Validation of SARS-CoV-2 pooled testing for surveillance using the Panther Fusion^® system: Impact of pool size, automation, and assay chemistry

by Rudolph Park, Prabha Chandrasekaran, Heather Hernandez, Ines Lakhal-Naouar, Kristina K. Peachman, Holly R. Hack, Dante Coleman, Jason Ouellette, Janice M. Darden, Oussama M’hamdi, Victor A. Sugiharto, Hua-Wei Chen, Megan A. Schilling, Mark P. Simons, Natalie D. Collins, Yuliya S. Johnson, Linda L. Jagodzinski, Sheila A. Peel

Combining diagnostic specimens into pools has been considered as a strategy to augment throughput, decrease turnaround time, and leverage resources. This study utilized a multi-parametric approach to assess optimum pool size, impact of automation, and effect of nucleic acid amplification chemistries on the detection of SARS-CoV-2 RNA in pooled samples for surveillance testing on the Hologic Panther Fusion^® System. Dorfman pooled testing was conducted with previously tested SARS-CoV-2 nasopharyngeal samples using Hologic’s Aptima^® and Panther Fusion^® SARS-CoV-2 Emergency Use Authorization assays. A manual workflow was used to generate pool sizes of 5:1 (five samples: one positive, four negative) and 10:1. An automated workflow was used to generate pool sizes of 3:1, 4:1, 5:1, 8:1 and 10:1. The impact of pool size, pooling method, and assay chemistry on sensitivity, specificity, and lower limit of detection (LLOD) was evaluated. Both the Hologic Aptima^® and Panther Fusion^® SARS-CoV-2 assays demonstrated >85% positive percent agreement between neat testing and pool sizes ≤5:1, satisfying FDA recommendation. Discordant results between neat and pooled testing were more frequent for positive samples with C_T>35. Fusion^® C_T (cycle threshold) values for pooled samples increased as expected for pool sizes of 5:1 (C_T increase of 1.92–2.41) and 10:1 (C_T increase of 3.03–3.29). The Fusion^® assay demonstrated lower LLOD than the Aptima^® assay for pooled testing (956 vs 1503 cp/mL, pool size of 5:1). Lowering the cut-off threshold of the Aptima^® assay from 560 kRLU (manufacturer’s setting) to 350 kRLU improved the assay sensitivity to that of the Fusion^® assay for pooled testing. Both Hologic’s SARS-CoV-2 assays met the FDA recommended guidelines for percent positive agreement (>85%) for pool sizes ≤5:1. Automated pooling increased test throughput and enabled automated sample tracking while requiring less labor. The Fusion^® SARS-CoV-2 assay, which demonstrated a lower LLOD, may be more appropriate for surveillance testing.