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Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.
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Background: Accurate measurement of antibodies is a necessary tool for assessing exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and facilitating an understanding of the role antibodies play in overall immunity. Most available assays are qualitative in nature and employ a threshold to determine the presence of antibodies, however some-quantitative assays are now available. Using cross-sectional data collected as part of an ongoing longitudinal cohort study, we aim to assess the seroprevalence of SARSCoV-2 antibodies using the Abbott AdviseDX SARS-CoV-2 IgG II (anti-S) assay and compare these results to previously measured seroprevalence of anti-nucleoprotein (anti-N) IgG in this cohort of health care workers (HCWs) at an academic medical center in Boston. Method(s): A total of 1,743 HCWs at Boston Medical Center (BMC) provided serum samples that were analyzed for SARS-CoV-2 anti-S IgG and IgM using the Abbott AdviseDx SARS-CoV-2 IgG II and Abbott AdviseDx SARS-CoV-2 IgM assay, respectively. These results were compared to previously assessed anti-N IgG seroprevalence. Precision, linearity, and positive and negative concordance with prior reverse transcription-polymerase chain reaction (RT-PCR) test were evaluated for the anti-S IgG II assay. Seroprevalence and its association with demographic variables was also assessed. Result(s): Linearity and precision results were clinically acceptable. The anti-S IgG positive and negative concordance with RT-PCR results were 88.2% (95% CI: 79.4-94.2%) and 97.4% (95% CI: 95.2-98.8%), respectively. Overall, 126 (7.2%) of 1,743 participants were positive for anti-S IgG. The original agreement in this population with the qualitative, anti-N IgG assay was 70.6%. Upon optimizing the threshold from 1.4 to 0.49 signal to cut-off ratio (S/CO) of the anti-N IgG assay, the positive agreement of the assay increased to 84.7%. Conclusion(s): The anti-S IgG II assay demonstrated reproducible and reliable measurements. Higher anti-S IgG to anti-N IgG seroprevalence highlights the present differences between serum antibodies to different epitopes of the SARS-CoV-2 virus. Further, the greater seroprevalence of anti-S IgG compared to positive RT-PCR results points to a potential for asymptomatic infection among this group of HCWs. Our results also highlight the potential utility in optimizing thresholds of the qualitative SARS-CoV-2 anti-N IgG assay for better agreement with the anti-S IgG II assay by the same vendor.Copyright © 2022 by the Author(s).
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Introduction. Health Technology Assessment (HTA) Process assists decision-making in health policies. The COVID-19 pandemic caused a high demand on protocol or guideline updates and incorporation of new drugs or therapies, overwhelming local agencies. A recent study reported that major HTA bodies in England, Scotland, Germany, and Canada reduced their number of drug recommendations in 2020, due to reprioritization of resources and COVID-related challenges. The present study aimed to evaluate the impact of the COVID-19 pandemic at the Brazilian National Committee for Health Technology Incorporation (Conitec) recommendation process. Methods. This descriptive study evaluated all official recommendation reports available on the Government website in 2020 and 2021, extracting the data of disease category, technology type, the aim of the report, Public Involvement, and final result for the recommendation. The results were presented in tabular and graphical form using the machine learning, through the software R studio and excel. Results. A total of 168 documents were evaluated, including guidelines and recommendation reports, with no reduction in the number of evaluations considering 2019. In 2020, there was a more significant evaluation of guidelines, and in 2021, a report on the nonincorporation of technologies. There were four specific documents about COVID 19, including vaccines and hospital care guidelines. The most incorporated and non-incorporated technologies were medication, targeting rare and highly prevalent diseases in balance. The Brazilian government was the main proposer. These results are part of the study A Survey about the core methods of the recommendation reports for Brazilian Ministry of Health carried out by Brazilian Health Technology Assessment Centers, which will characterize and analyze the core methods of the recommendation reports conducted by the Brazilian HTA Centers. Conclusions. The pandemic had a low impact on demands in the routine of the Conitec. Establish indicators and technological norms applicable to health services, contribute to the identification of possible new practices, methods or criteria.
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Background. Patients with hematological malignancy or other cancers as well as immunosuppression bear a high risk for severe COVID-19. Monoclonal antibodies (mAb) are efficient at early stages of the disease but may lose potency with new variants. Trials on plasma from convalescent donors in unselected patients have not shown clinical benefit. No randomized trials focussing on patients with underlying disease have been published. Methods. We conducted an open-label, multicenter, randomized controlled trial to evaluate efficacy of plasma (CVP - convalescent or after vaccination) in patients with COVID-19 at high risk for adverse outcome in Germany. We assessed the effect of high-titer CVP (2 units from different donors, 238-337 ml each, on subsequent days). Patients with hematological or other malignancy (group 1), immunosuppression (group 2), age >50 and <=75 years and lymphopenia and/or high D-dimers (group 3) or age >75 years (group 4) who were hospitalized with confirmed SARS-CoV-2 infection and with an oxygen saturation <=94% were included. Primary outcome measure was time to clinical improvement on a seven-point ordinal scale, secondary outcome was mortality (Janssen et al. Trials 2020 Oct 6;21(1):828). Results. Overall, 133 patients were randomized, 68 received CVP with an additional 10 patients as a crossover on day 10. Median age (range) was 68 years (39-95) in the CVP group and 70 (38-90) in controls. For the entire cohort, no significant difference was seen in time to improvement (median days: CVP 12.5 vs. control 18;HR 1.24 (95% confidence interval (CI) 0.83-1.85), p=0.29). Subgroup analysis (group 1+2) revealed shortened time to improvement (median days CVP 13 vs. control 32;HR 2.03 (95%CI 1.17-3.6), p=0.01) and mortality was reduced (mortality CVP n=6 (18%) vs. control n=10 (29%). No significant differences in time to improvement were observed in group 3 or 4 (HR 0.72 (95%CI 0.41-1.28), p=0.26). No relevant adverse events were observed. Conclusion. CVP improves time to clinical improvement and mortality for COVID-19 patients with underlying hematological disease/cancer or other reasons of impaired immune response. Even with new variants, high-titer CVP may offer a widely available and inexpensive therapy option in high-risk groups. Funding. BMBF FKZ 01KI20152;EudraCT 2020-001632-10.
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T cell immunity plays a pivotal role in the control of SARS-CoV-2 infections. Despite major efforts in quantifying SARSCoV-2-specific T cell responses, the quality of such responses, in particular of CD8+ T cells, has been marginally investigated so far, leaving the importance of CD8+ T cells during COVID-19 and for protective immunity unclear. To assess CD8+ T cell func-tionality, the identification and characterization of SARS-CoV-2- specific T cell receptors (TCRs) is indispensable. To first identify SARS-CoV-2-specific epitopes, we evaluated immunogenicity of candidate epitopes on convalescent donor material as functional and protective responses are supposed to be found. After a short in-vitro expansion and restimulation of SARS-CoV-2-specific CD8+ T cells, we were able to detect response rates ranging from 33 - 100 %. Importantly, SARS-CoV-2-specific CD8+ T cells were detected even one year after infection. For two HLA-restricted epitopes we subsequently identified TCRs by performing single-cell RNA sequencing. By re-expressing SARS-CoV-2 TCRs via CRISPR/Cas9-mediated orthotopic replacement (OTR), we confirmed functional avidity as well as cytotoxicity towards virus-infected cells. By combining experimental data with gene signatures of recent activation, we further defined a ''reactivity signature'' and a ''functionality signature'' to differentiate TCRs with high and low functionality. To test these gene signatures as a means of predicting TCR functionality in silico, we identified TCRs against nine additional immunodominant SARS-CoV-2 epitopes restricted to five different HLA class I molecules and could confirm TCR functionality. Finally, we showed that the SARS-CoV-2 TCR repertoire is highly polyclonal. In summary, our data demonstrate by single cell TCR identification in combination with OTR engineering that CD8+ T cell responses upon mild COVID-19 infection are polyclonal, long-lasting, and highly functional. We furthermore demonstrated for several TCRs highly effective cytotoxicity towards virus-infected cells, which might open options for therapeutic use with adoptive T cell therapy in COVID-19 patients.
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Background. SARS-CoV-2 continues to spread globally, including in limited resource settings. It is therefore important to derive general case definitions that can be useful and accurate in the absence of timely test results. We aim to validate the World Health Organization (WHO) case definition, a symptom-screening tool currently used to identify SARS-CoV-2 cases in a cohort of symptomatic health care providers (HCP) who completed a symptom survey interview and received a PCR test at Boston Medical Center (BMC) between March 13, 2020 and May 5, 2020. Methods. We classified each HCP as a probable or not probable case of SARSCoV-2 based on the WHO case definition. Using PCR test as gold standard, we computed the sensitivity and specificity of the WHO case definition. We used a stepwise logistic regression model on all PCR-tested HCP to identify symptoms predictive of PCR positivity. Results. Of 328 included HCP, 109 (33.2%) were PCR positive, 213 (64.9%) negative, and 6 (1.8%) had indeterminate test result. The sensitivity and specificity of the WHO case definition were 65.1% and 74.6%, respectively. The positive predictive value was 56.8% and the negative predictive value was 80.7%. Symptoms found to be predictive of PCR positivity were fever, headache, loss of smell and/or loss of taste, and muscle ache/joint pain. Sore throat was found to be predictive of PCR negativity. The area under the curve using the final model was 0.8412. All statistically significant symptoms included in the final model, were also included in the WHO case definition. Conclusion. In our largely symptomatic HCP cohort, our model yielded similar symptoms to those identified in the WHO probable case definition. As seen in similar studies, it is unlikely that further adjustment will improve the performance of a SARSCoV-2 case definition. However, it is concerning that 35% (38/109) of PCR positive SARS-CoV-2 HCP would have been classified as not probable cases by the WHO definition, given that this definition does not even include asymptomatic cases. This is further evidence for global building of laboratory capacity and development of affordable diagnostics to improve global pandemic control.
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Introduction: Acute respiratory tract infection is the major cause for morbidity in human coronavirus (CoV) infections including COVID-19. Approximately 30% of the patients hospitalized with COVID-19 also developed kidney injury. Although the molecular basis remains to be characterized, there is initial evidence to suggest that both indirect (hypoxia and hypotension, cytokine storm, etc.) and direct effects (infection via ACE2 present in the apical border of the proximal tubular cells and in podocytes) contribute to this kidney injury. A detailed understanding of the molecular biology and pathogenesis of coronaviruses is essential for providing better patient care and for facilitating the development of therapeutics. In this study, we sought to characterize a putative ion channel function of an accessory protein (ap) that is conserved in alphacoronaviruses and referred to as ap4 in human coronavirus 229E (HCoV-229E). We characterized the full-length ap4 protein encoded by clinical isolates of HCoV-229E and a C-terminally truncated version of this protein (ap4a) that is expressed by a cell culture-adapted strain of HCoV-229E. Proteins related to ap4 are also encoded in the genomes of other coronaviruses (between the S and E genes) and there is preliminary evidence that some of these proteins act as viroporins and have a role in viral replication and egress. Methods: Recombinant HCoV-229E strains with different forms of ap4 were propagated in Huh-7 cells, MRC-5 cells and human monocyte derived macrophages. Viral growth kinetics was determined and changes in the ap4 coding regions were studied by sequence analysis of virus progeny collected after serial passaging in these cell types. Also, a potassium uptake assay was performed in a Saccharomyces cerevisiae strain lacking the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) technology. Results: Serial passaging demonstrated the acquisition of frameshift mutations in ORF4 resulting in C-terminally truncated protein versions, indicating a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length accessory protein ap4 was expressed compared to the C-terminally truncated protein ap4a. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a nonselective channel or weak K+ channel with the following selectivity filter range: K+<Na+, Rb+<Cs+. Discussion: Our study provides the first evidence that ap4 acts as a viroporin. Further studies are warranted to (i) determine the precise role of ap4 in viral replication by modulating host cell ion homeostasis and (ii) explore if ap4 represents a potential antiviral drug target.
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Objective Liver injury has frequently been reported in COVID-19 patients. The clinical relevance of liver injury related to SARS-CoV-2 infection remains unclear with a need for prospective studies on the impact of liver function test (LFT) abnormalities at baseline. Design Data of 217 patients without pre-existing liver disease prospectively included in the COVID-19 registry of the LMU university hospital were analysed in order to assess the association of abnormal LFT at admission and course of the disease. Severe course was defined as admission to the intensive care unit (ICU) or as COVID-19-related death. Results Abnormal LFT at baseline was present in 58% of patients, with a predominant elevation of aspartate aminotransferase (AST) (42%), gamma-glutamyltransferase (GGT) (37%) and alanine aminotransferase (ALT) (27%), hypoalbuminaemia was observed in 33%. Elevation of ALT and GGT, as well as hypoalbuminaemia, was associated with higher proportions of patients requiring ICU treatment and mechanical ventilation. After adjusting for age, gender and comorbidities, hypoalbuminaemia combined with abnormal AST or GGT at hospital admission was a highly significant independent risk factor for ICU admission (OR 46.22 and 38.8, respectively) and for a composite endpoint of ICU admission and/or COVID-19-related death (OR 42.0 and 26.9, respectively). Conclusion Abnormal LFTs at hospital admission, in particular GGT and albumin, are associated with a severe course of SARS-CoV-2 infection.
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The ongoing outbreak of coronavirus disease of 2019 (COVID-19), triggered by the rapid community spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought forward the need for speedy testing and manufacturing of respiratory face masks for global use. Overwhelmed by the fast-growing demand for disposable face masks, many governments have recommended the use of, and elaborated design criteria for, reusable face masks that are meant to slow down the transmission of SARS-CoV-2 in the general population. Using aerosol particle size spectrometers, we performed particle size distribution measurements and calculated the fractional aerosol particle size-selective filtration efficiencies of over 300 fabrics and fabric-assemblies, including chiffon, cotton, synthetics, and various promising layered combinations. We suggest, and experimentally verify, a simple way for estimating the aerosol filtration efficiency of layered fabrics. The analysis herein investigates the relationship between the breathability and filtration properties of fabrics, assemblies of fabrics, and commercial reusable masks from various regions around the globe. In addition, we demonstrate how a hydrophobic coating can provide a statistically significant increase in the fabrics’ filtration efficiency. The insights of this work are crucial to developing non-woven, high-filtration-performance, reusable face masks that can be worn for extended periods of time. © The Author(s).
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COVID-19 is causing a health crisis and a decline in economic activity, forcing governments to take unprecedented measures. According to the IMF, Germany is one of the world leaders in terms of spending to mitigate the related economic consequences. This article provides an overview of the main tax and fiscal policy measures in Germany as well as the related effects on financial decision-making. © 2020, International Bureau of Fiscal Documentation (IBFD). All rights reserved.
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In most cases infection with the new coronavirus 2 involves the lung, but it also has the potential to affect other organ systems such as the heart, the intestines and especially the vascular system. In the context of diagnostics, computed tomography is the imaging method of choice, although it requires a hygienically problematic transport of the patient for examination, does not provide any information about functional restrictions in the organ systems affected by the infection, and, moreover, until today only limited experience in the diagnosis of the infected lungs is available. The concept of focused symptom-oriented multi-organ sonography describes a bedside imaging method ubiquitously available to all areas of acute medicine. The present work provides an overview of the cross-organ possibilities of sonographic imaging for diagnosis and therapy optimisation in patients who have to be hospitalised due to a coronavirus infection. © Anästh Intensivmed 2021.
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Providing medical care to patients suffering from the coronavirus disease 2019 (COVID-19) pandemic is a major challenge for government healthcare systems around the world. The new coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), shows a high organ specificity for the lower respiratory tract. Since there is so far no effective treatment or vaccination against the virus, early diagnostic recognition is of great importance. Due to the specific aspects of the infection, which mainly begins in the peripheral lung parenchyma, lung ultrasonography is suitable as a diagnostic imaging method to identify suspected cases as such in the early stages of the disease. Serial ultrasound examinations on patients with confirmed COVID-19 can promptly detect changes in the affected lung tissue at the bedside. This article summarizes the diagnostic potential of lung ultrasound with respect to screening and therapeutic decision-making in patients with suspected or confirmed SARS-CoV2 pneumonia.
Subject(s)
COVID-19/diagnostic imaging , Lung/diagnostic imaging , Ultrasonography/methods , COVID-19/diagnosis , COVID-19 Testing , Humans , PandemicsABSTRACT
In 2011, the German Society of Anaesthesiology and Intensive Care Medicine (DGAI) for the first time published standards for sonography training courses, with a focus on anaesthesiology related ultrasound techniques (AFS Course System). Meanwhile, perioperative sonography has become an essential component in the educational curriculum of anaesthesia, intensive care and emergency medicine. The need for re-designing the course system originally derived from the growing sonographic experience of the anaesthesiological community, but is currently strongly supported by the course restrictions associated with the Corona pandemic. The DGAI is now publishing the new standards for AFS training courses that implement various e-learning modules. The society leaves it to the course organisers, whether they offer a traditional course format or rather present a practical training course introduced by an e-learning course that can be attended from any place. In addition, a sonography certificate is introduced, based on the attendance of the AFS courses, and the successfull participation in e-learning tests. © Anästh Intensivmed 2020;