The distal gastrointestinal tract contains a large and BRIEF REVIEW. Therapeutic Potential of Fecal Microbiota Transplantation.

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1 GASTROENTEROLOGY 2013;145: BRIEF REVIEW Robert F. Schwabe and John W. Wiley, Section Editors Therapeutic Potential of Fecal Microbiota Transplantation LOEK P. SMITS, 1 KRISTIEN E. C. BOUTER, 1 WILLEM M. DE VOS, 2,3 THOMAS J. BORODY, 4 and MAX NIEUWDORP 1 1 Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2 Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands; 3 Department of Bacteriology and Immunology, Helsinki University, Helsinki, Finland; and 4 Centre for Digestive Diseases, New South Wales, Australia There has been growing interest in the use of fecal microbiota for the treatment of patients with chronic gastrointestinal infections and inflammatory bowel diseases. Lately, there has also been interest in its therapeutic potential for cardiometabolic, autoimmune, and other extraintestinal conditions that were not previously considered to be associated with the intestinal microbiota. Although it is not clear if changes in the microbiota cause these conditions, we review the most current and best methods for performing fecal microbiota transplantation and summarize clinical observations that have implicated the intestinal microbiota in various diseases. We also discuss case reports of fecal microbiota transplantations for different disorders, including Clostridium difficile infection, irritable bowel syndrome, inflammatory bowel diseases, insulin resistance, multiple sclerosis, and idiopathic thrombocytopenic purpura. There has been increasing focus on the interaction between the intestinal microbiome, obesity, and cardiometabolic diseases, and we explore these relationships and the potential roles of different microbial strains. We might someday be able to mine for intestinal bacterial strains that can be used in the diagnosis or treatment of these diseases. Keywords: Gutmicrobiota; Human Disease; Fecal Transplantation; Therapy. The distal gastrointestinal tract contains a large and diverse array of microorganisms, of which bacteria are the most dominant. 1 The community of at least bacteria is dominated by strict anaerobes and includes thousands of different species, many of which have not yet been cultured. These bacteria can interact with the intestinal mucosa and influence intestinal permeability; they are important for the absorption, distribution, metabolism, and excretion of nutrients 2 and can trigger (auto) immunity. 3 Recently developed high-throughput approaches, including metagenomic sequencing, have associated the composition of the microbiota with human health. 2,4,5 The composition of the intestinal microbiota can be altered with diet and prebiotics or probiotics, which produce mild temporary changes, or with antibiotics, which produce large changes in the bacterial composition of the intestines. 5 A new and underexplored method to alter the gastrointestinal microbiota involves fecal microbiota transplantation (FMT). The first known description of human donor feces as a therapeutic agent came from China. In the fourth century, Ge Hong, in the Handbook of Emergency Medicine, prescribed ingestion of feces for a variety of conditions. 6 Much later, Ralph Lewin reported that.consumption of fresh, warm camel feces has been recommended by Bedouins as a remedy for bacterial dysentery; its efficacy was confirmed by German soldiers in Africa during World War II. 7 The first use of FMT in mainstream medicine was described in 1958 for the treatment of pseudomembranous colitis (presumably due to Clostridium difficile infection [CDI]) by Eiseman et al. 8 FMT has since increased in popularity due to its efficacy and ease of use for the treatment of patients with CDI. We review the methodology and safety of modern FMT and the evidence to support its use in treating a variety of diseases (Figure 1). Methods of FMT FMT comprises the administration of a fecal solution from a donor into the intestinal tract of a recipient. How is the donor selected, how is the solution prepared, and how is the transplant material administered? Donor Selection Healthy donors are usually recruited from family members or friends as well as by newspaper advertisements. The risk of transmission of unknown pathogens via FMT cannot be excluded. 9 Therefore, it is important to carefully screen and select donors to avoid causing a new disease in the recipient. Potential donors should be questioned about their travel history, sexual behavior, previous operations, blood transfusions, and other factors that increase the risk of transmissible disease. 10 Potential Abbreviations used in this paper: CDI, Clostridium difficile infection; FMT, fecal microbiota transplantation; IBD, inflammatory bowel disease; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; SCFA, short-chain fatty acid; UC, ulcerative colitis by the AGA Institute /$

2 November 2013 FECAL MICROBIOTA TRANSPLANTATION 947 Figure 1. Disorders associated with alterations to the intestinal microbiota that could be treated by FMT. Green indicates disorders for which FMT has shown efficacy in randomized controlled trials (RCT), blue indicates disorders for which FMT has shown efficacy in case series studies, and black indicates disorders that have been associated with disruption of the intestinal microbiota. donors are also screened for a family history of autoimmune and metabolic diseases as well as malignancies (in first- and seconddegree family members). Once a donor is selected, blood and fecal samples must be screened for pathogens (see Table 1). 11 In a systematic review of 317 patients with recurrent CDI and pseudomembranous colitis, Gough et al found a slightly higher rate of disease resolution among recipients of transplanted fecal material from related donors (93%) than unrelated donors (84%). There were no significant differences in disease resolution when donors were of a different sex than the recipient. 12 Studies are under way to determine the composition of donor and recipient microbiota, including enterotypes 13 ; this information could increase the efficiency of FMT. Preparation of FMT Material There has been large heterogeneity among studies in the preparation of material for FMT. Although there have been several reviews on preparing infusions for FMT, 12,14 the lack of sufficient numbers and controls in most studies makes it difficult to draw solid conclusions. That said, infusions for FMT prepared using water have been reported to achieve higher rates of disease resolution than those using normal saline (98.5% vs 86%), although relapse of CDI was >2-fold higher with water than saline. 12 Other diluents, including milk and saline with psyllium, produced resolution rates of 94%. 12 A nonsignificant trend toward an improved outcome has also been observed with increasing weight of donor stool and volume of prepared solution; a 97% resolution rate was achieved in patients who received infusions >500 ml versus only 80% in those given <200 ml. 12 According to the Amsterdam protocol 10,15 (Table 1), donor stool ( g, dissolved in 500 ml sterile saline) is used, preferably within 6 hours of passage. In a recent case series, standardized frozen stool samples were used in FMT for CDI; the study reported results comparable to those of studies that used fresh fecal samples. 16 This finding indicates the feasibility of FMT in clinical practice, with establishment of fecal donor banks. It will be important to compare the effects of using the saline-based mixing and straining method versus an electrical blender to homogenize donor feces. These methods could affect the viability of strict intestinal anaerobes (blending can introduce air) and alter the bacterial load. Routes of Administration The fecal material for transplantation can be infused by various methods, including a nasogastric tube, a nasojejunal tube, upper tract endoscopy (esophagogastroduodenoscopy),

3 948 SMITS ET AL GASTROENTEROLOGY Vol. 145, No. 5 Table 1. Amsterdam Protocol for FMT via Gastroduodenoscopy Donor Screening for transmittable diseases Blood: for human immunodeficiency virus, human T-lymphocytic virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, cytomegalovirus, Epstein Barr virus, lues, Strongyloides, amoebiasis Fecal pathogens: bacteria (Helicobacter pylori antigen, Yersinia, Campylobacter, Shigella, Salmonella, enteropathogenic E coli), viruses (rotavirus, adenovirus, enterovirus, parechovirus, sapovirus, norovirus, and astrovirus), and parasites (triple feces test for ova and parasites, Giardia) Screening for other criteria Diarrhea Recent use of medications (within 3 months, mainly antibiotics or proton pump inhibitors) Risk factors for transmittable diseases Abnormal defecation patterns and symptoms of irritable bowel syndrome Feces Freshly produced (within 6 hours of treatment) At least 150 g (directly covered in 500 ml sterile saline 0.9% solution), subsequently filtered for a homogeneous solution Patient Placement of duodenal tube and small intestinal biopsies Bowel lavage with 1 2 L of macrogol through duodenal tube Administration of fecal solution through duodenal tube No antibiotics before procedure colonoscopy, or retention enema. 12 In a systematic review, Gough et al found that FMT via esophagogastroduodenoscopy, nasogastric tube, or nasoduodenal tube resolved CDI in 76% to 79% of patients compared with the mean worldwide rates of 91% for FMT via colonoscopy. 12,14,17 22 In a recent randomized controlled trial, duodenal infusion of donor feces was shown to be as effective as colonoscopic administration in the treatment of CDI. 10 Fewer studies, however, have compared routes of administration for other potential indications; the best route most likely depends on the anatomic location of the disease. In our experience, duodenal infusions of donor fecal material are safe and effective for the treatment of metabolic diseases. 15 Adverse Events To date, FMT appears to be safe; we have performed more than 200 fecal transplantation procedures at the Academic Medical Center in Amsterdam and more than 3000 at the Centre for Digestive Diseases in Sydney, Australia, without any serious adverse events. 10,15,23 Most patients treated with FMT experience diarrhea on the day of infusion, and a small percentage report belching and/or abdominal cramping or constipation. These observations are in line with published case reports and series of FMT for CDI 12,19 ; adverse events were reported for only 3 of 317 patients (upper gastrointestinal tract bleeding, peritonitis, or enteritis). In another case report, nasoduodenal FMT for Crohn s disease resulted in transient adverse effects, including fever and abdominal tenderness in 3 of 4 patients. However, these effects disappeared for all patients over the following 2 days. 24 In another case report, in which FMT was administered during colonoscopy, these side effects were not observed 25 ; most importantly, long-term follow-up studies have found that FMT is relatively free of adverse effects. 26 Despite the fact that FMT appears to be safe, there are insufficient long-term follow-up data; thus, a potential association between FMT and infection, inflammation, or gastrointestinal malignancies requires further study. Clinical Use of FMT To date, most clinical experience has focused on the use of FMT in patients with relapsing CDI or occasionally severe CDI. Subsequently, FMT was used to treat patients with inflammatory bowel disease (IBD) complicated by CDI. From there, FMT was used to treat patients with only IBD and later, via serendipitous observations, those with previously unconsidered conditions. Relapsing CDI The most frequently reported use of FMT has been in the treatment of patients with CDI. CDI occurs most commonly in patients with disruptions of the colonic microbiota by antibiotics. Tvede and Rask-Madsen (1989) 27 and Khoruts et al (2010) 17 reported that the intestinal microbiota of patients with relapsing CDI has lower proportions of Bacteroides and Firmicutes than that of healthy subjects. Unsurprisingly, antibiotics such as metronidazole or vancomycin do not eradicate CDI in a large proportion of patients and fail to correct the underlying microbial deficiencies. 17,27 FMT, in contrast, eradicates CDI and replaces missing components of the microbiota (such as Bacteroidetes species) 28 to increase bacterial diversity, similar to that of healthy donors. 10,28 Two systematic reviews concluded that FMT resolves recurrent CDI in approximately 90% of patients. 12,19 Duodenal infusion of healthy donor feces resolved recurrent CDI in 82% of patients (defined by the absence of CD-associated diarrhea, without relapse, within 10 weeks), whereas vancomycin resolved CDI in only 31% of patients. 10 Severe CDI In the largest retrospective case series of FMT for patients with severe or complicated CDI (n ¼ 13), 29 Aroniadis et al collected data on patients for an average of 15 months after the procedure. They found that 84% of patients were cured by FMT as a primary treatment (resolution of symptoms without recurrence within 90 days) and 92% of patients were cured by FMT as a secondary treatment (resolution of symptoms after a course of vancomycin or repeat FMT). 30 Other similar case studies reported rapid recovery after FMT in these gravely ill patients CDI in IBD Historically, the coexistence of CDI and IBD was uncommon; tests for Clostridium difficile were rarely recommended for patients with flares of IBD. However, CDI has been associated with increased disease severity of IBD and mortality. The latest American College of Gastroenterology guidelines on CDI recommend CDI testing of all patients hospitalized for IBD flares. 33 However, although treatment guidelines exist for CDI, there are none for patients with CDI and IBD. In our experience with this patient population, after FMT and eradication of CDI, the severity of IBD is gradually reduced, and patients have improved responses to medications for IBD. 25 Reddy

4 November 2013 FECAL MICROBIOTA TRANSPLANTATION 949 and Brandt recently extensively reviewed CDI in patients with IBD. 34 IBD The etiology of IBD is unknown, but interactions between bacterial and host cells seem to be involved in pathogenesis. The intestinal microbiota of patients with IBD appears to have reduced diversity compared with that of healthy subjects, based on studies reporting 25% fewer microbial genes. Patients with IBD have reduced numbers of the phyla Firmicutes and Bacteroidetes and increased numbers of Actinobacteria and Proteobacteria However, it is not clear whether these differences are a cause or consequence of the development of IBD. The rationale for using FMT to treat patients with ulcerative colitis (UC) dates back to when it was used to treat pseudomembranous enterocolitis; CDI was not known to be the cause at that time. 8,38,39 In 1988, the first patient with idiopathic UC was treated with FMT, resulting in durable clinical and histological cure. 40 Then, a case report published in 2003 documented the complete clinical, colonoscopic, and histological reversal of UC in 6 patients with severe, relapsing UC. 41 Recent studies have confirmed these findings; a meta-analysis of FMT for patients with IBD, conducted by Anderson et al, 42 found that 63% of patients with UC entered remission, 76% were able to stop taking medications for IBD, and 76% experienced a reduction in gastrointestinal symptoms. Similarly, Brandt and Aroniadis 26 performed a long-term follow-up study of FMT in 6 patients with UC and reported that all patients had reduced symptoms. Maximal benefits were observed in 2 patients with newly diagnosed UC and CDI and in one who had been taking antibiotics. These findings indicate that remission of UC is possible with (multiple) FMTs for a subgroup of patients. There are currently 6 registered trials testing FMT for patients with IBD (see Irritable Bowel Syndrome Some studies have associated altered composition of the intestinal microbiota (decreased diversity and decreased numbers of Bacteroidetes) with subsets of irritable bowel syndrome. 43,44 There are approximately 50 published cases of use of FMT to treat patients with diarrhea-predominant irritable bowel syndrome and constipation-predominant irritable bowel syndrome In a case series of 3 patients with chronic constipation, FMT greatly reduced constipation during the month after FMT; all patients defecated daily (or every other day) without the assistance of laxatives. In a long-term follow-up study, 45 patients with chronic, severe constipation were treated with a liquid culture comprising 20 cultured, nonpathogenic enteric anaerobes and aerobes, including Bacteroides, Escherichia coli, and Lactobacillus species. 47 Substantial improvements were reported in 30 patients (60%), who had improved defecation and an absence of bloating and abdominal pain during a follow-up period of 9 to 19 months. Chronic Fatigue Syndrome Intestinal dysfunction is a symptom in many patients with chronic fatigue syndrome. These patients have been reported to have alterations in the intestinal microbiota compared with healthy subjects (controls). 49 Patients with chronic fatigue syndrome were found to have a reduced proportion of gram-negative E coli (49% of all aerobic flora) compared with controls (92.3% of all aerobic flora), whereas lactic acid producing gram-positive facultative anaerobic Enterococcus and Streptococcus species seem to be overrepresented in patients with chronic fatigue syndrome. 50,51 In an uncontrolled study of 60 patients with chronic fatigue syndrome and gastrointestinal dysfunction treated with FMT, 50% had resolved sleep deprivation, lethargy, or fatigue during a 15- to 20-year follow-up period. 52 Metabolic and Cardiovascular Disorders Alterations in the composition of the intestinal microbiota have also been associated with obesity and type 2 diabetes mellitus. 5,53 However, the previously reported increase in Firmicutes phylum and decrease in Bacteroidetes observed in obese mice have not been confirmed in humans. 54 Alterations in the intestinal microbiota might contribute to the development of cardiometabolic diseases by increasing intestinal permeability; this would lead to metabolic endotoxemia and chronic inflammation, which contribute to metabolic and cardiovascular disease. 5 Obese subjects have increased postprandial plasma levels of bacteria or their proteins (mainly lipopolysaccharide or endotoxin), 55 most likely because of increased intestinal permeability. 56 Diet-derived fibers, which are metabolized and fermented to short-chain fatty acids (SCFAs; including acetate, propionate, and butyrate) by intestinal bacteria, might also be involved. The types and amounts of SCFAs produced are believed to vary with the microbial composition of the intestine (Figure 2). 5 In addition to serving as an energy source for intestinal epithelium and liver (SCFAs are predominantly transported via the portal vein after intestinal absorption), SCFAs are believed to have immunomodulatory effects in that they reduce intestinal permeability. 2 Translocation of lipopolysaccharide from the intestine to the portal vein is believed to be involved in the pathogenesis of obesity-associated low-grade inflammation and subsequent insulin resistance in mice. 57 These phenotypes were partly reversed on administration of the propionate-producing Akkermansia muciniphila, 58 which normalizes intestinal permeability and intestinal mucus layer thickness. In obese men, 15 FMT from lean donors significantly increased insulin sensitivity, most likely by increasing concentrations of butyrate-producing intestinal bacteria in the small and large intestine. These findings indicate that the intestinal microbiota could actually cause obesity and insulin resistance. There is a strong association among fatty liver disease (nonalcoholic fatty liver disease [NAFLD] and

5 950 SMITS ET AL GASTROENTEROLOGY Vol. 145, No. 5 Figure 2. Mechanisms by which alterations to the intestinal microbiota can contribute to disease. The composition of the intestinal microbiota is determined by genetic factors, diet, and medications (such as antibiotics) (left side). When these change, so can the intestinal microbiota (right side). Changes in the microbiota might induce production of specific autoantibodies (produced via molecular mimicry and can lead to autoimmune disorders) or allow expansion of pathogenic species such as C difficile. An altered intestinal microbiota could also change production of compounds such as SCFA. This affects lipid and glucose metabolism as well as inflammation. SCFAs can also increase intestinal permeability, which leads to endotoxemia. nonalcoholic steatohepatitis [NASH]), the intestinal microbiota, and obesity, which is not surprising because NAFLD and NASH are associated with obesity and insulin resistance. Henao-Mejia et al found that increased intestinal permeability and metabolic endotoxemia, by altering the composition of the intestinal microbiota, contribute to the progression of NASH in mice. 59 They found that transfer of specific microbiota to mice on methionine- and choline-deficient diets increased the severity of NAFLD and NASH via a process that involved TLR4. These findings indicated that the intestinal microbiota can contribute to the progression of NAFLD and NASH. Data from human studies also support the concept that changes in the intestinal microbiota contribute to the development of fatty liver diseases. Patients with NAFLD have increases in intestinal permeability, 60 endotoxemia, 61 and numbers of Gammaproteobacteria, whereas they have reduced numbers of Bacteroidetes 62,63 compared with healthy subjects. Moreover, epidemiological studies have associated NAFLD with cardiovascular disease. 64 Interestingly, the association between bacterial translocation (determined by

6 November 2013 FECAL MICROBIOTA TRANSPLANTATION 951 plasma levels of endotoxin) and risk of cardiovascular events was proposed 14 years ago. 65 It was supported by the detection of DNA from intestinal bacteria in inflamed atherosclerotic plaques as well as reported differences in the composition of the intestinal microbiota between patients with symptomatic atherosclerosis and healthy subjects. 66,67 Further support came from the finding that the atherogenic compound trimethylamine-n-oxide, produced by intestinal microbiota from choline and carnitine, contributes to vascular inflammation FMT-based intervention studies are currently under way to see if this technique can reduce features of fatty liver disease and vascular inflammation. Autoimmune Diseases The incidence of autoimmune diseases is increasing worldwide, but they remain among the most poorly understood conditions in terms of causes or cures. 3 Various environmental factors and infectious agents have been proposed to contribute to their pathogenesis. A number of bacteria-derived proteins have been identified that act as superantigens, inducing nonspecific activation of self-reactive B and T cells via molecular mimicry. Multiple sclerosis, which is caused by myelin-directed autoimmunity, might be treated by FMT. In 3 patients with multiple sclerosis who underwent FMT daily for 1 to 2 weeks for constipation, neurological symptoms resolved for 2 to 15 years. 71 Idiopathic thrombocytopenic purpura is believed to be caused by production of autoantibodies against platelet surface antigens 72 after viral or bacterial infection. 3 In a patient with this disorder who underwent FMT for UC, a progressive but significant increase in platelet levels was reported that lasted for several years. 73 Discussion The intestinal microbiota can be manipulated to treat diseases in humans; for example, FMT seems to be a safe and promising treatment for recurrent CDI. Welldesigned randomized controlled trials are needed to establish the efficacy of FMT for other diseases, and these studies should analyze the composition of the small intestinal and fecal microbiota before and after FMT so researchers can better understand the mechanisms of this therapy. 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8 November 2013 FECAL MICROBIOTA TRANSPLANTATION Borody T, Campbell J, Rorers M. Reversal of idiopathic thrombocytopenic purpura with fecal microbiota transplantation (FMT). Am J Gastroenterol 2011;106: De Vos WM, Nieuwdorp M. Genomics: a gut prediction. Nature 2013; 498: Received July 14, Accepted August 26, Reprint requests Address requests for reprints to: Max Nieuwdorp, MD, PhD, Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Room F , 1105 AZ Amsterdam, The Netherlands. m.nieuwdorp@amc.uva.nl; fax: (31) Conflicts of interest The authors disclose the following: Thomas J. Borody has a pecuniary interest in the Centre for Digestive Diseases, where fecal microbiota transplantation is a treatment option for patients and he has filed patents in this field. The remaining authors disclose no conflicts. Funding L. P. Smits, W. M. de Vos, and M. Nieuwdorp were supported by a grant from CVON (IN-CONTROL) (CVON number 2012). K. E. C. Bouter was supported by a Rembrandt Grant W. M. de Vos was also supported by grants from the Academy of Finland, European Research Council, and the Netherlands Scientific Research Organization (NWO).