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      Rapid Large-Scale COVID-19 Testing during Shortages

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          Abstract

          The Coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in economic and social lockdowns in most countries all over the globe. Early identification of infected individuals is regarded as one of the most important prerequisites for fighting the pandemic and for returning to a ‘New Normal’. Large-scale testing is therefore crucial, but is facing several challenges including shortage of sample collection tools and of molecular biological reagents, and the need for safe electronic communication of medical reports. We present the successful establishment of a holistic SARS-CoV-2 testing platform that covers proband registration, sample collection and shipment, sample testing, and report issuing. The RT-PCR-based virus detection, being central to the platform, was extensively validated: sensitivity and specificity were defined as 96.8% and 100%, respectively; intra-run and inter-run precision were <3%. A novel type of sample swab and an in-house-developed RNA extraction system were shown to perform as good as commercially available products. The resulting flexibility guarantees independence from the current bottlenecks in SARS-CoV-2 testing. Based on our technology, we offered testing at local, national, and global levels. In the present study, we report the results from approx. 18,000 SARS-CoV-2 tests in almost 10,000 individuals from a low-frequency SARS-CoV-2 pandemic area in a homogenous geographical region in north-eastern Germany for a period of 10 weeks (21 March to 31 May 2020). Among the probands, five SARS-CoV-2 positive cases were identified. Comparative analysis of corresponding virus genomes revealed a diverse origin from three of the five currently recognized SARS-CoV-2 phylogenetic clades. Our study exemplifies how preventive SARS-CoV-2 testing can be set up in a rapid and flexible manner. The application of our test has enabled a safe maintenance/resume of critical local infrastructure, e.g., nursing homes where more than 5000 elderlies and caretakers got tested. The strategy outlined by the present study may serve as a blueprint for the implementation of large-scale preventive SARS-CoV-2 testing elsewhere.

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          A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

          Summary Background An ongoing outbreak of pneumonia associated with a novel coronavirus was reported in Wuhan city, Hubei province, China. Affected patients were geographically linked with a local wet market as a potential source. No data on person-to-person or nosocomial transmission have been published to date. Methods In this study, we report the epidemiological, clinical, laboratory, radiological, and microbiological findings of five patients in a family cluster who presented with unexplained pneumonia after returning to Shenzhen, Guangdong province, China, after a visit to Wuhan, and an additional family member who did not travel to Wuhan. Phylogenetic analysis of genetic sequences from these patients were done. Findings From Jan 10, 2020, we enrolled a family of six patients who travelled to Wuhan from Shenzhen between Dec 29, 2019 and Jan 4, 2020. Of six family members who travelled to Wuhan, five were identified as infected with the novel coronavirus. Additionally, one family member, who did not travel to Wuhan, became infected with the virus after several days of contact with four of the family members. None of the family members had contacts with Wuhan markets or animals, although two had visited a Wuhan hospital. Five family members (aged 36–66 years) presented with fever, upper or lower respiratory tract symptoms, or diarrhoea, or a combination of these 3–6 days after exposure. They presented to our hospital (The University of Hong Kong-Shenzhen Hospital, Shenzhen) 6–10 days after symptom onset. They and one asymptomatic child (aged 10 years) had radiological ground-glass lung opacities. Older patients (aged >60 years) had more systemic symptoms, extensive radiological ground-glass lung changes, lymphopenia, thrombocytopenia, and increased C-reactive protein and lactate dehydrogenase levels. The nasopharyngeal or throat swabs of these six patients were negative for known respiratory microbes by point-of-care multiplex RT-PCR, but five patients (four adults and the child) were RT-PCR positive for genes encoding the internal RNA-dependent RNA polymerase and surface Spike protein of this novel coronavirus, which were confirmed by Sanger sequencing. Phylogenetic analysis of these five patients' RT-PCR amplicons and two full genomes by next-generation sequencing showed that this is a novel coronavirus, which is closest to the bat severe acute respiatory syndrome (SARS)-related coronaviruses found in Chinese horseshoe bats. Interpretation Our findings are consistent with person-to-person transmission of this novel coronavirus in hospital and family settings, and the reports of infected travellers in other geographical regions. Funding The Shaw Foundation Hong Kong, Michael Seak-Kan Tong, Respiratory Viral Research Foundation Limited, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund Limited, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, Sanming Project of Medicine (Shenzhen), and High Level-Hospital Program (Guangdong Health Commission).
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            Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

            Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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              Virological assessment of hospitalized patients with COVID-2019

              Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
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                Author and article information

                Journal
                Diagnostics (Basel)
                Diagnostics (Basel)
                diagnostics
                Diagnostics
                MDPI
                2075-4418
                08 July 2020
                July 2020
                : 10
                : 7
                : 464
                Affiliations
                Author notes
                [* ]Correspondence: christian.beetz@ 123456centogene.com ; Tel.: +49-381-8011-3400
                [†]

                These Authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0001-7061-2895
                Article
                diagnostics-10-00464
                10.3390/diagnostics10070464
                7399816
                32650631
                f56838b0-3b78-450f-a58b-e03868066770
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 22 June 2020
                : 07 July 2020
                Categories
                Article

                covid-19,preventive testing,rt-pcr,sars-cov-2,testing,validation

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