Methylation status of LDLR, PCSK9 and LDLRAP1 is associated with cardiovascular events in familial hypercholesterolemia.

Aim: Methylation of LDLR, PCSK9 and LDLRAP1 CpG sites was assessed in patients with familial hypercholesterolemia (FH). Methods: DNA methylation of was analyzed by pyrosequencing in 131 FH patients and 23 normolipidemic (NL) subjects. Results: LDLR, PCSK9 and LDLRP1 methylation was similar between FH patients positive (MD) and negative (non-MD) for pathogenic variants in FH-related genes. LDLR and PCSK9 methylation was higher in MD and non-MD groups than NL subjects (p < 0.05). LDLR, PCSK9 and LDLRAP1 methylation profiles were associated with clinical manifestations and cardiovascular events in FH patients (p < 0.05). Conclusion: Differential methylation of LDLR, PCSK9 and LDLRAP1 is associated with hypercholesterolemia and cardiovascular events. This methylation profile maybe useful as a biomarker and contribute to the management of FH.

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Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability.

OBJECTIVES: This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. METHODS: Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. RESULTS: A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. CONCLUSION: Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.

Lipidomic analysis identified potential predictive biomarkers of statin response in subjects with Familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a disorder of lipid metabolism that causes elevated low-density lipoprotein cholesterol (LDL-c) and increased premature atherosclerosis risk. Statins inhibit endogenous cholesterol biosynthesis, which reduces LDL-c plasma levels and prevent from cardiovascular events. This study aimed to explore the effects of statin treatment on serum lipidomic profile and to identify biomarkers of response in subjects with FH. Seventeen adult FH patients underwent a 6-week washout followed by 4-week treatment with atorvastatin (80 mg/day) or rosuvastatin (40 mg/day). LDL-c response was considered good (40-70 % reduction, n = 9) or poor (3-33 % reduction, n = 8). Serum lipidomic profile was analyzed by ultra-high-performance liquid chromatography combined with electrospray ionization tandem time-of-flight mass spectrometry, and data were analyzed using MetaboAnalyst v5.0. Lipidomic analysis identified 353 lipids grouped into 16 classes. Statin treatment reduced drastically 8 of 13 lipid classes, generating a characteristic lipidomic profile with a significant contribution of phosphatidylinositols (PI) 16:0/18:2, 18:0/18:1 and 18:0/18:2; and triacylglycerols (TAG) 18:2x2/18:3, 18:1/18:2/18:3, 16:1/18:2x2, 16:1/18:2/18:3 and 16:1/18:2/Arachidonic acid (p-adjusted <0.05). Biomarker analysis implemented in MetaboAnalyst subsequently identified PI 16:1/18:0, 16:0/18:2 and 18:0/18:2 as predictors of statin response with and receiver operating characteristic (ROC) areas under the curve of 0.98, 0.94 and 0.91, respectively. In conclusion, statins extensively modulate the overall serum lipid composition of FH individuals and these findings suggest that phosphatidyl-inositol molecules are potential predictive biomarkers of statin response.

LDLR and PCSK9 3´UTR variants and their putative effects on microRNA molecular interactions in familial hypercholesterolemia: a computational approach.

BACKGROUND: Familial hypercholesterolemia (FH) is caused by pathogenic variants in low-density lipoprotein (LDL) receptor (LDLR) or its associated genes, including apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and LDLR adaptor protein 1 (LDLRAP1). However, approximately 40% of the FH patients clinically diagnosed (based on FH phenotypes) may not carry a causal variant in a FH-related gene. Variants located at 3' untranslated region (UTR) of FH-related genes could elucidate mechanisms involved in FH pathogenesis. This study used a computational approach to assess the effects of 3'UTR variants in FH-related genes on miRNAs molecular interactions and to explore the association of these variants with molecular diagnosis of FH. METHODS AND RESULTS: Exons and regulatory regions of FH-related genes were sequenced in 83 FH patients using an exon-target gene sequencing strategy. In silico prediction tools were used to study the effects of 3´UTR variants on interactions between miRNAs and target mRNAs. Pathogenic variants in FH-related genes (molecular diagnosis) were detected in 44.6% FH patients. Among 59 3'UTR variants identified, LDLR rs5742911 and PCSK9 rs17111557 were associated with molecular diagnosis of FH, whereas LDLR rs7258146 and rs7254521 and LDLRAP1 rs397860393 had an opposite effect (p < 0.05). 3´UTR variants in LDLR (rs5742911, rs7258146, rs7254521) and PCSK9 (rs17111557) disrupt interactions with several miRNAs, and more stable bindings were found with LDLR (miR-4435, miR-509-3 and miR-502) and PCSK9 (miR-4796). CONCLUSION: LDLR and PCSK9 3´UTR variants disturb miRNA:mRNA interactions that could affect gene expression and are potentially associated with molecular diagnosis of FH.

Influence of polymorphisms in IRS1, IRS2, MC3R, and MC4R on metabolic and inflammatory status and food intake in Brazilian adults: An exploratory pilot study.

Polymorphisms in genes of leptin-melanocortin and insulin pathways have been associated with obesity and type 2 diabetes. We hypothesized that polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory markers and food intake composition in Brazilian subjects. This exploratory pilot study included 358 adult subjects. Clinical, anthropometric, and laboratory data were obtained through interview and access to medical records. The variants IRS1 rs2943634 A˃C, IRS2 rs1865434 C>T, MC3R rs3746619 C>A, and MC4R rs17782313 T>C were analyzed by real-time polymerase chain reaction. Food intake composition was assessed in a group of subjects with obesity (n = 84) before and after a short-term nutritional counseling program (9 weeks). MC4R rs17782313 was associated with increased risk of obesity (P = .034). Multivariate linear regression analysis adjusted by covariates indicated associations of IRS2 rs1865434 with reduced low-density lipoprotein cholesterol and resistin, MC3R rs3746619 with high glycated hemoglobin, and IRS1 rs2943634 and MC4R rs17782313 with increased high-sensitivity C-reactive protein (P < .05). Energy intake and carbohydrate and total fat intakes were reduced after the diet-oriented program (P < .05). Multivariate linear regression analysis showed associations of IRS2 rs1865434 with high basal fiber intake, IRS1 rs2943634 with low postprogram carbohydrate intake, and MC4R rs17782313 with low postprogram total fat and saturated fatty acid intakes (P < .05). Although significant associations did not survive correction for multiple comparisons using the Benjamini-Hochberg method in this exploratory study, polymorphisms in IRS1, IRS2, MC3R, and MC4R influence metabolic and inflammatory status in Brazilian adults. IRS1 and MC4R variants may influence carbohydrate, total fat, and saturated fatty acid intakes in response to a diet-oriented program in subjects with obesity.

Identification of pathogenic variants in the Brazilian cohort with Familial hypercholesterolemia using exon-targeted gene sequencing.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by high plasma low-density lipoprotein cholesterol (LDL-c) levels and increased risk of premature atherosclerotic cardiovascular disease. Mutations in FH-related genes account for 40% of FH cases worldwide. In this study, we aimed to assess the pathogenic variants in FH-related genes in the Brazilian FH cohort FHBGEP using exon-targeted gene sequencing (ETGS) strategy. FH patients (n = 210) were enrolled at five clinical sites and peripheral blood samples were obtained for laboratory testing and genomic DNA extraction. ETGS was performed using MiSeq platform (Illumina). To identify deleterious variants in LDLR, APOB, PCSK9, and LDLRAP1, the long-reads were subjected to Burrows-Wheeler Aligner (BWA) for alignment and mapping, followed by variant calling using Genome Analysis Toolkit (GATK) and ANNOVAR for variant annotation. The variants were further filtered using in-house custom scripts and classified according to the American College Medical Genetics and Genomics (ACMG) guidelines. A total of 174 variants were identified including 85 missense, 3 stop-gain, 9 splice-site, 6 InDel, and 71 in regulatory regions (3'UTR and 5'UTR). Fifty-two patients (24.7%) had 30 known pathogenic or likely pathogenic variants in FH-related genes according to the American College Medical and Genetics and Genomics guidelines. Fifty-three known variants were classified as benign, or likely benign and 87 known variants have shown uncertain significance. Four novel variants were discovered and classified as such due to their absence in existing databases. In conclusion, ETGS and in silico prediction studies are useful tools for screening deleterious variants and identification of novel variants in FH-related genes, they also contribute to the molecular diagnosis in the FHBGEP cohort.

Mechanical ventilation and death in pregnant patients admitted for COVID-19: a prognostic analysis from the Brazilian COVID-19 registry score.

BACKGROUND: The assessment of clinical prognosis of pregnant COVID-19 patients at hospital presentation is challenging, due to physiological adaptations during pregnancy. Our aim was to assess the performance of the ABC2-SPH score to predict in-hospital mortality and mechanical ventilation support in pregnant patients with COVID-19, to assess the frequency of adverse pregnancy outcomes, and characteristics of pregnant women who died. METHODS: This multicenter cohort included consecutive pregnant patients with COVID-19 admitted to the participating hospitals, from April/2020 to March/2022. Primary outcomes were in-hospital mortality and the composite outcome of mechanical ventilation support and in-hospital mortality. Secondary endpoints were pregnancy outcomes. The overall discrimination of the model was presented as the area under the receiver operating characteristic curve (AUROC). Overall performance was assessed using the Brier score. RESULTS: From 350 pregnant patients (median age 30 [interquartile range (25.2, 35.0)] years-old]), 11.1% had hypertensive disorders, 19.7% required mechanical ventilation support and 6.0% died. The AUROC for in-hospital mortality and for the composite outcome were 0.809 (95% IC: 0.641-0.944) and 0.704 (95% IC: 0.617-0.792), respectively, with good overall performance (Brier = 0.0384 and 0.1610, respectively). Calibration was good for the prediction of in-hospital mortality, but poor for the composite outcome. Women who died had a median age 4 years-old higher, higher frequency of hypertensive disorders (38.1% vs. 9.4%, p < 0.001) and obesity (28.6% vs. 10.6%, p = 0.025) than those who were discharged alive, and their newborns had lower birth weight (2000 vs. 2813, p = 0.001) and five-minute Apgar score (3.0 vs. 8.0, p < 0.001). CONCLUSIONS: The ABC2-SPH score had good overall performance for in-hospital mortality and the composite outcome mechanical ventilation and in-hospital mortality. Calibration was good for the prediction of in-hospital mortality, but it was poor for the composite outcome. Therefore, the score may be useful to predict in-hospital mortality in pregnant patients with COVID-19, in addition to clinical judgment. Newborns from women who died had lower birth weight and Apgar score than those who were discharged alive.

Sandwich-like electrochemical aptasensing of heat shock protein 70 kDa (HSP70): Application in diagnosis/prognosis of coronavirus disease 2019 (COVID-19).

In this research, by using aptamer-conjugated gold nanoparticles (aptamer-AuNPs) and a modified glassy carbon electrode (GCE) with reduced graphene oxide (rGO) and Acropora-like gold (ALG) nanostructure, a sandwich-like system provided for sensitive detection of heat shock protein 70 kDa (HSP70), which applied as a functional biomarker in diagnosis/prognosis of COVID-19. Initially, the surface of the GCE was improved with rGO and ALG nanostructures, respectively. Then, an aptamer sequence as the first part of the bioreceptor was covalently bound on the surface of the GCE/rGO/ALG nanostructures. After adding the analyte, the second part of the bioreceptor (aptamer-AuNPs) was immobilized on the electrode surface to improve the diagnostic performance. The designed aptasensor detected HSP70 in a wide linear range, from 5 pg mL-1 to 75 ng mL-1, with a limit of detection (LOD) of ∼2 pg mL-1. The aptasensor was stable for 3 weeks and applicable in detecting 40 real plasma samples of COVID-19 patients. The diagnostic sensitivity and specificity were 90% and 85%, respectively, compared with the reverse transcription-polymerase chain reaction (RT-PCR) method.

LDLR missense variants disturb structural conformation and LDLR activity in T-lymphocytes of Familial hypercholesterolemia patients.

Familial hypercholesterolemia (FH) is caused by deleterious mutations in the LDLR that increase markedly low-density lipoprotein (LDL) cholesterol and cause premature atherosclerotic cardiovascular disease. Functional effects of pathogenic LDLR variants identified in Brazilian FH patients were assessed using in vitro and in silico studies. Variants in LDLR and other FH-related genes were detected by exon-target gene sequencing. T-lymphocytes were isolated from 26 FH patients, and 3 healthy controls and LDLR expression and activity were assessed by flow cytometry and confocal microscopy. The impact of LDLR missense variants on protein structure was assessed by molecular modeling analysis. Ten pathogenic or likely pathogenic LDLR variants (six missense, two stop-gain, one frameshift, and one in splicing region) and six non-pathogenic variants were identified. Carriers of pathogenic and non-pathogenic variants had lower LDL binding and uptake in activated T-lymphocytes compared to controls (p < 0.05), but these variants did not influence LDLR expression on cell surface. Reduced LDL binding and uptake was also observed in carriers of LDLR null and defective variants. Modeling analysis showed that p.(Ala431Thr), p.(Gly549Asp) and p.(Gly592Glu) disturb intramolecular interactions of LDLR, and p.(Gly373Asp) and p.(Ile488Thr) reduce the stability of the LDLR protein. Docking and molecular interactions analyses showed that p.(Cys184Tyr) and p.(Gly373Asp) alter interaction of LDLR with Apolipoprotein B (ApoB). In conclusion, LDLR null and defective variants reduce LDL binding capacity and uptake in activated T-lymphocytes of FH patients and LDLR missense variants affect LDLR conformational stability and dissociation of the LDLR-ApoB complex, having a potential role in FH pathogenesis.

Effects of PCSK9 missense variants on molecular conformation and biological activity in transfected HEK293FT cells.

PCSK9 gain-of-function (GOF) variants increase degradation of low-density lipoprotein receptor (LDLR) and are potentially associated with Familial Hypercholesterolemia (FH). This study aimed to explore the effects of PCSK9 missense variants on protein structure and interactions with LDLR using molecular modeling analyses and in vitro functional studies. Variants in FH-related genes were identified in a Brazilian FH cohort using an exon-target gene sequencing strategy. Eight PCSK9 missense variants in pro- [p.(E32K) and p.(E57K)], catalytic [p.(R237W), p.(P279T) and p.(A443T)], and C-terminal histidine-cysteine rich (CHR) [p.(R469W), p.(Q619P) and p.(R680Q)] domains were identified. Molecular dynamics analyses revealed that GOF variants p.(E32K) and p.(R469W) increased extreme motions in PCSK9 amino acid backbone fluctuations and affected Hbond and water bridge interactions between the pro-domain and CM1 region of the CHR domain. HEK293FT cells transfected with plasmids carrying p.(E32K) and p.(R469W) variants reduced LDLR expression (8.7 % and 14.8 %, respectively) compared to wild type (p < 0.05) but these GOF variants did not affect PCSK9 expression and secretion. The missense variants p.(P279T) and p.(Q619P) also reduced protein stability and altered Hbond interactions. In conclusion, PCSK9 p.(E32K), p.(R469W), p.(P279T) and p.(Q619P) variants disrupt intramolecular interactions that are essential for PCSK9 structural conformation and biological activity and may have a potential role in FH pathogenesis.

In silico analysis of upstream variants in Brazilian patients with Familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a prevalent autosomal genetic disease associated with increased risk of early cardiovascular events and death due to chronic exposure to very high levels of low-density lipoprotein cholesterol (LDL-c). Pathogenic variants in the coding regions of LDLR, APOB and PCSK9 account for most FH cases, and variants in non-coding regions maybe involved in FH as well. Variants in the upstream region of LDLR, APOB and PCSK9 were screened by targeted next-generation sequencing and their effects were explored using in silico tools. Twenty-five patients without pathogenic variants in FH-related genes were selected. 3 kb upstream regions of LDLR, APOB and PCSK9 were sequenced using the AmpliSeq (Illumina) and Miseq Reagent Nano Kit v2 (Illumina). Sequencing data were analyzed using variant discovery and functional annotation tools. Potentially regulatory variants were selected by integrating data from public databases, published data and context-dependent regulatory prediction score. Thirty-four single nucleotide variants (SNVs) in upstream regions were identified (6 in LDLR, 15 in APOB, and 13 in PCSK9). Five SNVs were prioritized as potentially regulatory variants (rs934197, rs9282606, rs36218923, rs538300761, g.55038486A > G). APOB rs934197 was previously associated with increased rate of transcription, which in silico analysis suggests that could be due to reducing binding affinity of a transcriptional repressor. Our findings highlight the importance of variant screening outside of coding regions of all relevant genes. Further functional studies are necessary to confirm that prioritized variants could impact gene regulation and contribute to the FH phenotype.

Influence of antidepressant drugs on DNA methylation of ion channels genes in blood cells of psychiatric patients.

Aim: This study investigated the influence of antidepressant drugs on methylation status of KCNE1, KCNH2 and SCN5A promoters and ECG parameters in adult psychiatric patients. Materials & methods: Electrocardiographic evaluation (24 h) and blood samples were obtained from 34 psychiatric patients before and after 30 days of antidepressant therapy. Methylation of promoter CpG sites of KCNE1, KCNH2 and SCN5A was analyzed by pyrosequencing. Results: Three CpG and four CpG sites of KCNE1 and SCN5A, respectively, had increased % methylation after treatment. Principal component analysis showed correlations of the methylation status with electrocardiographic variables, antidepressant doses and patient age. Conclusion: Short-term treatment with antidepressant drugs increase DNA methylation in KCNE1 and SCN5A promoters, which may induce ECG alterations in psychiatric patients.

Predictors of venous thromboembolism in COVID-19 patients: results of the COVID-19 Brazilian Registry.

Previous studies that assessed risk factors for venous thromboembolism (VTE) in COVID-19 patients have shown inconsistent results. Our aim was to investigate VTE predictors by both logistic regression (LR) and machine learning (ML) approaches, due to their potential complementarity. This cohort study of a large Brazilian COVID-19 Registry included 4120 COVID-19 adult patients from 16 hospitals. Symptomatic VTE was confirmed by objective imaging. LR analysis, tree-based boosting, and bagging were used to investigate the association of variables upon hospital presentation with VTE. Among 4,120 patients (55.5% men, 39.3% critical patients), VTE was confirmed in 6.7%. In multivariate LR analysis, obesity (OR 1.50, 95% CI 1.11-2.02); being an ex-smoker (OR 1.44, 95% CI 1.03-2.01); surgery ≤ 90 days (OR 2.20, 95% CI 1.14-4.23); axillary temperature (OR 1.41, 95% CI 1.22-1.63); D-dimer ≥ 4 times above the upper limit of reference value (OR 2.16, 95% CI 1.26-3.67), lactate (OR 1.10, 95% CI 1.02-1.19), C-reactive protein levels (CRP, OR 1.09, 95% CI 1.01-1.18); and neutrophil count (OR 1.04, 95% CI 1.005-1.075) were independent predictors of VTE. Atrial fibrillation, peripheral oxygen saturation/inspired oxygen fraction (SF) ratio and prophylactic use of anticoagulants were protective. Temperature at admission, SF ratio, neutrophil count, D-dimer, CRP and lactate levels were also identified as predictors by ML methods. By using ML and LR analyses, we showed that D-dimer, axillary temperature, neutrophil count, CRP and lactate levels are risk factors for VTE in COVID-19 patients.

Genetic Variant ABCC1 rs45511401 Is Associated with Increased Response to Statins in Patients with Familial Hypercholesterolemia.

Statins are the first-line treatment for familial hypercholesterolemia (FH), but response is highly variable due to genetic and nongenetic factors. Here, we explored the association between response and genetic variability in 114 Brazilian adult FH patients. Specifically, a panel of 84 genes was analyzed by exon-targeted gene sequencing (ETGS), and the functional impact of variants in pharmacokinetic (PK) genes was assessed using an array of functionality prediction methods. Low-density lipoprotein cholesterol (LDL-c) response to statins (reduction ≥ 50%) and statin-related adverse event (SRAE) risk were assessed in carriers of deleterious variants in PK-related genes using multivariate linear regression analyses. Fifty-eight (50.8%) FH patients responded to statins, and 24 (21.0%) had SRAE. Results of the multivariate regression analysis revealed that ABCC1 rs45511401 significantly increased LDL-c reduction after statin treatment (p < 0.05). In silico analysis of the amino-acid change using molecular docking showed that ABCC1 rs45511401 possibly impairs statin efflux. Deleterious variants in PK genes were not associated with an increased risk of SRAE. In conclusion, the deleterious variant ABCC1 rs45511401 enhanced LDL-c response in Brazilian FH patients. As such, this variant might be a promising candidate for the individualization of statin therapy.

Diagnostic accuracy of a SARS-CoV-2 rapid test and optimal time for seropositivity according to the onset of symptoms.

Point-of-care serological tests for SARS-CoV-2 have been used for COVID-19 diagnosis. However, their accuracy over time regarding the onset of symptoms is not fully understood. We aimed to assess the accuracy of a point-of-care lateral flow immunoassay (LFI). Subjects, aged over 18 years, presenting clinical symptoms suggestive of acute SARS-CoV-2 infection were tested once by both nasopharyngeal and oropharyngeal RT-PCR and LFI. The accuracy of LFI was assessed in periodic intervals of three days in relation to the onset of symptoms. The optimal cut-off point was defined as the number of days required to achieve the best sensitivity and specificity. This cut-off point was also used to compare LFI accuracy according to participants' status: outpatient or hospitalized. In total, 959 patients were included, 379 (39.52%) tested positive for SARS-CoV-2 with RT-PCR, and 272 (28.36%) tested positive with LFI. LFI best performance was achieved after 10 days of the onset of symptoms, with sensitivity and specificity of 84.9% (95%CI: 79.8-89.1) and 94.4% (95%CI: 91.0-96.8), respectively. Although the specificity was similar (94.6% vs. 88.9%, p = 0.051), the sensitivity was higher in hospitalized patients than in outpatients (91.7% vs. 82.1%, p = 0.032) after 10 days of the onset of symptoms. Best sensitivity of point-of-care LFI was found 10 days after the onset of symptoms which may limit its use in acute care. Specificity remained high regardless of the number of days since the onset of symptoms.

Diagnostic accuracy of a SARS-CoV-2 rapid test and optimal time for seropositivity according to the onset of symptoms / Acurácia diagnóstica de um teste rápido para SARS-CoV-2 e tempo ótimo para a soropositividade a partir do início dos sintomas / Precisión diagnóstica del test rápido SARS-CoV-2 y período óptimo para la seropositividad según la aparición de síntomas

Point-of-care serological tests for SARS-CoV-2 have been used for COVID-19 diagnosis. However, their accuracy over time regarding the onset of symptoms is not fully understood. We aimed to assess the accuracy of a point-of-care lateral flow immunoassay (LFI). Subjects, aged over 18 years, presenting clinical symptoms suggestive of acute SARS-CoV-2 infection were tested once by both nasopharyngeal and oropharyngeal RT-PCR and LFI. The accuracy of LFI was assessed in periodic intervals of three days in relation to the onset of symptoms. The optimal cut-off point was defined as the number of days required to achieve the best sensitivity and specificity. This cut-off point was also used to compare LFI accuracy according to participants' status: outpatient or hospitalized. In total, 959 patients were included, 379 (39.52%) tested positive for SARS-CoV-2 with RT-PCR, and 272 (28.36%) tested positive with LFI. LFI best performance was achieved after 10 days of the onset of symptoms, with sensitivity and specificity of 84.9% (95%CI: 79.8-89.1) and 94.4% (95%CI: 91.0-96.8), respectively. Although the specificity was similar (94.6% vs. 88.9%, p = 0.051), the sensitivity was higher in hospitalized patients than in outpatients (91.7% vs. 82.1%, p = 0.032) after 10 days of the onset of symptoms. Best sensitivity of point-of-care LFI was found 10 days after the onset of symptoms which may limit its use in acute care. Specificity remained high regardless of the number of days since the onset of symptoms.

Os testes sorológicos no local de atendimento (point-of-care) para a infecção pelo SARS-CoV-2 têm sidos utilizados para o diagnóstico da COVID-19. Entretanto, não está plenamente elucidada a acurácia dos testes ao longo do tempo em relação ao início dos sintomas. Nosso objetivo foi de avaliar a acurácia, no local de atendimento, do imunoensaio de fluxo lateral (LFI). Pacientes com ≥ 18 anos de idade que apresentavam sintomas clínicos sugestivos de infecção aguda pelo SARS-CoV-2 foram testados uma vez com RT-PCR da nasofaringe e orofaringe, além do LFI. A acurácia do LFI foi avaliada com intervalos periódicos de 3 dias a partir do início dos sintomas. O ponto de corte ótimo foi definido como o número necessário de dias para atingir a melhor sensibilidade e especificidade. Esse ponto foi utilizado também para comparar a acurácia do LFI de acordo com a situação do paciente (ambulatorial ou hospitalizado). Foram incluídos 959 pacientes, dos quais 379 (39,52%) testaram positivos para SARS-CoV-2 pelo RT-PCR e 272 (28,36%) pelo LFI. Foi atingido o melhor desempenho para o LFI com 10 dias a partir do início dos sintomas, com sensibilidade e especificidade de 84,9% (IC95%: 79,8-89,1) e 94,4% (IC95%: 91,0-96,8), respectivamente. Embora a especificidade não tenha sido diferente entre os grupos de pacientes (94,6% vs. 88,9%, p = 0,051), a sensibilidade foi mais alta nos pacientes hospitalizados que nos ambulatoriais (91,7% vs. 82,1%, p = 0,032) no dia 10 depois do início dos sintomas. A melhor sensibilidade do LFI no local de atendimento ocorre 10 dias depois do início dos sintomas, o que pode limitar seu uso no atendimento agudo. A especificidade permanece alta, independentemente do número de dias desde o início dos sintomas.

Los puestos de atención para pruebas serológicas del SARS-CoV-2 han sido usado para la diagnosis de la COVID-19. No obstante, su precisión a lo largo del tiempo, en lo que respecta a la aparición de los síntomas, no se ha comprendido completamente. Nuestro objetivo fue evaluar la precisión de un puesto de atención de inmunoanálisis de flujo lateral (LFI). Se hizo pruebas a individuos ≥ 18 años, presentando síntomas clínicos compatibles con una infección aguda de SARS-CoV-2, tanto vía nasofaríngea y orofaríngea RT-PCR, como LFI. La precisión de LFI fue evaluada en intervalos periódicos de 3 días con respecto a la aparición de los síntomas. El punto óptimo de corte se definió como el número de días requerido para alcanzar la mejor sensibilidad y especificidad. Este punto también se usó para comparar la precisión del LFI, según el estatus de los participantes: ambulatorios u hospitalizados. Se incluyeron a 959 pacientes, 379 (39,52%) dieron positivo en las pruebas de SARS-CoV-2 RT-PCR, y 272 (28,36%) fueron positivos en los LFI. Se alcanzó el mejor rendimiento de los LFI tras 10 días de la aparición de los síntomas, con una sensibilidad y especificidad de un 84,9% (IC95%: 79,8-89,1) y 94,4% (IC95%: 91,0-96,8), respectivamente. A pesar de que la especificidad no fue diferente (94,6% vs. 88,9%, p = 0,051), la sensibilidad fue mayor en pacientes hospitalizados que en los ambulatorios (91,7% vs. 82,1%, p = 0,032) tras 10 días desde la aparición de los síntomas. La mejor sensibilidad LFI del puesto de cuidado se produce tras 10 días de la aparición de los síntomas, lo que quizás limite su uso en el cuidado de urgencias. La especificidad permanece alta independientemente del número de días desde la aparición de los síntomas.

Which outcomes are most important to measure in patients with COVID-19 and how and when should these be measured? Development of an international standard set of outcomes measures for clinical use in patients with COVID-19: a report of the International Consortium for Health Outcomes Measurement (ICHOM) COVID-19 Working Group.

OBJECTIVES: The COVID-19 pandemic has resulted in widespread morbidity and mortality with the consequences expected to be felt for many years. Significant variation exists in the care even of similar patients with COVID-19, including treatment practices within and between institutions. Outcome measures vary among clinical trials on the same therapies. Understanding which therapies are of most value is not possible unless consensus can be reached on which outcomes are most important to measure. Furthermore, consensus on the most important outcomes may enable patients to monitor and track their care, and may help providers to improve the care they offer through quality improvement. To develop a standardised minimum set of outcomes for clinical care, the International Consortium for Health Outcomes Measurement (ICHOM) assembled a working group (WG) of 28 volunteers, including health professionals, patients and patient representatives. DESIGN: A list of outcomes important to patients and professionals was generated from a systematic review of the published literature using the MEDLINE database, from review of outcomes being measured in ongoing clinical trials, from a survey distributed to patients and patient networks, and from previously published ICHOM standard sets in other disease areas. Using an online-modified Delphi process, the WG selected outcomes of greatest importance. RESULTS: The outcomes considered by the WG to be most important were selected and categorised into five domains: (1) functional status and quality of life, (2) mental functioning, (3) social functioning, (4) clinical outcomes and (5) symptoms. The WG identified demographic and clinical variables for use as case-mix risk adjusters. These included baseline demographics, clinical factors and treatment-related factors. CONCLUSION: Implementation of these consensus recommendations could help institutions to monitor, compare and improve the quality and delivery of care to patients with COVID-19. Their consistent definition and collection could also broaden the implementation of more patient-centric clinical outcomes research.

Absence of detection of RSV and influenza during the COVID-19 pandemic in a Brazilian cohort: Likely role of lower transmission in the community.

BACKGROUND: Respiratory syncytial virus (RSV) and influenza are prevalent seasonal community viruses. Although not completely understood, SARS-CoV-2 may have the same means of transmission. Preventive social measures aimed at preventing SARS-CoV-2 spread could impact transmission of other respiratory viruses as well. The aim of this study is to report the detection of RSV and influenza during the period of social distancing due to COVID-19 pandemic in a heavily affected community. METHODS: Prospective study with pediatric and adult populations seeking care for COVID-19-like symptoms during the fall and winter of 2020 at two hospitals in Southern Brazil. RT-PCR tests for SARS-CoV-2, influenza A (Flu A), influenza B (Flu B) and respiratory syncytial virus (RSV) was performed for all participants. RESULTS: 1435 suspected COVID-19 participants (1137 adults, and 298 children). were included between May and August. Median age was 37.7 years (IQR = 29.6-47.7), and 4.92 years (IQR = 1.96-9.53), for the adult and child cohorts, respectively. SARS-CoV-2 was positive in 469 (32.7%) while influenza and RSV were not detected at all. CONCLUSIONS: Measures to reduce SARS-CoV-2 transmission likely exerted a huge impact in the spread of alternate respiratory pathogens. These findings contribute to the knowledge about the dynamics of virus spread. Further, it may be considered for guiding therapeutic choices for these other viruses.

The Economic Impact of COVID-19 Treatment at a Hospital-level: Investment and Financial Registers of Brazilian Hospitals.

Background: The economic impact associated with the treatment strategies of coronavirus disease-2019 (COVID-19) patients by hospitals and health-care systems in Brazil is unknown and difficult to estimate. This research describes the investments made to absorb the demand for treatment and the changes in occupation rates and billing in Brazilian hospitals. Methods: This research covers the initial findings of "COVID-19 hospital costs and the proposition of a bundled reimbursement strategy for the health-care system," which includes 10 hospitals. The chief financial officer, the chief medical officer, and hospital executives of each participating hospital provided information regarding investments attributed to COVID-19 patient treatment. The analysis included variations in occupation rates and billing from 2019 to 2020 observed in each institution, and the investments for medical equipment, individual protection materials and building construction per patient treated. Results: The majority of hospitals registered a decrease in hospitalization rates and revenue from 2019 to 2020. For intensive care units (ICUs), the mean occupancy rate ranged from 88% to 83%, and for wards, it ranged from 85% to 73%. Monthly average revenue decreased by 10%. The mean hospital investment per COVID-19 inpatient was I$6800 (standard deviation 7664), with the purchase of ventilators as the most common investment. For this item, the mean, highest and lowest acquisition cost per ventilator were, respectively, I$31 468, I$48 881 and I$17 777. Conclusion: There was significant variability in acquisition costs and investments by institution for responding to the COVID-19 pandemic. These findings highlight the importance of continuing microeconomic studies for a comprehensive assessment of hospital costs. Only with more detailed analyses, will it be possible to define and drive sustainable strategies to manage and reimburse COVID-19 treatment in health-care systems.

Functional analysis of PCSK9 3'UTR variants and mRNA-miRNA interactions in patients with familial hypercholesterolemia.

Aim: Functional analysis of PCSK9 3'UTR variants and mRNA-miRNA interactions were explored in patients with familial hypercholesterolemia (FH). Materials & methods:PCSK9 3'UTR variants were identified by exon-targeted gene sequencing. Functional effects of 3'UTR variants and mRNA-miRNA interactions were analyzed using in silico and in vitro studies in HEK293FT and HepG2 cells. Results: Twelve PCSK9 3'UTR variants were detected in 88 FH patients. c.*75C >T and c.*345C >T disrupted interactions with miR-6875, miR-4721 and miR-564. Transient transfection of the c.*345C >T decreased luciferase activity in HEK293FT cells. miR-4721 and miR-564 mimics reduced PCSK9 expression in HepG2 cells. Conclusion:PCSK9 c.*345C >T has a possible role as loss-of-function variant. miR-4721 and miR-564 downregulate PCSK9 and may be useful to improve lipid profile in FH patients.