SARS-CoV-2 RNA quantification using droplet digital RT-PCR

Quantitative viral load assays have transformed our understanding of - and ability to manage - viral diseases. They hold similar potential to advance COVID-19 control and prevention, but SARS-CoV-2 viral load tests are not yet widely available. SARS-CoV-2 molecular diagnostic tests, which typically employ real-time reverse transcriptase-polymerase chain reaction (RT-PCR), yield semi-quantitative results only. Reverse transcriptase droplet digital PCR (RT-ddPCR), a technology that partitions each reaction into 20,000 nanolitre-sized droplets prior to amplification, offers an attractive platform for SARS-CoV-2 RNA quantification. We evaluated eight primer/probe sets originally developed for real-time RT-PCR-based SARS-CoV-2 diagnostic tests for use in RT-ddPCR, and identified three (Charite-Berlin E-Sarbeco and Pasteur Institute IP2 and IP4) as the most efficient, precise and sensitive for RT-ddPCR-based SARS-CoV-2 RNA quantification. Analytical efficiency of the E-Sarbeco primer/probe set, for example, was ~83%, while assay precision, as measured by the coefficient of variation, was ~2% at 1000 input copies/reaction. Lower limits of quantification and detection for this primer/probe set were 18.6 and 4.4 input SARS-CoV-2 RNA copies/reaction, respectively. SARS-CoV-2 RNA viral loads in a convenience panel of 48 COVID-19-positive diagnostic specimens spanned a 6.2log10 range, confirming substantial viral load variation in vivo. We further calibrated RT-ddPCR-derived SARS-CoV-2 E gene copy numbers against cycle threshold (Ct) values from a commercial real-time RT-PCR diagnostic platform. The resulting log-linear relationship can be used to mathematically derive SARS-CoV-2 RNA copy numbers from Ct values, allowing the wealth of available diagnostic test data to be harnessed to address foundational questions in SARS-CoV-2 biology.

Evaluation of nasopharyngeal swab collection techniques for nucleic acid recovery and participant experience: recommendations for COVID-19 diagnostics

Nasopharyngeal swabs are critical to the diagnosis of respiratory infections including COVID-19, but collection techniques vary. We compared two recommended nasopharyngeal swab collection techniques in adult volunteers and found that swab rotation following nasopharyngeal contact did not recover additional nucleic acid (as measured by human DNA/RNA copy number). Rotation was also less tolerable for participants. Notably, both discomfort and nucleic acid recovery were significantly higher in Asians, consistent with nasal anatomy differences. Our results suggest that it is unnecessary to rotate the swab in place following contact with the nasopharynx, and reveal that procedural discomfort levels can differ by ethnicity. summaryNasopharyngeal swabs are critical to COVID-19 diagnostics, but collection techniques vary. Comparison of two collection techniques revealed that swab rotation did not recover more nucleic acid and was more uncomfortable. Discomfort and biological material recovery also varied by participant ethnicity.

Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Improper nasopharyngeal swab collection could contribute to false-negative COVID-19 results. In support of this, specimens from confirmed or suspected COVID-19 cases that tested negative or indeterminate (i.e. suspected false-negatives) contained less human DNA (a stable molecular marker of sampling quality) compared to a representative pool of specimens submitted for testing.