Validation and Implementation of a Direct RT-qPCR Method for Rapid Screening of SARS-CoV-2 Infection by Using Non-Invasive Saliva Samples

Abstract

Background: There is an urgent need to curb COVID-19 pandemic through early identification of asymptomatic but infectious cases. We aimed to validate and implement an optimized screening method for detection of SARS-CoV-2 RNA combining use of self-collected raw saliva samples, single-step heat-treated virus inactivation and RNA extraction, and direct RT-qPCR.Methods: The study was conducted in Sant Joan de Déu University Hospital (Barcelona, Spain), including: i) method analytical validation against standard RT-qPCR in saliva samples; ii) method diagnostic validation against standard RT-qPCR in nasopharyngeal samples using a prospective cohort of asymptomatic teenagers and young adult players and staff in a youth sports academy; and iii) method implementation through pilot screening of asymptomatic health workers, students, and aid volunteers in the study site.Findings: The direct method had comparable performance to standard RT-qPCR in saliva and high intra- and inter-assay precision. Diagnostic validation included saliva and nasopharyngeal samples serially obtained from 173 participants during 9-12 weeks. At week 0, all saliva and nasopharyngeal samples were negative. In the following weeks, standard RT-qPCR yielded 23 positive results in nasopharyngeal samples. Paired saliva specimens yielded 22 (95·7%) positive and one inconclusive result. A total of 2,709 participants engaged in the pilot screening, with a high rate of participation (83·4% among health workers). Only 17 (0·6%) of saliva samples self-collected by participants were invalid. Saliva was positive in 24 (0·9%) out of 2,692 valid specimens and inconclusive in 27 (1·0%). All 24 saliva-positive and 4 saliva-inconclusive participants were positive by standard RT-PCR in nasopharyngeal samples. Use of a high throughput system allowed fast screening workflow (up to 384 samples in Interpretation: Direct RT-qPCT on self-collected raw saliva is a simple, rapid, and accurate method with potential to be scaled up for enhanced SARS-CoV-2 community-wide screening.Funding:This work was supported by the Kids Corona Project promoted by SJDH, which received donations from Stavros Niarchos Foundation and Banco Santander.Declaration of Interests: CMA reports past grants to her organization from BioMèrieux, Roche Diagnostics, Qiagen, BioFire Diagnostics, Alere, and Genomica, outside the submitted work and personal fees from BioMèrieux, Roche Diagnostics, and Qiagen for presentations in satellite symposiums outside the submitted work. PB reports personal fees from Roche Diagnostics for a presentation in a satellite symposium outside the submitted work. The rest of authors declare no conflicts of interest.Ethics Approval Statement: The study was approved by the Ethics Commitee of SJDH prior to the beginning of activities (ref. PIC-240-20). Use of samples collected from participants in the “Kids Corona Study of SARS-CoV-2 transmission at Football Club Barcelona Academy “La Masia” for the present and future studies was covered in the informed consent process and approval of that study (ref. PIC-200-20).