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      Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens

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          Abstract

          On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories.

          ABSTRACT

          On 31 December 2019, the World Health Organization was informed of a cluster of cases of pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel coronavirus, now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from the affected patients. Highly sensitive and specific laboratory diagnostics are important for controlling the rapidly evolving SARS-CoV-2-associated coronavirus disease 2019 (COVID-19) epidemic. In this study, we developed and compared the performance of three novel real-time reverse transcription-PCR (RT-PCR) assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay, which is used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel assay had the lowest limit of detection in vitro (1.8 50% tissue culture infective doses [TCID 50]/ml with genomic RNA and 11.2 RNA copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an additional 42 RdRp-P2-negative specimens (119/273 [43.6%] versus 77/273 [28.2%]; P < 0.001), including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract specimens. The mean viral load of these specimens was 3.21 × 10 4 RNA copies/ml (range, 2.21 × 10 2 to 4.71 × 10 5 RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis of COVID-19.

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          Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

          Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.
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            A Novel Coronavirus from Patients with Pneumonia in China, 2019

            Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.)
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              Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus–Infected Pneumonia in Wuhan, China

              In December 2019, novel coronavirus (2019-nCoV)-infected pneumonia (NCIP) occurred in Wuhan, China. The number of cases has increased rapidly but information on the clinical characteristics of affected patients is limited.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                J Clin Microbiol
                J. Clin. Microbiol
                jcm
                jcm
                JCM
                Journal of Clinical Microbiology
                American Society for Microbiology (1752 N St., N.W., Washington, DC )
                0095-1137
                1098-660X
                4 March 2020
                23 April 2020
                May 2020
                23 April 2020
                : 58
                : 5
                : e00310-20
                Affiliations
                [a ]State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China
                [b ]Department of Clinical Microbiology and Infection, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Guangdong Province, China
                [c ]Department of Microbiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China
                [d ]Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China
                [e ]Hainan Medical University-The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, Hainan Medical University, Haikou, Hainan, and The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, China
                [f ]Department of Microbiology, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China
                [g ]Department of Medicine, Queen Elizabeth Hospital, Hong Kong, Hong Kong Special Administrative Region, China
                [h ]Department of Pathology, Queen Elizabeth Hospital, Hong Kong, Hong Kong Special Administrative Region, China
                [i ]Department of Medicine, Queen Mary Hospital, Pokfulam, Hong Kong Special Administrative Region, China
                [j ]Department of Medicine and Geriatrics, Princess Margaret Hospital, Hong Kong, Hong Kong Special Administrative Region, China
                Boston Children’s Hospital
                Author notes
                Address correspondence to Kwok-Yung Yuen, kyyuen@ 123456hku.hk .

                Jasper Fuk-Woo Chan and Cyril Chik-Yan Yip contributed equally to this work as co-first authors. Author order was determined alphabetically.

                Citation Chan JF-W, Yip CC-Y, To KK-W, Tang TH-C, Wong SC-Y, Leung K-H, Fung AY-F, Ng AC-K, Zou Z, Tsoi H-W, Choi GK-Y, Tam AR, Cheng VC-C, Chan K-H, Tsang OT-Y, Yuen K-Y. 2020. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens. J Clin Microbiol 58:e00310-20. https://doi.org/10.1128/JCM.00310-20.

                Author information
                https://orcid.org/0000-0002-1921-5824
                Article
                00310-20
                10.1128/JCM.00310-20
                7180250
                32132196
                83ad97e2-77b4-4115-a0c4-2169d3bd8e8e
                Copyright © 2020 Chan et al.

                This article is made available via the PMC Open Access Subset for unrestricted noncommercial re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 20 February 2020
                : 26 February 2020
                : 3 March 2020
                Page count
                supplementary-material: 2, Figures: 1, Tables: 5, Equations: 0, References: 36, Pages: 10, Words: 7453
                Funding
                Funded by: Michael Seak-Kan Tong;
                Award Recipient :
                Funded by: Respiratory Viral Research Foundation Limited;
                Award Recipient :
                Funded by: Marina Man-Wai Lee;
                Award Recipient :
                Funded by: Theme-Based Research Scheme of the Research Grants Council, HKSARS;
                Award ID: T11/707/15
                Award Recipient :
                Funded by: The Hong Kong Hainan Commercial Association South China Microbiology Research Fund;
                Award Recipient :
                Funded by: High Level-Hospital Program, Health Commission of Guangdong Province, China;
                Award Recipient :
                Funded by: Richard Yu and Carol Yu;
                Award Recipient :
                Funded by: Hui Ming;
                Award Recipient :
                Funded by: Hui Hoy and Chow Sin Lan Charity Fund Limited;
                Award Recipient :
                Funded by: Chan Yin Chuen Memorial Charitable Foundation;
                Award Recipient :
                Categories
                Virology
                Custom metadata
                May 2020

                Microbiology & Virology
                coronavirus,covid-19,diagnostic,emerging,pcr,sars,wuhan,virus
                Microbiology & Virology
                coronavirus, covid-19, diagnostic, emerging, pcr, sars, wuhan, virus

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