Inflammatory Pathways Associated with Covid-19 Severity Differ Based on Obesity Status

Background: Severe COVID-19 and obesity are characterized by higher inflammation. We aimed to examine early inflammatory patterns in people with (Ob) and without (NOb) obesity and COVID-19 and how they relate to COVID-19 disease severity Methods: Ob (BMI >30 Kg/m2) and NOb with COVID-19 matched for age, sex and WHO disease severity provided blood early after diagnosis. Immunoassays measured 57 plasma biomarkers reflecting innate immune and endothelial activation, systemic inflammation, coagulation, metabolism and microbial translocation (Fig 1). Between-group differences were assessed by Mann- Whitney. Associations between subsequent maximal COVID-19 severity (mild vs moderate/severe/critical) and biomarkers were explored by logistic regression adjusted for age, sex, hypertension (HTN) and diabetes (DM). Data are median pg/mL [IQR] or n [%] unless stated Results: Of 100 subjects (50 Ob and 50 Nob) presenting between April 2020 and March 2021, characteristics (Ob vs Nob) included: age 65 [23-91] vs 65 [21-95];female sex 27 (48%) vs 28 (56%);BMI 33.7 [30.0-71.8] vs 23.3 [15.3-25.9];disease severity mild 22 [48%] vs 23 [46%], moderate 15 [30%] vs 13 [26%], severe 6 [12%] vs 7 [14%];HTN 30 (60%) vs 17 (34%);DM 19 [38%] vs 6 [12%];days from symptom onset 7 [2-17] vs 8 [1-15];vaccinated 3 (6%) vs 0 (0%). Compared to NOb, Ob had higher IFN-alpha (1.8 [0.6;11] vs 0.9 [0.1;4.7]), CRP (10 mAU/mL [9.6;10.2] vs 9.7 [7.2;10]), IL-1RA (197 [122;399] vs 138 [88;253]), IL-4 (288 AU/mL [161;424] vs 205 [82;333]), vWF (252 [166;383] vs 163 [96;318]), Zonulin (114 ng/mL [77;131] vs 57 [18;106]), Resistin (956 [569;1153] vs 727 [712;1525]), Leptin (3482 [1513;5738] vs 848 [249;2114]), and lower Adiponectin (1.12 mg/L [0.09;1.5] vs 1.5 [1.18;1.93]), all p< 0.05. In both groups higher, proinflammatory IL-18 and lower levels of antiinflammatory CCL22 and IL-5 were associated with higher odds of disease severity, and lower E-selectin with higher disease severity only in Ob. However, in NOb higher type 3 interferons (IL-28A), macrophage activation (sCD163, CCL3) and vascular inflammation markers (ICAM-1, VCAM-1), along with higher S100B, GM-CSF and leptin were also associated with disease severity, a pattern not observed in Ob (Fig 1) Conclusion(s): Although Ob had higher overall levels of inflammation than NOb, few biomarkers predicted subsequent COVID-19 severity in Ob. These differential inflammatory patterns suggest dysregulated immune responses in Ob with COVID-19. (Figure Presented).

Early Inflammatory Profiles Predict Maximal Disease Severity in Covid-19

Background: Better understanding of host inflammatory changes that precede development of severe COVID-19 could improve delivery of available antiviral and immunomodulatory therapies, and provide insights for the development of new therapies. Method(s): In plasma from individuals with COVID-19, sampled <=10 days from symptom onset from the All-Ireland Infectious Diseases Cohort study, we measured 61 biomarkers, including markers of innate immune and T cell activation, coagulation, tissue repair, lung injury, and immune regulation. We used principal component analysis (PCA) and k-means clustering to derive biomarker clusters, and univariate and multivariate ordinal logistic regression to explore association between cluster membership and maximal disease severity, adjusting for risk factors for severe COVID-19, including age, sex, ethnicity, BMI, hypertension and diabetes. Result(s): From March 2020-April 2021, we included 312 individuals, (median (IQR) age 62 (48-77) years, 7 (4-9) days from symptom onset, 54% male) in the analysis. PCA and clustering derived 4 clusters. Compared to cluster 1, clusters 2-4 were significantly older and of higher BMI but there were no significant differences in sex or ethnicity. Cluster 1 had low levels of inflammation, cluster 2 had higher levels of markers of tissue repair and endothelial activation (EGF, VEGF, PDGF, TGFalpha, serpin E1 and p-selectin). Cluster 3 and 4 were both characterised by higher overall inflammation, but compared to cluster 4, cluster 3 had downregulation of growth factors, markers of endothelial activation, and immune regulation (IL10, PDL1), but higher alveolar epithelial injury markers (RAGE, ST2). In univariate analysis, compared to cluster 1, cluster 3 had the highest odds of severe disease (OR (95% CI) 9.02 (4.62-18.31), followed by cluster 4: 5.59 (2.75-11.72) then cluster 2: 4.5 (2.38-8.81), all p < 0.05). Cluster 3 remained most strongly associated with severe disease in fully adjusted analyses;cluster 3: OR(95% CI) 5.99 (2.69-13.35), cluster 2: 3.14 (1.54-6.42), cluster 4: 3.13 (1.36-7.19), all p< 0.05). Conclusion(s): Distinct early inflammatory profiles predicted maximal disease severity independent of known risk factors for severe COVID-19. A cluster characterised by downregulation of growth factor and endothelial markers and early evidence of alveolar injury was associated with highest risk of developing severe COVID19. Whether this reflects a dysregulated inflammatory response that could improve targeted treatment requires further study. Heatmap of biomarker derived clusters and forest plot of association between clusters and disease severity. A: Heatmap demonstrating differences in biomarkers between clusters B: Forest plot demonstrating odds ratio of specific clusters for progressing to moderate or severe disease (reference Cluster 1), calculated using ordinal logistic regression. Odds ratio (95% CI) presented as unadjusted and fully adjusted (for age, sex, ethnicity, BMI, hypertension, diabetes, immunosuppression, smoking and baseline anticoagulant use). Maximal disease severity graded per the WHO severity scale.

Lower B-Cell Responses Rather Than Circulating Antibody Titres Are Associated With SARS-CoV-2 Infection Post Third Dose COVID-19 Vaccination

Background. Which components of the immune response to SARS-CoV-2 vaccination best protect against subsequent infection remains unclear. We explored SARS-CoV-2 specific antibody and B-cell responses post 3rd dose vaccine and their relationship to subsequent SARS-CoV-2 infection. Methods. In a multicentre prospective cohort, adult subjects provided samples before and 14 days (d14) post 3rd dose vaccine with Pfizer-BioNTech 162b2. At 18-22 weeks post vaccine, subjects self-reported SARS-CoV-2 infection (confirmed by PCR or antigen test). We used electrochemiluminescence assays to quantify antibodies to SARS-CoV-2 spike subunit 1 (S1), subunit 2 (S2) and receptor-binding domain (RBD) in plasma (reported inWHOIU/mL). In a subset of subjects, we assessed SARS-CoV-2 specific differentiated B-cell (plasma cell) and memory B-cell responses from peripheral blood mononuclear cells. Unstimulated plasma cells, and memory B cells stimulated with R848 and IL2, were seeded on plates coated with RBD or full Spike antigen and antigen-specific responses measured by ELISpot (Mabtech ELISpot, Sweden). We compared between group differences by Wilcoxon signed rank or Mann-Whitney tests. Data are median [IQR] unless specified. Results. Of 133 subjects (age 43 [32-50], 81.2% female (table 1), 77 subjects in the B-cell subgroup (table 2)), 47 (35.3%) reported SARS-CoV-2 infection post 3rd vaccine. Antibody titres, plasma cell and memory B-cell responses all increased significantly at d14 post 3rd vaccine (Table 1 & 2, all P< 0.001). Although d14 antibody titres did not differ in those with and without subsequent infection (table 1), those reporting subsequent infection had significantly lower d14 RBD-specific plasma cells and a lower proportion of RBD-specific memory B-cells (Figure 1a-b, both P< 0.05). Similar results were observed with full-spike-specific memory B-cell responses (Figure 1d). The differences persisted when the non-infected group was restricted only to those reporting a confirmed close contact (n=26). Conclusion. Infection following 3rd dose vaccine is associated with lower d14 circulating and memory B cell responses, but not antibody titres, suggesting B-cell responses better predict protection against subsequent SARS-CoV-2 infection.

Performance and validation of an adaptable multiplex assay for detection of serologic response to SARS-CoV-2 infection or vaccination

Background. A wide array of assays to detect the serologic response to SARS-CoV-2 have been developed since the emergence of the pandemic. The majority of these are either qualitative or semi-quantitative, detect antibodies against one antigenic target, and are not adaptable to new antigens. Methods. We developed a new, multiplex immunoassay to detect antibodies against the receptor binding domain, S1 and S2 spike subunits and nucleocapsid (N) antigens of SARS-CoV-2 (the CEPHR SARS-CoV-2 Serology Assay). This assay uses electrochemiluminescence technology which allows for a broad dynamic range, and a linker format which allows for the addition of new antigenic targets. We tested this assay on a series of biobanked samples and validated its performance against the Abbott SARS-CoV-2 IgG and Abbott SARS-CoV-2 IgG II assays, and the MesoScale Diagnostics V-PLEX SARS-CoV-2 Panel 2 Kit. Results. Participant demographics are shown in Table 1. The mean (standard deviation (SD)) intra-assay (within plate) coefficient of variation (CV) of 80 plasma samples run on the same plate was 3.9% (2.9) for N, 3.8% (6.2) for RBD, 3.8% (5.9) for S1 and 3.9% (5.3) for S2. The mean (SD) inter-assay CV derived from 5 samples run across 3 days by two different operators was 11% (6.5) for N, 13% (5.7) for RBD, 14% (8.9) for S1 and 13% (5.1) for S2. In the convalescent group (n=193), overall sensitivity for each assay was;RBD 82% (95% CI 76-87), S1 86% (81-91%), S2 88% (83 - 92%) and N 72% (64 - 78%). Sensitivity improved when analysis included only individuals who were sampled more than 14 days from onset of symptoms (n=166), RBD 87% (81 - 95%), S1 91% (85 - 95%), S2 91% (85 - 95%) but not for the N-target (73% (66-80%)). In vaccinated individuals (n = 58), 100% (94-100%) had both detectable RBD and S1 antibodies. Overall specificity was 96% (87-99%) for RBD, 90% (78-97%) for S1, 94% (84-99%) for S2 and 90% (78-97%) for N. There was excellent correlation between the Abbott IgG II and both CEPHR anti-RBD IgG (rho 0.91) and CEPHR anti-S1 IgG (rho 0.9, both p < 0.001, Figure 1.) and the V-PLEX full spike and both CEPHR RBD IgG (rho 0.83) and S1 IgG (rho 0.82, both p < 0.001, Figure 4). Conclusion. The CEPHR SARS-CoV-2 Serology Assay is a robust, customisable, multiplex serologic assay for the detection of several different IgG specific to SARS-CoV-2.

Impact of covid-19 on mediocre life-style and their psychology (Special emphasis on middle-class indian households)

India being the second most populated nation, observed the chronic outbreak of Novel Coronavirus as an instrumental factor for bringing about a major shift in the conduct of routine work and regular course of common life during the tragic year of 2020. The virus has given severe jolts to the economy, its distinct classes as well as its citizens alike, projecting far reaching effects across the country and disrupting the ordinary flow of the vital world. With special reference to the middle- class households in India, the purpose of this study is to highlight the impact of COVID-19 on the various facets of mediocre lifestyle including law and order, family and festivals, income and expenditure, mental and physical health, community welfare and social work, creativity and culture, citizen compliance and environmental concern etc. This research intends to provide a useful insight on how the virus is affecting the perspective of mediocre men under the exceptional circumstances of Pandemic and what are the surfacing pros and cons ascribed to the preventive measures like lock- down, quarantine and social distancing. © MEDIC SCIENTIFIC, All rights reserved.

ESTABLISHMENT of AN ANTI-RBD THRESHOLD THAT PREDICTS ROBUST NEUTRALIZING ACTIVITY

Background: Although presence of SARS-CoV-2 neutralising antibodies can provide protection against development of COVID-19, how reflective circulating anti-SARS-CoV-2 antibody levels are of underlying neutralising capacity, and whether a threshold exists to predict sufficient neutralising capacity remains unclear. Methods: In plasma from individuals with PCR-confirmed COVID-19 recruited to the All Ireland Infectious Diseases Cohort Study, we measured IgG concentrations against RBD, Spike protein sub-unit 1 and 2 (S1, S2) and Nucleocapsid (NC) using multiplex electrochemiluminescence (normalised to World Health Organisation reference serum as IU/mL). Neutralising capacity was measured against live SARS-CoV-2 virus (clinical isolate 2019-nCoV/Italy-INMI1) by determining the maximum plasma dilution required to maintain 50% inhibition of infection of Vero E6 cells (50% Neutralisation Titre (NT50)), by flow cytometry-based micro-neutralisation assay. Given that the Beta SARS-CoV-2 variant of concern (VOC) reduces neutralising activity up to six fold, we estimated a NT50 of 1:1000 against wild type SARS-CoV-2 would maintain neutralising activity against VOC. We used Spearman correlation and linear regression to model relationships between NT50 and IgG concentrations. Data are presented as median (IQR) unless specified. Results: In 190 individuals (age 50 (40-64) years, 55% female, time from symptom onset 98 (35-179) days), NT50 most highly correlated with anti-RBD IgG (Rho 0.81 p<0.001, Fig 1a) compared with other IgG classes (S1;Rho 0.8, S2;0.73, NC;0.72, all p<0.001). Median RBD titre was 246 (71-662) but trended lower over time, with a median of 319 (61-1012) IU/ml at 0-90 days, 244 (86-523) IU/ml at 90-180 days and 157 (80-364) IU/ml at >180 days post symptom onset respectively (p=0.08, Fig 1b). RBD IgG titres of 476 IU/ml corresponded to a NT50 of 1:1000. Overall, RBD ≥476 IU/ml predicted NT50 ≥1:1000 with a sensitivity of 77 (95% CI 65-87)% and specificity 89 (95% CI 82-93)%. This improved in an analysis restricted to convalescent samples (>30 days post symptom onset, n=148), with a sensitivity 88% (95% CI 74-96%) and specificity 90% (95%CI 82-95%) respectively. Conclusion: In convalescent plasma, RBD IgG titres ≥476IU/mL is sensitive and specific for predicting robust underlying neutralising capacity. Further research is required to validate these findings in other cohorts and confirm these thresholds in post-vaccinated individuals.

CIRCULATING LEVELS of TYPE I, TYPE II, and TYPE III INTERFERONS and COVID-19 SEVERITY

Background: Interferons play a pivotal role as a first line of the innate immune host response to viral infections, including COVID-19. Accumulating data suggests dysregulated interferon (IFN) responses in COVID-19. However, the clinical relevance of circulating levels of interferon to COVID-19 disease severity remains unclear Methods: In plasma from individuals with PCR confirmed SARS-CoV-2 infection recruited to the All Ireland Infectious Diseases Cohort, collected within 10 days from onset of symptoms, we measured levels of type I (IFN-α2a and IFN-β), type II (IFN-γ), and type III (IFN-a;1) interferons by electro-chemiluminescence immunoassays. Subsequent maximum COVID-19 disease severity was classified according to World Health Organization guidance (Critical, Severe, Moderate and Mild). We used Kruskal-Wallis tests to explore differences in IFN levels between COVID-19 severity groups, and logistic regression to determine associations, adjusting for demographics (age, sex at birth, ethnicity), comorbidities (obesity, hypertension, respiratory disease, heart disease) and medical management (antibiotics, immunosuppressants, anticoagulants, invasive ventilation) Results: Out of the 335 subjects with early infection and available samples, 319 had data on disease severity, 33 (10.3%) Critical, 37 (11.6%) Severe, 76 (23.8%) Moderate and 173 (54.2%) Mild. The population was predominantly Caucasian (79.3%), with a median [IQR] age of 64 [53,77] and male (52.7%). There was a significant difference between the 4 groups for the levels of Type I IFN-α2a (p=0.0028) and Type 3 IFN-a;1 (p=0.0001), both being higher in the critical group. In adjusted analyses, higher levels of Type I IFN-α2a but not Type 3 IFN-a;1 remained significantly associated with the development of Critical COVID-19 (Odds Ratio: 5.911/95% CI: 0.608, 52.388/p=0.029). (Fig 1) Conclusion: Increased circulating Type I IFN-α2a, but not other IFN classes, measured in the early stages of SARS-CoV-2 infection was associated with higher odds of Critical COVID-19 infection. These data point to specific differences in host responses that may lead to more targeted interventions to prevent development of severe COVID-19 infection.

Comparison of Sodium and Potassium Levels among COVID-19 Patients on Arterial Blood Gas Analysers and Clinical Chemistry Autoanalysers

Introduction: The electrolytes and Arterial Blood Gas (ABG) analysis are the crucial part of the evaluation in Coronavirus Disease 2019 (COVID-19) patients. Hyponatremia and hypokalemia are the electrolyte imbalance, commonly seen in COVID-19 and hence, patients require serial monitoring of electrolytes. Variations of sodium and potassium levels between arterial and venous blood are known, however as per existing literature, there are no previous studies on the comparison of electrolytes in COVID-19 patients, when analysed in arterial and venous blood, by different methods and its significance in clinical practice. Aim: To determine whether the sodium and potassium levels of COVID-19 patients are comparable when simultaneously analysed in arterial whole blood and venous serum sample, by ABG analyser and chemistry Autoanalyser (AA) respectively. Materials and Methods: This prospective observational study was conducted among COVID-19 positive patients admitted in Medical Intensive Care Unit at a Tertiary Care Super-specialty Hospital, Pune, Maharashtra, India, from February 2021 to June 2021. A total of 100 arterial and 100 venous blood samples of COVID-19 patients were analysed for sodium and potassium levels on Gem Premier 3000 Blood Gas Analyser and Auto-Quant 400i chemistry AA. The statistical analysis was done by the Bland Altman method to assess the agreement between the method of measurement for sodium and potassium levels in arterial and venous samples. Shapiro-Wilk's test was applied to check normal distribution and statistical variables in sodium and potassium values measured by two methods. Results: The mean values for sodium in arterial blood were 135.91 +/- 8.36 mmol/L and in serum was 140.26 +/- 8.49 mmol/L. The mean value of potassium in arterial blood was 4.12 +/- 0.76 mmol/L and in serum 4.41 +/- 0.67 mmol/L. Coefficients of variation for arterial and venous sample sodium level was 4.21 mmol/L and for potassium was 0.28, with bias (95% limits of agreement) of 4.96-3.46 mmol/L and 0.35-0.21 mmol/L, respectively. Conclusion: The present study found a significant difference in electrolyte levels when compared between arterial whole blood in ABG analyser and venous serum sample in chemistry autoanalyser in COVID-19 patients. So, the clinicians must be aware of these variations and the same has to be kept in mind, while interpreting the results in COVID-19.

Safe biochemical testing of COVID-19 samples: A clinical lab perspective

The current COVID-19 pandemic has not only greatly burdened healthcare system globally but also exposed the medical and paramedical staff to risk of infection. Although the major mode of transmission of this highly infectious disease is via close contact with an infected person, droplet infection due to coughing/sneezing and aerosol generation, few research articles have shown presence of SARS-CoV-2 in blood and serum. This poses a potential risk to health care professionals who are handling these samples. Once the suspected/confirmed case of COVID-19 is admitted in the hospital, setting it requires a battery of clinical chemistry investigations. Laboratory has a vital and indispensable role to play in the management of COVID-19 patients as several biochemical markers are used for prognostication as well as monitoring and guiding treatment in the critical patients. Hence, this evaluation was undertaken to have protocols based on robust recommendations and guidelines to be followed while handling the potentially infective samples in the clinical laboratories in order to ensure safety of the staff. However, these recommendations are based on the limited and rapidly evolving knowledge available at the moment and hence need to be reviewed periodically.