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(1) Background: early in the COVID-19 pandemic, reverse transcription polymerase chain reaction (RT-PCR) testing was limited. Assessing seroprevalence helps understand prevalence and reinfection risk. However, such data are lacking for the first epidemic wave in Belgian nursing homes. Therefore, we assessed SARS-CoV-2 seroprevalence and cumulative RT-PCR positivity in Belgian nursing homes and evaluated reinfection risk. (2) Methods: we performed a cross-sectional study in nine nursing homes in April and May 2020. Odds ratios (ORs) were calculated to compare the odds of (re)infection between seropositive and seronegative participants. (3) Results: seroprevalence was 21% (95% CI: 18-23): 22% (95% CI: 18-25) in residents and 20% (95% CI: 17-24) in staff. By 20 May 2020, cumulative RT-PCR positivity was 16% (95% CI: 13-21) in residents and 8% (95% CI: 6-12) in staff. ORs for (re)infection in seropositive (compared to seronegative) residents and staff were 0.22 (95% CI: 0.06-0.72) and 3.15 (95% CI: 1.56-6.63), respectively. (4) Conclusion: during the first wave, RT-PCR test programmes underestimated the number of COVID-19 cases. The reinfection rate in residents was 3%, indicating protection, while it was 21% in staff, potentially due to less cautious health behaviour. Future outbreaks should use both RT-PCR and serological testing for complementary insights into transmission dynamics.

During the SARS-CoV-2 pandemic, rapid and sensitive detection of SARS-CoV-2 has been of high importance for outbreak control. Reverse transcriptase polymerase chain reaction (RT-PCR) is the current gold standard, however, the procedures require an equipped laboratory setting and personnel, which have been regularly overburdened during the pandemic. This often resulted in long waiting times for patients. In contrast, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is a simple, cost-efficient, and fast procedure, allowing for rapid and remote detection of SARS-CoV-2. In the current study, we performed a clinical evaluation of a new point-of-care test system based on LAMP-technology for SARS-CoV-2 detection, providing a result within 25 min (1copy™ COVID-19 MDx Kit Professional system). We tested 112 paired nasopharyngeal swabs, collected in the COVID-19 Ghent University Hospital test center, using the 1copy™ COVID-19 MDx Kit Professional system, and RT-PCR as the reference method. The test system was found to have a clinical sensitivity of 93.24% (69/74) (95% confidence interval [CI]: 84.93%-97.77%) and specificity of 97.37% (37/38) (95% CI: 86.19%-99.93%). Due to its easy smartphone operation and ready-to-use reagents, it ought to be easily applied in for instance general practices, pharmacies, nursing homes, schools, and companies. This would facilitate an efficient SARS-CoV-2 outbreak control and quarantine policy, as diagnosis can occur sooner in a near-patient setting., (© 2023 Wiley Periodicals LLC.)

Dried Blood Spots (DBS) are broadly used in SARS-CoV-2 surveillance studies, reporting either the presence or absence of SARS-CoV-2 antibodies. However, quantitative follow-up has become increasingly important to monitor humoral vaccine responses. Therefore, we aimed to evaluate the performance of DBS for the detection of anti-spike SARS-CoV-2 antibody concentrations using a commercially available assay, reporting in a standardised unitage (International Units/mL; IU/mL). To assess the sensitivity and specificity of the ImmunoDiagnostics ELISA on serum and DBS for SARS-CoV-2 antibody detection, we analysed 72 paired DBS and serum samples. The SARS-CoV-2 S1 IgG ELISA kit (EUROIMMUN) on serum was used as the reference method. We performed a statistical assessment to optimise the cut-off value for DBS and serum and assessed the correlation between DBS and serum antibody concentrations. We found that anti-spike SARS-CoV-2 antibody concentrations detected in DBS are highly correlated to those detected in paired serum (Pearson correlation 0.98; p-value < 0.0001), allowing to assess serum antibody concentration using DBS. The optimal cut-off for antibody detection on DBS was found to be 26 IU/mL, with 98.1% sensitivity and 100% specificity. For serum, the optimal cut-off was 14 IU/mL, with 100% sensitivity and 100% specificity. Therefore, we conclude that the ImmunoDiagnostics ELISA kit has optimal performance in the detection of SARS-CoV-2 antibodies on both DBS and serum. This makes DBS ideal for large-scale follow-up of humoral SARS-CoV-2 immune responses, as it is an easy but valuable sampling method for quantification of SARS-CoV-2 antibodies, compared to serum., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2022. Published by Elsevier B.V.)

Introduction: The high variability of SARS-CoV-2 serological response after COVID-19 infection hampers its use as indicator of the timing of infection. A potential alternative method is the determination of affinity maturation of SARS-CoV-2 IgG, expressed as the SARS-CoV-2 IgG avidity., Methods: SARS-CoV-2 IgG concentration and avidity were measured in sera of hospitalized COVID-19 patients sampled at two weeks and ≥12 weeks post symptom onset using an in-house developed protocol based on EUROIMMUN (anti-spike) and EDI™ (anti-nucleocapsid) SARS-CoV-2 IgG ELISA protocols., Results: We included 68 confirmed COVID-19 patients that tested positive for SARS-CoV-2 IgG in both the initial and follow-up specimen sampled at a median of 14 (range 10-18) days and 120 (range 84-189) days, respectively, post symptom onset. The median anti-spike and anti-nucleocapsid SARS-CoV-2 IgG avidity response was 40% (range 9-93%) and 72% (range 27-104%), respectively, for the first sample, and 66% (range 28-90%) and 57% (range 25-94%), respectively, for the second sample. The proportion of SARS-CoV-2 IgG avidity results ≥60% was significantly lower for anti-spike compared to anti-nucleocapsid IgG for initial samples ( p < 0.01) and vice versa for follow-up samples ( p < 0.01)., Conclusion: Anti-nucleocapsid SARS-CoV-2 IgG maturation occurs faster and avidity decreases faster than anti-spike IgG, indicating different kinetics of anti-spike and anti-nucleocapsid IgG. Further, affinity maturation after SARS-CoV-2 infection is frequently incomplete.

Background: Molecular detection of SARS-CoV-2 in respiratory samples is the gold standard for COVID-19 diagnosis but it has a long turnaround time and struggles to detect low viral loads. Serology could help to diagnose suspected cases which lack molecular confirmation. Two case reports are presented as illustration., Objectives: The aim of this study was to evaluate the performance of several commercial assays for COVID-19 serology. We illustrated the added value of COVID-19 serology testing in suspect COVID-19 cases with negative molecular test., Study Design: Twenty-three sera from 7 patients with a confirmed molecular diagnosis of SARS-CoV-2 were tested using 14 commercial assays. Additionally, 10 pre-pandemic sera and 9 potentially cross-reactive sera were selected. We calculated sensitivity and specificity. Furthermore, we discuss the diagnostic relevance of COVID-19 serology in a retrospective cohort of 145 COVID-19 cases in which repetitive molecular and serological SARS-CoV-2 tests were applied., Results: The interpretation of the pooled sensitivity of IgM/A and IgG resulted in the highest values (range 14-71% on day 2-7; 88-94% on day 8-18). Overall, the specificity of the assays was high (range 79-100%). Among 145 retrospective cases, 3 cases (2%) remained negative after sequential molecular testing but positive on final SARS-CoV-2 serology., Conclusion: Sensitivity of COVID-19 serological diagnosis was variable but consistently increased at >7 days after symptom onset. Specificity was high. Our data suggest that serology can complement molecular testing for diagnosis of COVID-19, especially for patients presenting the 2 nd week after symptom onset or later.

We report on sample IS/17575 since it generated highly divergent results in the Belgian SARS-CoV-2 serology external quality assessment scheme. Sample IS/17575 was serum originating from a 30 years old male patient. 124 diagnostic laboratories analysed this sample. A total of 168 results was returned (including 5 doubles). Overall, 38 were positive. All tests against S1 were positive except the Euroimmun IgG ELISA and the Ortho clinical Diagnostics VITROS IgG CLIA. All tests against S1/S2 (Liaison, Diasorin) resulted in a signal above cutoff. Assays against RBD, mostly generate a negative result. An exception are the Wantai SARS-CoV-2 ELISA's. All tests targeting N protein were negative. The survey shows, when >6 months post-infection, assays targeting at least S1, and preferably S1 combined with S2, are the most sensitive. This finding accentuates the necessity of external quality assessment schedules and importance of antigenic composition of serologic SARS-CoV-2 assays., Competing Interests: Declaration of competing interest The author stated that there are no conflicts of interest regarding the publication of this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: The incidence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections in the Belgian community is mainly estimated based on test results of patients with coronavirus disease (COVID-19)-like symptoms. The aim of this study was to investigate the evolution of the SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positivity ratio and distribution of viral loads within a cohort of asymptomatic patients screened prior hospitalization or surgery, stratified by age category., Materials/methods: We retrospectively studied data on SARS-CoV-2 real-time RT-PCR detection in respiratory tract samples of asymptomatic patients screened pre-hospitalization or pre-surgery in nine Belgian hospitals located in Flanders over a 12-month period (1 April 2020-31 March 2021)., Results: In total, 255925 SARS-CoV-2 RT-PCR test results and 2421 positive results for which a viral load was reported, were included in this study. An unweighted overall SARS-CoV-2 real-time RT-PCR positivity ratio of 1.27% was observed with strong spatiotemporal differences. SARS-CoV-2 circulated predominantly in 80+ year old individuals across all time periods except between the first and second COVID-19 wave and in 20-30 year old individuals before the second COVID-19 wave. In contrast to the first wave, a significantly higher positivity ratio was observed for the 20-40 age group in addition to the 80+ age group compared to the other age groups during the second wave. The median viral load follows a similar temporal evolution as the positivity rate with an increase ahead of the second wave and highest viral loads observed for 80+ year old individuals., Conclusion: There was a high SARS-CoV-2 circulation among asymptomatic patients with a predominance and highest viral loads observed in the elderly. Moreover, ahead of the second COVID-19 wave an increase in median viral load was noted with the highest overall positivity ratio observed in 20-30 year old individuals, indicating they could have been the hidden drivers of this wave., Competing Interests: The authors have declared that no competing interests exist.

In the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, testing for SARS-CoV-2-specific antibodies is paramount for monitoring immune responses in postauthorization vaccination and seroepidemiological studies. However, large-scale and iterative serological testing by venipuncture in older persons can be challenging. Capillary blood sampling using a finger prick and collection on protein saver cards, i.e., dried blood spots (DBSs), has already proven to be a promising alternative. However, elderly persons have reduced cutaneous microvasculature, which may affect DBS-based antibody testing. Therefore, we aimed to evaluate the performance of DBS tests for the detection of SARS-CoV-2 antibodies among nursing homes residents. We collected paired venous blood and DBS samples on two types of protein saver cards (Whatman and EUROIMMUN) from nursing home residents, as well as from staff members as a reference population. Venous blood samples were analyzed for the presence of SARS-CoV-2 IgG antibodies using the Abbott chemiluminescent microparticle immunoassay (CMIA). DBS samples were analyzed by the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 IgG antibodies. We performed a statistical assessment to optimize the ELISA cutoff value for the DBS testing using Youden's J index. A total of 273 paired DBS-serum samples were analyzed, of which 129 were positive, as assessed by the reference test. The sensitivities and specificities of DBS testing ranged from 95.0% to 97.1% and from 97.1% to 98.8%, respectively, depending on the population (residents or staff members) and the DBS card type. Therefore, we found that DBS sampling is a valid alternative to venipuncture for the detection of SARS-CoV-2 antibodies among elderly subjects. IMPORTANCE Since the implementation of newly developed SARS-CoV-2 vaccines in the general population, serological tests are of increasing importance. Because DBS samples can be obtained with a finger prick and can be shipped and stored at room temperature, they are optimal for use in large-scale SARS-CoV-2 serosurveillance or postauthorization vaccination studies, even in an elderly study population.

Background: Early 2020, a COVID-19 epidemic became a public health emergency of international concern. To address this pandemic broad testing with an easy, comfortable and reliable testing method is of utmost concern. Nasopharyngeal (NP) swab sampling is the reference method though hampered by international supply shortages. A new oropharyngeal/nasal (OP/N) sampling method was investigated using the more readily available throat swab., Results: 35 patients were diagnosed with SARS-CoV-2 by means of either NP or OP/N sampling. The paired swabs were both positive in 31 patients. The one patient who tested negative on both NP and OP/N swab on admission, was ultimately diagnosed on bronchoalveolar lavage fluid. A strong correlation was found between the viral RNA loads of the paired swabs (r = 0.76; P < 0.05). The sensitivity of NP and OP/N analysis in hospitalized patients (n = 28) was 89.3% and 92.7% respectively., Conclusions: This study demonstrates equivalence of NP and OP/N sampling for detection of SARS-CoV-2 by means of rRT-PCR. Sensitivity of both NP and OP/N sampling is very high in hospitalized patients.

Vitamin D is an essential nutrient for various physiological functions, including immunity. While it has been suggested that higher vitamin D levels/supplementation are associated with a better immune response to COVID-19 vaccination, conflicting data exist. Therefore, we aimed to investigate the association between vitamin D (25-hydroxyvitamin D) deficiency/supplementation, and SARS-CoV-2 antibody responses post-vaccination in nursing home residents (NHRs) and staff (NHS). Blood samples were collected from 115 NHRs and 254 NHS at baseline and 14 days after primary course BNT162b2 vaccination. Baseline samples were assessed for serum 25-hydroxyvitamin D levels, while follow-up samples were analyzed for spike protein S1 receptor-binding domain (S1RBD) IgG antibody concentrations and 50% pseudoneutralization titers. Vitamin D supplementation status was obtained from NHRs medical records. We compared immune responses between (severe) vitamin D-deficient and -sufficient NHRs/NHS and between supplemented and non-supplemented NHRs, stratified for history of SARS-CoV-2 infection and participant type. No significant differences in either binding or neutralizing COVID-19 vaccine antibody response were found between groups. The prevalence of vitamin D deficiency (<20 ng/mL) was 45% (95% CI: 36–54%) among NHRs and 60% (95% CI: 54–66%) among NHS. Although we showed that vitamin D status may not be related to a better COVID-19 vaccine antibody response, addressing the high prevalence of vitamin D deficiency in the nursing home population remains important. [ABSTRACT FROM AUTHOR]

Background: Immunocompromised patients are at increased risk of severe COVID-19 and impaired vaccine response. In this observational prospective study, we evaluated immunogenicity of the BNT162b2 mRNA vaccine in cohorts of primary or secondary immunocompromised patients. Methods: Five clinical groups of immunocompromised patients [primary immunodeficiency (PID) (n=57), people living with HIV (PLWH) (n=27), secondary immunocompromised patients with a broad variety of underlying rheumatologic (n=23) and homogeneous (multiple sclerosis) neurologic (n=53) conditions and chronic kidney disease (CKD) (n=39)] as well as a healthy control group (n=54) were included. Systemic humoral and cellular immune responses were evaluated by determination of anti-SARS-CoV-2 Spike antibodies using a TrimericS IgG assay (Diasorin) and through quantification of interferon gamma release in response to SARS-CoV-2 antigen with QuantiFERON SARS-CoV-2 assay (Qiagen), respectively. Responses were measured at pre-defined time-points after complete vaccination. Results: All healthy controls, PLWH and CKD-patients had detectable antibodies 10 to 14 days (T2) and 3 months (T3) after administration of the second vaccination. In contrast, only 94.5% of the PID, 50.0% of the rheumatologic and 48.0% of neurologic patients developed antibodies at T2 and only 89.1% of the PID, 52.4% of the rheumatologic and 50.0% of neurologic patients developed antibodies at T3. At T3 no significant differences in cellular response between the healthy control group and the PLWH and CKD groups were found, while proportions of reactive subjects were lower in PID and rheumatologic patients and higher in neurologic patients. Humoral and cellular immune responses significantly correlated in the healthy control, PID, PLWH groups for all 3 antigens. Conclusion: Patients with acquired or inherited immune disorders may show variable immune responses to vaccination with the BNT162b2 mRNA vaccine against SARS-CoV-2. Whether humoral, cellular or both immune responses are delayed depends on the patient group, therapy and individual risk factors. These data may guide the counselling of patients with immune disorders regarding vaccination of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies, using dried blood spots, was determined in October–November 2020, among residents and staff randomly selected from 20 nursing homes (NH) geographically distributed in Flanders, Belgium. Sociodemographic and medical data [including coronavirus disease 2019 (COVID-19) symptoms and results of RT-PCR tests] were retrieved using questionnaires. The overall seroprevalence was 17.1% [95% confidence interval (CI) 14.9–19.5], with 18.9% (95% CI 15.9–22.2) of the residents and 14.9% (95% CI 11.9–18.4) of the staff having antibodies, which was higher than the seroprevalence in blood donors. The seroprevalence in the 20 NH varied between 0.0% and 45.0%. Fourteen per cent of the staff with antibodies, reported no typical COVID-19 symptoms, while in residents, 51.0% of those with antibodies had no symptoms. The generalised mixed effect model showed a positive association between COVID-19 symptoms and positive serology, but this relation was weaker in residents compared to staff. This study shows that NH are more affected by SARS-CoV-2 than the general population. The large variation between NH, suggests that some risk factors for the spread among residents and staff may be related to the NH. Further, the results suggest that infected people, without the typical COVID-19 symptoms, might play a role in outbreaks. [ABSTRACT FROM AUTHOR]

Despite the high efficacy of the BNT162b2 vaccine in the general population, data on its immunogenicity among frail elderly individuals are limited. Recently, levels of anti-SARS-CoV-2 spike IgG antibodies and serum neutralization titers were confirmed as good immune markers of protection against the virus, with evidence showing a reverse correlation between these two parameters and susceptibility to infection. Here we analyzed sera from 138 nursing home residents (median age of 88.9 years) and 312 nursing home staff (median age of 50.7 years) to determine the humoral response to two doses of the BNT162b2 vaccine, and found markedly decreased serum anti-spike antibody levels and neutralization titers in the nursing home resident (NHR) group, with over 11% non-responders compared to only 1.3% among the controls. Moreover, three months post-vaccination, a significant decrease in antibody titers was observed in COVID-19-naive nursing home residents. Subsequent flow cytometry and interferon gamma secretion analyses indicated that antibody non-responders among NHRs also failed to mount cellular responses. The presented data emphasize that additional measures are needed in the population of frail elderly individuals. Given the high proportion of non-responders among NHRs, continued monitoring should be considered in this group. [ABSTRACT FROM AUTHOR]

We retrospectively compared the long-term evolution of IgG anti-spike (S) and anti-nucleocapsid (N) levels (Abbott immunoassays) in 116 non-severe and 115 severe SARS-CoV-2 infected patients from 2 university hospitals up to 365 days post positive RT-PCR. IgG anti-S and anti-N antibody levels decayed exponentially up to 365 days after a peak 0 to 59 days after positive RT-PCR. Peak antibody level/cut-off ratio 0 to 59 days after positive RT-PCR was more than 70 for anti-S compared to less than 6 for anti-N (P < 0.01). Anti-S and anti-N were significantly higher in severe compared to non-severe patients up to 180 to 239 days and 300 to 365 days, respectively (P < 0.05). Despite similar half-lives, the estimated time to 50% seronegativity was more than 2 years for anti-S compared to less than 1 year for anti-N in non-severe and severe COVID-19 patients, due to the significantly higher peak antibody level/cut-off ratio for anti-S compared to anti-N. [ABSTRACT FROM AUTHOR]