Association of SARS-COV-2 viral RNAemia, IL- 6 gene polymorphism, serum IL-6 and peripheral blood lymphocytes and monocytes with disease severity in COVID-19 patients

Coronavirus disease 2019 (COVID-19) is a fatal pandemic viral disease caused by the severe acute respiratory syndrome corona virus type-2 (SARS-CoV-2). The aim of this study is to observe the associations of IL-6, SARS-COV-2 viral load (RNAemia), IL- 6 gene polymorphism and lymphocytes and monocytes in peripheral blood with disease severity in COVID-19 patients. This study was carried out from March 2021 to January 2022. RT-PCR positive 84 COVID-19 patients and 28 healthy subjects were enrolled. Blood was collected to detect SARS-COV-2 viral RNA (RNAemia) by rRT-PCR, serum IL-6 level by chemiluminescence method, SNPs of IL-6 by SSP-PCR, immunophenotyping of lymphocytes and monocyte by flow cytometry. Serum IL-6 level (pg/ml) was considerably high among critical patients (102.02 +/- 149.7) compared to severe (67.20 +/- 129.5) and moderate patients (47.04 +/- 106.5) and healthy controls (3.5 +/- 1.8). Serum SARS-CoV-2 nucleic acid positive cases detected mostly in critical patients (39.28%) and was correlated with extremely high IL-6 level and high mortality (R =.912, P < 0.001). Correlation between IL-6 and monocyte was statistically significant with disease severity (severe group, p < 0.001, and 0.867*** and critical group p < 0.001 and 0.887***). In healthy controls, moderate, severe and critically ill COVID-19 patients, IL-6 174G/C (rs 1800795) GG genotype was 82.14%, 89.20%, 67.85% and 53.57% respectively. CC and GC genotype had strong association with severity of COVID-19 when compared with GG genotype. Significant statistical difference found in genotypes between critical and moderate groups (p < 0.001, OR-10.316, CI-3.22-23.86), where CC genotype was associated with COVID-19 severity and mortality. The absolute count of T cell, B cell, NK cell, CD4+ T cells and CD8+ T cells were significantly decreased in critical group compared to healthy, moderate and severe group (P < 0.001). Exhaustion marker CD94/NKG2A was increased on NK cells and CD8+ cytotoxic T cell among critical and severe group. Absolute count of monocyte was significantly increased in critical group (P < 0.001). Serum IL-6, IL-6 174 G/C gene and SARS-CoV-2 RNAaemia can be used in clinical practice for risk assessment;T cell subsets and monocyte as biomarkers for monitoring COVID-19 severity. Monoclonal antibody targeting IL-6 receptor and NKG2A for therapeutics may prevent disease progression and decrease morbidity and mortality.Copyright © 2023 Elsevier Inc.

Winter-autumn air pollution control plan in North China modified the PM2.5 compositions and sources in Central China

The additional impact of emission-reduction measures in North China (NC) during autumn and winter on the air quality of downwind regions is an interesting but less addressed topic. The mass concentrations of routine air pollutants, the chemical compositions, and sources of fine particles (PM2.5) for January 2018, 2019, and 2020 at a megacity of Central China were identified, and meteorology-isolated by a machine-learning technique. Their variations were classified according to air mass direction. An unexpectedly sharp increase in emission-related PM2.5 by 22.7% (18.0 mug m-3) and 25.7% (19.4 mug m-3) for air masses from local and NC in 2019 was observed compared to those of 2018. Organic materials exhibited the highest increase in PM2.5 compositions by 6.90 mug m-3 and 6.23 mug m-3 for the air masses from local and NC. PM2.5 source contributions related to emission showed an upsurge from 1.39 mug m-3 (biomass burning) to 24.9 mug m-3 (secondary inorganic aerosol) in 2019 except for industrial processes, while all reduced in 2020. From 2018 to 2020, the emission-related contribution of coal combustion to PM2.5 increased from 10.0% to 19.0% for air masses from the local area. To support the priority natural gas quotas in northern China, additional coal in cities of southern China was consumed, raising related emissions from transportation activities and road dust in urban regions, as well as additional biofuel consumption in suburban or rural regions. All these activities could explain the increased primary PM2.5 and related precursor NO2. This study gave substantial evidence of air pollution control measures impacting the downwind regions and promote the necessity of air pollution joint control across the administration.Copyright © 2023 Elsevier Ltd

Third Booster Dose Vaccine against Sars-Cov-2 in Primary Sjogren's Syndrome: Longitudinal Cohort from the Safer Study

Objectives: Few studies evaluate the immunogenicity and safety of different COVID-19 vaccine platforms in patients with primary Sjogren's Syndrome (pSS). The present study aims to assess the immunogenicity through anti-spike IgG antibodies after the COVID-19 vaccine dose in heterologous groups compared to homologous regimen in patients with pSS. Method(s): These data are from the SAFER study: 'Safety and efficacy of the COVID-19 vaccine in rheumatic disease', a real-life phase IV multicenter longitudinal study, evaluating patients since before the first dose. Pregnant women, those with a history of serious adverse events prior to any vaccine, and those with other causes of immunosuppression were excluded. Patients with pSS > 18 years, classified according to ACR/EULAR 2016 classification criteria were included. Antibodies against the Receptor Binding Domain - RBD portion of the Spike protein of SARS-CoV-2 (IgG-S) were measured by chemiluminescence (Architect SARS-CoV-2 Quanti II, Abbott), before the first dose and 28 days after the 2nd and 3rd dose. Seropositivity was defined as IgG-Spike titers >=7.1 BAU/mL. Patients received adenoviral vector (ChAdOx1, Astrazeneca), mRNA (Pfizer) or inactivated SARS-COV-2 (Coronavac). Non-parametric methods were used. The alpha level of significance was set at 5%. Result(s): 56 participants received 3 doses, 46 +/- 11 years old, disease duration 7.62 years, 92.9% female, 41.1% White and 55.4% Mixed. The homologous third-booster dose group (n = 15, all ChAdOx1) and heterologous group (n = 41) were homogeneous for age, sex, ethnicity, comorbidities, medication and baseline IgG-S median [IQR] titers. After primary vaccination (2 doses) IgG-S median and titers [IQR] were similar in homologous and heterologous groups (373.03 [179.58, 843.92] vs. 473.36 [119.05, 1059.60], p = 0.705). Third-booster dose induced higher IgG-S median [IQR] titers compared to only 2 doses (1229.54 [333.55, 4365.47] vs 464.95 [140.42, 1015.25], p alpha 0.001). Heterologous 3rd-booster induced higher IgG-S median [IQR] titers than homologous scheme with ChAdOx1 (1779.52 [335.83, 4523.89] vs 730.76 [303.37, 1858.98], p = 0.150), Fig 1 and 2, although not statistically significant. Conclusion(s): Third booster dose induced higher humoral immune response compared to two doses whichmay improve protection against COVID-19 in patients with pSS. Although not statistically significant, the response to the heterologous scheme tended to be better than the response to the homologous booster vaccination, which heterologous booster scheme tended to respond better than homologous booster vaccination, which is relevant in this immunosuppressed population. Increasing the sample size will help clarify this issue. .

Safety and Efficacy of Covid-19 Immunization in Systemic Vasculitides - a Multicentric Cohort

Objectives: Patients with immune-mediated rheumatic diseases (IMRDs) develop more severe outcomes of Coronavirus disease 2019 (COVID-19). Recent studies have contributed to understand the safety and efficacy of COVID-19 vaccines in IMRDs, suggesting that different diseases and therapies may interfere on immunization efficacy. In this study we analyze the immunogenicity of COVID-19 vaccines in patients with Systemic Vasculitides (VASC), the rate of COVID-19 and the frequency of disease relapse following immunization. Method(s): We included patients with VASC (n = 73), a subgroup of the SAFER study (Safety and Efficacy on COVID-19 Vaccine in Rheumatic Disease), a longitudinal, multicenter, Brazilian cohort.We analyzed the geometric means of IgG antibody against receptor-biding domain of protein spike of SARS-CoV-2 (anti-RBD) after two shots of CoronaVac (Inactivated vaccine), ChadOx-1 (AstraZeneca) or BNT162b2 (Pfizer-BioNTech). IgG anti-RBD was measured by chemiluminescence test. We assessed new-onset COVID-19 episodes, adverse events (AE) and disease activity for each VASC. Result(s): The sample included Behcet's disease (BD) (n = 41), Takayasu arteritis (TAK) (n = 15), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) (n = 14), polyarteritis nodosa (n = 7) and other small vessel VASC(n = 6). The majority of patients were female (69%) without comorbidities (49%) and a median age of 37 years. The most common medication was conventional synthetic disease-modifying anti-rheumatic drugs, followed by biologic drugs. No patient received rituximab at baseline. Most patients received CoronaVac (n = 25) or ChadOx-1 (n = 36), while four received BNT162b2. Baseline IgG-RBD means were 1.34 BAU/mL. They increased to 3.89 and 5.29 BAU/mL after the 1st and 2nd vaccine dose, respectively. ChadOx-1 had higher antibody titers than CoronaVac (p = 0.002). There were no differences between different VASC. There were 3 cases of COVID-19 after immunization with CoronaVac. BD patients had a tendency for more cutaneous-articular activity following ChadOx-1. There were no severe relapses and no serious adverse events. Conclusion(s): Our results show the safety of different SARS-CoV-2 vaccines in VASC population. A progressive increase of IgG-RBD antibodies was observed after each dose. ChadOx-1 led to higher IgG-RBDgeometricmeans compared toCoronaVac. Finally, even though ChadOx-1 presented a tendency of triggering mild disease activity, there were no significant disease activity following vaccination in VASC patients. .

Effect of Covid-19 on nasal allergies: A study from hilly regions of Himachal Pradesh

Background: The World Health Organization (WHO) designated SARS-CoV-2 infection as coronavirus disease 2019 (Covid-19).Due to the government implication of Covid-19 specific guideline of using mask, there could be a significant decrease in the allergic rhinitis. Objective(s): Present study aims to analyze the changes in the trends of nasal allergies from hilly regions of Himachal Pradesh following Covid-19 pandemic. Method(s): The prospective data obtained from January 2022 to November 2022 was compared from the retrospective data available between January 2019 to November 2019. Prospectively, a total of 596 patients were included in the study. All these patients underwent Skin prick tests for common allergens. All these patients also underwent testing for total IgE levels in biochemistry lab of the hospital by chemiluminescence method.The results were compared with retrospective dataof 728 age sex match patients. Result(s): A significant difference in the allergen sensitivity was observed. The number of patients who were sensitized during Covid was comparatively less than those during Pre covid period.Dust mite, Cockroach, Peanut and Wheat revealed a non-significant odds ratio indicating that they were not true predictors for sensitization and non-sensitization. Whereas Grass pollen, Mould mix and Pine mix revealed a significant odds ratio. Usage of mask found to have an impact on improvement in symptoms. Majority of the patients who did not use mask had no improvement in symptoms. Majority of the patients had high IgE levels in pre covid period whereas it was normal for majority of them during covid. Conclusion(s): In our study, allergic rhinitis incidence decreased throughout the pandemic period. After pandemic, there was a noticeably decreased level of sensitivity to grass pollen, mould, and pine mix. Use of face masks lead to significant decrease in symptoms of allergic rhinitis.Copyright © 2022, Anka Publishers. All rights reserved.

Evaluation of Homologous and Heterologous Vaccine against Sars-Cov-2 in Systemic Lupus Erythematosus Patients: Data from Safer Study

Objectives: To evaluate the immunogenicity of ChAdOx1, Coronavac and BNT162B2 vaccines in SLE patients, including homologous and heterologous immunizations. Method(s): The 'Safety and efficacy on COVID-19 Vaccine in Rheumatic Disease-SAFER study' is a Brazilian multicentric longitudinal phase IV study to evaluate COVID-19 Vaccine in immune-mediated rheumatic diseases (IMRD) in real life, started on May 2021. SLE patients (according to the 2012 SLICC classification criteria), older than 18 years of age were recruited after 2 or 3 doses of vaccine against COVID-19 (ChAdOx1, BNT162b2 and CoronaVac) and were evaluated at baseline and on the 28th day after each dose. Homologous immunization was considered if they received three doses of the same vaccine and heterologous if a different one was applied. IgG antibody against SARS-CoV-2 spike receptor-binding domain were measured by chemiluminescence (SARS-CoV-2-IgG-II Quant assay, Abbott-Laboratories) at baseline and 28 days after the first, 2nd and 3rd doses (Seropositivity IgGSpike>= 7.1BAU/mL). Statistical analysis: ANOVA and pairwise comparisons tests Results: 316 SLE patients were included (255 heterologous and 61 homologous immunization), 89.2% were female and the mean age was 37.6 +/- 11.2 years. The two groups were homogeneous regarding demographical data, disease activity and immunosuppressive treatment. 49.7% used corticosteroids (alpha 5 mg/day in 52.3%), 83.5% antimalarials, 22.8% azathioprine and 20.3% mycophenolate mofetil. 207 patients received the first two doses with CoronaVac, 128 ChadOx-1 and 32 BNT162b2. Regarding the first two doses of the same vaccine, there was no difference in IgG titers over time between CoronaVac or ChadOx-1 (p = 0.313). IgG titers increased in all vaccine groups, with difference only after 2nd dose: 4.96 +/- 1.71BAU/mL CoronaVac vs. 6.00 +/- 1.99BAU/mL ChadOx-1 vs. 7.31 +/- 1.49BAU/mL BNT162b2 (p alpha 0.001). There was no difference in IgG titers over time between homologous or heterologous vaccine schedule (p = 0.872). IgG titers also increased in all groups, with difference only after 2nd dose: 5.49 +/- 1.96BAU/mL heterologous vs. 6.30 +/- 2.10BAU/mL homologous (p = 0.009). Conclusion(s): Induction of immunogenicity occurred in different vaccine regimens in SLE patients. Future research to explore different heterologous schemes in IMRD must be performed.

mRNA VACCINES INDUCES A HIGHER ANTIBODIES RESPONSE IN CHILDREN WITH PREVIOUS COVID-19

Background: mRNA vaccines elicit a durable humoral response to SARS-CoV-2 in adults, whereas evidence in children is lacking. This study aimed to evaluate the early and long-term immunological response after the BNT162b2 vaccine in children with or without a previous SARS-CoV-2 infection. Method(s): In a multicenter, prospective, observational study we profiled the immune response to the BNT162b2 vaccine in children aged 5-11 years attending the Pediatric Departments at the University of Padua and Bambino Gesu Children's Hospital in Rome (Italy). Forty-four healthy children (HC), 20 immune compromised (IC), and 18 children who previously developed MIS-C (MIS-C) were included in the study. Blood samples were collected pre-, 1, and 6 months after a 2-doses vaccination schedule. Neutralizing antibodies (NAbs) and anti-S-RBD IgG titers were analyzed through Plaque Reduction Neutralization Test (PRNT) and chemiluminescent immune-enzymatic assay (CLIA), respectively. B and T cell phenotypes were analyzed by flow cytometry. Geometric mean titers (GMTs) and 95% confidence intervals and median and interquartile range (IQR) of variables were evaluated according to pre-existing confirmed COVID-19. Result(s): Eighty-two children were studied;60 with a molecular-documented previous COVID-19 (Group A) and 22 without previous infection defined as the absence of antigen-specific antibodies before the vaccination (Group B). Overall, in Group A we observed higher NAbs GMTs, anti-S-RBD titers, and T- and B-reg cells than in Group A, at both 1 and 6 mo after vaccination (table);Nabs against the parental virus resulted to be greater in Group A than in Group B by a factor of 18 and 11, at 1 and 6 mo after vaccination, respectively. Both Groups recorded a decrease in antibody titers of approximately 50-70% between 1 and 6 months. A significant difference for Omicron NAbs (p=0.02) and anti-S-RBD (p=0.07) titers decay was observed between Group A and B;in contrast, Parental NAbs titers appeared to have similar trends in the 2 groups (p=0.47). Comparable antibody titers at 1 and 6 mo. (p=0.37) were detected across the three categories of HC, IC, and MIS-C (table). Conclusion(s): mRNA vaccination triggers a higher humoral response in children with a previous history of COVID-19, regardless of the immune deficiency or previous MIS-C, at least up to 6 mo, providing insight into boosting preexisting immunity with mRNA vaccines.

Interaction Between the Viral RNA Leader Sequence and nsp1 in SARS Coronavirus

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS-CoV), inhibits host translation thorough cleaving host mRNA and blocking the translation initiation site on the 40S ribosome. Stem-Loop-1 (SL-1) of the viral RNA leader sequence has been identified to bind to nsp1, allowing viral RNA to escape translation repression. However, the specific residues on nsp1 and the specific sequences on SL-1 important to binding have not been experimentally verified. To investigate this binding, we used gel-shift assay and RNA pull-down to verify binding between nsp1 and SL-1. By mutating SL- 1, we seek to identify the nucleotides of SL-1 that bind to nsp1. Based on recent literature, we hypothesized that disrupting the stem region of SL-1 will decrease binding between nsp1 and SL-1. Moreover, we seek to identify the residues important to binding to SL-1 by mutating specific amino acids of nsp1. Interestingly, nsp1 is a small protein (180 amino acids) with intrinsically unstructured regions at both C- and N-terminal ends of the protein. Based on recent literature we hypothesize that disrupting the R124 and K125 residues will decrease binding to SL-1. The results of this study will increase the knowledge of how viral RNA is able to escape suppression of host gene expression. To investigate the binding of nsp1 to SL1, we used nsp1 purified from bacterial lysate using glutathione beads followed by precision protease cleavage of GST-nsp1, and biotinylated RNA. LightShift Chemiluminescence RNA EMSA Kit (Promega) was used to detect the RNA in complex with nsp1 using a gel shift assay. Contrary to our hypothesis, we found an increase in nsp1 binding to the RNA carrying stem mutation, and a decrease in nsp1 binding to the RNA with the loop mutation. Moreover, we observed two distinct bands in the stem mutant indicating two possible binding sites on SL-1. Using an electrophoretic mobility shift assay, the loop region of SL-1 has been determined to be vital for binding to nsp1 in vitro. We hypothesize when the stem was mutated, we created a new binding site for nsp1. Currently we are further investigating several mutations in SL-1 to identify the actual binding site. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Prognostic value of initial KL-6 (Krebs von den Lungen 6) plasma level during COVID-19 pneumonia

Introduction: Krebs von den Lungen-6(KL-6) is useful in the diagnosis and severity assessment of diffuse interstitial lung disease. The objective of our study was to determine the prognostic value of the initial KL-6 plasma level in COVID-19 pneumonia Methods: All patients hospitalized between March 2020 and May 2020 for a suspected COVID-19 pneumonia in our Chest department of university hospital (Paris, France) were included. Initial referred as within 72 h of diagnostic suspicion, KL-6 plasma concentration in U/mL was measured by a ChemiLuminescent Enzyme Immunoassay (LUMIPULSE, Japan). Survival analyses were performed using a Cox regression and modeled by a Kaplan-Meier curve Results: 66 COVID-19 patients with initial KL-6 plasma measurement were analyzed, among whom 47 were men and average age was 64+/-14 yrs. Median KL-6 plasma concentration was 409+/-312 U/mL. KL-6 was significantly higher in men (p=0.003), elders (p=0.0001) and for higher Charlson's score (p=0.002). Higher KL-6 concentration was significantly associated with in-hospital mortality (HR: 8.66;95% CI:1.1-69.2), radiological extension of lesions on chest CT-scan (p=0.0040), higher WHO severity score (p=0.042), but not with admission in intensive care unit. In the 9 (14%) non surviving COVID-19 patients, KL-6 plasma concentration increased while it remained stable or decreased in survivors. At 3 months (n=48), DLCO was negatively correlated with the initial KL-6 value (r = -0.47, p=0.001) Conclusion(s): Initial KL-6 plasma concentration is associated with in-hospital mortality, unfavorable outcome, and persistent impairment of DLCO at 3 months. Initial KL-6 plasma determination appears as a prognostic biomarker in COVID-19 pneumonia.

Safety and immunogenicity of booster dose in patients with chronic liver disease

Background/Aims: Several studies showed that patients with liver cirrhosis have an immune dysregulation leading to poor immunological response to vaccination. However, in literature there are few data about the response to SARS-CoV-2 vaccination in patients with chronic liver disease (CLD). Aims of the study are (is) the evaluation of safety and immunogenicity of booster dose in patients with CLD. Methods: From September 2021 to April 2022, all consecutive outpatients with CLD who completed the primary vaccination course and the booster dose for anti-SARS-CoV-2 vaccination were enrolled. Blood samples were collected 12-16 weeks after second dose and after booster dose. Collected samples were analyzed for detecting anti-Spike protein IgG using LIAISON TrimericS IgG chemiluminescent assay (Diasorin, Italy). Results: We enrolled 340 patients (187 Males, mean age:64.32±17.34years). Stratified by the presence of cirrhosis, 249 had CLD and 91 were cirrhotic whose 57 (62.24%) had portal hypertension. At the end of the primary vaccination course, 60 patients (17.65%) did not develop a protective antibody titer, with no statistically significant differences between the two groups (19.7% in cirrhotic vs 16.8% in non-cirrhotic;p=0.076). The majority of them (53/60 patients;88.3%) developed a protective titer after booster dose, without differences between cirrhotics and non-cirrhotics (p=0.089). At multivariate analysis, factors associated with a higher humoral response after booster dose were young age (p=0.0098);porto-sinusoidal vascular disorder (p=0.005), none or a single comorbidity rather than two or more (p=0.05) and Spikevax booster dose compared with Comirnaty (p=0.001). Moreover, the antibody titer is inversely related to age (p=0.000). Conclusions: In a large cohort of patients with CLD booster dose of anti-Sars-CoV-2 vaccine has an excellent immunogenicity and leads to an adequate antibody response even in those who had not produced a protective titer after the primary vaccination course. Cirrhosis is not associated with a reduced humoral response.

Humoral and cellular immunogenicity against SARS-CoV- 2 mRNA vaccine in patients with inflammatory rheumatic disease, Comparison of mRNA-1273 vaccine and BNT162b2

Objectives: To assess humoral and cellular immune responses and safety profiles after two doses of different mRNA vaccine against SARS-CoV- 2;BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) in patients with rheumatic musculoskeletal disease (RMD). Method(s): We enrolled consecutive, previously uninfected RMD patients with inflammatory rheumatic diseases receiving mRNA vaccine including BNT162b2 and mRNA-1273. Healthy participants all receiving BNT162b2 were recruited as control. Blood samples were obtained 3weeks after second dose of vaccines. We measured titres of neutralizing antibodies against SARS-CoV- 2 with chemiluminescent enzyme immunoassay to evaluate humoral responses and assessed T-cell immunity responses with interferon releasing assay against SARS-CoV- 2 in a part of the patients. Adverse reaction symptoms were obtained from participants through questionnaire. Result(s): A total of 1040 RMD patients and healthy 621 control participants were enrolled. Among RMD patients with immunosuppressants, 704 were received BNT162b2 and 156 were received mRNA-1273. Neutralizing antibody titres 3 weeks after vaccination and positive seroconversion rates were significantly higher in healthy participants with BNT162b2 and RMD patients with mRNA-1273 compared with RMD patients with BNT162b2;neutralizing antibody titre, 23.9 +/- 14.2 IU/mL vs 29.4 +/- 33.9 IU/mL vs 10.8 +/- 16.5 IU/mL, p < 0.001;seroconversion rates, 99.5% vs 99.4% vs 80.2%, p < 0.001, respectively, We identified that age, glucocorticoid (prednisolone dose > 7.5mg/day), and use of immunosuppressants including methotrexate, mycophenolate and rituximab, are associated with attenuation of humoral responses in patients with BNT162b2. T cell reaction against SARS-CoV- 2 were also higher in patients with RMD vaccinated with mRNA-1273 than those with BNT162b2 (Interferon gamma levels for antigen 1, 3.2 +/- 6.5 IU/mL vs 0.6 +/- 1.3 IU/mL, p = 0.002;for antigen 2, 3.2 +/- 6.3 IU/mL vs 1.0 +/- 2.1 IU/mL, p = 0.021, respectively). Regarding adverse reaction of mRNA vaccine, the proportion of systemic adverse reactions including fever and general fatigue are significantly higher in healthy controls and RMD patients with mRNA-1273 than those with BNT162b2;fever, 46.2% vs 56.7% vs 14.3%, p < 0.001;general fatigue, 62.6% vs 73.0% vs 38.5%, p < 0.001, respectively, while the frequency of background RMD flare after vaccination were not significantly different between mRNA-1273 and BNT162b2 (5.2% [n = 8] vs. 3.7% [n = 26], p = 0.41) Conclusion(s): We demonstrated higher humoral, cellular immunogenicity of the SARS-CoV- 2 mRNA-1273 (Moderna) compared with the BNT162b2 (Pfizer-BioNTech) in RMD patients. Although reactogenicity including systemic adverse reaction including fever and fatigue were observed mRNA1273 vaccinated patients, proportion of RMD relapse were similar between the patients with mRNA-1273 and BNT162b2.

Normal Reference Values Evaluation of Rapid Chemiluminescent Adamts 13 Activity Assay

Introduction: Acquired thrombotic thrombocytopenic purpura (TTP) is a severe, rare, thrombotic microangiopathy (TMA). A diagnosis of acquired TTP is confirmed by a severe deficiency (< 10%) of ADAMTS13 activity. Recently, maybe because of Sars-COV2 Pandemic, in our laboratory we had the impression that normal reference ranges of our ADAMT13 activity assay HemosiL Acustar ADAMTS13 rapid immunoassay, could be larger than manufacturer's. The objective of this study was to evaluate whether manufacturer's are suitable normal reference values for this assay in our laboratory Methods: To evaluate manufacturer's reference limits (60-130.6%) in our laboratory we decided to assay with HemosIL Acustar ADAMTS13 activity test 30 plasma samples from normal subjects. Result(s): 3 out of 20 normal subjects tested showed ADAMTS13 activity outside manufacturer's limits (1 below 60.6% and 2 above 130.6%), therefore even if calculated in a small number of subjects (30 individuals) we decide to try to calculate in house ADAMTS13 reference values. They ranged from 47.5 (10th perc.), 72.5 (25th perc.) and 41.6 (mean- 2sd) to 150.0 (99th perce.), 119,6 (75th perc.) and 152.7 (mean+2sd). To investigate the analytical performance of manufacturer's and calculated cut-offs limits, we re-evaluate one year of ADAMTS13 activity results (95 subjects). ADAMTS13 activity was severely reduced (< 0.7%) in 10 acute TTPs;20 patients with Hemolytic Uremic Syndrome showed ADAMTS13 activity above manufacturer's and in all calculated cut-offs;21/45 patients with TTPs in remission phase showed ADAMTS activity below all in house calculated cut off and 24/45 showed ADAMTS activity below manufacturer's;12/20 other patients showed ADAMTS activity below all in house calculated and manufacturer's cut offs, all of them were Sars COV2 positive subjects Conclusion(s): In conclusion HemosiL Acustar ADAMTS13 activity calculated cut-offs in our laboratory were larger than manufacturer's reference range. By the analysis of one year ADAMTS13 activity dosages both manufacturer's and calculated cut-offs showed similar performances but our calculated reference ranges even if obtained by the analysis of a small number of normal subjects were found to be more similar to literature ELISA (40-130%) and FRET (45-147%) ADAMTS13 activity normal values.

High seroprevalence of anti-SARS-CoV-2 antibodies in the capital of Chad

Background. Since the start of the COVID-19 pandemic, Chad has had 7,417 confirmed cases and 193 deaths, one of the lowest in Africa. Objective. This study assessed SARS-CoV-2 immunity in N'Djamena. Methods. In August-October 2021, eleven N'Djamena hospitals col-lected outpatient data and samples. IgG antibodies against SARS-CoV-2 nucleocapsid protein were identified using ELISA. "Bambino Gesu" Laboratory, Rome, Italy, performed external quality control with chemiluminescence assay. Results. 25-34-year-old (35.2%) made up the largest age group at 31.9+/-12.6 years. 56.4% were women, 1.3 women/men. The 7th district had 22.5% and the 1st 22.3%. Housewives and students dominated. Overall seroprevalence was 69.5% (95% CI: 67.7-71.3), females 68.2% (65.8-70.5) and males 71.2% (68.6-73.8). >44-year-old had 73.9% seroprevalence. Under-15s were 57.4% positive. Housewives (70.9%), civil servants (71.5%), and health workers (9.7%) had the highest antibody positivity. N'Djamena's 9th district had 73.1% optimism and the 3rd district had 52.5%. Seroprevalences were highest at Good Samaritan Hospital (75.4%) and National General Referral Hospital (74.7%). Conclusion. Our findings indicate a high circulation of SARS-CoV-2 in N'Djamena, despite low mortality and morbidity after the first two COVID-19 pandemic waves. This high seroprevalence must be considered in Chad's vaccine policy. Copyright © 2022 The Authors and PAGEPRESS PUBLICATIONS.

Study of D-Dimer and Serum Ferritin Levels as Prognostic Biomarkers in Covid-19 Patients

Objective: Coronavirus disease 2019 (COVID-19) is primarily a respiratory illness causing thrombotic disorders. Pro-inflammatory cytokines are one of the responsible causes of cytokine storm syndrome in patients with COVID-19. Coagulopathy and inflammation are associated with COVID-19 severity. The coronavirus spike protein facilitates the entry of the virus into the target cells causing coagulopathy and inflammation.Other infections include direct viral toxicity, endothelial cell damage, inflammation, and deregulation of the immune response and renin-angiotensinaldosterone system. The study aims to estimate levels of D-Dimer and Serum Ferritin in symptomatic and asymptomatic COVID-19 patients and its comparison with healthy controls. Method(s): The study includes 30 healthy control and 30 symptomatic and 30 asymptomatic COVID-19 patients of both sexes. Analysis of serum ferritin was done on a fully automated immunology analyzer-SIEMENS based on the principle of chemiluminescence. D-dimer was estimated on mLab which is cartridge-based. Result(s): We observed that the levels of D-Dimer and Serum Ferritin significantly increased in symptomatic COVID-19 patients as compared to asymptomatic COVID-19 positive patients and healthy non-COVID-19 controls. Conclusion(s): The elevated serum ferritin and D-dimer were associated with a poor outcome and poor prognosis and could predict the worsening of COVID-19 patients. The significant increase showed that D-Dimer and serum ferritin accurately predicts patients developing severe COVID- infection. Copyright © 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Use cases for rapid antigen-detecting tests for COVID-19 screening and surveillance: a systematic review

Background. Testing remains critical to controlling the COVID-19 pandemic. Antigen-detecting rapid diagnostic tests (Ag-RDTs), which can be used at the point of care, have the potential to increase access to COVID-19 testing, particularly in settings with limited laboratory capacity. This systematic review synthesized literature on specific use cases and performance of Ag-RDTs for detecting SARS-CoV-2, for the first comprehensive assessment of Ag-RDT use in real-world settings. Methods. We searched three databases (PubMed, EMBASE and medRxiv) up to 12 April 2021 for publications on Ag-RDT use for large-scale screening and surveillance of COVID-19, excluding studies of only presumptive COVID-19 patients. We tabulated data on the study setting, populations, type of test, diagnostic performance, and operational findings. We assessed risk of bias using QUADAS-2 and an adapted tool for prevalence studies. Results. From 4313 citations, 39 studies conducted in asymptomatic and symptomatic individuals were included. Of 39 studies, 37 (94.9%) investigated lateral flow Ag-RDTs and 2 (5.1%) investigated multiplex sandwich chemiluminescent enzyme immunoassay Ag-RDTs. Six categories of testing initiatives were identified: mass screening (n=13), targeted screening (n=11), healthcare entry testing (n=6), at-home testing (n=4), surveillance (n=4) and prevalence survey (n=1). Sensitivity and specificity values by testing category are shown in the table. Ag-RDTs were noted as convenient, easy-to-use, and low cost, with a rapid turnaround time and high user acceptability. Risk of bias was generally low or unclear across studies. Conclusion. During the first year of the COVID-19 pandemic, Ag-RDTs were used across a wide range of real-world settings for screening and surveillance of COVID-19 in both symptomatic and asymptomatic individuals. Ag-RDTs were fast and simple to run, but due to their often low sensitivity, careful consideration must be given to their implementation and interpretation. Ag-RDTs have subsequently been rolled out more broadly and recommended for COVID-19 self-testing.

Clinical performance of Fujirebio Lumipulse G SARS-CoV-2 rapid antigen test

Introduction: SARS-CoV-2 infection has plagued the world for the past two years, during which time it has become necessary to use screening tests to prevent the spread of the virus, especially among high-risk patients. The gold standard for the detection of viral RNA is real-time PCR (RT-PCR). This technique requires special care when handling samples, as well as being time-consuming and costly. To meet the need for mass screening, antigenic tests were developed and continuously improved over the years. In our study, we examined the performance of Lumipulse G SARS-CoV-2 Ag (Fujirebio, Japan), a quantitative and automated chemiluminescence enzymeimmunoassay- based antigen test. Material(s) and Method(s): Our study includes 160 subjects (median age 38 years, interquartile range (IQR) 24-58 years;43% females) screened for SARS-COV-2 at the Service of Laboratory Medicine of Pederzoli Hospital (Peschiera del Garda, Verona, Italy), between August 16 and September 15, 2021. A nasopharyngeal swab was collected for each subject and analyzed at the same time by antigen test Fujirebio Lumipulse G SARS-CoV-2 Ag and by molecular test performed by Altona Diagnostics RealStar SARSCoV- 2 RT-PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Result(s): A significant Spearman's correlation was found between values of Fujirebio Lumipulse G SARS-CoV-2 Ag and measurable Ct values of SARSCoV- 2 of both the S (r= -0.94;p<0.001) and E genes (r= -0.95;p<0.001). The sensitivity and specificity at 1.0 pg/ mL (cut-off already used by other authors to discriminate samples positive for SARS-CoV-2) were 0.71 and 1.00. We also used a locally calculated threshold of 0.60 pg/mL (sensitivity 0.88;specificity 0.75). The exclusion of samples tested positive at molecular testing with Ct values comprised between 25-37 enabled the attainment of better diagnostic performance on the 103 residual samples using the 0.60 pg/mL cut-off. Conclusion(s): the results of our study confirm the good performance of Fujirebio Lumipulse G SARS-CoV-2 Ag (considering cutoff 1.0 pg/mL), especially in samples with high viral load (i.e., Ct value <25), which has proved even better using our locally-calculated cut-off (i.e., 0.60 pg/mL).

Evaluation of SARS-CoV-2 neutralizing antibodies before and after III dose of the vaccine

Introduction We all know that the most important task in combating COVID-19 pandemic is to produce enough effective vaccines and the greatest number of vaccinated subjects within a time frame. Nevertheless, the goal of this worldwide effort should be aligned to raise protective level of neutralizing antibodies (NAb) in vaccinees. It is clear that the NAb can block a viral invasion at the initial access to human receptors. Some methods for NAb measurement are commercially available.In this study, we evaluate the immune response of all employees receiving the III dose of BNT162b2 mRNA vaccine at November 2021. Serological NAb determination was performed before the administration of the III dose of vaccine (T0) and then 1 (T1) and 3 months (T2). Methods We collected serum samples of 46 laboratory workers at T0, T1 and T2. We determined the concentration of Anti-S IgG with SARS-CoV-2 IgG II Quant kit by chemiluminescence method on Alinity i Abbott (cutoff 50 AU/mL) in all samples and of NAb in 11 laboratory workers at T0, T1, T2 using SARS-CoV-2 Anticorpi Neutralizzanti kit (SGM Italia), an immunoturbidimetric method (high concentration >30 AU/mL, high % inibition >56%) on Alinity i Abbott platform. This method is able to detect NAb which bind specifically to the binding domain of the RBD receptor blocking the human ACE2 receptor. Statistical analysis was performed using MedCalc Software Ltd. Results At T0, T1 and T2, the mean+/-standard deviation concentration for anti-S IgG is 1016+/-1462, 22239+/-22480, 41777+/-30778 AU/mL and for NAb is 33+/-24, 100+/-100, 95+/-12, and the mean inibition percentage (%NAb) is 66+/-51, 309+/-105 and 320+/-164, respectively. Comparison between anti-S IgG and NAb at T0 showed a good correlation (R2=0.88);the NAb concentration was upper of linearity an all subjects at T1 and T2. Comparison between anti-S IgG and %NAb showed the same trend. Discussion The anti-S IgG are significantly reduced after 6 months from II dose of vaccine and increased about 30 times at T1. Concentration become half at T2 in all subjects who not affected by SARS-CoV-2 between T1 and T2. Although all subjects had a high %NAb at T0 that becomes 5 times higher at T1 and remains high at T2, 7 out of 11 subjects have been infected by SARS-CoV-2. Further studies are needed to define better the SARSCoV-2 neutralizing activity and the suitable routine test to measure them.

Antineutrophil Cytoplasmic Antibodies and Covid-19: A Manifestation of Immunological Dysregulation

Background: Coronavirus disease 2019 (COVID-19) is characterized by different manifestations, including an immune system imbalance. However, the specific mechanism that triggers a dysregulated immune response is not yet completely known. AntiNeutrophil Cytoplasmic Antibodies (ANCAs) are autoantibodies directed against various neutrophil antigens, including Myelo Per Oxidase (MPO) and PRoteinase 3 (PR3). In this study, we investigated the potential usefulness ofanti-MPO and anti-PR3 to elucidate whether the infection stimulates autoantibody production and contributes to autoimmunity activation in COVID-19 patients. Method(s): We assessedone hundred ten patients hospitalized for COVID-19, 62 (interquartile range [IQR], 52-72) years, admitted to COVID-19 Units at the University Hospital P. Giaccone of Palermo, Italy. Hematological, biochemical, and inflammatory parameters were evaluated. ANCA testing (anti-MPO and anti-PR3) was analyzed using a chemiluminescent assay (ACL AcuStar;Instrumentation Laboratory). Result(s): Laboratory results revealed a reduction in lymphocytes, higher levels of C Reactive Protein (CRP), and IL-6. In the great majority (76%) a moderate decrease in vitamin D levels was found. In addition, a weak increase in serum D-dimer and high-sensitive troponin T (hs-TnT) concentrations were observed in 37% and 51% of patients. Our analysis showed that anti-MPO and anti-PR3 antibodies were present in <2% and <5%, respectively, of study population. Conclusion(s): It has been known that SARS-CoV-2 can trigger a strong immune response in some individuals. Our results do not show greater activation of autoimmune response given the low rate of ANCAs positivity encountered. However, the study is ongoing for a long-term evaluation of patients.

Evaluation of SARS-CoV-2 antibodies dinamics after booster dose

Introduction At november 2021 in our public hospital, all employees have been received the III dose of SARS-CoV-2 vaccine with BNT162b2 mRNA. To evaluate the immune response to the vaccine, the antibodies measurement directed against the S protein or, more specifically, against the RBD domain stimulated by vaccination, was performed. The aim of this study is to evaluate SARS-CoV-2 antibody dinamics over 6 months after vaccine (at T0, T1, T2, T3: before III dose, at 1, 3 and 6 months, respectively). Methods We collected serum samples from 46 laboratory workers at T0, T1, T2, T3 (between november 2021 and may 2022). Serologic testing for specific SARS-CoV-2 anti-RBD IgG were performed by chemiluminescence method on Alinity Abbott instrument;according to manifacturer, the cut-off is 50 AU/mL. All samples were tested also for anti-N IgG with chemiluminescence assay on Alinity Abbott instrument (cutoff is 1.4 AU/mL) to evaluate subjects affected by COVID-19. Results At T0, the mean concentration of anti-S IgG is 581+/-303 AU/mL for 42 subjects (91%) that received II dosis until to April 2021 whereas the mean concentration is 3963+/-1386 AU/mL for 4 workers (9%) affected by SARS-CoV-2 between I dosis and november 2021. At T1, the mean concentration of anti-S IgG is 23326+/- 15905 AU/mL (min/ max value 4766 and 69816 AU/mL) without difference among subjects that had the upper concentration at T0. At T2, the mean concentration of anti-S IgG is 27374+/-27057 AU/mL (min/max value 1038 and 80000 AU/mL) with mean concentration of 60561+/-22281 AU/mL in 6 subjects affected by SARS-CoV-2 between T1 and T2.At T3, the mean concentration of anti-S IgG is 20610+/-12403 AU/mL (min/max value 1825 and 39796 AU/mL) with mean concentration of 22525+/-10121 AU/mL in 5 subjects affected by SARS-CoV-2 between T2 and T3. Discussion We found that booster dose of the vaccine triggers robust immune responses in healthy recipients, COVID-19 triggers an earlier and more intense immune response even after 2-3 months from III dose;in all cases, however, antibody titers remain at high levels in COVID-19 recovered patients. Although virus infection among vaccinated subjects is rare, this would seem to promote a more intense immune response after boosting dose, inducing antibody titers significantly higher and likely more durable.

A retrospective evaluation of seroconversion after COVID-19 during the early Omicron wave in fully vaccinated MS patients receiving B-cell depletion therapies

Introduction: Patients with multiple sclerosis (MS) are commonly treated with B-cell depletion therapies (BCDTs). Reduced seroconversion following COVID-19 vaccination in patients receiving certain BCDTs has been reported, however the immune response following natural infection is poorly understood. Objective(s): This study aimed to evaluate COVID-19 antibody responses after vaccination and natural infection in BCDT-treated patients. This single-centre study evaluated COVID-19 seroconversion and spike protein antibody titres for double-vaccinated MS or neuroinflammatory disease patients treated with BCDT (n=33) with confirmed COVID-19 infection (n=16) or uninfected by COVID-19 (control;n=17). Method(s): We performed a retrospective review of patients at the Yale MS Center who had systematically checked COVID-19 spike antibody levels among patients treated with BCDTs (ocrelizumab [OCR], n=24;rituximab [RTX], n=5;ofatumumab [OFT], n=4). Data were collected from Mar 2020 to Feb 2022. All patients had received >=2 doses of FDA-approved COVID-19 vaccine. Qualitative spike antibody seropositivity was determined based on test-specific lab reference ranges. For a subset of patients (n=18), quantitative spike antibody levels were assessed via DiaSorin Liaison chemiluminescence assay (positive titre of >=13;OCR, n=13;RTX, n=3;OFT, n=2). Vaccination and COVID-19 infection dates were also recorded. Patients were monitored for health effects following COVID-19. Result(s): Overall,16/33 (48%) patients seroconverted post full vaccination. After COVID-19 infection, 15/16 (94%) seroconverted, while 7/17 (41%) of uninfected patients seroconverted after vaccination. For the 18 patients with quantitative COVID-19 spike antibody titres, mean titres post-vaccination were 37.38. Mean antibody titres were significantly higher after COVID-19 infection;540.32 vs 20.1 in the control group (p<0.05). Of the 16 infected patients, 15 had mild COVID-19 symptoms and 1 was asymptomatic. No hospitalizations or deaths were reported. Conclusion(s): This study reports that COVID-19 spike antibody titres in fully vaccinated, BCDT-treated patients were significantly increased post-infection compared to the control group. BCDT-treated patients infected with COVID-19 displayed mild infection or were asymptomatic, with no hospitalizations or deaths. These results provide reassurance that BCDTs in doublevaccinated MS patients do not preclude an appropriate COVID-19 antibody response post infection.