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.