The U.S. Food and Drug Administration (FDA) announced the release of its FDA SARS-CoV-2 Reference Panel, which allows for a more precise comparison of the analytical performance of different molecular in vitro diagnostic (IVD) assays intended to detect SARS-CoV-2.
The Reference Panel contains common, independent, and well-characterized reference material that is available to developers of SARS-CoV-2 nucleic acid-based amplification tests (NAATs) for which Emergency Use Authorization (EUA) was requested.
During the early months of the COVID-19 pandemic, clinical specimens were not readily available to developers of IVDs to detect SARS-CoV-2. Therefore, the FDA authorized IVDs based on available data from contrived samples generated from a range of SARS-CoV-2 material sources (for example, gene specific RNA, synthetic RNA, or whole genome viral RNA) for analytical and clinical performance evaluation. While validation using these contrived specimens provided a measure of confidence in test performance at the beginning of the pandemic, it is not feasible to precisely compare the performance of various tests that used contrived specimens because each test validated performance using samples derived from different gene specific, synthetic, or genomic nucleic acid sources.
From February through the middle of May, the FDA issued a total of 59 EUAs for IVDs for the qualitative detection of nucleic acid from SARS-CoV-2 based on validation data using contrived specimens derived from SARS-CoV-2 viral RNA. As the pandemic progressed and more patient specimens became available, on May 11, 2020, the FDA recommended in the Policy for Coronavirus Disease-2019 Tests that developers obtain and use patient specimens to validate their tests.
Recognizing the value to healthcare professionals, laboratories, and patients in understanding the relative performance of NAATs for SARS-CoV-2, the FDA obtained live virus in February to develop a reference panel. Reference panels are a fundamental tool for performance assessment of molecular tests, and the use of the same reference material across different tests allows a direct comparison of analytical sensitivity performance across these tests. As was done for the evaluation of NAATs for Zika, the FDA is again providing a tool for a comparative analysis of the performance of different tests. Such comparison has shown to be useful to healthcare providers and laboratories using these tests.
The FDA SARS-CoV-2 Reference Panel is shared with developers who have interacted with FDA through the review process. To more precisely compare the performance of NAAT SARS-CoV-2 assays, through a collaboration between the Center for Devices and Radiological Health (CDRH) and the Center for Biologics Evaluation and Research (CBER), the FDA established a Reference Panel composed of standardized material, suitable for the determination and direct comparison of analytical sensitivity and cross-reactivity of nucleic acid-based SARS-CoV-2 assays.
The panel contains one heat-inactivated SARS-CoV-2 strain and one heat-inactivated MERS-CoV strain in cell culture media. The panel is composed of five tubes (T1 to T5): T1 contains the SARS-CoV-2 strain (2019-nCoV/USA-WA1/2020) at a concentration of ~1.8x108 RNA NAAT detectable units/mL (NDU/mL); T2, T3, T4, and T5 contain blinded samples, meaning that, although the FDA knows the concentration, the developer testing the samples does not. Based on a standard protocol provided by the FDA for T1, the developers are asked to perform a range finding Limit of Detection (LoD) study followed by a confirmatory study to further define and corroborate the LoD of their assay. The blinded samples (T2 to T5) are also tested per a protocol provided by the FDA, to confirm the LoD determined for T1 and evaluate cross-reactivity with MERS-CoV virus. Depending on the test, the number of tests performed on different amounts of viral replicates can range from over 40 to over 150.
The FDA SARS-CoV-2 Reference Panel was first provided to all developers of authorized IVD EUAs that used contrived samples to validate their assay and is provided to all developers who request an EUA for SARS-CoV-2 NAATs. In general, FDA's EUAs require developers to evaluate and submit the analytical limit of detection and assess traceability of their product with any FDA-recommended reference material as a condition of the authorization. As explained above, assessment of assay performance using the FDA SARS-CoV-2 Reference Panel allows for a consistent determination of the relative sensitivity of these tests and cross-reactivity with MERS-CoV virus.
While the FDA SARS-CoV-2 Reference Panel helps determine the comparative performance among authorized tests, the panel is not a replacement for the analytical and clinical validation recommendations the FDA has provided in the EUA templates. For example, the panel only includes one strain of SARS-CoV-2 and one cross-reactant, MERS-CoV. Recent mutations reported for SARS-CoV-2 (e.g., D614G), which may impact molecular testing, are not included.
FDA began distribution of the FDA SARS-CoV-2 Reference Panel in May 2020. As of September 23, 2020, the FDA has contacted developers of 186 authorized assays for shipping information and has sent the reference panel to developers of 161 authorized assays. The FDA is reviewing results as they are returned and continues to send the reference panel out to additional developers.
As of August 28, 2020, the FDA has contacted developers of 165 authorized assays for shipping information and by August 31, 2020 sent the reference panel to developers of 147 authorized assays which are included in the tables below. Developers who received the reference panel were asked to conduct testing and return results within two weeks of receiving the panel. Many developers returned data to the FDA by September 11, 2020, but in some cases, FDA did not receive the data, or the data was uninterpretable, or is still under interactive review. Confirmed results of the relative sensitivity of EUA authorized assays provided by developers as of September 11, 2020, are displayed in Tables 2A, 2B, and 2C. The results are presented in three tables according to the clinical matrix used in the study: swab in transport media, direct swabs (dry swabs), or saliva.
Following a protocol provided by the FDA with the reference panel, developers conducted testing with three replicates of serial dilutions of the SARS-CoV-2 virus provided in T1 in clinical negative specimens. The studies are performed by diluting the panel material in "negative patient clinical matrix" acquired and prepared by individual developers. The developers identified a provisional LoD and then performed confirmatory testing. To corroborate the LoD identified from testing T1, developers then diluted in clinical negative specimens and tested the blinded samples (T2-T5) according to the protocol provided by the FDA.
