RT-PCR detection of SARS-CoV-2 in nasopharyngeal and salivary specimens: contribution of alternative collection systems and extraction processes to cope with mass screening. Interpretation of low viral loads

Abstract

Objectives: Due to massive screening of the persistent coronavirus SARS-CoV-2, supply difficulties emerged for swabs and extraction reagents leading to test alternative choices. Quality sampling may have an impact on the result and a low RNA detection may be difficult to interpret because it does not necessarily mean that infectious particles are present in biological samples. There is a need to understand whether the Ct value information is relevant and informative. Methods: We compared the pre-analytical stability of RNA in saline solution, UTM®, Amies and Cary-Blair transport media. Expression profile of E, N and RdRp genes was assessed at various concentration levels with the Allplex™ 2019-nCoV Assay. Factors that may influence the determination of Ct were studied with several extraction reagents coupled to the GSD NovaPrime® SARS-CoV-2 RT-PCR testing kit. Results: Seventy two-hour RNA stability has been demonstrated for all the transport media assessed. A matrix effect was shown, leading to a decrease in the detection of E and RdRp genes, so that only N gene was often found for Ct greater than 35.0. A follow-up over more than 67,000 patients suggests that N gene may be a sensitive indicator to detect a new active viral circulation, but establishing a correlation between a positive threshold and a low risk of infection for a given method remains difficult. Conclusions: Several transport media and extraction processes are suitable for PCR-based SARS-CoV-2 detection. During periods of active virus circulation, any weakly positive results should be considered.