SARS-CoV-2 antigenemia and RNAemia in association with disease severity in patients with COVID-19 – Scientific Reports

The authors reporting experiments on the use of human and/or human tissue samples were all experiments conducted in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects and/or legal guardians. The study was conducted in accordance with relevant guidelines and regulations for all methods.

Participants

In our hospital, we performed a prospective cohort study from June 21, 2020, to September 30, 2023, with patients who possibly had COVID-19 using a preformed case record form. We enrolled 119 patients with confirmed COVID-19 who gave consent for comprehensive use of specimens, admitted from June 21, 2020 to October 22, 2021 at Chosun University Hospital, South Korea. Our aim was to examine the clinical association of antigenemia and RNAemia. All selected patients were clinically confirmed to be SARS-CoV-2 positive using more than one diagnostic methods, including real-time reverse transcriptase polymerase chain reaction (qRT-PCR) with the confirmation of more than two target genes, cell culture, or a ≥ fourfold increase or seroconversion in terms of SARS-CoV-2 antibody titer (enzyme-linked immunosorbent assay [ELISA] or indirect immunofluorescence antibody assay). Moreover, 81 serum/plasma samples of healthy individuals without clinical symptoms, no history of COVID-19, and negative nasopharyngeal qRT-PCR results were used as control samples for the sensitivity assay.

Sampling and RNA extraction

Peripheral blood was collected from all patients and 200 μL serum/plasma samples from fresh blood were used for ribonucleic acid (RNA) extraction. Concurrently self-collected sputum samples collected from the patients were diluted in phosphate-buffered saline (PBS), mixed by vortexing and pulse-centrifuged for 1 min, and 200 μL supernatant was subjected to RNA extraction. Nasopharynx swabs were directly collected into the commercial UTM™ kits containing 1 mL of a viral transport medium (NobleBio, Oldenzaal, The Netherlands) by a physician, and 200 μL were employed for RNA extraction. The viral RNA was extracted by Real-prep Viral DNA/RNA Kit (Biosewoom, Seoul, South Korea) using a fully automated instrument (Real-Prep system, Biosewoom).

Real-time reverse transcription-polymerase chain reaction (qRT-PCR) for SARS-CoV-2 detection

For the qRT-PCR assay of the nucleocapsid protein (NP) gene, primers and probes were designed in-house, nCov-NP_572F (5′-GCAACAGTTCAAGAAATTC-3′), nCov-NP_687R (5′-CTGGTTCAATCTGTCAAG-3′), and nCov-NP_661P (5′-FAM-AAGCAAGAGCAGCATCACCG-BHQ1-3′). Thermal cycling was performed as follows: 50 °C for 10 min for reverse transcription, one cycle of 95 °C for 30 s for preincubation, 95 °C for 5 s at 57 °C for amplification, and 45 cycles for data detection. For the target genes E (encoding envelope protein) and RdRp (encoding RNA-dependent RNA polymerase), the Kogene Kit (Kogene Biotech Co., Ltd., Seoul, South Korea) and SD Kit (SD Biotechnologies Co., Ltd., Seoul, South Korea) were used, and amplification was performed according to the manufacturer’s instructions. For the NP target, qRT-PCR was performed in an Exicycler™ 96 Real-Time Quantitative Thermal Block (Bioneer, Smiths Parish, Bermuda), and for Kogene and SD kits, the CFX96 Touch™ Real-Time PCR Detection System (Biorad, Hercules, CA, USA) was used. Cycle threshold (C_t) values were set to ≤ 40 for the reference gene and were assumed to denote a positive result.

Cell culture

For the identification of SARS-CoV-2 in culture, monolayers of Vero E6 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum and a 1 × penicillin–streptomycin antibiotic solution (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in an atmosphere containing 5% of CO₂ at 37 °C. Then, 200 μL of an unfrozen swab sample in viral transport medium (UTM™ kit, NobleBio) diluted with 1 mL of Dulbecco’s phosphate-buffered saline (Welgene, Taipei, Fujian, China) was inoculated to the monolayer of cultured Vero cells. After two passages, viral proliferation was confirmed by qRT-PCR with a confirmatory C_t value 16,17.

Sandwich ELISA for antigenemia

The nucleocapsid protein (N) antigenemia assay of patients with and without COVID-19 was carried out using single molecule array (SIMOA) technology with paramagnetic microbeads–based sandwich ELISA. The SIMOA SARS-CoV-2 N Protein Advantage kit assay (Quanterix Corp, Boston, MA, USA, PN/103806) is a digital immunoassay that quantitatively measures the SARS-CoV-2 nucleocapsid protein in human serum and plasma. Plasma or serum obtained from fresh blood was frozen after aliquoting to minimize protein degradation due to freeze–thaw cycles and thawed at room temperature before use for antigenemia assay. Briefly, each well of 96-well ELISA microplates (Quanterix® plates) was loaded with 4 × dilution of plasma or serum and assayed in Simoa HD-X instrument (Quanterix) using a two‐step immunoassay. For detection, incubation was performed with the target antibody coated with paramagnetic beads, sample, and biotinylated antibody (SIMOA Guide Quanterix). The nucleocapsid protein present in the sample was captured using antibody-coated beads bound to the biotinylated antibody, and detected simultaneously as described previously^18,19.

Indirect ELISA

Indirect ELISA for SARS-CoV-2 was performed using a recombinant nucleocapsid protein (Bioapp. Inc., Pohang, South Korea) to determine serological titers of immunoglobulin G (IgG), immunoglobulin M (IgM), and total antibodies. Frozen serum samples were thawed at room temperature and used for indirect ELISA. In brief, 100 µL of 2 µg/mL recombinant SARS-CoV-2 nucleocapsid protein (Bioapp. Inc.) was coated in a 96-well ELISA microplate (Thermo Fisher Scientific) with carbonate-bicarbonate buffer, with overnight incubation at 4 °C. The ELISA plates were washed with PBS containing 0.05% Tween 20, followed by 2-h blocking at 37 °C with 5% skim milk in blocking buffer. The plates were further washed incubated for another 2 h at 37 °C with the serum samples diluted 100-fold in blocking buffer. After washing, a secondary antibody (horseradish peroxidase-conjugated goat anti-human IgG antibody [1:6000, Invitrogen, Thermo Fisher Scientific, Cat A18805], anti-human IgM antibody [1:3000, Invitrogen, Thermo Fisher Scientific, Cat 31415], or anti-human total-antibody antibody [1:40,000; Thermo Fisher Scientific, Cat 31418]) was added, and incubated again for another 1 h at 37 °C. The plates were further washed and 50 µL of the 3,3′5,5’-tetramethylbenzidine substrate (Sigma-Aldrich, St. Louis, MO, USA) was added and incubated at room temperature (20–30 °C) for 30 min in dark. Moreover, 25 µL of 1 M H₂SO₄ was added for arresting the reaction and the optical density was measured using an Epoch™ two microplate spectrophotometer (Kitchener, ON, Canada) at 450 nm (OD₄₅₀). The cutoff values and positivity for SARS-CoV-2 were set as described previously²⁰.

Statistical methods

Statistical analyses were performed using MedCalc 20·013 software (Ostend, Belgium), and IBM SPSS Statistics for Windows, version 26.0. (IBM Corp., Armonk, NY, USA), and GraphPad Prism 9 (San Diego, CA, USA). The sensitivity, specificity, and accuracy of the test were evaluated using receiver operating characteristic (ROC) curve analysis. Confidence intervals for sensitivity, specificity and accuracy are “exact” Clopper–Pearson confidence intervals²¹. To determine the 40-day survival rate, Kaplan–Meier survival analysis was conducted based on the antigenemia concentration. Quantitative variables are presented as mean ± standard deviation and n (%) for normally distributed variables. Mean values were compared using t-tests for continuous variables and were found to be normally distributed. P-values comparing patients with COVID-19 with evidence of antigenemia and RNAemia to those without antigenemia and RNAemia were calculated using the Mann–Whitney U or Fisher’s exact test, as appropriate.