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.

Visualising SARS-CoV-2 infection of the lung in deceased COVID-19 patients.

BACKGROUND: SARS-CoV-2 is a single-stranded positive-sense RNA virus. Several negative-sense SARS-CoV-2 RNA species, both full-length genomic and subgenomic, are produced transiently during viral replication. Methodologies for rigorously characterising cell tropism and visualising ongoing viral replication at single-cell resolution in histological sections are needed to assess the virological and pathological phenotypes of future SARS-CoV-2 variants. We aimed to provide a robust methodology for examining the human lung, the major target organ of this RNA virus. METHODS: A prospective cohort study took place at the University Hospitals Leuven in Leuven, Belgium. Lung samples were procured postmortem from 22 patients who died from or with COVID-19. Tissue sections were fluorescently stained with the ultrasensitive single-molecule RNA in situ hybridisation platform of RNAscope combined with immunohistochemistry followed by confocal imaging. FINDINGS: We visualised perinuclear RNAscope signal for negative-sense SARS-CoV-2 RNA species in ciliated cells of the bronchiolar epithelium of a patient who died with COVID-19 in the hyperacute phase of the infection, and in ciliated cells of a primary culture of human airway epithelium that had been infected experimentally with SARS-CoV-2. In patients who died between 5 and 13 days after diagnosis of the infection, we detected RNAscope signal for positive-sense but not for negative-sense SARS-CoV-2 RNA species in pneumocytes, macrophages, and among debris in the alveoli. SARS-CoV-2 RNA levels decreased after a disease course of 2-3 weeks, concomitant with a histopathological change from exudative to fibroproliferative diffuse alveolar damage. Taken together, our confocal images illustrate the complexities stemming from traditional approaches in the literature to characterise cell tropism and visualise ongoing viral replication solely by the surrogate parameters of nucleocapsid-immunoreactive signal or in situ hybridisation for positive-sense SARS-CoV-2 RNA species. INTERPRETATION: Confocal imaging of human lung sections stained fluorescently with commercially available RNAscope probes for negative-sense SARS-CoV-2 RNA species enables the visualisation of viral replication at single-cell resolution during the acute phase of the infection in COVID-19. This methodology will be valuable for research on future SARS-CoV-2 variants and other respiratory viruses. FUNDING: Max Planck Society, Coronafonds UZ/KU Leuven, European Society for Organ Transplantation.

Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium.

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID-19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT-qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS-CoV-2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS-CoV-2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS-CoV-2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS-CoV-2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS-CoV-2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Sequencing directly from antigen-detection rapid diagnostic tests in Belgium, 2022: a gamechanger in genomic surveillance?

BackgroundLateral flow antigen-detection rapid diagnostic tests (Ag-RDTs) for viral infections constitute a fast, cheap and reliable alternative to nucleic acid amplification tests (NAATs). Whereas leftover material from NAATs can be employed for genomic analysis of positive samples, there is a paucity of information on whether viral genetic characterisation can be achieved from archived Ag-RDTs.AimTo evaluate the possibility of retrieving leftover material of several viruses from a range of Ag-RDTs, for molecular genetic analysis.MethodsArchived Ag-RDTs which had been stored for up to 3 months at room temperature were used to extract viral nucleic acids for subsequent RT-qPCR, Sanger sequencing and Nanopore whole genome sequencing. The effects of brands of Ag-RDT and of various ways to prepare Ag-RDT material were evaluated.ResultsSARS-CoV-2 nucleic acids were successfully extracted and sequenced from nine different brands of Ag-RDTs for SARS-CoV-2, and for five of these, after storage for 3 months at room temperature. The approach also worked for Ag-RDTs for influenza virus (n = 3 brands), as well as for rotavirus and adenovirus 40/41 (n = 1 brand). The buffer of the Ag-RDT had an important influence on viral RNA yield from the test strip and the efficiency of subsequent sequencing.ConclusionOur finding that the test strip in Ag-RDTs is suited to preserve viral genomic material, even for several months at room temperature, and therefore can serve as source material for genetic characterisation could help improve global coverage of genomic surveillance for SARS-CoV-2 as well as for other viruses.

Early SARS-CoV-2 Reinfections within 60 Days and Implications for Retesting Policies.

Illustrated by a clinical case supplemented by epidemiologic data, early reinfections with SARS-CoV-2 Omicron BA.1 after infection with Delta variant, and reinfection with Omicron BA.2 after Omicron BA.1 infection, can occur within 60 days, especially in young, unvaccinated persons. The case definition of reinfection, which influences retesting policies, should be reconsidered.

Evolution, geographic spreading, and demographic distribution of Enterovirus D68.

Worldwide outbreaks of enterovirus D68 (EV-D68) in 2014 and 2016 have caused serious respiratory and neurological disease. We collected samples from several European countries during the 2018 outbreak and determined 53 near full-length genome ('whole genome') sequences. These sequences were combined with 718 whole genome and 1,987 VP1-gene publicly available sequences. In 2018, circulating strains clustered into multiple subgroups in the B3 and A2 subclades, with different phylogenetic origins. Clusters in subclade B3 emerged from strains circulating primarily in the US and Europe in 2016, though some had deeper roots linking to Asian strains, while clusters in A2 traced back to strains detected in East Asia in 2015-2016. In 2018, all sequences from the USA formed a distinct subgroup, containing only three non-US samples. Alongside the varied origins of seasonal strains, we found that diversification of these variants begins up to 18 months prior to the first diagnostic detection during a EV-D68 season. EV-D68 displays strong signs of continuous antigenic evolution and all 2018 A2 strains had novel patterns in the putative neutralizing epitopes in the BC- and DE-loops. The pattern in the BC-loop of the USA B3 subgroup had not been detected on that continent before. Patients with EV-D68 in subclade A2 were significantly older than patients with a B3 subclade virus. In contrast to other subclades, the age distribution of A2 is distinctly bimodal and was found primarily among children and in the elderly. We hypothesize that EV-D68's rapid evolution of surface proteins, extensive diversity, and high rate of geographic mixing could be explained by substantial reinfection of adults. Better understanding of evolution and immunity across diverse viral pathogens, including EV-D68 and SARS-CoV-2, is critical to pandemic preparedness in the future.

Two Separate Clusters of SARS-CoV-2 Delta Variant Infections in a Group of 41 Students Travelling from India: An Illustration of the Need for Rigorous Testing and Quarantine.

We report two clusters of SARS-CoV-2 B.1.617.2 (Delta variant) infections in a group of 41 Indian nursing students who travelled from New Delhi, India, to Belgium via Paris, France. All students tested negative before departure and had a second negative antigen test upon arrival in Paris. Upon arrival in Belgium, the students were quarantined in eight different houses. Four houses remained COVID-free during the 24 days of follow-up, while all 27 residents of the other four houses developed an infection during quarantine, including the four residents who were fully vaccinated and the two residents who were partially vaccinated. Genome sequencing revealed two distinct clusters affecting one and three houses, respectively. In this group of students, vaccination status did not seem to prevent infection nor decrease the viral load. No severe symptoms were reported. Extensive contact tracing and 3 months of nationwide genomic surveillance confirmed that these outbreaks were successfully contained and did not contribute to secondary community transmission in Belgium. These clusters highlight the importance of repeated testing and quarantine measures among travelers coming from countries experiencing a surge of infections, as all infections were detected 6 days or more after arrival.

Fast detection of SARS-CoV-2 variants including Omicron using one-step RT-PCR and Sanger sequencing.

SARS-CoV-2 has kept the world in suspense for almost 2 years now. The virus spread rapidly worldwide and several variants of concern have emerged: Alpha, Beta, Gamma, Delta and recently Omicron. A rapid method to detect key mutations is needed because these variants may jeopardize the effectiveness of immune protection following vaccination or past infection. This article describes an easy, cheap and fast method for the detection of mutations in the spike protein that are indicative for specific variants. This method can easily distinguish Omicron from other variants.

Closing schools for SARS-CoV-2: a pragmatic rapid recommendation.

Background: In Belgium, schools closed during the first lockdown in March 2020, with a partial reopening in May. They fully reopened in September. During the summer, infections started to increase in the general population, speeding up in September. Some measures were taken to limit social contacts but those were insufficient to mitigate the exponential rise of infections in October. Children were still receiving all lessons at school at that time and it was questioned whether this position was tenable. We systematically compared the benefits and harms of closing primary and secondary schools and developed a recommendation. Methods: A multidisciplinary panel, including school pupils and teachers, educational experts, clinicians and researchers, produced this recommendation in compliance with the standards for trustworthy rapid guidelines. The recommendation is based on data collected through national surveillance or studies from Belgium, and supported by a rapid literature review. Results: Closing schools during the first lockdown probably resulted in a large learning delay and possibly led to more cases of child abuse. We are uncertain about the effect on the infection rate, hospitalisations, transmission rates, mental health of children, teachers and parents. The panel concluded that the balance of benefits and harms of closing schools clearly shifts against closing schools. Detrimental effects are even worse for vulnerable children. This recommendation is affected by the local virus circulation. Conclusion: The guideline panel issues a strong recommendation against closing schools when the virus circulation is low to moderate, and a weak recommendation against closing schools when the virus circulation is high. It does not apply when the school system cannot function due to lack of teachers, too many children who are at home or a shortage of support services. As the results of international studies are consistent with Belgian study results, this recommendation may also be relevant internationally.