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

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.

Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for a pandemic-scale infection surveillance

“…These data suggest that our surveillance system is likely to be sufficiently sensitive to detect all infectious SARS-CoV-2 carriers, given that an individual’s viral load beneath our limit of detection is generally thought not to be contagious, given multiple reported failures to cultivate any SARS-CoV-2 virus using in vitro cell culture from samples with Ct values of over minimum reported thresholds ranging from 24 to 33 26-28 . Indeed, sequencing of SARS-CoV-2 transcripts from infected cells recently established that full genome sequence coverage was not observed in samples with a Ct greater than 32 (measured with the same Perkin Elmer RT-PCR assay which we used here as our comparator 29 ), suggesting that any RT-PCR detection of small SARS-CoV-2 genomic fragments in samples beyond this threshold would not likely reflect intact viable virus with potential to be infectious.…”

COVID-19 Diagnostic Assay Sensitivity: Lessons for the Upcoming Wave Or Next Pandemic

“…We had previously sequenced 827 samples ranging from 10 to 42 Ct values, of which 95 samples were with Ct value >37 with PE RT-PCR assay [ 10 ]. Of the 827-sample sequenced, the percentage of sequencing reads that aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) ranged from 5.1 to 100%.…”

“…All samples sequenced showed high sequence specificity to the SARS-CoV-2 virus. Low-Ct samples showed complete uniform coverage across the entire 29 kb SAR-CoV-2 genome, whereas the coverage in samples with high-Ct (>37) ranged from 5.1% to 99.9%, and a gradual decrease in coverage uniformity was observed with increasing Ct values [ 10 ]. The results of our study confirmed that samples with >37 Ct value reported with PE RT-PCR assay were true positive results and not PCR artifacts or contamination.…”