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Clinician guide to COVID-19 diagnostics
  1. Mildred A Iro1,2,
  2. Helen Umpleby3,
  3. Emanuela Pelosi3
  1. 1 Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK
  2. 2 Paediatric Immunology and Infectious Diseases, Southampton University Hospitals NHS Trust, Southampton, UK
  3. 3 Southampton Specialist Virology Centre, Southampton University Hospitals NHS Trust, Southampton, UK
  1. Correspondence to Dr Mildred A Iro, Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK; m.a.iro{at}

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The novel SARS-CoV-2 virus responsible for COVID-19 has resulted in a worldwide pandemic. To curtail the ongoing pandemic, emphasis has been placed on enhanced testing. Consequently, there has been an exponential rise in the number of diagnostic platforms that are available. This review gives an overview of the diagnostic methods for COVID-19, highlighting key considerations needed when interpreting the test results.

Physiological background

The aims of SARS-CoV-2 testing are to (1) diagnose acute infection in symptomatic patients, (2) screen for and diagnose asymptomatic infection and (3) identify past infection. The detection assays used include nucleic acid amplification tests (NAATs), antigen-based tests and antibody (serological) tests.

NAATs are aimed at identifying the presence of SARS-CoV-2 ribonucleic acid (RNA) in a sample. Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard NAAT used for the diagnosis of COVID-19. In RT-PCR, repeated, automated cycles of heating and cooling (thermocycling) are used to amplify specific targets, that is, segments of the virus’ genome. The targets for SARS-CoV-2 include open reading frame 1ab/RdRp, envelope, nucleocapsid (N) and spike (S) genes (figure 1). The amount of virus in a sample is then quantified in real time using fluorescently labelled probes. A test is positive if the fluorescence goes above a certain threshold level. The number of thermocycles required to get over this threshold is termed the cycle threshold (Ct) value. The lower the Ct value, the higher the quantity of viral genetic material in the sample; this is used as a proxy for viral load and infectivity. A 3.3-point increase in Ct value is equivalent to a 10-fold decrease in the quantity of viral genetic material. Roughly speaking, a Ct value of <35 (across all targets) indicates active infection and strongly correlates with cultivable virus,1 whereas a Ct value of >35 is considered a low-level positive …

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  • MAI and HU are joint first authors.

  • Contributors MAI generated the idea for the paper. MAI and HU performed the literature review and wrote the article. EP provided comments on the manuscript. MAI acts as guarantor of the content.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. MAI was supported by the University of Southampton National Institute for Health Research Academic Clinical Lecturer Programme.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.