Testing Strategy

FDA to SARS-CoV-2 Test Developers: Evaluate Impact of Genetic Mutations on Test Performance

Evolution and mutation of SARS-CoV-2 virus has made COVID-19 diagnosis a moving target. On Feb. 22, 2021, the U.S. Food and Drug Administration issued new guidance telling developers of SARS-CoV-2 tests how to evaluate the impact of viral mutations on test performance. The guidance covers all three types of SARS-CoV-2 tests—molecular, serology and antigen—including both tests seeking and that have already received Emergency Use Authorization (EUA).

The Diagnostic Challenge

Like other living organisms, viruses evolve over time. And they evolve quickly. Virus evolution often outpaces diagnostics development. As a result, diagnostics designed to detect current iterations may be incapable of detecting the mutated virus by the time they are put into use. This creates the risk that the test will produce false negative results.

The concern is that SARS-CoV-2 virus evolution may be following this pattern. Since the original virus was first detected over a year ago, variants of it have turned up across the globe and in the U.S., including the B117 variant first isolated in the U.K., the B1351 South African variant and the P1 Brazilian variant.

To keep up with the pace of evolution, developers of SARS-CoV-2 tests must evaluate how viral mutations may affect test performance. Last month, the FDA issued an alert warning that current tests may be missing the B117 and possibly other variants. The new guidance lays out a series of recommendations about what developers should do to evaluate viral mutation impact on test performance.

Molecular SARS-CoV-2 Tests

Molecular SARS-CoV-2 tests are designed to detect the virus by targeting one or more specific region(s) of the viral RNA genome. But if the genome mutates, the test may seek the wrong target. And when it fails to detect that target, it may produce a false negative finding. The susceptibility of a particular test depends in part on how it is designed. Thus, for example, tests designed to detect multiple genetic targets are less susceptible than tests that detect a single target. Other factors affecting the impact of genetic variants on molecular test performance include the variant’s sequencing and prevalence in the patient population.

The guidance calls on molecular test developers to account for all these factors by:

  • Designing their tests so as to minimize the impact of viral mutations on test performance;
  • Routinely monitoring for viral mutations that may impact test performance; and
  • Clearly communicating any test limitations in the test’s labeling.

Developers seeking EUA for a new test should include in their submission a description of how they evaluated test performance across all known variants having mutations in the targeted region, along with a discussion of how test design mitigates the risk of future viral mutations impacting the test performance. The submission should also address whether the labeling should include statements or limitations indicating when the specimens used in clinical evaluations were collected and noting that performance may vary depending on the variants. Developers of tests that have already received EUA should reach out to the FDA to determine whether they need to update the labeling.

The guidance suggests that developers of tests with multiple targets include a “highly conserved pan-SARS-CoV target,” that is not specific to SARS-CoV-2 to improve performance with a new genetic variant. If they do, there may be a need for more information on appropriate result interpretation.

The agency also calls on developers to conduct sequence alignment of their primer and probe sequences with available SARS-CoV-2 genomes to determine whether the mutations will impact test performance. If there is an impact, developers should calculate the percentage by which the mutations could reduce performance. If they determine that the impact is 5 percent or more, they should notify the FDA via a supplemental EUA request.

If there is a mutation expected to result in a mismatch within the target primer and probe binding sites, developers should evaluate hybridization changes and give the FDA:

  • Information on the results from melting temperature calculations with the primers and probes;
  • An analysis on how the melting temperature changes as the salt and primer concentration changes;
  • An analysis of the likelihood that the mutation will impact performance;
  • An evaluation of that impact on benefits and risks for the tests;
  • A justification for any actions taken based on the analysis outcomes.

Serology & Antigen SARS-CoV-2 Tests

Serology and antigen tests detect SARS-CoV-2 indirectly by detecting, respectively, the antibodies the body produces to fight off the virus and the antigens those antibodies secrete. Mutations in the viral genome may also produce changes to the viral proteins the test targets and result in false negatives.

The guidance says that serology and antigen test developers should touch base with the agency early in the development process and consider the potential impact of genetic mutations and variants already in circulation. Specifically, they should develop a plan to:

  • Routinely monitor for new genetic mutations and viral variants; and
  • Assess how mutations or viral variants impact test performance, as needed, considering the potential of a given mutation or viral variant to impact their test.

As with molecular tests, developers should measure the potential impact of mutations on test performance and notify the FDA of potential reductions of 5 percent or more.


As of now, the recommendations contained in the guidance are just that—recommendations. However, that is likely to change. The guidance notes that the agency is considering evaluation of virus mutation impact on test performance a mandatory element of the EUA submission. If the FDA goes that route, it will presumably incorporate many if not most of the guidance’s recommendations. In any event, the agency promises to immediately notify test developers if viral mutation analysis becomes a requirement of EUA submission.


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