Vaccine-induced binding and neutralizing antibodies against Omicron 6 months after a homologous BNT162b2 booster.

Evidence about the long-term persistence of the booster-mediated immunity against Omicron is mandatory for pandemic management and deployment of vaccination strategies. A total of 155 healthcare professionals (104 COVID-19 naive and 51 with a history of SARS-CoV-2 infection) received a homologous BNT162b2 booster. Binding antibodies against the spike protein and neutralizing antibodies against Omicron were measured at several time points before and up to 6 months after the booster. Geometric mean titers of measured antibodies were correlated to vaccine efficacy (VE) against symptomatic disease. Compared to the highest response, a significant 10.2- and 11.5-fold decrease in neutralizing titers was observed after 6 months in participants with and without history of SARS-CoV-2 infection. A corresponding 2.5- and 2.9-fold decrease in binding antibodies was observed. The estimated T1/2 of neutralizing antibodies in participants with and without history of SARS-CoV-2 infection was 42 (95% confidence interval [CI]: 25-137) and 36 days (95% CI: 25-65). Estimated T1/2 were longer for binding antibodies: 168 (95% CI: 116-303) and 139 days (95% CI: 113-180), respectively. Both binding and neutralizing antibodies were strongly correlated to VE (r = 0.83 and 0.89). However, binding and neutralizing antibodies were modestly correlated, and a high proportion of subjects (36.7%) with high binding antibody titers (i.e., >8434 BAU/ml) did not have neutralizing activity. A considerable decay of the humoral response was observed 6 months after the booster, and was strongly correlated with VE. Our study also shows that commercial assays available in clinical laboratories might require adaptation to better predict neutralization in the Omicron era.

Peri-infection titers of neutralizing and binding antibodies as a predictor of COVID-19 breakthrough infections in vaccinated healthcare professionals: importance of the timing.

OBJECTIVES: The BNT162b2 messenger RNA vaccine is highly effective in reducing COVID-19 infection, hospitalization and death. However, many subjects developed a breakthrough infection despite a full vaccination scheme. Since the waned efficacy of mRNA vaccines is correlated with the decrease of antibodies occurring over time, we aimed at evaluating whether lower levels of antibodies were associated with an increased risk of breakthrough infection in a cohort of breakthrough subjects that received three vaccine doses. METHODS: Total binding antibodies against the RBD of the S1 subunit (Roche Diagnostics, Machelen, Belgium) and neutralizing antibodies using the Omicron B.1.1.529 variant pseudovirus were measured. Based on individual kinetic curves, the antibody titer of each subject was interpolated just before the breakthrough infection and compared to a matched-control group that did not develop a breakthrough infection. RESULTS: Lower levels of total binding and neutralizing antibodies were observed compared to the control group (6.900 [95% CI; 5.101-9.470] vs. 11.395 BAU/mL [8.627-15.050] [p=0.0301] and 26.6 [18.0-39.3] vs. 59.5 dilution titer-1 [32.3-110] [p=0.0042], respectively). The difference between breakthrough and control subjects was mostly observed for neutralizing antibodies before three months after the homologous booster administration (46.5 [18.2-119] vs. 381 [285-509] [p=0.0156]). Considering the measurement of total binding antibodies before 3 months, there was no significant difference (p=0.4375). CONCLUSIONS: In conclusion, our results showed that subjects that developed a breakthrough infection had lower levels of neutralizing and total binding antibodies compared to controls. The difference was mostly noticeable considering neutralizing antibodies, especially for infections occurring before 3 months after the booster administration.

Kinetics and ability of binding antibody and surrogate virus neutralization tests to predict neutralizing antibodies against the SARS-CoV-2 Omicron variant following BNT162b2 booster administration.

OBJECTIVES: To assess the long-term humoral immunity induced by booster administration, as well as the ability of binding antibody and surrogate virus neutralization tests (sVNT) to predict neutralizing antibodies (NAbs) against the SARS-CoV-2 Omicron variant. METHODS: A total of 269 sera samples were analyzed from 64 healthcare workers who had received a homologous booster dose of BNT162b2. Neutralizing antibodies assessed by sVNT and anti-RBD IgG measured with the sCOVG assay (Siemens Healthineers®) were analyzed at five timepoints; before and up to 6 months following the booster. Antibody titers were correlated with neutralizing antibodies against the Omicron BA.1 variant obtained by pseudovirus neutralization test (pVNT) as a reference method. RESULTS: While Wild-type sVNT percentage of inhibition (POI) remained above 98.6% throughout the follow-up period after booster administration, anti-RBD IgG and NAbs assessed by Omicron BA.1 pVNT showed respectively a 3.4-fold and 13.3-fold decrease after 6 months compared to the peak reached at day 14. NAbs assessed by Omicron sVNT followed a steady decline until reaching a POI of 53.4%. Anti-RBD IgG and Omicron sVNT assays were strongly correlated (r=0.90) and performed similarly to predict the presence of neutralizing antibodies with Omicron pVNT (area under the ROC: 0.82 for both assays). In addition, new adapted cut-off values of anti-RBD IgG (>1,276 BAU/mL) and Omicron sVNT (POI>46.6%) were found to be better predictors of neutralizing activity. CONCLUSIONS: This study showed a significant drop in humoral immunity 6 months after booster administration. Anti-RBD IgG and Omicron sVNT assays were highly correlated and could predict neutralizing activity with moderate performance.

The underestimated issue of non-reproducible cardiac troponin I and T results: case series and systematic review of the literature.

Cardiac troponins (cTn) are the preferred biomarkers for the evaluation of myocardial injury and play a key role in the diagnosis of acute myocardial infarction (MI). Pre-analytical or analytical issues and interferences affecting troponin T and I assays are therefore of major concern given the risk of misdiagnosis. False positive troponin results have been related to various interferences including anti-troponin antibodies, heterophilic antibodies, or elevated alkaline phosphatase level. On the other hand, false negative results have been reported in the case of a large biotin intake. These interferences are characterized with erroneous but reproducible troponin results. Of interest, non-reproducible results have also been reported in the literature. In other words, if the sample is reanalyzed a second time, a significant difference in troponin results will be observed. These interferences have been named "fliers" or "outliers". Compared to the biotin interference that received major attention in the literature, troponin outliers are also able to induce harmful clinical consequences for the patient. Moreover, the prevalence of outliers in recent studies was found to be higher (0.28-0.57%) compared to the biotin interference. The aim of this systematic review is to warn clinicians about these non-reproducible results that may alter their clinical judgment. Four case reports that occurred in the Clinique of Saint-Luc Bouge are presented to attest this point. Moreover, we aimed at identifying the nature of these non-reproducible troponin results, determining their occurrence, and describing the best way for their identification.

How pre-analytical conditions impact glucose measurement and (gestational) diabetes diagnosis: A real-world stability study and a call for harmonization.

BACKGROUND AND AIMS: Since 2023, guidelines of the AACC/ADA recommend the use of citrate buffer-containing tubes as a first option for glucose measurement. This study aims to assess the pre-analytical stability of glucose under various conditions (room temperature (RT) or at 4 °C) and the potential real-world impact of introducing these tubes on (gestational) diabetes and IFG prevalence. MATERIALS AND METHODS: 25 healthy volunteers were sampled to assess glucose stability across time, at 4 °C and at RT, before and following centrifugation. 701 patients undergoing fasting plasma glucose analysis and 109 women having OGTT were collected according to current procedures (NaFl K2C2O4 (NaFl) tubes) as well as with citrate-containing tubes (FC Mix). RESULTS: The mean glucose concentration bias between FC Mix and NaFl tubes when centrifugation occurred within 5 min was 0.53 % and this difference raised slowly to reach 2.3 %, six-hours post-centrifugation. When centrifugation was delayed, a rapid decrease in glucose concentrations was observed for NaFl tubes (4.9 % at 30 min) and this trend was only partially reduced by placing samples at 4 °C (3.1 %). The decrease reached 10.8 % (RT) and 7.8 % (4 °C) at 2 h, before reaching a plateau. Samples collected on citrate remained stable during 24 h. In real-life conditions, the mean bias between FC Mix and NaFl tubes increased progressively over time and reached 8.59 % for samples centrifuged between two- and four-hours following sampling. Compared to widespread practices, the use of citrate-containing tubes increased IFG, DM and GDM prevalences by 84.0 %, 36.7 % and 150 %, respectively. CONCLUSIONS: Glucose concentrations rapidly decrease in NaFl tubes following collection and placing samples at 4 °C reduces only marginally the decay. Citrate-containing tubes offer a valuable solution for direct and long-lasting glucose stabilization but, before wider adoption, large epidemiologic studies should confirm or redefine current diabetes diagnostic thresholds.

Phosphorylated tau 181 (p-tau181) as an innovative, fast and robust biomarker for cerebrospinal fluid leaks.

BACKGROUND: Cerebrospinal fluid (CSF) leaks can lead to serious complications if left untreated, making rapid and accurate diagnosis essential. Biomarkers such as ß2-transferrin (B2TRF) and ß-trace protein are used to detect CSF leaks, but their limitations warrant the exploration of alternative markers. This study investigates the potential of phosphorylated tau at threonine 181 (p-tau181) as a biomarker for CSF leaks. METHODS: Samples from 56 subjects were analyzed for B2TRF and p-tau181 using immunoaffinity blotting and chemiluminescent enzyme immunoassay, respectively. Data analysis included Mann-Whitney test to assess the overall difference in median p-tau181 concentrations between B2TRF positive and negative patients and a receiver operating characteristic (ROC) curve analysis to determine optimal p-tau181 cutoff values for predicting B2TRF positivity. RESULTS: p-tau181 levels were significantly higher in B2TRF positive samples compared to negative samples (p < 0.001). ROC analysis showed high diagnostic performance for p-tau181, with an optimal cutoff of 13.22 pg/mL providing 92.0% sensitivity and 93.1% specificity. Excluding hemolyzed samples improved further the diagnostic performances, maintaining high sensitivity (90.9%) and achieving perfect specificity (100.0%). CONCLUSIONS: This study highlights the potential of p-tau181 as a valuable biomarker for the detection of CSF leaks due to its high diagnostic accuracy and practical advantages over the current biomarkers. The characteristics of p-tau181 assay being both quantitative and rapid, with high diagnostic accuracy, suggest that it could be a valuable tool for the detection of CSF leaks. Further research are now needed to validate these findings.

Double trouble: Unmasking two hook effects on Siemens Atellica® - Total PSA and total hCG assays.

The "hook effect" or "prozone phenomenon" occurs when the concentration of a particular analyte saturates the antibodies used in the test, resulting in falsely low or negative results despite the presence of high analyte concentrations. We report two recent cases of hook effect encountered with a widely used immunoassay analyzer, the Siemens Atellica® IM1600. The first case involves a patient with advanced metastatic prostate cancer whose total PSA (tPSA) concentration dropped dramatically from his last biological control. The second case concerns a pregnant woman whose total HCG (ThCG) levels were also subject to the hook effect and who was found to have a molar pregnancy. In both cases, a dilution step enabled to overcome this analytical concern and to obtain a correct result. In addition, a comparison of the sensitivity of different immunoassay analyzers to this phenomenon was carried out. To avoid this analytical error, an additional dilution step should automatically be performed when there is a clinical suspicion of elevated levels of tumor or hormone markers. Finally, the most affected manufacturers should adapt their assays, accordingly.

Evaluation of Neutralizing Capacity of Tixagevimab plus Cilgavimab (AZD7442) against Different SARS-CoV-2 Variants: A Case Report Study with Comparison to a Vaccinated Population.

AZD7442 (150 mg of tixagevimab plus 150 mg of cilgavimab) has been approved for the preexposure prophylaxis of COVID-19 and for the treatment of adults and adolescents with COVID-19 who do not require supplemental oxygen and who are at increased risk of severe COVID-19. Thus, the aim of the present study is to evaluate the neutralizing capacity of tixagevimab and cilgavimab across different SARS-CoV-2 variants in two patients who received AZD7442 for immunoprophylaxis. A cohort of subjects (n = 45) who had received the BNT162b2 mRNA COVID-19 vaccine has been included to compare these two preventive strategies. Neutralizing antibody (NAb) titers against several variants were assessed against the wild-type, alpha, beta, gamma, delta, omicron BA.5, and XBB.1.5 variants. Binding antibodies have also been measured. NAbs T 1/2 for AZD7442 was 8.1 days (95% CI: 5.1-19.5 days) and was 11.8 days (95% CI: 7.9-23.7 days) for the primo-vaccination cohort. The time to reach neutralization negativity was 108.3 days (95% CI: 66.9-130.7) for AZD7442 compared to 95.4 days (95% CI: 31.0-119.7 days) for the primo-vaccination cohort. The time to reach NAbs' negativity differs between variants with the maximum value obtained for alpha (i.e., 101.1 days (95% CI: 30.0-135.4 days)) and the minimum obtained for beta (i.e., 61.2 days (95% CI: 37.8-77.1 days)). Our results reinforce the need of reviewing the use of AZD7442 in relation to variants of concern and potentially adapting its administration schedule. AZD7442 could be indicated for short-term prophylaxis in frail patients who may be acutely exposed to SARS-CoV-2.

Vaccine-induced humoral response of BNT162b2 and mRNA-1273 against BA.1, BA.5, and XBB.1.5. (sub)variants 6 months after a homologous booster: is immunogenicity equivalent?

Introduction: Some studies suggest that the monovalent mRNA-1273 vaccine is more effective than BNT162b2 in producing higher levels of antibodies. However, limited data are available, and the methods used are not directly comparable. Material and methods: Blood samples were obtained before the booster (third dose) and after 14, 90, and 180 days in two similar cohorts who received the original BNT162b2 or mRNA-1273 vaccine designed to target wild type SARS-CoV-2. The aim of our study is to compare their effectiveness by assessing the levels of binding and neutralizing antibodies specifically against each of the BA.1 variant, BA.5 variant, and the XBB.1.5 subvariant. Results: Once the peak was reached after two weeks, a drastic decline in binding and neutralizing antibodies was observed up to 6 months after the homologous booster administration. The humoral response was however more sustained with the mRNA-1273 booster, with half-lives of 167, 55, and 48 days for binding, BA.1, and BA.5 neutralizing antibodies compared to 144, 30, and 29 days for the BNT162b2 booster, respectively. Compared to the BA.1 variant, the neutralizing capacity was significantly decreased at 6 months with the BA.5 variant (fold-decrease: 1.67 to 3.20) and the XBB.1.5. subvariant (fold-decrease: 2.86 to 5.48). Conclusion: Although the decrease in the humoral response was observed with both mRNA vaccines over time, a more sustained response was observed with the mRNA-1273 vaccine. Moreover, the emergence of Omicron-based variants causes a reduced neutralizing capacity, notably with the XBB.1.5. subvariant. The administration of subsequent boosters would therefore be needed to restore a sufficiently high neutralizing response.

Clinical performance evaluation of the Fluorecare® SARS-CoV-2 & Influenza A/B & RSV rapid antigen combo test in symptomatic individuals.

BACKGROUND: A SARS-CoV-2+Flu A/B+RSV Combo Rapid test may be more relevant than Rapid Antigen Diagnostic (RAD) tests targeting only SARS-CoV-2 since we are facing a concurrent circulation of these viruses during the winter season. OBJECTIVES: To assess the clinical performance of a SARS-CoV-2+Flu A/B+RSV Combo test in comparison to a multiplex RT-qPCR. STUDY DESIGN: Residual nasopharyngeal swabs issued from 178 patients were included. All patients, adults and children, were symptomatic and presented at the emergency department with flu-like symptoms. Characterization of the infectious viral agent was done by RT-qPCR. The viral load was expressed as cycle threshold (Ct). Samples were then tested using the multiplex RAD test Fluorecare®à¸ SARS-CoV-2 & Influenza A/B & RSV Antigen Combo Test. Data analysis was carried out using descriptive statistics. RESULTS: The sensitivity of the test varies according to the virus, with the highest sensitivity observed for Influenza A (80.8.% [95%CI: 67.2 - 94.4]) and the lowest sensitivity observed for RSV (41.5% [95%CI: 26.2 - 56.8]). Higher sensitivities were observed for samples with high viral loads (Ct < 20) and decrease with low viral loads. The specificity for SARS-CoV-2, RSV and Influenza A and B was >95%. CONCLUSIONS: The Fluorecare® combo antigenic presents satisfying performance in real-life clinical setting for Influenza A and B in samples with high viral load. This could be useful to allow a rapid (self-)isolation as the transmissibility of these viruses increase with the viral load. According to our results, its use to rule-out SARS-CoV-2 and RSV infection is not sufficient.

Analytical Sensitivity of Six SARS-CoV-2 Rapid Antigen Tests for Omicron versus Delta Variant.

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to <4.9 log10 copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.

Lung Transplant Recipients Immunogenicity after Heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA Vaccination.

(1) Background: High immunosuppressive regimen in lung transplant recipients (LTRs) hampers the immune response to vaccination. We prospectively investigated the immunogenicity of heterologous ChAdOx1 nCoV-19-BNT162b2 mRNA vaccination in an LTR cohort. (2) Methods: Forty-nine COVID-19 naïve LTRs received a two-dose regimen ChAdOx1 nCoV-19 vaccine. A subset of 32 patients received a booster dose of BNT162b2 mRNA vaccine 18 weeks after the second dose. (3) Results: Two-doses of ChAdOx1 nCoV-19 induced poor immunogenicity with 7.2% seropositivity at day 180 and low neutralizing capacities. The BNT162b2 mRNA vaccine induced significant increases in IgG titers with means of 197.8 binding antibody units per milliliter (BAU/mL) (95% CI 0-491.4) and neutralizing antibodies, with means of 76.6 AU/mL (95% CI 0-159.6). At day 238, 32.2% of LTRs seroconverted after the booster dose. Seroneutralization capacities against Delta and Omicron variants were found in only 13 and 9 LTRs, respectively. Mycophenolate mofetil and high-dose corticosteroids were associated with a weak serological response. (4) Conclusions: The immunogenicity of a two-dose ChAdOx1 nCoV-19 vaccine regimen was very poor in LTRs, but was significantly enhanced after the booster dose in one-third of LTRs. In immunocompromised individuals, the administration of a fourth dose may be considered to increase the immune response against SARS-CoV-2.

Serum SARS-CoV-2 Antigens for the Determination of COVID-19 Severity.

The diagnostic of SARS-CoV-2 infection relies on reverse transcriptase polymerase chain reactions (RT-PCRs) performed on nasopharyngeal (NP) swabs. Nevertheless, false-negative results can be obtained with inadequate sampling procedures, making the use of other biological matrices worthy of investigation. This study aims to evaluate the kinetics of serum N antigens in severe and non-severe patients and compare the clinical performance of serum antigenic assays with NP RT-PCR. Ninety patients were included in the study and monitored for several days. Disease severity was determined according to the WHO clinical progression scale. Serum N antigen levels were measured with a chemiluminescent assay (CLIA) and the Single Molecular Array (Simoa) assay. Viremia thresholds for severity were determined and proposed. In severe patients, the peak antigen response was observed 7 days after the onset of symptoms, followed by a decline. No real peak response was observed in non-severe patients. Severity thresholds for the Simoa and the CLIA provided positive likelihood ratios of 30.0 and 10.9 for the timeframe between day 2 and day 14, respectively. Sensitive detection of N antigens in serum may thus provide a valuable new marker for COVID-19 diagnosis and evaluation of disease severity. When assessed during the first 2 weeks since the onset of symptoms, it may help in identifying patients at risk of developing severe COVID-19 to optimize better intensive care utilization.

A colossal, enigmatic, and long-lasting high-sensitivity cardiac troponin T elevation.

This case describes the incidental finding of a massive and persistent elevation of troponin T in a patient with end-stage renal disease. This high troponin T value was not consistent with the patient's clinical condition and the laboratory was called in to investigate this discrepancy. After exclusion of analytical interference and discovery of a discordance between troponin T and troponin I, a clinical investigation including cardiac and whole-body magnetic resonance imaging was performed. Magnetic resonance imaging results allowed us to exclude a cardiac origin of troponin elevation but revealed a skeletal muscle pathology. This case constitutes the first description of high-sensitivity cardiac troponin T elevation due to musculoskeletal pathology without cardiac involvement in a patient with end-stage renal disease.

Early antibody response in health-care professionals after two doses of SARS-CoV-2 mRNA vaccine (BNT162b2).

OBJECTIVES: Data on the immune response after two doses of BNT162b2 are so far limited. Previously infected individuals were excluded from pivotal clinical trials and the optimum dose regimen in this population has not been clearly studied. The CRO-VAX HCP study aims to investigate the early antibody response in a population of health-care professionals having received two doses of the BNT162b2 mRNA coronavirus disease 2019 (COVID-19) vaccine. METHODS: The CRO-VAX HCP study is a multicentre, prospective, interventional study conducted in several sites in Belgium. The study included 231 health-care professional volunteers who received the two-dose regimen of the BNT162b2 mRNA COVID-19 vaccine. Of these, 73 were previously infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 158 were uninfected and seronegative. In the first group, blood samples were collected at baseline and after 2, 4, 7, 10, 14, 21 and 28 days. In the second group, samples were obtained at baseline and after 14 and 28 days. Antibodies against the SARS-CoV-2 nucleocapsid and the receptor binding domain of the S1 subunit of the spike protein were measured in all individuals at different time-points. RESULTS: In uninfected individuals, 95.5% (95% CI 91.0%-98.2%) developed anti-spike antibodies after 14 days and a 24.9-fold rise (95% CI 21.4%-28.9%) in antibody titre was observed after the second dose. In previously infected individuals, peak antibody response was reached after 7 days (i.e. 6347 U/mL) and the second dose did not lead to significantly higher antibody titres (i.e. 8856-11 911 U/mL). Antibody titres were higher in previously infected individuals. CONCLUSIONS: This study supports the concept that a single dose of BNT162b2 would be sufficient in previously infected individuals.