Saliva as a testing specimen with or without pooling for SARS-CoV-2 detection by multiplex RT-PCR test

Testing for SARS-CoV-2 remains a critical tool in managing the ongoing pandemic. Most diagnostic methods rely on nasopharyngeal or oropharyngeal swabs, which can be uncomfortable and pose risks to healthcare workers. Alternative specimen types are being explored to improve patient comfort and reduce exposure. Saliva has emerged as a potential alternative due to its ease of collection and lower risk of aerosol generation. However, the performance of saliva-based testing compared to traditional methods has not been fully established. The need for high-throughput, accurate, and safe testing platforms remains unmet. Prior research has shown that saliva can be used for molecular detection, but its sensitivity and feasibility in pooled testing require further validation. This gap motivated the evaluation of a saliva-based RT-PCR test for SARS-CoV-2. The study aimed to determine whether saliva can serve as a reliable and efficient specimen type for large-scale testing.

The study aimed to assess the performance of a saliva-based RT-PCR test for SARS-CoV-2 detection. A key objective was to determine the test’s limit of detection and compare its accuracy to traditional nasal swab methods. The researchers also wanted to evaluate the feasibility of pooling saliva samples to increase testing throughput. They tested the QuantiVirus™ SARS-CoV-2 assay using saliva as the specimen type. The study included both reference panel testing and clinical sample analysis. The goal was to confirm the test’s sensitivity and specificity in real-world conditions. The researchers compared results with an established PCR method, the Abbott m2000 assay. They also examined the impact of pooling on test performance. This work aimed to provide evidence for the use of saliva in large-scale testing strategies.

The researchers validated the QuantiVirus™ SARS-CoV-2 test using a combination of reference panels and clinical samples. They used the SeraCare Accuplex SARS-CoV-2 reference panel to assess the limit of detection. Clinical performance was evaluated using 85 positive and 90 negative nasopharyngeal swab samples. Paired saliva and nasal swab samples from 20 patients were also tested. The test was compared to the Abbott m2000 PCR assay. Community-collected saliva samples from 389 individuals were analyzed for screening performance. The researchers evaluated the feasibility of pooling saliva samples. They tested up to six samples per pool to assess sensitivity and specificity. Statistical methods, including the Wilcoxon matched pairs signed rank test, were used to compare results. The study followed FDA-EUA and molecular assay validation guidelines to ensure accuracy and reliability.

The QuantiVirus™ SARS-CoV-2 test demonstrated a limit of detection of 100–200 copies/mL in saliva. The positive percentage agreement was 100% at 1xLOD, 1.5xLOD, and 2.5xLOD in contrived saliva samples. Clinical testing showed a positive percentage agreement of 100% (95% CI: 94.6% to 100%) and a negative percentage agreement of 98.9% (95% CI: 93.1% to 99.9%). No cross-reactivity was observed with other respiratory pathogens. Paired saliva and nasal swab samples showed 80% concordance, with no significant difference (p = 0.13). The positive test rate in community-collected samples was 1.79%. Pooled saliva testing with up to six samples showed 94.8% sensitivity and 100% specificity. These results indicate that saliva-based testing is as accurate as nasal swab testing. The study supports the use of saliva for high-throughput SARS-CoV-2 detection.

The QuantiVirus™ SARS-CoV-2 test using saliva as a specimen type demonstrated strong performance in both reference and clinical settings. The test achieved a limit of detection of 200 copies/mL in contrived saliva samples. Clinical results showed high agreement with nasal swab testing, with no significant difference in concordance. The absence of cross-reactivity with other pathogens supports the test’s specificity. Preliminary data suggest that saliva pooling up to six samples is feasible without significant loss of sensitivity. The study confirms that saliva-based testing is a viable alternative to traditional swab methods. The test offers a safe and comfortable option for patients and reduces exposure risks for healthcare workers. The results support the use of saliva in large-scale testing strategies. The QuantiVirus™ SARS-CoV-2 test provides a reliable platform for high-throughput SARS-CoV-2 detection.