Integration of sequencing and epidemiological data for surveillance of SARS-CoV-2 infections in a tertiary-care hospital.

BACKGROUND: The ongoing COVID-19 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of SARS-CoV-2 is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020. METHODS: Between calendar weeks 41/2020 and 1/2021 40% of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness. RESULTS: Epidemiologic data and contact tracing readily recognize most healthcare-associated patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in healthcare-associated infections at the end of the second/beginning of the third wave despite high community transmissions. CONCLUSIONS: While epidemiologic analysis is critical for immediate containment of healthcare-associated SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2-associated infections in healthcare settings.

Remdesivir-induced emergence of SARS-CoV2 variants in patients with prolonged infection.

We here investigate the impact of antiviral treatments such as remdesivir on intra-host genomic diversity and emergence of SARS-CoV2 variants in patients with a prolonged course of infection. Sequencing and variant analysis performed in 112 longitudinal respiratory samples from 14 SARS-CoV2-infected patients with severe disease progression show that major frequency variants do not generally arise during prolonged infection. However, remdesivir treatment can increase intra-host genomic diversity and result in the emergence of novel major variant species harboring fixed mutations. This is particularly evident in a patient with B cell depletion who rapidly developed mutations in the RNA-dependent RNA polymerase gene following remdesivir treatment. Remdesivir treatment-associated emergence of novel variants is of great interest in light of current treatment guidelines for hospitalized patients suffering from severe SARS-CoV2 disease, as well as the potential use of remdesivir to preventively treat non-hospitalized patients at high risk for severe disease progression.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4+ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA- CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127- virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Molecular consequences of SARS-CoV-2 liver tropism.

Extrapulmonary manifestations of COVID-19 have gained attention due to their links to clinical outcomes and their potential long-term sequelae1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) displays tropism towards several organs, including the heart and kidney. Whether it also directly affects the liver has been debated2,3. Here we provide clinical, histopathological, molecular and bioinformatic evidence for the hepatic tropism of SARS-CoV-2. We find that liver injury, indicated by a high frequency of abnormal liver function tests, is a common clinical feature of COVID-19 in two independent cohorts of patients with COVID-19 requiring hospitalization. Using autopsy samples obtained from a third patient cohort, we provide multiple levels of evidence for SARS-CoV-2 liver tropism, including viral RNA detection in 69% of autopsy liver specimens, and successful isolation of infectious SARS-CoV-2 from liver tissue postmortem. Furthermore, we identify transcription-, proteomic- and transcription factor-based activity profiles in hepatic autopsy samples, revealing similarities to the signatures associated with multiple other viral infections of the human liver. Together, we provide a comprehensive multimodal analysis of SARS-CoV-2 liver tropism, which increases our understanding of the molecular consequences of severe COVID-19 and could be useful for the identification of organ-specific pharmacological targets.

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants.

BACKGROUND: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. METHODS: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. RESULTS: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9-132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7-334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3-158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7-129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). CONCLUSION: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.

Comparing susceptibility and contagiousness in concurrent outbreaks with a non-VOC and the VOC SARS-CoV-2 variant B.1.1.7 in daycare centers in Hamburg, Germany.

We describe two outbreaks of SARS-CoV-2 in daycare centers in the metropolitan area of Hamburg, Germany. The outbreaks occurred in rapid chronological succession, in neighborhoods with a very similar sociodemographic structure, thus allowing for cross-comparison of these events. We combined classical and molecular epidemiologic investigation methods to study infection entry, spread within the facilities, and subsequent transmission of infections to households. Epidemiologic and molecular evidence suggests a superspreading event with a non-variant of concern (non-VOC) SARS CoV-2 strain at the root of the first outbreak. The second outbreak involved two childcare facilities experiencing infection activity with the variant of concern (VOC) B.1.1.7 (Alpha). We show that the index cases in all outbreaks had been childcare workers, and that children contributed substantially to secondary transmission of SARS-CoV-2 infection from childcare facilities to households. The frequency of secondary transmissions in households originating from B.1.1.7-infected children was increased compared to children with non-VOC infections. Self-reported symptoms, particularly cough and rhinitis, occurred more frequently in B.1.1.7-infected children. Especially in light of the rapidly spreading VOC B.1.617.2 (Delta), our data underline the notion that rigorous SARS-CoV-2 testing in combination with screening of contacts regardless of symptoms is an important measure to prevent SARS-CoV-2 infection of unvaccinated individuals in daycare centers and associated households.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection.

BACKGROUND: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. METHODS: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. RESULTS: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. CONCLUSION: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Dying of VOC-202012/01 - multimodal investigations in a death case of the SARS-CoV-2 variant.

The current pandemic with Severe acute respiratory syndrome-coronavirus-2 has been taking on new dynamics since the emergence of new variants last fall, some of them spreading more rapidly. Many countries currently find themselves in a race to ramp up vaccination strategies that have been initiated and a possible third wave of the pandemic from new variants, such as the Variant of Concern-202012/01 from the B.1.1.7 lineage. Until today, many investigations in death cases of Coronavirus-disease-19 have been conducted, revealing pulmonary damage to be the predominant feature of the disease. Thereby, different degrees of macroscopic and microscopic lung damage have been reported, most of them resembling an Acute Respiratory Distress Syndrome. Far more, systemic complications of the disease such as pulmonary embolisms have been described. However, neither morphologic nor virologic findings of patients dying of the new variants have yet been reported. Here, we report on a comprehensive analysis of radiologic, morphologic, and virologic findings in a fatal case of this variant.

What happened to financially sustainable firms in the Corona crisis?: SMF

Purpose: Financial sustainability is underrepresented in both research on and the practice of sustainability management and reporting. In this article, we examine empirically how financially sustainable firms performed in the Corona crisis.Methods: We measure financial sustainability by four conditions: (1) firm growth, (2) the company’s ability to survive, (3) an acceptable overall level of earnings risk exposure, and (4) an attractive earnings risk profile. We apply this measurement to investment portfolios of a broad sample of firms from 15 European countries of the MSCI Europe using typical investment portfolio characteristics.Results: We find that financially sustainable firms outperform both the broad market and firms with low financial sustainability for the time span July 2019 to March 2020.Conclusion: An investment strategy that invests in financially sustainable firms seems to be better capable of overcoming economic breakdowns such as the Corona crisis. We find that the returns increase with each of the four conditions that are included in the investment strategy. This underlines that considering financial sustainability is interesting for financial management, corporate governance and management control.

SARS Coronavirus-2 variant tracing within the first Coronavirus Disease 19 clusters in northern Germany.

OBJECTIVES: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany. METHODS: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg. RESULTS: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters. CONCLUSIONS: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level.

SARS-CoV-2 outbreak investigation in a German meat processing plant.

We describe a multifactorial investigation of a SARS-CoV-2 outbreak in a large meat processing complex in Germany. Infection event timing, spatial, climate and ventilation conditions in the processing plant, sharing of living quarters and transport, and viral genome sequences were analyzed. Our results suggest that a single index case transmitted SARS-CoV-2 to co-workers over distances of more than 8 m, within a confined work area in which air is constantly recirculated and cooled. Viral genome sequencing shows that all cases share a set of mutations representing a novel sub-branch in the SARS-CoV-2 C20 clade. We identified the same set of mutations in samples collected in the time period between this initial infection cluster and a subsequent outbreak within the same factory, with the largest number of confirmed SARS-CoV-2 cases in a German meat processing facility reported so far. Our results indicate climate conditions, fresh air exchange rates, and airflow as factors that can promote efficient spread of SARS-CoV-2 via long distances and provide insights into possible requirements for pandemic mitigation strategies in industrial workplace settings.

Complete Genome Sequence of a SARS-CoV-2 Strain Isolated in Northern Germany.

Here, we describe the complete genome sequence of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab sample from a female patient with COVID-19 who was infected in Hamburg, northern Germany.

Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections.