COVID-19 Diagnostics

Written by: Apoorva Kakkilaya and Jina Zhou
Edited by: Esther Melamed
Last Updated: 10/27/2020

Nucleic Acid-Based Diagnostic Tests

For COVID-19 acute infection, most diagnostic tests utilize a molecular technique, reverse transcriptase polymerase chain reaction (RT-PCR), to amplify viral RNA from the patient sample.

• Samples are typically collected by placing a thin sterile swab (~3mm in diameter) at the back of the patient’s nasal passage for several seconds to absorb secretions
• The FDA has recently granted emergency use authorizations to tests that utilize saliva specimen as the primary test biomaterial
  • Using saliva would allow for broader population screening and easy at-home collection compared to the other approved methods for testing. 
• The main difference between RT-PCR test kits is the targeted genetic regions of the SARS-CoV-2 genome, which dictates the primers that are used for the test. 
  • Most CDC-approved kits target regions that code for the protein making up the virus’s nucleocapsid (N gene). 
  • The kit used by the WHO targets the envelope protein encoding gene (E gene). 
• Typically, these tests take hours to days to process. However, the FDA has granted EUAs to several companies that are working on developing rapid testing technologies.  One example is the isothermal nucleic acid amplification-based test developed by Abbott Diagnostics, which gives a positive result in as little as five minutes. The efficacy of this rapid-test however has recently come under scrutiny with 126 adverse reports of “adverse events” reported to the FDA, specifically citing a high return of false-negative tests. Studies have shown Abbot’s test missing as much as a third of positive COVID-19 tests  (Basu et al. 2020). Abbot has agreed to conduct post-market studies with at least 150 COVID-19 patients. 

What Makes a Good Test?

In general, a good diagnostic test should have high sensitivity (the ability for the test to correctly identify people with the disease; the true positive rate) and high specificity (the ability for the test to correctly identify people without the disease; the true negative rate). Maximizing specificity at the expense of sensitivity can lead to underdiagnosis of those with the disease, while maximizing sensitivity at the expense of specificity can mean that precious resources are being unnecessarily used on those who have never been exposed to SARS-CoV-2. Most of the diagnostic test manufacturers given EUAs have reported the clinical specificity of their product, comparing the results of their test to the expected result. 

As of 10/27/2020, the FDA has granted EUAs to 186 molecular-based diagnostic tests.

Table 1 – Select nucleic acid-based diagnostic tests given EUAs. The full list from the FDA can be found here.

Antigen-Based Diagnostic Tests

Antigen tests are rapid diagnostic tests that utilize lateral flow technology, similar to pregnancy tests. Samples are collected with nasal or throat swab. According to FDA guidelines, antigen tests are more likely to miss an active coronavirus infection compared to molecular tests. As such, test results do not definitively rule out active coronavirus infection, and negative results may need to be confirmed with a molecular test. However, positive results are usually highly accurate. These tests provide utility in point-of-care settings as the diagnostic technology is easily portable and results are returned within 30 minutes. 

Table 2 – Antigen Tests given EUAs

Test Name	Manufacturer	Notes	Sensitivity	Specificity
Sofia 2 SARS Antigen FIA	Quidel Corporation	-Detects nucleocapside antigen -NP and NS swabs -Immunofluorescence-based lateral flow technology -results in 15 minutes	96.7% (29/30)	100% (179/179)
BD Veritor System for Rapid Detection of SARS-CoV-2	Becton, Dickinson and Company (BD)	-Detects nucleocapside antigen -nasal swabs within first five days on onset of symptoms -Immunochromatrographic assay -Results in 15 minutes -Highly portable; approximate size of a cell phone	85% (26/31)	100% (195/195)
LumiraDx SARS-CoV-2 Ag Test	LumiraDx UK Ltd.	-results in 15 minutes -nasal swab -fluorescence immunoassay	97.6% (81/87)	96.6% 168/170
BinaxNOWTM COVID-19 Ag Card	Abbott Diagnostics Scarborough, Inc.	- lateral flow immunoassay -nasal swab -intended for use within first 7 days of symptom onset -results visible on test card in 15 minutes	97.1% (34.35)	98.5% (66/67)
Sofia 2 Flu + SARS Antigen FIA	Quidel Corporation	-Lateral Flow -NP and NS swabs -Multi-analyte: differentiation of SARS-CoV-2, influenza A and influenza B -results in 15 minutes	SARS-CoV-2 95.2% (40/42) Influenza A 100% (72/72) Influenza B 100% (14/14)	SARS-CoV-2 100% (122/122) Influenza A 96.6% (56/58) Influenza B 98.2% (56/57)
CareStart™ COVID-19 Antigen	Access Bio, Inc.	-lateral flow -NP swab -results interpretable from test strip in 10 minutes	88.37% (38/43)	100% (63/63)

 

Antibody Tests

Serological tests can identify antibodies to SARS-CoV-2 in a patient’s bloodstream to determine whether the patient had previously been infected.Current serological tests are not designed for diagnosis of an acute infection, though in the future expansion of testing for SARS-CoV-2 IgM may accomplish the latter goal. 

When are antibodies against SARS-CoV-2 produced? 

Most patients have been found to start developing antibodies from two to 15 days post symptoms. However, asymptomatic carriers may also have developed antibodies as an immune response. Early after infection (usually after the first week), a class of antibodies known as immunoglobulin M (IgM) develops, although these are not typically long-lasting. After the first 2-4 weeks following infection, a larger quantity of IgG, a more durable antibody, is produced. Detection of virus-specific IgM and IgG in circulating blood can help determine whether a person has been infected with that pathogen, either recently (IgM) or more distantly (IgG).

How do antibody tests work? 

Most of the serological tests given FDA EUAs are either lateral flow immuno-assays, enzyme-linked immunosorbent assays (ELISA), or chemiluminescence assays (CLIA). SARS-CoV-2 antigens (typically spike protein) are immobilized on a surface to which serum samples are applied. An enzymatic substrate produces a visible signal if antibody interaction/binding is detected, essentially signaling qualitative detection of the antibodies of interest. Most tests utilize serum, plasma, or venous whole blood as the primary testing specimen. Currently, the only FDA EUA approved test for fingerstick blood is Vibrant America’s COVID-19 Ab Assay. Testing is limited to Vibrant America Lab in San Carlos, CA. 

Why are these tests important?

The FDA has emphasized that results from these tests can help identify those who have already been infected (i.e. have SARS-CoV-2 IgG), those who have developed antibodies that may protect them from future SARS-CoV-2 infection (i.e. have neutralizing antibodies), as well as potentially those individuals who are still at risk for infection Serological tests can also help inform who may qualify to donate blood that can be used to manufacture convalescent plasma, an investigational product for use by patients who are seriously ill from COVID-19. Additionally, seroprevalence studies utilize antibody tests to identify people in a population or community that have antibodies against an infectious disease. The CDC is conducting seroprevalence surveys called “large-scale geographic seroprevalence surveys” in locations across the United States.This is useful to estimate the number of people who have been previously infected with SARS-CoV-2 and were not included in official case counts and guide public health measures. 

Table 3 – Antibody Tests given EUAs. More information from the FDA can be found here.

CRISPR-Based Diagnostic Tests

CRISPR systems in conjunction with Cas proteins (most commonly Cas9) have been widely used to recognize and modify genetic material in living cells. Researchers at Mammoth Biosciences and UCSF have recently developed a rapid (<40 min), easy-to-implement and accurate CRISPR–Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts, called DETECTR. The test carries out simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP) of RNA, followed by Cas12 detection of predefined coronavirus sequences, after which cleavage of a reporter molecule confirms the detection of the virus. Guide RNAs were designed to detect the N (nucleoprotein) gene and E (envelope) gene in SARS-CoV-2, which Cas12 then cuts. Color change due to cleavage of the reporter molecule indicates a positive result. 

A similar technology, termed SHERLOCK, has been developed by researchers at the Broad Institute, although the institute has emphasized that their initial research protocols are not diagnostic tests and have not been tested on patient samples. The targets for this test were chosen from the S gene and Orf1ab gene in the SARS-CoV-2 genome. Instead of Cas12, the SHERLOCK protocol uses the Cas13 protein to cleave the molecular targets.

References

Center for Devices and Radiological Health. “Emergency Use Authorizations.” U.S. Food and Drug Administration, FDA, www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-authorizations.

Broughton, J.P., Deng, X., Yu, G. et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0513-4

Kellner, M.J., Koob, J.G., Gootenberg, J.S. et al. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 14, 2986–3012 (2019). https://doi.org/10.1038/s41596-019-0210-2

Long, Q., Liu, B., Deng, H. et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med (2020). https://doi.org/10.1038/s41591-020-0897-1

Basu, Atreyee et al. 2020. Performance of Abbott ID NOW COVID-19 Rapid Nucleic Acid Amplification Test in Nasopharyngeal Swabs Transported in Viral Media and Dry Nasal Swabs, in a New York City Academic Institution. Microbiology. preprint. http://biorxiv.org/lookup/doi/10.1101/2020.05.11.089896 (July 25, 2020).

Commissioner, Office of the. 2020. “Coronavirus (COVID-19) Update: FDA Informs Public About Possible Accuracy Concerns with Abbott ID NOW Point-of-Care Test.” FDA. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-informs-public-about-possible-accuracy-concerns-abbott-id-now-point (July 25, 2020).