5.1 General procedure and patient consent
This study extends the COVID-COG project. It utilizes existing data from the COVID-COG project, which includes samples from hospitalized and intensive care patients (all patients were hospitalised for SARS-CoV-2 infection), along with neuropsychological, sociodemographic, and clinical data (see section 5.2, 5.3 and 2.1).
In the present study we specifically extracted and retrospectively analysed acute cytokine data from hospitalized and intensive care patients with viable venous blood samples (see figure 3 and section 5.6 and 2.2). We analysed the relationship and predictability of episodic memory raw scores measured at 6-9 months and 12-15 months post-infection by acute cytokine levels. The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the cantonal ethics committee of Geneva (CER-02186). Each patient included in the study was informed and freely consented in writing to participate in the study.
5.2 COVID-COG cohort
The aim of the COVID-COG project was to observe whether SARS-CoV-2 infection could lead to cognitive, mood and emotional difficulties 6-9 months and 12-15 months after infection. People included in the COVID-COG cohort had been infected with SARS-COV-2, confirmed by positive polymerase chain reaction (PCR) results using the LightCycler 480 system (Roche, Switzerland) from a nasopharyngeal swab between March 2020 and May 2021 at the Geneva University Hospitals (HUG). Approximately 300 people were eligible according to the exclusion criteria: no neurological, psychiatric, cognitive and medical history that may affect cognition (e.g., cancer, HIV), no neurodevelopmental pathologies, no pregnancy, no substance use, no treatment affecting cognition and no age over 80. Of these 300 people, 121 agreed to take part in the COVID-COG project and gave their free and informed consent. A complete and validated neuropsychological test battery was administered to the participants 6-9 months and 12-15 months post-infection (see section 6.3 and Voruz, et al. 3). The 121 people included in the COVID-COG project were divided into subgroups according to the severity of the infection in the acute phase. A first "mild" subgroup requiring no hospitalisation (N=49), a second "moderate" subgroup requiring hospitalisation without mechanical ventilation (N=48) and finally a "severe" subgroup requiring hospitalisation in intensive care with mechanical ventilation (N=24).
5.3 Measurement of episodic memory
Data from objective measures of episodic memory were extracted from the COVID-COG protocol. Based on the MNESIS model (Eustache, et al. 36), we measured two dimensions of episodic memory. We used two different episodic memory tasks to highlight verbal and visuospatial episodic memory processes. We used the following tools: free/cued recall paradigm (RLRI 16) (Grober and Buschke 37) measuring verbal episodic memory (we collected free recall and total recall scores) and the Rey–Osterrieth Complex Figure test (Meyers and Meyers 38) measuring visuospatial episodic memory (we collected the scores for the copy, the 3-minutes recall and the 20-minutes recall) (see supplementary materials 1 and 2). In clinical practice, these tests are used on a daily basis to assess different memory processes. The Behavior Rating Inventory of Executive Function (Abeare et al., 2021) enabled us to measure the validity of symptoms and non-credible symptoms; these were good for all participants in the cohort (Voruz, et al. 3).
5.4 Participants included in the study
As described above, we extracted, from the COVID-COG cohort, the patients who benefited from a venous blood sample during the acute phase of the infection as well as a neuropsychological examination at the two measurement times (6-9 months and 12-15 months post-infection). Thus, as shown in the following flowchart, we retained 33 patients in this study, 22 from intensive care and 11 from intermediate care during the acute phase of the disease (see figure 3 and table 1).
The figure is positioned here
Figure 3. Study flowchart.
Note. ICU: intensive care unit
5.5 Statistical power
We set the type 2 β error at 0.80, the α threshold was set at 0.025 in view of our hypotheses. Finally, we estimated a correlation coefficient on the observed relationship between TNFα levels and post-COVID cognitive symptoms obtained in Nuber-Champier, et al. 17,39.
The standard normal deviate for α = Zα = 1.9600
The standard normal deviate for β = Zβ = 0.8416
C = 0.5 * ln[(1+r)/(1-r)] = 0.6625
Total sample size = N = [(Zα+Zβ)/C]2 + 3 = 21
Thus, based on the calculation made by Sb, et al. 40, the necessary sample size is estimated at 21 participants.
5.6 Retrospective analysis of cytokines
Cytokine measurements were conducted on the first day of hospitalization, prior to the administration of any therapies that could affect cytokine level variations. Blood samples were collected, on average, 1.26 ± 2.85 days following a positive PCR test (see table 2). Cytokines (TNFα, IL-1β, IL-6) were measured (pg/ml) using commercially available multiplex bead immunoassays (Fluorokine MAP Multiplex Human Cytokine Panel, R&D Systems, Minneapolis, USA) and read using a Bioplex 200 array reader (Bio-Rad Laboratories, Hercules, CA, USA) and Luminex xMAP Technology (Luminex Corporation, Austin, TX, USA).
5.7 Statistical analysis
Given the distribution of our cytokine data and the proximity of certain scores to zero, we performed a logarithmic (log) transformation of our data41. In addition, as the raw cognitive data were not normally distributed either, we used non-parametric tests, namely Spearman correlation tests and generalised linear mixed models (GLMM) gamma with log link or linear depending on data distribution 41,42. We present the descriptive clinical and socio-demographic data of the sample in Table 1.
In relation to the hypothesis of an association between levels of cytokine secretion in the acute phase and memory performance at 6-9 months and 12-15 months post-infection, we performed Spearman correlations with a significance threshold set at .05. A total of 4 correlation matrices were produced: i) relationship between cytokines levels in the acute phase of COVID-19 and verbal episodic memory scores measured at 6-9 months; ii) relationship between cytokines levels in the acute phase of COVID-19 and visuospatial episodic memory scores measured at 6-9 months; iii) relationship between cytokines levels in the acute phase of COVID-19 and verbal episodic memory scores measured at 12-15 months and iv) relationship between cytokines levels in the acute phase of COVID-19 and visuospatial episodic memory scores measured at 12-15 months.
Concerning the hypothesis that memory performance measured at 6-9 months and 12-15 months could be predicted by cytokines levels in the acute phase of COVID-19, we performed GLMM. We performed a regression model for each verbal and visuospatial episodic memory raw score measured at 6-9 months and 12-15 months with log TNFα, log IL-6 and log IL-1β levels as predictors and gender, age and educational level as covariates.
Despite the hypothesis-driven nature of this study and the fact that we only investigated a limited number of variables (inflammatory and cognitive) related to our hypotheses and that our inclusion criteria were very selective, we decided to apply a correction for the false discovery rate (FDR) in order to limit statistical error on the prediction analyses.