Standardization and Comparison of Emergency Use Authorized COVID-19 Assays and Testing Laboratories

SARS-CoV-2, the causal agent of the global COVID-19 pandemic, made its appearance at the end of 2019 and is still circulating in the population. The pandemic led to an urgent need for fast, reliable, and widely available testing. After December 2020, the emergence of new variants of SARS-CoV-2 led to additional challenges since new and existing tests had to detect variants to the same extent as the original Wuhan strain. When an antigen-based test is submitted to the Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) consideration it is benchmarked against PCR comparator assays, which yield cycle threshold (CT) data as an indirect indicator of viral load—the lower the CT, the higher the viral load of the sample and the higher the CT, the lower the viral load. The FDA mandates that 10-20% of clinical samples used to evaluate the antigen test have to be low positive. Low positive, as defined by the FDA, are clinical samples in which the CT of the SARS-CoV-2 target gene is within 3 CT of the mean CT value of the approved comparator test's Limit of Detection (LOD). While all comparator tests are PCR-based, the results from different PCR assays used are not uniform. Results vary depending on assay platform, target gene, LOD and laboratory methodology. The emergence and dominance of the Omicron variant further challenged this approach as the fraction of low positive clinical samples dramatically increased as compared to earlier SARS-CoV-2 variants. This led to 20-40% of clinical samples having high CT values and therefore assays vying for an EUA were failing to achieve the 80% Percent Positive Agreement (PPA) threshold required. Here we describe the methods and statistical analyses used to establish a predefined cutoff, based on genome copies per ml (GE/ml) to classify samples as low positive (less than the cutoff GE/ml) or high positive (greater than the cutoff GE/mL). CT 30 for the E gene target using Cobas® SARS-CoV-2-FluA/B platform performed at TriCore Reference Laboratories, and this low positive cutoff value was used for two EUA authorizations. Using droplet digital PCR and methods described here, a value 49,447.72 was determined as the GE/ml equivalent for the low positive cutoff. The CT cutoff corresponding to 49447.72 GE/ml was determined across other platforms and laboratories. The methodology and statistical analysis described here can now be used for standardization of all comparators used for FDA submissions with a goal towards establishing uniform criteria for EUA authorization. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the National Institute of Biomedical Imaging and Bioengineering under the Atlanta Center for Microsystems Engineered Point-of-Care Technologies (ACME POCT). This work was supported by the NIBIB at the NIH under awards 3U54 EB027690-03S1, 3U54 EB027690-03S2, 3U54 EB027690-04S1 and the National Center for Advancing Translational Sciences of the NIH under award UL1TR002378. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study used ONLY data that were openly available to the public, including purchased pooled nasal wash samples from Lee Biosolutions (Catalog number 991-26-P), and the publicly available Heat-Inactivated SARS-Related Coronavirus 2, Isolate USA-WA1/2020 from BEI resources (catalog number NR-52286). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data and code are available at: https://github.com/rbparso/Calibration-Curve