Study On The Effect And Safety Of The Intensive Treatment Of Fecal Microbiota Transplantation In Inflammatory Bowel Disease

PART 1 Establishment and optimization of donor screening criteria and processes in fecal bacteria transplantation Background and AimsFecal bacteria transplantation(fecal microbiota transplantation,FMT)is a new type of "organ transplant" that regards the largest immune organ digestive tract in the human body as a mediated pathway through a series of filtration and enrichment of the faeces of healthy people(donor),The beneficial flora and functional flora were transplanted into the patient's gastrointestinal tract so that they were implanted in the digestive tract in order to repair,improve or reshape the intestinal microecological environment of the patients,and to further develop the normal intestinal flora function in order to participate in important regulation and metabolism,so as to achieve the treatment of microbial related intestinal and parenteral diseases.was officially listed by the U.S.Food and Drug Administration(FDA)in 2013 as an alternative therapy for CDI and RCDI to incorporate treatment guidelines.At present,it has been studied and applied continuously in the treatment of refractory Clostridium difficile infection,inflammatory bowel disease,irritable bowel syndrome,chronic diarrhea,chronic constipation,autism,hepatic encephalopathy and some parenteral diseases related to metabolic diseases(SARS).With the development of microbial systematics and the progress of sequencing technology,the symbiosis between flora and participation in the regulation of biological metabolism is gradually recognized.A large number of studies have shown that flora disorders are associated with a range of diseases,including Clostridium difficile infection,inflammatory bowel disease,obesity,asthma,depression,and so on.Fecal bacteria transplantation can change the structure and composition of the original intestinal flora of the patient,and make it tend to be composed of the flora of the dung donor.Therefore,in the course of transplantation,there is a risk of the host's new disease caused by the potential threat flora of the original donor.While there are currently internationally agreed guidelines to guide the clinical application of FMT,further high-quality research is needed on the donor.In this study,the concept of standardization and multidimensional of donor screening is proposed for the first time,and it is proposed to explore the standard and optimize donor screening standards and processes in order to realize the establishment of uniform standard process,and to provide higher evidence for the effectiveness and security evaluation of FMT.Methods and Results1.Establish a standardized screening process to determine strict and comprehensive screening criteriaBy retrieving databases such as PubMed,Medline,and so on,set up search keywords such as"FMT","fecal transplant","fecal microbiota transplantation","faecal transplant","donor","fecal donation","fecal donor","faecal donor" and so on,after screening from the Communist Party of China to obtain 48 results,a total of 215 cases involving donor,statistical analysis based on the donor characteristics in all studies and the standard of to improve,and add new consideration elements,establish a unique screening process and standards,in the form of personal information registration form,series of questionnaires,enrich the laboratory testing screening project,as far as possible overall care,in the existing research on intestinal microbial mechanism,as far as possible to avoid the factors that may bring the risk of transplantation.2.Screening of qualified donor bodiesWidely publicize recruitment,in strict accordance with the procedures and standards set out in 1 to screen,in line with the screening conditions to the alternative donor detailed explanation of the principle of fecal bacteria transplantation,until it is fully understood to sign the donation of informed consent,can become a qualified faecal donation donor in the project.3.Collect faecal samples from the donor for 16sRNA sequencing According to the observation point specification,the faecal samples of the donor were collected and frozen in the-80? refrigerator,and their DNA was extracted and sent to the I-sanger platform(Shanghai Majorbio Biomedical Technology Co.,Ltd.LTD)for 16sRNA sequencing.Results1.Establishing standardized donor screening conditions and process donors in this study by literature analysis.2.The most frequent blood tests were human immunodeficiency virus(HIV)(98%),hepatitis C virus(98%),hepatitis B virus(98%),hepatitis A virus(86%)and syphilis(84%).Other pathogens were screened in less than one-third of studies,for example cytomegalovirus(CMV)(31%),human T-lymphotropic virus(28%),Epstein-Barrvirus(EBV)(28%).Full blood count(27%),liver enzymes(27%),C-reactive protein(18%)and renal function tests(11%)were also commonly tested.Fewer Asian studies screened vancomycin-resistant enterococci(VRE)(6%vs 28%)and Giardia(28%vs 58%)than North American studies,whereas fewer North American studies screened CMV(14%vs 53%)and EBV(11%vs 55%)than European studies.with all tests sorted by continents in Appendix S7Characteristics of faecal flora in qualified donor bodies The intestinal flora of healthy donor has high diversity and species richness,among which the abundance of bacillus genus,Clostridium,Bifidobacterium and lactic acid bacteria is significantly higher than that of other fungi,while the genus Enterobacter,Enterococcus and Clostridium have lower abundance.In the results of 6 months of fecal bacteria sampling point,although the abundance of bacteria has changed,but the variation is still small,and its diversity remains stable,showing the dynamic stability of faecal flora over a period of time.PART 2 Chapter ? Efficacy evaluation of intensive treatment of fecal bacteria transplantation in inflammatory bowel disease Background and AimsInflammatory bowel disease(inflammatory bowel disease,IBD)has increased dramatically over the past 60 years and is a chronic non-specific disease with unclear causes.Epidemiological investigation and a large number of clinical mechanisms show that there are obvious flora disorders and heterogeneity in the intestinal flora of IBD patients,and the diversity and abundance of their flora are lower than those of healthy population,which is thought to be related to the abnormal regulation of metabolism,which triggers immune injury or defect in intestinal tract.FMT,as an intervention therapy with the repair or remodeling of intestinal flora,has received more and more attention,and it is superior to the efficiency and remission rate of traditional standard antibiotic treatment in Clostridium difficile infection,so it is thought that it may induce patients with inflammatory bowel disease to achieve clinical remission or endoscopic mucosal improvement.To date,there have been more than thousands of cases of FMT application and IBD clinical treatment in the world,but due to the absence of uniform norms on FMT methodology,most of the results of FMT in the effectiveness of IBD treatment are not consistent.Therefore,in the whole FMT treatment system,the focus of donor selection,the filtration and extraction of fecal bacteria,the method of transplantation,the route of introduction,the optimal dosage of FMT,the frequency of transplantation and many other aspects still need further in-depth study,and on the long-term safety of FMT,there is still a lack of adequate clinical data support.Based on the establishment and optimization of donor screening,this study is intended to replace the traditional method of enema or colonoscopy spraying,and on this basis,to strengthen the frequency of FMT transplantation,to FMT intensive treatment to observe and evaluate its clinical potency for patients with inflammatory bowel disease.Methods1.SubjectsIn strict accordance with the established inclusion criteria and exclusion criteria,into the group by the Southern Hospital Digestive medicine diagnosed as ulcerative colitis or Crohn's disease patients.2.MethodsAll subjects stopped all routine treatment for inflammatory bowel disease(including 5-asa,antibiotics,hormones,immunosuppressants,probiotics,etc.)at least 4 days before treatment.After endoscopic tet tube catheterization,FMT intensive treatment,that is,in the treatment cycle of the first and the third and the fifth days respectively through the Tet tube infusion of faecal fungus liquid.Use the 12th week after treatment as the end of the test to assess the clinical potency of the treatment intervention.The clinical improvement or effect of FMT intensive therapy was observed and evaluated from the aspects of clinical symptoms,psychological state and laboratory examination indexes,and the follow-up and evaluation of adverse events were carried out at the same time,after and after treatment,the 1th week,week 4th,week 12th and week 24th were followed up.All subjects were required to follow up until 24 weeks after treatment.The changes of fecal flora structure and components before and after FMT intensive therapy were analyzed by 16sRNA sequencing of fecal genome,and the functional pathways which played a key role were explored.3.Results3.1.Basic characteristics of the subjects In this experiment,13 patients with inflammatory bowel disease,including ulcerative colitis,10 cases,Crohn's disease in 3 cases.All subjects completed 24 weeks of follow-up follow-up.The average age of the subjects was 37.3 years(ranging from 18?56 ?),with 7:6 per cent for men and women.Smokers 3 people.The average duration of the subjects' illness was 4.77 years(1-11 years),the improved Mayo rating of the UC subjects was divided into 7.9 points,and the CD subjects ' best Cdai was divided into 205.50 points.3.2.Intensive treatment with faecal bacteria transplantation can significantly improve the clinical symptoms of the subjectsAmong the 13 subjects of IBD,10 subjects of UC and 3 subjects of CD all completed the observation record and evaluation of the end point of the test at week 12,and completed the 24-week follow-up without any lost cases.Among them,4 UC subjects achieved clinical remission(4/10,40%).4 UC subjects achieved clinical efficacy(4/10,40%).One CD subject achieved clinical remission,while the other two CD subjects were clinically ineffective.Fecal bacteria transplantation after intensive treatment,effective response to treatment or to relieve clinical subjects in test at the end,the laboratory indexes such as white blood cell count,neutrophil count,red blood cells sedimentation rate was significantly decreased(P<0.05),hemoglobin content was significantly increased(P<0.05),c-reactive protein has dropped significantly,but with the FMT fully improved correlation is not possible.Weight gain was significant before and after treatment.At the same time in the test at the end of the endoscopy results of subjects and the intestinal mucosa pathological compared with before treatment,4 cases of clinically ease of subjects,the colonoscopy examination showed the mucosa is smooth,blood vessels,texture clear,no active inflammation ",intestinal mucosa tissue pathology showed:basic normal mucosa,submucosa a few inflammatory cells infiltration,mild vascular congestion;The colonoscopy of the 4 subjects who responded effectively showed"mild mucosal hyperemia,no ulcers and new neoplasms".The pathological examination of the intestinal mucosa showed that part of the mucosa was shed by the compound epithelium,the adjacent mucosa was basically normal,and the infiltration of inflammatory cells in the submucosa was significantly reduced.In addition to the above clinical indicators,at the end test,achieve alleviate clinical or producing effective response of the subjects,the Mayo scores(P<0.05),the Best CDAI score(P=0.43),abdominal pain score(P<0.05),number of stools(P=0.171)and depression score(P<0.05)were significantly reduced,but the change of the number of stools and FMT intensive treatment failed to determine the correlation.3.3.Inflammatory cytokines were significantly altered after intensive treatment with faecal transplantationBefore and after intensive treatment of FMT,proinflammatory factors il-1(p<0.05),TNF-(p<0.05)showed a significant downward trend,while anti-inflammatory factors il-10(p<0.05)and TGF-(p<0.05)showed a significant increase trend.3.4.The structure and components of intestinal flora of the subjects changed significantly After FMT transplantation,the proportion of thick-walled bacteria in the subjects increased(p<0.05),the proportion of proteobacteria decreased(p<0.05),and the proportion of Fusobacterium nucleatum decreased significantly(p<0.05).In the gut microbiota analysis of subjects who responded and achieved clinical remission,the species richness and diversity of the gut flora showed a significant increase compared to the pre-treatment itself,and in each stool specimen The collection time point was significantly higher than that of non-responders.3.5.Function pathway abundance and metabolic information that play a significant role in intensive treatment of fecal bacteria transplantation The main COG functions of the subject's fecal microbes include:energy production and conversion,amino acid transport and metabolism,nucleotide transport and metabolism,carbohydrate transport and metabolism,coenzyme transport and metabolism,lipid transport and metabolism,and inorganic ion transport.metabolism.The most important of these are carbohydrate transport and metabolism.The analysis results showed that the microbial metabolic function in the fecal samples of the subjects was enriched after intensive treatment with fecal bacteria.After comparing the information to the KEGG database,the dominant metabolic pathway obtained is the glycolysis/gluconeogenesis pathway.4.DiscussionsThis experimental study shows that intensive treatment of fecal bacteria transplantation is an effective means for treating patients with inflammatory bowel disease.This study further confirmed that reshaping the intestinal environment can effectively lay a strong foundation for the treatment of fecal bacteria transplantation.At the same time,it further clarifies the importance of the donor as the basic source of the whole therapy.This research will greatly promote the clinical application of the fecal transplant,and the donor will inevitably be further studied in the future with the in-depth study of intestinal microbes.For long-term progress and improvement.At the same time,the results of this study show that FMT may provide a preliminary clinical basis for the exploration of FMT development mechanism by participating in functional metabolic pathways to regulate the expression of related inflammatory factors.