3. Results
3.1 GI complaints before, during and after acute SARS-CoV-2 infection is
associated with PCS Fatigue
PCS and ME/CFS patients reported a higher susceptibility to infections
(Table 2 ). A higher number of PCS and ME/CFS patients reported
signs of hypermobility. Pre-existing food intolerances were more common
in PCS and ME/CFS patients. Notably, only PCS patients had more
pre-existing GI complaints before acute infection. A significant
association between GI symptoms prior to SARS-CoV-2 infection compared
to current GI complaints was detected (chi-square test value = 13.8;
p<0.001). Also, a significant association was found between
pre-existing GI symptoms and PCS development (chi-square test value =
5.9; p=0.015). During acute infection, PCS patients reported more often
respiratory, cardiovascular and GI symptoms, but also head ache,
myalgia, back pain, hair loss and sleep disorders (Supplementary
Table 1 ). In general, multisystemic symptoms during acute infection
were more commonly found in PCS patients compared to those who fully
recovered. 100% of PCS patients still suffer from exercise intolerance,
and 50% were unable to work. Significant association were also observed
for GI complaints (chi-square test value = 6.6; p=0.01) and
cardiovascular symptoms (chi-square test value = 9.8; p=0.002) during
acute infection and their persistence in PCS. In terms of current
symptoms, respiratory syndromes, neurological symptoms, cardiovascular
symptoms, GI complaints, pain symptoms, signs of cognitive impairment,
dysautonomia symptoms, sleeping disorders, as well as signs of Fatigue
and PEM (reduced mental and physical activity, ability to leave the
house or go to work) occurred more often in PCS and ME/CFS patients.
(Supplementary Table 2 ).
3.2 No signs of viral shedding were observed by determining SARS-CoV-2
RNA levels in body fluids
We evaluated SARS-CoV-2 E-protein RNA levels to examine viral shedding
evidenced by viral RNA in body fluids. Plasma, stool and throat washing
samples were analyzed by RT-qPCR, but no viral particles were found
(data not shown).
3.3 Comparison of SARS-CoV-2 IgG and IgA antibody titers and
immunological events in the study cohort
Measurements of SARS-CoV-2 specific IgG and IgA antibodies against
S1/RBD in plasma by ELISA showed no statistical differences in IgG
titers between the test groups. SARS-CoV-2 convalescent study
participants had significantly higher IgA titers than the healthy
control group (Supplementary Figure 1A & 1B ). To
include data on the participants’ vaccination state and provide a better
context of the results, anamnestic information on immunological events
was analyzed (Supplementary Figure 1C & 1D ). The term
”immunological event” refers to either a SARS-CoV-2 infection or a
SARS-CoV-2 vaccination with a single event in a 14-day period. No
statistical differences were observed when comparing the time between
the last immunological event and the day of study inclusion and
sampling. SARS-CoV-2 convalescent participants had a higher number of
immunological events, although, not significant.
3.4 PCS patients reveal signs of intestinal barrier leakage
PCS patients showed significantly higher serum
lipopolysaccharide-binding protein (LBP) levels compared to convalescent
SARS-CoV-2 participants and healthy controls (Figure 1A ).
Levels of sCD14 in serum samples were significantly lower compared to
all other groups (Figure 1B ). As sCD14 is needed as a co-factor
together with LBP to mediate innate immunity against LPS to in the
immune system, the LBP/sCD14 ratio was calculated. We were able to
detect a significantly higher ratio in PCS patients compared to
convalescent SARS-CoV-2 participants and healthy controls
(Figure 1C ). Serum I-FABP, which is released by enterocytes
upon cell damage, did not differ between all groups (Figure
1D ).
3.5 Evaluation of disease-related marker associated with low-grade
inflammation and intestinal barrier disruption
To detect signs of chronic low-grade inflammation in PCS patients, the
pro-inflammatory and intestinal barrier integrity- related cytokines
IFN-γ, TNF-α, IL-6, IL-1-β, IL-8, and IL-33 were measured in serum
samples. A significant elevation in IL-6 levels and a reduction in serum
TNF-α and IL-1-β levels was observed in PCS patients (Figure 2
A-C ). However, some results of the pro-inflammatory cytokine ELISAs
were below the Limit of Detection (LOD) stated by the manufacturer and
had to be excluded for the analysis. The means (± SD) of the respective
parameters’ remaining values were under or close to the LOD
(Supplementary Table 4 ). Serum IL-33 levels were significantly
decreased in PCS patients when compared to the SARS-CoV-2 negative
control participants (Figure 2D ). IL-8 and IFN-γ levels did
neither show statistical significance, nor a disease- specific pattern
(data not shown).
3.6 No significant differences in fecal pro-inflammatory marker levels
were detected
To assess whether participants show signs of intestinal inflammation
levels of calprotectin, β-defensin-2, zonulin family peptides and
serotonin (5-HT) were evaluated (Table 3 ) by ELISA. No
significant differences between the groups were found when examining
levels of calprotectin, β-defensin-2 and serotonin (5-HT).
3.7 Association analysis between the development of PCS and intestinal
barrier marker
To assess influence of gastrointestinal barrier markers on the
development of PCS, we used univariate binary logistic regression.
Statistical outliers were removed from the analysis before calculating
odds ratios (OR) and p-values (Supplementary Table 5 ). We
analyzed all SARS-CoV-2 positive participants to investigate differences
in intestinal barrier markers related to the development of PCS (binary,
dependent variable). A higher LBP level (OR=1.065) was associated with
6.5% increased odds of developing PCS. Lower levels of I-FABP
(OR=0.513) suggested 48.7% reduced odds of PCS, while higher levels of
sCD14 (OR=0.774) indicated a 22.6% decrease in PCS probability. No
significant associations were found between serum IL-33 levels or fecal
zonulin family peptide levels and the outcomes studied.