Exploring Urinary Extracellular Vesicles and Immune Mediators as Biomarkers of Kidney Injury in COVID-19 Hospitalized Patients.

Kidney injury is an important outcome associated with COVID-19 severity. In this regard, alterations in urinary extracellular vesicles (uEVs) could be detected in the early phases of renal injury and may be reflective of the inflammatory process. This is an observational study performed with a case series of COVID-19 hospitalized patients presenting mild-to-critical disease. Total and podocyte-derived uEVs were identified by nanoscale flow cytometry, and urinary immune mediators were assessed by a multiplex assay. We studied 36 patients, where 24 (66.7%) were considered as mild/moderate and 12 (33.3%) as severe/critical. Increased levels of total uEVs were observed (p = 0.0001). Importantly, total uEVs were significantly higher in severe/critical patients who underwent hemodialysis (p = 0.03) and were able to predict this clinical outcome (AUC 0.93, p = 0.02). Severe/critical patients also presented elevated urinary levels (p < 0.05) of IL-1ß, IL-4, IL-6, IL-7, IL-16, IL-17A, LIF, CCL-2, CCL-3, CCL-11, CXCL-10, FGFb, M-CSF, and CTAcK. Lastly, we observed that total uEVs were associated with urinary immune mediators. In conclusion, our results show that early alterations in urinary EVs could identify patients at higher risk of developing renal dysfunction in COVID-19. This could also be relevant in different scenarios of systemic and/or infectious disease.

Cathepsin B and Plasma Kallikrein Are Reliable Biomarkers to Discriminate Clinically Significant Hepatic Fibrosis in Patients with Chronic Hepatitis-C Infection.

We aimed to determine the biomarker performance of the proteolytic enzymes cathepsin B (Cat B) and plasma kallikrein (PKa) and transforming growth factor (TGF)-ß to detect hepatic fibrosis (HF) in chronic hepatitis C (CHC) patients. We studied 53 CHC patients and 71 healthy controls (HCs). Hepatic-disease stage was determined by liver biopsies, aminotransferase:platelet ratio index (APRI) and Fibrosis (FIB)4. Hepatic inflammation and HF in CHC patients were stratified using the METAVIR scoring system. Cat-B and PKa activities were monitored fluorometrically. Serum levels of TGF-ß (total and its active form) were determined using ELISA-like fluorometric methods. Increased serum levels of Cat B and PKa were found (p < 0.0001) in CHC patients with clinically significant HF and hepatic inflammation compared with HCs. Levels of total TGF-ß (p < 0.0001) and active TGF-ß (p < 0.001) were increased in CHC patients compared with HCs. Cat-B levels correlated strongly with PKa levels (r = 0.903, p < 0.0001) in CHC patients but did not correlate in HCs. Levels of Cat B, PKa and active TGF-ß increased with the METAVIR stage of HF. Based on analyses of receiver operating characteristic (ROC) curves, Cat B and PKa showed high diagnostic accuracy (area under ROC = 0.99 ± 0.02 and 0.991 ± 0.007, respectively) for distinguishing HF in CHC patients from HCs. Taken together, Cat B and PKa could be used as circulating biomarkers to detect HF in HCV-infected patients.

Worsening Renal Function and Congestion in Patients with Acute Heart Failure: A Study with Bioelectrical Impedance Vector Analysis (BIVA) and Neutrophil Gelatinase-Associated Lipocalin (NGAL). / Agravamento da Função Renal e Congestão em Pacientes com Insuficiência Cardíaca Aguda: Estudo com Análise Vetorial de Bioimpedância Elétrica (BIVA) e Lipocalina Associada à Gelatinase Neutrofílica (NGAL).

BACKGROUND: Worsening renal function (WRF) is frequently observed in the setting of aggressive diuresis for the treatment of acute decompensated heart failure (ADHF) and is associated with poor outcomes in some studies. OBJECTIVE: We sought to assess the relationship of WRF and congestion at discharge with events (cardiac death or heart failure hospitalization). METHODS: Eighty patients with ADHF were studied. WRF was defined by an absolute increase in serum creatinine of ≥0.5 mg/dL from the values measured at the time of admission. B-type natriuretic peptide (BNP) and plasma neutrophil gelatinase-associated lipocalin (NGAL) were measured at admission and at discharge. Congestive state at discharge was assessed using bioelectrical impedance vector analysis (BIVA). Primary endpoint was time to first event defined as a combination of cardiac death or heart failure hospitalization. Receiver operating characteristic (ROC) curve analysis was used to determine the best hydration index cutoff to predict events. Kaplan-Meier event-free survival curves were constructed and compared using the log-rank test. Cox proportional hazards models were used to investigate the association with events. The criterion for determining statistical significance was p<0.05. RESULTS: Mean age was 60.6±15 years, and 48 (60%) were male. Mean ejection fraction was 35.3±7.8%. WRF occurred in 37.5% of the sample. Baseline creatinine was associated with WRF (p<0.001), but neither admission BNP (p=0.35) nor admission NGAL (p=0.18) was predictor of WRF. Using Cox proportional hazard models, hydration index at discharge calculated with BIVA was significantly associated with events (HR 1.39, 95% CI 1.25-1.54, p<0.0001) but not WRF (HR 2.14, 95% CI 0.62-7.35, p=0.22). CONCLUSION: Persistent congestion at discharge was associated with worse outcomes. WRF seems to be related to hemodynamic changes during the decongestion process but not to kidney tubular injuries.

FUNDAMENTO: O agravamento da função renal (AFR) é frequentemente observado na terapia agressiva com diuréticos para o tratamento de insuficiência cardíaca aguda descompensada (ICAD) e está associado com piores desfechos em alguns estudos. OBJETIVO: Avaliar a relação de AFR e congestão na alta hospitalar com ocorrência de eventos (morte cardíaca ou internação por insuficiência cardíaca). MÉTODOS: Oitenta pacientes com ICAD foram estudados. O AFR foi definido por um aumento absoluto (≥0,5 mg/dL) nos níveis séricos de creatinina a partir dos valores obtidos na admissão. Concentrações de peptídeo natriurético do tipo B (BNP) e lipocalina associada à gelatinase neutrofílica (NGAL) foram medidas na admissão e na alta hospitalar. Congestão foi avaliada na alta utilizando a análise vetorial de bioimpedância elétrica (BIVA). O desfecho primário foi o tempo para o primeiro evento, definido como uma combinação de morte cardíaca ou hospitalização por insuficiência cardíaca. Análise de curva Característica de Operação do Receptor (curva ROC) foi realizada para determinar o ponto de corte de IH mais adequado para predição de eventos. Curvas Kaplan-Meier de sobrevida livre de eventos foram construídas e comparadas usando o teste de log-rank. Modelos de riscos proporcionais de Cox foram usados para investigar a associação com eventos. O critério para se estabelecer significância estatística foi um p<0.05. RESULTADOS: A idade média foi 60,6 ± 15,0 anos, e 48 (60%) pacientes eram do sexo masculino. A fração de ejeção média foi 35,3±7,8%. O AFR ocorreu em 37,5% da amostra. A creatinina basal associou-se com AFR (p<0,001), mas nem BNP (p=0,35) nem NGAL (p=0,18) na admissão foram preditores de AFR. Usando modelos de riscos proporcionais de Cox, o índice de hidratação na alta, estimado por BIVA, associou-se significativamente com ocorrência de eventos (HR 1,39; IC95% 1,25-1,54, p<0,0001), mas não com AFR (HR 2,14; IC95% 0,62-7,35, p=0,22). CONCLUSÃO: A congestão persistente na alta associou-se com piores desfechos. O AFR parece estar relacionado com alterações hemodinâmicas durante o processo de descongestionamento, mas não com lesões renais.

Agravamento da Função Renal e Congestão em Pacientes com Insuficiência Cardíaca Aguda: Estudo com Análise Vetorial de Bioimpedância Elétrica (BIVA) e Lipocalina Associada à Gelatinase Neutrofílica (NGAL) / Worsening Renal Function and Congestion in Patients with Acute Heart Failure: A Study with Bioelectrical Impedance Vector Analysis (BIVA) and Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Resumo Fundamento: O agravamento da função renal (AFR) é frequentemente observado na terapia agressiva com diuréticos para o tratamento de insuficiência cardíaca aguda descompensada (ICAD) e está associado com piores desfechos em alguns estudos. Objetivo: Avaliar a relação de AFR e congestão na alta hospitalar com ocorrência de eventos (morte cardíaca ou internação por insuficiência cardíaca). Métodos: Oitenta pacientes com ICAD foram estudados. O AFR foi definido por um aumento absoluto (≥0,5 mg/dL) nos níveis séricos de creatinina a partir dos valores obtidos na admissão. Concentrações de peptídeo natriurético do tipo B (BNP) e lipocalina associada à gelatinase neutrofílica (NGAL) foram medidas na admissão e na alta hospitalar. Congestão foi avaliada na alta utilizando a análise vetorial de bioimpedância elétrica (BIVA). O desfecho primário foi o tempo para o primeiro evento, definido como uma combinação de morte cardíaca ou hospitalização por insuficiência cardíaca. Análise de curva Característica de Operação do Receptor (curva ROC) foi realizada para determinar o ponto de corte de IH mais adequado para predição de eventos. Curvas Kaplan-Meier de sobrevida livre de eventos foram construídas e comparadas usando o teste de log-rank. Modelos de riscos proporcionais de Cox foram usados para investigar a associação com eventos. O critério para se estabelecer significância estatística foi um p<0.05. Resultados: A idade média foi 60,6 ± 15,0 anos, e 48 (60%) pacientes eram do sexo masculino. A fração de ejeção média foi 35,3±7,8%. O AFR ocorreu em 37,5% da amostra. A creatinina basal associou-se com AFR (p<0,001), mas nem BNP (p=0,35) nem NGAL (p=0,18) na admissão foram preditores de AFR. Usando modelos de riscos proporcionais de Cox, o índice de hidratação na alta, estimado por BIVA, associou-se significativamente com ocorrência de eventos (HR 1,39; IC95% 1,25-1,54, p<0,0001), mas não com AFR (HR 2,14; IC95% 0,62-7,35, p=0,22). Conclusão: A congestão persistente na alta associou-se com piores desfechos. O AFR parece estar relacionado com alterações hemodinâmicas durante o processo de descongestionamento, mas não com lesões renais.

Abstract Background: Worsening renal function (WRF) is frequently observed in the setting of aggressive diuresis for the treatment of acute decompensated heart failure (ADHF) and is associated with poor outcomes in some studies. Objective: We sought to assess the relationship of WRF and congestion at discharge with events (cardiac death or heart failure hospitalization). Methods: Eighty patients with ADHF were studied. WRF was defined by an absolute increase in serum creatinine of ≥0.5 mg/dL from the values measured at the time of admission. B-type natriuretic peptide (BNP) and plasma neutrophil gelatinase-associated lipocalin (NGAL) were measured at admission and at discharge. Congestive state at discharge was assessed using bioelectrical impedance vector analysis (BIVA). Primary endpoint was time to first event defined as a combination of cardiac death or heart failure hospitalization. Receiver operating characteristic (ROC) curve analysis was used to determine the best hydration index cutoff to predict events. Kaplan-Meier event-free survival curves were constructed and compared using the log-rank test. Cox proportional hazards models were used to investigate the association with events. The criterion for determining statistical significance was p<0.05. Results: Mean age was 60.6±15 years, and 48 (60%) were male. Mean ejection fraction was 35.3±7.8%. WRF occurred in 37.5% of the sample. Baseline creatinine was associated with WRF (p<0.001), but neither admission BNP (p=0.35) nor admission NGAL (p=0.18) was predictor of WRF. Using Cox proportional hazard models, hydration index at discharge calculated with BIVA was significantly associated with events (HR 1.39, 95% CI 1.25-1.54, p<0.0001) but not WRF (HR 2.14, 95% CI 0.62-7.35, p=0.22). Conclusion: Persistent congestion at discharge was associated with worse outcomes. WRF seems to be related to hemodynamic changes during the decongestion process but not to kidney tubular injuries.

Successful DAA therapy for chronic hepatitis C reduces HLA-DR on monocytes and circulating immune mediators: A long-term follow-up study.

INTRODUCTION: After DAA treatment for chronic hepatitis C infection, peripheral monocyte subsets from patients who achieved sustained virological response (SVR) reduced compared to healthy control. Improvement in inflammatory parameters and liver stiffness has been observed. However, little is known about the long-term impact of DAA treatment on peripheral monocyte subsets and immune mediators levels. OBJECTIVES: We aimed to examine peripheral monocyte subsets and immune mediators levels in Brazilian chronic HCV patients after long-term successful IFN-free SOF-based treatment. MATERIAL AND METHODS: We analyzed CD14++CD16-, CD14++CD16+ and CD14+CD16++ monocytes and 27 immune mediators by flow cytometry and analysis of multiple secreted proteins assay, respectively, in monoinfected chronic HCV patients receiving IFN-free sofosbuvir-based regimens followed before treatment, at SVR and one year after the end of treatment (1y). RESULTS: Twenty-one biomarkers decreased significantly at 1y and 55-80 % of patients this reduction at 1y. Experimented patients presented a greater modulation of immune mediators at 1y. HLA-DR expression significantly decreased on CD14++CD16- and CD14++CD16+ monocytes at 1y when compared to SVR. CONCLUSIONS: Successful DAA therapy did not modify monocyte subsets frequency but reduced monocyte activation at 1y and sustained the downregulation and restoration of circulating immune mediators, indicating that long-term reversal of inflammation status could occur after HCV eradication.

Predictors of adverse drug reactions associated with ribavirin in direct-acting antiviral therapies for chronic hepatitis C.

PURPOSE: To identify factors associated with the development of adverse drug reactions (ADR) in ribavirin therapeutic regimens. METHODS: A multicenter, prospective study was conducted in three public health hospitals in Rio de Janeiro between November 2015 and March 2018. Inclusion criteria were defined by patient follow-up at pharmaceutical consultation at the time of drug dispensing as those who used sofosbuvir in combination with simeprevir, daclatasvir, and/or ribavirin. All patients were invited to participate in the study during the first interview. Adverse drug reactions were reported according to the treatment regimen and frequency of occurrence. Statistical analysis was used to compare adverse reactions between treatments and their associated factors. RESULTS: A total of 405 patients were included in the study (mean age 59.6 ± 9.6 years); 61.0% were female, 88.1% were infected with genotype 1, and 65.4% were cirrhotic. The most prescribed treatment was the combination of sofosbuvir, daclatasvir, and ribavirin (55.3%). The majority of patients reported at least one ADR during treatment (83.2%), of which fatigue, anemia, and headache were the most common. Being female (OR = 1.86, [1.08-3.20]) and use of ribavirin (OR: 2.39; 95% CI [1.38-4.13]) were predictors for the development of ADR, which was also associated with development of anemia (OR: 10.28; 95% CI: [5.78-18.30]). Treatment efficacy was 98.1%. CONCLUSIONS: Direct-acting antiviral has been shown to be safe and effective. Therefore, use of ribavirin is questionable due to associated adverse reactions and similar efficacy to other treatments.

Effects of Heart Rate Reduction With Either Pyridostigmine or Ivabradine in Patients With Heart Failure: A Randomized, Double-Blind Study.

BACKGROUND: Heart rate (HR) reduction with ivabradine has been proved to reduce hospitalization and death from heart failure (HF). We sought to investigate whether pyridostigmine would effectively reduce HR in patients with chronic HF as compared with ivabradine. METHODS: Twenty-one patients with HF who were in sinus rhythm with a resting HR over 70 bpm, despite optimal medical treatment, were included in a randomized, double-blind study comparing pyridostigmine versus ivabradine. The initial dose of ivabradine was 5 mg twice daily to reach a target HR between 50 and 60 bpm and could be titrated to a maximum of 7.5 mg twice daily. Pyridostigmine was used in a fixed dose of 30 mg 3 times daily. RESULTS: The baseline HR for ivabradine and pyridostigmine groups was 89.1 (13.5) and 80.1 (7.2) bpm, respectively (P = .083). After 6 months of treatment, HR was significantly reduced to 64.8 (8.3) bpm in the ivabradine group (P = .0014) and 63.6 (5.9) bpm in the pyridostigmine group (P = .0001). The N-terminal pro-B-type natriuretic peptide was reduced in the ivabradine group (median: 1308.4 [interquartile range: 731-1896] vs 755.8 [134.5-1014] pg/mL; P = .027) and in the pyridostigmine group (132.8 [89.9-829] vs 100.7 [38-360] pg/mL; P = .002). Inflammatory markers interleukin-1, interleukin-6, and tumor necrosis factor were reduced in both groups. Exercise capacity was improved in both groups, with increments in volume of oxygen utilization (V˙O2; ivabradine: 13.1 vs 15.6, P = .048; pyridostigmine: 13.3 vs 16.7, P = .032). Heart rate recovery in the first minute postexercise was improved with pyridostigmine (11.8 [3.9] vs 18 [6.5]; P = .046), but not with ivabradine (13.3 [6.9] vs 14.1 [8.2]; P = .70). No differences in either group were observed in the myocardial scintigraphy with 123-iodine-metaiodobenzylguanidine. CONCLUSION: Both drugs significantly reduced HR, with improvements in exercise capacity and in neurohormonal and inflammatory profiles.

Restoring Inflammatory Mediator Balance after Sofosbuvir-Induced Viral Clearance in Patients with Chronic Hepatitis C.

This study aimed at analyzing circulating levels of inflammatory and profibrogenic cytokines in patients with hepatitis C virus (HCV) chronic infection undergoing therapy with direct-acting antiviral agents (DAA) and correlating these immune biomarkers with liver disease status. We studied 88 Brazilian monoinfected chronic hepatitis C patients receiving interferon- (IFN-) free sofosbuvir-based regimens for 12 or 24 weeks, followed-up before therapy initiation and three months after the end of treatment. Liver disease was determined by transient elastography, in addition to APRI and FIB-4 indexes. Analysis of 30 immune mediators was carried out by multiplex or enzymatic immunoassays. Sustained virological response rate was 98.9%. Serum levels of cytokines were increased in HCV-infected patients when compared to control group. CCL-2, CCL-3, CCL-4, CXCL-8, CXCL-10, IL-1ß, IL-15, IFN-γ, IL-4, IL-10, TGF-ß, FGFb, and PAI-1 decreased significantly after antiviral therapy, reaching values similar to noninfected controls. TGF-ß and suPAR levels were associated with fibrosis/cirrhosis. Also, we observed amelioration in hepatic parameters after DAA treatment. Together, our results suggest that viral control induced by IFN-free DAA therapy restores inflammatory mediators in association with improvement in liver function.

Exploring lipid and apolipoprotein levels in chronic hepatitis C patients according to their response to antiviral treatment.

BACKGROUND: Hepatitis C virus is known to be highly dependent of lipid metabolism to infect new cells and replicate. AIMS: To investigate lipid and apolipoprotein profile in chronic HCV patients according to treatment response. METHODS: Patients recruited from the Hepatitis Treatment Center at Niteroi (Brazil) who received interferon (IFN)-based therapies were separated into two groups, those who achieved sustained virological response (SVR) or not (non-SVR). Another group of patients treated with IFN-free direct-acting antiviral (DAA) therapies was followed from before starting the treatment until one year after therapy. Triglycerides, total cholesterol and fractions were determined by colorimetric and/or electrophoresis techniques. Lecithin cholesterol acyltransferase (LCAT) activity and serum levels of apolipoproteins A1, A2, B, C2, C3 and E were assessed by enzymatic and multiplex assays, respectively. RESULTS: We studied 114 patients, and SVR was reached in 28 (39.4%) patients treated with IFN-therapy and in all (100%) patients who received DAA. Non-SVR patients (n = 43) presented altered liver parameters post-treatment. Levels of total cholesterol, LDL-C, VLDL-C and triglycerides were significant higher in SVR group. In contrast, LCAT activity and HDL-C levels were elevated in non-SVR patients. Only apolipoproteins B, C2 and C3 levels were increased in SVR group. The follow-up of SVR-DAA patients (n = 43) revealed a significant and progressive increase in serum levels of total cholesterol, LDL-C, VLDL-C and triglycerides. CONCLUSIONS: After a successful treatment, chronic hepatitis C patients experienced a reestablishment of lipid metabolism. Our results suggest that the monitoring of serum lipids could be a practical and routine laboratory tool to be applied during the treatment follow-up.

Increased Cytokeratin 19 Fragment Levels Are Positively Correlated with Adenosine Deaminase Activity in Malignant Pleural Effusions from Adenocarcinomas.

Adenosine deaminase (ADA) and cytokeratin 19 (CK19) are known pleural biomarkers. Although ADA in humans functions mainly in the immune system, it also appears to be associated with the differentiation of epithelial cells. Keratin filaments are important structural stabilizers of epithelial cells and potent biomarkers in epithelial differentiation. This study aimed to investigate the simultaneous presence of the ADA enzyme and CK19 fragments to assess epithelial differentiation in malignant and benign pleural fluids. Diagnosis of the cause of pleural effusion syndrome was confirmed by means of standard examinations and appropriate surgical procedures. An ADA assay, in which ADA irreversibly catalyzes the conversion of adenosine into inosine, was performed using a commercial kit. The CK19 assay was performed using a CYFRA 21-1 kit, developed to detect quantitative soluble fragments of CK19 using an electrochemiluminescence immunoassay. One hundred nineteen pleural fluid samples were collected from untreated individuals with pleural effusion syndrome due to several causes. ADA levels only correlated with CK19 fragments in adenocarcinomas, with high significance and good correlation (rho = 0.5145, P = 0.0036). However, further studies are required to understand this strong association on epithelial differentiation in metastatic pleural fluids from adenocarcinomas.