SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Abstract

Background: Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA).

Methods: We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues.

Findings: From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (<0.05%) results.

Conclusions: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/).

Funding: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.

Keywords: COVID-19; SARS-CoV-2; molecular testing; population screening; saliva.

Graphical abstract

Figure 1

SalivaDirect is a simplified method for SARS-CoV-2 detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com. (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4°C, room temperature (RT; ∼19°C), and 30°C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARS-CoV-2 copies/μL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30°C as compared to fresh specimens (Kruskal-Wallis; p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon; p < 0.01). (D) Testing extracted nucleic acid from saliva with the N1 primer-probe set (singleplex) as compared to a multiplex assay showed stronger N1 detection in multiplex (Wilcoxon; p < 0.01). The dotted line in (B)–(D) indicates the limit of detection. Data used to make this figure can be found in Data S1.

Figure 2

SalivaDirect is validated for use with reagents and instruments from multiple vendors We determined the lower limit of detection of SalivaDirect with a 2-fold dilution series (400, 200, 100, 50, 25, 12, and 6 copies/μL) of positive saliva spiked-in negative saliva. Initially, each concentration and negative saliva were tested in triplicate to determine the preliminary limit of detection (dark-colored dots). The limit of detection was confirmed with 20 additional replicates (light-colored dots) for which 19 out of 20 needed to be detected. Limit of detection when tested with (A–C) proteinase K, (D–F) RT-qPCR kits, and (G–I) qRT-PCR instruments from different vendors, while keeping the other conditions constant. (A) and (D), as well as (F) and (G), are duplicates to enable comparisons between the different combinations of reagents or instruments within a single row. Shown are the Ct values for the N1 primer-probe set. The horizontal bars indicate the median and the dotted line indicates the limit of detection. Data used to make this figure can be found in Data S1.

Figure 3

Sensitivity of SalivaDirect is comparable to a standard approach for SARS-CoV-2 detection in saliva We compared Ct values for N1 between the modified CDC assay (nucleic acid extraction and singleplex qRT-PCR) and SalivaDirect for 41 saliva specimens tested with both methods. Overall, detection of SARS-CoV-2 with SalivaDirect is weaker (median 1.2 Ct, Wilcoxon; p < 0.001) than the modified CDC assay, but with a high agreement in outcomes of both tests of (93%). Shown are the Ct values for the N1 primer-probe set and the dotted line indicates the limit of detection. Data used to make this figure can be found in Data S1.

Figure 4

SalivaDirect is highly comparable to standard qRT-PCR tests with nucleic acid extraction from nasopharyngeal swabs and saliva We selected 37 paired positive and 30 paired negative nasopharyngeal swabs and saliva specimens. Paired samples were collected a maximum 4 days apart. Nasopharyngeal swabs and saliva specimens were tested with the ThermoFisher Scientific TaqPath COVID-19 combo kit, and average Ct values for N, S, and ORF1ab were compared to N1 Ct values for saliva specimens tested with SalivaDirect. (A) Comparison of 37 paired nasopharyngeal swabs and saliva tested with the TaqPath COVID-19 combo kit showed 84% positive agreement and no significant differences in each of the three virus targets (Wilcoxon; N: p = 0.51, S: p = 0.72, ORF1ab: p = 0.39). (B) Comparison of nasopharyngeal swabs tested with the TaqPatch COVID-19 combo kit and saliva tested with SalivaDirect showed 94% positive agreement. Median N1 Ct values were 3.3 Ct higher for SalivaDirect (Wilcoxon; p < 0.01). (C) Comparison of saliva tested with TaqPath COVID-19 combo kit and SalivaDirect again shows that SalivaDirect showed 97% positive agreement. Median N1 Ct values were 5.0 Ct higher for SalivaDirect (Wilcoxon; p < 0.001). (D) 30 paired nasopharyngeal swabs and saliva specimens tested negative with both the TaqPath COVID-19 combo kit and SalivaDirect. Shown are average Ct values for N, S, and ORF1ab for the TaqPath combo kit and N1 Ct values for SalivaDirect. The dashed line indicates the limit of detection for the TaqPath combo kit (37 Ct) and the dotted line indicates the limit of detection for SalivaDirect (40 Ct). Data used to make this figure can be found in Data S1.