**3.2 Exclusion criteria**

Exclusion criteria include exposure to toxics such as alcohol abuse with heavy drinking more than 40 g/day in men and 30 g/day in women, over the past 10 years, or exposure to industrial toxic substances, as well as to several groups of drugs with liver toxicity, and other liver conditions either inherited or acquired like hemochromatosis, alpha1 antitrypsin deficiency, Wilson disease, autoimmune hepatitis, primary biliary cirrhosis, infection with viral B, D, or C hepatitis. Many other conditions such as organ insufficiency (heart, lungs, liver, or kidney), cancer, recent trauma and surgery, burning, myocadial infarction and cardiogenic shock, stroke, bacterial infectious diseases and sepsis, pancreatic diseases, thyroid diseases, long-standing parenteral nutrition, inflammatory bowel disease, and other entities resulting in malnutrition syndromes, as well as recent treatment with antibiotics or probiotics have been ruled out.

### **3.3 Examination approach and laboratory work-up**

Patients underwent measurements of waist circumference and blood pressure (BP), body mass index (BMI) assessment, as well as thoroughly clinical examination. *Laboratory work-up:* complete blood count (CBC), routine liver tests including hepatitis B surface (HBs) antigen, Delta antigen, anti-HCV antibodies, plasma iron and copper, alpha 1 antitrypsin, antinuclear antibodies (ANA), antimitochondrial antibodies (AMA), lipase, thyroid stimulating hormone, as well as C-reactive protein (CRP), procalcitonin (PCT), fasting plasma glucose (FPG), HbA1c, total

cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), triglycerides, creatinine and uric acid, microproteinuria, urine and stool microbiology were run, using standardized, accredited methods.

*Stool's microbiological assessment:* Sterile containers with collected stool samples were frozen at −20°C and initially processed in order to determine possible aerobe, anaerobe, or microaerophiles species [18]. After identifying different types of stool species by the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method, they were expressed as colony formatting units (CFU)/gram stool and the severity of gut microbiota DB was semiquantitative scored as follows: 0 = absent, 1 = mild, 2 = medium, 3 = severe [19]. In the case of dysbiosis, frozen stools were further processed by the 16S rRNA next-generation sequencing (NGS) method in order to assess the enterotype, H index of alpha-biodiversity, and several bioindicators of the gut microbiome [20].
