Factors of Severity in Patients with COVID-19: Cytokine/Chemokine Concentrations, Viral Load, and Antibody Responses

Patients and clinical samples.

We prospectively enrolled 31 confirmed cases of COVID-19 admitted to four university-affiliated hospitals in the Republic of Korea—Asan Medical Center, Chung-Ang University Hospital, Soonchunhyang University Seoul Hospital, and Inje University Sanggye Paik Hospital—from February 2020 to April 2020. COVID-19 was confirmed by real-time reverse transcription–PCR (RT-PCR) analysis for the RdRp gene of SARS-CoV-2. The severity of COVID-19 was categorized into mild, moderate, severe, and critical according to the WHO guidance.⁹ Peripheral blood was collected at admission and every 2–3 days thereafter until discharge. Plasma samples were immediately separated by centrifugation and stored at −70°C until further analysis; for cytokine and antibody analysis, approximately 1 mL of plasma was irradiated with up to 6 million rad from a⁶⁰Co gamma source according to the method used in our previous study.¹⁰ The study was approved by the respective institutional review boards of each participating hospital.

SARS-CoV-2 RT-PCR.

Viral RNA was extracted from the upper respiratory tract swab samples using the MagNA Pure 96 system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. RNA was analyzed with the PowerChek 2019-nCoV Real-Time PCR Kit (KogeneBiotech, Seoul, Republic of Korea) that targets the RdRp gene of SARS-CoV-2 and the E gene of beta-coronavirus, and the Allplex^™ 2019-nCoV assay (Seegene) that targets the RdRp gene and N gene of SARS-CoV-2 and the E gene of beta-coronavirus. Ct values < 40 for RdRp gene were considered as positive results.

SARS-CoV-2 serology.

We measured the levels of human anti–SARS-CoV-2 IgG and IgM by using laboratory-developed ELISA. SARS-CoV-2 S1-His protein (Sino Biological, Beijing, China) was coated onto 96-well plates (MaxiSorp, Thermo Fisher Scientific, Waltham, MA) at a concentration of 2 μg/mL in PBS. Plasma samples were used at dilutions of 1:100, 1:1,000, and 1:10,000. horseradish peroxidase-conjugated antihuman IgG (Jackson Immunoresearch, West Grove, PA) and IgM (MilliporeSigma, Burlington, MA) were used as secondary antibodies. The optical density (OD) value of 450 nm (OD₄₅₀) was measured. If OD₄₅₀ of 1/100 or 1/1,000 diluted sample exceeds 2.5, the OD₄₅₀ of next diluted sample was multiplied by dilution factor (OD₄₅₀ ratio) and determined as the endpoint titer. The OD₄₅₀ of plasma specimens that were not previously exposed to SARS-CoV-2 were used to determine the cutoff value (i.e., OD₄₅₀ = 0.4) for both IgG and IgM.

Cytokine analysis.

Collected plasma samples were stored at −70°C until analysis and analyzed at once, on the same day. We simultaneously measured plasma concentrations of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17A, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation and normally T-cell expressed and secreted, monokine induced by IFN-γ (MIG), IFN-γ–induced protein (IP)-10, and vascular endothelial growth factor (VEGF) using a cytometric bead array based on microspheres for detecting cytokine/chemokine according to the manufacturer’s instruction (BD Bioscience, San Jose, CA). Data were acquired using the FACS CANTO II flow cytometer, FACSDiva software (BD Bioscience), and FlowJo software (FlowJo LLC, Ashland, OR), as described in our previous study.^10,11

Statistical analyses.

Categorical variables were compared using the Fisher’s exact test or the χ² test, and continuous variables were compared with the Kruskal–Wallis test, Mann–Whitney U test, or unpaired Student’s t-test. All tests of significance were two-tailed and P-values less than 0.05 were considered statistically significant. Statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, Inc., LA Jolla, CA).

Clinical characteristics and outcomes.

A total of 31 patients with COVID-19 confirmed by SARS-CoV-2–specific RT-PCR of Nasopharyngeal (NP) swab specimen were enrolled in this study. The patients were classified according to disease severity: five (16%) mild, 17 (55%) moderate, six (19%) severe, and two (7%) critical patients; one (3%) patient did not have any signs or symptoms and was therefore classified as an asymptomatic case (Table 1); for analysis, the patients were categorized into three groups: asymptomatic and mild (19%), moderate (55%), and severe and critical (26%). Of the 31 patients, 18 (58%) were female, and the mean age (±SD) was 50.0 (±3.3) years. The median time interquartile range (IQR) from symptom onset to admission was 6 days (3–8). The median time (IQR) from admission to discharge was 24 (18–33) days. Old age, initial low WBC count, low platelet count, high CRP level, and fever were identified as factors associated with severity. Detailed baseline characteristics, laboratory tests, and outcomes are shown in Tables 1 and 2.

Viral load kinetics.

The detailed kinetics of viral loads in NP swab specimens are shown in Figure 1A. The viral load within 16 days from symptom onset was lowest in asymptomatic and mild group compared with those of moderate group and severe and critical group (Figure 1A and Table 2, P = 0.044 and 0.034, respectively). The viral load at 11–16 days from symptom onset was significantly lower in asymptomatic and mild group than that in severe and critical group (Figure 1A and Table 2, P = 0.009). The viral load gradually decreased over time in all patients.

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Viral load and antibody kinetics in COVID-19 patients during the course of disease. The kinetics of viral load of the upper respiratory tract from symptom onset (A) and the viral load at the first day of hospitalization (B). The kinetics of plasma IgG (C) and IgM (D) titer against SARS-CoV-2 S1 protein. If OD₄₅₀ of 1/100 or 1/1,000 diluted sample exceeds 2.5, the endpoint titer (OD₄₅₀ ratio) of IgG and IgM was determined by multiplication of dilution factor and OD₄₅₀. Black significance bars compare two groups; asymptomatic and mild vs moderate, asymptomatic and mild vs severe and critical, or moderate vs severe and critical. Red bars indicate statistical differences among three groups. Blue dot = moderate; green dot = asymptomatic and mild; red dot = severe and critical. *P < 0.05, **P < 0.01, ***P < 0.001.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 6; 10.4269/ajtmh.20-1110

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The viral load on the day of admission was measured in 26 (84%) patients, and there was no significant difference in the viral load according to disease severity (Figure 1B). The initial viral load was compared at 5–10 days from symptom onset because unlike the other groups, patients in the severe and critical group were hospitalized at approximately a week later from the day of symptom onset compared with other groups. The mean (±SD) initial viral load at 5–10 days from symptom onset in asymptomatic and mild group, moderate group, and severe and critical group was 32.65 (±7.62), 27.68 (±6.98), and 26.52 (±4.82) cycles, respectively (P for trend = 0.038). The median time (IQR) to negative conversion of the RT-PCR result for SARS-CoV-2 from the day of symptom onset was 18 (14–24) days. The duration of positive RT-PCR results was significantly shorter in the asymptomatic and mild group than in the other groups (P = 0.021; Supplemental Figure 1A).

Antibody analysis.

The plasma concentrations of SARS-CoV-2–specific IgG and IgM were measured, and the detailed kinetics of antibody titers are shown in Figure 1C and D. The median time (IQR) to seroconversion of IgG from symptom onset in the asymptomatic and mild group, moderate group, and the severe and critical group was 3 days (1.5–12.0), 11 days (7.0–13.0), and 14 days (10.0–15.8), respectively (P = 0.033, Supplemental Figure 1B). The plasma IgG was gradually increased and maintained during hospitalization in all groups. By contrast, the plasma IgM in the asymptomatic and mild group peaked at around 3 weeks after symptom onset, and then almost disappears and not in other groups (P = 0.004). Although the severe and critical group showed a relatively delayed response in terms of IgG and IgM, the response size after 25 days since symptom onset was significantly higher than those in other groups (P < 0.001 and P = 0.004, respectively).

Cytokine analysis.

A total of 131 plasma specimens were available for multiplex cytokine bead array analysis. Among the 21 cytokines/chemokines measured, the plasma concentrations of IFN-α, IP-10, MIG, IL-6, IL-8, MCP-1, IFN-γ, VEGF, and IL-10 were found to be significantly higher in the severe and critical group than those in other groups. The detailed kinetic profiles of cytokines/chemokines in response to COVID-19 during hospitalization are shown in Figure 2 and Supplemental Figure 2. In particular, the plasma concentrations of IFN-α, IFN-γ, IP-10, MIG, and IL-6 were elevated in the severe and critical group at 5–10 days from symptom onset. Although the plasma concentrations of VEGF and IP-10 gradually decreased with time, their levels were significantly higher in the severe and critical group throughout hospitalization (Figure 2). The concentrations of other cytokines/chemokines were not significantly different according to disease severity (Supplemental Figure 2). Among 21 cytokines/chemokines, only MCP-1 had correlation with viral load (P = 0.034, Supplemental Figure 3). In addition, IFN-α and IP-10 had a trend to correlate with viral load (P for trend = 078 and 0.061, respectively).

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Plasma cytokines and chemokine concentrations in COVID-19 patients during the course of disease, according to the severity of illness. Black significance bars compare two groups; asymptomatic and mild vs. moderate, asymptomatic and mild vs. severe and critical, or moderate vs. severe and critical. Red bars indicate statistical differences among three groups. Blue dot = moderate; green dot = asymptomatic and mild; red dot = severe and critical. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IFN = interferon; IL = interleukin; IP = IFN-γ-induced; MCP = monocyte chemoattractant protein; MIG = monokine induced by IFN-γ; VEGF = vascular endothelial growth factor.

Citation: The American Journal of Tropical Medicine and Hygiene 103, 6; 10.4269/ajtmh.20-1110

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