Patients and specimen collection
The study population was composed of 29 autopsy cases (17 forensic autopsies and 12 clinical autopsies) investigated between October 2020 and February 2021 at the Fondazione Policlinico Universitario Agostino Gemelli IRCCS in Rome, Italy. Twenty-two of 29 cases died while hospitalized. In our morgue, postmortem swabs are routinely performed, and we included only cases in which postmortem swabs tested positive for SARS-CoV-2. All the hospitalized cases (H) had antemortem positive tests, while no antemortem microbiological information was available for non-hospitalized cases (NH). We included cases who died outside hospital (NH) only if death was witnessed and thus, the time since death was known (and reported by police and/or public health authorities). The mean age of the NH was 65 years, while the H were older (mean age: 73 years). Swab samples were collected from the nasopharyngeal tract (28 swabs: 6 in NH and 22 in H) and/or lungs (27 swabs: 6 in NH and 21 in H). They were preserved in universal transport medium (UTM, Copan S.p.A., Italy) and stored at 2–8 °C until testing by real-time reverse transcriptase‒polymerase chain reaction (rRT-PCR) assay for total and replicative SARS-CoV-2 RNA and mRNA detection, respectively, according to previously described protocols . The interval between death and postmortem swab(s) was registered: In NH, the mean value is 5.50 days (ranging from 0 to 14 days), and in H, 3.98 days (ranging from 0 to 16 days). Samples were processed on Seegene NIMBUS Automated Liquid Handling Workstations, from nucleic acid extraction (using STARMag Universal Cartridge kit) to PCR setup, according to the manufacturer’s directions (Arrow Diagnostics, Genova, Italy). Procedures to prevent specimen contamination and PCR carryover were in accordance with standard laboratory practices. Every 2 weeks, naso-pharyngeal swabs were performed on all the members of the teams that performed the autopsies, and these swabs were processed following the same procedures as used for the postmortem samples. All procedures were in accordance with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Data were processed in compliance with the European Union General Data Protection Regulation.
Total SARS-CoV-2 RNA detection and quantification
Total viral RNA was detected by the commercially available Seegene Allplex™ SARS-CoV2 Assay kit (Arrow Diagnostics, Genova, Italy), a multiplex real-time PCR for simultaneous detection of 4 target genes of SARS-CoV-2 in a single tube: the RdRP/S and N genes specific for SARS-CoV-2, and the E gene for all Sarbecoviruses, including SARS-CoV-2, as in the WHO-recommended protocols. Multiplex RT-PCR was performed on a Bio-Rad CFX96™ real-time detection system (Bio-Rad, Italy), and each RT-PCR provided a Ct value, the number of cycles required for the fluorescent signal to cross the threshold for a positive assay. Seegene automated data analysis software (Seegene Viewer) was used to identify positive detections. In particular, a positive result (i.e., a Ct value lower than 40) for at least one of two viral genes (i.e., RdRP/S and N) or for the E gene alone indicated the certain or presumptive presence of SARS-CoV-2 RNA in the sample, respectively.
Total viral RNA quantification was performed using the Quanty COVID-19 assay (Clonit S.r.l, Milan, Italy). Separate PCR microplate wells were each filled with 5 μL of sample extracted RNA (i.e., derived from the Nimbus RNA extraction step), positive control, negative control, and standards. The viral load in the swab sample was calculated by interpolation of the corresponding Ct value with a standard curve, which was previously built with the Ct values obtained following PCR amplification of samples containing 101, 102, 103, 104, and 105 copies/μL of synthetic viral N1-encoding RNA .
Replicative SARS-CoV-2 mRNA detection
Postmortem swab samples were also analyzed for subgenomic viral RNA (i.e., E gene replicative/intermediate RNA), which is intended as a surrogate for virus replication, using an in-house RT-PCR assay, as described elsewhere . Specifically, the QIAGEN® oneStep RT-PCR Kit (Qiagen, Valencia, CA, USA), 600 nM of each of two primers (sgE_SARS-CoV2_F 5′-CGATCTCTTGTAGATCTGTTCTC-3′; sgE_SARS-CoV2_R 5′-ATATTGCAGCAGTACGCACACA-3′), and 200 nM of probe (sgE_SARS-CoV2_P 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ-3′) were used in a 25-μL reaction volume. Thermal cycling consisted of 30 min at 50 °C for reverse transcription, followed by 15 min at 95 °C and 45 subsequent cycles of 10 s at 95 °C, 15 s at 55 °C, and 5 s at 72 °C.