Fecal Microbiota Transplantation Slows the Progression of HBV-Related Liver Diseases and Induces Virologic Response in Patients with HBV Infection

Research Article

Austin J Gastroenterol. 2021; 8(2): 1117.

Fecal Microbiota Transplantation Slows the Progression of HBV-Related Liver Diseases and Induces Virologic Response in Patients with HBV Infection

Guo Q1#, Huang S-S1#, Li J1, Tian Y1, Zhou Y1,2* and Li X-A1,2*

¹The Gastroenterology Tumor and Microenvironment Laboratory, Department Of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, Sichuan, P.R. China

²Department of Gastroenterology, Mianyang central hospital, Mianyang, Sichuan, P.R. China

#Contributed Equally to this Work

*Corresponding author: Xiao-An Li, Department of Gastroenterology, Mianyang central hospital, Mianyang, Sichuan, P.R. China

Yan Zhou, Department of Gastroenterology, Mianyang central hospital, Mianyang, Sichuan, P.R. China

Received: September 16, 2021; Accepted: October 14, 2021; Published: October 21, 2021


Aims: Healthy gut microbiome plays a crucial role in the treatment of Hepatitis B Virus (HBV) infection and chronic liver disease. Based on existing studies, we aimed to explore the difference in the efficacy of Fecal Microbiota Transplantation (FMT) in patients with different stages of HBV-related chronic liver disease.

Methods: In this study, 10 HBV patients with HBeAg-negative infection, 8 patients with Chronic Hepatitis B (CHB) infection, and 8 patients with hepatitis B cirrhosis Child-Pugh score A, 9 patients with hepatitis B cirrhosis Child-Pugh score B/C were treated with FMT.

Results: Our results demonstrated that continuous FMT treatment improved liver function, controlled the replication of HBV-DNA, enhanced the intestinal mucosal barrier function, relieve the degree of liver fibrosis and postponed the progression of HBV-related chronic liver disease. The result of 16S ribosomal RNA (rRNA) sequencing indicated that the individual genus and composition of the bacteria in the feces of patients gradually approached the structure seen in case of the feces of healthy donors; the number of specific Operational Taxonomic Units (OTUs) in the stool samples of patients gradually decreased.

Conclusion: Our study further confirmed that FMT could be a novel and effective treatment strategy for patients with chronic HBV infection.

Keywords: Hepatitis B virus; Fecal microbiota transplantation; Chronic hepatitis B; Cirrhosis


Continuous HBV infection and repeated inflammation of the liver could easily progress to end-stage liver diseases, such as liver failure, cirrhosis, and primary liver cancer, having a great impact on the quality of life and lifespan of patients [1,2]. In China, HBV infection is the most common cause of cirrhosis and primary liver cancer [3], with the proportion of patients with HBV infection-induced cirrhosis being as high as 60% [4], thereby posing a prominent public health problem in China. Therefore, it is particularly important to study HBV-related chronic liver diseases.

With the implementation of the Intestinal Microbial Genome Project, research on the microecology of the digestive tract has received extensive attention [5]. Animal studies have shown that pathways of HBV immune tolerance, particularly through the Toll-Like Receptor 4 (TLR4)-dependent pathway [6,7], exist in young mice before the establishment of intestinal bacteria, while the microbiota in the mature intestinal tract of adult mice have been reported to stimulate the immune response of the liver, leading to rapid HBV clearance [8]. A previous study has shown that the gut microbiome appears to play an important role in the pathophysiological process of chronic liver disease, and the severity of disorders in the flora was shown to be related to the severity of liver disease [9]. Another study has shown that the gut microbiome could regulate immune homeostasis, thereby protecting the liver from viral hepatitis infections [10]. Due to the importance of the intestinal microecological changes on the prognosis of liver diseases [11], effectively improving the intestinal microecological status of patients with chronic HBV infection, understanding the mechanisms governing its occurrence and development, and exploring appropriate interventions and treatment measures for the management of these diseases is urgently needed.

Fecal Microbiota Transplantation (FMT), which refers to the processing of fresh feces from selected healthy donors into a fecal suspension introduced into the gut of patients, has been studied as a method for the treatment of intestinal and extra-intestinal diseases [12,13]. In recent years, as the incidence of Clostridium Difficile Infection (CDI) has increased year by year, FMT has been used increasely in clinical practice. For this reason, the European Conference proposed a consensus on FMT clinical practice and published it in Gut magazine in 2017 [14]. FMT is widely used in intestinal diseases, but there are still relatively few clinical studies on chronic liver diseases, especially HBV-related chronic liver diseases. Therefore, this study will explore the efficacy of FMT in the treatment of HBV-related chronic liver disease and the changes in intestinal microbes.

Materials and Methods

Diagnostic criteria of patients

Patients were diagnosed with chronic HBV infection or hepatitis B cirrhosis according to the Chinese Medical Association Liver Diseases Branch and the Chinese Medical Association Infectious Diseases Branch October 2015 Guidelines for Prevention and Treatment of Chronic Hepatitis B (updated version 2015).

Inclusion criteria

The following criteria needed to be met by patients to be included in our study:

• Patients who have been diagnosed with chronic HBV infection without taking any treatment measures.

• Patients meeting the diagnostic criteria for hepatitis B cirrhosis, with no improvement in the past 3 months through routine treatment of liver (Glutathione, Polyene phosphatidylcholine), diuretic (Furosemide, Spironolactone), or nutritional support.

• Patients who were 18–85 years old, without gender limitations, and a follow-up time greater than 3 months.

• All patients refused to use antiviral drugs due to their own reasons, and at the same time, it was fully evaluated that no antiviral drugs would not cause serious consequences. Therefore, no antiviral drugs were used during treatment and follow-up.

• Patients suitable for and undergoing gastroscopy and FMT treatment.

• Patients who agreed to participate in this clinical study and signed an informed consent form to receive follow-up, as well as consented to the retention of their specimens on time.

Exclusion criteria

Any of the following criteria met by patients, led to their exclusion from our study:

• Pregnant or lactating women; drug users; suspected or confirmed history of alcohol/drug abuse.

• Patients with a history of large abdominal surgery, gastrointestinal bleeding, genetic or metabolic liver diseases.

• Patients suffering from diseases that might cause gastrointestinal tract damage, such as diabetes, thyroid disease, intestinal obstruction, inflammatory bowel disease, etc.

• Patients taking antibiotics, non-steroidal anti-inflammatory drugs, microecological preparations, gastrointestinal motility drugs, acid suppression drugs, and other drugs affecting the intestinal flora 2 weeks before treatment.

• Patients with other serious diseases (including serious infections, severe heart, kidney, respiratory, blood, neuropsychiatric diseases, etc.) that might affect their survival or combined with other types of tumors.

Research design

Due to the complexity of clinical diagnosis and treatment, the clinical implementation process of FMT, and the poor acceptance of FMT in some patients, the random double-blind design could not be effectively applied in clinical practice. Therefore, we conducted a prospective study. According to the clinical diagnosis, the included patients were divided into four groups: Group A1 comprised HBeAgnegative infection. Group A2 included patients with CHB. The included HBV patients with HBeAg-negative infection and CHB had not took antiviral drugs for various reasons, and it is fully assessed that without antiviral drugs would not lead to serious consequences. Therefore, In order to understand the effect of FMT on hepatitis B virus, only a single FMT treatment (1 time/month, 3 times) was accepted during treatment. Group B1 included Child A patients with hepatitis B cirrhosis. Group B2 comprised Child B/C patients with hepatitis B cirrhosis. The included patients with liver cirrhosis who are ineffective in conventional hepatoprotection and diuresis, in order to fully benefit the patients, receive both conventional treatment (comprehensive treatment of routine liver protection, diuresis, nutritional support) and FMT treatment (1 time/month, 3 times). All participants had healthy diet during treatment and follow-up. All the Patients signed an informed consent form. The study was approved by the Ethics Committee of the First Affiliated Hospital of Chengdu Medical College and passed the American Clinical Trial Registration Certification on 01/09/2017 (ClinicalTrials.gov ID: NCT03014505).

Donor screening

We choose healthy men between the ages of 18 and 30 and have a body fat index between 18.5 and 23.9 as donors for FMT. Donors were healthy individuals without diseases or pathologic conditions potentially associated with changes in the gut microbiota. Donors were not use of antibiotics, immunosuppressive medications, systemic antineoplastic agents, and recent ingestion of a potential allergen in the preceding 3 months before screening. After the initial screening, they were subjected to strict examinations (serology and stool tests). Serological tests include blood routine, liver and kidney function, electrolytes, blood sugar, blood lipids, syphilis, HIV, HAV, HBV, HCV. Stool examination includes stool routine, human rotavirus, ova and parasites, Cryptosporidium, Cyclospora, Isospora, Clostridium difficile toxin, and Giardia, Blastocystis hominis and Dientamoeba fragilis. They are considered healthy blood donors only if all serological and stool tests are normal. Taking into account the incubation period of the disease, screening and inspection are repeated every 6 months. In addition, the fecal donors with healthy diet, regular exercise every day, no smoking, no alcohol, and no other unhealthy foods, such as preserved and canned food. In the end, 4 healthy people who met the requirements successfully donated their stools.

Preparation of fecal suspensions

In the morning, the fecal sample of the donor that was not contaminated by urine was placed in a sterile closed container. Immediately after receiving the specimen, the fecal suspension was prepared (completed within 2h). A 50g stool sample was added to 250ml of 0.9 % physiological saline, and a glass rod was used to homogenize it. The homogenized suspension was then filtered through a stainless-steel molecular sieve having a diameter of 2.0, 1.0, 0.5, and 0.25 mm, with the larger particulate matter being filtered out. Subsequently, the filtered slurry was centrifuged at 6000 g for 15 min, discard the supernatant and added 50 ml physiological saline solution to homogenize and resuspended the pellet.

FMT infusion

Patients were required to be fasting for at least 8 h before FMT treatment. For the FMT treatment, freshly prepared bacterial solution was used. All patients underwent gastroscope FMT treatment: the patient was placed in the left lateral position under propofol intravenous anesthesia, and after the gastroscope reached the duodenal level, a sterile catheter was inserted from the gastroscopic biopsy hole, and then, the prepared bacterial solution (50ml) was slowly injected from the catheter with a syringe. After completion of the injection of the solution, the catheter and the gastroscope were slowly withdrawn to avoid aspirating the air, and patients were observed for potential back flow of the fecal suspension. After FMT treatment, the patient was required to maintain a half-sitting position of 45 degrees for at least 4h, follow a light liquid diet after fasting for 2-4 h, and stay in the hospital for at least 8-10 h.


Blood samples: 5ml of peripheral venous blood was collected from patients 2 days before FMT treatment and 3 months after the last FMT treatment. We evaluate liver function and HBV-DNA indicators before and after treatment. The amount of Diamine Oxidase (DAO), D-lactic acid, interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), Collagen Type IV (COL IV), Procollagen III Propeptide (PIIIP), Hyaluronic Acid (HA) and Laminin (LN) were measured in the serum of patients using the human ELISA kit, according to the manufacturer’s instructions.

Intestinal flora analysis: 3-5 g of early morning feces were collected 2 days before FMT treatment and one month after each treatment of FMT, and 2 and 3 months after the last treatment, placed in sterile EP tubes, and then stored in a refrigerator at -80°C. We used the fecal genomic DNA extraction kit to extract total bacterial DNA from the stool samples. The concentration and purity of DNA samples were verified by nanodrop 2000 instrument and sent to Tianjin novo Zhiyuan sequencing Co., Ltd. for 16S sequencing. The Illumina Hiseq- PE250 technology-sequencing platform was used to perform 16S V3- V4 region sequencing of the sample using the double-end sequencing method, and corresponding data were statistically analyzed according to the sequencing results.

Statistical analysis

Obtained data were expressed as the mean ± standard error (se). Mean values were compared using the t-test. The Mann-Whitney test was used to compare the measurement data between two or more groups. The Wilcoxon rank sum test was used for comparison between two or more groups, and the chi-square test was used for comparison of the count data. A P value <0.05 indicated a statistically significant difference.


Baseline conditions of the subjects in this study

A total of 35 patients were enrolled in this study: 18 patients with chronic HBV infection, including 14 males and 4 females, aged between 24 to 71 y, with an average age of 44.83 ± 12.24 y (Among these, there were 10 cases of HBeAg-negative infection, and 8 cases of patients with CHB). 17 patients with hepatitis B cirrhosis, including 12 males and 5 females, aged between 41 to 82 y, with an average age of 54.59 ± 11.09 y (among these, there were 8 cases of Child-Pugh A grade, 8 cases of B grade, and 1 case of C grade) (Table 1).