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COVID-19 causes immune perturbations which may persist long-term, and patients frequently report ongoing symptoms for months after recovery. We assessed the extent and nature of immune activation at 3 months post hospital admission in patients with mild, moderate or severe COVID-19 and investigated whether immune activation associates with disease severity and long COVID. Patients with severe disease displayed persistent activation of CD4+ and CD8+ T-cells, based on expression of HLA-DR, CD38, Ki67 and granzyme B, but they lacked activation of other immune subsets. Elevated plasma levels of IL-4, IL-7, IL-17 and TNF- were present in patients with severe compared to mild and/or moderate disease. Plasma from severe patients caused T-cells from healthy donors to upregulate IL-15R, suggesting that factors in the plasma of severe patients may increase T-cell responsiveness to IL-15-driven bystander" activation, which may drive persistent T-cell activation after severe COVID-19. Patients with severe disease reported a higher number of long COVID symptoms which correlated with the frequency of two subsets of activated CD4+ and CD8+ T cells (CD4+ T-cell population 2 and CD8+ T-cell population 4; FDR p<0.05), however these associations were lost after adjusting for age, sex and disease severity. Our data suggests that persistent immune activation and long COVID correlate independently with severe disease.
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BackgroundPatients with coronavirus disease-19 (COVID-19) are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. ObjectivesWe aimed to characterise the mechanism of altered platelet function in COVID-19 patients. MethodsThe platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalised with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry. ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered out of 5773 quantified). Levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKC, were lowered. Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin IIb{beta}3 activation and P-selectin expression. Agonist-stimulated integrin IIb{beta}3 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. ConclusionsOverall, our data suggests the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets. Platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients. Essentials- COVID-19 patient platelet function and platelet proteins were compared with healthy controls - Proteomic analysis of platelets indicated that COVID-19 decreased platelet activation proteins - Agonist induced PS exposure and integrin IIb{beta}3 activation were impaired in COVID-19 - COVID-19 led to maximal levels of P-selectin dependent platelet-neutrophil aggregates
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Neutrophils are vital in defence against pathogens but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome (ARDS). COVID-19 is associated with systemic expansion of immature neutrophils but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10- subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable three months post symptom onset, indication long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients downregulate the chemokine receptor CXCR2, while neutrophils from severely ill individuals failed to do so, suggesting altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10-and CXCR2hi neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for identification of individuals at high risk of progressing to severe COVID-19.
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Aerosol generating procedures (AGPs) are defined as any procedure releasing airborne particles <5 m in size from the respiratory tract. There remains uncertainty about which dental procedures constitute AGPs. We quantified the aerosol number concentration generated during a range of periodontal, oral surgery and orthodontic procedures using an aerodynamic particle sizer, which measures aerosol number concentrations and size distribution across the 0.5-20 m diameter size range. Measurements were conducted in an environment with a sufficiently low background to detect a patients cough, enabling confident identification of aerosol. Phantom head control experiments for each procedure were performed under the same conditions as a comparison. Where aerosol was detected during a patient procedure, we assessed whether the size distribution could be explained by the non-salivary contaminated instrument source in the respective phantom head control procedure using a two-sided unpaired t-test (comparing the mode widths (log({sigma})) and peak positions (DP,C)). The aerosol size distribution provided a robust fingerprint of aerosol emission from a source. 41 patients underwent fifteen different dental procedures. For nine procedures, no aerosol was detected above background. Where aerosol was detected, the percentage of procedure time that aerosol was observed above background ranged from 12.7% for ultrasonic scaling, to 42.9% for 3-in-1 air + water syringe. For ultrasonic scaling, 3-in-1 syringe use and surgical drilling, the aerosol size distribution matched the non-salivary contaminated instrument source, with no unexplained aerosol. High and slow speed drilling produced aerosol from patient procedures with different size distributions to those measured from the phantom head controls (mode widths log({sigma})) and peaks (DP,C), p< 0.002) and, therefore, may pose a greater risk of salivary contamination. This study provides evidence for sources of aerosol generation during common dental procedures, enabling more informed evaluation of risk and appropriate mitigation strategies.
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There is widespread interest in the capacity for SARS-CoV-2 evolution in the face of selective pressures from host immunity, either naturally acquired post-exposure or from vaccine acquired immunity. Allied to this is the potential for long perm persistent infections within immune compromised individuals to allow a broader range of viral evolution in the face of sub-optimal immune driven selective pressure. Here we report on an immunocompromised individual who is hypogammaglobulinaemic and was persistently infected with SARS-CoV-2 for over 290 days, the longest persistent infection recorded in the literature to date. During this time, nine samples of viral nucleic acid were obtained and analysed by next-generation sequencing. Initially only a single mutation (L179I) was detected in the spike protein relative to the prototypic SARS-CoV-2 Wuhan-Hu-1 isolate, with no further changes identified at day 58. However, by day 155 the spike protein had acquired a further four amino acid changes, namely S255F, S477N, H655Y and D1620A and a two amino acid deletion ({Delta}H69/{Delta}V70). Infectious virus was cultured from a nasopharyngeal sample taken on day 155 and next-generation sequencing confirmed that the mutations in the virus mirrored those identified by sequencing of the corresponding swab sample. The isolated virus was susceptible to remdesivir in vitro, however a 17-day course of remdesivir started on day 213 had no effect on the viral RT-PCR cycle threshold (Ct) value. On day 265 the patient was treated with the combination of casirivimab and imdevimab. The patient experienced progressive resolution of all symptoms over the next 8 weeks and by day 311 the virus was no longer detectable by RT-PCR. The {Delta}H69/{Delta}V70 deletion in the N-terminus of the spike protein which arose in our patient is also present in the B.1.1.7 variant of concern and has been associated with viral escape mutagenesis after treatment of another immunocompromised patient with convalescent plasma. Our data confirms the significance of this deletion in immunocompromised patients but illustrates it can arise independently of passive antibody transfer, suggesting the deletion may be an enabling mutation that compensates for distant changes in the spike protein that arise under selective pressure.
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Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the Spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known whether heart vascular pericytes (PCs) are infected by SARS-CoV-2, and if the S protein alone provokes PC dysfunction. Here, we aimed to investigate the effects of the S protein on primary human cardiac PC signalling and function. Results show, for the first time, that cardiac PCs are not permissive to SARS-CoV-2 infection in vitro, whilst a recombinant S protein alone elicits functional alterations in PCs. This was documented as: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors responsible for EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation and rescued PC function in the presence of the S protein. In conclusion, our findings suggest that circulating S protein prompts vascular PC dysfunction, potentially contributing to establishing microvascular injury in organs distant from the site of infection. This mechanism may have clinical and therapeutic implications. Clinical perspectiveO_LISevere COVID-19 manifests as a microvascular syndrome, but whether SARS-CoV-2 infects and damages heart vascular pericytes (PCs) remains unknown. C_LIO_LIWe provide evidence that cardiac PCs are not infected by SARS-CoV-2. Importantly, we show that the recombinant S protein alone elicits cellular signalling through the CD147 receptor in cardiac PCs, thereby inducing cell dysfunction and microvascular disruption in vitro. C_LIO_LIThis study suggests that soluble S protein can potentially propagate damage to organs distant from sites of infection, promoting microvascular injury. Blocking the CD147 receptor in patients may help protect the vasculature not only from infection, but also from the collateral damage caused by the S protein. C_LI
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The SARS-CoV-2 virus causes COVID-19, an infection capable of causing severe disease and death but which may also be asymptomatic or oligosymptomatic in many individuals. While several risk factors, including age, have been described, the mechanisms of this variation are poorly understood. Several studies have described associations between blood group and COVID-19 severity, while others do not. Expression of ABO glycans on secreted proteins and non-erythroid cells is controlled by a fucosyltransferase (FUT2). Inactivating mutations result in a non-secretor phenotype which is known to protect against some viral infections. We investigated whether ABO or secretor status was associated with COVID-19 severity. Data combined from healthcare records and laboratory tests (n=275) of SARS-CoV-2 PCR positive patients hospitalised with COVID-19, confirmed higher than expected numbers of blood group A individuals compared to O (RR=1.24, CI 95% [1.05,1.47], P=0.0111). There was also a significant association between group A and COVID-19-related cardiovascular complications (RR=2.56, CI 95% [1.43,4.55], P=0.0011) which is independent of gender. Molecular analysis of phenotype revealed that group A patients who are non-secretors are significantly less likely to be hospitalised than secretors. In a larger cohort of 1000 convalescent plasma donors, among whom the majority displayed COVID-19 symptoms and only a small minority required hospitalisation, group A non-secretors were slightly over-represented. Our findings indicate that group A non-secretors are not resistant to infection by SARS-CoV-2, but they are likely to experience a less severe form of its associated disease. Key PointsO_LIBlood group type A is associated with an increased risk of cardiovascular complications in COVID-19 patients. C_LIO_LIFUT2 "non-secretor" status reduces the risk of severe COVID-19 outcomes in patients with blood group A. C_LI
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ObjectivesTo assess the performance (sensitivity and specificity) of the Abbott Architect SARS-CoV-2 IgG antibody assay across three clinical settings. MethodsAntibody testing was performed on three clinical cohorts of COVID-19 disease: hospitalised patients with PCR confirmation, hospitalized patients with a clinical diagnosis but negative PCR, and symptomatic healthcare workers (HCWs). Pre-pandemic respiratory infection sera were tested as negative controls. The sensitivity of the assay was calculated at different time points (<5 days, 5-9 days, 10-14 days, 15-19 days, >20 days, >42 days), and compared between cohorts. ResultsPerformance of the Abbot Architect SARS-CoV-2 assay varied significantly between cohorts. For PCR confirmed hospitalised patients (n = 114), early sensitivity was low: <5 days: 44.4% (95%CI: 18.9%-73.3%), 5-9 days: 32.6% (95%CI, 20.5%-47.5%), 10-14 days: 65.2% (95% CI 44.9%-81.2%), 15-20 days: 66.7% (95% CI: 39.1%-86.2%) but by day 20, sensitivity was 100% (95%CI, 86.2-100%). In contrast, 17 out of 114 symptomatic healthcare workers tested at >20 days had negative results, generating a sensitivity of 85.1% (95%CI, 77.4% - 90.5%). All pre-pandemic sera were negative, a specificity of 100%. Seroconversion rates were similar for PCR positive and PCR negative hospitalised cases. ConclusionsThe sensitivity of the Abbot Architect SARS-CoV-2 IgG assay increases over time, with sensitivity not peaking until 20 days post symptoms. Performance varied markedly by setting, with sensitivity significantly worse in symptomatic healthcare workers than in the hospitalised cohort. Clinicians, policymakers, and patients should be aware of the reduced sensitivity in this setting.
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IntroductionCOVID-19 has an unpredictable clinical course so prognostic biomarkers would be invaluable when triaging patients on admission to hospital. Many biomarkers have been suggested using large observational datasets but sample timing is crucial to ensure prognostic relevance. The DISCOVER study prospectively recruited patients with COVID-19 admitted to a UK hospital and analysed a panel of putative prognostic biomarkers on the admission blood sample to identify markers of poor outcome. MethodsConsecutive patients admitted to hospital with proven or clinicoradiological suspected COVID-19 were recruited. Admission bloods were extracted from the clinical laboratory. A panel of biomarkers (IL-6, suPAR, KL-6, Troponin, Ferritin, LDH, BNP, Procalcitonin) were performed in addition to routinely performed markers (CRP, neutrophils, lymphocytes, neutrophil:lymphocyte ratio). Age, NEWS score and CURB-65 were included as comparators. All biomarkers were tested in logistic regression against a composite outcome of non-invasive ventilation, intensive care admission, or death, with Area Under the Curve (AUC) figures calculated. Results155 patients had 28-day outcomes at the time of analysis. CRP (AUC 0.51, CI:0.40-0.62), lymphocyte count (AUC 0.62, CI:0.51-0.72), and other routine markers did not predict the primary outcome. IL-6 (AUC: 0.78,0.65-0.89) and suPAR (AUC 0.77, CI: 0.66-0.85) showed some promise, but simple clinical features alone such as NEWS score (AUC: 0.74, 0.64-0.83) or age (AUC: 0.70, 0.61-0.78) performed nearly as well. DiscussionAdmission blood biomarkers have only moderate predictive value for predicting COVID-19 outcomes, while simple clinical features such as age and NEWS score outperform many biomarkers. IL-6 and suPAR had the best performance, and further studies should validate these biomarkers in a prospective fashion.
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Hydroxychloroquine(HCQ) has been widely used to treat SARS-CoV-2 infection however HCQ pharmacokinetics in this condition have not been studied in non-critical care patient groups. Here we report the serum concentrations of HCQ in a small cohort of patients treated with HCQ as part of the RECOVERY trial.
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ObjectivesTo develop a regional model of COVID-19 dynamics, for use in estimating the number of infections, deaths and required acute and intensive care (IC) beds using the South West of England (SW) as an example case. DesignOpen-source age-structured variant of a susceptible-exposed-infectious-recovered (SEIR) deterministic compartmental mathematical model. Latin hypercube sampling and maximum likelihood estimation were used to calibrate to cumulative cases and cumulative deaths. SettingSW at a time considered early in the pandemic, where National Health Service (NHS) authorities required evidence to guide localised planning and support decision-making. ParticipantsPublicly-available data on COVID-19 patients. Primary and secondary outcome measuresThe expected numbers of infected cases, deaths due to COVID-19 infection, patient occupancy of acute and IC beds and the reproduction ("R") number over time. ResultsSW model projections indicate that, as of the 11th May 2020 (when lockdown measures were eased), 5,793 (95% credible interval, CrI, 2,003 - 12,051) individuals were still infectious (0.10% of the total SW England population, 95%CrI 0.04 - 0.22%), and a total of 189,048 (95%CrI 141,580 - 277,955) had been infected with the virus (either asymptomatically or symptomatically), but recovered, which is 3.4% (95%CrI 2.5 - 5.0%) of the SW population. The total number of patients in acute and IC beds in the SW on the 11th May 2020 was predicted to be 701 (95%CrI 169 - 1,543) and 110 (95%CrI 8 - 464) respectively. The R value in SW England was predicted to be 2.6 (95%CrI 2.0 - 3.2) prior to any interventions, with social distancing reducing this to 2.3 (95%CrI 1.8 - 2.9) and lockdown/ school closures further reducing the R value to 0.6 (95CrI% 0.5 - 0.7). ConclusionsThe developed model has proved a valuable asset for local and regional healthcare services. The model will be used further in the SW as the pandemic evolves, and - as open source software - is portable to healthcare systems in other geographies. Future work/ applicationsO_LIOpen-source modelling tool available for wider use and re-use. C_LIO_LICustomisable to a number of granularities such as at the local, regional and national level. C_LIO_LISupports a more holistic understanding of intervention efficacy through estimating unobservable quantities, e.g. asymptomatic population. C_LIO_LIWhile not presented here, future use of the model could evaluate the effect of various interventions on transmission of COVID-19. C_LIO_LIFurther developments could consider the impact of bedded capacity in terms of resulting excess deaths. C_LI