Development of the RealTime SARS-CoV-2 quantitative Laboratory Developed Test and correlation with viral culture as a measure of infectivity

Abstract

While diagnosis of COVID-19 relies on qualitative molecular testing for the absence or presence of SARS-CoV-2 RNA, quantitative viral load determination for SARS-CoV-2 has many potential applications in antiviral therapy and vaccine trials as well as implications for public health and quarantine guidance. To date, no quantitative SARS-CoV-2 viral load tests have been authorized for clinical use by the FDA. In this study, we modified the FDA emergency use authorized qualitative RealTi m e SARS-CoV-2 assay into a quantitative SARS-CoV-2 Laboratory Developed Test (LDT) using newly developed Abbott SARS-CoV-2 calibration standards. Both analytical and clinical performance of this SARS-CoV-2 quantitative LDT was evaluated using nasopharyngeal swabs (NPS). We further assessed the correlation between Ct and the ability to culture virus on Vero CCL81 cells. The SARS-CoV-2 quantitative LDT demonstrated high linearity with R 2 value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ±0.08-0.14 log 10 copies/mL for inter-assay reproducibility and ±0.09 to 0.19 log 10 copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log 10 copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log 10 copies/mL. Quantitative tests, validated against viral culture capacity, may allow more accurate identification of individuals with and without infectious viral shedding from the respiratory tract.

Emergence of SARS-CoV-2 Resistance with Monoclonal Antibody Therapy

“…CoV-2 quantitative LDT 31 . In this assay, 2 calibrator levels (3 log10 RNA copies/mL and 6 log10 RNA copies/mL) tested in triplicate were used to generate a calibration curve and 3 control levels 326 (negative, low positive at 3 log10 RNA copies/mL and high positive at 5 log10 RNA copies/mL) were included in each run for quality management.…”

“…SARS-CoV-2 quantitative Laboratory Developed Test (LDT) was developed utilizing open mode functionality on m2000sp/rt (Abbott, Chicago, IL) by using EUA Abbott SARS-CoV-2 qualitative reagents 30 . Identical extraction and amplification protocols developed for RealTime SARS-CoV-2 qualitative EUA assay were also used for the development of the RealTime SARS-CoV-2 quantitative LDT 31 . In this assay, 2 calibrator levels (3 log10 RNA copies/mL and 6 log10 325 RNA copies/mL) tested in triplicate were used to generate a calibration curve and 3 control levels (negative, low positive at 3 log10 RNA copies/mL and high positive at 5 log10 RNA copies/mL) 327 were included in each run for quality management.…”

Rapid Antigen Tests for Voluntary Screen Testing

“…Figure 5 shows the estimated range of Ct values at a viral load of 100,000 Copies/mL from 42 identified standard curves. [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27), with 46% of Ct values below 25, and 83% below 30. [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27).…”

“…Figure 5. Ct Values at a Viral Load of 100,000 Copies/mL Estimated From 42 Standard Curves Box and Whisker plot of Ct values at a viral load of 100,000 Copies/mL from 42 identified PCR standard curves reported in 23 original articles [12][13][14]20,22,23,[27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]. The median Ct value at this viral load was 25.82 (interquartile range 23.03 to 29.27).…”

SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

“…There are 10 unread ("dark") cycles on the Abbott Molecular platform, in which case C t values are 10 less than most other manufacturer assays; the C t values reported in this study are adjusted by 10 to include the dark cycles. Viral loads were approximated based on equivalent C t values obtained with a RealTime SARS-CoV-2 quantitative assay on the m2000sp/rt that uses calibrators and controls (Abbott Molecular, Des Plaines, IL) (22)(23)(24).…”