Immunovirological and environmental screening reveals actionable risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks.

Coronavirus Disease 2019 (COVID-19) vaccination has resulted in excellent protection against fatal disease, including in older adults. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases among residents) by combining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aerosol monitoring, whole-genome phylogenetic analysis and immunovirological profiling of nasal mucosa by digital nCounter transcriptomics. Phylogenetic investigations indicated that each outbreak stemmed from a single introduction event, although with different variants (Delta, Gamma and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 d after the initial infection. Combining demographic, immune and viral parameters, the best predictive models for mortality comprised IFNB1 or age, viral ORF7a and ACE2 receptor transcripts. Comparison with published pre-vaccine fatal COVID-19 transcriptomic and genomic signatures uncovered a unique IRF3 low/IRF7 high immune signature in post-vaccine fatal COVID-19 outbreaks. A multi-layered strategy, including environmental sampling, immunomonitoring and early antiviral therapy, should be considered to prevent post-vaccination COVID-19 mortality in nursing homes.

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.

Evaluation of Screening Program and Phylogenetic Analysis of SARS-CoV-2 Infections among Hospital Healthcare Workers in Liège, Belgium.

Healthcare workers (HCWs) are known to be at higher risk of developing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections although whether these risks are equal across all occupational roles is uncertain. Identifying these risk factors and understand SARS-CoV-2 transmission pathways in healthcare settings are of high importance to achieve optimal protection measures. We aimed to investigate the implementation of a voluntary screening program for SARS-CoV-2 infections among hospital HCWs and to elucidate potential transmission pathways though phylogenetic analysis before the vaccination era. HCWs of the University Hospital of Liège, Belgium, were invited to participate in voluntary reverse transcriptase-polymerase chain reaction (RT-PCR) assays performed every week from April to December 2020. Phylogenetic analysis of SARS-CoV-2 genomes were performed for a subgroup of 45 HCWs. 5095 samples were collected from 703 HCWs. 212 test results were positive, 15 were indeterminate, and 4868 returned negative. 156 HCWs (22.2%) tested positive at least once during the study period. All SARS-CoV-2 test results returned negative for 547 HCWs (77.8%). Nurses (p < 0.05), paramedics (p < 0.05), and laboratory staff handling respiratory samples (p < 0.01) were at higher risk for being infected compared to the control non-patient facing group. Our phylogenetic analysis revealed that most positive samples corresponded to independent introduction events into the hospital. Our findings add to the growing evidence of differential risks of being infected among HCWs and support the need to implement appropriate protection measures based on each individual's risk profile to guarantee the protection of both HCWs and patients. Furthermore, our phylogenetic investigations highlight that most positive samples correspond to distinct introduction events into the hospital.

A 2-month field cohort study of SARS-CoV-2 in saliva of BNT162b2 vaccinated nursing home workers.

Background: Nursing home (NH) residents have been severely affected during the COVID-19 pandemic because of their age and underlying comorbidities. Infection and outbreaks in NHs are most likely triggered by infected workers. Screening for asymptomatic NH workers can prevent risky contact and viral transmission to the residents. This study examined the effect of the BNT162b2 mRNA COVID­19 (Comirnaty®; BioNTech and Pfizer) vaccination on the saliva excretion of SARS-CoV-2 among NH workers, through weekly saliva RT-qPCR testing. Methods: A 2-month cohort study was conducted among 99 NHs in the Walloon region (Belgium), at the start of February 2021. Three groups of workers, i.e., non-vaccinated (n = 1618), one-dosed vaccinated (n = 1454), and two-dosed vaccinated (n = 2379) of BNT162b2 mRNA COVID­19 vaccine, were followed-up weekly. Their saliva samples were used to monitor the shedding of SARS-CoV-2. All positive samples were sequenced and genotyped to identify the circulating wild-type virus or variants of concern. Results: The protection fraction against the excretion of the SARS-CoV-2 in the saliva samples of the workers after the second dose is estimated at 0.90 (95% CI: 0.18; 0.99) at 1 week and 0.83 (95% CI: 0.54; 0.95) at 8 weeks. We observe more circulating SARS-CoV-2 and a greater variability of viral loads in the unvaccinated group compared to those of the vaccinated group. Conclusions: This field cohort study advances our knowledge of the efficacy of the mRNA BNT162b2 COVID-19 vaccine on the viral shedding in the saliva specimens of vaccinated NH workers, contributing to better decision-making in public health interventions and management.

Transmission of SARS-CoV-2 After COVID-19 Screening and Mitigation Measures for Primary School Children Attending School in Liège, Belgium.

Importance: Recent data suggest a relatively low incidence of COVID-19 among children. The possible role that children attending primary school may play in the transmission of SARS-CoV-2 remains poorly understood. Objective: To gain a better understanding of the possible role of children in the transmission of SARS-CoV-2. Design, Setting, and Participants: This prospective cohort study was conducted from September 21 to December 31, 2020, in a primary school in Liège, Belgium, among a volunteer sample of 181 children, parents, and school employees. Exposures: Participants were tested for SARS-CoV-2 infection once a week for 15 weeks through throat washing, performed with 5 mL of saline and collected in a sterile tube after approximately 30 seconds of gargling. Quantitative reverse transcription-polymerase chain reaction was performed to detect SARS-CoV-2 infection. Main Outcomes and Measures: In case of test positivity, participants were asked to complete a questionnaire aimed at determining the timing of symptom onset and symptom duration. SARS-CoV-2 genetic sequencing was also performed. Confirmed cases were linked based on available information on known contacts and viral sequences. Results: A total of 181 individuals participated in this study, including 63 children (34 girls [54.0%]; mean [SD] age, 8.6 [1.9] years [range, 5-13 years]) and 118 adults (75 women [63.6%]; mean [SD] age, 42.5 [5.7] years [range, 30-59 years]). Forty-five individuals (24.9%) tested positive: 13 children (20.6%; 95% CI, 10.6%-30.6%) and 32 adults (27.1%; 95% CI, 19.1%-35.7%) (P = .34). Children were more often asymptomatic compared with adults (6 [46.2%; 95% CI, 19.1%-73.3%] vs 4 of 31 [12.9%; 95% CI, 1.3%-24.5%]; P = .04). The median duration of symptoms was shorter in children than in adults (0.00 days [IQR, 0.00-1.00 days] vs 15.00 days [IQR, 7.00-22.00 days]). A reconstruction of the outbreak revealed that most transmission events occurred between teachers and between children within the school. Of the observed household transmission events, most seemed to have originated from a child or teacher who acquired the infection at school. Conclusions and Relevance: Despite the implementation of several mitigation measures, the incidence of COVID-19 among children attending primary school in this study was comparable to that observed among teachers and parents. Transmission tree reconstruction suggests that most transmission events originated from within the school. Additional measures should be considered to reduce the transmission of SARS-CoV-2 at school, including intensified testing.

Genomic sequencing of SARS-CoV-2 in Rwanda reveals the importance of incoming travelers on lineage diversity.

COVID-19 transmission rates are often linked to locally circulating strains of SARS-CoV-2. Here we describe 203 SARS-CoV-2 whole genome sequences analyzed from strains circulating in Rwanda from May 2020 to February 2021. In particular, we report a shift in variant distribution towards the emerging sub-lineage A.23.1 that is currently dominating. Furthermore, we report the detection of the first Rwandan cases of the B.1.1.7 and B.1.351 variants of concern among incoming travelers tested at Kigali International Airport. To assess the importance of viral introductions from neighboring countries and local transmission, we exploit available individual travel history metadata to inform spatio-temporal phylogeographic inference, enabling us to take into account infections from unsampled locations. We uncover an important role of neighboring countries in seeding introductions into Rwanda, including those from which no genomic sequences were available. Our results highlight the importance of systematic genomic surveillance and regional collaborations for a durable response towards combating COVID-19.

Exploiting genomic surveillance to map the spatio-temporal dispersal of SARS-CoV-2 spike mutations in Belgium across 2020.

At the end of 2020, several new variants of SARS-CoV-2-designated variants of concern-were detected and quickly suspected to be associated with a higher transmissibility and possible escape of vaccine-induced immunity. In Belgium, this discovery has motivated the initiation of a more ambitious genomic surveillance program, which is drastically increasing the number of SARS-CoV-2 genomes to analyse for monitoring the circulation of viral lineages and variants of concern. In order to efficiently analyse the massive collection of genomic data that are the result of such increased sequencing efforts, streamlined analytical strategies are crucial. In this study, we illustrate how to efficiently map the spatio-temporal dispersal of target mutations at a regional level. As a proof of concept, we focus on the Belgian province of Liège that has been consistently sampled throughout 2020, but was also one of the main epicenters of the second European epidemic wave. Specifically, we employ a recently developed phylogeographic workflow to infer the regional dispersal history of viral lineages associated with three specific mutations on the spike protein (S98F, A222V and S477N) and to quantify their relative importance through time. Our analytical pipeline enables analysing large data sets and has the potential to be quickly applied and updated to track target mutations in space and time throughout the course of an epidemic.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Genome Sequencing from Post-Mortem Formalin-Fixed, Paraffin-Embedded Lung Tissues.

Implementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, paraffin-embedded (FFPE) samples. So far, one study reported the feasibility of SARS-CoV-2 genome sequencing on FFPE tissues with only one contributory case of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (COVID-19) patients. SARS-CoV-2 was detected in all FFPE blocks using a real-time RT-qPCR targeting the E gene with crossing point (Cp) values ranging from 16.02 to 34.16. Sequencing was considered as contributory (i.e. with a uniformity >55%) for 17 FFPE blocks. Adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values allowed optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for blocks with a Cp value <28 and 25 PCR cycles for blocks with a Cp value between 28 and 30). Most blocks with a Cp value >30 were non-contributory. Comparison of matched frozen and FFPE tissues revealed discordance for only three FFPE blocks, all with a Cp value >28. Variant identification and clade classification was possible for 13 patients. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions.

A Phylodynamic Workflow to Rapidly Gain Insights into the Dispersal History and Dynamics of SARS-CoV-2 Lineages.

Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of SARS-CoV-2 have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analyzed with gold-standard phylogeographic approaches. To tackle this practical limitation, we here describe and apply a rapid analytical pipeline to analyze the spatiotemporal dispersal history and dynamics of SARS-CoV-2 lineages. As a proof of concept, we focus on the Belgian epidemic, which has had one of the highest spatial densities of available SARS-CoV-2 genomes. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay.

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.