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      False-negative results of initial RT-PCR assays for COVID-19: A systematic review

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          Abstract

          Background

          A false-negative case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is defined as a person with suspected infection and an initial negative result by reverse transcription-polymerase chain reaction (RT-PCR) test, with a positive result on a subsequent test. False-negative cases have important implications for isolation and risk of transmission of infected people and for the management of coronavirus disease 2019 (COVID-19). We aimed to review and critically appraise evidence about the rate of RT-PCR false-negatives at initial testing for COVID-19.

          Methods

          We searched MEDLINE, EMBASE, LILACS, as well as COVID-19 repositories, including the EPPI-Centre living systematic map of evidence about COVID-19 and the Coronavirus Open Access Project living evidence database. Two authors independently screened and selected studies according to the eligibility criteria and collected data from the included studies. The risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool. We calculated the proportion of false-negative test results using a multilevel mixed-effect logistic regression model. The certainty of the evidence about false-negative cases was rated using the GRADE approach for tests and strategies. All information in this article is current up to July 17, 2020.

          Results

          We included 34 studies enrolling 12,057 COVID-19 confirmed cases. All studies were affected by several risks of bias and applicability concerns. The pooled estimate of false-negative proportion was highly affected by unexplained heterogeneity (tau-squared = 1.39; 90% prediction interval from 0.02 to 0.54). The certainty of the evidence was judged as very low due to the risk of bias, indirectness, and inconsistency issues.

          Conclusions

          There is substantial and largely unexplained heterogeneity in the proportion of false-negative RT-PCR results. The collected evidence has several limitations, including risk of bias issues, high heterogeneity, and concerns about its applicability. Nonetheless, our findings reinforce the need for repeated testing in patients with suspicion of SARS-Cov-2 infection given that up to 54% of COVID-19 patients may have an initial false-negative RT-PCR (very low certainty of evidence).

          Systematic review registration

          Protocol available on the OSF website: https://tinyurl.com/vvbgqya.

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          Most cited references62

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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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            Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area

            There is limited information describing the presenting characteristics and outcomes of US patients requiring hospitalization for coronavirus disease 2019 (COVID-19).
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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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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: SupervisionRole: ValidationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: InvestigationRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                10 December 2020
                2020
                10 December 2020
                : 15
                : 12
                : e0242958
                Affiliations
                [1 ] Clinical Biostatistics Unit, Hospital Universitario Ramón y Cajal- IRYCIS, Madrid, Spain
                [2 ] CIBER of Epidemiology and Public Health, Madrid, Spain
                [3 ] Institute of Social and Preventive Medicine (ISPM), University of Bern, Bern, Switzerland
                [4 ] Graduate School for Health Sciences, University of Bern, Bern, Switzerland
                [5 ] Centro de Investigación en Salud Pública y Epidemiología Clínica (CISPEC), Facultad de Ciencias de la Salud “Eugenio Espejo”, Universidad UTE, Quito, Ecuador
                [6 ] Department of Microbiology, Ramón y Cajal University Hospital, Ramón y Cajal Health Research Institute (IRYCIS), Madrid, Spain
                [7 ] Instituto de Efectividad Clínica y Sanitaria (IECS-CONICET), Buenos Aires, Argentina
                [8 ] Fundación Huésped, Buenos Aires, Argentina
                [9 ] Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Amsterdam University Medical Centres, University of Amsterdam, Amsterdam, The Netherlands
                [10 ] Infectious Diseases Department, National Referral Centre for Tropical Diseases, Hospital Universitario Ramón y Cajal, Madrid, Spain
                [11 ] Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain
                [12 ] Institute of Applied Health Research, University of Birmingham, Birmingham, United Kingdom
                Universidad Nacional de la Plata, ARGENTINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-7326-4504
                https://orcid.org/0000-0001-5142-6122
                https://orcid.org/0000-0003-4817-8986
                https://orcid.org/0000-0003-4427-0128
                Article
                PONE-D-20-14317
                10.1371/journal.pone.0242958
                7728293
                33301459
                6d76634d-a7eb-4a5e-b2fa-0aa071d5501e
                © 2020 Arevalo-Rodriguez et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 May 2020
                : 12 November 2020
                Page count
                Figures: 3, Tables: 2, Pages: 19
                Funding
                The author(s) received no specific funding for this work.
                Categories
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                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
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