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

  1. Nikhil Shri Sahahjpal
  2. Ephrem Chin Lip Hon
  3. Stephanie Dallaire
  4. Colin Williams
  5. Sudha Ananth
  6. Ashis K Mondal
  7. Amyn M Rojiani
  8. Madhuri Hegde
  9. Ravindra Kolhe

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Abstract

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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  1. ScreenIT

    SciScore for 10.1101/2021.03.24.21254271: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: The samples were processed under an approved HAC by the IRB Committee A (IRB REGISTRATION # 611298), Augusta University, GA. Based on the IRB approval, all PHI was removed and all data was anonymized before accessing for the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    BLASTn analysis queries of the PerkinElmer® New Coronavirus Nucleic Acid Detection Kit primers and probes were performed against public domain nucleotide sequences with default settings.
    BLASTn
    suggested: (BLASTN, RRID:SCR_001598)
    genome-specific ARTIC v3 primer set (3) along with all reagents needed for reverse transcription, amplicon-based enrichment, and NGS library preparation using Unique Dual Index Barcodes (UDI, PerkinElmer).
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    The final library pool was then prepared and loaded on to an Illumina® NovaSeq® 6000, per manufacturer’s instructions, using an Illumina® NovaSeq® 6000 SP Reagent Kit v1.5 (200 cycles) and flow cell and performing a paired-end run (2×75 bp) targeting ∼7M reads per library (Figure 3) Bioinformatics analysis: Data output in the form of FASTQ files were trimmed of adapters via Trimmomatic and aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) via Bowtie2 for bioinformatics analyses.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Alignment rates were plotted in a heatmap using Python scripting to visualize any potential signaling with species other than SARS-CoV-2.
    Python
    suggested: (IPython, RRID:SCR_001658)

    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:
    14,15 However, a significant caveat in extrapolating these studies to all RT-PCR methods is that these studies have not been able to take into account the variability in specimen collection techniques, collection types, and sample types used for collection and transport of the samples. Further, repeat studies on high Ct samples, which have undergone a freeze-thaw cycle, are likely to affect the RT-PCR assay sensitivity due to sample degradation. It is nearly impossible to compare the different kits/ assays due to their unique formulation, LoD claims, and the number of targets in the assay (1-4 targets that include combinations of N, E, ORF1ab, and S genes). Multiple publications have demonstrated the correlation of the severity of COVID-19 disease to low Ct values and high viral loads, 4,6,7 whereas, the clinical utility of high Ct and low viral load is still being debated in the literature. Conflicting reports have emerged in the literature on the utility of high Ct results, with some reports demonstrating high Ct results as false positives, while others arguing the need to report high Ct results to prevent transmission. Recent data have shown that at these high Ct values retesting after 24 hours will very likely yield a negative result.16 Similar studies performed on samples with Ct >30 about 3- 25% of the samples retested as negative depending on the kit used.17-19 However, a recent study by Arnaut et al., highlights that the sensitivity of currently approved assay varies ...

    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.

    About SciScore

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  2. Version published to 10.1101/2021.03.24.21254271v1 on medRxiv