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.

SARS-Cov-2 infection and neuropathological findings: a report of 18 cases and review of the literature.

INTRODUCTION: COVID-19-infected patients harbour neurological symptoms such as stroke and anosmia, leading to the hypothesis that there is direct invasion of the central nervous system (CNS) by SARS-CoV-2. Several studies have reported the neuropathological examination of brain samples from patients who died from COVID-19. However, there is still sparse evidence of virus replication in the human brain, suggesting that neurologic symptoms could be related to mechanisms other than CNS infection by the virus. Our objective was to provide an extensive review of the literature on the neuropathological findings of postmortem brain samples from patients who died from COVID-19 and to report our own experience with 18 postmortem brain samples. MATERIAL AND METHODS: We used microscopic examination, immunohistochemistry (using two different antibodies) and PCR-based techniques to describe the neuropathological findings and the presence of SARS-CoV-2 virus in postmortem brain samples. For comparison, similar techniques (IHC and PCR) were applied to the lung tissue samples for each patient from our cohort. The systematic literature review was conducted from the beginning of the pandemic in 2019 until June 1st, 2022. RESULTS: In our cohort, the most common neuropathological findings were perivascular haemosiderin-laden macrophages and hypoxic-ischaemic changes in neurons, which were found in all cases (n = 18). Only one brain tissue sample harboured SARS-CoV-2 viral spike and nucleocapsid protein expression, while all brain cases harboured SARS-CoV-2 RNA positivity by PCR. A colocalization immunohistochemistry study revealed that SARS-CoV-2 antigens could be located in brain perivascular macrophages. The literature review highlighted that the most frequent neuropathological findings were ischaemic and haemorrhagic lesions, including hypoxic/ischaemic alterations. However, few studies have confirmed the presence of SARS-CoV-2 antigens in brain tissue samples. CONCLUSION: This study highlighted the lack of specific neuropathological alterations in COVID-19-infected patients. There is still no evidence of neurotropism for SARS-CoV-2 in our cohort or in the literature.

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.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

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.

Unspecific post-mortem findings despite multiorgan viral spread in COVID-19 patients.

BACKGROUND: Post-mortem studies can provide important information for understanding new diseases and small autopsy case series have already reported different findings in COVID-19 patients. METHODS: We evaluated whether some specific post-mortem features are observed in these patients and if these changes are related to the presence of the virus in different organs. Complete macroscopic and microscopic autopsies were performed on different organs in 17 COVID-19 non-survivors. Presence of SARS-CoV-2 was evaluated with immunohistochemistry (IHC) in lung samples and with real-time reverse-transcription polymerase chain reaction (RT-PCR) test in the lung and other organs. RESULTS: Pulmonary findings revealed early-stage diffuse alveolar damage (DAD) in 15 out of 17 patients and microthrombi in small lung arteries in 11 patients. Late-stage DAD, atypical pneumocytes, and/or acute pneumonia were also observed. Four lung infarcts, two acute myocardial infarctions, and one ischemic enteritis were observed. There was no evidence of myocarditis, hepatitis, or encephalitis. Kidney evaluation revealed the presence of hemosiderin in tubules or pigmented casts in most patients. Spongiosis and vascular congestion were the most frequently encountered brain lesions. No specific SARS-CoV-2 lesions were observed in any organ. IHC revealed positive cells with a heterogeneous distribution in the lungs of 11 of the 17 (65%) patients; RT-PCR yielded a wide distribution of SARS-CoV-2 in different tissues, with 8 patients showing viral presence in all tested organs (i.e., lung, heart, spleen, liver, colon, kidney, and brain). CONCLUSIONS: In conclusion, autopsies revealed a great heterogeneity of COVID-19-associated organ injury and the remarkable absence of any specific viral lesions, even when RT-PCR identified the presence of the virus in many organs.