Estimating Antigen Test Sensitivity via Target Distribution Balancing: Development and Validation Study.

Background: Sensitivity-expressed as percent positive agreement (PPA) with a reference assay-is a primary metric for evaluating lateral-flow antigen tests (ATs), typically benchmarked against a quantitative reverse transcription polymerase chain reaction (qRT-PCR). In SARS-CoV-2 diagnostics, ATs detect nucleocapsid protein, whereas qRT-PCR detects viral RNA copy numbers. Since observed PPA depends on the underlying viral load distribution (proxied by the number of cycle thresholds [Cts], which is inversely related to load), study-specific sampling can bias sensitivity estimates. Cohort differences-such as enrichment for high- or low-Ct specimens-therefore complicate cross-test comparisons, and real-world datasets often deviate from regulatory guidance to sample across the full concentration range. Although logistic models relating test positivity to Ct are well described, they are seldom used to reweight results to a standardized reference viral load distribution. As a result, reported sensitivities remain difficult to compare across studies, limiting both accuracy and generalizability. Objective: The aim of this study was to develop and validate a statistical methodology that estimates the sensitivity of ATs by recalibrating clinical performance data-originally obtained from uncontrolled viral load distributions-against a standardized reference distribution of target concentrations, thereby enabling more accurate and comparable assessments of diagnostic test performance. Methods: AT sensitivity is estimated by modeling the PPA as a function of qRT-PCR Ct values (PPA function) using logistic regression on paired test results. Raw sensitivity is the proportion of AT positives among PCR-positive samples. Adjusted sensitivity is calculated by applying the PPA function to a reference Ct distribution, correcting for viral load variability. This enables standardized comparisons across tests. The method was validated using clinical data from a community study in Chelsea, Massachusetts, demonstrating its effectiveness in reducing sampling bias. Results: Over a 2-year period, paired ATs and qRT-PCR-positive samples were collected from 4 suppliers: A (n=211), B (n=156), C (n=85), and D (n=43). Ct value distributions varied substantially, with suppliers A and D showing lower Ct (high viral load) values in the samples, and supplier C skewed toward higher Ct values (low viral load). These differences led to inconsistent raw sensitivity estimates. To correct for this, we used logistic regression to model the PPA as a function of Cts and applied these models to a standardized reference Ct distribution. This adjustment reduced bias and enabled more accurate comparisons of test performance across suppliers. Conclusions: We present a distribution-aware framework that models PPA as a logistic function of Ct and reweights results to a standardized reference Ct distribution to produce bias-corrected sensitivity estimates. This yields fairer, more consistent comparisons across AT suppliers and studies, strengthens quality control, and supports regulatory review. Collectively, our results provide a robust basis for recalibrating reported sensitivities and underscore the importance of distribution-aware evaluation in diagnostic test assessment.