Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

Abstract

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Analysis by real‐time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID‐19 diagnosis in Santiago of Chile

“…This is a strong chaotropic agent with the ability to deactivate viruses and preserve nucleic acids by disabling the action of nucleases and protein structures 21 . The use of this viral stabilizing and inactivating agent has been widely used in the study of several viruses 22,23 including SARS‐CoV‐2 for their detection and diagnosis during the pandemic 12,24,25 . For example, Carvalho et al 26 analyzed NPSs kept in the medium with guanidine salts, obtaining Cq values like those reported in our current study.…”

Evaluation of Alternative Transport Media for RT-qPCR-Based SARS-CoV-2 Testing

“…Thus, during the COVID-19 pandemic, the FDA allowed laboratories to consider the use of alternative transport media [ 12 ]. Among these, the guanidine thiocyanate-based eNAT™ medium reportedly inactivates SARS-CoV-2 and stabilizes its nucleic acid [ 19 , 20 , 30 ]. In this study, we demonstrated the efficacy of GeneTM (another guanidine thiocyanate-based medium) by comparing it with eNAT™, using PBS as a control.…”

“…There are studies on guanidine thiocyanate-based media that are suitable for virus inactivation and RT-qPCR detection assays [ 19 ]. eNAT™ (Copan, Brescia, Italy) is a guanidine thiocyanate-based medium that can help nucleic acids remain stable for a long period and is suitable for sample collection and transport [ 20 ]. Similarly, GeneTM is a guanidine thiocyanate-based medium that is suitable for collecting nasal (nasopharyngeal) and oral (oropharyngeal or salivary) samples for respiratory infection testing; it can also be used for the transportation and preservation of samples for COVID-19 testing.…”

Evaluation and Clinical Validation of Guanidine-Based Inactivation Transport Medium for Preservation of SARS-CoV-2

“…Guanidinium thiocyanate or guanidinium hydrochloride has shown to inactivate SARS-CoV-2 and can be used for RT-PCR applications [ 52 – 54 ] since inactivation reagents were effective at reducing viral titers [ 55 ]. It was suggested to be used as part of SARS-CoV-2 NAAT at high-risk locations (schools, workplaces, prisons, skilled nursing facilities, homeless shelters, etc.)…”

“…It was suggested to be used as part of SARS-CoV-2 NAAT at high-risk locations (schools, workplaces, prisons, skilled nursing facilities, homeless shelters, etc.) [ 52 ]. In this research, VITPAD®, a guanidine-based inactivation transport media (ITM) formulated to maintain the RNA quality of SARS-CoV-2 during transportation without cold chains, was evaluated.…”