• Clinical Validation of a Sensitive Test for Saliva Collected in Healthcare and Community Settings with Pooling Utility for Severe Acute Respiratory Syndrome Coronavirus 2 Mass Surveillance

      Sahajpal, Nikhil S.; Mondal, Ashis K.; Ananth, Sudha; Njau, Allan; Ahluwalia, Pankaj; Kota, Vamsi; Caspary, Kevin; Ross, Ted M.; Farrell, Michael; Shannon, Michael P.; et al. (Elsevier, 2021-07)
      The clinical performance of saliva compared with nasopharyngeal swabs (NPSs) has shown conflicting results in healthcare and community settings. In the present study, a total of 429 matched NPS and saliva sample pairs, collected in either healthcare or community setting, were evaluated. Phase-1 (protocol U) tested 240 matched NPS and saliva sample pairs; phase 2 (SalivaAll protocol) tested 189 matched NPS and saliva sample pairs, with an additional sample homogenization step before RNA extraction. A total of 85 saliva samples were evaluated with both protocols. In phase-1, 28.3% (68/ 240) samples tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from saliva, NPS, or both. The detection rate from saliva was lower compared with that from NPS samples (50.0% versus 89.7%). In phase-2, 50.2% (95/189) samples tested positive for SARS-CoV-2 from saliva, NPS, or both. The detection rate from saliva was higher compared with that from NPS samples (97.8% versus 78.9%). Of the 85 saliva samples evaluated with both protocols, the detection rate was 100% for samples tested with SalivaAll, and 36.7% with protocol U. The limit of detection with SalivaAll protocol was 20 to 60 copies/mL. The pooled testing approach demonstrated a 95% positive and 100% negative percentage agreement. This protocol for saliva samples results in higher sensitivity compared with NPS samples and breaks the barrier to using pooled saliva for SARS-CoV-2 testing.
    • SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

      Sahajpal, Nikhil S.; Mondal, Ashis K.; Ananth, Sudha; Njau, Allan; Ahluwalia, Pankaj; Newnam, Gary; Lozoya-Colinas, Adriana; Hud, Nicholas V.; Kota, Vamsi; Ross, Ted M.; et al. (MDPI, 2021-05-19)
      Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 ◦C for 30-min 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 agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-samplepooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).