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. 2021 Nov 26;7(48):eabj5629.
doi: 10.1126/sciadv.abj5629. Epub 2021 Nov 26.

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality

Elsa Brunet-Ratnasingham  1   2 , Sai Priya Anand  1   3 , Pierre Gantner  1   2 , Alina Dyachenko  1 , Gaël Moquin-Beaudry  1   4 , Nathalie Brassard  1 , Guillaume Beaudoin-Bussières  1   2 , Amélie Pagliuzza  1 , Romain Gasser  1 , Mehdi Benlarbi  1 , Floriane Point  1 , Jérémie Prévost  1   2 , Annemarie Laumaea  1 , Julia Niessl  1   2 , Manon Nayrac  1   2 , Gérémy Sannier  1   2 , Catherine Orban  2   5 , Marc Messier-Peet  1   5 , Guillaume Butler-Laporte  6   7 , David R Morrison  6 , Sirui Zhou  6   7 , Tomoko Nakanishi  6   8   9   10 , Marianne Boutin  1   2 , Jade Descôteaux-Dinelle  1   2 , Gabrielle Gendron-Lepage  1 , Guillaume Goyette  1 , Catherine Bourassa  1 , Halima Medjahed  1 , Laetitia Laurent  6 , Rose-Marie Rébillard  1   4 , Jonathan Richard  1   2 , Mathieu Dubé  1 , Rémi Fromentin  1 , Nathalie Arbour  1   4 , Alexandre Prat  1   4 , Catherine Larochelle  1   4 , Madeleine Durand  1   5 , J Brent Richards  6   7   8   11 , Michaël Chassé  1   5 , Martine Tétreault  1   4 , Nicolas Chomont  1   2 , Andrés Finzi  1   2   3 , Daniel E Kaufmann  1   2   5   12
Affiliations

Integrated immunovirological profiling validates plasma SARS-CoV-2 RNA as an early predictor of COVID-19 mortality

Elsa Brunet-Ratnasingham et al. Sci Adv. .

Abstract

Despite advances in COVID-19 management, identifying patients evolving toward death remains challenging. To identify early predictors of mortality within 60 days of symptom onset (DSO), we performed immunovirological assessments on plasma from 279 individuals. On samples collected at DSO11 in a discovery cohort, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA), low receptor binding domain–specific immunoglobulin G and antibody-dependent cellular cytotoxicity, and elevated cytokines and tissue injury markers were strongly associated with mortality, including in patients on mechanical ventilation. A three-variable model of vRNA, with predefined adjustment by age and sex, robustly identified patients with fatal outcome (adjusted hazard ratio for log-transformed vRNA = 3.5). This model remained robust in independent validation and confirmation cohorts. Since plasma vRNA’s predictive accuracy was maintained at earlier time points, its quantitation can help us understand disease heterogeneity and identify patients who may benefit from new therapies.

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Figures

Fig. 1.
Fig. 1.. High quantity of SARS-CoV-2 RNA in plasma at DSO11 is associated with increased risk of mortality.
(A) Pie charts representing the fractions of assessed samples that had undetectable (aviremic, light shades, <13 copies/ml) or detectable SARS-CoV-2 vRNA (dark shades, ≥13 copies/ml). Numbers in parts refer to the number (and percentages) of patients within each cohort. Noncritical and critical subgroups compared by χ2 test. (B) Quantities of SARS-CoV-2 N copies detected per milliliter of plasma in each cohort. Dotted line is the limit of detection (13 copies/ml). Empty shapes have undetectable vRNA (arbitrarily set at 5 copies/ml for representation). (C and D) Amounts of SARS-CoV-2 N copies detected per milliliter of plasma in patients who survived (white column) or died (gray column) by DSO60 for (C) total cohort or (D) critical subgroup only. Red circles represent critical patients, and blue circles are noncritical. (E) HR with 95% CI calculated using Cox regression for an increase of 1 U of log10-transformed vRNA (copies/ml). (F and G) Modelization of the predicted survival curves of patients with high [orange; upper interquartile range (IQR)], low (purple; lower IQR), or undetectable (gray) plasma vRNA in (F) all patients with COVID-19 or (G) critical cases only. (B) Kruskal-Wallis with Dunn’s multiple comparisons test. (C) Mann-Whitney test. n = 61 COVID-19 subjects (13 mortalities) or 29 critical COVID-19 cases (11 mortalities) and 10 UC. IQR: calculated among detectable vRNA quantities only.
Fig. 2.
Fig. 2.. High cytokine titers in plasma at DSO11 discriminates critical disease and is associated with increased risk of mortality.
(A) PCA representation of critical and noncritical patients (at DSO11), and UC (at baseline), on the basis of the 26 plasma analytes. Color-coded squares represent the mean PC (principal component) coordinates for each group. Length of arrow indicates the contribution of analytes to PCs. Numbers in parentheses along axes are the percentage of variance that PC accounts for. (B) Heatmap analysis of log-transformed concentrations of all 26 plasma analytes (yellow: high relative expression; blue: low relative expression), with unsupervised hierarchical clustering of the analytes (top dendrogram) or of patients (left dendrogram). The leftmost column represents outcome at DS60 (white: survival; black: deceased). The following column is the severity of the patient at DSO11. (C) Table showing the Spearman R values and corresponding P values of correlation of each plasma analyte with plasma vRNA. Values shaded in gray are nonsignificant. (D) Comparison of CytoScore of each cohort (see Materials and Methods for details on CytoScore). (E) Correlation between plasma vRNA and CytoScore. Empty shapes are aviremics (<13 copies of SARS-CoV-2 N copies/ml of plasma). (F and G) CytoScore of patients who survived (white column) or deceased (gray column) by DSO60 for (F) all patients with COVID-19 or (G) critical subgroup only. (H) HR with 95% CI calculated using Cox regression for a 1-U increase in the log10-transformed concentration of each plasma analyte with robust detection (see Materials and Methods for details) and CytoScore. ns: not significant. (I and J) Modelization of the predicted survival curves of patients with high (orange; upper IQR) or low (purple; lower IQR) CytoScore in (I) all patients with COVID-19 or (J) critical subgroup only. (C and E) Spearman correlations. (D) Kruskal-Wallis with Dunn’s multiple comparisons test. (F) Mann-Whitney test. For (A), (B), and (D) to (F), color-coded dots represent severity of the patient at DSO11 (red: critical; blue: noncritical) or UC cohort (green). (B and D) Cytokines with titles annotated by ∅ are poorly detected (see Materials and Methods for details). n = 61 COVID-19 subjects (13 mortalities) or 29 critical COVID-19 cases (11 mortalities) and 43 UC. IQR: calculated within the CytoScores of the COVID-19 discovery cohort.
Fig. 3.
Fig. 3.. Limited IgG responses against SARS-CoV-2 Spike at DSO11 are associated with mortality.
(A) ELISA-based relative quantification of SARS-CoV-2 RBD-specific antibodies’ isotypes IgM (left), IgA (middle), or IgG (right) in relative light units (RLU) normalized to an internal control (CR3022). (B to D) Comparison of functional properties of the plasma of all three groups, namely, (B) plasma capacity to recognize the SARS-CoV-2 full Spike (Spike Ig) using a flow cytometry–based assay [median fluorescence intensity (MFI)], (C) plasma neutralization activity [unit: half of maximal inhibitory plasma dilution (ID50)], and (D) plasma ADCC activity (unit: % of ADCC-mediated killing). (E and F) Correlation matrices with colors representing the Spearman R value (blue: negative association −1; red: positive association 1) and P values indicated as * in the circles, (E) between all serology measurements or (F) of serology measurements versus plasma vRNA and plasma analytes. (G to J) Comparison of serology measurements in patients who survived (white column) or deceased (gray column) by DSO60 for (G) RBD-specific IgM (left), IgA (middle), or IgG (right) or (H) full Spike binding, (I) neutralization, or (J) ADCC. (K) Hazard ratio with 95% CI calculated using Cox regression for an increase of 1 U of log10-transformed (square) or 10 U (diamond) of serology measurements. (L to N) Modelization of the predicted survival curves of patients with high (orange; upper IQR) or low (purple; lower IQR) (L) RBD-specific IgG, (M) Spike Ig, or (N) ADCC activity in all patients with COVID-19. (A to D) Kruskal-Wallis with Dunn’s multiple comparisons test. (E and F) Spearman R correlation. (F) Cytokines with titles annotated by ∅ are poorly detected. (G to J) Mann-Whitney test. For (G) to (J), color-coded dots represent severity of the patient at DSO11 (red: critical; blue: noncritical), and the dotted line represents the limit of detection. (E, F, and L) *P < 0.05; **P < 0.01; ***P < 0.001. n = 61 COVID-19 subjects (13 mortalities) or 29 critical COVID-19 cases (11 mortalities) and 43 UC. IQR: calculated within the COVID-19 discovery cohort.
Fig. 4.
Fig. 4.. Time-dependent ROC curves reveal plasma vRNA as reproducibly associated with mortality in the discovery, validation, and confirmation cohorts.
(A to C) Time-dependent ROC curves measured within the discovery cohort for (A) plasma vRNA, age, and sex; (B) cytokines and tissue insult markers; or (C) anti–SARS-CoV-2 antibody responses. (D) Time-dependent ROC curves of top multivariate models selected by Bayesian information criterion (BIC) stepwise selection in the discovery (left), validation (middle), and confirmation cohorts (right). (E) Time-dependent AUC of multivariate models over time in the discovery cohort. (F) Hazard ratio of plasma vRNA when sampled at DSO5, DSO9, or DSO13. HR adjusted for age and sex or not. Discovery: n = 61; validation: n = 87; confirmation: n = 69. For (F), all three cohorts were combined, complemented by 62 patients sampled before DSO7 (total n = 279).
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