SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

• Type of Material: Article
• Authors: Nikhil S Sahajpal, Augusta University; Ashis K Mondal, Augusta University; Sudha Ananth, Augusta University; Allan Njau, Aga Khan Univ Hosp; Pankaj Ahluwalia, Augusta University; Gary Newnam, Georgia Institute of Technology; Adriana Lozoya-Colinas, Georgia Institute of Technology; Nicholas V Hud, Georgia Institute of Technology; Vamsi Kota, Emory University; Ted M Ross, University of Georgia; Michelle Reid, Emory University; Sadanand Fulzele, Augusta University; Alka Chaubey, Augusta University; Madhuri Hegde, Emory University; Amyn M Rojiani, Augusta University; Ravindra Kolhe, Augusta University
• Language: English
• Date: 2021-05-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2021 by the authors.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/diagnostics11050904
• Title of Journal or Parent Work: DIAGNOSTICS
• Volume: 11
• Issue: 5
• Grant/Funding Information: This project has been funded by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under contract 75N93019C00052.
• Keywords: RT-PCR; Science & Technology; pooling; SPECIMENS; Medicine, General & Internal; extraction-free; General & Internal Medicine; Life Sciences & Biomedicine; saliva
• Research Categories: Health Sciences, Immunology; Health Sciences, Pathology

Abstract

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-sample-pooling 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).