How the SARS-CoV-2 EUA Antigen Tests Work
As of Aug. 27, 2020, the U.S. Food and Drug Administration (FDA) has issued 3 emergency use authorizations (EUAs) for COVID-19 assays that test for the presence of SARS-CoV-2 antigens in patient samples. The 2 major testing methods currently used for SARS-CoV-2 in the U.S. include molecular (PCR) testing (used to detect viral RNA during active infections) and antibody testing (used to detect host antibodies developed against prior infections). Antigen testing, which tests for the presence of viral proteins, rather than viral RNA or host antibodies, offers a third option.
The Role of Antigen Testing in COVID-19The use of diagnostic assays to detect COVID-19 in the United States has been immensely challenging for various reasons including supply chain shortages, funding and staffing challenges and overburdening of the healthcare system. It is important to understand that different SARS-CoV-2 test methodologies serve unique purposes in this pandemic, and there is not a one-size-fits-all solution to the crisis. While PCR assays are highly sensitive and excellent for diagnosing acute infection, they are expensive and require the use of special reagents, equipment and specially trained clinical laboratory scientists, all of which have been stretched thin during the pandemic so far. Serology testing is useful for determining if someone had a previous SARS-CoV-2 infection, but since it takes 1-2 weeks for antibodies to develop, it is not recommended for the diagnosis of acute disease.
Antigen tests are cheaper to produce than PCR tests and can be performed very quickly, making them an option worth considering as a screening tool. Once additional reliable antigen assays enter the market, it may be possible to scale antigen testing up to millions of tests a day in the United States, which could help ease the testing burden in some locations. While molecular tests can be performed in real time, supply chain and throughput challenges have made this unrealistic during the COVID-19 pandemic. Implementing antigen testing in the COVID-19 testing algorithm could reduce strain on laboratories and reserve molecular testing for situations where high test sensitivity is required.
The advantages and disadvantages of antigen testing for SARS-CoV-2 need to be considered to determine if and when it is a reliable method for COVID-19 diagnosis.
- Antigen testing is cheap and fast. Knowing that molecular assays cannot continue to carry the burden of all acute COVID-19 testing in the United States, antigen testing offers a solution to the problem of needing tests that can be produced cheaply and run quickly.
- Antigen testing may be a useful public health tool. Since antigen tests are generally considered very accurate when they are positive, they may be helpful in quickly identifying highly infectious individuals within a community. However, it is important to note that antigen test performance in areas with low disease prevalence may be poor and data on test performance in asymptomatic individuals are limited. Since antigen tests are more likely to be positive when a person has a higher viral load, super-spreader events or outbreaks may be prevented by identifying these individuals early and isolating them.
- Potential for use in the point-of-care setting. The rapid nature of antigen assays, as well as less reliance on expensive equipment and reagents, make them good candidates for point-of-care testing. This may reduce the burden of testing on laboratories and provide rapid results at the location where the patient is tested. It is important to note that point-of-care testing sites must have a CLIA certificate in order to run these tests, so the locations where they can be offered will vary.
- Lower sensitivity than PCR tests. Antigen tests are not as sensitive as PCR tests and false negatives pose a real problem. A negative antigen test should always be treated as presumptive, but can still give the patient and care provider a false sense of security. These tests may not be appropriate in settings where a positive result cannot be missed, such as hospitals or settings with high-risk patients or staff. While they occur less frequently with this test type, false positives are also problematic and can lead to major workflow problems within medical facilities, as well as missed days of work and school among the general public.
- Less expert interpretation in point-of-care settings. Although point-of-care testing is convenient, it is important to remember that the test is often performed and interpreted by healthcare professionals that are not trained in clinical laboratory science. Appropriate messaging around the interpretation of results is imperative, including the possibility of false negative and false positive results, and the continued use of public health measures, such as masking and distancing, are essential.
- Limited evidence on performance and use. There is very little literature available that discusses test performance of antigen testing for COVID-19. Guidance on the use of these tests in the pandemic setting may be limited.
- Laboratory space, supplies and staff are still required. Just because antigen testing can be done more rapidly than most PCR assays does not mean it can be used outside of a certified laboratory or without proper training. Additionally, 2 of the 3 antigen assays that have received emergency use authorization (EUA) by the U.S. Food and Drug Administration (FDA) require their manufacturers' automated systems to be used, which would require laboratories to purchase new instrumentation and supplies if they do not already own those systems.
SARS-CoV-2 Antigen Tests With Emergency Use AuthorizationThe 3 assays that have received emergency use authorization from the FDA include the Becton Dickinson (BD) Veritor™ System, the Quidel Sofia 2 SARS antigen FIA and the Abbott Diagnostics BinaxNOW COVID-19 Ag Card. All 3 assays function in a similar way by detecting the nucleocapsid protein (N protein) of SARS-CoV-2 from upper respiratory samples. The N protein plays an important role in both SARS-CoV and SARS-CoV-2 infection by packaging viral RNA and aiding in the release of additional viral particles from infected cells.
Abbott BinaxNow™ COVID-19 Ag Card
This is a lateral flow immunoassay that qualitatively detects the presence of the N protein in nasal swab samples. Instrumentation is not needed for this test, and all necessary reactions take place on a test card. First, 6 drops of test reagent are added to the top well of the test card. Then, the swab containing the patient sample is inserted into the second well and rotated three times. The card is sealed. Within 15 minutes, a result will be displayed on a paper strip (similar to a pregnancy test) that can be visualized through a small window on the front of the test card.
Becton Dickinson (BD) Veritor™ SystemThis system uses chromatogenic digital immunoassay technology to detect the N protein (antigen) in respiratory specimens. The system includes a lateral flow test strip that is covered in SARS-CoV-2 antibodies that specifically bind to the N antigen. These antibodies are conjugated to detector particles that allow binding to be visualized if the N antigen is detected. The BD instrument detects this binding and accounts for non-specific binding, providing an objective result.
Quidel Sofia 2 SARS Antigen SystemThis is a lateral flow immunofluorescent sandwich assay that is used with the Sofia and
Sofia 2 instrument made by Quidel. This assay detects SARS-CoV and SARS-CoV-2. The patient sample is placed in a reagent tube, which disrupts the virus and exposes the N protein inside. When this solution containing patient specimen and reagent is added to the testing cassette, the sample moves along a test strip. If SARS-CoV or SARS-CoV-2 viral antigen is present, it is trapped in a specific location on the test strip, and this is detected by the Sofia instrument.
Comparison of Test Characteristics and Performance
|Becton Dickinson (BD) Veritor™ System||Quidel Sofia 2 SARS Antigen System||Abbott BinaxNOW™ COVID-19 Ag Card|
|Sample Type||Nasal swab specimens obtained by the dual nares collection method.||Nasal swabs or nasopharyngeal swabs.||Nasal swabs inserted less than 1 inch into the nostril. One swab should be collected from each nostril.|
|Limitations||Test performance was determined in symptomatic patients only. Performance in asymptomatic patients is unknown.
||The use of viral transport media may result in decreased test sensitivity. Since nasopharyngeal swabs are often collected in viral media, this is important to consider. Additionally, if viral transport media is not used, delivery to the laboratory should be as rapid as possible.
||False negative results can occur if the swab is not rotated in the second well prior to closing the test card, if an inadequate amount of extraction buffer is used (e.g., < 6 drops) and if the test is performed > 1 hour after collection. Additionally, the presence of mupirocin may interfere with the test and may cause false negative results.|
|Evaluation of Test Performance||Performance was evaluated for nasal swabs on the BD Veritor™ system compared to PCR testing on nasal swabs.||Performance was evaluated for nasal swabs on the Quidel Sofia system compared to PCR testing on nasopharyngeal swabs.||Clinical performance was evaluated during a multi-site prospective study in the U.S. in patients within 7 days of onset of symptoms. Testing was performed by operators with no laboratory experience and who are representative of the intended users at CLIA-waived testing sites. The comparator method for this study was RT-PCR.|
|Positive Percent Agreement||84% (C.I. 67%–93%)||96.7% (CI 83.3%, 99.4%)||97.1% (95% CI: 85.1% - 99.9%)|
|Positive Predictive Value||100% (C.I. 89%, 100%)||100.0% (CI 88.3% 100.0%)|
|Negative Percent Agreement||100% (C.I. 98%–100%)||100.0% (CI 97.9%, 100.0%)||98.5% (95% CI: 92.0% - 100%)|
|Negative Predictive Value||97.5% (C.I. 95%, 99%)||99.4% (CI 96.9%, 99.9%)|
|Overall Percent Agreement||98%||99.5%|
|Where the Test Can Be Used||Testing is limited to CLIA-certified laboratories and point of care (POC), i.e., inpatient care settings operating under a CLIA Certificate of Waiver, Certificate of Compliance or Certificate of Accreditation.|
|Who Should Be Tested||People who are suspected of having COVID-19 disease.|
|When the Test Should Be Used||Patients should be tested within the first 5-7 days of symptoms.|
|Clinical Considerations||The highest number of SARS-CoV-2 antigens are present in the upper respiratory tract during the acute phase of infection. A positive result means antigen was detected, but clinical correlation with patient history and other diagnostic information is necessary to determine infection status. Negative results should be treated as presumptive and do not rule out infection. If necessary for patient management, negative results should be confirmed with a molecular (PCR) assay.|
Positive percent agreement: true positives/true positives + false negatives
Positive predictive value: true positives/ true positives + false positives
Negative percent agreement: true negatives/ true negatives + false positives
Negative predictive value: true negatives/ true negatives + false negatives
Overall percent agreement: true positives + true negatives / total samples
Additional Testing Considerations Moving ForwardDiscussions about best practices in testing during a pandemic have continued to evolve, and now include the idea of cheap at-home antigen testing. While not currently authorized by the FDA, some scientists believe these tests take more of a public health approach to getting ahead of the virus, as they could potentially allow for consistent mass testing of millions of people.
Experts like Dr. Anthony Fauci and scientists at The Center for Ethics at Harvard University have expressed the importance of scaling up testing to over 1 million tests a day in order to safely re-engage in normal life in the United States. While the idea seems great and more tests are needed, there are numerous factors that must be considered with respect to these tests. Some of these include equitable access to the tests, cost, pre-analytical challenges and test reliability. If all else fails, disease sniffing dogs are always an option.
Additional assays will be developed and receive approval from the FDA. It is critical to consider both the clinical diagnostic and public health implications of each test in order to effectively implement them in settings that offer the most benefit.
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