The Limit of Detection Matters: The Case for Benchmarking Severe Acute Respiratory Syndrome Coronavirus 2 Testing

Abstract

Background: Resolving the coronavirus disease 2019 (COVID-19) pandemic requires diagnostic testing to determine which individuals are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The current gold standard is to perform reverse-transcription polymerase chain reaction (PCR) on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of approximately 100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss infected patients. However, the relative clinical sensitivity of these assays remains unknown.

Methods: Here we model the clinical sensitivities of assays based on their LoD. Cycle threshold (Ct) values were obtained from 4700 first-time positive patients using the Abbott RealTime SARS-CoV-2 Emergency Use Authorization test. We derived viral loads from Ct based on PCR principles and empiric analysis. A sliding scale relationship for predicting clinical sensitivity was developed from analysis of viral load distribution relative to assay LoD.

Results: Ct values were reliably repeatable over short time testing windows, providing support for use as a tool to estimate viral load. Viral load was found to be relatively evenly distributed across log10 bins of incremental viral load. Based on these data, each 10-fold increase in LoD is expected to lower assay sensitivity by approximately 13%.

Conclusions: The assay LoD meaningfully impacts clinical performance of SARS-CoV-2 tests. The highest LoDs on the market will miss a majority of infected patients. Assays should therefore be benchmarked against a universal standard to allow cross-comparison of SARS-CoV-2 detection methods.

Keywords: SARS-CoV-2; antigen detection; cycle threshold; limit of detection; viral load.

Figure 1.

Cycle threshold (Ct) values are highly repeatable. Data points shown are Ct values for severe acute respiratory syndrome coronavirus 2 testing of pairs of nasopharyngeal samples obtained within either 6 hours (A) or 12 hours (B) of each other from the same patient, represented by the X and Y coordinates of each data point. Shaded areas indicate 95% confidence intervals for Theil-Sen linear regression fit. Abbreviations: CI, confidence interval; LR, linear regression fit; TS, Theil-Sen linear regression fit.

Figure 2.

Viral load distribution and limit of detection (LoD). A, Fraction of positive tests binned by 0.5 log₁₀ bins of viral load. B, Cumulative histogram distribution of viral loads showing percentage detected as a function of LoD: actual (solid line) and trend line (dotted line). For purposes of discussion, examples of LoD for several other methods, obtained from Emergency Use Authorization package inserts or the scientific literature, are overlaid. Abbreviations: Ag, antigen; CDC, Centers for Disease Control and Prevention; EUA, Emergency Use Authorization.