Development and Evaluation of a duo SARS-CoV-2 RT-qPCR Assay Combining Two Assays Approved by the World Health Organization Targeting the Envelope and the RNA-Dependant RNA Polymerase (RdRp) Coding Regions.

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide has highlighted the importance of reliable and rapid diagnostic testing to prevent and control virus circulation. Dozens of monoplex in-house RT-qPCR assays are already available; however, the development of dual-target assays is suited to avoid false-negative results caused by polymorphisms or point mutations, that can compromise the accuracy of diagnostic and screening tests. In this study, two mono-target assays recommended by WHO (E-Sarbeco (enveloppe gene, Charite University, Berlin, Germany) and RdRp-IP4 (RdRp, Institut Pasteur, Paris, France)) were selected and combined in a unique robust test; the resulting duo SARS-CoV-2 RT-qPCR assay was compared to the two parental monoplex tests. The duo SARS-CoV-2 assay performed equally, or better, in terms of sensitivity, specificity, linearity and signal intensity. We demonstrated that combining two single systems into a dual-target assay (with or without an MS2-based internal control) did not impair performances, providing a potent tool adapted for routine molecular diagnosis in clinical microbiology laboratories.

Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples.

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 minute contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.

Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

Prolonged Infectivity of SARS-CoV-2 in Fomites.

We spotted severe acute respiratory syndrome coronavirus 2 on polystyrene plastic, aluminum, and glass for 96 hours with and without bovine serum albumin (3 g/L). We observed a steady infectivity (<1 log10 drop) on plastic, a 3.5 log10 decrease on glass, and a 6 log10 drop on aluminum. The presence of proteins noticeably prolonged infectivity.

Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solutions for Standardization of Real-Time PCR and RT-qPCR Diagnostics

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process involving the preparation of oligonucleotide primers and a hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared to the classic protocol using extemporaneously prepared liquid reagents, assaying (i) sensitivity, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C, mimicking transportation without a cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). The results of our study demonstrate that (i) Lyoph-P&P is stable for at least four days at 37 °C, supporting shipping without the need of a cold chain, (ii) Lyoph-P&P rehydrated solution is stable at 4 °C for at least two weeks, (iii) the sensitivity observed with Lyoph-P&P is at least equal to, and often better than, that observed with liquid formulation, and (iv) the validation of results observed with low-copy specimens is rendered easier by higher fluorescence levels. In conclusion, Lyoph-P&P holds several advantages over extemporaneously prepared liquid formulations and merits consideration as a novel real-time molecular assay for implementation into a laboratory with routine diagnostic activity. Since the meeting, this concept has been applied to the COVID-19 situation: two diagnostic assays (E gene and RdRp) have been developed and can be ordered on the European Virus Archive catalog (https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-2019-ncov-e-gene;https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-sars-cov-2-rdrp-gene).