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1.
iScience ; 25(9): 104926, 2022 Sep 16.
Article in English | MEDLINE | ID: covidwho-1983263

ABSTRACT

Secondary infections contribute significantly to covid-19 mortality but driving factors remain poorly understood. Autopsies of 20 covid-19 cases and 14 controls from the first pandemic wave complemented with microbial cultivation and RNA-seq from lung tissues enabled description of major organ pathologies and specification of secondary infections. Lethal covid-19 segregated into two main death causes with either dominant diffuse alveolar damage (DAD) or secondary pneumonias. The lung microbiome in covid-19 showed a reduced biodiversity and increased prototypical bacterial and fungal pathogens in cases of secondary pneumonias. RNA-seq distinctly mirrored death causes and stratified DAD cases into subgroups with differing cellular compositions identifying myeloid cells, macrophages and complement C1q as strong separating factors suggesting a pathophysiological link. Together with a prominent induction of inhibitory immune-checkpoints our study highlights profound alterations of the lung immunity in covid-19 wherein a reduced antimicrobial defense likely drives development of secondary infections on top of SARS-CoV-2 infection.

2.
Frontiers in psychology ; 13, 2022.
Article in English | EuropePMC | ID: covidwho-1980784

ABSTRACT

The SARS-CoV-2 pandemic has highlighted the interdependency of healthcare systems and research organizations on manufacturers and suppliers of personnel protective equipment (PPE) and the need for well-trained personnel who can react quickly to changing working conditions. Reports on challenges faced by research laboratory workers (RLWs) are rare in contrast to the lived experience of hospital health care workers. We report on experiences gained by RLWs (e.g., molecular scientists, pathologists, autopsy assistants) who significantly contributed to combating the pandemic under particularly challenging conditions due to increased workload, sickness and interrupted PPE supply chains. RLWs perform a broad spectrum of work with SARS-CoV-2 such as autopsies, establishment of virus cultures and infection models, development and verification of diagnostics, performance of virus inactivation assays to investigate various antiviral agents including vaccines and evaluation of decontamination technologies in high containment biological laboratories (HCBL). Performance of autopsies and laboratory work increased substantially during the pandemic and thus led to highly demanding working conditions with working shifts of more than eight hours working in PPE that stressed individual limits and also the ergonomic and safety limits of PPE. We provide detailed insights into the challenges of the stressful daily laboratory routine since the pandemic began, lessons learned, and suggest solutions for better safety based on a case study of a newly established HCBL (i.e., BSL-3 laboratory) designed for autopsies and research laboratory work. Reduced personal risk, increased resilience, and stress resistance can be achieved by improved PPE components, better training, redundant safety measures, inculcating a culture of safety, and excellent teamwork

3.
J Clin Microbiol ; 60(7): e0037622, 2022 07 20.
Article in English | MEDLINE | ID: covidwho-1874498

ABSTRACT

Measuring SARS-CoV-2 neutralizing antibodies after vaccination or natural infection remains a priority in the ongoing COVID-19 pandemic to determine immunity, especially against newly emerging variants. The gold standard for assessing antibody-mediated immunity against SARS-CoV-2 are cell-based live virus neutralization assays. These assays usually take several days, thereby limiting test capacities and the availability of rapid results. In this study, therefore, we developed a faster live virus assay, which detects neutralizing antibodies through the early measurement of antibody-mediated intracellular virus reduction by SARS-CoV-2 qRT-PCR. In our assay, Vero E6 cells are infected with virus isolates preincubated with patient sera and controls. After 24 h, the intracellular viral load is determined by qRT-PCR using a standard curve to calculate percent neutralization. Utilizing COVID-19 convalescent-phase sera, we show that our novel assay generates results with high sensitivity and specificity as we detected antiviral activity for all tested convalescent-phase sera, but no antiviral activity in prepandemic sera. The assay showed a strong correlation with a conventional virus neutralization assay (rS = 0.8910), a receptor-binding domain ELISA (rS = 0.8485), and a surrogate neutralization assay (rS = 0.8373), proving that quantifying intracellular viral RNA can be used to measure seroneutralization. Our assay can be adapted easily to new variants, as demonstrated by our cross-neutralization experiments. This characteristic is key for rapidly determining immunity against newly emerging variants. Taken together, the novel assay presented here reduces turnaround time significantly while making use of a highly standardized and sensitive SARS-CoV-2 qRT-PCR method as a readout.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/diagnosis , Humans , Neutralization Tests/methods , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus
4.
N Biotechnol ; 70: 19-27, 2022 Sep 25.
Article in English | MEDLINE | ID: covidwho-1778384

ABSTRACT

The outbreak of the SARS-CoV-2 pandemic created an unprecedented requirement for diagnostic testing, challenging not only healthcare workers and laboratories, but also providers. Quantitative RT-PCR of various specimen types is considered the diagnostic gold standard for the detection of SARS-CoV-2, both in terms of sensitivity and specificity. The pre-analytical handling of patient specimens is a critical factor to ensure reliable and valid test results. Therefore, the effect of storage duration and temperature on SARS-CoV-2 RNA copy number stability was examined in various commercially available specimen collection, transport and storage devices for naso/oropharyngeal swabs and saliva. The swab specimen transport and storage devices tested showed no significant alteration of viral RNA copy numbers when stored at room temperature, except for one system when stored for up to 96 h. However, at 37 °C a significant reduction of detectable RNA was found in 3 out of 4 of the swab solutions tested. It was also found that detectability of viral RNA remained unchanged in all 7 saliva devices as well as in unstabilized saliva when stored for 96 h at room temperature, but one device showed marked RNA copy number loss at 37 °C. All tested saliva collection devices inhibited SARS-CoV-2 infectivity immediately, whereas SARS-CoV-2 remained infectious in the swab transport systems examined, which are designed to be used for viral or bacterial growth in cell culture systems.


Subject(s)
COVID-19 , COVID-19/diagnosis , COVID-19 Testing , Clinical Laboratory Techniques/methods , Humans , Polymerase Chain Reaction , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2 , Specimen Handling/methods
5.
Clin Chem Lab Med ; 60(5): 778-785, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1731617

ABSTRACT

OBJECTIVES: Rapid antigen tests (RAT) can provide valuable information on the presence or absence SARS-CoV-2 within 15 min without the need of a laboratory. The analytical and diagnostic characteristics of available RATs has led to the question whether they can safely distinguish between infectious and non-infectious patients in an acute care setting. METHODS: Three nasopharyngeal swabs for the analysis by RAT, reverse transcriptase real time polymerase chain reaction (RT-qPCR), and a cell culture based infection assay were collected from 67 patients that presented to the emergency department of the University Hospital of Graz (Austria). The first swab was used for on-site RAT testing in the emergency department using the Roche SARS-CoV-2 RAT. The second swab was sent to the central laboratory of the hospital for RT-qPCR with two independent methods (Cepheid Xpert® Xpress SARS-CoV-2 assay and Roche Cobas SARS-CoV-2 Test) and repeat RAT testing using the same commercial test. With the third swab a cell culture-based infection assay was performed. RESULTS: The RATs performed from independent samples showed substantial agreement (Cohen's-kappa: 0.73, p<0.001). All patients with a positive RAT had positive RT-qPCR with cycle threshold (ct) values <25. Fifteen out of 55 RAT-negative samples were RT-qPCR positive with ct values between 25 and 40. The inoculation of cell cultures with RT-qPCR negative swabs and RT-qPCR positive swabs with ct values >25 did not induce cytopathic effects that were related to SARS-CoV-2. The infection assays from four RAT-negative patients showed cytopathic effects that were induced by other pathogens. CONCLUSIONS: The SARS-CoV-2 RAT from Roche Diagnostics is a valuable tool for managing symptomatic patients. RAT-negative patients may be regarded as non-contagious.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , COVID-19 Testing , Humans , Sensitivity and Specificity , Specimen Handling
6.
Phytomedicine ; 98: 153970, 2022 Jan 30.
Article in English | MEDLINE | ID: covidwho-1655019

ABSTRACT

BACKGROUND: The COVID-19 pandemic will continue to threaten our health care systems in the next years. In addition to vaccination there is a need for effective tools for prevention and treatment. Products from natural sources, like standardized plant extracts offer a wide range of antiviral effects and possible applications. PURPOSE: The aim of this study was to investigate, whether a sorbitol/lecithin-based throat spray containing concentrated green tea extract (sGTE) interacts with SARS-CoV-2 viral particles and additionally is capable to block the virus replication. STUDY DESIGN AND METHODS: The antiviral effect was studied in a VeroE6 cell culture model, including concentration/effect correlations and the biological mechanism of virus blockade, using the Wuhan type of SARS CoV-2 as well as its beta- and delta-mutations. In addition, the qualitative and quantitative tannin profile present on the oral mucosa after spray application has been investigated by LC-MS/MS and HPLC-DAD analyses of (-)-epigallocatechin-3-O-gallate (EGCG) and related catechin derivatives. RESULTS: The findings of this study demonstrate, that sGTE has strong neutralizing activity on SARS-CoV-2 resulting in an up to 6,3E+04-fold reduction of infectivity independent from the strain. The type of interaction of sGTE with surface proteins seems to be direct and non-specific concerning the viral surface protein structures and resembles the general non-specific activity of polyphenols. By HPLC-DAD analysis, eight catechins were identified in sGTE, with EGCG and (-)-epicatechin-3-O-gallate as the most abundant ones. The total content of catechin derivatives, calculated as catechin, was 76 g/100 g. LC-MS/MS and HPLC-DAD analyses of throat swabs after application of a sGTE spray have shown that the concentrations of green tea tannins in the pharyngeal mucosa are higher than the effective dose found in the in vitro studies with SARS-CoV-2, even 1 h after the last application. CONCLUSION: The findings of this study suggest that sGTE has strong neutralizing activity on SARS-CoV-2 independent from the strain (Wuhan strain, beta- or delta-variants). sGTE might be relevant for reduction of corresponding viral infections when periodically applied to mouth and throat.

7.
Elife ; 102021 12 20.
Article in English | MEDLINE | ID: covidwho-1592091

ABSTRACT

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Molecular Dynamics Simulation , Polysaccharides/metabolism , Receptors, Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , COVID-19/prevention & control , COVID-19/virology , Glycosylation , Humans , Polysaccharides/chemistry , Protein Binding , Protein Engineering , Receptors, Virus/chemistry , Receptors, Virus/genetics , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Virus Internalization
8.
Biomedicines ; 10(1)2021 Dec 27.
Article in English | MEDLINE | ID: covidwho-1581028

ABSTRACT

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600-680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50-500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

9.
N Biotechnol ; 66: 36-45, 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1521430

ABSTRACT

The COVID-19 pandemic has generated a major need for non-destructive and environmentally friendly disinfection methods. This work presents the development and testing of a disinfection process based on gaseous ozone for SARS-CoV-2-contaminated porous and non-porous surfaces. A newly developed disinfection chamber was used, equipped with a CeraPlas™ cold plasma generator that produces ozone during plasma ignition. A reduction of more than log 6 of infectious virus could be demonstrated for virus-contaminated cotton and FFP3 face masks as well as glass slides after exposure to 800 ppm ozone for 10-60 min, depending on the material. In contrast to other disinfectants, ozone can be produced quickly and cost-effectively, and its environmentally friendly breakdown product oxygen does not leave harmful residues. Disinfection with ozone could help to overcome delivery difficulties of personal protective equipment by enabling safe reuse with further applications, thereby reducing waste generation, and may allow regular disinfection of personal items with non-porous surfaces.


Subject(s)
Disinfection/methods , Ozone , SARS-CoV-2/drug effects , Virus Inactivation/drug effects , Equipment Contamination , Masks/virology , Ozone/pharmacology , Porosity
10.
Emerg Infect Dis ; 27(6): 1734-1737, 2021 Jun.
Article in English | MEDLINE | ID: covidwho-1181334

ABSTRACT

Detecting severe acute respiratory syndrome coronavirus 2 in deceased patients is key when considering appropriate safety measures to prevent infection during postmortem examinations. A prospective cohort study comparing a rapid antigen test with quantitative reverse transcription PCR showed the rapid test's usability as a tool to guide autopsy practice.


Subject(s)
COVID-19 , SARS-CoV-2 , Autopsy , Humans , Prospective Studies , Real-Time Polymerase Chain Reaction
11.
Pathobiology ; 88(1): 37-45, 2021.
Article in English | MEDLINE | ID: covidwho-967862

ABSTRACT

Information obtained from autopsies of patients infected with high-risk pathogens is an important pillar in managing a proper response to pandemics, particular in the early phase. This is due to the fact that autopsy allows efficient evaluation of comorbidities for risk assessment, as well as identification of key pathophysiological and molecular mechanisms in organs driving the severity of disease which might be important targets for therapeutic interventions. In the case of patients who have died of infection with unknown pathogens, isolation and culture of pathogens from the affected organs is another important opportunity for a proper response to (re)emerging infectious diseases. However, the situation of COVID-19 demonstrated that there were concerns about performing autopsies because of biosafety risks. In this review we compare requirements for biosafety level 3 (BSL-3) laboratories from the European Commission and the World Health Organization and summarize specific recommendations for postmortem analysis of COVID-19-deceased patients from the Centers for Disease Control and Prevention. Furthermore, we describe in detail a BSL-3 facility with enhanced protection of personnel and an environment that has been designed for performing autopsies, biobanking of collected tissue specimens, and culture of pathogens in cases of high-risk pathogen infections and report on the experience obtained in operating this facility in the context of COVID-19.


Subject(s)
Autopsy , COVID-19/pathology , COVID-19/virology , SARS-CoV-2/pathogenicity , Autopsy/methods , Biological Specimen Banks , Containment of Biohazards , Humans , Laboratories
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