COVID-19 Testing Explained

By this point I believe we are all tired of reading and talking about COVID. However based on reading comments on social media, it’s quite clear that there are a lot of misconceptions about COVID testing. For starters COVID-19 is the disease caused by the SARS-CoV-2 virus. So all of the tests we are using to assist in the diagnosis of COVID-19 are really looking for signs that the person was infected with SARS-CoV-2. There are also 3 main categories of tests for SARS-CoV-2 based on the target of the assay: RNA, antigen, and antibody.

Diagnosis of COVID-19 should be based on clinical symptoms, risk of exposure, test results and timeline. The diagnostic tests based on detection of SARS-CoV-2 RNA are the most commonly used and reliable for diagnosis of COVID-19.1 All of these assays are based on amplifying the viral RNA to detect the presence of the RNA. Most assays use some form of PCR to amplify the virus, however because the virus is RNA-based it has to be converted to cDNA with reverse transcriptase PCR before amplification and detection. TMA or transcription-mediated amplification is another chemistry that can be used to amplify the RNA to a detectable level. Both PCR and TMA based assays are very sensitive at detecting the virus especially within the first week after symptoms develop.1,2 Due to the RNA-based nature of the SARS-CoV-2 genome, the mutation rate is anticipated to be high. Most of the RNA-based assays have adopted a strategy to target 2 different areas of the viral genome to prevent missing the presence of the virus due to a mutation in the primer binding site.

A SARS-CoV-2 antigen test received EUA in early May. The test is designed with immunofluorescence-based lateral flow. This type of test is designed to detect SARS-CoV-2 proteins present on the outside of the virus. In general, this class of test is cheaper and faster than RNA-based testing however it is less sensitive (80% clinical sensitivity).3 The clinical specificity of antigen assays is shown to be 100%,3 therefore a positive result is reliable. These tests can be used for screening; however patients with negative results may still need to proceed to testing by an RNA-based method. Antigen based tests is typically more sensitive during the same timeframe when PCR testing is more sensitive, ie earlier in the course of disease.

SARS-CoV-2 antibody tests are the last class of tests. Seroconversion appears to occur within 7-14 days of symptom onset2 or 15-20 days post exposure to the virus.4 There are many different tests to choose from to determine if the patient has previously been exposed to SARS-CoV-2. The assays range from lateral flow cassettes to high throughput chemiluminescent based assays. Some of the SARS-CoV-2 antibody assays detect IgG, IgM, IgA or some combination of the 3 including total antibody without differentiating between the three. The latest studies have shown that some patients develop IgM first, some with IgG, and others had both IgG and IgM develop at the same time.5 Therefore differentiating IgG from IgM is not providing a timeline for acute infection as we have seen in response to other viruses. Although sensitivity and specificity vary widely between manufacturers total antibody detection appears to be more sensitive than IgG or IgM detection alone.4 The FDA recently pulled numerous assays off of the market due to poor performance.

It is important to note that even with the most sensitive and specific antibody test, these tests cannot determine if a patient has protective immunity. Unfortunately we don’t know enough about immunity with regards to COVID yet. Early studies are promising, showing that some level of antibody will likely provide protection from future exposure. We don’t know if there is a threshold of antibody that needs to be present before a patient is immune, will the immunity only decrease the severity and not prevent reinfection, and how long the antibodies are maintained after exposure. These will be important questions to answer before the clinical utility of antibody testing can be realized. Right now the test is useful to determine is a patient was previously exposed to SARS-CoV-2 and is helpful to address epidemiological questions with regards to prevalence of COVID-19 in the community. The antibody test should not be used for diagnosis of current infection due to the delay to seroconvert after exposure.


  1. Sethuraman, N., Jeremiah, S. S., & Ryo, A. (2020). Interpreting Diagnostic Tests for SARS-CoV-2. JAMA. doi:10.1001/jama.2020.8259
  2. Wolfel, R., Corman, V. M., Guggemos, W., Seilmaier, M., Zange, S., Muller, M. A., . . . Wendtner, C. (2020). Virological assessment of hospitalized patients with COVID-2019. Nature, 581(7809), 465-469. doi:10.1038/s41586-020-2196-x
  3. Quidel Sofia®2 SARS Antigen FIA. 5/29/2020.
  4. Lou, B., Li, T. D., Zheng, S. F., Su, Y. Y., Li, Z. Y., Liu, W., . . . Chen, Y. (2020). Serology characteristics of SARS-CoV-2 infection since exposure and post symptom onset. Eur Respir J. doi:10.1183/13993003.00763-2020
  5. Long, Q. X., Liu, B. Z., Deng, H. J., Wu, G. C., Deng, K., Chen, Y. K., . . . Huang, A. L. (2020). Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. doi:10.1038/s41591-020-0897-1

-Tabetha Sundin, PhD, HCLD (ABB), MB (ASCP)CM,  has over 10 years of laboratory experience in clinical molecular diagnostics including oncology, genetics, and infectious diseases. She is the Scientific Director of Molecular Diagnostics and Serology at Sentara Healthcare. Dr. Sundin holds appointments as Adjunct Associate Professor at Old Dominion University and Assistant Professor at Eastern Virginia Medical School and is involved with numerous efforts to support the molecular diagnostics field. 

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