. We therefore aimed to evaluate the relationships between fasting GLP-1 levels, body fat mass and other obesity markers in a large sample of young and healthy adults. DESIGN AND PATIENTS: Our population-based study included 2096 individuals aged 24-44. Exclusion criteria were prevalent cardiovascular disease, diabetes or a body mass index (BMI) >35 kg/m(2) . Body fat mass was obtained by bioelectrical impedance analysis. Multivariable linear regression models were constructed to assess the relationships of GLP-1 with various measures of body composition. RESULTS: Median age of our population was 37 years, median BMI 24·1 kg/m(2) and median body fat 25·1%. A strong positive correlation was observed in age-adjusted models between GLP-1 and fat mass in men (ß (95% confidence interval) 1·38 (0·69; 2·07), P < 0·001) and women (1·27 (0·65; 1·89), P < 0·001) as well as fully adjusted models including BMI in men [0·87 (0·27; 1·46), P < 0·01] but not women [0·29 (-0·07; 0·64), P = 0·11]. The relationships of GLP-1 with BMI for men and women [0·00 (-0·34; 0·34), P = 0·99] [-0·02 (-0·28; 0·25), P = 0·91] and waist circumference [0·43 (-0·45; 1·30), P = 0·34] [0·37 (-0·44; 1·18), P = 0·37], respectively, were not significant after multivariable adjustment including fat mass. CONCLUSION: Among young and healthy adults, GLP-1 levels are strongly and independently related to body fat mass especially in men but not BMI or waist circumference. These results raise the hypothesis that GLP-1 may be implicated in body fat mass regulation.

The influence of age on serum concentrations of cardiac troponin I: results in rats, monkeys, and commercial sera.

Cardiac troponins serve as serum biomarkers of myocardial injury. The current study examined the influence of age on serum concentrations of cardiac troponin I (cTnI). An ultrasensitive immunoassay was used to monitor cTnI concentrations in Sprague-Dawley (SD) rats and Erythrocebus patas monkeys of different ages. The mean cTnI concentrations were highest in 10-day-old rats compared to 25-, 40-, and 80-day-old SD rats. Cardiomyocyte remodeling was apparent in hearts from 10-day-old SD rats as evident by hypercellularity, irregularly shaped nuclei, and moderate numbers of myocytes undergoing mitosis and apoptosis. The mean concentration of cTnI in 5 newborn monkeys was considerably higher than that of three 1-year-old monkeys. Evidence of cardiomyocyte remodeling was also observed in these newborn hearts (loss of myofibrils and cytoplasmic vacuolation). Commercial animal serum samples were also analyzed. The concentrations of cTnI detected in fetal equine and porcine serum were considerably higher than that found in adult equine and porcine serum samples Likewise, fetal bovine serum had higher cTnI concentrations (>2,400 pg/ml) than did adult caprine and laprine samples (2.5-2.7 pg/ml). The present study found age-related differences in cTnI concentrations, with higher levels occurring at younger ages. This effect was consistent across several animal species.

Reference range and short- and long-term biological variation of interleukin (IL)-6, IL-17A and tissue necrosis factor-alpha using high sensitivity assays.

Interleukin (IL)-6, and tissue necrosis factor alpha (TNF-α) are established biomarkers for clinical practice and use in clinical trials of patients with cardiovascular disease. IL-17A may be an emerging marker for atherosclerosis disease progression. We measured IL-6, TNF-α, and IL-17A using a high sensitivity immunoassay (Erenna, Singulex, Inc.) to determine the reference range and to calculate the weekly (25 subjects over 6weeks) and monthly (17 subjects over 9months) biological variation (BV) from apparently healthy subjects and those attending a cardiovascular disease clinic. As a validation for the experimental and statistical approach taken, the weekly BV for high sensitivity C-reactive protein was also determined and result compared to previous reports. The upper 95th percentile reference limit for IL-6, TNF-α, and IL17-A was 4.45, 2.53, and 1.93pg/mL, respectively. The intra-individual variability ranged from 21% to 57% and the inter-individual variation ranged from 22% to 53%. The corresponding index of individuality was 0.65-1.6 and reference change values from 63% to 161%. The BV for IL-6 and TNF-α are similar to previous reports, documenting their diagnostic utility in clinical practice. Up until now, the biological variation of IL-17A and upper reference limit has not been previously reported, thereby limiting the use of this marker in clinical trials.

Baseline serum cardiac troponin I concentrations in Sprague-Dawley, spontaneous hypertensive, Wistar, Wistar-Kyoto, and Fisher rats as determined with an ultrasensitive immunoassay.

Cardiac troponins have proved to be reliable blood biomarkers for identifying a variety of myocardial alterations in humans and animals. Recently, an ultrasensitive cTnI assay (Erenna IA) has been used to demonstrate increases in baseline cTnI resulting from drug-induced myocardial injury in rats, dogs, and monkeys, as well as to document baseline cTnI ranges in Sprague-Dawley (SD) rats. The present study was initiated to use the Erenna cTnI assay to further document baseline cTnI concentrations in normal control animals from multiple strains, including SD, Spontaneous Hypertensive (SHR), Wistar, Wistar-Kyoto (WKY), and Fisher strains. Baseline cTnI concentrations were quantified in all rats tested, and males had higher mean cTnI concentrations than females of the same strain. SHR males had the highest mean cTnI concentrations and the largest cTnI variability. Interestingly, cTnI concentrations increased in castrated SHR compared with unaltered male SHR, whereas cTnI concentrations decreased in ovariectomized SHR compared with unaltered female SHR. These results show significant differences in cTnI concentrations between strains, sexes, and noncardiac surgical alterations in control animals, and identify these as potential contributing factors to cTnI baseline variability that should be taken into account when using ultrasensitive cTnI as a biomarker to assess preclinical cardiotoxicity.

A multifaceted evaluation of imatinib-induced cardiotoxicity in the rat.

Cardiotoxicity was an unanticipated side effect elicited by the clinical use of imatinib (Imb). This toxicity has been examined in only a limited number of experimental studies. The present study sought, by a variety of approaches, to identify important characteristics of Imb-induced cardiac alterations. Male spontaneously hypertensive rats (SHRs) received oral doses of 10, 30, or 50 mg/kg Imb or water daily for 10 d. Cardiac lesions, detected at all doses, were characterized by cytoplasmic vacuolization and myofibrillar loss. In a second experiment, cardiac lesions were found in Sprague Dawley (SD) and SHR rats given 50 or 100 mg/kg Imb for 14 d. Mean cardiac lesion scores and serum levels of cardiac troponin I were higher in SHRs than in SD rats. Imb induced myocyte death by necrosis, autophagy, and apoptosis. Dose-related increases in cardiac expression were observed for several genes associated with endoplasmic reticulum stress response, protein folding, and vascular development and remodeling. Imb caused alterations in isolated myocytes (myofibrillar loss, highly disrupted and disorganized sarcomeric α-actinin, apoptosis, and increased lactate dehydrogenase release) at low concentrations (5 mM). The authors conclude that Imb exerts cardiotoxic effects that are manifest through a complex pattern of cellular alterations, the severity of which can be influenced by arterial blood pressure.

Assessment of the toxicity of hydralazine in the rat using an ultrasensitive flow-based cardiac troponin I immunoassay.

The purpose of this study was to correlate the histologic changes in the heart to serum cardiac troponin I (cTnI) concentrations assayed with the Erenna Immunoassay System in Wistar rats (Crl:Wi[Han]) using the hydralazine model of cardiotoxicity. A single dose of hydralazine caused an increase of cTnI concentrations at six hours post-dose, followed by a sharp decrease at twenty-four hours and a return to baseline at forty-eight hours. The second dose of hydralazine caused a smaller magnitude increase in cTnI concentrations at six hours as compared to the first dose. Also, cTnI concentrations returned to baseline at twenty-four hours after the second dose. The increased cTnI concentrations coincided with acute myocardial necrosis at histology. However, increased cTnI concentrations in the absence of microscopic lesions were identified in several rats. As cTnI concentrations decreased, microscopic changes in the heart matured to cardiomyophagy. In conclusion, the increases in cTnI concentrations six hours after the administration of hydralazine were indicative of a myocardial damage that did not consistently have a microscopic correlate. However, the window of increased cTnI concentrations was short, and only microscopic evaluation of the heart detected the damage at twenty-four to forty-eight hours after the episode of acute myocardial necrosis.

Effect of Health and Training on Ultrasensitive Cardiac Troponin in Marathon Runners.

PURPOSE: Cardiac troponin (cTn) is the gold standard biomarker for assessing cardiac damage. Previous studies have demonstrated increases in plasma cTn because of extreme exercise, including marathon running. We developed an easy-to-use, ultrasensitive assay for cardiac troponin I (cTnI) by combining single-molecule counting (SMC™) technology with dried blood spot (DBS) collection techniques and validated the assay on a cohort of marathon runners by correlating postmarathon cTnI elevations with training or risk variables. METHODS: An SMC-DBS method was developed for accurate and reproducible measurement of cTnI in fingerstick whole blood. Samples were collected from 42 runners both before and immediately after running a marathon. A similar collection was obtained from 22 non-running control individuals. Pre- and postrace questionnaires containing health and training variables were correlated with cTnI concentration. RESULTS: The assay quantified cTnI in all controls and marathon runners, both before and after the race. Prerace concentrations were significantly higher in marathon runners vs controls (median 3.1 vs 0.4 pg/mL; P < 0.0001). Immediate postmarathon concentrations were increased in 98% of runners (median elevation, 40.5 pg/mL; P < 0.001), including many above traditional cutoffs for acute myocardial infarction. Several health and training variables trended toward significant correlation with cTnI elevations. CONCLUSION: While further studies are needed to better understand the mechanisms and clinical implications of exercise-induced cTnI elevations, the present study suggests several variables that may be associated with such elevations and demonstrates a simple, cost-effective method for monitoring cTnI during exercise, managing chronic disease, and/or for assessing risk in large populations.

Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms.

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin-/PCR+, GDH+/toxin-/PCR-, and GDH-/toxin- samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

Relationships of kidney injury molecule-1 with renal function and cardiovascular risk factors in the general population.

BACKGROUND: Kidney injury molecule-1 (KIM-1) has been associated with kidney damage in patients with preexisting renal disease. However, little is known about the relationships of KIM-1 with renal function and cardiovascular risk factors in healthy individuals from the general population. METHODS: Healthy individuals aged 25-41years were enrolled in a population-based study. Main exclusion criteria were a BMI >35kg/m2, preexisting kidney disease or established cardiovascular disease. KIM-1 was measured from frozen plasma samples using a high-sensitivity assay. Multivariable linear regression models were constructed to assess the relationships of KIM-1 with renal function and various cardiovascular risk factors. RESULTS: We included 2060 individuals (47% men, median (interquartile range) age: 37 (31-40) years) in this analysis. Median KIM-1 levels were 82.5 (IQR 59.4-112.7) pg/ml. We found no significant relationship of KIM-1 with creatinine (adjusted ß-coefficient (95% confidence interval) 0.0005 (-0.002; 0.003), p=0.61) and cystatin C (-0.02 (-0.21; 0.17), p=0.84). There were significant linear relationships of log-transformed KIM-1 with systolic blood pressure (adjusted ß-coefficient (95% confidence interval) 0.07 (0.04; 0.09), p<0.0001), diastolic blood pressure (0.04 (0.02; 0.07), p=0.001), low-density lipoprotein cholesterol (0.09 (0.06; 0.11), p<0.0001), high-density lipoprotein cholesterol (0.07 (0.05; 0.1), p<0.0001), high-sensitivity C-reactive protein (0.05 (0.03; 0.07), p<0.0001), age (0.09 (0.07; 0.11), p<0.0001), BMI (0.04 (0.01; 0.06), p=0.005) and current smoking (0.12 (0.07; 0.17), p<0.0001). CONCLUSION: Among healthy adults from the general population, plasma levels of KIM-1 were not associated with renal function but were independently related to multiple cardiovascular risk factors.

Ultrasensitive quantification of cardiac troponin I by a Single Molecule Counting method: analytical validation and biological features.

AIM: To evaluate analytical and biological characteristics of the Singulex Clarity® cTnI assay, based upon Single Molecule Counting technology. METHODS: Assay's analytical sensitivity, precision, linearity, hook effect, cross-reactivity or interference by endogenous and exogenous substances, stability, 99th reference percentile [p99th] in EDTA plasma were evaluated in single or multi-site studies. RESULTS: Detection limit was 0.12 ng/L. Sensitivity was 0.14 ng/L at 20% CV (functional sensitivity) and 0.53 ng/L at 10% CV. Imprecision was 3.16%-10.0% in a multi-lot, single-site study, and 5.5%-12.0% in a single-lot, multi-site study; assay was linear from 0.08 to 25,000 ng/L. No hook effect was observed; any cross-reactivity/interference exceeded the 10%. Healthy subjects were recruited using clinical history, normal NT-proBNP and eGFR (n = 560) or plasma creatinine (n = 535) as inclusion criteria. cTnI was detectable in 96.8% of healthy subjects. The p99th were 8.01 (eGFR used) and 8.15 ng/L (plasma creatinine); both were measured with ≤5.7% CV. Median cTnI were significantly higher in older and male than in young and female subjects. CONCLUSIONS: The Singulex Clarity cTnI assay show analytical features and % detection in healthy subjects that improve the corresponding values of most of the existing high-sensitivity cTnI assays.