Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices

Abstract

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.

Figure 1.. Stability of SARS-CoV-2 RNA detection in saliva.

SARS-CoV-2 RNA detection in (A) saliva (n=20) on day of sample collection (fresh) or after storage at −80°C, 30°C for 3 days or room temperature (RT, recorded as ~19°C) for 5 days. The detection of RNA remained stable regardless of starting Ct value (Pearson’s r = −0.085, p = 0.518). At room temperature (B), detection remained stable for up to 25 days. Ct values from the same sample in different conditions are connected by a dotted line. The black dashed line represents Ct 38 which we applied as the cut-off to determine sample positivity. Samples that remained not detected (ND) after 45 cycles are depicted as Ct 42.

Figure 2.. Viral load of saliva samples tested for infectious SARS-CoV-2.

Starting viral load (calculated from RT-qPCR detection of N1) of saliva samples incubated with Vero-E6 cells for 72 hours. Orange diamonds depict samples in which a reduction in Ct value of >2 at 72 hours post-inoculation was observed as compared to 1 hour post-inoculation. Plaque assays with the cellular lysate from 72 hours post-inoculation however, resulted in no plaque forming units (PFU) after 48 hours post-infection.