Effects of Different Types of Recombinant SARS-CoV-2 Spike Protein on Circulating Monocytes' Structure.

This study investigated the biological effects on circulating monocytes after challenge with SARS-CoV-2 recombinant spike protein. Whole blood collected from seven ostensibly healthy healthcare workers was incubated for 15 min with 2 and 20 ng/mL final concentration of recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Samples were analyzed with Sysmex XN and DI-60 analyzers. Cellular complexity (i.e., the presence of granules, vacuoles and other cytoplasmic inclusions) increased in all samples challenged with the recombinant spike protein of the Ancestral, Alpha, and Delta variants, but not in those containing Omicron. The cellular content of nucleic acids was constantly decreased in most samples, achieving statistical significance in those containing 20 ng/mL of Alpha and Delta recombinant spike proteins. The heterogeneity of monocyte volumes significantly increased in all samples, achieving statistical significance in those containing 20 ng/mL of recombinant spike protein of the Ancestral, Alpha and Delta variants. The monocyte morphological abnormalities after spike protein challenge included dysmorphia, granulation, intense vacuolization, platelet phagocytosis, development of aberrant nuclei, and cytoplasmic extrusions. The SARS-CoV-2 spike protein triggers important monocyte morphological abnormalities, more evident in cells challenged with recombinant spike protein of the more clinically severe Alpha and Delta variants.

SARS-CoV-2 antibodies in inflammatory neurological conditions: a multicentre retrospective comparative study.

It is well established that neurological and non-neurological autoimmune disorders can be triggered by viral infections. It remains unclear whether SARS-CoV-2 infection induces similar conditions and whether they show a distinctive phenotype. We retrospectively identified patients with acute inflammatory CNS conditions referred to our laboratory for antibody testing during the pandemic (March 1 to August 31, 2020). We screened SARS-COV-2 IgA/IgG in all sera by ELISA and confirmed the positivity with additional assays. Clinical and paraclinical data of SARS-COV-2-IgG seropositive patients were compared to those of seronegative cases matched for clinical phenotype, geographical zone, and timeframe. SARS-CoV-2-IgG positivity was detected in 16/339 (4%) sera, with paired CSF positivity in 3/16. 5 of these patients had atypical demyelinating disorders and 11 autoimmune encephalitis syndromes. 9/16 patients had a previous history of SARS-CoV-2 infection and 6 of them were symptomatic. In comparison with 32 consecutive seronegative controls, SARS-CoV-2-IgG-positive patients were older, frequently presented with encephalopathy, had lower rates of CSF pleocytosis and other neurological autoantibodies, and were less likely to receive immunotherapy. When SARS-CoV-2 seropositive versus seronegative cases with demyelinating disorders were compared no differences were seen. Whereas seropositive encephalitis patients less commonly showed increased CSF cells and protein, our data suggest that an antecedent symptomatic or asymptomatic SARS-CoV-2 infection can be detected in patients with autoimmune neurological conditions. These cases are rare, usually do not have specific neuroglial antibodies.

Clinical assessment of SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay.

OBJECTIVES: Given that SARS-CoV-2 antigen tests will represent a pillar for supporting or surrogating molecular testing in the endemic period, we report here the clinical performance of the new SNIBE Maglumi SARS-CoV-2 antigen fully-automated chemiluminescent immunoassay (MAG-CLIA SARS-CoV-2 Ag). METHODS: The study population consisted of 181 subjects (mean age 61 ± 21 years; 92 females) undergoing coronavirus disease 2019 (COVID-19) testing at the local diagnostic facility, from December 2022 to February 2023. Routine diagnostic practice involved the collection of a double nostril nasopharyngeal swab, analyzed in duplicate with SARS-CoV-2 antigen (MAG-CLIA SARS-CoV-2 Ag) and molecular (Altona Diagnostics RealStar SARS-CoV-2 RT-PCR Kit) tests. RESULTS: A significant Spearman's correlation was found between MAG-CLIA SARS-CoV-2 Ag and mean Ct values of SARS-CoV-2 E and S genes (r=-0.95; p<0.001). In all nasopharyngeal samples, the area under the curve (AUC) of MAG-CLIA SARS-CoV-2 Ag was 0.86 (95% CI, 0.81-0.90), with 0.71 sensitivity and 1.00 specificity at 7 ng/L cut-off, increasing to 0.98 (95% CI, 0.96-1.00) AUC and 0.96 sensitivity (with 0.97 specificity) in high viral load samples. When SARS-CoV-2 N protein concentration was replaced with raw instrumental readings (i.e., relative light units [RLU]), the AUC in all samples increased to 0.94. A RLU value of 945 was associated with 88.4% accuracy, 0.85 sensitivity, 0.95 specificity, 0.77 negative predictive value (NPV) and 0.97 positive predictive value (PPV), respectively. CONCLUSIONS: We found satisfactory analytical performance of MAG-CLIA SARS-CoV-2 Ag, which could be used as surrogate of molecular testing for identifying high viral load samples. Broadening the reportable range of values may generate even better performance.

Positivization time of a COVID-19 rapid antigen self-test predicts SARS-CoV-2 viral load: a proof of concept.

OBJECTIVES: This proof of concept study was aimed to validate the hypothesis that the time of positivization of SARS-CoV-2 self-performed rapid diagnostic tests (RDTs) may reflect the actual viral load in the specimen. METHODS: A SARS-CoV-2 positive sample with high viral load was diluted and concomitantly assayed with molecular assay (Xpert Xpress SARS-CoV-2) and RDT (COVID-VIRO ALL IN RDT). The (mean cycle threshold; Ct) values and RDT positivization times of these dilutions were plotted and interpolated by calculating the best fit. The parameters of this equation were then used for converting the positivization times into RDT-estimated SARS-CoV-2 Ct values in routine patient samples. RESULTS: The best fit between measured and RDT-estimated Ct values could be achieved with a 2-degree polynomial curve. The RDT-estimated Ct values exhibited high correlation (r=0.996) and excellent Deming fit (y=1.01 × x - 0.18) with measured Ct values. In 30 consecutive patients with positive RDT test, the correlation between RDT positivization time and measured Ct value was r=0.522 (p=0.003). The correlation of RDT-estimated and measured Ct values slightly improved to 0.577 (Deming fit: y=0.44 × x + 11.08), displaying a negligible bias (1.0; 95% CI, -0.2 to 2.2; p=0.105). Concordance of RDT-estimated and measured Ct values at the <20 cut-off was 80%, with 0.84 sensitivity and 0.73 specificity. CONCLUSIONS: This proof of concept study demonstrates the potential feasibility of using RDTs for garnering information on viral load in patients with acute SARS-CoV-2 infection.

Assessment of humoral and cellular immunity after bivalent BNT162b2 vaccination and potential association with reactogenicity.

OBJECTIVES: This study investigated the feasibility and clinical value of using a novel, automated and high-throughput SARS-CoV-2 Interferon Gamma Release Assay (IGRA), combined with total anti-SARS-CoV-2 antibodies assessment, for evaluating the immune response after bivalent BNT162b2 vaccination. METHODS: A cohort of healthcare workers, who already underwent primary vaccination and boosting with monovalent BNT162b2 vaccine, received a booster dose of the new BNT162b2 bivalent formulation. Blood samples were taken immediately before vaccination (T0) and 1 month afterwards (T1). Humoral and cellular immunity were assayed with Roche Elecsys Anti-SARS-CoV-2 and Roche Elecsys IGRA SARS-CoV-2, respectively. RESULTS: The study population consisted of 51 subjects (median age: 43 years; 51% females). Total anti-SARS-CoV-2 antibodies and IGRA SARS-CoV-2 values increased at T1 from 9,050 to 25,000 BAU/mL (p<0.001), and from 0.44 to 0.78 IU/mL (p=0.385), accounting for median increase of 2.0 and 1.6 folds, respectively. Increased T1 values of total anti-SARS-CoV-2 antibodies and IGRA SARS-CoV-2 were recorded in 100% and 68.6% subjects, respectively. In those with baseline values below the median, post-vaccine levels displayed larger increases of 3.3 and 5.1 folds for anti-SARS-CoV-2 total antibodies and IGRA SARS-CoV-2, respectively. The variation of total anti-SARS-CoV-2 antibodies was inversely associated with their T0 values (r=-0.97; p<0.001), whilst that of IGRA SARS-CoV-2 was inversely associated with its T0 value (r=-0.58; p<0.001). No other signifcant associations were found with demographical or clinical variables, including side effects. CONCLUSIONS: The bivalent BNT162b2 vaccine booster enhances humoral and cellular immunity against SARS-CoV-2, especially in recipients with lower baseline biological protection.

Clinical performance of Fujirebio Lumipulse G SARSCoV- 2 Ag chemiluminescent immunoassay.

We evaluated the performance of Fujirebio Lumipulse G SARS-CoV-2 Ag chemiluminescent immunoassay. A nasopharyngeal swab was collected from 160 subjects and assayed simultaneously with Fujirebio Lumipulse G SARS-CoV-2 Ag and Altona Diagnostics RealStar SARS-CoV-2 RT-PCR assays. Using 0.60 pg/mL diagnostic threshold, Fujirebio Lumipulse G SARS-CoV-2 Ag displayed 0.88 area under the curve, 0.88 sensitivity and 0.75 specificity compared to molecular testing. The area under the curve increased to 1.00 after excluding samples with low viral load (i.e., cycle threshold values between 25-37). Thus, this chemiluminescent immunoassay could be used for rapid identification of many subjects with high nasopharyngeal SARS-CoV-2 viral load.

Correlation Between Time to Positive Result of SARS-CoV-2 Rapid Antigen Self-Test and Viral Antigen Concentration

Background This study was planned to investigate how the positivization time of a SARS-CoV-2 rapid antigen self-test may correlate with SARS-CoV-2 nucleocapsid (N) antigen concentration measured with a quantitative laboratory-based immunoassay. Methods Paired nasopharyngeal (healthcare-collected) and nasal (self-collected) samples were taken from patients undergoing routine SARS-CoV-2 testing. The concentration of SARS-CoV-2 antigen nucleocapsid (N) was assayed with Liaison SARS-CoV-2 Antigen test, whilst the time of positivization of COVID-VIRO ALL rapid diagnostic test (RDT) was concomitantly measured and then compared SARS-CoV-2 viral load measured with Liaison SARS-CoV-2 Antigen test and expressed as Median Tissue Culture Infectious Dose (TCID50)/mL. Results The study sample consisted of 32 paired specimens which tested positive with COVID-VIRO ALL IN RDT and had SARS-CoV-2 N protein concentration measured with Liaison SARS-CoV-2 Antigen test. A highly significant correlation was found between SARS-CoV-2 viral antigen concentration and RDT positivization time (r=-0.64;95%CI, -0.81 to -0.38;p<0.001). At the >1500 TCID50/mL threshold of the Liaison SARS-CoV-2 Antigen test, the positivization time of the COVID-VIRO ALL IN RDT displayed high accuracy (93.7%). A positivization time <42 sec enabled to identify patients with high SARS-CoV-2 antigen concentration (i.e., >1500 TCID50/mL) with 91.3% negative and 100% positive predictive values. Conclusion Self-testing using COVID-VIRO ALL IN RDT could be reliably used for garnering valuable information on the actual SARS-CoV-2 viral antigen concentration in respiratory samples.

Real-world assessment of the clinical performance of COVID-VIRO ALL IN rapid SARS-CoV-2 antigen test.

OBJECTIVES: Since the external validation of severe acute respiratory syndrome coronavirus 2 antigen rapid diagnostic tests (SARS-CoV-2 RDT-Ags) is a necessary requisite before they can be introduced into routine clinical practice, this study reports the results of a real-world assessment of the clinical performance of the new COVID-VIRO ALL IN device. METHODS: The study population consisted in 165 outpatients (median age: 43 years, range: 14-68 years; 66.1% females) who had paired nasal and nasopharyngeal samples collected upon hospital presentation. The samples were concomitantly tested with the AAZ-LMB COVID-VIRO ALL IN SARS-CoV-2 RDT-Ag and with Cepheid Xpert Xpress SARS-CoV-2 real-time reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The number of subjects with positive RT-PCR results (i.e., mean Ct value <45) was 116 (70.3%), 109 (66.1%) and 86 (52.1%) with mean Ct values <37 and <30, respectively. In all RT-PCR positive samples, COVID-VIRO ALL IN displayed 78.8% agreement, 0.698 sensitivity, 1.000 specificity, 0.583 negative predictive value (NPV) and 1.000 positive predictive value (PPV) compared to RT-PCR. The median Ct value of samples testing positive with COVID-VIRO ALL IN was significantly lower than those testing negative (22.8 vs. 32.2; p<0.001). In samples with high viral load (i.e., Ct value <30), COVID-VIRO ALL IN displayed 92.1% agreement, 0.895 sensitivity, 0.949 specificity, 0.983 NPV and 0.951 PPV compared to RT-PCR. CONCLUSIONS: Although the diagnostic performance of COVID-VIRO ALL IN do not exactly match those of the manufacturer, its high NPV in high viral load samples would enable fast-track and rapid identification of highly contagious subjects.

The strength of association between pre-and post-booster BNT162b2 anti-SARS-CoV-2 antibodies levels depends on the immunoassay.

OBJECTIVES: Reliable evidence suggests that anticipating the humoral response to coronavirus disease 2019 (COVID-19) vaccines is essential for predicting their clinical effectiveness. In this work, we sought to determine the extent to which the response of anti-SARS-CoV-2 antibodies BNT162b2 booster measured with four different commercial immunoassays could be predicted after initial homologous vaccination. METHODS: This observational study enrolled 181 SARS-CoV-2 baseline seronegative healthcare workers (mean age 42±13 years; 59.7% females), who received two doses of the BNT162b2 vaccine. Antibodies levels were assessed with Roche Elecsys Anti-SARS-CoV-2 S, ACCESS SARS-CoV-2 IgG II, Snibe S-RBD IgG, and LIAISON SARS-CoV-2 TrimericS IgG. The correlation of anti-SARS-CoV-2 serum antibodies 21 days after the first vaccine dose and 30 days after the second dose was assessed with Pearson's test. RESULTS: A significant correlation was found between serum anti-SARS-CoV-2 antibodies levels after the first (T1) and second (T2) BNT162b2 vaccine dose with all immunoassays, though the strength of such association depended on the immunoassay. Briefly, the highest correlation was found for LIAISON SARS-CoV-2 TrimericS IgG (r=0.71), followed by ACCESS SARS-CoV-2 IgG II (r=0.65), Snibe S-RBD IgG (r=0.52), and then Roche Elecsys Anti-SARS-CoV-2 S (r=0.40). CONCLUSION: The value of predicting post-booster values of anti-SARS-CoV-2 antibodies levels from pre-booster levels significantly depends on the immunoassay used.

Variation of Total Anti-SARS-CoV-2 Antibodies After Primary BNT162b2 Vaccination and Homologous Booster

Background In this serosurveillance study, we investigated the variation of total anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antibodies in healthcare workers receiving primary BNT162b2 vaccination and homologous booster. Methods A total number of 524 subjects (median age, 46 years;65.3% females), were studied. All received primary BNT162b2 vaccination (two doses) and homologous booster (one dose) >8 months after completing the primary cycle. Blood samples were collected before the first and second vaccine doses, at 1, 3 and 6 months after the second dose, as well as before and 1 month after booster. Total anti-SARS-CoV-2 neutralizing antibodies were assayed with Roche Elecsys Anti-SARS-CoV-2 S chemiluminescent immunoassay. Results Overall, 65.1% subjects were baseline (i.e., pre-vaccination) SARS-CoV-2 seronegative and always tested SARS-CoV-2 negative (“N/N”), 16.2% were baseline SARS-CoV-2 seronegative but tested SARS-CoV-2 positive after receiving the vaccine booster dose (“N/P”), whilst 18.7% were baseline SARS-CoV-2 seropositive and always tested SARS-CoV-2 negative afterwards (“P/N”). All groups displayed a similar trend of total anti-SARS-CoV-2 S antibodies throughout the study period, though the P/N cohort exhibited higher values compared to the other two groups until receiving the booster, after which the levels become similar in all cohorts. Significant differences in total anti-SARS-CoV-2 S antibodies values were not found between N/N and N/P groups, neither 1 month after booster. The rate of subjects with protective antibodies values become 100% in all groups after booster. Conclusions Although baseline seropositivity is associated with more pronounced humoral immune response following primary vaccination compared to never infected subjects, SARS-CoV-2 infection after booster does not significantly foster antibody titers.

Association between viral load and positivization time of a SARS-CoV-2 rapid antigen test in routine nasopharyngeal specimens.

Background: Rapid SARS-CoV-2 antigen tests are potentially useful tools for screening carriers with high viral load. This study was aimed to assess the potential association between viral load and positivization time of a manual SARS-CoV-2 commercial antigen test in routine nasopharyngeal specimens. Methods: In a sample of subjects undergoing routine diagnostic testing, SARS-CoV-2 positivity of nasopharyngeal samples was assayed with both molecular (Altona Diagnostics RealStar SARS-CoV-2 RT-PCR Kit) and antigenic (Roche SARS-CoV-2 Rapid Antigen Test) tests. Positivization time of rapid antigen test was correlated and compared with viral load expressed as mean of SARS-CoV2 E/S genes cycle threshold (Ct) values. Results: The study sample consisted of 106 patients (median age 48 years, 55 women) with positive results of rapid SARS-CoV-2 antigen testing. A highly significant Spearman's correlation was found between mean SARSCoV-2 E/S genes Ct values and positivization time of manual antigen test (r= 0.70; p<0.001). The positivization time of rapid SARS-CoV-2 antigen test displayed an area under the curve of 0.82 (95%CI, 0.74-0.89) for predicting nasopharyngeal samples with high viral load (i.e., mean Ct <20). A positivization time cut-off of 32 SEC had 94.9% sensitivity and 58.2% specificity for detecting specimens with high viral load. The overall agreement between mean Ct value <20 and positivization time <32 SEC was 70.8%. Conclusions: Positivization time of rapid SARS-CoV-2 antigen tests may provide easy and rapid information on viral load, thus making this type of manual assay potentially suitable for quick and reliable detection and isolation of supercarriers.

Impact of BNT162b2 primary vaccination and homologous booster on anti-SARS-CoV-2 IgA antibodies in baseline seronegative healthcare workers.

Objectives: We investigated here the response of anti-SARS-CoV-2 IgA antibodies to BNT162b2 primary vaccination followed by administration of a homologous booster dose in baseline seronegative healthcare workers. Methods: The study population included 69 healthy recipients of primary BNT162b2 vaccination (two doses) followed by administration of a single homologous booster after 8 months. Blood samples were collected throughout the study, starting before the first vaccine dose, up to 1 month after the booster. The serum levels of anti-SARS-CoV-2 IgA were assayed with Euroimmun Anti-SARS-CoV-2 spike S1 ELISA IgA. Results: A first peak of serum anti-SARS-CoV-2 IgA was seen 1 month after the second BNT162b2 dose, after which values gradually declined, with stabilization after 6 months. The BNT162b2 booster (third dose) elicited a second peak, comparable to that observed 1 month after the second dose (p=0.100). Highly significant correlation was found between pre- and post-booster anti-SARS-CoV-2 IgA serum values (r=0.41; p<0.001), whilst no significant correlation was observed with age (r=0.10; p=0.416) or sex (r=0.04; p=0.729). The rate of SARS-CoV-2 IgA seropositive recipients increased from 0% before vaccination to 80 and 97% after the first and second vaccine dose, but then declined becoming 74% at 3 months and 54% at 6 months, respectively, after which stabilization was reached. The BNT162b2 booster dose restored the seropositivity rate to 99%. Conclusions: These results support the suggestion that vaccine boosters may be advisable after 3 months from primary vaccination to restore IgA to protective levels, especially in those at higher risk of SARS-CoV-2 infection and complications.

Correlation between Anti-SARS-CoV-2 Total Antibodies and Spike Trimeric IgG after BNT162b2 Booster Immunization.

OBJECTIVE: In this work we monitored both total and IgG anti-SARS-CoV-2 antibodies responses after BNT162b2 vaccine booster immunization in a cohort of ostensibly healthy healthcare workers. METHODS: The study population consisted of 266 subjects (median age, 46 years and interquartile range (IQR), 35-52 years; 168 females) undergoing homologous 30-µg BNT162b2 booster administration. Serum samples were collected immediately before the booster dose and 1 month after. RESULTS: The concentration of anti-SARS-CoV-2 RBD total antibodies and anti-SARS-CoV-2 spike trimeric IgG increased by 31 (IQR, 16-53) and 22 (IQR, 11-43) folds, respectively, after receiving the BNT162b2 vaccine booster. A highly significant Spearman's correlation was found between the relative increase (i.e., ratio of post-booster and pre-booster serum values) of anti-SARS-CoV-2 RBD total antibodies and anti-SARS-CoV-2 spike trimeric IgG (r = 0.86; p < 0.001). CONCLUSION: These results suggest that monitoring of post-BNT162b2 booster immunization for purposes of identifying "low responders" could be conducted almost interchangeably with either total or IgG anti-SARS-CoV-2 antibodies.

Unbalanced IDO1/IDO2 Endothelial Expression and Skewed Keynurenine Pathway in the Pathogenesis of COVID-19 and Post-COVID-19 Pneumonia.

Despite intense investigation, the pathogenesis of COVID-19 and the newly defined long COVID-19 syndrome are not fully understood. Increasing evidence has been provided of metabolic alterations characterizing this group of disorders, with particular relevance of an activated tryptophan/kynurenine pathway as described in this review. Recent histological studies have documented that, in COVID-19 patients, indoleamine 2,3-dioxygenase (IDO) enzymes are differentially expressed in the pulmonary blood vessels, i.e., IDO1 prevails in early/mild pneumonia and in lung tissues from patients suffering from long COVID-19, whereas IDO2 is predominant in severe/fatal cases. We hypothesize that IDO1 is necessary for a correct control of the vascular tone of pulmonary vessels, and its deficiency in COVID-19 might be related to the syndrome's evolution toward vascular dysfunction. The complexity of this scenario is discussed in light of possible therapeutic manipulations of the tryptophan/kynurenine pathway in COVID-19 and post-acute COVID-19 syndromes.