SARS-CoV-2 Subgenomic RNA Kinetics in Longitudinal Clinical Samples
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Abstract
Background
Given the persistence of viral RNA in clinically recovered coronavirus disease 2019 (COVID-19) patients, subgenomic RNAs (sgRNAs) have been reported as potential molecular viability markers for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, few data are available on their longitudinal kinetics, compared with genomic RNA (gRNA), in clinical samples.
Methods
We analyzed 536 samples from 205 patients with COVID-19 from placebo-controlled, outpatient trials of peginterferon Lambda-1a (Lambda; n = 177) and favipiravir (n = 359). Nasal swabs were collected at 3 time points in the Lambda (days 1, 4, and 6) and favipiravir (days 1, 5, and 10) trials. N-gene gRNA and sgRNA were quantified by quantitative reverse transcription polymerase chain reaction. To investigate the decay kinetics in vitro, we measured gRNA and sgRNA in A549ACE2+ cells infected with SARS-CoV-2, following treatment with remdesivir or dimethylsulfoxide control.
Results
At 6 days in the Lambda trial and 10 days in the favipiravir trial, sgRNA remained detectable in 51.6% (32/62) and 49.5% (51/106) of the samples, respectively. Cycle threshold (Ct) values for gRNA and sgRNA were highly linearly correlated (marginal R2 = 0.83), and the rate of increase did not differ significantly in the Lambda trial (1.36 cycles/d vs 1.36 cycles/d; P = .97) or the favipiravir trial (1.03 cycles/d vs 0.94 cycles/d; P = .26). From samples collected 15–21 days after symptom onset, sgRNA was detectable in 48.1% (40/83) of participants. In SARS-CoV-2-infected A549ACE2+ cells treated with remdesivir, the rate of Ct increase did not differ between gRNA and sgRNA.
Conclusions
In clinical samples and in vitro, sgRNA was highly correlated with gRNA and did not demonstrate different decay patterns to support its application as a viability marker.
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SciScore for 10.1101/2021.04.26.21256131: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Ethics statement: All participants were >18 years of age and provided written informed consent. Sex as a biological variable not detected. Randomization Overview and Study Population: This was a sub-study of two Phase 2 randomized, placebo-controlled trials of peginterferon-Lambda-1a (Lambda) (NCT04331899) and favipiravir (NCT04346628) for treatment of COVID-19. Blinding The favipiravir trial is an ongoing study and remains blinded. Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources We estimated copies/sample from a standard curve using a pET21b+ plasmid (GenScript, USA) with the N-gene. pET21b+suggested: RRID:Addgene_92208)Results from OddPub: We did not detect …
SciScore for 10.1101/2021.04.26.21256131: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Consent: Ethics statement: All participants were >18 years of age and provided written informed consent. Sex as a biological variable not detected. Randomization Overview and Study Population: This was a sub-study of two Phase 2 randomized, placebo-controlled trials of peginterferon-Lambda-1a (Lambda) (NCT04331899) and favipiravir (NCT04346628) for treatment of COVID-19. Blinding The favipiravir trial is an ongoing study and remains blinded. Power Analysis not detected. Table 2: Resources
Recombinant DNA Sentences Resources We estimated copies/sample from a standard curve using a pET21b+ plasmid (GenScript, USA) with the N-gene. pET21b+suggested: RRID:Addgene_92208)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:Our study has several limitations, which include lack of viral culture data and samples at later timepoints. Additionally, to study the sgRNA decay kinetics, we targeted a single and highly abundant gene to compare and quantify gRNA and sgRNA, though we found high correlation between E-gene and N-gene sgRNA copy numbers. Analyzing additional targets would help us understand the stability of other sgRNAs. Another limitation our study is lack of unblinded data from the favipiravir study, precluding analysis by study arm. However, our findings of sgRNA persistence in clinical samples and lack of difference in its decline compared with gRNA are notable regardless of whether favipiravir reduced levels in one arm. For remdesivir, a RDRP inhibitor like favipiravir, we found no differential effect on sgRNA, compared with gRNA, in cell culture, despite effective reduction in viral replication and cytopathic effects. In summary, we found that SARS-CoV-2 sgRNAs are persistently detectable in clinical samples, correlate strongly with gRNA, and decline at indistinguishable rates in clinical samples and cell culture. We find little evidence to support the premise that sgRNA detection is a reliable marker of transcriptionally active virus or that it provides additional information beyond detection of gRNA in clinical samples. We advise caution against using sgRNA assays to inform decisions concerning treatment or medical isolation.
Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04331899 Active, not recruiting Single-Blind Study of a Single Dose of Peginterferon Lambda-… NCT04346628 Completed Oral Favipiravir Compared to Placebo in Subjects With Mild C… 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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