Two Stage Hierarchical Group Testing Strategy to Increase SARS-CoV-2 Testing Capacity at an Institution of Higher Education: A Retrospective Analysis

Abstract

Population testing for severe acute respiratory syndrome 2 (SAR-CoV-2) is necessary owing to the possibility of viral transmission from asymptomatic cases, yet scarcity of reagents and equipment has added to the cost-prohibitive implementation of screening campaigns at institutions of higher education. The high analytical sensitivities of leading nucleic acid amplification diagnostic methods allow for group testing to increase testing capacity. A feasibility study was performed using an optimized testing configuration model for pooling three, five, and ten samples. Following the standard RNA extraction and purification workflow for quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) method using Thermo Fisher TaqPath COVID-19 multiplex primers and probes for the ORF1ab, N, and S genes, matrix and dilution effects were assessed using pooled negative samples as the diluent. Probit analysis produced a limit of detection of 16075 (ORF1ab), 1308 (N), and 1180182 (S) genomic copy equivalents per milliliter. Trials comparing neat to 1:5 dilution for 34 weak-to-strongly positive samples demonstrated average threshold cycle (CT) shifts of 2.31{+/-}1.16 (ORF1ab), 2.23{+/-}1.12 (N), and 2.79{+/-}1.40 (S). Notwithstanding observed S gene dropouts, the false negative rate was unaffected. As the ratio of asymptomatic positive to symptomatic positive SARS-CoV-2 infected individuals was approximately 4:1 and the average prevalence was 0.16% since we started testing in August 2020, pooled testing was identified as a viable, cost-effective option for monitoring the Northeastern University community.