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

Ramy Arnaout, Rose A. Lee, Ghee Rye Lee, Cody Callahan, Annie Cheng, Christina F. Yen, Kenneth P. Smith, Rohit Arora, James E. Kirby

Research output: Contribution to journalArticlepeer-review

55 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)E3042-E3046
JournalClinical Infectious Diseases
Issue number9
StatePublished - Nov 1 2021
Externally publishedYes


  • SARS-CoV-2
  • antigen detection
  • cycle threshold
  • limit of detection
  • viral load

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases


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