January 8, 2021
The SARS-CoV-2 virus can mutate over time, resulting in genetic variation in the population of circulating viral strains. The U.S. Food and Drug Administration (FDA) is alerting clinical laboratory staff and health care providers that false negative results may occur with any molecular test for the detection of SARS-CoV-2 if a mutation occurs in the part of the virus’ genome assessed by that test. The FDA monitors the potential effects of genetic variation in molecular tests that have received Emergency Use Authorization (EUA) and has been doing so on an ongoing basis throughout the pandemic. The FDA’s analysis to date has identified three molecular tests that have received EUAs whose performance could be impacted by SARS-CoV-2 genetic variants (see FDA Analysis of Molecular Tests below).
The FDA is providing recommendations to address possible false negative results for clinical laboratory staff and health care providers who use molecular tests for the detection of SARS-CoV-2 (see Recommendations below).
Impact of Genetic Variation on Diagnostic Tests
The presence of SARS-CoV-2 genetic variants in a patient sample can potentially change the performance of the SARS-CoV-2 test. Molecular tests designed to detect multiple SARS-CoV-2 genetic targets are less susceptible to the effects of genetic variation than tests designed to detect a single genetic target. The FDA reminds clinical laboratory staff and health care providers about the risk of false negative results with all laboratory tests, including molecular tests. No test is perfect. Laboratories should expect some false results to occur even when very accurate SARS-CoV-2 tests are used. The prevalence of genetic variants may vary in any population and may lead to more false negative results than otherwise expected.
FDA Analysis of Molecular Tests
Based on the FDA’s analysis to date, the FDA is providing the following information about three tests that have been issued an EUA:
- Accula SARS-Cov-2 Test: The FDA’s analysis, including additional information provided by the manufacturer, Mesa Biotech Inc., indicates that the Accula SARS-Cov-2 Test performance may be impacted when a SARS-CoV-2 virus patient sample having a genetic variant at position 28881 (GGG to AAC) is tested. While the impact does not appear to be significant, the FDA is providing this alert to health care providers out of an abundance of caution while we continue to gather additional data and work with the manufacturer.
- TaqPath COVID-19 Combo Kit: The FDA’s analysis, including additional information provided by the manufacturer, Thermo Fisher Scientific, Inc., and multiple reports from clinical laboratories, indicates that one of three targets of the TaqPath COVID-19 Combo Kit (which may also be labeled as the TaqPath COVID-19 Combo Kit Advanced) has significantly reduced sensitivity due to certain mutations, including one of the mutations in the recently identified B.1.1.7 variant (UK VOC-202012/01). Since this test is designed to detect multiple genetic targets, the overall test sensitivity should not be impacted. The pattern of detection when certain mutations are present may help with early identification of new variants in patients to reduce further spread of infection.
- Linea COVID-19 Assay Kit: The FDA’s analysis, including additional information provided by the manufacturer, Applied DNA Sciences, Inc., indicates that one of the two targets of the Linea COVID-19 Assay Kit has significantly reduced sensitivity due to certain mutations, including one of the mutations in the recently identified B.1.1.7 variant. Since this test is designed to detect multiple genetic targets, the overall test sensitivity should not be impacted. The pattern of detection when certain mutations are present may help with early identification of new variants in patients to reduce further spread of infection.
The FDA continues to monitor the effects of different variants on authorized molecular tests for the detection of SARS-CoV-2 and will update clinical laboratory staff and health care providers accordingly.
The FDA recommends clinical laboratory staff and health care providers who use molecular tests for the detection of SARS-CoV-2 to:
- Be aware that genetic variants of SARS-CoV-2 arise regularly and false negative test results can occur.
- Be aware that tests that use multiple genetic targets to determine a final result are less likely to be impacted by increased prevalence of genetic variants.
- Consider negative results in combination with clinical observations, patient history, and epidemiological information.
- Consider repeat testing with a different test (with different genetic targets) if COVID-19 is still suspected after receiving a negative test result.
For clinical laboratory staff and health care providers using these specific tests:
- Accula SARS-Cov-2 Test: Be aware of the updated instructions for use for the Accula SARS-Cov-2 Test that now include the exact variant location, in silico analysis of the primer binding, and observed performance impact when compared with the perfect match target.
- TaqPath COVID-19 Combo Kit and Linea COVID-19 Assay Kit: Be aware of the pattern of detection associated with certain mutations, including the B.1.1.7 variant, specifically a pattern of 2/3 positive targets showing the S-gene drop out (reduced sensitivity with the S-gene target), when using the TaqPath COVID-19 Combo Kit, and a pattern of 1/2 positive targets showing the S-gene drop out when using the Linea COVID-19 Assay Kit. If local or state clinical laboratories have access to quick turnaround whole genome sequencing services, such as those using the EUA-authorized Illumina COVIDSeq Test, these labs should consider further characterizing the specimen with genetic sequencing when this pattern is identified. If such services are not readily available, local or state clinical laboratories should consider reaching out to the Centers for Disease Control and Prevention at EOCenter177@cdc.gov for additional information.
Most molecular tests for SARS-CoV-2 are designed to detect specific RNA sequences found in the viral genome. However, the SARS-CoV-2 virus mutates regularly, resulting in several genetically unique variants, each with different RNA sequences, that may be found in infected individuals as the pandemic progresses. The FDA has been monitoring the SARS-CoV-2 virus mutations, and potential impact on testing, throughout the pandemic.
Since molecular tests look for a specific sequence, it is possible for certain virus mutations to affect the performance of certain tests if the mutation is in a region of the genome targeted by the test. The FDA monitors these potential performance changes by comparing the sequence of genetic variants of the SARS-CoV-2 virus with the RNA sequences targeted by authorized SARS-CoV-2 tests. The clinical impact of genetic variants on test sensitivity is influenced by the sequence of the variant, the design of test and the prevalence of the variant in the patient population. Tests that rely on the detection of multiple regions of the genome may be less impacted by genetic variation in the SARS-CoV2 genome than tests that rely on detection of only a single region.
The recently identified B.1.1.7 variant has been associated with an increased risk of transmission; therefore, early identification of this variant in patients may help reduce further spread of infection. The B.1.1.7 variant carries many mutations, including a double deletion at positions 69 and 70 on the spike protein gene (S-gene), which is the mutation that appears to impact the pattern of detection when using the TaqPath COVID-19 Combo Kit and the Linea COVID-19 Assay Kit.
In addition to the FDA’s monitoring, periodic in silico assessment (computer simulations) of molecular tests performed by test developers assesses the potential impact of genetic variation. This is critical to identify impacts on performance and protect patients. Based on the FDA’s in silico analysis, the FDA requested and received reports from test developers that showed reduced analytical sensitivity associated with certain SARS-CoV-2 molecular tests. While the evaluation of analytical sensitivity has demonstrated some impact, the FDA believes the risk that these mutations will impact clinical sensitivity is low. The FDA continues to monitor and evaluate these reports and other available information about device safety and performance.
Molecular tests are typically highly sensitive for the detection of the SARS-CoV-2 virus. However, all diagnostic tests may be subject to false negative results, and the risk of false negative results may increase when testing patients with genetic variants of SARS-CoV-2. Health care providers should always carefully consider diagnostic test results in the context of all available clinical, diagnostic and epidemiological information.
- The FDA is monitoring for new viral mutations and their impact on authorized SARS-CoV-2 molecular tests and is taking action to ensure tests remain accurate.
- The FDA continues to monitor the emerging B1.1.7 variant and evaluate authorized molecular test performance.
- The FDA is working with sponsors whose authorized tests are impacted to update their labeling to reflect potential changes in performance of their tests, and to consider modifications to the test if needed.
- The FDA will continue to keep health care providers and the public informed if new or additional information becomes available.
Reporting Problems to the FDA
The FDA encourages stakeholders to report any adverse events or suspected adverse events as well as performance issues experienced with molecular tests for detection of SARS-CoV-2.
- Voluntary reports can be submitted through MedWatch, the FDA Safety Information and Adverse Event Reporting program.
- Generally, as specified in a test's EUA, device manufacturers must comply with applicable Medical Device Reporting (MDR) regulations.
- Health care personnel and clinical laboratory staff employed by facilities that are performing COVID-19 testing should follow the reporting requirements for authorized laboratories as specified in the test's EUA.
Prompt reporting of adverse events can help the FDA identify and better understand the risks associated with medical devices.
If you have questions about this letter, contact COVID19DX@fda.hhs.gov.