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

• Published in: Viruses 2023-05, Vol.15 (5), p.1115
• Main Authors: Moisan, Alice, Soares, Anaïs, De Oliveira, Fabienne, Alessandri-Gradt, Elodie, Lecoquierre, François, Fourneaux, Steeve, Plantier, Jean-Christophe, Gueudin, Marie
• Format: Article
• Language: English
• Subjects: Automation; Coronaviruses; COVID-19; COVID-19 - diagnosis; COVID-19 Testing; Evaluation; Gene deletion; Genomes; Glycoproteins; Hospitals; Humans; in-house RT-PCR; Laboratories; Medical research; Medicine, Experimental; Methods; Mutation; mutations of interest; Next-generation sequencing; Polymerase chain reaction; Public health; Retrospective Studies; Reverse transcriptase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral - genetics; SARS-CoV-2 - genetics; SARS-CoV-2 diversity; Severe acute respiratory syndrome coronavirus 2; Software; Thermal cycling; variants of concern; Whole genome sequencing
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v15051115

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.