Detection of SARS-CoV-2 neutralizing antibodies: reliability of serological tests

Serological assays allow to explore the synthesis of antibodies against SARS-CoV-2 proteins. The gained knowledge finds applications in sero-epidemiology studies, to develop accurate forecasts of protective immunity spreading within a population. Indeed, last February, the European Centre for Disease prevention and Control (ECDC) recommended the use of serological tests for population surveys, in the context of epidemiological studies. The up-to-date reference method to determine the amount of protective antibodies is the in vitro neutralization test, which is expensive, timeconsuming and requires biosafety level 3 laboratories. The current study aims at highlighting the correlation between the results of the reference method and four different serological assays. Three immunoassays (Elecsys Anti SARS-CoV-2 S, MAGLUMI SARSCoV- 2 S-RBD IgG and MAGLUMI 2019-nCoV IgG test) and an ELISA surrogate viral neutralization test (EUROIMMUN SARS-CoV-2 NeutraLISA assay) have been studied on 83 patients, previously infected by SARS-CoV-2. The correlation between each serological test and the reference method is reported, as well as the predictive performances to distinguish serum samples with neutralizing antibody titers higher than 160. All the assays achieved good performances in terms of correlation with the reference method, as well as concerning the predictive ability over NAb titers > 160. The best correlation (Spearman coefficient = 0.784) and predictive features (area under curve = 0.921) have been observed for EUROIMMUN SARS-CoV-2 NeutraLISA assay, as displayed by ROC curve analyses.

THIRD COVID-19 VACCINE DOSE MAXIMIZES ANTIBODY TITER IN KIDNEY TRANSPLANT RECIPIENTS

BACKGROUND AND AIMS: COVID-19 infection in solid organ transplant recipients (SOT) is associated with increased morbidity and mortality due to comorbidities and immunosuppression state (Chaudhry ZS et al, 2020). Although vaccines represent the greatest hope to control COVID-19 pandemic, several studies showed the low immunogenicity of a two-dose mRNA COVID-19 vaccine regimen in SOT as compared with general population (Boyarsky BJ et al, 2021). Based on this evidence, on September 2021, the Italian Medicine Agency (AIFA) authorized a third vaccine administration as additional primary dose to immunocompromised patients. The aim of this study is to evaluate the seroconversion rate after the third dose of BNT162b2 (Pfizer-BioNTech) SARS-CoV-2 mRNA vaccine in kidney transplant recipients (KTRs) and to investigate the baseline factors associated with the absence of the antibody response. METHOD: we performed a prospective and observational study on a monocentric cohort of 329 consecutive Caucasian KTRs given three doses of the BNT162b2 COVID-19 vaccine. Key exclusion criteria were a previous history of COVID-19 infection and transplantation or having underwent chemotherapy treatment within the last year. Antibody response against the spike protein was tested on blood sample collected before the administration of vaccine (T0), at 15 and 90 days after the second dose (T2 and T3, respectively) and one month after the third dose (T5). The level of antibodies was assessed using the Roche Elecsys anti-SARS-CoV-2 S enzyme immunoassay (positive cut-off ≥ 0.8 U/mL). A total of 22 patients were excluded from the analysis because categorized as SARS-CoV-2-pre-immunized according to the antibodies' baseline status (T0) above the positivity cut-off. The Local Ethics Committees approved the study protocol and written informed consent was obtained before enrolment. RESULTS: The study population of 307 KTRs was 57.10 ± 13.10 years, with a predominance of male sex (64.2%). Median time from transplantation to vaccine was 10 [IQR 5-17] years. Blood analysis at baseline revealed mean eGFR assessed by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation to be 56.95 ± 23.04 mL/min/1.73 m2. The standard immunosuppressive regimen consisted of glucocorticoids in all patients, calcineurin inhibitors (88.6% of patients), antimetabolites (73.3% of patients) and mTOR inhibitors (in 15.6% of patients). The first two doses were administered 21 days apart, and the third dose was administrated 172 ± 4 days after the second dose. In our cohort, 43.3% patients (133/307) responded to the vaccine at T2. The proportion of responders increased to 68.4% (186/272) at T3 (median antibody level: 5.2 [0.40-74.07]). One month after the third dose, a positive antibody titer was detected in 251 of 307 patients (81.8%) (median antibody titre: 1137.50 [9.32- 4189.75]). The response curve starting at T2 and increasing at T3 makes apparent that there is a distinctive kinetic of humoral response in immunocompromised patients compared to immunocompetent individuals (Walsh EE et al., 2020). A multivariate analysis showed that the negative response to the third primary dose was associated with antimetabolite immunosuppressants (P = .001), lower estimated glomerular filtration rate (P < .001) and female sex (P = .04) (Figure 1). No serious adverse events were reported. Neither De novo DSAs nor change in proteinuria were reported after vaccination. The limitation of this study is the absence of assays for cellular immune response. CONCLUSION: Although the exact threshold of antibody titer for protection against SARS-CoV-2 infection remains unclear, the ability of the additional mRNA COVID- 19 vaccine dose to increase both immune response (Figure 2A) and the prevalence of seroconversion rate (Figure 2B) associated with the acceptable safety profile supports its use after an initial 2-dose mRNA COVID-19 primary vaccine series in immunocompromised patients. (Table Presented).

Evaluation of il-6 levels in covid-19 patients whit smell disorders

Smell dysfunction is one of the most frequent symptoms in COVID-19 patients. In the early stages of the disease it allows to identify positive subjects. The odorous substances recognize two different systems in the olfactory epithelium: the olfactory and the trigeminal systems that coexist and interact in the processing of sensory information. In COVID-19 patients there is an inflammatory reaction of the nasal mucosa. Infected supporting cells of the nasal mucosa release molecules that activate the local antiviral innate immune response. In fact, macrophages spread inflammatory mediators, in particular TNF-η , IL-6 and IL-1. In this study we compared IL-6 levels with the degree of olfactory disorders and with the type of unperceived odour.Materials and methodsFrom 15 March to 30 November 2020 have been selected 82 patients (45 men age 62.3 ±14.2 and 37 women age 57.1± 12.8) with only smell dysfunctions were divided into mild and moderate patients. The evaluation of the smell disorder was carried out with a 14 questionnaire relating to the perception of domestic odorous: 6 questions for olfactory sensitivity (own perfume usually sprayed, oregano, olive oil, nutella, coffee aroma, orange juice) and 8 for olfactory-trigeminal sensitivity (alcohol, fish odor, vinegar, mint (gum), toothpaste, shampoo, cheese, ammonia).The IL-6 (v.n. 0 - 7 pg/ml) was measured with chemiluminescence assay using Cobas e801 (Roche Instrumentation). Statistical analyses were performed with Wilcoxon Rank test, and Mann-Whitney test (p <0.05). ResultsThe trigeminal and olfactory sensitivity are more compromised in moderate than mild patients (p <0.05). The statistically significant differences there were in IL6 levels in moderate versus mild patients when there was an impairment of trigeminal sensitivity (p <0.05). Conclusion In this study suggested that the smell disorders in Covid-19 patients couldn't be a deficit of the olfactory central nervous pathways but could be rather than mainly associated with the inflammatory process of the nasal mucosa and that deficit of the type of domestic unperceived odour ('olfactory' or 'trigeminal' sensitivity) could indicate the degree of severity of the disease.

The university as a safe environment during the SARS-COV-2 pandemic: the experience of Bari Politecnico.

the SARS-CoV-2 pandemic started in December 2019 and still remains a major global health issue. Every country in the world has adopted drastic measures to contain the virus, although their stringency varies among countries, ranging from increased surveillance and focused interventions to strict lockdown (1). Italy was the second country where the disease had a major impact early in the pandemic, such that a strict nationwide lockdown was declared from March 9 to May 3, 2020. Nonetheless, between January and May 2020, there were 210,000 COVID-19 cases in Italy and 29,000 deaths were recorded (2). Due to the lockdown, universities (and in general all educational services) shifted to online classes, with students attending lessons and taking their exams from home. On-site activities were reduced to those considered indispensable. Research activities also had to be modified, such as by the adoption of a smart-working model (3). Between May and August 2020, the number of SARS-CoV-2 infections in Italy decreased. In response, the lockdown was loosened and some activities were restarted, albeit with specific safety protocols (social distancing, use of masks, temperature checks at the workplace entry, environmental disinfection, mixed models of smart and in-office work). These actions were accompanied by periodic serological and PCR screening tests (4).

Vitamin D and Interleukine 6 evaluation in Covid-19 patients: Our experience

Background and aim. Coronavirus disease 2019 (COVID-19) is a viral pandemic that emerged from East Asia and quickly spread to the rest of the world due to Coronavirus 2 SARS-CoV-2. The clinical manifestations are accompanied by the onset of an inflammatory storm (cytokine release syndrome CRS). The key role in the cytokine storm is played by interleukin 6 (IL-6) owing to its robust pro-inflammatory function. It induces a variety of acute-phase proteins, increasing body temperature. Vitamin D signaling has emerged as a key regulator of immunity in humans. Studies have shown that Vitamin D, acting in an intracrine fashion, is able to induce expression of antibacterial proteins. The net effect of these actions is to support increased bacterial killing in a variety of cell types. Aim of this study was to evaluate the correlation between Vitamin D and IL-6 in patients with laboratory-confirmed COVID-19. Methods. 160 patients with laboratory-confirmed COVID-19 (85 males-median age 62y, and 75 female-median age 66y) were recruited (between March and may 2020). Patients did not require intensive care admission and they aren't in treatment with drugs Vitamin D homologous before the access to the hospital. Vitamin D (ng/ml) and IL-6 (pg/ml) were measured using chemiluminescence method on TGSTSTechno Genetics and Cobas e 8100-Roche respectively in three consecutive days. Statistical analysis was obtained using MedCalc software and p-value threshold of 5% was adopted. Results. Linear regression line: Y=121,16-0,98 (x);intercept 121,16 (95% CI =99,42 to 151.15, P<0.0001);slope =-0,98 (95% CI =-3,29 to-0,35, P<0.0001). Pearson correlation coefficient: R=-0.62 (95% CI=-0.71 to-0.33, P <0.0001) show a statistically significant inverse correlation. Conclusions. In Covid-19 patients, the increase of Vitamin D concentration, mainly in the third day, correlated to IL-6 lowering may be considered a signal of better clinical outcome of these patients. Further studies are needed to confirm the Vitamin D utility in Covid-19 patient management and to better explain its role in immunity response.

THE HbA1c in COVID 19 patients

Purpose of the study: During the emergency period Covid-19 the glycosilate hemoglobin has been required for patients admitted to specialist wards suitable for metabolic control in diabetics and non-diabetics. Material and methods: The test HbA1c has been performed on Variant II Biorad, calibrated with standard Biorad for glycate hemoglobin, using EDTA whole blood samples after running two-tier Biorad controls. Results: The patients have been studied in the wave of coronavirus infection corresponding to the two months of loackdown. Thirty patients that have been hospitalized in covid area have been tested in our laboratory . In 12 females and 6 males with average age 50 years old the HbA1c has been resulted in the limits (up to 42 mmoli/moles) and in 10 patients ( 6 females and 4 males average age 48 years old ) higher values ( between 51 and 79 mmoli/molioli ) have been found. The patients have been so followed for the care of the case. Discussion and conclusion: Thus it has been possible to discern among patients with comorbidity and patients with lower degree of exposure to risks because in return to metabolic improvement and it has been possible to collaborate for the health of the sicks.

Correlation between Interleukin 6 (IL-6) and Human Epididymis Protein 4 (HE4) in Covid-19 patients: Two new potential biomarkers

Background and aim: Coronavirus disease 2019 (COVID-19) is a systemic illness, that recognizes the throat and nose as a gateway, due to Coronavirus 2 (SARS-CoV-2). In the lower respiratory tract, it is responsible of interstitial pneumonia developing into a severe acute respiratory distress syndrome. Human Epididymis Protein 4 (HE4) is present in many normal human tissues but also in the affected tissue of the SARSCoV-2 (oral cavity tissue, nasopharynx, respiratory tract). Many studies have shown the presence of HE4 in the lining fluid of the airway surface, secreted by the submucous glands. Its function is not fully clarified but HE4 seems to play an important role in the immune defense. Instead, in the bloodstream of COVID-19 patients, Interleukin 6 (IL-6) is the main inflammatory cytokine with a key role in the inflammatory storm (cytokine release syndrome, CRS) that accompanies the clinical onset of COVID-19 and thus represents a valid biomarker of the acute phase. Aim of this study was to evaluate the correlation between HE4 and IL-6 concentrations in patients with laboratory-confirmed COVID-19. Methods: 190 patients with laboratory-confirmed COVID-19 (115 males-median age 65y, and 75 female-median age 67y) were recruited (between March and May 2020). Patients did not require intensive care admission and they had no diagnosis of ovaric tumor, lung tumor and renal and/or lung fibrosis. HE4 (pmol/L) and IL-6 (pg/mL) were measured using chemiluminescence method by Architect i1000SR (Abbott) and Cobas e8100 (Roche) respectively in three consecutive days. Statistical analysis was obtained using MedCalc software and a p-value threshold of 5% was adopted. Results: Linear regression line: Y=11,87(x) + 78,16;intercept 11,87 (95% CI=7,22 to 16,21;P<0,05);slope=78,16 (95% CI=34,41 to 96,22;P<0,05). Pearson correlation coefficient: R=0.58 (95% CI=0,25 to 0,66;P<0,05) shows a statistically significant correlation. Conclusions: Based on our results, HE4 could play an important role in the inflammatory response in COVID-19 patients and it could be used as a potential biomarker with IL-6 in the management of patient COVID-19. Further studies are needed to confirm the usefulness of the HE4 assay and to better explain its possible role in the immune response.