Laboratory analyses

The QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used for RNA isolation in swabs, according to the manufacturer’s instructions. Before RNA extraction, an aliquot of 140 µL of the viral transport medium was inactivated in lysis buffer (Buffer AVL, Qiagen; 560 µL). In each sample, 10 µL of internal control RNA (MS2 Phage Control included in TaqPath COVID-19 Kit) and an RNA carrier were added. The purified RNA was eluted in 50 µL and immediately stored at −80°C.

To detect SARS-CoV-2 RNA, a multiplex real-time RT-PCR test (TaqPath COVID-19 CE-IVD RT-PCR Kit, Thermo Fisher Scientific) was applied.8 Five microlitres of RNA was reverse transcribed into cDNA and amplified using the QuantStudio 12K Flex Real-Time PCR Instruments (Applied Biosystems, California, USA). The reaction was run in a 384-well plate prepared by a Microlab Starlet robot (Hamilton Robotics, Bonaduz, Switzerland).

In the PCR reaction, the probes anneal to three specific SARS-CoV-2 sequences: (1) ORF1ab with reporter dye FAM; (2) N protein (nucleocapsid) with reporter dye VIC; and (3) S protein (spike) with reporter dye ABY. In addition, a bacteriophage MS2 Control (with reporter dye JUN) is also amplified to verify the efficacy of the sample preparation and the absence of inhibitors. The reaction mix added to each RNA sample (5 µL) is the following: 5 µL of TaqPath 1-Step Multiplex Master Mix (No ROX) (4X), 1 µL of TaqPath COVID-19 Assay Multiplex and 9 µL nuclease-free water.

In each run, a standard curve was added to check the efficiency of the amplification; the standard TaqPath COVID-19 Control (1×104 copies/µL) was diluted serially in TaqPath COVID-19 Control Dilution Buffer 1/4-fold per dilution to produce six concentrations of copies, ranging from 312.5 to 0.305 copies/µL. Five microlitres of each standard was then distributed in the ‘standard curve’ 1000 wells. The plates were then sealed, centrifuged briefly at 800 × g and located in QuantStudio 12K Flex Real-Time PCR Instruments (Applied Biosystems) for the run. The passive reference was set to ‘None’, and ‘Absolute Quantification’ as run type.

The following thermal protocol was applied: 2 min at 25°C for UNG (Uracil-DNA glycosylase) activation, 10 min at 53°C for the reverse transcription reaction, 2 min at 95°C for activation, 40 cycles of denaturation at 95°C for 3 s and anneal/extension at 60°C for 30 s.

The data analysis was performed using the ‘Design and Analysis Software’ (V.2.3.3, Applied Biosystems) setting ‘Automatic Threshold’. The reaction was considered only if MS2 Ct ≤38. If any two of the three SARS-CoV-2 genes were positive (Ct ≤38) the sample was classified as positive; if only one of the assays was positive, the test was repeated. Once the test was repeated, and the result was again positive, the sample was classified positive for SARS-CoV-2. If all three of the assays were negative (Ct=undetermined), the subject was classified as negative.

Blood EDTA was centrifuged at 1200 g for 15 min to obtain cell-free plasma. Immunoglobulin M (IgM) and IgG against SARS-CoV-2 in plasma samples were tested.

The Wantai anti-SARS-CoV-2 IgM ELISA (Beijing Wantai Biological Pharmacy Enterprise, Beijing, China)9 was performed according to the manufacturer’s instructions. Reported sensitivity is 86% and specificity is 100%. The assays detect antibodies binding SARS-CoV-2 spike protein receptor-binding domain (RBD) in human serum or plasma. Briefly, 10 μL plasma samples and 100 μL of specimen diluent were added to wells coated with antibodies directed against the human IgM proteins and incubated for 30 min at 37°C. Each well was aspirated and washed five times using an automatic microplate washer (MicroFill Dispenser, BioTek Instruments, Winooski, Vermont, USA). Then, a recombinant horseradish peroxidase (HRP)-conjugated SARS-CoV-2 antigen was added and incubated for 30 min at 37°C. After further washing, a chromogen solution was added. The reaction was stopped after 15 min at 37°C, and the resultant absorbance was read on a microplate reader (Synergy HT, BioTek Instruments) at 450 nm with reference at 620 nm. The cut-off value for a positive result was calculated according to the manufacturer’s instruction and equal to 0.105 for anti-SARS-CoV-2 IgM ELISA.

To perform RBD ELISA IgG, ELISA plates were coated with 1 µg/mL of purified recombinant spike-RBD HEK-derived protein (Sino Biological, Beijing, China); the assay was set and qualified as already reported.10 After overnight incubation at +4°C, coated plates were washed three times with 300 µL/well of ELISA washing solution containing Tris-buffered saline (TBS)-0.05% Tween 20, then blocked for 1 hour at 37°C with a solution of TBS containing 5% of non-fat dry milk (Euroclone, Pero, Italy).

Human serum samples were heat inactivated at 56°C for 1 hour to reduce the risk of the presence of intact and infectious viruses in the sample, then diluted 1:100 in TBS-0.05% Tween 20 5%. Plates were washed three times as previously then 100 µL of each serum dilution was added to the coated plates and incubated for 1 hour at 37°C. Next, after the washing step, 100 µL/well of Goat anti-Human IgG-Fc HRP-conjugated antibody diluted 1:100.000 (Bethyl Laboratories, Montgomery, Texas, USA) was added. Plates were incubated at 37°C for 30 min. Following incubation, the plates were washed and 100 µL/well of 3,3′,5,5′-tetramethylbenzidine substrate (Bethyl Laboratories) was added and incubated in the dark at room temperature for 20 min. The reaction was stopped by adding 100 µL of ELISA stop solution (Bethyl Laboratories) and read at 450 nm. The cut-off value was established as three times the average of optical density (OD) values from blank wells (background—no addition of analyte). Samples with the ODs under the cut-off value at the first (1:100) dilution were assigned as negative; samples where the ODs at 1:100 dilution were above the cut-off value were assigned as positive. Borderline samples were defined where one replicate was under the cut-off and the other was above. The reported sensitivity of this method is 85.7% and specificity is 98.1%.