Starlet

All samples were stored at 2–6 °C at the test site before transportation to the department for Clinical Microbiology either at Zealand University Hospital, Herlev Hospital or Rigshospitalet depending on the site of inclusion and testsing.
All samples from Zealand University hospital were tested with Allplex^TM SARS-CoV-2 real-time PCR assay (Seegene, Seoul, South Korea). The system is automated using the STARlet (Seegene, South Korea) for extraction of RNA and PCR setup, and Seegene Viewer for analyzing the data. The Allplex^TM SARS-CoV-2 assay includes internal control (IC) and four targets: E gene, N gene, RdRP gene and S gene. RdRP and S genes use the same fluorophore and cannot be distinguished. The assay has a software algorithm with cycle threshold (Ct) cut-off values ≤ 40 for IC and all four targets. Samples that are only positive for E gene are scored as inconclusive by the software, because the E gene is not SARS-CoV-2 specific, but a pan-Sarbecovirus target.
The respiratory specimens collected at Herlev Hospital or Rigshospitalet were analyzed on the RT-PCR systems available for the qualitative detection of SARS-CoV-2 RNA at the sample time, and therefore many different systems were used (See Supplementary Table S1). However, all samples collected from a participant were tested with the same RT-PCT assay.

For the detection of SARS-CoV-2, two automated equipments were used to extract RNA (EliTe InGenius® (ELITech Group, Dieren, The Netherlands) for blood samples, Seegene STARlet (Seegene Inc. Seoul, South Korea) for nasopharyngeal swabs). For RNA isolation, manufacturer-validated reagents (SP200 extraction reagent, STARMAg isolation kit, Seegene Inc, Seoul, South Korea) were used. Allplex™ SARS-CoV-2 Assay (Seegene Inc., Seoul, South Korea) was chosen for multiplex real-time PCR, due to its capability to detect 4 different SARS-CoV-2 genes simultaneously (E-gene, N-gene, RdRP gene, S-gene). PCR tests were considered negative at 40 cycle threshold (Ct) or more.