An Evaluation of Pooling Strategies for RT-qPCR testing for SARS-CoV-2 Infection: A Pragmatic Multi-site Parallel Operational Study

Abstract

Background and Objectives. Sample pooling of COViD-19 PCR tests has been recently proposed as a low-cost alternative to individual tests. This multi-site, laboratory-based, proof-of-concept study explores the feasibility of pooled SARS-CoV-2 RT-qPCR testing, by demonstrating the effect of pooling on sensitivity, specificity, accuracy, number of tests saved, and turnaround time. Methodology. The research was conducted in two experiments. In Experiment 1, archival nasopharyngeal (NPS) and oropharyngeal (OPS) swab samples were diluted to simulate 5, 10, and 20 sized pools, and tested for SARS-CoV-2 RNA using RT-qPCR. In Experiment 2, actual nasopharyngeal and oropharyngeal swab samples were collected from asymptomatic low-risk volunteers. Aliquots of the samples were pooled following the 5, 10-5, and 20-10-5 multi-staged Dorfman pooling methods and tested. The sensitivity, specificity, accuracy, test savings, and turnaround time for each pooling method were documented. Results and Conclusions. The study provided evidence that pooling of NP and OP samples for SARS-CoV-2 RNA detection using RT-qPCR is feasible and can be implemented in the Philippines. A 2-stage Dorfman 5 pooling strategy appears to be the best method, because it has the highest over-all accuracy, while still achieving acceptable test savings, and turnaround time. Pooling of nasopharyngeal and oropharyngeal swab samples prior to RT-qPCR testing may be considered by select molecular diagnostic laboratories to further increase testing capacity and at the same time reduce the cost of testing.