Repeated Coronavirus Disease 2019 Molecular Testing: Correlation of Severe Acute Respiratory Syndrome Coronavirus 2 Culture With Molecular Assays and Cycle Thresholds
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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.
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SciScore for 10.1101/2020.08.05.20168963: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The study was approved by the Johns Hopkins University School of Medicine Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The humanized monoclonal antibody D-006 (Sino Biological) was used as the primary antibody to detect Spike or S protein, followed by Alexa Fluor 488-conjugated goat anti-human IgG. anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2 Virus Isolation: VeroE6 cells (ATCC CRL-1586) were cultured at 37°C with 5% carbon dioxide in a … SciScore for 10.1101/2020.08.05.20168963: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: The study was approved by the Johns Hopkins University School of Medicine Institutional Review Board. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The humanized monoclonal antibody D-006 (Sino Biological) was used as the primary antibody to detect Spike or S protein, followed by Alexa Fluor 488-conjugated goat anti-human IgG. anti-human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2 Virus Isolation: VeroE6 cells (ATCC CRL-1586) were cultured at 37°C with 5% carbon dioxide in a humidified chamber using complete medium (CM) consisting of Dulbecco’s modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco), 1mM glutamine (Invitrogen), 1mM sodium pyruvate (Invitrogen), 100μg/mL penicillin (Invitrogen) and 100 μg/mL streptomycin (Invitrogen). VeroE6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources The fluorescence intensity of each droplet was measured in FAM and HEX, and droplets were determined to be positive or negative for each target within the Bio-Rad SARS-CoV-2 ddPCR Test: N1, N2 and RP. N2suggested: NoneSoftware and Algorithms Sentences Resources Coverage was normalized across the genome and variant calling was performed with Nanopolish v0.13.2 (52). Nanopolishsuggested: (Nanopolish, RRID:SCR_016157)The fluorescence data was then analyzed by QuantaSoft 1.7 and QuantaSoft Analysis Pro 1.0 Software to determine the presence of SARS-CoV-2 N1 and N2 in the specimen. QuantaSoftsuggested: NoneQuantaSoft Analysis Prosuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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