Pools were constructed before nucleic acid extraction by combining 500 μL from each of the individual samples. For a pool size of 8, this resulted in a total volume of 4 mL and a dilution factor of 1:8. For a pool size of 4, this resulted in a total volume of 2 mL and a dilution factor of 1:4.

Subsequently, total nucleic acids were extracted from 500 μL taken from each pool and each individual specimen by using QIAsymphony and the QIAsymphony DSP Virus/Pathogen Midi Kit (QIAGEN, https://www.qiagen.com) and eluted into 60 μL of AVE buffer according to manufacturer’s instructions. Real-time reverse transcription PCR (rRT-PCR) was performed by using an emergency use authorization laboratory-developed test (LDT) targeting the envelope gene with the Rotor-Gene Q Instrument (QIAGEN) as described (1416), with pooled samples tested on the same run as component individual samples. A Ct result of 40–45 was considered an indeterminate result, which was adjudicated by repeat testing and resulted as positive if reproducible with an acceptable amplification curve. Specimens were only reported as negative if the internal control human RNase P gene was detected at a Ct<35.

On the same day as QIAsymphony extraction, another 500 μL from each pool was transferred to a Hologic Panther Specimen Lysis Tube (Hologic, https://www.hologic.com) and tested by using the Panther Fusion SARS-CoV-2 Assay (Hologic) and Panther Aptima SARS-CoV-2 assay (Hologic) per the manufacturer’s recommendations (17,18). In addition to the manufacturer-set cutoff value, receiver operating characteristic (ROC) curve analysis of pooled relative light unit (RLU) values, with individual test results as the reference method, was used to determine the optimal RLU discrimination threshold. A focused electronic medical record review was conducted for all samples.

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