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.

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.

Comparative longitudinal variation of total IgG and IgA anti-SARS-CoV-2 antibodies in recipients of BNT162b2 vaccination.

Objectives: This article aims to summarize the 6-month variation of a vast array of anti-SARS-CoV-2 antibodies in recipients of BNT162b2 mRNA-based vaccination. Methods: The study population consisted of 84 baseline SARS-CoV-2 seronegative healthcare employees (median age 45 years, 53.6% females), receiving mRNA-based BNT162b2 primary vaccination cycle. Blood was collected before the first and second BNT162b2 vaccine doses, as well as 1, 3 and 6 months afterwards. The serum titers of the following anti-SARS-CoV-2 antibodies were assayed: total anti-RBD (receptor binding domain), anti-spike trimeric IgG, anti-RBD IgG and anti-spike S1 IgA. Results: All antibodies' levels peaked 1 month after vaccination, but then displayed a considerable decrease. The median rates of 6-month decline were -95% for IgG anti-SARS-CoV-2 RBD, -85% for IgG anti-SARS-CoV-2 trimeric spike, -73% for IgA anti-SARS-CoV-2 S1 and -56% for total anti-SARS-CoV-2 RBD antibodies, respectively. The median time of seronegativization was estimated at 579 days for total anti-SARS-CoV-2 RBD antibodies, 271 days for IgG anti-SARS-CoV-2 trimeric spike, 264 days for IgG anti-SARS-CoV-2 RBD and 208 days for IgA anti-SARS-CoV-2 S1, respectively. The rate of seropositive subjects declined from 98-100% at the peak to 50-100% after 6 months. The inter-individual variation of anti-SARS-CoV-2 antibodies reduction at 6 months was 3-44% from the peak. Conclusions: The results of this longitudinal serosurvey demonstrate that the titer of anti-SARS-CoV-2 antibodies declined 6 months after BNT162b2 vaccination, with median time of IgG/IgA seronegativization estimated between 7 and 9 months, thus supporting the opportunity of administering vaccine boosters approximately 5 to 6 months after the last dose of the primary vaccination cycle.

Total anti-SARS-CoV-2 antibodies measured 6 months after Pfizer-BioNTech COVID-19 vaccination in healthcare workers.

Background: This study aimed at monitoring the kinetics of serum total anti-SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antibodies in a cohort of healthcare workers after voluntary vaccination with Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA-based vaccine. Methods: The study population consisted of 787 healthcare workers (mean age 44±12 years; 66% females), who received two 30 mg doses of Pfizer-BioNTech COVID-19 vaccine, 3 weeks apart. Venous blood was drawn before the first vaccine dose, immediately before the second vaccine dose, and then at 1, 3 and 6 months after the second vaccine dose. Serological testing employed the total antiSARS-CoV-2 antibodies measurement with Roche Elecsys Anti-SARS-CoV-2 S chemiluminescent immunoassay. Results: The median serum levels of total anti-SARS-CoV-2 antibodies reached the peak (1762 kU/L) 1 month after the second vaccine dose, but tended to progressively decline at the 3-month (1086 kU/L) and 6-month (802 kU/L) follow-up points. Overall, the values after 3and 6months were 37% and 57% lower than the corresponding concentrations measured at the peak. No healthcare worker had total anti-SARS-CoV-2 antibodies below the method-dependent cut-off after 6 months. The decline compared to the peak was more accentuated in baseline seropositive persons than in those who were baseline seronegative (74% vs. 52%) cohort. The 6-month post-vaccination anti-SARS-CoV-2 antibodies in subjects aged <65 years remained over 2-fold higher than in those aged ≥65 years (813 vs. 343 kU/L) and also remained consistently higher in women than in men. Conclusions: Gradual decline of total anti-SARS-CoV-2 antibodies occurred 6 months after Pfizer-BioNTech COVID-19 vaccination, though values remained higher than the method-dependent cut-off, with no case of sero-negativization.

Effect of BNT162b2 booster dose on anti-SARS-CoV-2 spike trimeric IgG antibodies in seronegative individuals.

OBJECTIVES: We provide here an updated analysis of an ongoing serosurveillance study, presenting data on the effect of a third dose of Pfizer/BioNTech BNT162b2 vaccine on serum anti-SARS-CoV-2 IgG antibodies. METHODS: We tested baseline SARS-CoV-2 seronegative healthcare workers undergoing primary vaccination with the mRNA-based COVID-19 Comirnaty vaccine, followed by administration of homologous vaccine booster (third dose). Venous blood was collected before either dose of primary vaccination, at 1, 3 and 6 months afterwards, as well as before and 1 month after receiving the vaccine booster. The serum concentration of anti-SARS-CoV-2 IgG was assayed with DiaSorin Trimeric spike IgG immunoassay. RESULTS: The final study population included 53 SARS-CoV-2 seronegative healthcare workers (median age 46 years; 60% females). A first peak of anti-SARS-CoV-2 spike trimeric IgG values was reached 1 month after completing primary vaccination, after which the levels gradually declined until before receiving the vaccine booster. A second peak of anti-SARS-CoV-2 spike trimeric IgG concentration was observed 1 month after receiving the vaccine booster dose (8,700 kBAU/L), which was 39-fold higher than before receiving the vaccine booster (221 kBAU/L; p<0.001), but was also nearly threefold higher compared to values seen at the first peak (2,990 kBAU/L; p<0.001). The rate of subjects with protective anti-SARS-CoV-2 spike trimeric IgG values (i.e., >264 kBAU/L) increased from 47.2% to 100% after 1 month from vaccine booster. CONCLUSIONS: These results support current policies fostering COVID-19 vaccine boosters to reinforce humoral immunity against SARS-CoV-2.

Clinical performance of the Roche Elecsys SARS-CoV-2 antigen fully automated electrochemiluminescence immunoassay.

OBJECTIVE: We assessed the clinical performance of novel Roche Elecsys SARS-CoV-2 Antigen fully automated electrochemiluminescence immunoassay (ECLIA). DESIGN AND METHODS: We tested 160 subjects, 110 (68.8%), with positive molecular test for SARS-CoV-2 infection in nasopharyngeal samples, with Altona Diagnostics RealStar SARS-CoV-2 RT-PCR Kit and Roche Elecsys SARS-CoV-2 Antigen. RESULTS: Highly significant correlation was found between Elecsys SARS-CoV-2 Antigen ECLIA and cycle threshold (Ct) values of SARS-CoV-2 S and E genes (both r = -0.91; p < 0.001). The area under the curve (AUC), sensitivity and specificity of Elecsys SARS-CoV-2 Antigen ECLIA were 0.83, 0.43 and 1.00 in all samples, 0.99, 0.87 and 0.99 in those with both Ct values < 30, as well as 1.00, 1.00 and 0.89 in samples with both Ct values < 25. CONCLUSION: Roche Elecsys SARS-CoV-2 Antigen ECLIA may be a surrogate of molecular testing for identification of super-spreaders.