Study design
The study design was a randomized controlled trial (Fig. 1). Briefly, the NAFLD subjects were defined as H. pylori positive or negative by 13C-UBT. The H. pylori-positive subjects were randomized into two different groups by a random number table: treatment or no treatment. Four weeks after treatment, the presence of H. pylori infection was assessed again in the treated subjects by 13C-UBT. Metabolic and inflammatory parameters and FibroScan were measured in all subjects at baseline and 1 year after the treatment.
Study participants
Participants who voluntarily underwent a general health screening from January to June 2017 were recruited from the International Health Care Centre of the Second Affiliated Hospital of Zhejiang University School of Medicine. The inclusion criteria were as follows: 1) ultrasonography for NAFLD; 2) alanine aminotransferase (ALT) levels less than 2 times the UNL; and 3) no symptoms of gastrointestinal tract disease. The exclusion criteria were as follows: 1) the alcoholic consumption of 3 or more drink units per week; 2) any chronic liver disease; 3) a history of gastric surgery; 4) the use of bismuth, antibiotics, proton pump inhibitors or H2 blockers within the prior 4 weeks; 5) a significant mental or neurological disorder; 6) a history of cancer; and 7) patients on steatogenic medications, such as methotrexate and corticosteroids. All subjects underwent a detailed physical examination, including 13C-UBT detection of H. pylori infection, ultrasonography and FibroScan for NAFLD.
Ethics approval and consent to participate
All participants provided written informed consent before the examination. The present study was reviewed and approved by the Ethics Committee of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University (2016-322).
Registration
The trial was registered on Chinese Clinical Trials Registry, with the name of the registry being “Helicobacter pylori Infection Eradication on Nonalcoholic Fatty Liver Disease: A Randomized Controlled Trial.” The trial registration number was ChiCTR2200061243.
Questionnaires
The medical history of each participant was obtained using a questionnaire that included the history of the present illness, previous diagnoses of H. pylori infection, history of anti-H. pylori therapy, history of gastric surgery, history of significant mental or neurological disorders, history of cancer(s), use of bismuth, antibiotics, PPIs or H2 blockers within the previous 4 weeks, alcohol intake, and cigarette smoking.
Diagnosis of H. pylori infection
After fasting for at least 2 hours, all participants underwent 13C-UBT at our centre. After a baseline breath sample was collected, the participants ingested a 13C-urea reagent that was dissolved in water. The second breath sample was collected 30 minutes later and analysed. A delta over baseline (DOB) value ≥4.0 indicated a positive result for H. pylori infection.
Definition of NAFLD
NAFLD was defined according to the guidelines published in 2012 by the American Association for the Study of Liver Diseases (AASLD), American College of Gastroenterology (ACG), and American Gastroenterological Association (AGA)33. In this study, the diagnosis of NAFLD required (1) hepatic steatosis detected by ultrasonography; (2) no significant alcohol consumption (to strictly exclude the influence of alcohol, we chose individuals with alcohol consumption of less than 3 drink units per week); and (3) no coexisting causes of chronic liver disease, such as hepatitis C, medications, parenteral nutrition, Wilson’s disease or severe malnutrition.
Treatments
The enrolled NAFLD subjects were randomized to H. pylori eradication treatment or untreated groups based on standard instructions for diet and exercise according to the guidelines. The H. pylori eradication treatment consisted of 14 days of quadruple therapy (Esomeprazole 20 mg given twice daily + colloidal bismuth pectin 200 mg twice daily + amoxicillin 1000 mg twice daily + furazolidone 100 mg twice daily). If the patient was allergic to amoxicillin, it was substituted with clarithromycin (500 mg twice daily). Eradication was assessed by 13C-UBT 4 weeks after the end of treatment. Eradication was considered successful when the UBT was negative.
The untreated groups only accepted health education and lifestyle guidance.
Health education and lifestyle guidance
Two general practitioners (unknown about the research group) provided full-time guidance and monthly telephone or network follow-up supervision.
Specific content was based on the recommendations of the guidelines.
1) Moderate-intensity calorie restriction and personalized daily calorie intake according to the body mass index are recommended;
2) Changes in the diet composition to a low-sugar and low-fat balanced diet and reductions of sucrose beverages and saturated fat and intake of trans fats and increase fibre content are recommended;
3) A moderate amount of aerobic exercise is recommended 5 times a week, and the cumulative exercise time should be at least 150 min.
Data collection
Blood pressure measurements were obtained after at least 10 minutes of rest. Body mass index (BMI) was defined as weight divided by height squared (kg/m2). The waist circumference (WC) was measured while standing with a measuring tape midway between the lowest rib and the iliac crest. The fasting plasma WBC, hs-CRP, fasting blood glucose, glycosylated haemoglobin (HbA1c), alanine aminotransferase (ALT), γ-glutamyltranspeptidase (γ-GT), aspartate aminotransferase (AST), total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), and triglyceride (TG) levels were measured after an 8-hour overnight fast (Beckman Coulter AU 5400). The HOMA-IR was calculated according to the following formula: HOMA-IR=[FINS (µIU/mL)×FPG (mmol/L)]/22.525.
IL-6 and TNF-α
Venous blood (4 ml) was collected into a serum separation tube. After the blood clot formed, the sample was centrifuged, and the serum was collected. The total IL-6 and TNF-α contents in plasma were determined using ELISA (Abcam, USA).
FibroScan
Liver stiffness was evaluated using a FibroScan-502 touch (Echosense, Paris, France). The operation was carried out according to the user manual by two specially trained doctors who obtained the FibroScan operator certificate.
- Fasting patients were in the supine position, and they held their head with their right hand to maximize the expansion of the intercostal space.
- The probe was placed on the right lobe of the liver in the intercostal position.
- The probe was kept perpendicular to the skin, and when the pressure indicator displayed in green, the M waveform on the display screen was consistent and evenly distributed and the A waveform was linear; subsequently, the detection process started.
- The unit of liver steatosis was dB/m, which was achieved in 10 replications with a success rate of 60% and an interquartile range of <30%.
Statistical analysis
The statistical analysis was performed using SPSS 13.0 software. Measurement data are expressed as the mean ± standard deviation. A paired t test was used for comparisons before and after treatment within groups; a single factor analysis of variance was used for multigroup comparisons, and SNK was used for pairwise comparisons between groups. All p values were based on a two-sided test of statistical significance. Significance was accepted at the level of p<0.05.