Analytical validation of quantitative SARS-CoV-2 subgenomic and viral load laboratory developed tests conducted on the Panther Fusion® (Hologic) with preliminary application to clinical samples

Abstract

Objective: Validate the performance characteristics of two analyte specific, laboratory developed tests (LDTs) for the quantification of SARS-CoV-2 subgenomic RNA (sgRNA) and viral load on the Hologic Panther Fusion® using the Open Access functionality.

Methods: Custom-designed primers/probe sets targeting the SARS-CoV-2 Envelope gene (E) and subgenomic E were optimized. A 20-day performance validation following laboratory developed test requirements was conducted to assess assay precision, accuracy, analytical sensitivity/specificity, lower limit of detection and reportable range.

Results: Quantitative SARS-CoV-2 sgRNA (LDT-Quant sgRNA) assay, which measures intermediates of replication, and viral load (LDT-Quant VLCoV) assay demonstrated acceptable performance. Both assays were linear with an R2 and slope equal to 0.99 and 1.00, respectively. Assay precision was evaluated between 4-6 Log10 with a maximum CV of 2.6% and 2.5% for LDT-Quant sgRNA and LDT-Quant VLCoV respectively. Using negative or positive SARS-CoV-2 human nasopharyngeal swab samples, both assays were accurate (kappa coefficient of 1.00 and 0.92). Common respiratory flora and other viral pathogens were not detected and did not interfere with the detection or quantification by either assay. Based on 95% detection, the assay LLODs were 729 and 1206 Copies/mL for the sgRNA and VL load LDTs, respectively.

Conclusion: The LDT-Quant sgRNA and LDT-Quant VLCoV demonstrated good analytical performance. These assays could be further investigated as alternative monitoring assays for viral replication; and thus, medical management in clinical settings which could inform isolation/quarantine requirements.

Fig 1. Summary of calibration runs.

For every calibration run, a standard curve (shown as S1 to S6, S is for standard curve) for the Ct values obtained for 7 serial dilutions of armored RNA quantified panel (1E9 to 1E3 Copies/mL) was plotted in Prism.

Fig 2

Precision of (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays positive extraction run controls. The precision of the High (in black) and Low (in blue) extraction controls is shown in a Levey-Jennings plot for 20 assay runs on log10-transformed values. Solid and dotted lines indicate ±3SD and ±2SD respectively.

Fig 3. Viral load and sgRNA precision.

Three concentrations of analyte spanning the reportable range were used to evaluate the precision of the LDT-Quant VLCoV and LDT-Quant sgRNA assays. Samples at each concentration (1E6, 1E5 and 1E4 Copies/mL) were tested in duplicate in one run per day over 20 days using two operators.

Fig 4

Plot of results from the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays linearity experiment to determine reportable range. Assigned values (converted to Log₁₀) were plotted on the x-axis versus measured values (converted to Log₁₀) on the y-axis using EP Evaluator CLSI EP6 Linearity module based upon Log₁₀ (Copies/mL).

Fig 5

Probit Analysis for the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA assays. The LLOD results were submitted to a Python script that performs a Linear Regression fit of the probit score in terms of the quantities for VL or sgRNA (in Copies/mL). The LLOD is extrapolated from the curve and as indicated by a black arrow.

Fig 6

Quantitative Method Comparison for the (A) LDT-Quant VLCoV and (B) LDT-Quant sgRNA. Results of the Manual Assay (RUO only) were compared to the Panther Fusion^® LDT and plotted in EP Evaluator (Alternate Method comparison module). Deming regressions are shown below the graphs with 95% CI between parentheses.

Fig 7. Viral trajectories of sgRNA and genomic viral loads.

Viral Load and sgRNA were quantified on the Panther Fusion^® and average quantities per day (Log 10 Copies/mL) were plotted (black triangle for Viral Load and blue circle for sgRNA). The curves were generated in GraphPad with exponential growth model and indicate the dynamic tendency of viral load in analyzed samples.