Evaluation of Analytical and Clinical Performance and Usefulness in a Real-Life Hospital Setting of Two in-House Real-Time RT-PCR Assays to Track SARS-CoV-2 Variants of Concern

Abstract

Since the end of 2020, multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have emerged and spread worldwide. Tracking their evolution has been a challenge due to the huge number of positive samples and limited capacities of whole-genome sequencing. Two in-house variant-screening RT-PCR assays were successively designed in our laboratory in order to detect specific known mutations in the spike region and to rapidly detect successively emerging VOCs. The first one (RT-PCR#1) targeted the 69-70 deletion and the N501Y substitution simultaneously, whereas the second one (RT-PCR#2) targeted the E484K, E484Q, and L452R substitutions simultaneously. To evaluate the analytical performance of these two RT-PCRs, 90 negative and 30 positive thawed nasopharyngeal swabs were retrospectively analyzed, and no discordant results were observed. Concerning the sensitivity, for RT-PCR#1, serial dilutions of the WHO international standard SARS-CoV-2 RNA, corresponding to the genome of an Alpha variant, were all detected up to 500 IU/mL. For RT-PCR#2, dilutions of a sample harboring the E484K substitution and of a sample harboring the L452R and E484Q substitutions were all detected up to 1000 IU/mL and 2000 IU/mL, respectively. To evaluate the performance in a real-life hospital setting, 1308 and 915 profiles of mutations, obtained with RT-PCR#1 and RT-PCR#2, respectively, were prospectively compared to next-generation sequencing (NGS) data. The two RT-PCR assays showed an excellent concordance with the NGS data, with 99.8% for RT-PCR#1 and 99.2% for RT-PCR#2. Finally, for each mutation targeted, the clinical sensitivity, the clinical specificity and the positive and negative predictive values showed excellent clinical performance. Since the beginning of the SARS-CoV-2 pandemic, the emergence of variants-impacting the disease's severity and the efficacy of vaccines and therapies-has forced medical analysis laboratories to constantly adapt to the strong demand for screening them. Our data showed that in-house RT-PCRs are useful and adaptable tools for monitoring such rapid evolution and spread of SARS-CoV-2 VOCs.

Keywords: SARS-CoV-2 diversity; in-house RT-PCR; mutations of interest; next-generation sequencing; variants of concern.

Figure 1

(A) Schematic of SARS-CoV-2 genome structure. The genes are located relative to the numbering of the Wuhan-Hu 1 SARS-CoV-2 reference strain (Genbank accession number MN908947.3). (B) Schematic of SARS-CoV-2 spike protein structure. Different domains are shown: NTD = N-terminal domain, RBD = receptor-binding domain; FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain and CT = cytoplasmic tail (derived from [7,8]). The substitutions targeted by the two RT-PCR are located relative to the spike aminoacid numbering. The location of the amplicon is represented by grey boxes, with the hybridization location of probes and primers for each RT-PCR.

Figure 2

Algorithm used to characterize and rapidly detect SARS-CoV-2 VOCs which have successively emerged (derived from the French guidelines [30,31]).

Figure 3

Frequencies of detection of the SARS-CoV-2 variants in France (colored by clade and normalized to 100% at each time point) [22] and evolution of the strategy of variant screening in our laboratory.

Figure 4

(A) Linear regression curves for the evaluation of linearity for the three fluorophores FAM, Cy5, and JOE of the RT-PCR#1. (B) Linear regression curve for the evaluation of linearity for the fluorophore JOE of the RT-PCR#2. Ct = cycle threshold, IC = internal control.