Pooling of Upper Respiratory Specimens Using a SARS-CoV-2 Real-time RT-PCR Assay Authorized for Emergency Use in Low-Prevalence Populations for High-Throughput Testing

Abstract

Nucleic acid amplification testing is a critical tool for addressing the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Specimen pooling can increase throughput and conserve testing resources but requires validation to ensure that reduced sensitivity does not increase the false-negative rate. We evaluated the performance of a real time reverse transcription PCR (RT-PCR) test authorized by the U.S. Food and Drug Administration (FDA) for emergency use for pooled testing of upper respiratory specimens. Positive specimens were selected from three prevalence groups, 1-3%, >3-6%, and >6-10%. Positive percent agreement (PPA) was assessed by pooling single-positive specimens with three negative specimens; performance was assessed using Passing-Bablok regression. Additionally, we assessed the distributions of RT-PCR cycle threshold (Ct) values for 3,091 positive specimens. PPA was 100% for the 101 pooled specimens. There was a linear relationship between Ct values for pooled and single-tested specimens (r: 0.96-0.99, slope ≈ 1). The mean pooled Ct shifts at 40 cycles were 2.38 and 1.90 respectively for the N1 and N3 targets. The median Cts for 3,091 positive specimens were 25.9 (N1) and 24.7 (N3). The percent of positive specimens with Cts between 40 and the shifted Ct was 1.42% (N1) and 0.0% (N3). Pooled and individual testing of specimens positive for SARS-CoV-2 demonstrated 100% agreement and demonstrate the viability of pooled specimens for SARS-COV-2 testing using a dual-target RT-PCR system. Pooled specimen testing can help increase testing capacity for SARS-CoV-2, with a low risk of false-negative results.