SalivaSTAT: Direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance.

Sahajpal, N. S. S.; Mondal, A. K.; Ananth, S.; Njau, A.; Ahluwalia, P.; Newnam, G.; Lozoya-Colinas, A.; Hud, N. V.; Kota, V.; Ross, T. M.; Reid, M. D.; Fulzele, S.; Chaubey, A.; Hegde, M.; Rojiani, A. M.; Kolhe, R.

Background: The limitations of widespread current COVID-19 diagnostic testing lie at both pre-analytical and analytical stages. Collection of nasopharyngeal swabs is invasive and is associated with exposure risk, high cost, and supply-chain constraints. Additionally, the RNA extraction in the analytical stage is the most significant rate-limiting step in the entire testing process. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction free RT-PCR test using any of the commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment and homogenization. The effect of homogenization on saliva samples for extraction-free RT-PCR assay was determined by evaluating samples with and without homogenization and preforming viscosity measurements. Saliva samples (872) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. Further, a five-sample pooling strategy was evaluated as per FDA guidelines using the SalivaSTAT protocol. Results: The saliva collection (done without any media) performed comparable to the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95C for 30-minutes and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreement (NPA) of 95.8% and 100%, respectively. The LoD was established as ~20-60 copies/ml by absolute quantification. Further, a five-sample pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free direct RT-PCR assay for saliva samples that demonstrated comparable performance to FDA-EUA assay (Extraction and RT-PCR). The SalivaSTAT protocol is a rapid, sensitive, and cost-effective method that can be adopted globally, and has the potential to meet testing needs and may play a significant role in management of the current pandemic.