Surveillance and Correlation of Severe Acute Respiratory Syndrome Coronavirus 2 Viral RNA, Antigen, Virus Isolation, and Self-Reported Symptoms in a Longitudinal Study With Daily Sampling
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Abstract
The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of US Food and Drug Administration emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, that is, the presence of replicable virus. As the number of tests conducted increased, persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA positivity by reverse-transcription polymerase chain reaction (RT-PCR) in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike, nucleocapsid, membrane, envelope, and ORF8. Viral RNA abundances calculated from these assays were compared with antigen presence, self-reported symptoms, and culture outcome (virus isolation) using samples from a 14-day longitudinal household transmission study. By characterizing the clinical and molecular dynamics of infection, we show that sgRNA detection has higher predictive value for culture outcome compared to detection of gRNA alone. Our findings suggest that sgRNA presence correlates with active infection and may help identify individuals shedding culturable virus.
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SciScore for 10.1101/2021.12.23.21268319: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The household transmission study protocol was approved by the Vanderbilt University Institutional Review Board. Sex as a biological variable not detected. Randomization : Complete genomes absent of ambiguous nucleotides were randomly sampled from 01 July 2021 using the NCBI Nucleotide SARS-CoV-2 Data hub (https://www.ncbi.nlm.nih.gov/sars-cov-2/). Blinding not detected. Power Analysis not detected. Cell Line Authentication Authentication: The top performing primer set, determined by amplification efficiency, was selected for further validation and multiplex optimization (Table 1; Supplemental Figure 1B-E; Supplemental Table 3). Table 2: Resources
Experimental Models: Cell Lines Sentences Resources In… SciScore for 10.1101/2021.12.23.21268319: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The household transmission study protocol was approved by the Vanderbilt University Institutional Review Board. Sex as a biological variable not detected. Randomization : Complete genomes absent of ambiguous nucleotides were randomly sampled from 01 July 2021 using the NCBI Nucleotide SARS-CoV-2 Data hub (https://www.ncbi.nlm.nih.gov/sars-cov-2/). Blinding not detected. Power Analysis not detected. Cell Line Authentication Authentication: The top performing primer set, determined by amplification efficiency, was selected for further validation and multiplex optimization (Table 1; Supplemental Figure 1B-E; Supplemental Table 3). Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Inoculation of cells with clinical specimens and virus titration: Vero E6 cells (JCRB1819 Vero E6suggested: NoneSerial dilutions of virus prepared in virus diluent were transferred to 6-well plates containing 95% confluent Vero E6-TMPRSS2 cells. Vero E6-TMPRSS2suggested: NoneRecombinant DNA Sentences Resources DNA fragments synthesized and cloned into pUC57 (GenScript) were designed with the T7 promoter upstream of the SARS-CoV-2 leader sequence, followed by the truncated ORF for a given transcript, and ended with an RNaseP gene fragment. pUC57suggested: RRID:Addgene_40306)Software and Algorithms Sentences Resources Sequences were analyzed using Geneious “Test with Saved Primers”, an adaptation to Primer3 using the primers in Supplemental Table 2. Geneioussuggested: (Geneious, RRID:SCR_010519)Results 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: We detected the following sentences addressing limitations in the study:Of course, these conclusions may have some limitations based on the following assumptions: 1) virus isolation in culture directly correlates to a person’s infectivity; 2) viral RNA abundance correlates with infectious units; 3) all sampling and storage methods lead to similar quantities of specimen available for testing; 4) the relationship between viral RNA abundance and infectivity is consistent and robust to factors negatively influencing RNA or virion integrity. While one or more of the foregoing might not hold true, sgRNA is nonetheless produced during viral replication and would likely indicate active infection. Detection of sgRNAs can vary dramatically depending on specimen and tissue source [20], storage conditions [30], symptom presence and severity at the time of sampling [32], patient demographics, and overall medical history [33]. In patient specimens, sgRNA can be detected for more than two weeks after initial detection and has been found for up to 162 days by PCR [20, 23]. Some specimens we tested were Cx-, yet positive for sgRNA. One proposed mechanism ascribes nuclease-resistance and structural stability as the basis of sgRNA [24]. Replication of SARS-CoV-2 and sgRNA production likely occur in or on double-membrane vesicles [25], which when associated with sgRNA can promote nuclease-resistance. Alternatively, extended, or recurrent sgRNA detection has been reported in immunocompromised individuals and therefore can be a sign of a persistent, active infection [...
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.
Results from scite Reference Check: We found no unreliable references.
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