Repeated Coronavirus Disease 2019 Molecular Testing: Correlation of Severe Acute Respiratory Syndrome Coronavirus 2 Culture With Molecular Assays and Cycle Thresholds

Clin Infect Dis. 2021 Aug 16;73(4):e860-e869. doi: 10.1093/cid/ciaa1616.


Background: Repeated coronavirus disease 2019 (COVID-19) molecular testing can lead to positive test results after negative results and to multiple positive results over time. The association between positive test results and infectious virus is important to quantify.

Methods: A 2-month cohort of retrospective data and consecutively collected specimens from patients with COVID-19 or patients under investigation were used to understand the correlation between prolonged viral RNA positive test results, cycle threshold (Ct) values and growth of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in cell culture. Whole-genome sequencing was used to confirm virus genotype in patients with prolonged viral RNA detection. Droplet digital polymerase chain reaction was used to assess the rate of false-negative COVID-19 diagnostic test results.

Results: In 2 months, 29 686 specimens were tested and 2194 patients underwent repeated testing. Virus recovery in cell culture was noted in specimens with a mean Ct value of 18.8 (3.4) for SARS-CoV-2 target genes. Prolonged viral RNA shedding was associated with positive virus growth in culture in specimens collected up to 21 days after the first positive result but mostly in individuals symptomatic at the time of sample collection. Whole-genome sequencing provided evidence the same virus was carried over time. Positive test results following negative results had Ct values >29.5 and were not associated with virus culture. Droplet digital polymerase chain reaction results were positive in 5.6% of negative specimens collected from patients with confirmed or clinically suspected COVID-19.

Conclusions: Low Ct values in SARS-CoV-2 diagnostic tests were associated with virus growth in cell culture. Symptomatic patients with prolonged viral RNA shedding can also be infectious.

Keywords: COVID-19; SARS-CoV-2; infectious virus; repeat testing.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19*
  • Humans
  • RNA, Viral / genetics
  • Retrospective Studies
  • SARS-CoV-2*
  • Virus Shedding


  • RNA, Viral