After the calibration of the computational models, a parametric study is then conducted to assess the influence of the FRP axial stiffness (thickness and modulus of elasticity) on the interfacial normal and shear stresses. The numerical analysis results show that increasing the thickness or the elastic modulus of the FRP strengthening affects the efficiency of the FRP bonding and makes it susceptible to earlier debonding failures. A tapering technique is proposed in wet lay-up applications since multiple FRP layers are often required. It is shown that by gradually decreasing the thickness of the FRP strengthening, the concentration of stress along the plate end can be reduced, and thus, the overall strengthening performance is maximized. The tapering is successful in reducing the bond stress concentrations by up to 15%, which can be sufficient to prevent concrete rip-off and peel-off debonding failure modes. This article contributes towards a better understanding of the debonding phenomena in FRP-strengthened elements in flexure and towards the development of more efficient computational tools to analyze such structures.This article examines numerically the behavior of prestressed reinforced concrete slabs strengthened with externally bonded reinforcement (EBR) consisting of fiber-reinforced polymer (FRP) sheets. The non-linear finite element (FE) program Abaqus The need to increase service loads, changes or updates in design codes, design errors or aging make the strengthening of reinforced concrete elements necessary. Due to several advantages over more traditional techniques, externally bonded reinforcement (EBR) in the form of fiber-reinforced polymers (FRP) has gained a special interest in strengthening applications of reinforced concrete (RC) structures ,2,3,4. TPrevious research indicates that EBR FRP strengthening can increase the flexural capacity of elements in both service and ultimate conditions ,6,7,8. HIn order to prevent debonding failures at the end anchorage of a plate or sheet of an EBR FRP-strengthened slab a, severaSeveral models/guidelines exist to predict the bond behavior of FRP-concrete systems, but they are still unable to give accurate predictions ,7. This Past studies have adopted different approaches to simulate debonding phenomena. For example, smeared and discrete approaches can be used to simulate cracking and debonding in plain and FRP-strengthened concrete structures ,27. OtheThis article examines numerically the end-plate behavior of prestressed concrete slabs strengthened in flexure with EBR FRP sheets. This is achieved by using previous data from four-point bending tests on EBR FRP-strengthened prestressed concrete slabs. Non-linear FE analyses was first performed to calibrate computational models of the strengthened slabs. Subsequently, a parametric study was conducted to assess the influence of the FRP axial stiffness  on the normal and shear interfacial stresses. The article also provides some recommendations to enhance the capacity of FRP bonded to prestressed concrete slabs, as well as to improve the effectiveness of FRP plate bonding techniques. This article contributes towards a better understanding of the debonding phenomena in FRP-strengthened elements in flexure and towards the development of more efficient computational tools to analyze such structures. 2  were used as flexural bottom reinforcement. Such tendons complied with the ASTM A421 [Five prestressed concrete slabs tested previously by the authors  were tak2 b. Four fSTM A421  specificft, of 1.4 mm, was used to strengthen the slabs’ soffit. The FRP sheets were bonded with the slabs in an upside-down position, as shown in PS-EBR-1-250-TP: EBR CFRP-strengthened slab, strip width 250 mm, tapered at one end anchorage (tapering length = 250 mm).PS-EBR-2-100-TP: EBR CFRP-strengthened slab, strip width 100 mm each, tapered at one end anchorage (tapering length = 250 mm).PS-EBR-1-250 and PS-EBR-2-100: same as above but without tapering.PS-C: unstrengthened control slab.One layer of carbon FRP (CFRP) strip with single or double layouts, having a thickness fE = 200 GPa, Poisson ratio = 0.29, ultimate strength fuf = 2590 MPa and rupture strain εfu = 0.015, as given by the manufacturer. The FRP was bonded to the slabs’ soffit using a two-part epoxy adhesive bonding agent. The properties of the epoxy resin reported by the producer were modulus of elasticity mE = 5 GPa, tensile strength mf = 20 MPa and Poisson ratio = 0.35. The slabs were cast using C30 concrete with a mean compressive strength of cf = 30.8 MPa, tensile splitting strength crf = 3.6 MPa and modulus of elasticity cE = 22 GPa, which were calculated according to fib Model Code [The properties of the CFRP were modulus of elasticity The five slabs were subjected to monotonic load until failure. The mid-span deflection behavior of the slabs was monitored using vertical displacement transducers located at the soffit. As expected, the ultimate failure of the slabs was controlled by debonding of the CFRP sheets at the free end anchorage for all of the strengthened specimens. ® FE software [®. The internal reinforcement was modeled using 1D elements and is embedded in the concrete matrix, , ode 2010 .For CFRP-strengthened specimens with tapering CFRP sheets at the end anchorage (PS-EBR-1-250-TP and PS-EBR-2-100-TP), it was found that plate end bonding failure was well prevented, and instead, multiple intermediate cracks induced interfacial debonding failures i.e., . This isThe results in ft = 0.7, 1.4, 2.0 and 4.0 mm), whereas fE = 150, 200 and 250 GPa). The results in these figures indicate that, as fE increases, the overall stiffness of the slab also increases, and less deflection is recorded . recorded b. When tmax) and shear stress (S11) of the concrete–adhesive interface of the CFRP-strengthened slabs without end-tapering (PS-EBR-250) and with end-tapering (PS-EBR-250-TP). In these plots, the thickness of the CFRP varied from 1.0 to 1.4 mm in PS-EBR-250-TP to compare with the 1.4 mm of CFRP in PS-EBR-250. The development of the principal stresses in the FE model can be used to identify the concentration of stresses in the concrete, CFRP sheets and adhesive resin. As a failure ratio of 0.037 was used , a diagonal crack in the concrete would occur when the tensile stress in the concrete (at the end of the plate) reaches its ultimate strength. However, the full composite action is assumed in this modeling exercise, which makes the analysis run completely until a compressive failure of concrete occurs. At this point, the maximum tensile stress in the CFRP sheets can be used to examine the effectiveness of the FRP strengthening before the beam starts to develop diagonal cracking, loses its composite action and eventually fails due to FRP debonding  are used to investigate the principal and shear stresses of the slabs and the FRP plates. ft = 1.4 mm) is considered. The concentration of these stresses is found to be the cause of debonding due to rip-off or peel-off. Conversely, the tapering reduces the principal stresses (by up to 15%) and shear stresses (by up to 10%) along the anchorage length, thus, re-distributing stresses towards the beam mid-span. Therefore, tapering is extremely successful in reducing the stresses by up to 15%, which can be sufficient to prevent concrete rip-off and peel-off FRP debonding failures.In all CFRP-strengthened prestressed slabs, the ultimate failure was controlled by debonding of the CFRP sheets at the end anchorage. End debonding failure and intermediate crack-induced interfacial debonding failure were observed in the slabs without and with tapered CFRP sheets, respectively.The results from the tests showed that an end tapering technique improved the ultimate load by up to 5% compared to slabs without tapering. The tapering also prevented end debonding failure by allowing multiple interfacial debonding failures.For CFRP-strengthened slabs, the FE models predicted better the results compared to unstrengthened specimens up to a load level of approximately 75% of the ultimate load. However, the numerical predictions showed approximately 20–25% fewer deflections at ultimate loads. Further research is necessary to achieve better predictions at ultimate loads.Results from a parametric study showed that increasing the thickness or the elastic modulus of the CFRP strengthening sheets can affect the efficiency of the FRP plate bonding technique, thus, making it susceptible to potential premature debonding failures.The FE results confirmed that a tapering technique at the end anchorage of the CFRP sheets can increase the capacity of a CFRP strengthening system. For the CFRP-strengthened slabs analyzed in this study, the end tapering reduced the stresses by up to 15%, which can be sufficient to prevent concrete rip-off and peel-off debonding failures. Nonetheless, further numerical analyses are necessary to extend the validity of these observations to other structural elements strengthened with EBR CFRP sheets.This article numerically examines the behavior of prestressed reinforced concrete slabs strengthened with EBR CFRP sheets in flexure. Computer simulations based on finite element (FE) analysis were conducted on five specimens tested previously by the authors to investigate stress concentrations in the slabs, as well as at the CFRP–concrete bonded interface. The simulation is then used to carry out a parametric study that shows that increasing the thickness or the elastic modulus of the FRP plate affects the efficiency of the FRP plate bonding. Further analysis investigated the efficiency of tapering the EBR FRP on reducing the stress concentrations at the end anchorage to the strengthening system. Based on the results of this study, the following conclusions can be drawn:


The use of High-flow nasal cannula (HFNC) is increasing in admitted COPD-patients and could provide a step in between non-invasive ventilation (NIV) and standard oxygen supply. Recent studies demonstrated that HFNC is capable of facilitating secretion removal and reduce the work of breathing. Therefore, it might be of advantage in the treatment of acute exacerbations of COPD (AECOPD). No randomized trials have assessed this for admitted COPD-patients on a regular ward and only limited data from non-randomized studies is available.The aim of our study was to identify the reasons to initiate treatment with HFNC in a group of COPD-patients during an exacerbation, further identify those most likely to benefit from HFNC treatment and to find factors associated with treatment success on the pulmonary ward.This retrospective study included COPD-patients admitted to the pulmonary ward and treated with HFNC from April 2016 until April 2019. Only patients admitted with severe acute exacerbations were included. Patients who had an indication for NIV-treatment where treated with NIV and were included only if they subsequently needed HFNC, e.g. when they did not tolerate NIV. Known asthma patients were excluded.A total of 173 patients were included. Stasis of sputum was the indication most reported to initiate HFNC-treatment. Treatment was well tolerated in 83% of the patients. Cardiac and vascular co-morbidities were significantly associated with a smaller chance of successful treatment . Clinical assessment judged HFNC-treatment to be successful in 61% of the patients. Furthermore, in-hospital treatment with NIV was associated with a higher chance of HFNC failure afterwards .This large retrospective study showed that HFNC-treatment in patients with an AECOPD was initiated most often for sputum stasis as primary reason. Factors associated with improved outcomes of HFNC-treatment was the absence of vascular and/or cardiac co-morbidities and no need for in-hospital NIV-treatment. During severe exacerbations, patients need admission to a hospital . Additio dry air . In addiMoreover, in patients with acute acidotic hypercapnic respiratory failure, non-invasive ventilation (NIV) is a proven effective therapy. It reduces the work of breathing, improves gas exchange, reduces the length of hospital stay, decreases the need for endotracheal intubation and improves mortality –8. HowevIf NIV fails, in clinical practice usually two options remain, step-up to invasive ventilation or accept to step-down to standard oxygen therapy. If hypercapnic respiratory failure persists, invasive ventilation is recommended. However, invasive ventilation in COPD-patients is associated with worse outcomes, prolonged hospitalization and mortality as compared to NIV . Next toTo address these problems, an alternative treatment option positioned in between NIV and standard oxygen could be of additional value. Potentially, such a treatment could be used to treat COPD-patients with more severe hypoxemic respiratory failure needing more than standard oxygen therapy and patients with a mild to moderate combined hypoxemic/hypercapnic respiratory failure who do not tolerate NIV, especially those with mucus-related problems. Treatment with high-flow nasal cannula (HFNC) could be considered as this step in between standard oxygen and NIV. With HFNC heated and humidified air optionally supplemented with additional oxygen can be delivered at high flow rates up to 60L/min and this may have multiple advantages for the treatment of COPD-exacerbations.aCO2 [First, the humidification and heating of the delivered air may facilitate secretion removal, reduce airway inflammation and avoid epithelial injury , and potaCO2 –19.While awaiting large randomized controlled trials, HFNC is already being used in clinical practice for COPD-patients. From these clinical practices, valuable information can be obtained with regard to reasons for initiating treatment with HFNC in COPD-patients with an exacerbation and selection of patients that benefit most from treatment. We retrospectively analyzed data of admitted COPD-patients treated with HFNC at a pulmonary ward in order to address these questions. Furthermore, we aimed to describe clinical outcomes with regard to used settings of the HFNC, treatment tolerance and factors associated with successful treatment.We performed a retrospective study including COPD patients >18 years) admitted with a COPD exacerbation to the pulmonary ward of a Dutch teaching hospital and treated with HFNC in the period of April 2016 until April 2019. The definition of a COPD-exacerbation by the Global initiative for chronic obstructive lung diseases was used [8 years aData were retrieved from the hospital-electronic patient systems. Demographic data such as age, gender, COPD GOLD-stage, presence of co-morbidities , stable state lung function (forced expiratory volume in 1 second (FEV1)) and concomitant treatments were recorded. Recording of co-morbidities was limited to other lung diseases  cardiac- , vascular- , neurologic co-morbidities and/or diabetes. Also, the number of COPD exacerbations for which treatment with antibiotics and/or corticosteroids was required in the year before admission was recorded, as well as the number of admissions in the previous year.2) were retrieved from 3 different time points: before the start of HFNC-treatment (obtained within 24 hours before the start), during HFNC-treatment, and within 24 hours after ending HFNC-treatment. Arterial blood gasses (ABG) values were used for analysis if obtained within 48 hours before and 48 hours after HFNC-treatment. The settings of the HFNC-treatment; (inspired oxygen fraction (FiO2), flow rate and temperature of the humidified air), were recorded at start and during treatment. We assessed the need for NIV or invasive ventilation, length of hospital stay and mortality.Vital signs, such as respiratory rate (RR), heart rate (HR) and oxygen saturation . The HFNC was set according to decision of the treating physician.The judgement whether HFNC was successful or not was made by the attending consultant respiratory medicine based on the clinical examination of the patient, independent of the study team. Treatment was deemed non-successful when patients showed no clinical improvement, or did not tolerate the treatment. Successful treatment was defined as the absence of treatment failure.Descriptive data were expressed as mean ± standard deviation or as percentage (%). The distribution of data was assessed by performing a Shapiro-Wilk test and examining a histogram and Q-Q plot for every independent value. Median and interquartile ranges (Q1 and Q3) were used for non-normal distributed data and percentage (%) was used for categorical data.A Chi-square or Fisher’s-exact test was used to compare nominal/categorical variables. When comparing values from the same patient a paired T-test was used when the data was normally distributed and a sign-test when the data was non-normally distributed.For comparison between groups of continuous variables that were normally distributed an unpaired T-test was used. When these values were non-normally distributed a Mann Whitney U-test was used to calculate significance. All tests were two sided and considered significant if the P-value was below 0.05. Analysis was performed using SPSS statistics version 24.For comparison of factors associated with treatment success a multiple logistic regression was performed. First of a univariate analysis was done for all the variables, next the variables which were shown to have a slight association (p<0.15) were taken into account in the multiple regression analysis. Backward selection was used to further narrow down the variables. The variables were considered statistically significant if the P-value was below 0.05.The Medical Ethics Committee of our institution (RTPO Leeuwarden) confirmed the conduct of this retrospective study and the institutional board approved the execution of the study without the need for consent in accordance with Dutch regulations (ID NMWMO356). All data was handled confidentially and anonymously.1 of 1.19L (46% of predicted). Almost half of the patients were known with cardiac (48.6%) and/or vascular (54.9%) co-morbidities. The patients had a median exacerbation rate of 1.00 (0–12) exacerbations in the previous year with 0 (0–6) admissions in the previous year.Among all the patients treated with HFNC at the pulmonary ward, 173 patients met the inclusion criteria . The cha2-suppletion  and a median temperature of 31°C .HFNC-settings were recorded properly in the majority of the treated patients at start of treatment. The median flow rate used was 30 L/min , with a median FiOIn 69 patients (39.9%), the primary reason for treatment was stasis of sputum. Eleven patients (6.4%) were treated because of hypoxemia despite regular oxygen suppletion and 14 patients (8.1%) because of hypercapnia with normal pH. In 13 patients (7.5%) HFNC-treatment was a step-down after NIV-treatment, in 15 patients (8.7%) HFNC-treatment was initiated after NIV-treatment failed, mostly because NIV-treatment was not tolerated and in only 1 patient (0.6%) HFNC-treatment was initiated after detubation. In 46 patients (26.6%) HFNC was started for relief of symptoms related to dyspnea and in 4 patients (2.3%) the primary reason for starting treatment was not retrievable .2 was increased after treatment with HFNC  as well as the respiratory rate   and remained lower after treatment . SO= 0.001) .The mean length of hospital stay for all patients was 9.5 ± 6.2 days. Treatment outcome is shown in Comparing the patients that were treated successfully to those who were not , there w2 .Vital signs at the start of HFNC-treatment did not differ significantly. During and after HFNC-treatment, patients treated successfully showed significant lower respiratory rates  and a significant higher SO2, PaO2 and pH before, during or after HFNC-treatment. Patients treated successfully were treated longer on average than the non-successful treatment group . There was no difference observed between patients treated non-successfully and patients treated successfully in intubation rate or progression to O2-treatment at home, although numbers for analysis were small  and to relief symptoms of dyspnea  as primary goal. In 61% of the patients treated the treatment was successful suggesting a possible role for HFNC-treatment in COPD-patients. HFNC-treatment was tolerated well, in only 17% of the treated patients treatment was stopped because it was not tolerated. This 17% is lower than the reported incidence of discomfort in treatment with NIV; 30–50% , 9. ThisTo our knowledge this is one of the first large real-life studies investigating HFNC-treatment in a population with severe exacerbations of COPD. This study was also performed on the regular ward rather than on the ICU. In our study clinicians choose for HFNC most often because of sputum clearance problems. In most COPD-patients chronic sputum production as well as decrease in mucociliary clearance is a challenging problem –25. By dIn our cohort, the presence of vascular and/or cardiac co-morbidities and treatment with NIV were independently associated with HFNC-treatment failure. We hypothesize that both factors are more common in more vulnerable or severely ill patients with an AECOPD influencing HFNC success rates.Perhaps due to the fact that these co-morbidities often remain untreated. Heart failure for example can mimic or present itself concomitantly with an AECOPD , which maCO2 and that there was no difference in 30-day mortality, intubation-rate or treatment failure compared to NIV-treatment [Furthermore, patients who did not tolerate NIV (n = 15), often also not tolerated HFNC-treatment (N = 8) potentially due to the severity of respiratory failure. Moreover NIV-treatment is often needed in patients who are severely ill and are in need of reducing their work of breathing. As in NIV, HFNC is able to reduce work of breathing by reducing minute ventilation , 33. Furreatment –38. The reatment .Our study suggests that HFNC-treatment might be less suitable for patients who received NIV-treatment as is seen in the multivariate logistic regression analysis. However no differences were seen between patients treated successfully and non-successfully when looking at step-down after NIV-treatment or NIV-failure as reason for HFNC-treatment see . Only a The stable state lung function was not associated with a better or worse outcome of HFNC-treatment. FEV1 has been known to show high variability among individuals and therefore FEV1 might not be such a good predictor for outcomes during exacerbations , 41. PotIn our study COPD-patients showed a significant reduction in respiratory rate and improved saturation after HFNC-treatment. This is consistent with a study by Jeong et al. in which an improved respiratory rate and saturation in hypercapnic patients (33 patients with AECOPD) was seen after HFNC-treatment . The red2-suppletion alone is not enough. As is also stated in a recent published practical guideline for the use of HFNC [Our study suggests that HFNC could also play a role in the treatment of AECOPD in patients in which O of HFNC . The gui2-suppletion are often less suitable for this indication due to the fact that the O2- or NIV-mask needs to be removed to clear sputum. Resolving sputum stasis might reduce the work of breathing as well; as it can reduce dyspnea and lower the respiratory rate and thereby work of breathing, but further research is needed to confirm this notion.Moreover, our study implies a possible role for HFNC in COPD-patients with sputum-related problems. As was stated before by Crimi et al. whom showed that HFNC was capable of increasing mucus production in patients with AECOPD and coexisting bronchiectasis . NIV andOur study has some limitations. This study is a retrospective study, performed in a single center without a control group. Therefore, it is difficult to completely value data about clinical efficacy. Furthermore, the group of patients was mixed as HFNC was used for several indications and aims. However, there were no differences in reasons for HFNC-treatment between the success and non-success group. Third, the definition of successful treatment could be debated as this was decided on judgement of the clinicians without strict criteria. Since this is a retrospective study a standard operating procedure was not available, we defined successful treatment as the absence of treatment failure combined with the clinical aim of treatment and whether this goal was achieved based on the judgement of the treating clinical physician to obtain the most objective outcome. The main advantage of such a clinical endpoint is however that it reflects actual care for this severely exacerbated COPD-patients. For future prospective studies we would recommend to predefine such criteria. Another weakness of this study is that arterial blood gasses analysis data is not available. During clinical practice these are not routinely performed during HFNC-treatment. This reflects the aims and position of the HFNC-treatment, since HFNC was not used as treatment for respiratory acidosis.This large retrospective study showed that real world HFNC-treatment in patients with an AECOPD was initiated most often for sputum stasis as primary reason , 61% of all patients ended the treatment successfully based on clinical judgement, and were treated longer. Factors associated with improved outcomes of HFNC-treatment was the absence of vascular and/or cardiac co-morbidities and a longer duration of HFNC-treatment. In clinical practice HFNC-treatment reduces respiratory rate and improves saturation in patients with a COPD-exacerbation. Based on the now reported results, future trials should consider to add mucus related outcomes to their designs, to address the apparent position of HFNC use in the clinic.


This retrospective multi-center matched cohort study assessed the risk for severe COVID-19 (combination of severity indicators), intensive care unit (ICU) admission, and in-hospital mortality in hospitalized patients when infected with the Omicron variant compared to when infected with the Delta variant. The study is based on a causal framework using individually-linked data from national COVID-19 registries. The study population consisted of 954 COVID-19 patients  above 18 years old admitted to a Belgian hospital during the autumn and winter season 2021–2022, and with available viral genomic data. Patients were matched based on the hospital, whereas other possible confounders  were adjusted for by using a multivariable logistic regression analysis. The estimated standardized risk for severe COVID-19 and ICU admission in hospitalized patients was significantly lower  and RR = 0.56; 95% CI , respectively) when infected with the Omicron variant, whereas in-hospital mortality was not significantly different according to the SARS-CoV-2 variant ). This study demonstrates the added value of integrated genomic and clinical surveillance to recognize the multifactorial nature of COVID-19 pathogenesis. The clinical spectrum of coronavirus disease 19 (COVID-19), resulting from infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranges from asymptomatic infections or mild respiratory symptoms to severe pneumonia and acute respiratory distress syndrome (ARDS) ,4. In adContinuous genomic surveillance is essential to detect, monitor, and assess emerging virus variants. Infection with a SARS-CoV-2 variant can be confirmed using genomic detection techniques, such as whole-genome sequencing (WGS), or can be suspected based on diagnostic screening nucleic acid amplification technique (NAAT)-based assays  assays) in a context of known circulating variants . The surSeveral SARS-CoV-2 variants have become dominant globally over the course of the pandemic. In Belgium, the “wild-type” SARS-CoV-2 variant was predominantly present from the beginning of the pandemic, followed by the emergence of the Alpha variant in December 2020 . The risIn the present study, we assess the clinical severity of the Omicron versus the Delta variant in the Belgian hospitalized COVID-19 population of the autumn-winter season of 2021–2022, by accounting for both patient characteristics, including demographics, underlying comorbidities, and preexisting natural and vaccine-induced immunity, as well as organizational hospital characteristics.The study is conducted within a causal conceptual framework to assess the effect of SARS-CoV-2 variants on COVID-19 disease severity among hospitalized patients . This frst of September 2021 marks the start of the booster campaign in Belgium. Moreover, no Alpha variant  has been detected in Belgium after this date. The data were extracted on 11 April 2022 to allow for at least two weeks of follow-up of recently admitted patients. The analysis was restricted to adults (≥18 years) with a laboratory-confirmed COVID-19 infection (RT-PCR and/or rapid antigen test) who were tested because of COVID-19 symptoms, and with available viral genetic/genomic data (see further). Patients that were transferred, readmitted, or hospitalized in a psychiatric hospital or in a hospital without an intensive care unit (ICU) were excluded from the study population, in order to avoid incomplete clinical episodes within the analysis. Further, patients with a missing date of diagnosis, date of admission, or date of discharge were excluded, as well as patients with a missing national registry number, which is required to link information from the different COVID-19 registries.The study population consisted of hospitalized symptomatic COVID-19 patients who were admitted to a Belgian hospital between 1 September 2021 and 28 March 2022, and for whom an admission form was reported in the Clinical Hospital Survey (CHS) . The 1stViral genetic or genomic information indicating infection with the Omicron or Delta variant was obtained through the linkage of the national registry number of hospitalized patients meeting the inclusion criteria with the COVID-19 TestResults Database. Both confirmed variant results, i.e., identified through WGS, and compatible variant results, e.g., identified through a probe PCR assay, were considered for the analysis. The variant results were obtained from both the baseline and active genomic surveillance. All Omicron sublineages were considered for the analysis. However, at the time of data extraction, only, B.A.1, BA.1.1, and, B.A.2 were circulating among hospitalized COVID-19 patients in Belgium. Therefore, no other sublineages were detected in hospitalized COVID-19 patients registered in the CHS. To assure that the hospital admission was related to the detected infection, patients with their sample collected more than 20 days before hospital admission or after hospital discharge were excluded from the study population. MatchIt package (matchit function with method = “exact”) [Patients in the Omicron group were matched to patients in the Delta group based on the hospital to which they were admitted using the “exact”)  in R. St“exact”) , were acThe clinical severity among the hospitalized study population was assessed by comparing the development of severe COVID-19  between both exposure groups (Delta or Omicron). In addition, we specifically looked at the difference in risk for ICU admission and in-hospital mortality. Vaccination status was defined as ‘not or partially vaccinated’ when cases diagnosed with COVID-19 had not received a dose of vaccine, had received only one dose of a two-dose schedule, or had not reached 14 days after the complete primary vaccination ; ‘primary course completed’ when diagnosed ≥ 14 days after the complete primary vaccination; and ‘primary course completed plus booster’ when diagnosed ≥ 14 days after the booster dose. Reinfections were defined as two positive tests in the same individual taken more than 90 days apart . Patient2 or Fisher’s exact tests, and continuous variables were compared using the Wilcoxon–Mann–Whitney rank-sum test.Demographic and clinical information of the matched study population was presented by variant. The categorical variables were described as frequency rates, and percentages and continuous variables were described using the median and interquartile range (IQR). Univariate comparisons of categorical variables were performed using χmice package [Twenty-fold multiple imputation of missing values was performed using the  package  for all A weighted logistic regression model (using matching weights) was fitted with the following covariates : age, gRegression standardization , which iAll analyses were conducted in R 4.0.1 . Three sensitivity analyses were performed by conducting the same modeling procedure as described above, but applied to (1) a study population excluding patients with a documented previous infection, to (2) a study population only including patients with a confirmed Omicron or Delta infection (obtained through WGS from both baseline and active genomic surveillance), and to (3) a study population only including patients with their sample selected in the context of the representative baseline surveillance. As recorded on 11 April 2022, the CHS database contained a total of 109,233 case records of COVID-19 patients . After dThe median age of the matched study population (n = 954) was 74 years (IQR: 63–83), 59% (564/954) were men, and 10% (90/942) were nursing home residents. The patients stayed for a median of 9 days (IQR: 5–17) in the hospital, and the median ICU bed occupancy rate averaged over the patient’s hospital stay was 19% (IQR: 13–27). More than half of the patients  had at least one underlying comorbidity associated with a high risk for severe COVID-19 , whereasThe estimated standardized risks (with respect to the model and the covariate distribution) for severe COVID-19, ICU admission, and/or in-hospital mortality among hospitalized patients are presented for both variants in The occurrence of a previous infection was not included as a covariate in the logistic regression model given the low frequency  and the subsequent high probability of obtaining bootstrap resamples with zero counts for one of the exposure groups. Therefore, a sensitivity analysis assessed whether excluding patients with a documented previous infection from the study population would influence the results. The standardized risks and causal effect estimates within this subgroup (433 patients infected with the Omicron variant and 508 patients infected with the Delta variant) were similar as compared to the main analysis results .A second sensitivity analysis was performed by only including the confirmed (through WGS) Omicron and Delta infections. The causal effect estimates within this subgroup (251 patients infected with the Omicron variant and 368 patients infected with the Delta variant) showed similar trends as those in the main analysis, albeit with higher standardized risks and wider confidence intervals .at random by one of the sentinel laboratories). The causal effect estimates that were obtained within this subgroup (183 patients infected with the Omicron variant and 265 patients infected with the Delta variant) were no longer significant for any of the outcomes  than in the Delta group (71 year). There were more nursing home residents among the Omicron patients than the Delta patients. One of the possible factors could be the vaccination campaign in Belgium that targeted nursing home residents as the first group to receive the anti-SARS-CoV-2 vaccines in January 2021, followed by a booster campaign in September 2021. The first signs of a waning effect of the booster may have coincided with the emergence of the Omicron variant in November 2021. Another possibility is the immunosenescence in this frail patient group , which cA number of limitations need to be considered. First, it is important to acknowledge the study selection procedures when interpreting the risk estimates. Restricting the analysis to hospitalized patients may hamper both external and internal validity ,59,60. FA general limitation when assessing the severity of consecutive SARS-CoV-2 variants circulating during different time periods is the fact that the exposure groups differ considerably in terms of patient demography and contextual factors, such as vaccination rollout, testing strategy, and dynamics of the respective waves. Having exhaustive information on the vaccination status, and performing a sensitivity analysis excluding patients with a documented previous infection, enabled us to account, to a large extent, for the gradually changing background immunity in the population. However, the number of documented reinfections, defined as two positive tests in the same individual taken more than 90 days apart, is probably an underestimation given the larger antigenic diversity of Omicron . The effA third limitation is the potential measurement bias related to the inclusion of compatible Omicron and Delta cases in the main analysis. Indeed, multiplex mutation-specific PCR-based assays are not always able to definitively distinguish different SARS-CoV-2 variants. Therefore, a sensitivity analysis only including the confirmed Omicron and Delta infections through WGS was conducted. This analysis revealed similar conclusions (lower standardized risks for the severe outcomes when infected with Omicron as compared to when infected with Delta), albeit with different values for the standardized risks, potentially due to a selection bias when considering samples for WGS.Fourth, the, B.A.2 sublineage reached more than 80% in the baseline surveillance during the week of 22th of March 2022 . Still, Lastly, conducting the analysis during the fifth wave dominated by Omicron might have led to an overrepresentation of recently admitted Omicron patients with a short hospital stay. We partly accounted for this limitation by applying a data cutoff of two weeks to ensure sufficient follow-up time. The key strengths of this study are: (1) the availability of detailed information on the characteristics and outcomes of hospitalized patients; (2) the linkage with socio-economic data, sequencing results, and the exhaustive vaccination register; and (3) the multi-center nature of the study providing a nation-wide coverage, increasing the generalizability. Another important strength of the study design is the matching of patients based on the hospital to which they were admitted: this accounts for between-center variability in patient outcomes and differences that may exist in the decision-making process to admit (severe) COVID-19 patients and possibly in treatment protocols ,64. MoreWe observed a lower risk for severe COVID-19 (based on a combination of severity indicators) and ICU admission in Belgian hospitalized COVID-19 patients when infected with the Omicron variant as compared to when infected with the Delta variant. Inferring the intrinsic severity of SARS-CoV-2 variants is challenging and requires detailed information on potential confounders as identified in a causal model. This study demonstrates the added value of genomic surveillance during the COVID-19 pandemic and the subsequent individual-level linkage with other surveillance registries to better understand the complex and multifactorial nature of COVID-19 disease severity, and to deliver evidence-based results to policy-makers.


Chest CT has been proposed as a screening test to rule out SARS-CoV-2 lung infection in acute stroke. Our objectives are to analyze the predictive value of neck CT angiography (CTA) source images compared with conventional chest CT, the interobserver concordance and the reliability of the diagnosis using a mobile app.A retrospective observational study that included acute stroke patients admitted to a stroke center. Two raters blinded to the clinical data evaluated and classified the pulmonary findings in chest CT and neck CTA source images according to the COVID-19 Reporting and Data System (CO-RADS). CTA findings were evaluated using a conventional workstation and the JOIN mobile app. Scores of 3–5 were grouped as appearing typical or indeterminate for COVID-19 lung involvement and 0–2 as appearing atypical or negative for pneumonia. SARS-CoV-2 infection was confirmed by polymerase chain reaction (PCR).A total of 242 patients were included (42 with PCR-confirmed COVID-19). In the cohort of 43 patients with both neck CTA and chest CT, the predictive value for COVID-19 was equivalent . The interobserver agreement in the classification into CO-RADS 3–5 or 1–2 in CTA was good . In the cohort of 242 patients with neck CTA, the intraobserver agreement between the workstation and the JOIN app was perfect .Neck CTA enables the accurate identification of COVID-19-associated lung abnormalities in acute stroke. CO-RADS evaluations through mobile applications have a predictive value similar to the usual platforms. The need to confirm or rule out SARS-CoV-2 infection during acute stroke management in order to activate the specific pathways for infected patients might compromise the speed of workflows in stroke care protocols in the pandemic era. The COVID-19 pandemic is far from over; however, in many countries reverse transcription polymerase chain reaction (RT-PCR) testing is being eliminated from general screening in emergency settings or replaced with rapid detection methods such as point-of-care antigen tests, which are not universally available. According to a recent review from the Cochrane Library, the average sensitivity for the detection of SARS-CoV-2 infection using rapid tests in asymptomatic patients is 54.7% (95%CI 47.7‒61.6%), with wider confidence intervals depending on the brand, which may lead to misdiagnosis in some cases. In fact, none of the assays evaluated by the Cochrane Library met the World Health Organization acceptable performance standards. This translates into one in two or two in three asymptomatic COVID-19 cases missed when diagnosis is made based on point-of-care antigen tests [Expert consensus has recommended chest CT as an additional test in acute stroke to identify radiological abnormalities suggestive of COVID-19 –5. HowevCTA is one of the most commonly used non-invasive diagnostic tests for patients with acute ischemic stroke and suspected large vessel occlusion on arrival at the emergency room , 8. SourTelemedicine and mobile apps are progressively being incorporated into stroke protocols for the rapid communication and visualization of images, with positive results and excellent acceptance by practitioners. Several studies have shown good interobserver concordance and no differences in the accuracy of diagnosis between conventional radiology workstations (PACS) and these apps, as well as an acceleration of stroke code workflows when using the apps –10. ThesOur aim was to evaluate whether the assessment of lung apices usually included in neck CTA, performed as part of the routine diagnostic workup on arrival at the emergency department in patients with acute stroke, provides reliable information on the probability of an underlying COVID-19 infection. Our secondary aim was to evaluate whether the assessment of lung apices in neck CTA using the mobile app, JOIN, is comparable to the evaluation performed in the typical workstation.We conducted a retrospective, single-center cohort study that included patients with acute stroke admitted to a European Stroke Organization-certified stroke center.Inclusion criteria were as follows: aged older than 18, diagnosis of acute stroke, and undergoing a neck CTA upon arrival to the emergency room as part of the acute stroke diagnostic workup. We excluded patients transferred from other hospitals who had already undergone neck CTA at the referring hospital, those with a clinical diagnosis of COVID-19 without PCR confirmation, as well as those with no apical lungs visible in the neck CTA. Confirmed diagnoses of COVID-19 were based on detecting SARS-CoV-2 nucleic acid by polymerase chain reaction (PCR) assay from nasopharyngeal/oropharyngeal swabs.The study was conducted in two separate stages. The first stage, which included patients from the first pandemic wave , evaluated the intraobserver and interobserver concordance between chest CT findings and CTA for the diagnosis of COVID-19-related lung findings using conventional workstations. The second stage, which added patients with CTA from the second pandemic wave , analyzed the concordance between the diagnosis of COVID-19-related lung findings in CTA source images created in the conventional workstations or using a mobile app.Lung findings were classified using the COVID-19 Reporting and Data System (CO-RADS) . Scores For the second stage, we used the mobile app, JOIN , a certified communication app for health professionals designed to speed and optimize clinical communication and workflows, enabling the upload of radiology images to an encrypted server reachable by a standard smartphone connected to the Internet. The app also offers dedicated medical features such as an integrated DICOM viewer, timestamps, patient cases and chat and video telephony. JOIN is compliant with the European Union’s General Data Protection Regulation and ISO27001 and is certified by the US Food and Drug Administration .A medical graduate analyzed the CTA source images using the JOIN app and PACS to evaluate COVID-19 apical lung findings, ranking the findings according to the CO-RADS scale. Image assessment using JOIN and the workstation was performed independently for each source, separated in time and in random order. These evaluators were also blinded to the clinical data and PCR results.The study data were collected and managed using Research Electronic Data Capture (REDCap)  tools hoThis study was approved by the Ethics Committee of La Paz University Hospital (PI-4660). As a retrospective study, the committee was exempted from requiring patient consent.Patient and Public Involvement: The development of the research objectives and outcomes are based on the neurologist´s experience treating this profile of patients and the desire to optimise stroke care pathways in the COVID-19 pandemic waves. Patients and their advisors were not involved in the design, recruitment or conduct of this study.A total of 323 patients with acute stroke were screened for inclusion in the various stages of the study, 81 of whom were excluded from the study due to various criteria. The sample selection is shown in the flowchart .For the comparison between neck CTA and chest CT, 101 patients with acute stroke were included (24 with PCR-confirmed COVID-19). The cohort’s baseline characteristics are shown in Both neck CTA and chest CT were performed on 43 patients, with 11 (25%) presenting lung findings suggestive of COVID-19. There was complete concordance in the classification according to CO-RADS between the two tests, with a COVID-19 diagnosis sensitivity and specificity of 53.8% and 92.9%, respectively, with a predictive positive value of 77.8% and negative predictive value of 81.3%.As a sensitivity analysis, we confirmed the predictive value in the cohort of 71 patients with neck CTA . The interobserver agreement in classifying the neck CTA lung findings into CO-RADS 3–5 or 1–2 was good  .To compare the diagnosis performed in the workstations with that from the JOIN app, we included 242 patients with acute stroke who underwent neck CTA (29 with PCR-confirmed COVID-19). The cohort’s baseline characteristics are shown in The agreement in classifying the lung findings using the JOIN app and workstation with PACS into CO-RADS 3–5 or 1–2 was perfect  .Several expert-based recommendations proposed performing chest CT combined with head-and-neck CTA in patients with acute stroke to rule out early COVID-19 and prevent contagion of attending personnel , 14, 15.To our knowledge, few studies have published the results of performing chest CT in patients with stroke during the pandemic. The correlation between acute neuroimaging abnormalities and a higher CT lung severity score has already been studied; however, the clinical implications of these findings have yet to be established . DespiteSome authors have underscored the need to recognize COVID-19 features on radiographic images that may be acquired for other purposes to avoid overwhelming the healthcare systems in general, and radiology departments in particular . To thisAlthough PCR tests are currently performed much faster, these diagnostic tests are not always available in certain settings, and, for a time-dependent disease such as stroke, any type of information that allows for the rapid detection of COVID-19 infection while waiting for PCR confirmation is of great utility in ensuring the proper protection of healthcare staff and other patients , 20, 24.This practice is especially important today, given that many hospitals are considering abandoning systematic screening tests for every patient who arrives at the emergency department, particularly if they show no symptoms of SARS-COV-2 infection. Considering that a number of COVID-19 patients are asymptomatic but can transmit the disease and that these patients would not be considered for PCR testing, our results highlight the value of apical lung evaluation in CTA source images in patients with stroke , 23. HowAlthough a number of studies have shown the benefit of implementing mobile apps into the stroke code pathways , 27, 28,This study has certain limitations. First, this was a retrospective and unicentric study with a small sample size. Second, one of the raters was not an expert radiologist, which could have led to an underestimated interrater agreement. Third, to evaluate the concordance with Cohen’s kappa, the CO-RADS scale was reduced to a dichotomic option (possible or not possible COVID-19). Fourth, there is time-dependent variability in lung abnormalities caused by COVID-19, and the stroke date might not match the peak of lung involvement. Fifth, the involvement of upper lobes in COVID-19 is less frequent than in lower ones , and theNeck CTA is useful for the early detection of COVID-19-associated lung alterations in patients with acute stroke, with a predictive value comparable to that of chest CT and good interrater agreement. This evaluation can be performed through mobile apps such as JOIN, with almost complete agreement with the diagnosis reached in workstations. Although the absence of apical lung involvement in CTA cannot be used to rule out an asymptomatic SARS-CoV-2 infection, lung involvement findings using this readily available tool in stroke patients could be a red flag for healthcare workers to institute personal protective measures while waiting for the results of a confirmatory RT-PCR. Further studies are warranted to investigate the impact of implementing mobile apps to aid in the diagnosis of SARS-CoV-2 infection and timely communication to all stakeholders.


This study on the Roche ElecsysThe ACOV2S assay quantifies antibodies to the receptor-binding domain of the SARS-CoV-2 spike protein. The assigned units and the underlying standardization were compared to the international reference standard in BAU/mL. Assay specificity was assessed in samples (n=5981) collected prior to the COVID-19 pandemic and in samples from patients with non-COVID-19 respiratory infections (n=697) or other infectious diseases (n=771). Sensitivity was measured in 1313 samples from patients with mild COVID-19 and 297 samples from patients hospitalized with COVID-19. Comparison of results was performed to a comparator semi-quantitative anti-S1 assay of indirect detection format as well as a commercially available and an in-house version of a surrogate neutralization assay (ACE2-RBD).The originally assigned units for the ACOV2S assay were shown to be congruent to the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Overall specificity was 99.98% with no geographical differences noted and no loss of specificity in samples containing potentially cross-reacting antibodies. High sensitivity was observed, with 98.8% of samples reported to be reactive >14 days after infection and sustained detection of antibodies over time. For all samples, ACOV2S titers and neutralization capacities developed with comparable dynamics. Robust standardization and assay setup enable excellent reproducibility of results, independent of lot or analyzer used.The results from this study confirmed that ACOV2S is a highly sensitive and specific assay and correlates well with surrogate neutralization assays. The units established for ACOV2S are also interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins. Worldwide availability of the assay and analyzers render ACOV2S a highly practical tool for population-wide assessment and monitoring of the humoral response to SARS-CoV-2 infection or vaccination. The RBD binds to the angiotensin-converting enzyme-2 (ACE2) receptor and mediates the initial step of virus fusion with the host cell . Thus, aWe developed the Elecsys Anti-SARS-CoV-2-S (ACOV2S) assay in order to quantitatively determine the humoral response to SARS-CoV-2 infection or vaccination. Here, we describe the design of the assay and present performance data, including standardization and correlation to the first international WHO standard on anti-SARS-CoV-2 immunoglobulins, specificity and sensitivity in a large cohort of samples acquired either pre-pandemic or from patients with a polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection, and correlation with a surrogate neutralization assay.in vitro diagnostic test for this indication. The immunoglobulin isotype was IgG unless indicated otherwise. To assess sensitivity, 1610 PCR-confirmed single or sequential samples from 402 different patients with a native SARS-CoV-2 infection that occurred between February and April 2020  were tested. This included 1313 samples from 159 patients with mild COVID-19  and 297 samples taken from 243 patients who had been hospitalized with COVID-19. Hospitalization typically occurred in response to severe symptoms such as respiratory distress or critically low oxygenation of peripheral blood, with all clinical decision-making at the discretion of the local treating physician. All samples had a known time difference between positive PCR test and blood draw. The date of PCR diagnosis served as the reference point for infection as memory recall of symptom onset can vary significantly between individuals, particularly in cases with a mild course of disease. The majority of the samples from mild cases were taken >20 days post-PCR, conversely, the majority of the samples from hospitalized patients were taken within 20 days of a positive PCR result. A representative subset of the sensitivity samples were also tested using the Siemens SARS-CoV-2 immunoglobulin G (IgG) (COV2G) semi-quantitative assay according to the manufacturer’s instructions. Analytical studies on precision and reproducibility required considerable sample volumes. To fulfil these needs, pooled samples from two different donors with a native infection had to be used.The performance of the Elecsys ACOV2S assay was evaluated at Roche Diagnostics . All samples were collected and tested in accordance with applicable regulations, including relevant European Union directives and regulations, and the principles of the Declaration of Helsinki. Anonymized frozen serum or plasma samples were used for this study and included residual samples from blood donation centers or routine laboratory diagnostics and commercially available samples. A statement was obtained from the Ethics Committee of the Landesärztekammer Bayern confirming that there are no objections to the transfer and coherent use of anonymized leftover samples. For specificity analyses, samples from blood donors (n=2713 from the USA and n=740 from Africa) or routine diagnostic samples (n=2528 from Europe) collected before December 2019, and therefore presumed to be negative for SARS-CoV-2, were tested. In addition, cross-reactivity of the ACOV2S assay was assessed in 697 samples from patients with a respiratory infection and 771 samples from patients with other infectious, auto-immune, or non-infectious hepatic disease. These samples were obtained prior to October 2019 and were therefore considered to be seronegative for SARS-CoV-2-specific antibodies. Presence of specific immunoglobulins for other infections was confirmed using an approved The Elecsys Anti-SARS-CoV-2 S (ACOV2S) immunoassay is a quantitative electrochemiluminescence immunoassay (ECLIA) that detects antibodies to the SARS-CoV-2 S protein RBD. The candidate antigens were generated from HEK293 culture and transient transfection. The expression constructs included a leader signal for secretion and a His-tag for purification. Recombinant protein was then harvested from culture supernatants and purified by affinity and size exclusion chromatography. Labelling of proteins with biotin and ruthenium was realized by NHS-mediated amide bond formation of labels and protein of interest. The assay applies the double antigen sandwich format detecting immunoglobulins from the sample when bridged between two specifier antigens provided by the assay. Results are automatically read off a lot-specific standard curve and reported as the analyte concentration of each sample in U/mL. As the ACOV2S assay is a quantitative and standardized assay, no specific cut-off formula was applied. A medical decision point (MDP) indicative of the significant presence of RBD-specific antibodies was postulated at 0.8 U/mL based on a preliminary sample set from infected and non-infected samples (data not shown). This MDP was then found to fit well in the design verification studies shown in this manuscript and previous publications  antibody test is an automated two-step indirect chemiluminescent sandwich immunoassay that detects IgG antibodies against the spike RBD of the SARS-CoV-2 virus. Here the detected antibodies bind with one or both of their paratopes to the immobilized antigen, while counterstaining with anti-human IgG then indirectly detects the bound antibodies. Indirect detection methods are less able to reflect antibody maturation compared to double antigen sandwich formats .COV2G results are reported in index values, with <1.0 interpreted as non-reactive (negative) for anti-SARS-CoV-2 antibodies and ≥1.0 interpreted as reactive (positive) for anti-SARS-CoV-2 antibodies . Of noteThe GenScript cPass™ SARS-CoV-2 neutralization antibody detection kit is a blocking enzyme-linked immunosorbent assay (ELISA) intended for the qualitative and semi-quantitative direct detection of immunoglobulins that neutralize the interaction between RBD and hACE2. In samples that do not contain SARS-CoV-2 neutralizing antibodies, RBD conjugated to HRP can bind to ACE-2 without any impairment, generating a strong signal comparable to the reference reaction. If SARS-CoV-2 neutralizing antibodies are present, they will bind to RBD-HRP and prevent the interaction with ACE-2, resulting in impaired signal. Results are reported as ratio of sample result to reference result which equals percentage binding inhibition, with <30% interpreted as negative and ≥30% interpreted as positive for SARS-CoV-2 neutralizing antibodies .We also developed an in-house version of a surrogate neutralization assay for application on cobas e analyzers (Elecsys ACE2-RBD assay). Similar to cPass, but performed in solution rather than solid phase, this method measures the potential of samples containing SARS-CoV-2 antibodies to compete with ACE2-RBD binding.Potential interference was investigated for commonly used drugs , 14, druThe software R, version 3.4.0, was used for regression analyses (Passing-Bablock) to the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins and for the assessment of reproducibility and precision of results across different aliquots, days, analyzers, and assay lots. The experimental setup and statistical analyses followed the recommendations of the respective CLSI guideline (EP05-A3) . In brieThe RBD antigen could be expressed in high yields and reproducibly high quality and was highly amenable to purification and labeling procedures. We also evaluated trimeric spike and monomeric spike S1 subdomain antigens, but these could be expressed with lower yields only, and additionally required higher purification stringency. Moreover, an initial functional assessment with pre-pandemic samples suggested that better specificity was achieved for antibodies targeting the RBD compared with the trimeric spike or monomeric spike S1 antigens Figure 1The Double Antigen Sandwich (DAGS) immunoassay format is well established for the detection of immunoglobulins using Elecsys assays on cobas e analyzers. Elecsys assays have a short turnaround time of typically 18 minutes, which requires a highly sensitive detection system and, most importantly, rapid formation of detectable immunocomplexes including the analyte. Successful complex formation requires the provision of all assay components in solution (no precoated solid phase) to benefit from free diffusion. This supports low thresholds for detectable concentrations and, together with the sensitive electrochemiluminescence (ECL) detection method coming with a broad linear range, enables strong signal dynamics of the DAGS method used in Elecsys assays. The DAGS format per se does not differentiate between immunoglobulin classes. However, bridging of two antigens is required for signal generation, which leads by design to the predominant detection of high-affinity antibodies, i.e., IgG. We sought to further sustain this tendency by the provision of well-defined and monomeric antigens as well as rather stringent buffer conditions, such that single binding events, as mediated by IgG, are the major signal driver. RBD was ultimately selected as the ideal candidate to fulfill these requirements. By design, DAGS assays feature strong specificity and do not require sample predilution to reduce unspecific reactivity. The application of a highly specific antigen further corroborates this feature. Together with the adjustment of buffer stringency, these aspects serve to balance the required specificity versus sensitivity.At the time of the development and launch of the ACOV2S assay, no standard reference material existed for qualitative SARS-CoV-2 antibody assays. We developed an internal reference standard to enable reproducible and lot independent test results in arbitrary units. Parallel assessment of a medical decision point (MDP) for differentiation of samples being non-reactive or reactive for RBD-specific antibodies indicated that such an MDP might lie close to the numeric unit of 1 on this scale. In addition, established working calibrators based on pools of native human sample material were adjusted to cover critical supporting points enabling the generation of a robust standard curve. The primary measuring range of the ACOV2S assay was defined from 0.4 to 250 U/mL, determined by optimal linear detection of a dilution series of human samples and with the majority of results within this range following native infection with a mild course of disease. While retrospectively a larger measuring range had been desirable, automated dilution provides a convenient workaround. We subsequently observed that the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins (NIBSC code 20/136) were congruent to the originally assigned units based on the internal standard Figure 2Specificity was extensively assessed in pre-pandemic samples obtained from blood donors in the USA and Africa and routine diagnostic samples acquired in Europe. One presumably false-positive result was observed from >5000 samples in total, giving an overall specificity of 99.98%. No differences in specificity were observed between samples from different geographical locations and no differences were observed between different serological backgrounds Table 1.Sensitive detection of seroconversion was observed for patients with a native infection with SARS-CoV-2, with most samples determined “reactive” by ACOV2S within 14 days of a positive PCR test. Sensitivity increased with the onset of the humoral immune response, with 98.8% of samples reported to be reactive >14 days after infection. By day 28, all samples for patients in this cohort were determined to be reactive Figure 3Samples from patients with mild COVID-19 exhibited average rising titers over time as indicated by the heat map  of all samples per timepoint  has already been described . Here, wWe observed a good correlation of cPass results with the in-house Elecsys ACE2-RBD surrogate neutralization assay  independently of the analyzer used, with the high throughput analyzer of the latest generation (cobas e 801) having slightly better precision than a benchtop analyzer of the previous generation  , 19, 20.in vitro and surrogate neutralization assays . In this and previous studies, the results from the ACOV2S assay have been shown to correlate well with n assays , 20, 24.No standard reference material existed for quantitative SARS-CoV-2 antibody assays at the time that the ACOV2S assay was initially developed. However, we have shown here that the units that were established for ACOV2S are interchangeable with the units of the First International WHO Standard for anti-SARS-CoV-2 immunoglobulins, signifying that data generated with ACOV2S can be assessed retrospectively without the need for restandardization.in vivo are probably also comparable in both scenarios. In contrast, indirect formats do not differentiate if an antibody is bound with one or both paratopes to the antigen; either leads to the same signal. Antibody quality in terms of affinity is therefore more difficult to reflect in an indirect format assay.Longitudinal monitoring of patients with mild course disease shows relatively stable or rising antibody titers over time when determined with ACOV2S and in contrast to the competitor method. These findings are in line with other reports on reduced waning of antibody titers with DAGS format assays , 25. Reiin vitro neutralization capacities and even the in vivo efficacy of immunity . In this study, the ACOV2S assay demonstrated excellent precision and reproducibility, which are prerequisites for reliable titer monitoring over time. Virtually worldwide availability of the assay and analyzers renders ACOV2S a suitable candidate for population-wide assessment and monitoring of the humoral response elicited by the SARS-CoV-2 virus.ACOV2S is a highly sensitive and specific assay for the detection of antibodies to the RBD of the SARS-CoV-2 S protein. In this analysis, we have demonstrated standardization and correlation to the first international WHO standard of anti-SARS-CoV-2 immunoglobulins and correlation with surrogate neutralization assays. An analysis of absolute antibody titers and dynamics suggested that the assay can have value in determining disease severity. Furthermore, the DAGS format allows for the detection of low concentrations of high avidity antibodies, meaning that the ACOV2S assay may retain sensitivity during the process of antibody maturation. The ability of ACOV2S to predict the risk of severe disease in vaccinated or convalescent patients remains unknown but is of great interest in the current phase of the pandemic.simon.jochum@roche.com.The study was conducted in accordance with applicable regulations. For more information on the study and data sharing, qualified researchers may contact the corresponding author, All samples were collected and tested in accordance with applicable regulations, including relevant European Union directives and regulations, and the principles of the Declaration of Helsinki. Anonymized frozen serum or plasma samples were used for this study and included residual samples from blood donation centers or routine laboratory diagnostics and commercially available samples. Approval was granted by the Ethics Committee of the Landesaerztekammer Bayern who confirmed that there are no objections to the transfer and coherent use of anonymized leftover samples.Study concept/design: SJ, MW, PM, and KT. Data acquisition: KT, JK, MW, and PM. Data analysis and interpretation: KT, MW, PM, JK, SJ, and SP. Review and final approval of manuscript: SJ, MW, KT, and SP. All authors contributed to the article and approved the submitted version.ELECSYS and COBAS are trademarks of Roche. All other product names and trademarks are the property of their respective owners. The Elecsys Anti-SARS-CoV-2 S assay is approved under Emergency Use Authorisation in the US. Editorial support was provided by Steph Carter and Jade Drummond of inScience Communications, Springer Healthcare Ltd, UK, and was funded by Roche Diagnostics International Ltd .This study received funding from Roche Diagnostics. The funder was involved in the study design, analysis, interpretation of data, the writing of this article and the decision to submit it for publication. Editorial support was provided by Steph Carter and Jade Drummond of inScience Communications, Springer Healthcare Ltd, UK, and was funded by Roche Diagnostics International Ltd . ELECSYS and COBAS are trademarks of Roche. All other product names and trademarks are the property of their respective owners. All authors  are employees of Roche Diagnostics.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A more-and-more-accepted alternative to the time-consuming and technique-sensitive, classic, incremental-layering technique of resin-based composites (RBCs) is their placement in large increments. The so-called bulk-fill RBCs had to be modified for a higher polymerization depth and already have a 20-year history behind them. From the initial simple mechanisms of increasing the depth of cure by increasing their translucency, bulk-fill RBCs have evolved into complex materials with novel polymerization mechanisms and bioactive properties. However, since the materials are intended to replace the tooth structure, they must be comparable in mechanical behavior to the substance they replace. The study compares already established bulk-fill RBCs with newer, less-studied materials and establishes their relationship to dentin with regard to basic material properties such as hardness and indentation modulus. Instrumented indentation testing enables a direct comparison of tooth and material substrates and provides clinically relevant information. The results underline the strong dependence of the measured properties on the amount of filler in contrast to the small influence of the material classes into which they are classified. The main difference of RBCs compared to dentin is a comparable hardness but a much lower indentation modulus, emphasizing further development potential. The constant expansion of scientific knowledge, underpinned by the further development of existing filling materials and the introduction of completely new ones, leads to changes in dental treatment concepts. In the chronological development of resin-based composites (RBCs), bulk-fill RBCs represent the most recent development. They were developed with the aim of significantly reducing the treatment time by no longer having to build up RBC fillings in 2 mm layers, but instead applying them in 4 to 5 mm thick layers. In addition, the risk of introducing defects such as voids or contaminants between two-layered increments is also reduced. Since the classic incremental-layering technique is time-consuming and technique-sensitive, especially in large-volume posterior cavities, the bulk-fill application has attracted interest as an alternative. Today the concept is established, and a clinical time saving of approx. 20% compared to the conventional, incremental restoration technique is documented .This alternative bulk-filling concept originally arose from the idea of combining bulk-fill RBCs in two different viscosities. Although the first light-curing, high-viscosity bulk-fill RBC   came ontλ) and relates to a high probability of light absorption at a given wavelength. Germanium-based initiators can be partially activated by the blue light, but they need shorter wavelength light (violet) to reach their full potential [The described aesthetic deficit has been addressed in following materials either by adding suitable opacifiers (nanoparticles of zirconium oxide or ytterbium trifluoride) or by focusing the polymerization process on mechanisms that are less dependent on reduced translucency. These include the addition of new, more efficient photo-initiators (germanium-based)  to supplotential . This asotential .Another advancement, which also relies less on reduced translucency, includes a modification of the polymerization mechanism, namely reversible addition-fragmentation chain transfer (RAFT) polymerization ,10. It rBulk-fill RBCs with a different, optically perceptible translucency than the tooth in the uncured state were also developed, to offer an additional clinical simplification of the application. This allows for a clearer distinction between the RBC and the tooth when applied in the cavity, while the translucency of the RBC decreases after curing  and is nAdditional features to support simplified restoration concepts, such as bioactive properties ,16,17, hClinical evaluation of bulk-fill RBC restorations to date shows statistically similar failure rates to conventional restorations that have been filled incrementally (2 mm increments) . While dThe present study aims to offer a direct comparison of bulk-fill RBCs belonging to the different development and clinical application concepts described above, and to relate them to the properties of dentin, the tooth structure they are intended to replace. This direct comparison is possible through instrumented indentation techniques.It is therefore hypothesized that bulk-fill RBCs will perform similarly to dentin in terms of elastic–plastic mechanical behavior when measured under similar conditions and within the material category for which they are marked.A total of 18 commercially available bulk-fill resin-based composites (bulk-fill RBCs) were characterized in terms of their micromechanical properties and related to properties measured under identical conditions in human dentin . The mat® Style, Ivoclar Vivadent, Schaan, Liechtenstein) was used for curing. Immediately after demolding, the samples were stored in distilled water at 37 °C for 24 h. After that, the surface that had received the first exposure was wet-ground with silicon carbide paper  and polished with a diamond suspension (average grain size: 1 µm) until the surface was shiny . In addition, tooth samples of 2 mm thickness were obtained from 10 caries-free human molars. For this purpose, a slice was cut per tooth perpendicular to the longitudinal axis of the tooth and polished as described above.A total of 108 samples (2 mm × 2 mm × 18 mm) were prepared, corresponding to six samples for each of the 18 bulk-fill RBCs analyzed, following the recommendation of ISO 4049:2019 . The unp® HM2000, Helmut Fischer, Sindelfingen, Germany) equipped with a Vickers diamond tip. Six indentations were performed in each bulk-fill RBC and tooth specimen. Measurements were performed under force control by recording the indentation depth and the indentation force during each indentation cycle, consisting of increasing the force from 0.4 mN to 1000 mN within 20 s at constant speed, holding the maximum force for 5 s and finally reducing it to zero within 20 s. Each indentation created an impression, and the projected contact area of the indenter (Ac) and the surface area of the indentation under the applied test load (As) were determined for further parameter calculations. The projected indenter contact area (Ac) was determined from the force–indentation depth curve considering the indenter correction according to the model of Oliver and Pharr and described in ISO 14,577 [s(h)) and was calculated by dividing the test load by the surface area of the indentation under the applied test load (As). As a measure of the resistance to plastic deformation, the indentation hardness was calculated from the maximum indentation force and the projected indenter contact area (HIT = Fmax/Ac). This parameter was then converted to the more familiar Vickers hardness (HV = 0.0945 × HIT). Finally, the indentation modulus (EIT) was calculated from the slope of the tangent of the indentation–depth curve at maximum force.The micromechanical properties were evaluated using an automated microindenter  evaluated the influence of the parameters filler volume, filler weight and material category on the analyzed properties. The partial eta-squared statistic reported the practical significance of each term, based on the ratio of the variation attributed to the effect. Larger values of partial eta-squared (ηP2) indicate a greater amount of variation accounted for by the model .All variables were normally distributed, allowing a parametric approach to be used. A multifactor analysis of variance was applied to compare the parameters of interest . Results were compared using one-way and multiple-way analysis of variance (ANOVA) and Tukey honestly significant difference (HSD) p < 0.001) and very strong effect of the parameters filler volume and filler weight on the measured properties, while the effect of material category was consistently lower. The filler volume had the greatest impact on indentation modulus EIT (ηP2 = 0.918), closely followed by Martens HM (ηP2 = 0.900) and Vickers hardness HV (ηP2 = 0.851). A similar ranking of the parameters was also observed with regards to the effect strength of the filler weight . For material category, e.g., low- and high-viscosity light-cured and dual-cured bulk-fill RBCs, the ranking of the measured parameters with regard to the effect strength exerted remained the same, but the effect was considerably lower .A multifactorial analysis indicates a significant  with a range of 64.5% (Filtek™ Bulk Fill Flowable Restorative and Filtek Bulk Fill) to 75% (x-tra base) for the filler weight and 38% (Venus Bulk Fill) to 61% (x-tra base) for the filler volume. For bulk-filled high-viscosity light-cured RBCs, filler weight ranged from 76.5 (Filtek One and SonicFill 2) to 87% (Beautifil Bulk restorative), while the filler volume ranged from 58.5 (Filtek One and SonicFill 2) to 74.5% (Beautifil Bulk restorative).Within a material category, a one-way ANOVA showed that only the material x-tra base achieved a significantly higher HM value compared to dentin in the low-viscosity light-cured bulk-fill RBC category a, while The statistical analysis is somewhat similar for the Vickers hardness, while dentin has similar HV values as Filtek Supreme XT flow and Tetric EvoFlow bulk-fill in the light-cured low-viscosity bulk-fill RBCs a and lowA completely different situation was observed for the indentation modulus. Dentin ranked highest among all light-cured low-viscosity bulk-fill RBCs a and wasThe health challenge posed by tooth decay places an enormous burden on healthcare systems around the world . ResearcSince dental materials are intended to replace the tooth structure, their properties should be designed accordingly. However, test methods that allow a direct comparison of both substrates are limited due to the small size and the heterogeneity of the tooth samples. In the present study, we opted for the quasi-static micro-indentation method, which, with indentation depths of 4–10 µm and standardized methods, not only enables a direct material and tooth substrate comparison, but also comparison with data collected over time in large databases that contain many clinically successful materials . The latThe classification of RBCs into material categories is rightly controversial and is very often based on advertising strategies. While the low-viscosity bulk-fill RBCs are generally characterized by lower filler content compared to the high-viscosity bulk-fill category, individual materials may not reflect their classification system . This stThe dentin substrate tested in the present study should not only allow a comparison with the analyzed materials, but also a ranking of all analyzed materials, since it serves as a reference in all graphic representations . General® SDR™ flow, aimed to create a material with excellent rheological properties to allow flow in narrow and deep cavities where condensing a material is not possible, and/or to serve as a flowable lining. The claimed that it reduced the polymerization shrinkage required for proper adaptation to the cavity after curing was confirmed as it was found that the shrinkage stress after polymerization not only compared to regular flowable materials but also compared to higher-filled RBCs, or the low shrinkage silorane was lower [The analyzed light-cured low-viscosity bulk-fill RBCs reflect a time scale in the development of the materials. The initial development of such materials, starting chronologically with SureFilas lower . This efas lower . With reAnother distinctive feature of many bulk-fill RBCs—e.g., SDR, Venus Bulk Fill, Venus Bulk One, Beautifil Bulk Flow—is that the base monomer bisphenol A-glycidyl methacrylate (Bis-GMA) has been completely replaced by less-viscous dimethacrylates such as urethane dimethacrylate (UDMA) and its derivatives, triethylene glycol dimethacrylate (TEGDMA) or ethoxylated bisphenol-A-dimethacrylate (EBPDMA), which can form more flexible polymers than Bis-GMA ,37.+, Sr2+, Al3+, BO33−, and SiO33−. Silicate and fluoride are known to act as strong inducers of the remineralization of the dentin matrix [The progressive developments observed in regular RBCs are increasingly reflected in bulk-fill materials as well. Here, too, there is a trend of modifying RBCs to meet the increased demand for bioactive materials that can prevent the recurrence of carious lesions. This was conducted in Beautifil Bulk Flow and Beautifil Bulk Restorative  by addinn matrix . Strontin matrix . The dif2-CaO-CaF2-Na2O glass) capable of raising the pH and forming fluoroapatite in phosphate-containing media, which explains the low incidence of secondary caries in Cention N [The ability to leach out caries-protective ions such as phosphate, fluorite, and especially alkali and alkaline earth ions such as calcium ,42, was ention N . In addiention N . An addiention N ,17. The ention N . The bioention N ,45,46 alention N ,17. DireThe most recently launched bulk-fill RBC is Venus Bulk Flow One, a material that meets two criteria at once to speed up the restoration process as it is a universal chromatic material that can be applied in bulk . DespiteSince materials have individual properties that differ not only between material categories but also within the material categories to which they belong and the tooth substance, all null hypotheses are rejected.Bulk-fill RBCs should not necessarily be classified into material categories but should be considered as materials to be evaluated individually as the properties measured relate to the amount of inorganic filler and not to the material category to which they belong. The ratio of the tested materials to dentin is also individual, with the general conclusion that the hardness of the tested RBCs is somewhat in the range of dentin, but dentin is characterized by an indentation modulus superior to most of them. This aspect can be useful in further material development, for a better adaptation of the material properties to those of the dentin.