Fig 1.
Flow chart showing recruitment sites, eligibility criteria for selecting volunteers, and methodologies used for sample processing.
Table 1.
Sociodemographic and clinical characteristics of the study population.
Fig 2.
COVID-19 cellular response test.
(A, B) Mean marginal IFN-γ production to assess cytokine production in response to PHA and SARS-CoV-2 S Protein, respectively, in whole blood COVID-19-cellular immunity test among the study population. (C) Anti-S protein IgG serology of healthy unexposed, and confirmed volunteers for COVID-19. Green circles indicate positive individuals (responders) for the cellular immune response, while red solid circles indicate those who were negative (non-responders). Solid squares represent individuals in the unexposed group, solid circles represent individuals in the COVID-19 group, and the box plots represent the interquartile range and the sample median (solid gray centerline). The larger black solid circles and central bars represent the fitted means estimated by the linear model, and the marginal mean values and their upper 95% confidence intervals were estimated for both unexposed individuals and for COVID-19 controlled for sex, BMI, and age for IFN-γ and PHA (mitogen) after stimulation with a specific biomarker for SARS-CoV-2 S protein. **, P<0.01.
Fig 3.
Plasma cytokine profile by SARS-CoV-2 Spike protein stimulation in whole blood CRA test.
The cytokines (A) IFN-γ, (B) TNF, (C) IL-1β, (D) IL-2, (E) IL-6, (F) IL-8, (G) IP-10, (H) IL-12, (I) IL-4, (J) IL-10, and (K) IL-17A were evaluated. The levels obtained from each inflammatory mediator were analyzed on a logarithmic transformed scale and illustrated using box plots to compare unexposed and COVID-19 groups. Squares represent individuals in the unexposed group, solid circles represent individuals in the COVID-19 group, and the box plots represent the interquartile range and the sample median (solid gray centerline). The larger black solid circles and central bars represent the fitted means estimated by the linear model, and the marginal mean values and their upper 95% confidence intervals were estimated for both unexposed individuals and for COVID-19 controlled for sex, BMI, and age for IFN-γ and PHA (mitogen) after stimulation with a specific biomarker for SARS-CoV-2 S protein. **, P<0.01; *, P<0.05.
Fig 4.
Plasma cytokine profile of the unexposed, recovered COVID-19, and long-COVID-19 groups.
Cytokines (A) IFN-γ, (B) TNF, (C) IL-1β, (D) IL-2, (E) IL-6, (F) IL-8, (G) IP-10, (H) IL-12, (I) IL-4, (J) IL-10, and (K) IL-17A were evaluated. The obtained levels of each inflammatory mediator were analyzed on a logarithmic transformed scale and illustrated using box plots to compare the unexposed, recovered, and long-COVID-19 groups. Squares represent individuals from the unexposed group, solid circles represent individuals from the Recovered COVID-19 subgroup, triangles represent individuals from the Long-COVID-19 subgroup, and the box plots represent the interquartile range and the sample median (solid gray centerline). The larger black solid circles and central bars represent the fitted means estimated by the linear model, and the marginal mean values and their upper 95% confidence intervals were estimated for both unexposed individuals and for COVID-19 controlled for sex, BMI, and age for IFN-γ and PHA (mitogen) after stimulation with a specific biomarker for SARS-CoV-2 S protein. **, P<0.01; *, P<0.05.
Fig 5.
Principal component analysis (PCA) discriminates cytokines between COVID-19 and healthy unexposed groups.
Analysis of variance of cytokine concentrations IFN-γ, TNF, IL-1β, IL-2, IL-6, IL-8, IP-10, IL-12, IL-4, IL-10, and IL-17A were evaluated for COVID-19 patients (N = 26) and unexposed healthy volunteers (N = 21). (A) A 2D representation, given by the first two principal components with 67.6% and 16.7% explained variance (84.3% cumulative percentage explained), of Unexposed (blue solid circles), Recovered COVID-19 (yellow solid circles) and Long COVID-19(gray solid circles), with point sizes proportional to the average individual contribution to any principal component. The variables (biomarkers) are expressed by colored vectors, indicating their average contribution to the principal components. (B) A representation where the vector represents the correlation between a variable (biomarker) and a principal component (PC) is used as the coordinates of the variable in the PC. The variables are colored according to the results of a divisive clustering of k-means (k = 2). (C) Bar graph indicating the seven variables (biomarkers) according to their average contribution to each main component. (D) Bar graph indicating the top 10 individuals according to their average contribution to any major component.
Table 2.
Principal-component analysis of inflammatory biomarkers in plasma from patients with COVID-19 and unexposed group.