Analytical evaluation of the novel Mindray high sensitivity cardiac troponin I immunoassay on CL-1200i.

OBJECTIVES: The current study was designed to evaluate the analytical performance of the new Mindray highly sensitive cardiac troponin I (hs-cTnI) chemiluminescent immunoassay on Mindray CL-1200i, as a thorough validation of novel hs-cTnI methods is required before introduction into clinical practice. METHODS: The evaluation of the analytical performance of this hs-cTnI immunoassay encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, imprecision, linearity, 99th percentile upper reference limit (URL) and concordance with another previously validated hs-cTnI chemiluminescent immunoassay. RESULTS: The LOB and LOD were 0.32 and 0.35 ng/L, whilst the functional sensitivity (expressed as cTnI value with <10 % imprecision), was 0.35 ng/L. The linearity was excellent throughout a wide range of clinically measurable values (r=1.00 between 0.8 and 9,726.9 ng/mL). The intra-assay, inter-assay and total imprecision were 1.1-1.3 %, 5.5-8.1 % and 5.6-8.2 %, respectively. The 99th percentile URL calculated using residual plasma from 246 ostensibly healthy blood donors was 9.2 ng/L (4.3 ng/L in women vs. 12.3 ng/L in men). The Spearman's correlation between Mindray hs-cTnI and Access hs-TnI was 0.97, with mean bias of 7.2 % (95 % CI, 2.6-11.9 %). CONCLUSIONS: Although we failed to confirm the very optimistic analytical characteristics previously reported for this method, our evaluation of the novel Mindray hs-cTnI immunoassay on CL-1200i demonstrated that the overall performance is comparable to that of other commercially available hs-cTnI techniques, making it a viable alternative to other methods.

Vaccination time does not influence total anti-SARS-CoV-2 antibodies response.

We measured total anti-SARS-CoV-2 receptor-biding domain (RBD) antibodies in 249 healthcare workers (mean age: 44 ± 13 years; 151 women), who received the first dose of mRNA-based Comirnaty COVID-19 vaccine at different times of the day. Compared with the reference vaccination time point (i.e. <10:00h), vaccine injection at the following times of day elicited a comparable response (all p > 0.05). Under our experimental conditions, we can therefore exclude a possible influence of the timing of primary mRNA-based COVID-19 vaccination on the levels of total anti-SARS-CoV-2 antibodies.

Are anti-SARS-CoV-2 S/N IgG/IgM antibodies always predictive of previous SARS-CoV-2 infection?

Objectives: We planned this study to verify whether immunoassays for quantifying anti-SARS-CoV-2 IgG/IgM antibodies against both spike (S) and nucleocapsid (N) proteins may be used for identifying previous SARS-CoV-2 infections. Methods: The study population consisted of a cohort of fully vaccinated healthcare workers. All study subjects underwent regular medical visits and molecular testing for diagnosing SARS-CoV-2 infections every 2-4 weeks between 2020-2022. Venous blood was drawn for measuring anti-SARS-CoV-2 antibodies with MAGLUMI 2019-nCoV lgG/IgM CLIA Assays directed against both SARS-CoV-2 S and N proteins. Results: Overall, 31/53 (58.5%) subjects had tested positive for SARS-CoV-2 by RT-PCR throughout the study (24 once, 7 twice). No positive correlation was found between anti-SARS-CoV-2 S/N IgM antibodies and molecular test positivity. In univariate regression analysis, both a molecular test positivity (r=0.33; p=0.015) and the number of positive molecular tests (r=0.43; p=0.001), but not vaccine doses (r=-0.12; p=0.392), were significantly correlated with anti-SARS-CoV-2 S/N IgG antibodies. These two associations remained significant in multiple linear regression analysis (p=0.029 and p<0.001, respectively) after adjusting for sex, age, body mass index, and vaccine doses. In ROC curve analysis, anti-SARS-CoV-2 S/N IgG antibodies significantly predicted molecular test positivity (AUC, 0.69; 95% CI; 0.55-0.84), with the best cutoff of 0.05 AU/mL displaying 67.9% accuracy, 0.97 sensitivity, and 0.27 specificity. Conclusions: Although anti-SARS-CoV-2 S/N IgG antibodies provide helpful information for identifying previous SARS-CoV-2 infections, a lower cutoff than that of sample reactivity should be used. Anti-SARS-CoV-2 S/N IgM antibodies using conventional cutoffs seem useless for this purpose.

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.

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.

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.

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.

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.

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.

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.

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.