Evaluation of PCR cycle threshold values by patient population with the quidel lyra SARS-CoV-2 assay

Abstract

The Lyra SARS-CoV-2 assay was the primary method for molecular testing performed at Barnes-Jewish Healthcare System in St. Louis, Missouri during the initial COVID-19 surge from mid-March to late-April 2020. We performed a retrospective analysis of 1,043 positive Lyra SARS-CoV-2 results during these 36 days to investigate associations between cycle threshold (C_T) value and patient characteristics. Total RNA were extracted from NP or OP swabs using either the EasyMag or KingFisher automated extraction systems and quantified with RotorGene Q (Qiagen) or Applied Biosystems 7500 Fast Dx thermocyclers respectively. Notably, we found lower a significant median lower C_T for samples tested on the KingFisher-ABI 7500 fastDX (KF/ABI) system compared to the EasyMag/RotorGene (EM/RGQ) platform. Since 77.5% of our tests were ran on the EM/RGQ pipeline we then perform additional analysis on these values and found that C _T values in outpatient care settings compared to samples obtained in the emergency department or inpatient had significantly lower C _T values. These collective findings suggests a difference in viral load amongst various patient populations.

Keywords: Clinical virology; Molecular assays; SARS-CoV-2.

Fig. 1

Comparison of C_T values for both testing platforms and distribution of EM/RGQ Lyra SARS-CoV-2 test C_T values from March-April, 2020. (A) Scatter dots depict each positive C_T value as a point. Red line depicts the median C_T value. We find differences in C_T values obtained between platform (unpaired parametric t-test p value .0012). (B) Histogram depicting number of samples binned to their lowest whole number for EM/RGQ samples. Distribution of C_T values indicates our data is not normally distributed as the Shapiro-Wilk test (p value <.0001) did not pass normality.

Fig. 2

C_T values by age, setting, and gender. Dotplot for EM/RGQ C_T values depicting depict each positive C_T value as a point for test order setting (A),age (B), and gender (C). Red line depicts the median C_T value. Overall difference (ANOVA p <0.0001) in C_T for age (A) and test order setting (B) but not gender (C). Individual pairwise comparisons significant by unpaired parametric t-test denoted in figure.

Fig. 3

Association of repeat C_T values in unique patients and the time between results. Repeat XY plot showing the EM/RGQ ΔC_T (C_T1-C_T2) as a function of days elapsed between the two tests for patients who consecutively tested positive for SARS-CoV-2. A negative ΔC_T value indicates a decrease in viral load between tests.