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

Author:

Gniazdowski Victoria1,Paul Morris C12,Wohl Shirlee3,Mehoke Thomas4ORCID,Ramakrishnan Srividya5,Thielen Peter4ORCID,Powell Harrison6,Smith Brendan6,Armstrong Derek T1,Herrera Monica6,Reifsnyder Carolyn6,Sevdali Maria6,Carroll Karen C1,Pekosz Andrew7ORCID,Mostafa Heba H1

Affiliation:

1. Department of Pathology, Division of Medical Microbiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

2. National Institutes of Allergy and Infectious Disease, Bethesda, Maryland, USA

3. Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

4. Johns Hopkins University Applied Physics Laboratory, Laurel, Maryland,USA

5. Department of Computer Science, Johns Hopkins University, Baltimore, Maryland, USA

6. Bio-Rad Laboratories, Pleasanton, California, USA

7. W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

Abstract

Abstract 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.

Funder

National Institutes of Health

Johns Hopkins Center of Excellence in Influenza Research and Surveillance

Publisher

Oxford University Press (OUP)

Subject

Infectious Diseases,Microbiology (medical)

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