Environmental circulation of adenovirus 40/41 and SARS-CoV-2 in the context of the emergence of acute hepatitis of unknown origin (preprint)

The recent surge of hepatitis of unknown origin in children is hypothesized to be caused by adenovirus 41 and/or SARS-CoV-2 infections. A relatively high proportion of patients testing positive for these viruses concomitantly with the development of acute hepatitis supports this hypothesis. To formally incriminate these viral infections as causative agents of hepatitis, both a plausible physiopathological pathway and supporting epidemiological dynamics in the community need demonstration. In this study, we measured the level of circulation of adenovirus 40/41 and SARS-CoV-2 in the general population of the city of Leuven in Belgium using wastewater monitoring between December 2020 and May 2022 and indoor air sampling in day care centers between November 2021 and May 2022. We also retrospectively analyzed medical records of 12.672 children attending a tertiary hospital draining the same region between January 2019 and April 2022. Our results demonstrate a recent but modest increase in hepatitis of unknown origin concomitant with a surge of circulating adenovirus 41 and SARS-CoV-2 in the general population, including in children under 5.

Early SARS-CoV-2 reinfections within 60 days highlight the need to consider antigenic variations together with duration of immunity in defining retesting policies (preprint)

The emergence of the SARS-CoV-2 Omicron variant, characterized by a significant antigenic diversity compared to the previous Delta variant, had led to a decrease in antibody efficacy in both convalescent and vaccinees sera resulting in high number of reinfections and breakthrough cases worldwide. However, to date, reinfections are defined by the ECDC as two positive tests >/= 60 days apart, influencing retesting policies after an initial positive test in several European countries. In our manuscript, we illustrate by a clinical case supplemental by epidemiological data that early reinfections do occur within 60 days especially in young, unvaccinated individuals. In older patient groups, unvaccinated and patients with a basic vaccination scheme are more vulnerable to reinfections compared to patients who received a first booster vaccine. For this reason, we consider that the duration of protection offered by a previous infection should be reconsidered, in particular when a shift between consecutive SARS-CoV-2 variants occurs.

Comprehensive immunovirological and environmental screening reveals risk factors for fatal COVID-19 during post-vaccination nursing home outbreaks (preprint)

COVID-19 vaccination has resulted in excellent protection against fatal disease, including in the elderly. However, risk factors for post-vaccination fatal COVID-19 are largely unknown. We comprehensively studied three large nursing home outbreaks (20-35% fatal cases) by combining SARS-CoV-2 aerosol monitoring, whole-genome phylogenetic analysis, and immunovirological profiling by digital nCounter transcriptomics. Phylogenetic investigations indicated each outbreak stemmed from a single introduction event, though with different variants (Delta, Gamma, and Mu). SARS-CoV-2 was detected in aerosol samples up to 52 days 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 signatures and reanalysis of single-cell RNAseq data highlights the unique 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.

Molecular Detection of SARS-Cov-2 in Exhaled Breath Using a Portable Sampler (preprint)

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, a cornerstone for any disease-control strategy, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It potentially measures contagiousness directly, the sample is easy to obtain, its production can be standardized between patients, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an in-situ RT-qPCR molecular assay to detect SARS-CoV-2 shedding. We demonstrate that sampling subjects using a one-minute breathing protocol, yields sufficient viral RNA to detect infections with a sensitivity comparable to standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test is the first to consistently report a negative result, putatively signaling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Genetic diversity and evolution of SARS-CoV-2 in Belgium during the first wave outbreak (preprint)

SARS-CoV-2, the causative agent of COVID-19 was first detected in Belgium on 3rd February 2020, albeit the first epidemiological wave started in March and ended in June 2020. One year after the first epidemiological wave hit the country data analyses reveled the temporal and variant distribution of SARS-CoV-2 and its implication with Belgian epidemiological measures. In this study, 766 complete SARS-CoV-2 genomes of samples originating from the first epidemiological were sequenced to characterize the temporal and geographic distribution of the COVID-19 pandemic in Belgium through phylogenetic and variant analysis. Our analysis reveals the presence of the major circulating SARS-CoV-2 clades (G, GH and GR) and lineages circulating in Belgium at that time. Moreover, it contextualizes the density of SARS-CoV-2 cases over time with non-intervention measures taken to prevent the spread of SARS-CoV-2 in Belgium, specific international case imports and the functional implications of the most representative non-synonymous mutations present in Belgium between February to June 2020.

The Emergence of COVID-19 Associated Mucormycosis: Analysis of Cases From 18 Countries (preprint)

Coronavirus disease 2019 (COVID-19)–associated mucormycosis (CAM) has recently been increasingly reported, particularly among patients with uncontrolled diabetes. Patients with diabetes and hyperglycemia often display an inflammatory state that may be potentiated by the activation of antiviral immunity to SARS-CoV-2, and thus may favor secondary infections. We analyze 80 published and unpublished cases of CAM, with a predominance (42/80) of cases from India. Uncontrolled diabetes mellitus as well as systemic corticosteroid treatment represented major comorbid predisposing factors and rhino-orbital cerebral mucormycosis was the most frequent presentation of disease. Mortality was high at 49%, driven particularly by those with pulmonary or disseminated mucormycosis and those with cerebral involvement. Furthermore, a significant proportion of surviving patients suffered life-changing morbidities (loss of vision in 46% of survivors). Our review indicates that CAM may be a relevant complication of severe COVID-19, particularly in those with uncontrolled diabetes. Funding: Martin Hoenigl received funding from Astellas for two investigator initiated studies (ISR005824 and ISR005838), and was supported by the National Institutes of Health, Grant UL1TR001442. Agostinho Carvalho was supported by the Fundação para a Ciência e a Tecnologia (FCT) (UIDB/50026/2020 and UIDP/50026/2020), the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000039), the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HR17/52190003.Declaration of Interest: MH received research funding from Gilead, Pfizer, Astellas, Scynexis and NIH. JPG received speaker and expert advice fees from Pfizer and Gilead. NK has received research grants or honoraria as a speaker or advisor from Astellas, Gilead, MSD, and Pfizer, outside the submitted work. KL received consultancy fees from SMB Laboratoires Brussels, MSD and Gilead, travel support from Pfizer, speaker fees from FUJIFILM WAKO, Pfizer and Gilead, a service fee from Thermo fisher Scientific. OAC is supported by the German Federal Ministry of Research and Education, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388 and has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra Therapeutics, Al-Jazeera Pharmaceuticals, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, Immunic, IQVIA, Janssen, Matinas, Medicines Company, MedPace, Melinta Therapeutics, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, Scynexis, and Shionogi. PLW performed diagnostic evaluations and received meeting sponsorship from Bruker, Dynamiker, and Launch Diagnostics; Speakers fees, expert advice fees and meeting sponsorship from Gilead; and speaker and expert advice fees 489 from F2G and speaker fees MSD and Pfizer. Is a founding member of the European Aspergillus PCR Initiative. ACh received funding support from educational grant of Pfizer, MSD Pharmaceutical Ltd, and Gilead. All other authors no conflicts.Ethical Approval: MISSING

Cov-MS: a community-based template assay for clinical MS-based protein detection in Sars-Cov-2 patients (preprint)

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Although this is efficient, automatable and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using Mass Spectrometry (MS). We established the Cov-MS consortium, consisting of fifteen academic labs and several industrial partners to increase applicability, accessibility, sensitivity and robustness of this kind of SARS-CoV-2 detection. This in turn gave rise to the Cov-MS Digital Incubator that allows other labs to join the effort, navigate and share their optimizations, and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR, using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity (preprint)

Epidemiological and clinical reports have indicated that the host immune response to SARS-CoV-2, more so than viral factors, determines COVID-19 disease severity. To elucidate the immunopathology underlying COVID-19 severity, cytokine and multiplex immune profiling was performed in mild-moderate and critically-ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, and was more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels followed by deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presenting machinery was reduced in critical disease, while also neutrophils contributed to disease severity and local tissue damage by amplifying hypercytokinemia and neutrophil extracellular trap formation. We suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.