Two Years of Genomic Surveillance in Belgium during the SARS-CoV-2 Pandemic to Attain Country-Wide Coverage and Monitor the Introduction and Spread of Emerging Variants.

An adequate SARS-CoV-2 genomic surveillance strategy has proven to be essential for countries to obtain a thorough understanding of the variants and lineages being imported and successfully established within their borders. During 2020, genomic surveillance in Belgium was not structurally implemented but performed by individual research laboratories that had to acquire the necessary funds themselves to perform this important task. At the start of 2021, a nationwide genomic surveillance consortium was established in Belgium to markedly increase the country's genomic sequencing efforts (both in terms of intensity and representativeness), to perform quality control among participating laboratories, and to enable coordination and collaboration of research projects and publications. We here discuss the genomic surveillance efforts in Belgium before and after the establishment of its genomic sequencing consortium, provide an overview of the specifics of the consortium, and explore more details regarding the scientific studies that have been published as a result of the increased number of Belgian SARS-CoV-2 genomes that have become available.

Change in COVID19 outbreak pattern following vaccination in long-term care facilities in Flanders, Belgium.

INTRODUCTION: Long term care facilities for elderly (LTCFs) in Europe encountered a high disease burden at the start of the COVID-19 pandemic. Therefore, these facilities were the first to receive COVID-19 vaccines in many European countries. A limited COVID-19 vaccine supply early 2021 resulted in a majority of residents and healthcare workers (HCWs) in LTCFs being vaccinated compared to a minority in the general population. This study exploits this imbalance to assess the efficiency of COVID-19 vaccination in containing outbreaks in LTCFs. METHODS: Exploratory statistics were performed using data from a COVID-19 surveillance system covering all 842 LTCFs in Flanders (the northern region of Belgium). The number and size of COVID-19 outbreaks in LTCFs were compared (1) before and after introducing vaccines and (2) with the status of the pandemic in the general population. Based on individual data from 15 LTCFs, the infection rate and symptoms of vaccinated and unvaccinated residents and HCWs were compared during a COVID-19 outbreak. RESULTS: 95.8% of the residents and 90.9% of the HCWs in Flemish LTCFs were vaccinated before May 30, 2021. Before vaccine introduction, residents in LTCFs were 10 times more likely to test positive for COVID-19 than the general population of Flanders. This ratio reversed after vaccination. Furthermore, after vaccination fewer and shorter outbreaks were observed involving fewer residents. During these outbreaks, vaccinated and unvaccinated residents were equally likely to test positive, but positive vaccinated residents were less likely to develop severe symptoms. In contrast, unvaccinated HCWs were more likely to test positive. CONCLUSION: In the first half of 2021, two-dose vaccination was highly efficient in preventing and containing outbreaks in LTCFs, reducing COVID-19 hospitalizations and deaths. The high likelihood of unvaccinated HCWs to be involved in COVID-19 outbreaks in vaccinated LTCFs emphasizes the importance of vaccinating HCWs.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Evaluation of Humoral and Cellular Responses in SARS-CoV-2 mRNA Vaccinated Immunocompromised Patients.

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.

Variant Analysis of SARS-CoV-2 Genomes from Belgian Military Personnel Engaged in Overseas Missions and Operations.

More than a year after the first identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of the 2019 coronavirus disease (COVID-19) in China, the emergence and spread of genomic variants of this virus through travel raise concerns regarding the introduction of lineages in previously unaffected regions, requiring adequate containment strategies. Concomitantly, such introductions fuel worries about a possible increase in transmissibility and disease severity, as well as a possible decrease in vaccine efficacy. Military personnel are frequently deployed on missions around the world. As part of a COVID-19 risk mitigation strategy, Belgian Armed Forces that engaged in missions and operations abroad were screened (7683 RT-qPCR tests), pre- and post-mission, for the presence of SARS-CoV-2, including the identification of viral lineages. Nine distinct viral genotypes were identified in soldiers returning from operations in Niger, the Democratic Republic of the Congo, Afghanistan, and Mali. The SARS-CoV-2 variants belonged to major clades 19B, 20A, and 20B (Nextstrain nomenclature), and included "variant of interest" B.1.525, "variant under monitoring" A.27, as well as lineages B.1.214, B.1, B.1.1.254, and A (pangolin nomenclature), some of which are internationally monitored due to the specific mutations they harbor. Through contact tracing and phylogenetic analysis, we show that isolation and testing policies implemented by the Belgian military command appear to have been successful in containing the influx and transmission of these distinct SARS-CoV-2 variants into military and civilian populations.

Different long-term avidity maturation for IgG anti-spike and anti-nucleocapsid SARS-CoV-2 in hospitalized COVID-19 patients.

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.

Combined oropharyngeal/nasal swab is equivalent to nasopharyngeal sampling for SARS-CoV-2 diagnostic PCR.

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.

Sensitivity and specificity of 14 SARS-CoV-2 serological assays and their diagnostic potential in RT-PCR negative COVID-19 infections.

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 2nd week after symptom onset or later.