A multiplexed immunochemical microarray for the determination of cardiovascular disease biomarkers.

A fluorescence antibody microarray has been developed for the determination of relevant cardiovascular disease biomarkers for the analysis of human plasma samples. Recording characteristic protein molecular fingerprints to assess individual's states of health could allow diagnosis to go beyond the simple identification of the disease, providing information on its stage or prognosis. Precisely, cardiovascular diseases (CVDs) are complex disorders which involve different degenerative processes encompassing a collection of biomarkers related to disease progression or stage. The novel approach that we propose is a fluorescent microarray chip has been developed accomplishing simultaneous determination of the most significant cardiac biomarkers in plasma aiming to determine the CVD status stage of the patient. As proof of concept, we have chosen five relevant biomarkers, C-reactive protein (CRP) as biomarker of inflammation, cystatin C (CysC) as biomarker of renal failure that is directly related with heart failure, cardiac troponin I (cTnI) as already established biomarker for cardiac damage, heart fatty acid binding protein as biomarker of ischemia (H-FABP), and finally, NT-proBNP (N-terminal pro-brain natriuretic peptide), a well-established heart failure biomarker. After the optimization of the multiplexed microarray, the assay allowed the simultaneous determination of 5 biomarkers in a buffer solution reaching LODs of 15 ± 5, 3 ± 1, 24 ± 3, 25 ± 3, and 3 ± 1 ng mL-1, for CRP, CysC, H-FABP, cTnI, and NT-proBNP, respectively. After solving the matrix effect, and demonstrating the accuracy for each biomarker, the chip was able to determine 24 samples per microarray chip. Then, the microarray has been used on a small pilot clinical study with 29 plasma samples from clinical patients which suffered different CVD and other related disorders. Results show the superior capability of the chip to provide clinical information related to the disease in terms of turnaround time (1 h 30 min total assay and measurement) and amount of information delivered in respect to reference technologies used in hospital laboratories (clinical analyzers). Despite the failure to detect c-TnI at the reported threshold, the microarray technology could be a powerful approach to diagnose the cardiovascular disease at early stage, monitor its progress, and eventually providing information about an eminent potential risk of suffering a myocardial infarction. The microarray chip here reported could be the starting point for achieving powerful multiplexed diagnostic technologies for the diagnosis of CVDs or any other pathology for which biomarkers have been identified at different stages of the disease.

Pre-analytical considerations in biomarker research: focus on cardiovascular disease.

Clinical biomarker research is growing at a fast pace, particularly in the cardiovascular field, due to the demanding requirement to provide personalized precision medicine. The lack of a distinct molecular signature for each cardiovascular derangement results in a one-size-fits-all diagnostic and therapeutic approach, which may partially explain suboptimal outcomes in heterogeneous cardiovascular diseases (e.g., heart failure with preserved ejection fraction). A multidimensional approach using different biomarkers is quickly evolving, but it is necessary to consider pre-analytical variables, those to which a biological sample is subject before being analyzed, namely sample collection, handling, processing, and storage. Pre-analytical errors can induce systematic bias and imprecision, which may compromise research results, and are easy to avoid with an adequate study design. Academic clinicians and investigators must be aware of the basic considerations for biospecimen management and essential pre-analytical recommendations as lynchpin for biological material to provide efficient and valid data.

Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator.

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).

Predictive biomarkers for death and rehospitalization in comorbid frail elderly heart failure patients.

BACKGROUND: Heart failure (HF) is associated with a high rate of readmissions within 30 days post-discharge and in the following year, especially in frail elderly patients. Biomarker data are scarce in this high-risk population. This study assessed the value of early post-discharge circulating levels of ST2, NT-proBNP, CA125, and hs-TnI for predicting 30-day and 1-year outcomes in comorbid frail elderly patients with HF with mainly preserved ejection fraction (HFpEF). METHODS: Blood samples were obtained at the first visit shortly after discharge (4.9 ± 2 days). The primary endpoint was the composite of all-cause mortality or HF-related rehospitalization at 30 days and at 1 year. All-cause mortality alone at one year was also a major endpoint. HF-related rehospitalizations alone were secondary end-points. RESULTS: From February 2014 to November 2016, 522 consecutive patients attending the STOP-HF Clinic were included (57.1% women, age 82 ± 8.7 years, mean Barthel index 70 ± 25, mean Charlson comorbidity index 5.6 ± 2.2). The composite endpoint occurred in 8.6% patients at 30 days and in 38.5% at 1 year. In multivariable analysis, ST2 [hazard ratio (HR) 1.53; 95% CI 1.19-1.97; p = 0.001] was the only predictive biomarker at 30 days; at 1 year, both ST2 (HR 1.34; 95% CI 1.15-1.56; p < 0.001) and NT-proBNP (HR 1.19; 95% CI 1.02-1.40; p = 0.03) remained significant. The addition of ST2 and NT-proBNP into a clinical predictive model increased the AUC from 0.70 to 0.75 at 30 days (p = 0.02) and from 0.71 to 0.74 at 1 year (p < 0.05). For all-cause death at 1 year, ST2 (HR 1.50; 95% CI 1.26-1.80; p < 0.001), and CA125 (HR 1.41; 95% CI 1.21-1.63; p < 0.001) remained independent predictors in multivariable analysis. The addition of ST2 and CA125 into a clinical predictive model increased the AUC from 0.74 to 0.78 (p = 0.03). For HF-related hospitalizations, ST2 was the only predictive biomarker in multivariable analyses, both at 30 days and at 1 year. CONCLUSIONS: In a comorbid frail elderly population with HFpEF, ST2 outperformed NT-proBNP for predicting the risk of all-cause mortality or HF-related rehospitalization. ST2, a surrogate marker of inflammation and fibrosis, may be a better predictive marker in high-risk HFpEF.

Impact of diabetes on the predictive value of heart failure biomarkers.

BACKGROUND: Patients with diabetes mellitus (DM) have an increased risk of developing heart failure (HF). Further, DM is associated with poor prognosis in patients with HF. Our aim was to determine whether DM has any impact on the predictive value of a multi-biomarker panel in patients with HF. METHODS: We included 1069 consecutive ambulatory HF patients in the study: age 66.2 ± 12.8 years, 33.5 ± 13.3 left ventricular ejection fraction, 36% diabetic patients. We measured serum concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP), high-sensitivity troponin T (hs-TnT), ST2, galectin-3, high-sensitivity C reactive protein (hs-CRP), cystatin-C, soluble transferrin receptor (sTfR), and neprilysin and followed patients for 4.9 ± 2.8 years. Primary endpoints were all-cause and cardiovascular death. RESULTS: During follow-up, 534 patients died; 283 died of cardiovascular causes. Diabetic subjects had higher mortality (57.7 vs. 45.6%, p < 0.001). NTproBNP (p = 0.07), hs-TnT (p < 0.001), galectin-3 (p < 0.001), and cystatin-C (p = 0.001) concentrations were higher in diabetic patients, whereas sTfR levels were lower (p = 0.005). There were no interactions between DM and NTproBNP, hs-TnT, galectin-3, hs-CRP, cystatin-C, sTfR, and neprilysin relative to risk prediction for all-cause or cardiovascular death. By contrast, ST2 significantly interacted with DM for all-cause (p = 0.02) and cardiovascular (p = 0.03) death. In diabetic patients, HRs for ST2 were 1.27 (95% CI 1.16-1.40, p < 0.001) and 1.23 (95% CI 1.09-1.39, p = 0.001) for all-cause and cardiovascular death, respectively. In nondiabetic patients, HRs for ST2 were 1.53 (95% CI 1.35-1.73, p < 0.001) and 1.64 (95% CI 1.31-2.05, p < 0.001) for all-cause and cardiovascular death, respectively. The multivariable Cox regression analysis showed that hs-TnT and ST2 were the only markers that were independently associated with both all-cause and cardiovascular mortality in patients with HF and diabetes. Moreover, in these patients, the combination of these two markers significantly increased discrimination as assessed by the area under the curve. CONCLUSIONS: Biomarkers used in the general population to predict the clinical course of heart failure are also useful in patients with diabetes. In these patients, among all the biomarkers analysed only hs-TnT and ST2 were independently associated with both all-cause and cardiovascular mortality.

Lack of transferability between two automated immunoassays for serum IGF-I measurement.

BACKGROUND: IGF-I is a clinically relevant protein in the diagnosis and monitoring of treatment of growth disor- ders. The Growth Hormone Research Society and the International IGF Research Society have encouraged the adoption of a universal calibration for immunoassays to improve standardization of IGF-I measurements, but currently commercial assays are calibrated either against the old WHO IRR 87/518 or the new WHO 02/254. We compared two IGF-I immunochemiluminescent assays: IMMULITE® 2000 (Siemens) and LIAISON® (DiaSorin), which differ in their standardization, and verified their precision according to quality specifications based on biological variation and their linear range. METHODS: 62 patient serum samples were analyzed for both assays and compared according to standards of the Clinical and Laboratory Standards Institute (CLSI), EP9-A2-IR. Precision was verified according to CLSI EP15- A2. Optimal coefficient of variation (CVo) and desirable coefficient of variation (CVd) for IGF-I assays were calculated as quality specifications based on the biological variability, in order to assess if the interassay analytical CV (CVa1) in the two methods were appropriate. Two dilution series using the 1st WHO International Standard (WHO IS) for IGF-I 02/254 were used to verify and compare the linearity range. RESULTS: The regression analysis showed constant and proportional differences for serum samples (slope b = 0.8115 (CI 95% CI; 0.7575-0.8556); intercept a = 33.6873 (95% CI: 23.3613-44.0133) between assays and similar pro- portional differences for WHO IS 02/254 standard dilutions series (slope b = 0.8024 (CI 95% CI; 0.7560-0.8616); intercept a = 6.9623 (95% CI: -2.0819-18.4383) between assays. Within-laboratory coefficients of variation for low and high levels were 2.82% and 3.80% for IMMULITE® 2000 and 3.58% and 2.14% for LIAISON®, respecttively. CONCLUSIONS: IGF-I concentrations measured by both assays are not transferable. The results emphasize the need to express IGF-I concentrations in standard deviation score (SDS) according to a matched normal population of the same age and gender. Within-laboratory precision in both methods met quality specifications derived from biological variation.

Chronoamperometric magneto immunosensor for myeloperoxidase detection in human plasma based on a magnetic switch produced by 3D laser sintering.

In this work, an amperometric immunosensor for detection of myeloperoxidase (MPO) in human plasma is reported. Detection is based on the immobilization of anti-MPO antibodies onto magnetic beads (MBs). Following MPO immunocapture and washing steps, MBs are transferred to a customized modular detector device produced by 3D laser sintering. This tool integrates electrodes, electrical connectors, and a novel magnetic switch, whose functioning is founded on the vertical displacement of a permanent magnet. In this way, magnetic switching makes possible the confinement of MBs over the working electrode for electrochemical detection, followed by the release of MBs for electrode washing and reutilization. Notably, electrochemical detection is based on the endogenous MPO activity, which reduces reagent consumption and assay time compared to sandwich assays using enzyme-labeled antibodies. After optimization, the assay could be completed in 45 min and displayed a linear response between 0.9 and 60 ng mL(-1) for MPO and a limit of detection of 0.4 ng mL(-1). The real applicability of this approach is demonstrated by the ability to carry out the successful analysis of MPO in human plasma samples. Furthermore, the results allowed the classification of patients into three groups at risk of suffering cardiac events (i.e., low, medium, or high) and correlated well with data provided by a commercially available standardized method.

Biological signatures of asymptomatic extra- and intracranial atherosclerosis: the Barcelona-AsIA (Asymptomatic Intracranial Atherosclerosis) study.

BACKGROUND AND PURPOSE: Intracranial atherosclerotic disease (ICAD) remains a challenge for stroke primary and secondary prevention. Molecular pathways involved in the development of ICAD from its asymptomatic stages are largely unknown. In our population-based study, we aimed to compare the risk factor and biomarker profiles associated with intracranial and extracranial asymptomatic cerebral atherosclerosis. METHODS: The Asymptomatic Intracranial Atherosclerosis (AsIA) study cohort includes a random sample population of 933 white subjects >50 years with a moderate to high vascular risk (based on REGICOR score) and without a history of stroke (64% males; mean age, 66 years). Carotid and intracranial atherosclerosis were screened by cervical and transcranial color-coded Duplex ultrasound, being moderate to severe stenoses confirmed by MR angiography. We registered clinical and anthropometric data and created a biobank with blood samples at baseline. A panel of biomarkers involved in atherothrombogenesis was determined: C-reactive protein, asymmetric-dimethylarginine, resistin, and plasminogen activator inhibitor-1. Insulin resistance was quantified by Homeostasis Model Assessment index. RESULTS: After multinomial regression analyses, male sex, hypertension, smoking, and alcoholic habits were independent risk factors of isolated extracranial atherosclerotic disease. Diabetes and metabolic syndrome conferred a higher risk for ICAD than for extracranial atherosclerotic disease. Moreover, metabolic syndrome and insulin resistance were independent risk factors of moderate to severe ICAD but were not risk factors of moderate to severe extracranial atherosclerotic disease. Regarding biomarkers, asymmetric-dimethylarginine was independently associated with isolated ICAD and resistin with combined ICAD-extracranial atherosclerotic disease. CONCLUSIONS: Our findings show distinct clinical and biological profiles in subclinical ICAD and extracranial atherosclerotic disease. Insulin resistance emerged as an important molecular pathway involved in the development of ICAD from its asymptomatic stage.

Follow up of the Humoral Response in Healthcare Workers after the Administration of Two Dose of the Anti SARS-CoV-2 Vaccines-Effectiveness in Delta Variant Breakthrough Infections.

The implementation of vaccination among healthcare workers (HCWs) allowed the management of the pandemic in a manner that differed from that in the first waves. It has been demonstrated that the mRNA vaccines elicit good humoral responses but that there are still breakthrough infections. In summer 2021, a fifth wave emerged, despite the good coverage of HCWs in Spain. We aimed to study the SARS-CoV-2 IgG antibody levels as a marker to predict the possibility of Delta variant infections after vaccination after a seroepidemiological campaign. Of the 5000 participants, a total of 4902 (98.04%) showed a positive result in the serological anti-S test and only 98 (1.96%) were negative. Among the 4368 fully vaccinated participants, only in five cases was the serology negative. Of the total number of participants that received antibody results during the study, 162 were PCR positive in the subsequent two months. Among these, 151 were fully vaccinated (two doses). Significant differences between antibody BAU/mL levels were found between PCR positive and non-PCR positive participants (p < 0.01). The median of BAU/mL was higher in those vaccinated patients with no infection (1260 BAU/mL; 465−2080) versus infected patients (661 BAU/mL; 361−2080). These data support the idea that vaccines play an important role in the control of the pandemic, especially among HCWs at the time of the Delta variant circulation. More studies with other variants of concern must be performed in order to establish a correlation between the levels of IgG and the new infections.

Finding a reliable assay for soluble neprilysin.

BACKGROUND: Lack of validation and standardization of research-use-only (RUO) immunoassays brings with it inherent threats to authenticity and functional quality. Poor correlation between different commercial neprilysin RUO immunoassays is concerning and discordant findings need to be resolved. We seek to identify and validate reliable neprilysin immunoassays to strengthen the scientific rigor and reproducibility of neprilysin-related investigation and of biomarker research in general. METHODS: Soluble neprilysin (sNEP) concentrations were determined in cohorts (n = 532) from Spain (Cohort 1), New Zealand (NZ, Cohort 2) and Singapore (Cohort 3), using commercial kits from six vendors. Apparent sNEP concentrations were correlated between different assays and with plasma neprilysin activity. Assay reliability was further validated by performance verification, MS analysis and cross-reactivity tests. RESULTS: sNEP in Cohorts 1 and 2 measured concurrently in Spain and NZ showed significant inter-laboratory correlation only for the Aviscera Bioscience sNEP ELISA SK00724-01. Neprilysin concentrations obtained with the R&D systems and SK00724-01 ELISAs correlated with each other but not with neprilysin activity. In Cohort 3, sNEP concentrations from the Perkin Elmer AlphaLISA and Biotechne ELLA assays agreed (r = 0.89) and both correlated with neprilysin activity (r = 0.87, 0.77 respectively). MS analysis detected authentic neprilysin in the AlphaLISA kit calibrator and in antibody pull-down material from human plasma. The AlphaLISA assay performed within acceptable limits (spike and recovery, dilutional linearity, inter- and intra-assay CV) and showed no cross-reactivity against neprilysin substrates and closely-related analogues. CONCLUSION: AlphaLISA and ELLA assays provide reliable measures of sNEP concentrations. Reliability of other commercial neprilysin assays remains in question.

Low Levels of Few Micronutrients May Impact COVID-19 Disease Progression: An Observational Study on the First Wave.

We report an observational study performed between March and May 2020 in a Spanish university hospital during the SARS-CoV-2 pandemic. The main objective was to analyse the association between the levels of micronutrients in severe COVID-19 patients and their outcome. Adult patients with a positive polymerase-chain-reaction (PCR) for SARS-CoV-2 in the nasopharyngeal swab or in tracheal aspirate culture in the case of intubation were included. Micronutrient data were obtained from plasma analysis of a standard nutritional assessment performed within the first 24 h of hospital admission. Vitamins A, B6, C and E were analysed with HPLC methods; 25-OH-vitamin D by immunoassay and zinc by colorimetric measurements. One hundred and twenty patients were included. We found that 74.2% patients had low levels of zinc (normal levels >84 µg/dL) with a mean value of 63.5 (SD 13.5); 71.7% patients had low levels of vitamin A (normal levels >0.3 mg/L) with a mean value of 0.17 (SD 0.06); 42.5% patients had low levels of vitamin B6 (normal levels >3.6 ng/mL) with a mean value of 2.2 (SD 0.9); 100% patients had low levels of vitamin C (normal levels >0.4 mg/dL) with a mean value of 0.14 (SD 0.05); 74.3% patients had low values of vitamin D (normal levels >20 ng/mL) with mean value of 11.4 (SD 4.3); but only 5.8% of patients had low levels of vitamin E (normal levels >5 mg/L) with a mean value of 3.95 (SD 0.87). The variables associated with the need for ICU admission were low levels of zinc (standard error 0.566, 95% CI 0.086 to 0.790, p = 0.017), low levels of vitamin A (standard error 0.582, 95% CI 0.061 to 0.594, p = 0.004), age over 65 (standard error 0.018, 95% CI 0.917 to 0.985, p = 0.005) and male gender (standard error 0.458, 95% CI 1.004 to 6.040, p = 0.049). The only variable that was independently associated with the need for orotracheal intubation was low levels of vitamin A (standard error 0.58, 95% CI 0.042 to 0.405, p = 0.000). Conclusions: Low levels of vitamin A and zinc are associated with a greater need for admission to the ICU and orotracheal intubation. Patients older than 65 years had higher mortality. Randomized clinical trials are needed to examine whether micronutrient supplementation could be beneficial as an adjunctive treatment in COVID-19.

Analytical Performance of Quantitative DiaSorin Liaison SARS-COV-2 Antigen Test for the Asymptomatic Population.

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigen (Ag) tests have been widely employed to identify patients for a rapid diagnosis and pandemic control. Rapid lateral-flow techniques are currently the most used, but automated technologies have emerged as another viable alternative to molecular methods. We aimed to evaluate the analytical performance of the DiaSorin Liaison SARS-CoV-2 Ag test in asymptomatic population and close contacts, for its use as a tool in pandemic control efforts. Material and Methods: A retrospective study was conducted. A total of 861 samples were included, 291 (34%) were positive for SARS-CoV-2 with cycle threshold (Ct) <40, and 570 (66%) were negative. Results: A strong correlation was observed between reverse transcriptase-PCR (RT-PCR) Ct and Ag 50% Tissue Culture Infectious Dose per milliliter (TCID50/ml; r = 0.6486; p < 0.0001) and all RT-PCR negative samples tested negative for the 200 TCID50/ml SARS-Cov-2 Ag cutoff, i.e., a specificity of 100% was reached (95% CI: 99.4-100.0%). Samples with <25 Ct and/or >106 extrapolated copies/ml were reached a sensitivity of 100% (95% IC 97.0-100.0%). For intermediate viral loads (>105 extrapolated copies/ml or <30 Ct), the sensitivity value still exceeded 80%. As with other Ag methods, samples between 30 and 40 Ct could not be detected with a reliable sensitivity. Conclusions: The LIAISON® SARS-CoV-2 Ag assay displays an acceptable sensitivity and a very high specificity that is useful for detecting the presence of SARS-CoV-2 in nasal swabs (NPS) of asymptomatic population or to regular monitoring of risk groups in controlled settings. Additionally, the flexibility in processing different samples and in the sampling preparation process makes this test an option for its use in high throughput laboratories. Automated tests may facilitate result reporting and yield consistent data, while avoiding some of the pitfalls of rapid lateral-flow techniques, such as observer variability.

The influence of sex and body mass index on the association between soluble neprilysin and risk of heart failure hospitalizations.

A higher neprilysin activity has been suggested in women. In this retrospective analysis, we evaluated the association of sex and body mass index (BMI) with soluble neprilysin (sNEP) and recurrent admissions among 1021 consecutive HF outpatients. The primary and secondary endpoints were the number of HF hospitalizations and all-cause mortality, respectively. The association between sNEP with either endpoint was evaluated across sex and BMI categories (≥ 25 kg/m2 vs. < 25 kg/m2). Bivariate count regression (Poisson) was used, and risk estimates were expressed as incidence rates ratio (IRR). During a median follow-up of 6.65 years (percentile 25%-percentile 75%:2.83-10.25), 702 (68.76%) patients died, and 406 (40%) had at least 1 HF hospitalization. Median values of sNEP and BMI were 0.64 ng/mL (0.39-1.22), and 26.9 kg/m2 (24.3-30.4), respectively. Left ventricle ejection fraction was < 40% in 78.9% of patients, and 28% were women. In multivariable analysis, sNEP (main effect) was positively associated with HF hospitalizations (p = 0.001) but not with mortality (p = 0.241). The predictive value of sNEP for HF hospitalizations varied non-linearly across sex and BMI categories (p-value for interaction = 0.003), with significant and positive effect only on women with BMI ≥ 25 kg/m2 (p = 0.039). For instance, compared to men, women with sNEP of 1.22 ng/mL (percentile 75%) showed a significantly increased risk (IRRs: 1.26; 95% CI: 1.05-1.53). The interaction analysis for mortality did not support a differential prognostic effect for sNEP (p = 0.072). In conclusion, higher sNEP levels in overweight women better predicted an increased risk of HF hospitalization.

Elevated Neopterin Levels Predict Fatal Outcome in SARS-CoV-2-Infected Patients.

Highlights: Innate immune activation during Covid-19 infection is associated with pernicious clinical outcome. Background: Coronavirus disease 2019 (Covid-19) is a worldwide threat that has already caused more than 3 000 000 deaths. It is characterized by different patterns of disease evolution depending on host factors among which old-age and pre-existing comorbidities play a detrimental role. Previous coronavirus epidemics, notably SARS-CoV, were associated with increased serum neopterin levels, which can be interpreted as a sign of acute innate immunity in response to viral infection. Here we hypothesize that neopterin may serve as a biomarker of SARS-CoV-2 viral infection and Covid-19 disease severity. Methods: We measured neopterin blood levels by ELISA. Seric concentration was quantified from 256 healthy donors and 374 Covid-19 patients at hospital admission. Enrolled Covid-19 patients were all symptomatic and displayed a large spectrum of comorbidities. Patients were followed until disease resolution or death. Results: Severe and critically ill SARS-CoV-2 infected patients were characterized by a profound exacerbation of immune activation characterized by elevated neopterin blood levels. Systemic neopterin levels above 19nM stratified healthy individuals from Covid-19 patients with 87% specificity and 100% sensitivity. Moreover, systemic neopterin levels above 53nM differentiated non-survivors from survivors with 64% specificity and 100% sensitivity. Conclusion: We propose that neopterin concentration measured at arrival to hospital is a hallmark of severe Covid-19 and identifies a high-risk population of pernicious clinical outcome with a need for special medical care.

[Prediction of bacteremia in patients with suspicion of infection in emergency room]. / Predicción de bacteriemia en los pacientes con sospecha de infección en urgencias.

BACKGROUND AND OBJECTIVES: To evaluate the relationship between some clinical and analytical data and the presence of bacteremia in order to establish a clinical decision rule. PATIENTS AND METHODS: All the patients with blood cultures obtained from the emergency room in a two months period were analyzed. Patients were randomly assigned to derivation or validation sets. A logistic regression of the significant values in the univariate analysis was performed and a score obtained. The prevalence of bacteraemia for every score was calculated. The diagnostic efficacy curves and the performance of the predictive model were calculated. RESULTS: 412 patients were enrolled. The blood cultures were positive in 12.8% of them. The significant values in the univariate analysis were Charlson index ≥2 and PCT > 0.4ng/ml. Four groups of increasing risk of bacteraemia were designed, from 0 to 35% in the derivation set and from 2.9% to 27.2% in the validation set. In the diagnostic efficacy curve, the AUC was 0.8 in the derivation set and 0.74 in the validation set. The model presented a negative predictive value of 95.2% in the derivation set and 95.3% in the validation set. CONCLUSIONS: A model that includes Charlson index and PCT makes possible to define a group of patients with a very low risk of bacteremia.

Seroprevalence of SARS-CoV-2 IgG specific antibodies among healthcare workers in the Northern Metropolitan Area of Barcelona, Spain, after the first pandemic wave.

BACKGROUND: The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) around the world has caused a global pandemic, infecting millions of individuals, with an unprecedented impact in health care systems worldwide. Healthcare workers are one of the risk groups that need to be well protected, due to their strategic role in patient management, presently and in prevention of healthcare needs for future outbreaks. Here, we present the results of the first SARS-CoV-2 seroprevalence study in the Northern Metropolitan Area of Barcelona, Spain. METHODS: IgG SARS-CoV-2 antibodies were analyzed in serum samples from 7563 healthcare workers of the Northern Metropolitan Area of Barcelona. Samples were collected after the first pandemic wave (from May 4th to May 22nd, 2020) and were analyzed by automated chemiluminescence assays. All samples were tested for IgG anti-S1/S2. Participant samples with negative or equivocal results but with analytical signals above the limit of detection and/or previously confirmed COVID-19 diagnosis were also tested for IgG anti-Nucleocapsid. RESULTS: A total of 779 of 7563 (10.3%) healthcare workers were positive for anti-SARS-CoV-2 IgG (specific for either S1/S2 or N antigens). No significant differences were observed between those working at primary care or at the reference hospital. Interestingly, among 341 participants with a confirmed COVID-19 diagnosis, 36 (10.55%) tested negative for SARS-CoV-2 IgG (both S1/S2 and recombinant N antigen). CONCLUSION: Seroprevalence of anti-SARS-CoV-2 IgG in the healthcare workers of the North Metropolitan Area of Barcelona was higher than in the general population in the same geographical area. Safety measures have to be stressed in order to protect these essential workers from future pandemic waves.

Clinical Role of CA125 in Worsening Heart Failure: A BIOSTAT-CHF Study Subanalysis.

OBJECTIVES: The aim of this study was to evaluate the association between antigen carbohydrate 125 (CA125) and the risk of 1-year clinical outcomes in patients with worsening heart failure (HF). BACKGROUND: CA125 is a widely available biomarker that is up-regulated in patients with acute HF and has been postulated as a useful marker of congestion and risk stratification. METHODS: In a large multicenter cohort of patients with worsening HF, either in-hospital or in the outpatient setting, the independent associations between CA125 and 1-year death and the composite of death/HF readmission (adjusted for outcome-specific prognostic risk score [BIOSTAT risk score]) were determined by using the Royston-Parmar method (N = 2,356). In a sensitivity analysis, the prognostic implications of CA125 were also adjusted for a composite congestion score (CCS). Data were validated in the BIOSTAT-CHF (Biology Study to Tailored Treatment in Chronic Heart Failure validation) cohort (N = 1,630). RESULTS: Surrogates of congestion, such as N-terminal pro-B-type natriuretic peptide and CCS, emerged as independent predictors of CA125. In multivariable survival analyses, higher CA125 was associated with an increased risk of mortality and the composite of death/HF readmission (p < 0.001 for both comparisons), even after adjustment for the CCS (p < 0.010 for both comparisons). The addition of CA125 to the BIOSTAT score led to a significant risk reclassification for both outcomes (category-free net reclassification improvement = 0.137 [p < 0.001] and 0.104 [p = 0.003] respectively). All outcomes were confirmed in an independent validation cohort. CONCLUSIONS: In patients with worsening HF, higher levels of CA125 were positively associated with parameters of congestion. Furthermore, CA125 remained independently associated with a higher risk of clinical outcomes, even beyond a predefined risk model and clinical surrogates of congestion.