By Jessica Lee, Biochemistry & Molecular Biology ‘21

Author’s Note: Inspired by the success of the asymptomatic testing at UC Davis, I wrote this article exploring the different types of diagnostic and antibody tests for SARS-CoV-2, focusing on mechanisms and relative sensitivities and specificities. 

The COVID-19 pandemic has demonstrated the importance of widespread and accurate diagnostic testing in controlling community spread. Together with mask mandates, social distancing, and quarantining, COVID-19 testing can slow the spread of a disease that has killed over 2.5 million people [1]. Approximately 80 million COVID-19 tests have been reported at this time, and yet, many people are confused as to how COVID-19 tests work and how each type of test differs in mechanism, sensitivity, and specificity [2]. 

Depending on the type of COVID-19 diagnostic test, they will either detect SARS-CoV-2 nucleic acid, protein, or antibodies generated as a consequence of infection [3]. Samples can be collected from patients via nasal swab, saliva collection, or blood collection [3]. This article will review current COVID-19 tests as well as address potential confusion arising from false negative and false positive results. 

Diagnostic Tests

Diagnostic tests are administered to patients to determine if they are infected with SARS-CoV-2 at time of sampling. Such tests may be administered to either symptomatic or asymptomatic patients as a diagnostic tool or preventative public health measure. Various types of diagnostic tests have been developed since the emergence of SARS-CoV-2 as an infectious agent; however, all tests rely on one of two underlying technologies [4]. Molecular tests detect segments of the viral genome while antigen tests detect the presence of viral proteins [4]. However, both molecular tests and antigen tests differ from antibody tests which detect previous SARS-CoV-2 infections. 

Molecular Tests

Molecular tests primarily rely on polymerase chain reaction (PCR) technology to detect relatively low quantities of SARS-CoV-2 genome [4]. A PCR has four primary components: DNA template, DNA-dependent DNA polymerase, primers, and nucleotides [5]. SARS-CoV-2 has a positive-sense single stranded RNA genome meaning the genome can directly be translated into protein by host translation machinery [6]. However, since PCRs require a DNA template, the genome must be converted from RNA to DNA via the enzyme reverse transcriptase [5]. Due to this reverse transcription step, this specific type PCR is called reverse transcription, or RT-qPCR. The resulting DNA product then serves as the template for PCR during which a DNA-dependent DNA polymerase recognizes the template and synthesizes the complementary strand via incorporation of nucleotides. The primers, short DNA fragments about 20 nucleotides in length, are the component of PCR that confers specificity to the assay through their complementarity to a specific region in the DNA template [5]. When all reaction components are combined and cycled through specific temperatures, the result is the exponential increase in the number of copies of the target DNA [5]. Thus, the assay can produce a positive result with very small quantities of original template. 

The U.S. Centers for Disease Control (CDC) has established two sets of oligonucleotide primers for the detection of the nucleocapsid (N) gene of SARS-CoV-2 [7]. Thus, if SARS-CoV-2 genome is present in a sample, the primers will hybridize to the N gene, resulting in the amplification of the region and yielding a positive result. Other primers for the detection of the RNA-dependent RNA polymerase and envelope genes have also been developed by the World Health Organization (WHO) [7]. Since the primers are essential to establishing high specificity, it is important that the primers only bind to the intended target. If the primer is too promiscuous, the assay might produce a false positive. Alternatively, if the primer is too stringent, the assay might produce a false negative. There is also concern that if the region in which the primers bind mutates, the assay may no longer consistently detect such mutants [7].

Samples for molecular tests may be collected via nasal swab or saliva and may be pooled for more efficient testing [8]. If a sample pool is positive for SARS-CoV-2, the individual samples are then tested to determine which individuals are positive. Care must be taken, however, with pooled sampling because samples with low viral loads may not be detected due to decreased sensitivity [8]. 

Molecular tests are generally characterized by high sensitivity, high specificity, and moderate price (~$100/test), earning their status as the “gold standard” of COVID-19 testing [9]. However, depending on the laboratory, results may take up to a week to be returned to the patient [3].

Antigen Tests

Antigen tests are immunoassays that detect viral proteins by binding viral protein to SARS-CoV-2 antibodies [10]. Generally, antibodies specific to the N protein are produced and purified for use in antigen tests [10]. There is more variation in how antigen tests work as compared to molecular tests; however, generally, the antibodies are conjugated to a tag which can either be read by a machine or is visible to the naked eye. The Abbott BinaxNow COVID-19 Ag Card is even similar to an over-the-counter pregnancy test–a positive result is indicated by a line in a test window [10]. Samples are collected via nasal or nasopharyngeal swab and are flowed over test strips loaded with conjugated antibodies [3,9]. 

Antigen tests often produce results within 15-30 minutes–much faster than many of the molecular tests [3]. Thus, many health care providers use antigen tests as point-of-care “rapid” tests [9]. Although antigen tests are generally cheaper than molecular tests at about $5-50 per test and are highly specific, they are moderately less sensitive than molecular tests [9]. If the antigen level of a specimen is low due to collection before symptom onset or in late infection, a false negative may be more likely than if a molecular test was administered [9]. However, for both molecular and antigen tests, the probability of a false positive is low due to their high specificity [9]. 

Antibody Tests

Instead of diagnosing active SARS-CoV-2 infections, antibody tests indicate if the patient has an adaptive immune response to a SARS-CoV-2 infection [3]. Samples are collected via fingerstick–a device which pricks the finger to produce a few drops of blood–or blood draw and detect the presence of antibodies generated by SARS-CoV-2 infection [3]. There are many different types of antibody tests, but all can be sorted into two categories: binding antibody detection or neutralizing antibody detection tests [11]. Assays that detect antibodies through binding of an antigen use purified spike or nucleocapsid protein from SARS-CoV-2 to determine if the patient has previously been infected [11]. The mechanism of these tests is very similar to antigen tests which also depend on the selective binding of antibodies to antigens. Neutralizing antibody detection tests determine the ability of antibodies in a sample to prevent infection in cell culture [11]. This category of tests give a more accurate assessment of a patient to resist reinfection.    

 The CDC’s binding antibody test uses purified spike protein for the detection of IgG and IgM antibodies [12, 13]. While both IgG and IgM antibodies are usually generated 1-3 weeks after exposure to an antigen, IgG antibodies persist longer–often months post infection [11]. Due to the lag between peak viral production and peak antibody production, antibody tests cannot be used as diagnostic tools; however, they are useful for identifying recovered individuals for surveillance purposes[13]. Furthermore, widespread serology surveillance allows public health officials to monitor where COVID-19 cases are concentrated and inform public health policy [14].  

Some antibody tests have high specificity and sensitivity; although, this is highly dependent on the type of test and period of time post infection. Tests may result in a false negative if there has not been sufficient antibody production or if antibody levels have decreased below the limit of detection [13,11]. For example, if one compares a test that only detects IgM antibodies to a test that only detects IgG antibodies, one might see differential results if administered months after infection. Some antibody tests have a slight cross-reactivity with SARS1 and MERS-CoV sera which could result in false positives; however, there is minimal cross-reactivity with commonly circulating coronaviruses [15]. While nucleic acid detection tests remain the gold standard for diagnosis of acute SARS-CoV-2 infection, antibody tests can be valuable tools for clinical and surveillance efforts [12]. 

Asymptomatic Testing 

Many U.S. colleges have implemented regular, asymptomatic testing of their students and employee populations [16]. Regular testing can allow early cases of COVID-19 to be identified and allow for efficient contact tracing, limiting the spread of COVID-19. At UC Davis, students and employees who go on campus are required to get tested at least every week at the free asymptomatic testing clinic [17]. With the ability to screen thousands of samples each day, UC Davis has expanded its saliva testing program to include the greater Davis community, catching at least 850 asymptomatic cases that otherwise might have spread to other individuals [17]. 

Clearly, the importance of widespread testing of both symptomatic and asymptomatic individuals cannot be understated. Testing is one of the most important tools public health officials have to monitor and control the ongoing pandemic.

References

1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Accessed April 13, 2021. Available from: https://covid19.who.int/. 
2. Centers for Disease Control and Prevention. COVID Data Tracker: United States Laboratory Testing. Accessed April 13, 2021. Available from: https://covid.cdc.gov/covid-data-tracker/#testing_totalpositivity.
3. U.S. Food and Drug Administration. Coronavirus Disease 2019 Testing Basics. Accessed April 13, 2021. Available from: https://www.fda.gov/consumers/consumer-updates/coronavirus-disease-2019-testing-basics#:~:text=There%20are%20two%20different%20types,tests%20and%20antibody%20tests.
4. U.S. Food and Drug Administration. A Closer Look at COVID-19 Diagnostic Testing. Accessed April 13, 2021. Available from: https://www.fda.gov/health-professionals/closer-look-covid-19-diagnostic-testing.
5. NCBI. Polymerase Chain Reaction (PCR). Accessed April 13, 2021. Available from: https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/.
6. Ahlquist P, Noueiry AO, Lee WM, Kushner DB, Dye BT. 2003. Host factors in positive-strand RNA virus genome replication. J Virol [Internet]. 77(15), 8181–8186. https://doi.org/10.1128/jvi.77.15.8181-8186.2003.
7. Wang R, Hozumi Y, Yin C, Wei GW. 2020. Mutations on COVID-19 diagnostic targets. Genomics [Internet]. 112(6):5204-5213. doi:10.1016/j.ygeno.2020.09.028.
8. Centers for Disease Control and Prevention. 2020. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Available from: https://www.fda.gov/media/134922/download.
9. Centers for Disease Control and Prevention. 2020. Interim Guidance for Antigen Testing for SARS-CoV-2. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html.
10. Andrea Prinzi, MPH. 2020. How the SARS-CoV-2 EUA Antigen Tests Work. American Society for Microbiology. Available from: https://asm.org/Articles/2020/August/How-the-SARS-CoV-2-EUA-Antigen-Tests-Work.
11. Centers for Disease Control and Prevention. 2021. Interim Guidelines for COVID-19 Antibody Testing. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antibody-tests-guidelines.html.
12. Centers for Disease Control and Prevention. 2020. Serology Testing for COVID-19 at CDC. Available from: https://www.cdc.gov/coronavirus/2019-ncov/lab/serology-testing.html. 
13. U.S. Food and Drug Administration. 2020. Serology/Antibody Tests: FAQs on Testing for SARS-CoV-2. Available from: https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/serologyantibody-tests-faqs-testing-sars-cov-2. 
14. Centers for Disease Control and Prevention. 2021. COVID-19 Serology Surveillance Strategy. Available from: https://www.cdc.gov/coronavirus/2019-ncov/covid-data/serology-surveillance/index.html.
15. Freeman B, Lester S, Mills L, et al. 2020. Validation of a SARS-CoV-2 spike protein ELISA for use in contact investigations and serosurveillance. Preprint. bioRxiv. 2020;2020.04.24.057323. doi:10.1101/2020.04.24.057323.
16. Anderson, N. 2020. Welcome to college. Now get tested for the coronavirus — again and again. The Washington Post. Available from: https://www.washingtonpost.com/local/education/welcome-to-college-now-get-tested-for-the-coronavirus–again-and-again/2020/09/04/2d087722-ed2f-11ea-b4bc-3a2098fc73d4_story.html.
17. Hubler, S. 2021. A California University Tries to Shield an Entire City From Coronavirus. The New York Times. Available from:  https://www.nytimes.com/2021/01/30/us/college-coronavirus-california.html.

Online References

1. World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Accessed 2021. 
2. Centers for Disease Control and Prevention. COVID Data Tracker: United States Laboratory Testing. Accessed 2021. 
3. U.S. Food and Drug Administration. Coronavirus Disease 2019 Testing Basics. Accessed 2021.
4. U.S. Food and Drug Administration. A Closer Look at COVID-19 Diagnostic Testing. Accessed 2021. 
5. NCBI. Polymerase Chain Reaction (PCR). Accessed 2021. 
6. Ahlquist P, et al. 2003. J Virol [Internet]. 77(15), 8181–8186. 
7. Wang R, et al. 2020. Genomics [Internet]. 112(6):5204-5213. 
8. Centers for Disease Control and Prevention. 2020. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. 
9. Centers for Disease Control and Prevention. 2020. Interim Guidance for Antigen Testing for SARS-CoV-2. 
10. Andrea Prinzi, MPH. 2020. American Society for Microbiology. 
11. Centers for Disease Control and Prevention. 2021. Interim Guidelines for COVID-19 Antibody Testing. 
12. Centers for Disease Control and Prevention. 2020. Serology Testing for COVID-19 at CDC. 
13. U.S. Food and Drug Administration. 2020. Serology/Antibody Tests: FAQs on Testing for SARS-CoV-2. 
14. Centers for Disease Control and Prevention. 2021. COVID-19 Serology Surveillance Strategy. 
15. Freeman B, et al. 2020. Preprint. bioRxiv. 2020;2020.04.24.057323. 
16. Anderson, N. 2020. The Washington Post.
17. Hubler, S. 2021. The New York Times.