2. Methods
2.1. Study Cohort
All participants were recruited between April 2021 and August 2022 and provided written informed consent before study inclusion (ethic vote number: 2281/2020). The study cohort consists of PCS patients, as well as sex- and age-matched SARS-CoV-2 convalescent participants (SARS-CoV-2, convalescent), SARS-CoV-2 naïve ME/CFS patients (ME/CFS) and SARS-CoV-2 naïve healthy participants (Healthy) as controls(Table 1 ). ME/CFS was diagnosed based on the Institute of Medicine (IOM) criteria [15]. All included ME/CFS patients suffered from an Epstein-Barr Virus (EBV)-related onset of the disease. For study participation, PCS patients had to suffer from Fatigue, PEM and additionally from clinical signs of autonomous dysregulation, orthostatic dysregulation and/or neurocognitive dysregulation. SARS-CoV-2 infected study participants had an asymptomatic or mild disease course, with 4 exceptions in the PCS group and 2 exceptions in the convalescent SARS-CoV-2 control group. Furthermore, previously SARS-CoV-2 positive participants were excluded if their acute COVID-19 diseases required intensive medical care. All participants were at least 10 weeks past an acute SARS-CoV-2 infection, at least 12 weeks past an acute EBV infection and at least 2 weeks after any other respiratory or GI infection. Participants were excluded if they suffered from preexisting malignant diseases, diabetes mellitus, inflammatory bowel diseases, an acute sepsis or if they underwent medical treatment with antibiotics, analgesics or antacids one month prior to study participation. PCR or SARS-CoV-2 antigen test was performed to screen for clinically inapparent SARS-CoV-2 infections prior to study inclusion.
2.2 Sampling Procedure and Self-reported Medical Record Data
Before the sampling, participants were asked to give a detailed medical record on their disease course and individual symptoms before, during and after the SARS-CoV-2 infection, or their general health status for the SARS-CoV-2 naïve control participants (Table 2 ,Supplementary Table 1 & 2 ). Serum and plasma/EDTA samples were collected on the day of study participation, after centrifugation with 2000× g for 10 min. Stool samples were collected and delivered by the patients on the day of study participation. Participants were asked to cool their samples in the fridge for storage prior to delivery. If the collection of a stool sample was not possible on the day of study participation, it was collected by the patient within two weeks after study participation. Stool extracts for further use were prepared according to the respective manufacturer’s protocols (Supplementary Table 3 ). Saliva and throat flushing samples were collected by the patients on the day of study participation, before tooth brushing and eating or drinking. Saliva samples were collected pure, throat flushing samples were collected by gurgling for 1 minute with 10 ml 0.9% NaCl. All samples were stored at -20°C until further processing.
2.3 SARS-CoV-2 detection in blood, stool and saliva samples
SARS-CoV-2 viral load in body fluids was evaluated by real-time RT-PCR using primers targeting the SARS-CoV-2 E-gene as previously published [16, 17]. Briefly, RNA was extracted from stool, plasma and saliva samples using the NucliSens EasyMag extractor system (BioMérieux, Marcy-l’Étoile, France). SARS-CoV-2 RNA was eluted and a reaction was prepared with the Superscript III one step RT-PCR system with Platinum Taq Polymerase (Invitrogen, Darmstadt, Germany) and reverse transcriptase/Taq mixture from the kit. Thermal cycling was performed at 55 °C for 10 min for reverse transcription, followed by 95 °C for 3 min and then 45 cycles of 95 °C for 15 s, 58 °C for 30 s.
2.4 Measurement of disease-related parameters.
Parameters for evaluating SARS-CoV-2 specific immune responses, systemic and local inflammation and intestinal barrier disruption were measured by commercially available Enzyme-linked immunosorbent assays (ELISAs). All markers and respective ELISA Kits are listed in the supplementary materials (Supplementary Table 3 ). ELISAs were performed according to the respective manufacturer’s protocols. Absorbance was measured at 450 nm using ELISA Reader Infinite m200 PRO (Tecan, Männedorf, Switzerland). A four-parameter logistic (4PL) curve was used to analyse antibody concentrations after subtracting levels detected in blank wells as background values.
2.5 Statistical analysis
Data sets were tested for normal distribution with Kolmogorov-Smirnov test. Significance between groups was assessed by Kruskal-Wallis-test and Dunn-Bonferroni-test for multiple comparison were performed. Anamnestic data was described using descriptive statistical methods. Characteristics were either given in absolute numbers or percentages for categorical variables or in medians/quartiles for continuous variables. To investigate possible associations between different biomarkers, cytokines and anamnestic characteristics and the development of PCS Fatigue chi-squared test (nominal and nominal variables) and Mann-Whitney-U test (nominal and metric variables) were used. Univariate binary logistic regression was performed for all patients after a SARS-CoV-2 infection (PCS and SARS-Cov-2, convalescent) with “developed PCS – yes/no” being the binary, dependent variable and LBP, sCD14, I-FABP, IL-33 and zonulin family peptides being the independent variable. In advance, the data used for regression was screened for statistical outliers using the ROUT method, as well as linear correlation. Through binary logistic regression odds ratios (OR) and p-values were calculated. Two tailed p-value of <0,05 was taken as statistically significant. Statistical analyses were performed by using Graph Pad Prism 9 or IBM SPSS Statistics 27. No adjustment for multiplicity was performed, p-values were interpreted descriptively.