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.