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 RealTime 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 R² value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ± 0.08-0.14 log₁₀ copies/mL for inter-assay reproducibility and ± 0.09 to 0.19 log₁₀ copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log₁₀ copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log₁₀ 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.

Fig. 1

RealTime SARS-CoV-2 quantitative LDT linearity assessed with (A) BEI SARS-CoV-2 viral stock (B) Abbott SARS-CoV-2 viral stock and (C) SeraCare Accuplex SARS-CoV-2 verification panel.

Fig. 2

(A) Ct correlation between the EUA CDC 2019-nCoV assay and the RealTime SARS-CoV-2 quantitative LDT; (B) Ct correlation between the cobas SARS-CoV-2 EUA assay and the RealTime SARS-CoV-2 quantitative LDT; (C) Ct correlation between the Alinity m SARS-CoV-2 EUA assay and the RealTime SARS-CoV-2 quantitative LDT (D) Inter-laboratory comparison of RealTime SARS-CoV-2 quantitative LDT.

Fig. 3

A. COVID-19 positive nasopharyngeal swabs in VTM (n = 36) with Cts ranging from 7.5 to 32 determined by the non-quantitative assay were cultured on Vero cells. After adjusting for sample dilution and PCR input, the highest Ct where cytopathic effects (CPE) were detected was 18.27. B. SARS-CoV-2 calibrator stocks were cultured in 96-well format and cytopathic effects were quantified with the Viral Tox Glo system, comparing overnight co-plating (Method 1) or 2 h infection (Method 2). The highest Ct producing CPE was 18.49, corresponding to 4.2 log₁₀ copies/ml. C. Clinical samples (n = 459) were screened with the RealTime SARS-CoV-2 quantitative LDT and identified 51 positives that were tested for their ability to infect in culture. A total of 9/51 (11.1%) demonstrated CPE, the majority having a Ct < 12 (> 1000,000 copies/mL). The lowest titer demonstrating CPE in culture had a Ct = 18.21 (28.21 w/o dark cycles), corresponding to 4.31 log₁₀ copies/mL.