SARS-CoV-2 Viral Load in Saliva Rises Gradually and to Moderate Levels in Some Humans

Winnett, Alexander; Cooper, Matthew M.; Shelby, Natasha; Romano, Anna E.; Reyes, Jessica A.; Ji, Jenny; Porter, Michael K.; Savela, Emily S.; Barlow, Jacob T.; Akana, Reid; Tognazzini, Colten; Feaster, Matthew; Goh, Ying-Ying; Ismagilov, Rustem F.

doi:10.1101/2020.12.09.20239467

Cited by 24 publications

(37 citation statements)

References 22 publications

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“…These findings seem to be in contrast with those published by Kim et al [11], who found that the N RNA is the most represented during the viral replication; nevertheless, these data were obtained from SARS-CoV-2 viral RNA extracted from Vero cells infected with BetaCoV/Korea/KCDC03/2020, the latter being an experimental setting that was different from ours. In support of our considerations, we can provide evidence by Winnett et al [21] stating that low-sensitivity tests are comparable to high-sensitivity tests in detecting early infections when the viral load rises quickly (within hours) after infection and reaches high levels (>10 5 -10 6 RNA copies/mL). However, although there are no human data testing these assumptions, they provided evidence that, in at least some human cases of SARS-CoV-2, the viral load rises slowly (over days, not hours) and not to such high levels as to be detectable reliably by any low-sensitivity test.…”

Section: Discussionsupporting

confidence: 84%

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

et al. 2021

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The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19–positive patients’ swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients’ swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

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Section: Discussionsupporting

confidence: 84%

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

et al. 2021

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“…On the contrary, the symptomatic population expectedly contains higher percentage of positive subjects, as symptoms usually timely correlates with the highest viral load, which allow an easier comparison of both sampling procedures. Anyhow, viral load in saliva of presymptomatic subjects remains in the range of detection of the RT-PCR test for several days both in saliva [ 68 ] and nasopharyngeal samples [ 69 ]. In addition, both asymptomatic and symptomatic subjects appear to be contagious [ 1 ] with similarities in their viral load evolution [ 70 – 72 ].…”

Section: Discussionmentioning

confidence: 99%

Screening for SARS-CoV-2 by RT-PCR: Saliva or nasopharyngeal swab? Rapid review and meta-analysis

et al. 2021

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Background Diagnosis of COVID-19 in symptomatic patients and screening of populations for SARS-CoV-2 infection require access to straightforward, low-cost and high-throughput testing. The recommended nasopharyngeal swab tests are limited by the need of trained professionals and specific consumables and this procedure is poorly accepted as a screening method In contrast, saliva sampling can be self-administered. Methods In order to compare saliva and nasopharyngeal/oropharyngeal samples for the detection of SARS-CoV-2, we designed a meta-analysis searching in PubMed up to December 29th, 2020 with the key words “(SARS-CoV-2 OR COVID-19 OR COVID19) AND (salivary OR saliva OR oral fluid)) NOT (review[Publication Type]) NOT (PrePrint[Publication Type])” applying the following criteria: records published in peer reviewed scientific journals, in English, with at least 15 nasopharyngeal/orapharyngeal swabs and saliva paired samples tested by RT-PCR, studies with available raw data including numbers of positive and negative tests with the two sampling methods. For all studies, concordance and sensitivity were calculated and then pooled in a random-effects model. Findings A total of 377 studies were retrieved, of which 50 were eligible, reporting on 16,473 pairs of nasopharyngeal/oropharyngeal and saliva samples. Meta-analysis showed high concordance, 92.5% (95%CI: 89.5–94.7), across studies and pooled sensitivities of 86.5% (95%CI: 83.4–89.1) and 92.0% (95%CI: 89.1–94.2) from saliva and nasopharyngeal/oropharyngeal swabs respectively. Heterogeneity across studies was 72.0% for saliva and 85.0% for nasopharyngeal/oropharyngeal swabs. Interpretation Our meta-analysis strongly suggests that saliva could be used for frequent testing of COVID-19 patients and “en masse” screening of populations.

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“…17 Further, a study by Winnet et al, identifies a case of pre-symptomatic household transmission from a healthy young adult to a sibling and a parent, with high Ct (low viral load) results in the early phase (pre-symptomatic) of the infection that could prevent transmission in the family. 10 Furthermore, an analysis of serial RT-PCR assays and chest CT scans has demonstrated that the mean interval between the initial negative to positive RT-PCR results was 5.1 days ± 1.5, and the mean interval between initial positive to subsequent negative RT-PCR results was 6.9 days ± 2.3. 20 Thus, highly sensitive RT-PCR methods might be able to detect individuals in the early pre-symptomatic phase and prevent transmission in the community.…”

Section: Discussionmentioning

confidence: 99%

“…Further, the reporting of high Ct ranges has been questioned and debated as the importance of these values with regards to viral load is not yet completely understood. The early detection of infected individuals (high Ct results) in the pre-symptomatic phase of the disease using highly sensitive RT-PCR methods has been argued as a strategy to prevent transmission, 10 while on the contrary, the reporting of high Ct has been criticized as false-positive results causing unnecessary testing and having negative implications. 12 To date, the verification of the presence of the SARS-CoV-2 virus in the high Ct samples has not been demonstrated.…”

Section: Introductionmentioning

confidence: 99%

COVID-19 RT-PCR diagnostic assay sensitivity and SARS-CoV-2 transmission: A missing link?

Ecl

Dallaire

et al. 2021

Preprint

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Background The sensitivity of commercially available RT-PCR assays varies over 10,000 fold, ranging from 10 to 20,000 viral copies/ml. The reporting of high Ct value results has been under scrutiny, as the clinical significance of these values is not yet completely understood. The early detection of infected individuals (high Ct results) in the pre-symptomatic phase of the disease using highly sensitive RT-PCR methods has been argued as a strategy to prevent transmission, while on the contrary, the reporting of high Ct has been criticized as false-positive results causing unnecessary testing and having several negative implications. The purpose of this study was to verify the presence of SARS-CoV-2 genomes in samples with a wide range of RT-PCR Ct values including samples with high Ct (37 to 42) using next-generation sequencing (NGS). Methods The study evaluated a total of 547 previously positive samples tested with the PerkinElmer New Coronavirus Nucleic Acid Detection RT-PCR kit. The samples included in this study ranged from Ct values of 17-42, with 44 samples having a Ct > 37. Of the 547 samples, 149 were sequenced using PerkinElmer NEXTFLEX Variant-Seq SARS-CoV2 assay on NovaSeq 6000, and 398 samples were sequenced using Illumina SARS-CoV-2 respiratory viral panel kits using the NextSeq 500/550 system. Results Between the two clinical laboratories, a total of ~1.95 million samples were tested using the FDA-EUA PerkinElmer New Coronavirus RT-PCR assay. Of the 1.95 million samples, ~1.72 million were negative, ~250,000 positive, and ~16,500 in the range of 37-42. Of the 547 samples sequenced, the percentage of sequencing reads that aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) ranged from 25.5% to 99.69%. All samples sequenced showed high sequence specificity to the SARS-CoV-2 virus. Low Ct samples showed complete uniform coverage across the entire 29kb SAR-CoV-2 genome. The average coverage in samples with high Ct (>37) was found to be 55.5% (range 16.1-99.2%). However, as sample Ct increased, a gradual decrease in coverage uniformity was observed for few samples. Conclusion This study demonstrates for the first time that the viral RNA is present in the high Ct value range of 37- 42 and the sequence is unique to SARS-CoV-2 confirmed using two separate sequencing assays. This confirms that the detected Ct values are reflective of the presence of the SARS-CoV-2 virus and they are not an artifact or contamination. In light of the recent work highlighting the majority of transmission being pre-symptomatic/ asymptomatic, and high Ct results being observed at both the early and late phases of infection warrants further investigation into the clinical utility of high Ct results to curtail the spread of the virus.

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SARS-CoV-2 Viral Load in Saliva Rises Gradually and to Moderate Levels in Some Humans

Cited by 24 publications

References 22 publications

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

Screening for SARS-CoV-2 by RT-PCR: Saliva or nasopharyngeal swab? Rapid review and meta-analysis

COVID-19 RT-PCR diagnostic assay sensitivity and SARS-CoV-2 transmission: A missing link?

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