Study design

We tested the analytical sensitivity of a large number of commercially available AgPOCTs by applying pooled samples from nasopharyngeal swabs with defined SARS-CoV-2 viral loads (given in cycle threshold (Ct)) including Ct16, Ct21, Ct25, and Ct28 (~ 9.8× 10⁸ to ~ 1.8 × 10⁵ genome copies per ml) as well as a pooled sample obtained from SARS-CoV-2 negative tested persons. Pools were generated using anonymized remnant swab sample material collected for the clinical diagnosis of a SARS-CoV-2 infection by RT-qPCR carried out by the Center for Infectious Diseases, Virology, Heidelberg University Hospital, Germany. Pharyngeal swab specimens were collected through the nose (nasopharyngeal) and contained in viral transport medium (VTM). Per test, 50 µl of the samples were mixed with the provided lysis buffer of each AgPOCT, and the tests were performed strictly according to the manufacturer’s instructions. After the recommended incubation time, we acquired images of the test chambers using a Panasonic Lumix DMC-G70 camera equipped with a Panasonic H-FS12060 objective. We tested AgPOCTs at least in duplicates with the corresponding test samples. We quantified test results by measuring the background-corrected signal intensities of the test (T) band versus control (C) band in ImageJ (v1.53c) using the Gels analysis function usually used for quantification of Western Blot bands. For qualitative evaluation of the visibility of the test bands (positive versus negative score), RGB pictures of AgPOCT results from randomly chosen replicates were evaluated independently by three individuals in a blinded manner. Furthermore, all test results were scored independently by another person.