Interim Guidance for Antigen Testing for SARS-CoV-2

Summary of Recent Changes

Revisions were made on December 5, 2020 including:

• The word “rapid” has been deleted because FDA has authorized laboratory-based antigen tests.
• New section on processing of antigen tests, reflecting what has been learned on how to minimize the risk of false results.
• Revised section on evaluating the results of antigen tests, introducing a new testing algorithm, and reflecting what has been learned about the performance of antigen tests and the need to implement confirmatory testing.

Antigen Testing for SARS-CoV-2

General Guidance
Antigen tests are commonly used in the diagnosis of respiratory pathogens, including influenza viruses and respiratory syncytial virus. The U.S. Food and Drug Administration (FDA) has granted emergency use authorization (EUA) for antigen tests that can identify SARS-CoV-2. See FDA’s list of In Vitro Diagnostics EUAsexternal icon.

Antigen tests are immunoassays that detect the presence of a specific viral antigen, which implies current viral infection. Antigen tests are currently authorized to be performed on nasopharyngeal or nasal swab specimens placed directly into the assay’s extraction buffer or reagent. The currently authorized antigen tests are not restricted to use on persons of a certain age. See Table 1 for additional information about antigen tests.

Antigen tests are relatively inexpensive, and most can be used at the point of care. Most of the currently authorized tests return results in approximately 15 minutes. Antigen tests for SARS-CoV-2 are generally less sensitive than real-time reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests (NAATs) for detecting the presence of viral nucleic acid. However, RT-PCR can detect levels of viral nucleic acid that cannot be cultured, suggesting that the presence of viral nucleic acid does not always indicate contagiousness.

Proper interpretation of both antigen test results and confirmatory testing when indicated is important for accurate clinical management of patients with suspected COVID-19, or for identification of infected persons when used for screening.

The clinical performance of antigen diagnostic tests largely depends on the circumstances in which they are used. Both antigen tests and NAATs perform best when the person is tested when viral load is generally highest. They also may be informative in diagnostic testing situations in which the person has a known exposure to a person with COVID-19.

There are increasing amounts of dataexternal icon to help guide the use of antigen tests as screening tests on asymptomatic persons to detect or exclude COVID-19, or to determine whether a person who previously was diagnosed with COVID-19 remains infectious. See FDA’s Recommendations for healthcare providers using SARS-CoV-2 diagnostic tests for screening asymptomatic individuals for COVID-19external icon. Also see information from the Centers for Medicare & Medicaid Services (CMS) on Enforcement discretion for the use of SARS-CoV-2 point-of-care testing on asymptomatic individuals.pdf iconexternal icon.

Antigen tests can be used for screening testing in high-risk congregate settings in which repeat testing could quickly identify persons with a SARS-CoV-2 infection to inform infection prevention and control measures, thus preventing transmission. In this case, and especially in settings where a rapid test turnaround time is required, there is value in providing immediate results with antigen tests, even though they may have lower sensitivity than NAATs.

Clinicians and public health practitioners should understand test performance characteristics to recognize potentially false negative or false positive test results and to guide additional confirmatory testing and patient management. Laboratory and testing professionals who perform antigen tests should understand the factors that affect the accuracy of antigen testing, as described in this guidance. Clinicians, laboratory and testing professionals, and public health practitioners should also understand the differences among diagnostic, screening, and surveillance testing. See CDC’s Overview of Testing for SARS-CoV-2, and Testing Strategies for SARS-CoV-2. Also see FDA’s FAQs on Testing for SARS-CoV-2external icon.

Regulatory Requirements for Using Antigen Tests for SARS-CoV-2

FDA regulates in vitro diagnostic devices and has provided recommendations and information regarding EUA requests for COVID-19 diagnostic tests in the Policy for Coronavirus Disease-2019 Tests During the Public Health Emergency (Revised) (“Policy for COVID-19 Tests”)external icon and the EUA templates referenced in that policy. COVID-19 assays and test systems used for diagnostic or screening testing, including those for antigen testing, must have received an EUA from FDA or be offered under the policies in FDA’s Policy for COVID-19 Testsexternal icon. Any antigen test for SARS-CoV-2 authorized for use by FDA is included on FDA’s list of In Vitro Diagnostics EUAsexternal icon.

Laboratory and testing professionals who conduct diagnostic or screening testing for SARS-CoV-2 with antigen tests must also comply with Clinical Laboratory Improvement Amendments (CLIA) regulations. Any laboratory or testing site that intends to report patient-specific test results must first obtain a CLIA certificate and meet all requirements to perform that testing. For more information, see CMS’ How to Obtain a CLIA Certificatepdf iconexternal icon. CMS has provided additional information on Enforcement discretion for the use of SARS-CoV-2 point-of-care testing on asymptomatic individuals.pdf iconexternal icon.

Analytical Performance of Antigen Tests for SARS-CoV-2

It is important for clinicians and testing personnel to understand the analytic performance characteristics, including sensitivity, specificity, and positive and negative predictive values, of the particular antigen test being used, and to follow the manufacturer’s instructions and package insert. See FDA’s In Vitro Diagnostics EUAsexternal icon for detailed information about the performance of specific authorized tests.

The “gold standard” for clinical diagnostic detection of SARS-CoV-2 remains NAATs, such as RT-PCR. Thus, it may be necessary to confirm an antigen test result with a nucleic acid amplification test, especially if the result of the antigen test is inconsistent with the clinical context. Table 1 summarizes the differences between NAATs and antigen tests. Analytic performance may differ from overall performance when considering issues of test availability, quality of specimen collection and transport, and turnaround times of results.

The sensitivity of antigen tests varies but is generally lower than most NAATs. The antigen level in specimens collected either before symptom onset, or late in the course of infection, may be below the limit of detection of virus of the test. This may result in a negative antigen test result, while a more sensitive test, such as most NAATs, may return a positive result.

The specificity of antigen tests is generally as high as most NAATs, which means that false positive test results are unlikely when an antigen test is used according to the manufacturer’s instructions. Despite the high specificity of antigen tests, false positive results will occur, especially when used in communities where the prevalence of infection is low – a circumstance that is true for all in vitro diagnostic tests. CDC considers low prevalence to be when NAAT positivity over the last 14 days is less than 5% or when there are fewer than 20 new cases of COVID-19 per 100,000 persons within the last 14 days. See CDC’s Indicators and thresholds for risk of introduction and transmission of COVID-19 in schools. In general, the lower the prevalence of infection in the community, the higher the rate of false positive test results.

Positive and negative predictive values of all in vitro diagnostic tests (e.g., NAAT and antigen assays) vary depending upon the pretest probability. Pretest probability considers both the prevalence of the target infection in the community as well as the clinical context of the individual being tested. If the prevalence of infection in the community is high, and the person being tested is symptomatic, then the pretest probability is generally considered high. If the prevalence of infection in the community is low, and the person being tested is asymptomatic and has not had any known contact to a person with COVID-19, then the pretest probability is generally considered low. See CDC’s Interpreting Results of Diagnostic Tests for additional information on the relationship between pretest probability and the likelihood of positive and negative predictive values.

To help estimate pretest probability, CDC recommends that laboratory and testing professionals who perform antigen testing determine infection prevalence based on a rolling average of the positivity rate of their own SARS-CoV-2 testing over the previous 7–10 days. If a specific testing site, such as a nursing home, has a test positivity rate near zero, the prevalence of disease in the community (e.g., cases among the population) should instead be used to help determine pretest probability. State health departments generally publish COVID-19 data on testing positivity rates and case rates for their communities.

Processing of Antigen Tests for SARS-CoV-2

The Conditions of Authorization in the antigen EUAs specify that CLIA-certified laboratories and testing sites are to follow the manufacturer’s instructions for use, typically found in the package insert, when performing the test and reading test results. The authorized instructions for use for each test can also be found at FDA’s In Vitro Diagnostics EUAsexternal icon.

For example, the performance of antigen tests can be affected if the test components are not stored and handled properly. They should never be frozen and should always be brought to room temperature (15-30°C) before use. The package insert for these tests includes instructions for handling of the test cartridge/card, such as ensuring it remains in its sealed pouch until immediately before use.

The package insert for antigen tests also includes instructions about how to read the test results, including the appropriate time to read the results and whether the results should be interpreted visually or with an instrument analyzer. Reading the test before or after the specified time could result in false positive or false negative test results.

Processing multiple specimens successively or in batch mode may make it more challenging to ensure that each specimen is incubated for the correct amount of time before the result is read. Refer to the package insert for the correct incubation time for that test, and then monitor and ensure proper timing for each specimen during testing and when reading results.

All testing for SARS-CoV-2, including antigen testing, depends on the integrity of the specimen, which is affected by procedures for both specimen collection and handling. Improper specimen collection, such as swabbing the nostril too quickly, may cause insufficient specimen collection, resulting in limited amounts of viral genetic or antigenic material for detection. Time from sample collection to testing should be minimized, and the temperature of the specimen during this time must be controlled. See CDC’s Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens for COVID-19.

Quality assurance procedures should be followed to prevent cross-contamination and inaccurate test results. For example, users should follow the manufacturer’s instructions, as well as state and local guidance, for when and how often to perform testing on control specimens. If antigen testing returns multiple unexpected positive results, it may be appropriate to stop testing patient specimens, review all procedures, disinfect all surfaces, change gloves, and run control specimens before restarting the testing of patient specimens.

Decontaminate work surfaces and equipment with appropriate disinfectants by using an EPA-approved disinfectant for SARS-CoV-2, following the manufacturer’s recommendations for use, such as dilution, contact time, and safe handling. See EPA’s List of Disinfectants for COVID-19external icon. Gloves should be changed before collecting, handling, and processing a new specimen in the antigen test system. Failing to change gloves can increase the risk of cross-contamination and false antigen test results. See CDC’s guidance on Point-of-Care Testing, and Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19).

Some antigen assays have explored the use of viral transport medium (VTM) during sample collection, but the use of VTM may dilute the specimen and may decrease the sensitivity of the assay (possibly causing false test results). Laboratories and testing sites should refer to the instructions for use and the package insert that are specific for the test that they are using regarding the use of VTM.

Also see FDA’s Letter to Clinical Laboratory Staff and Health Care Providersexternal icon on the potential for false positive results with antigen tests, and CDC’s guidance on Point-of-Care Testing.

Evaluating the Results of Antigen Testing for SARS-CoV-2

Evaluating the results of an antigen test for SARS-CoV-2 should take into account the performance characteristics (e.g., sensitivity, specificity) and the instructions for use of the FDA-authorized assay, the prevalence of SARS-CoV-2 infection in that particular community (positivity rate over the previous 7–10 days or the rate of cases in the community), and the clinical and epidemiological context of the person who has been tested.

The evaluation of an antigen test result should consider whether, and if so the length of time, the patient has experienced symptoms. Generally, clinicians can rely upon a positive antigen test result for a symptomatic patient because the specificity of current FDA-authorized antigen tests is high.

The sensitivity of current FDA-authorized antigen tests varies, and thus negative diagnostic testing results should be handled differently depending on the test, its stated performance characteristics, and intended application (e.g., clinical diagnosis, screening). In most cases, the manufacturers’ instructions for use of antigen tests indicate that negative test results should be considered “presumptive,” meaning that they are preliminary results. See FDA’s In Vitro Diagnostics EUAsexternal icon.

It may be appropriate to confirm antigen test results with another test. CDC recommends following its antigen testing algorithm (Figure 1 below, also available as PDF pdf icon[PDF – 457 KB]) to determine when confirmatory testing is recommended.

Figure 1. Antigen Test Algorithm

Close

download iconDownload Image[PNG]

Testing a symptomatic person – high pretest probability

Currently, the antigen tests that have received EUAs from FDA are authorized for diagnostic testing in symptomatic persons. The specific authorizations vary from test to test. See FDA’s In Vitro Diagnostics EUAsexternal icon.

When testing a person who has symptoms associated with COVID-19, indicating that pretest probability is high, the healthcare provider generally can interpret a positive antigen test to indicate that the person is infected with SARS-CoV-2. A negative antigen test result for a symptomatic person should be confirmed with an FDA-authorized NAAT. CDC recommends using a NAAT that has been evaluated against the FDA reference panel for analytical sensitivity. See FDA’s SARS-CoV-2 Reference Panel Comparative Dataexternal icon. See the antigen testing algorithm when pretest probability is high, Figure 2, which is excerpted directly from the full antigen testing algorithm in Figure 1. If the person has a low likelihood of SARS-CoV-2 infection (e.g., no known exposure), clinical judgement should be used to determine whether a confirmatory NAAT should be performed.

Figure 2. Antigen Testing Algorithm – High Pretest Probability

Close

download iconDownload Image[PNG]

When a symptomatic person receives a negative antigen test result followed by a negative confirmatory NAAT, the healthcare provider should take into consideration whether the person has had exposure to a person with COVID-19 within the past 14 days. If the person has had exposure, that person should follow infection control measures for 14 days after their most recent exposure to a person with COVID-19.

Testing an asymptomatic person who has had close contact with a person with COVID-19 – moderate pretest probability

When testing a person who is asymptomatic and has had exposure to a person with COVID-19 within the last 14 days, indicating that the pretest probability is moderate, the healthcare provider should confirm a positive antigen test result with an FDA-authorized NAAT. See the antigen testing algorithm when pretest probability is moderate, Figure 3, which is excerpted directly from the full antigen testing algorithm in Figure 1. Persons who receive a positive antigen test result that should undergo confirmatory testing should isolate while awaiting results of the confirmatory testing.

Figure 3. Antigen Testing Algorithm – Moderate Pretest Probability

Close

download iconDownload Image[PNG]

In instances of higher pretest probability, such as high incidence of infection in the community, or a person with household or continuous contact to a person with COVID-19, clinical judgement should determine if a positive antigen result for an asymptomatic person should be followed by a confirmatory NAAT.

The healthcare provider should direct the person who received a negative antigen test result, or a negative confirmatory NAAT result, to quarantine for 14 days after the last known exposure to a person with COVID-19. Clinical judgement should determine if and when additional testing is necessary.

In this moderate pretest probability scenario, the healthcare provider should consider performing a NAAT first if short test turnaround time is available, if the person cannot be effectively and safely quarantined, or if there are barriers to possible confirmatory testing (e.g., travel barriers for follow-up testing, tolerance of multiple specimen collections).

Testing an asymptomatic person with no known exposure to a person with COVID-19 – low pretest probability

Healthcare providers should consider pretest probability when using antigen tests as screening tests, and confirmatory testing may be required for clinical management and public health decision-making. See each test’s instructions for use at FDA’s In Vitro Diagnostics EUAsexternal icon, and see FDA’s Recommendations for healthcare providers using SARS-CoV-2 diagnostic tests for screening asymptomatic individuals for COVID-19external icon. Also see CMS’ Enforcement discretion for the use of SARS-CoV-2 point-of-care testing on asymptomatic individualspdf iconexternal icon.

When testing a person who is asymptomatic and has not had known exposure to a person with COVID-19 within the last 14 days, indicating that the pretest probability is low, the healthcare provider generally can interpret a negative antigen test to indicate that the person is not infected with SARS-CoV-2. If the prevalence of SARS-CoV-2 infection is not low in the community, clinical judgement should consider whether this negative antigen test result should be followed by a confirmatory NAAT. See the antigen testing algorithm when pretest probability is low, Figure 4, which is excerpted directly from the full antigen testing algorithm in Figure 1.

Figure 4. Antigen Testing Algorithm – Low Pretest Probability

Close

download iconDownload Image[PNG]

Because of concerns about false positive results when pretest probability is low, a positive antigen test result in this circumstance should be followed by a confirmatory NAAT, recognizing that the person will be tested at a later timepoint in their illness if truly infected. Persons who receive a positive antigen test result that should undergo confirmatory testing should quarantine while awaiting results of the confirmatory testing.

Confirmatory testing when using antigen tests

As the antigen testing algorithm indicates, confirmatory testing may be needed regardless of the symptom or exposure status of the person being tested, or the pretest probability. Confirmatory testing should take place as soon as possible after the antigen test, and not longer than 48 hours after the initial antigen testing. If more than 48 hours separate the two specimen collections, or if there have been opportunities for new exposures, a NAAT should be considered a separate test – not a confirmation of the earlier test. If the results are discordant between the antigen test and the confirmatory NAAT, in general the confirmatory test result should be interpreted as definitive for the purpose of clinical diagnosis.

However, several studies have documented persistent detection of virus using RT-PCR after recovery; in these cases, the persons did not seem to be infectious to others. Thus, if the person being tested has recently had COVID-19, it is possible for that person to receive a negative antigen test result and a positive confirmatory NAAT, potentially indicating a persistent detection of SARS-CoV-2 after recovery from COVID-19. See CDC’s Clinical Questions about COVID-19: Questions and Answers.

If confirmatory testing is not available, clinical discretion can determine whether to recommend that the patient isolate or quarantine. See CDC’s guidance on Testing in Nursing Homes, Quarantine and Isolation, Discontinuation of Isolation for Persons with COVID-19 Not in Healthcare Settings, Discontinuation of Transmission-Based Precautions of Patients in Healthcare Settings, and Return to Work for Healthcare Personnel.

Serial testing when using antigen tests

Depending on the circumstances and setting, it may be useful to implement serial antigen testing for persons who receive a negative antigen test result. Serial antigen testing within a closed congregate setting, such as a long-term care facility or a correctional or detention facility, could quickly identify someone with a SARS-CoV-2 infection and prevent further transmission. It may not be necessary to perform confirmatory testing with a NAAT when conducting serial antigen testing on those who have received a negative antigen test result. Serial testing, particularly in congregate settings when it has been possible to quarantine persons for 14 days, should not continue indefinitely.

Modeling evidenceexternal icon shows that outbreak control depends largely on the frequency of testing, the speed of reporting, and the application of interventions, and is only marginally improved by the sensitivity of the test. Additional evidencepdf iconexternal icon shows the value of repeat testing, using NAATs with fast turnaround times, for informing clinical and public health decision-making. For this reason, serial antigen testing may have benefits for early identification and controlling outbreaks in some situations, such as congregate living, compared to laboratory-based NAATs with prolonged turnaround times. See CDC’s guidance on Testing in Nursing Homes and FDA’s Recommendations for healthcare providers using SARS-CoV-2 diagnostic tests for screening asymptomatic individuals for COVID-19external icon.

CDC will update this guidance as more data become available.

Reporting Antigen Test Results for SARS-CoV-2 to Health Departments and Patients

A CLIA-certified laboratory or testing site must report antigen diagnostic test results to the local, state, tribal, or territory health department in accordance with Public Law 116-136, § 18115(a), the Coronavirus Aid, Relief, and Economic Security (CARES) Act. The CARES Act requires “every laboratory that performs or analyzes a test that is intended to detect SARS-CoV-2 or to diagnose a possible case of COVID-19” to report the results of each such test. Antigen test results that are reported to public health departments must be clearly distinguished from other COVID-19 tests, such as NAATs and antibody tests.

On June 4, 2020, the U.S. Department of Health and Human Services published guidance on COVID-19 Pandemic Response, Laboratory Data Reporting: CARES Act Section 18115pdf iconexternal icon that specifies what additional data should be collected and electronically reported to health departments along with COVID-19 diagnostic or screening test results. Laboratory and testing professionals should collect and report complete patient demographic information and ensure that they report antigen test results using the proper LOINC code for their particular FDA-authorized assays. Facilities should refer to CDC’s LOINC In Vitro Diagnostic (LIVD) Test Code Mapping for SARS-CoV-2 Tests.

A CLIA-certified laboratory or testing site must report antigen test results to the individual or the individual’s healthcare provider according to the instructions for use of the FDA-authorized SARS-CoV-2 in vitro diagnostic device that was used. Depending on the stipulations of the FDA authorization, the laboratory or testing site may be required to report negative test results to patients as “presumptive negative.”

For long-term care facilities that are enrolled in CDC’s National Healthcare Safety Network (NHSN), the preferred method for reporting point-of-care SARS-CoV-2 testing data, including antigen test results, is through the NHSN.

Summary Tables