Clinical Gastroenterology and Hepatology 2014;-:-–-
Development of Fecal Microbiota Transplantation Suitable for Mainstream Medicine Alexander Khoruts,*,‡,a Michael J. Sadowsky,‡,§,a and Matthew J. Hamilton‡ *Division of Gastroenterology, Department of Medicine, Center for Immunology, ‡BioTechnology Institute, §Department of Soil, Water, and Climate, Microbial and Plant Genomics Institute, University of Minnesota, Minneapolis, Minnesota Fecal microbiota transplantation has emerged as an increasingly common treatment for patients with refractory Clostridium difficile infection. Although it can be relatively simple to perform, a number of challenges need to be overcome before this procedure is widely accepted in mainstream clinical practice. Most of the solutions to these challenges already exist, but some need further optimization and testing. Standardized fecal microbiota is being developed as a therapeutic agent, although it clearly challenges some of the existing paradigms of drug development, delivery, and regulation.
via endoscopy typically are driven by the availability of specialty expertise and the relative potential for reimbursement, rather than data on safety and efficacy. None of these issues represent insurmountable hurdles, and the development of an efficient, safe, and reliable transplant mechanism is certainly within technological reach.
Keywords: Microbiota; Fecal Transplant; Microbiome; Clostridium difficile infection.
Historically, an ideal FMT donor was considered a close family member, an intimate partner, or a trusted friend. Such donors were thought to carry less infection risk because presumably they already had the opportunity to share different pathogens, known and unknown. In addition, there was an implicit assumption that commensal organisms were more likely to be shared among close household members than outsiders, which might improve their engraftment in case there was an immunologic barrier. Today, we know that microbiota from unrelated donors efficiently engrafts into patients treated for refractory CDI and there is no evidence that relatedness of a donor impacts the clinical outcome.1,3,4,6 Importantly, however, a physician’s responsibility in providing safe transplant material is not removed by tasking the patient with identifying the donor. Recent scientific advancements have reframed our understanding of distal gut commensal microbiota, which now generally is recognized as a metabolic organ integral to human physiology.10 This organ plays important roles in energy metabolism, and development and function of the immune and nervous systems. FMT provides an opportunity to restructure the patient’s microbiota to approximate that of the donor, which is why the procedure is being investigated as a potential treatment for
ecal microbiota transplantation (FMT) increasingly has become part of clinical practice in dealing with Clostridium difficile infection (CDI) that is otherwise unresponsive to standard antibiotic therapies. In contrast to antibiotic-based approaches, which have major damaging bystander effects on commensal gut microbiota, FMT results in restoration of the normal distal gut microbial community structure.1–6 The clinical efficacy of FMT in curing refractory CDI has been shown in multiple case series performed across the world,7,8 and the one controlled randomized study performed to date had to be stopped prematurely because of the clear superiority of FMT compared with the standard therapy group.3 However, even though FMT is now listed in some expert guidelines of standard practice for treating refractory CDI,9 it is not yet mainstream medicine. In part, this is because of a number of practical barriers that are associated with the procedure in a typical clinical setting. Chief among these is that many patients are unable to identify a suitable donor. Moreover, the effort and expense involved in qualifying the donor by history, physical examination, and laboratory testing are not reimbursed adequately. Preparation of the material suitable for administration is intrinsically and aesthetically challenging and ideally should be performed in the laboratory setting with appropriate safety precautions for the staff involved in processing the material. The choices of specific routes of administration, whether by retention enema, nasogastric or nasoduodenal tube, or
F
Roadblock Donor Selection
a
Authors share co-first authorship.
Abbreviations used in this paper: CDI, Clostridium difficile infection; CGMP, Current Good Manufacturing Practices; FDA, Food and Drug Administration; FMT, fecal microbiota transplantation; PCR, polymerase chain reaction. © 2014 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2014.11.014
2
Khoruts et al
inflammatory bowel disease, metabolic syndrome, irritable bowel syndrome, and even autism.11,12 Therefore, it is appropriate to consider the general health of the donor, much beyond mere infectious risk. In practice, it is difficult for patients, already exhausted by their disease, to find a donor who would qualify under strict criteria of health (eg, ideal weight and no history of food intolerances or allergies; lack of medications; and no elements of metabolic syndrome, gastrointestinal symptoms, abdominal surgeries, and so forth). Of course, these considerations may seem insignificant in the context of an elderly patient drained by repeated bouts of CDI and facing possible death. However, the patient also may be middle-aged, or even a young teenager or a child, perhaps with underlying inflammatory bowel disease. In our clinical experience, virtually all patients, regardless of age or background, are relieved when they are told that they do not need to bring in their own donor, even before criteria for donor selection are explained. The donor problem can be solved by the development of a dedicated, standardized, donor program. Such effort has certain parallels to blood donation, and it is less common in current medical practice to be restricted to related donors anymore. In fact, related donors may carry a higher infectious risk, possibly because they may not be able to reveal potential risk factors and may have shared exposure events.13 Screening and testing criteria for stool donors in standardized donor programs generally incorporate all the elements used in qualifying blood donors. However, exclusion criteria for stool donors also are based on the results of screening and testing for additional systemic problems, such as metabolic syndrome, diabetes, autoimmunity, irritable bowel syndrome, food intolerances, allergies, neurologic and psychiatric problems, and so forth.14,15 Obviously, potential donors cannot have a history of recent antibiotic exposure. In our center, we were only able to qualify a minority of recruited candidates (w10%). However, the program is feasible because qualified donors can provide repeated donations, supplying sufficient material for an extensive FMT program.
Technological Primer Material Processing Stool is a complex substance containing undigested food elements, cellular and chemical materials shed from the host intestinal tract, and large numbers of microorganisms. FMT can be performed using whole homogenized stool, although such a procedure can be very challenging aesthetically. However, it is possible to separate the microbial fraction from fecal material using a series of sieves, centrifugation, and filtration steps in the laboratory.14 Even more importantly, it is possible to freeze this microbial fraction in a cryoprotectant, while
Clinical Gastroenterology and Hepatology Vol.
-,
No.
-
maintaining the viability of the different microbial taxa and clinical efficacy of the preparation.4,14 There are several important advantages in using the frozen microbial fraction instead of the freshly prepared stool in FMT. First, the material is no longer aesthetically challenging, having lost most of the potent pungent odor associated with stool. Second, the preparation can be quantified in terms of numbers and types of bacteria present rather than the crude measure of stool weight, which can vary in bacterial content by an order of magnitude between individual donations. Most importantly, freezing and storing the preparation is critical to enabling up-to-date testing of the donor and the actual material intended for clinical use. A number of stool tests require significant periods of time, including stool cultures for bacterial pathogens and polymerase chain reaction (PCR) tests for viral pathogens, such as rotavirus and norovirus. In addition, seroconversion toward viral infections, such as human immunodeficiency virus and hepatitis, may lag by several weeks after acquiring the infection. The ability to bank the microbiota material before release allows performance of rigorous testing and elimination of uncertainty associated with freshly prepared material. The entire process of producing fecal microbiota material can be standardized in accordance with the Current Good Manufacturing Practices (CGMPs), which can be used and enforced by the US Food and Drug Administration (FDA) to ensure proper design, monitoring, and control of the manufacturing processes and facilities. All human pharmaceuticals are required to follow CGMPs to prevent product contamination and process deviations that may alter safety or therapeutic activity. At first glance, it may seem absurd to require adherence to CGMPs in the manufacture of fecal microbiota. Although it may be illogical to require CGMPs for FMTs practiced today by individual physicians or even tested in pilot academic clinical trials, adherence to CGMPs is absolutely critical to large-scale manufacture of fecal microbiota preparation that may enter routine clinical practice. The process includes rigorous protocols in securing the raw materials, maintenance of robust operating procedures, reliable laboratory testing procedures, detecting product quality deviations, and providing release of materials. The ultimate purpose in the manufacture of a product as complex as fecal microbiota is not to obtain precise compositional consistency, which is impossible given that composition of every donation is somewhat different. However, CGMPs do ensure that the manufacturing process is consistent between different batches and any possibility of introducing risk is minimized.
Next Challenges in Fecal Microbiota Transplantation Although the ready availability of liquid or frozen suspensions of microbiota would greatly simplify the
-
2014
clinical practice of FMT today, these preparations require ultra-low-temperature freezer facilities and specialized medical personnel for administration, regardless of the chosen route. The development of lyophilized preparations that can be stored at room temperature and potentially encapsulated clearly would constitute the next step forward. This requires careful testing of cryopreservation protocols suitable for this application. Although the preservation of different, individual microorganisms in lyophilized form has been accomplished for decades, the protocols need to be optimized for preserving the myriad of the different, but essential, taxa present in the complete fecal microbiota. The encapsulation process requires a number of special considerations unique to FMT. Obviously, it should not introduce toxic substances into the microbiota preparation to ensure that viability is not diminished. It also is desirable to release the capsule contents in the distal intestine because premature release may introduce a risk of aspiration and possible loss of viability of some microorganisms during their journey through the harsh digestive tract environment. The latter currently may not seem to be a critical problem because liquid preparations of homogenized stool appear clinically successful when administered by a nasoduodenal route,3 and oral administration of encapsulated frozen liquid microbiota remains curative of CDI.16 Nevertheless, the issue becomes more important for optimizing dosing of freeze-dried preparations, considering that the volume requirements within capsules need to accommodate both microorganisms and cryoprotectant agents. The stomach pH is likely to be a major variable in designing capsule release. Many patients take acidsuppressing medications and some may have neutral stomach pH as a result of autoimmune gastritis, which can result in premature dissolution of a pH-sensitive shell. Gastroparesis and severe esophageal reflux may constitute relative future contraindications to FMT capsules. Uncertain risks also may exist in patients with small-bowel Crohn’s disease, which may lead to increased dwell time of the capsule in areas of stricture and content release over ulcerated areas. An important issue in the development of nextgeneration FMT products likely will concern material composition. At this time, donor selection is based primarily on clinical history, physical examination, and laboratory tests. Notably, tests used currently for enteric pathogen detection were developed originally for the diagnosis of patients with symptomatic diarrhea. Their current use in testing asymptomatic donors is logical, but not validated for sensitivity with respect to pathogens that may be present in low abundance or clinical importance in the context of healthy microbial communities. Laboratory protocols undoubtedly will evolve and increasingly use molecular diagnostics tools, including multiplex PCR, microfluidic (digital PCR), and microarray technology.17 These methods may increase the efficiency, sensitivity, and range of pathogen detection, although we
FMT for Mainstream Medicine
3
also may discover that the pathogenicity of some microorganisms may be dependent on the rest of the microbial community composition and specific host factors. Ultimately, we also may apply metagenomics and other “omics” technologies to identify microbiota optimal for the recipient’s health.18 This currently is not quite possible, in large part because our understanding of normal is very limited. Concern over infectious risk intrinsic to fecal microbiota used for FMT has been one of the main articulated reasons for the drive to develop defined, minimal, consortia of microorganisms, which can be propagated outside the human host, yet retain the curative properties of the far more complex microbiota present in stool. Several such highly simplified microbial assemblies, selected primarily on the basis of favorable ex vivo growth characteristics, were shown to be clinically effective in animal models and human patients with CDI.19,20 However, the long-term effects of this inoculum is unknown and large-scale manufacturing of defined microbiota products has its own set of challenging problems, including the potential loss of efficacy associated with adaptation of microorganisms to the in vitro environment. The development of defined microbial consortia undoubtedly will continue, although the selection of microbiota likely will become increasingly mechanistically informed. For example, microorganisms involved in secondary bile acid metabolism may be especially critical for the prevention of CDI recurrence.4,21 It will be highly desirable to identify compositional features that enhance the stability and resilience of microbial communities in the gut, characteristics likely to be critically important in patients with refractory CDI who often are exposed to antibiotic treatments. In addition, defined, simplified microbiota may function as short-term scaffolding in rebuilding more complex microbial communities in the gut19; therefore, because such products are introduced into patients, it will be important to determine whether in the long term the resulting composition of microbiota will approximate normal microbiota or deviate in some unexpected way. The spectacular success of FMT in treating refractory CDI has provided a boost to various biotechnology startup manufacturing companies that are attempting to harness the power of the microbiome for novel therapeutics development.22 CDI remains one of the foundational targets, and different companies are taking varying approaches to develop fecal microbiota–based products, including standardized whole donor-derived microbiota; highly simplified, defined microbial consortia; and hybrid products such as SER-109, the lead spore-based compound from Seres Health (Cambridge, MA), which recently was reported to have promising early results in recurrent CDI.23 The pace of discovery and clinical validation is accelerating, and it appears very likely that a range of highly effective therapeutic options for CDI and perhaps other indications soon will be introduced into mainstream medicine.
4
Khoruts et al
However, in additional to the many scientific and technical hurdles, developers also are challenged by the fluid regulatory framework and uncertainties in the intellectual property landscape. The FDA has determined that fecal microbiota fits the legal definition of a drug under US law. FMT has not yet been approved by the FDA for any indication, and currently constitutes an investigational agent. Given the heavy regulatory burden associated with the use of such agents and the lack of therapeutic alternatives for too many patients, the FDA issued a policy of “enforcement discretion”23 regarding investigational new drug requirements for use of FMT by individual physicians in the treatment of CDI not responding to standard therapies. However, the typical requirement for an investigational new drug application in formal clinical trials also has been applied unevenly to commercial entities.24 In addition, the enforcement discretion policy, perhaps unintentionally, has opened the possibility of establishing nonprofit stool banks, such as OpenBiome at the Massachusetts Institute of Technology, which is able to ship standardized fecal microbiota across state lines with only limited regulatory oversight.24 The FDA, charged with the incredibly difficult task of protecting patients today and in the future, as well as guiding the development of better therapeutics, continues to adjust its policies to accommodate the new paradigms intrinsic to this field. As a drug, live microbiota is unparalleled in its compositional and metabolic complexity. Moreover, its composition is intrinsically highly dynamic and responsive to external factors such as diet.25 This complexity may prove too unsettling for the regulators and may require paradigm shifts as it once did for other cellular therapies. Functionally, gut microbiota fit the description of an organ, and given its tight co-evolutionary linkage to its specific host species, it can be considered a human organ composed of microbial cells.10,26 Therefore, it may be reasonable to borrow elements of regulation for microbiota products from those applied to tissue transplantation.27 Most importantly, therapeutics development in this area should be guided by the best science. Undoubtedly, that will require reworking of longestablished paradigms of the germ theory of disease and acceptance of a new conceptual framework that is grounded in microbial ecology and the growing understanding of microbiota-host interactions. Ultimately, we all—physicians, scientists, developers, and regulators—need to be informed by continued research, basic and clinical, to allow establishment of this promising new class of therapeutics into mainstream medicine.
References 1. Hamilton MJ, Weingarden AR, Unno T, et al. High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria. Gut Microbes 2013;4:125–135. 2. Shahinas D, Silverman M, Sittler T, et al. Toward an understanding of changes in diversity associated with fecal
Clinical Gastroenterology and Hepatology Vol.
-,
No.
-
microbiome transplantation based on 16S rRNA gene deep sequencing. MBio 2012;3. 3. van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 2013;368:407–415. 4. Weingarden AR, Chen C, Bobr A, et al. Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection. Am J Physiol Gastrointest Liver Physiol 2014;306:G310–G319. 5. Fuentes S, van Nood E, Tims S, et al. Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection. ISME J 2014;8:1621–1633. 6. Shankar V, Hamilton MJ, Khoruts A, et al. Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal microbiota transplantation. Microbiome 2014;2:13. 7. Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis 2011;53: 994–1002. 8. Kassam Z, Lee CH, Yuan Y, et al. Fecal microbiota transplantation for Clostridium difficile infection: systematic review and meta-analysis. Am J Gastroenterol 2013;108: 500–508. 9. Surawicz CM, Brandt LJ, Binion DG, et al. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am J Gastroenterol 2013;108:478–498; quiz 499. 10. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science 2005;307:1915–1920. 11. Aroniadis OC, Brandt LJ. Fecal microbiota transplantation: past, present and future. Curr Opin Gastroenterol 2013;29:79–84. 12. Smits LP, Bouter KE, de Vos WM, et al. Therapeutic potential of fecal microbiota transplantation. Gastroenterology 2013; 145:946–953. 13. Starkey JM, MacPherson JL, Bolgiano DC, et al. Markers for transfusion-transmitted disease in different groups of blood donors. JAMA 1989;262:3452–3454. 14. Hamilton MJ, Weingarden AR, Sadowsky MJ, et al. Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection. Am J Gastroenterol 2012;107:761–767. 15. Bakken JS, Borody T, Brandt LJ, et al. Treating Clostridium difficile infection with fecal microbiota transplantation. Clin Gastroenterol Hepatol 2011;9:1044–1049. 16. Youngster I, Russell GH, Pindar C, et al. Oral, capsulized, frozen fecal microbiota transplantation for relapsing Clostridium difficile infection. JAMA 2014;312:1772–1778. 17. Guarino A, Giannattasio A. New molecular approaches in the diagnosis of acute diarrhea: advantages for clinicians and researchers. Curr Opin Gastroenterol 2011;27:24–29. 18. Lamendella R, VerBerkmoes N, Jansson JK. ‘Omics’ of the mammalian gut–new insights into function. Curr Opin Biotechnol 2012;23:491–500. 19. Lawley TD, Clare S, Walker AW, et al. Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog 2012;8:e1002995. 20. Petrof EO, Gloor GB, Vanner SJ, et al. Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut. Microbiome 2013;1:3.
-
2014
FMT for Mainstream Medicine
5
21. Buffie CG, Bucci V, Stein RR, et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 2014 Epub ahead of print.
26. Ochman H, Worobey M, Kuo CH, et al. Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol 2010;8:e1000546.
22. Olle B. Medicines from microbiota. Nat Biotechnol 2013; 31:309–315.
27. Smith MB, Kelly C, Alm EJ. Policy: how to regulate faecal transplants. Nature 2014;506:290–291.
23. Guidance for Industry: Enforcement Policy Regarding Investigational New Drug Requirements for Use of Fecal Microbiota for Transplantation to Treat Clostridium difficile Infection Not Responsive to Standard Therapies. Available from: http://www. fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory Information/Guidances/Vaccines/ucm361379.htm. 24. Ratner M. Fecal transplantation poses dilemma for FDA. Nat Biotechnol 2014;32:401–402. 25. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2014; 505:559–563.
Reprint requests Address requests for reprints to: Alexander Khoruts, MD, Department of Medicine, Division of Gastroenterology, University of Minnesota, 2101 6th Street SE, Medical Biosciences Building, Room 3-184, Minneapolis, Minnesota 55414. e-mail: [email protected]; fax: (612) 625-2199. Conflicts of interest The authors disclose the following: Alexander Khoruts, Michael Sadowsky, and Matthew Hamilton have received funding from CIPAC, LLC, to perform research on frozen fecal microbiota; and Michael Sadowsky and Matthew Hamilton have provided consulting services for CIPAC.
Development of Fecal Microbiota Transplantation Suitable for Mainstream Medicine Alexander Khoruts,*,‡,a Michael J. Sadowsky,‡,§,a and Matthew J. Hamilton‡ *Division of Gastroenterology, Department of Medicine, Center for Immunology, ‡BioTechnology Institute, §Department of Soil, Water, and Climate, Microbial and Plant Genomics Institute, University of Minnesota, Minneapolis, Minnesota Fecal microbiota transplantation has emerged as an increasingly common treatment for patients with refractory Clostridium difficile infection. Although it can be relatively simple to perform, a number of challenges need to be overcome before this procedure is widely accepted in mainstream clinical practice. Most of the solutions to these challenges already exist, but some need further optimization and testing. Standardized fecal microbiota is being developed as a therapeutic agent, although it clearly challenges some of the existing paradigms of drug development, delivery, and regulation.
via endoscopy typically are driven by the availability of specialty expertise and the relative potential for reimbursement, rather than data on safety and efficacy. None of these issues represent insurmountable hurdles, and the development of an efficient, safe, and reliable transplant mechanism is certainly within technological reach.
Keywords: Microbiota; Fecal Transplant; Microbiome; Clostridium difficile infection.
Historically, an ideal FMT donor was considered a close family member, an intimate partner, or a trusted friend. Such donors were thought to carry less infection risk because presumably they already had the opportunity to share different pathogens, known and unknown. In addition, there was an implicit assumption that commensal organisms were more likely to be shared among close household members than outsiders, which might improve their engraftment in case there was an immunologic barrier. Today, we know that microbiota from unrelated donors efficiently engrafts into patients treated for refractory CDI and there is no evidence that relatedness of a donor impacts the clinical outcome.1,3,4,6 Importantly, however, a physician’s responsibility in providing safe transplant material is not removed by tasking the patient with identifying the donor. Recent scientific advancements have reframed our understanding of distal gut commensal microbiota, which now generally is recognized as a metabolic organ integral to human physiology.10 This organ plays important roles in energy metabolism, and development and function of the immune and nervous systems. FMT provides an opportunity to restructure the patient’s microbiota to approximate that of the donor, which is why the procedure is being investigated as a potential treatment for
ecal microbiota transplantation (FMT) increasingly has become part of clinical practice in dealing with Clostridium difficile infection (CDI) that is otherwise unresponsive to standard antibiotic therapies. In contrast to antibiotic-based approaches, which have major damaging bystander effects on commensal gut microbiota, FMT results in restoration of the normal distal gut microbial community structure.1–6 The clinical efficacy of FMT in curing refractory CDI has been shown in multiple case series performed across the world,7,8 and the one controlled randomized study performed to date had to be stopped prematurely because of the clear superiority of FMT compared with the standard therapy group.3 However, even though FMT is now listed in some expert guidelines of standard practice for treating refractory CDI,9 it is not yet mainstream medicine. In part, this is because of a number of practical barriers that are associated with the procedure in a typical clinical setting. Chief among these is that many patients are unable to identify a suitable donor. Moreover, the effort and expense involved in qualifying the donor by history, physical examination, and laboratory testing are not reimbursed adequately. Preparation of the material suitable for administration is intrinsically and aesthetically challenging and ideally should be performed in the laboratory setting with appropriate safety precautions for the staff involved in processing the material. The choices of specific routes of administration, whether by retention enema, nasogastric or nasoduodenal tube, or
F
Roadblock Donor Selection
a
Authors share co-first authorship.
Abbreviations used in this paper: CDI, Clostridium difficile infection; CGMP, Current Good Manufacturing Practices; FDA, Food and Drug Administration; FMT, fecal microbiota transplantation; PCR, polymerase chain reaction. © 2014 by the AGA Institute 1542-3565/$36.00 http://dx.doi.org/10.1016/j.cgh.2014.11.014
2
Khoruts et al
inflammatory bowel disease, metabolic syndrome, irritable bowel syndrome, and even autism.11,12 Therefore, it is appropriate to consider the general health of the donor, much beyond mere infectious risk. In practice, it is difficult for patients, already exhausted by their disease, to find a donor who would qualify under strict criteria of health (eg, ideal weight and no history of food intolerances or allergies; lack of medications; and no elements of metabolic syndrome, gastrointestinal symptoms, abdominal surgeries, and so forth). Of course, these considerations may seem insignificant in the context of an elderly patient drained by repeated bouts of CDI and facing possible death. However, the patient also may be middle-aged, or even a young teenager or a child, perhaps with underlying inflammatory bowel disease. In our clinical experience, virtually all patients, regardless of age or background, are relieved when they are told that they do not need to bring in their own donor, even before criteria for donor selection are explained. The donor problem can be solved by the development of a dedicated, standardized, donor program. Such effort has certain parallels to blood donation, and it is less common in current medical practice to be restricted to related donors anymore. In fact, related donors may carry a higher infectious risk, possibly because they may not be able to reveal potential risk factors and may have shared exposure events.13 Screening and testing criteria for stool donors in standardized donor programs generally incorporate all the elements used in qualifying blood donors. However, exclusion criteria for stool donors also are based on the results of screening and testing for additional systemic problems, such as metabolic syndrome, diabetes, autoimmunity, irritable bowel syndrome, food intolerances, allergies, neurologic and psychiatric problems, and so forth.14,15 Obviously, potential donors cannot have a history of recent antibiotic exposure. In our center, we were only able to qualify a minority of recruited candidates (w10%). However, the program is feasible because qualified donors can provide repeated donations, supplying sufficient material for an extensive FMT program.
Technological Primer Material Processing Stool is a complex substance containing undigested food elements, cellular and chemical materials shed from the host intestinal tract, and large numbers of microorganisms. FMT can be performed using whole homogenized stool, although such a procedure can be very challenging aesthetically. However, it is possible to separate the microbial fraction from fecal material using a series of sieves, centrifugation, and filtration steps in the laboratory.14 Even more importantly, it is possible to freeze this microbial fraction in a cryoprotectant, while
Clinical Gastroenterology and Hepatology Vol.
-,
No.
-
maintaining the viability of the different microbial taxa and clinical efficacy of the preparation.4,14 There are several important advantages in using the frozen microbial fraction instead of the freshly prepared stool in FMT. First, the material is no longer aesthetically challenging, having lost most of the potent pungent odor associated with stool. Second, the preparation can be quantified in terms of numbers and types of bacteria present rather than the crude measure of stool weight, which can vary in bacterial content by an order of magnitude between individual donations. Most importantly, freezing and storing the preparation is critical to enabling up-to-date testing of the donor and the actual material intended for clinical use. A number of stool tests require significant periods of time, including stool cultures for bacterial pathogens and polymerase chain reaction (PCR) tests for viral pathogens, such as rotavirus and norovirus. In addition, seroconversion toward viral infections, such as human immunodeficiency virus and hepatitis, may lag by several weeks after acquiring the infection. The ability to bank the microbiota material before release allows performance of rigorous testing and elimination of uncertainty associated with freshly prepared material. The entire process of producing fecal microbiota material can be standardized in accordance with the Current Good Manufacturing Practices (CGMPs), which can be used and enforced by the US Food and Drug Administration (FDA) to ensure proper design, monitoring, and control of the manufacturing processes and facilities. All human pharmaceuticals are required to follow CGMPs to prevent product contamination and process deviations that may alter safety or therapeutic activity. At first glance, it may seem absurd to require adherence to CGMPs in the manufacture of fecal microbiota. Although it may be illogical to require CGMPs for FMTs practiced today by individual physicians or even tested in pilot academic clinical trials, adherence to CGMPs is absolutely critical to large-scale manufacture of fecal microbiota preparation that may enter routine clinical practice. The process includes rigorous protocols in securing the raw materials, maintenance of robust operating procedures, reliable laboratory testing procedures, detecting product quality deviations, and providing release of materials. The ultimate purpose in the manufacture of a product as complex as fecal microbiota is not to obtain precise compositional consistency, which is impossible given that composition of every donation is somewhat different. However, CGMPs do ensure that the manufacturing process is consistent between different batches and any possibility of introducing risk is minimized.
Next Challenges in Fecal Microbiota Transplantation Although the ready availability of liquid or frozen suspensions of microbiota would greatly simplify the
-
2014
clinical practice of FMT today, these preparations require ultra-low-temperature freezer facilities and specialized medical personnel for administration, regardless of the chosen route. The development of lyophilized preparations that can be stored at room temperature and potentially encapsulated clearly would constitute the next step forward. This requires careful testing of cryopreservation protocols suitable for this application. Although the preservation of different, individual microorganisms in lyophilized form has been accomplished for decades, the protocols need to be optimized for preserving the myriad of the different, but essential, taxa present in the complete fecal microbiota. The encapsulation process requires a number of special considerations unique to FMT. Obviously, it should not introduce toxic substances into the microbiota preparation to ensure that viability is not diminished. It also is desirable to release the capsule contents in the distal intestine because premature release may introduce a risk of aspiration and possible loss of viability of some microorganisms during their journey through the harsh digestive tract environment. The latter currently may not seem to be a critical problem because liquid preparations of homogenized stool appear clinically successful when administered by a nasoduodenal route,3 and oral administration of encapsulated frozen liquid microbiota remains curative of CDI.16 Nevertheless, the issue becomes more important for optimizing dosing of freeze-dried preparations, considering that the volume requirements within capsules need to accommodate both microorganisms and cryoprotectant agents. The stomach pH is likely to be a major variable in designing capsule release. Many patients take acidsuppressing medications and some may have neutral stomach pH as a result of autoimmune gastritis, which can result in premature dissolution of a pH-sensitive shell. Gastroparesis and severe esophageal reflux may constitute relative future contraindications to FMT capsules. Uncertain risks also may exist in patients with small-bowel Crohn’s disease, which may lead to increased dwell time of the capsule in areas of stricture and content release over ulcerated areas. An important issue in the development of nextgeneration FMT products likely will concern material composition. At this time, donor selection is based primarily on clinical history, physical examination, and laboratory tests. Notably, tests used currently for enteric pathogen detection were developed originally for the diagnosis of patients with symptomatic diarrhea. Their current use in testing asymptomatic donors is logical, but not validated for sensitivity with respect to pathogens that may be present in low abundance or clinical importance in the context of healthy microbial communities. Laboratory protocols undoubtedly will evolve and increasingly use molecular diagnostics tools, including multiplex PCR, microfluidic (digital PCR), and microarray technology.17 These methods may increase the efficiency, sensitivity, and range of pathogen detection, although we
FMT for Mainstream Medicine
3
also may discover that the pathogenicity of some microorganisms may be dependent on the rest of the microbial community composition and specific host factors. Ultimately, we also may apply metagenomics and other “omics” technologies to identify microbiota optimal for the recipient’s health.18 This currently is not quite possible, in large part because our understanding of normal is very limited. Concern over infectious risk intrinsic to fecal microbiota used for FMT has been one of the main articulated reasons for the drive to develop defined, minimal, consortia of microorganisms, which can be propagated outside the human host, yet retain the curative properties of the far more complex microbiota present in stool. Several such highly simplified microbial assemblies, selected primarily on the basis of favorable ex vivo growth characteristics, were shown to be clinically effective in animal models and human patients with CDI.19,20 However, the long-term effects of this inoculum is unknown and large-scale manufacturing of defined microbiota products has its own set of challenging problems, including the potential loss of efficacy associated with adaptation of microorganisms to the in vitro environment. The development of defined microbial consortia undoubtedly will continue, although the selection of microbiota likely will become increasingly mechanistically informed. For example, microorganisms involved in secondary bile acid metabolism may be especially critical for the prevention of CDI recurrence.4,21 It will be highly desirable to identify compositional features that enhance the stability and resilience of microbial communities in the gut, characteristics likely to be critically important in patients with refractory CDI who often are exposed to antibiotic treatments. In addition, defined, simplified microbiota may function as short-term scaffolding in rebuilding more complex microbial communities in the gut19; therefore, because such products are introduced into patients, it will be important to determine whether in the long term the resulting composition of microbiota will approximate normal microbiota or deviate in some unexpected way. The spectacular success of FMT in treating refractory CDI has provided a boost to various biotechnology startup manufacturing companies that are attempting to harness the power of the microbiome for novel therapeutics development.22 CDI remains one of the foundational targets, and different companies are taking varying approaches to develop fecal microbiota–based products, including standardized whole donor-derived microbiota; highly simplified, defined microbial consortia; and hybrid products such as SER-109, the lead spore-based compound from Seres Health (Cambridge, MA), which recently was reported to have promising early results in recurrent CDI.23 The pace of discovery and clinical validation is accelerating, and it appears very likely that a range of highly effective therapeutic options for CDI and perhaps other indications soon will be introduced into mainstream medicine.
4
Khoruts et al
However, in additional to the many scientific and technical hurdles, developers also are challenged by the fluid regulatory framework and uncertainties in the intellectual property landscape. The FDA has determined that fecal microbiota fits the legal definition of a drug under US law. FMT has not yet been approved by the FDA for any indication, and currently constitutes an investigational agent. Given the heavy regulatory burden associated with the use of such agents and the lack of therapeutic alternatives for too many patients, the FDA issued a policy of “enforcement discretion”23 regarding investigational new drug requirements for use of FMT by individual physicians in the treatment of CDI not responding to standard therapies. However, the typical requirement for an investigational new drug application in formal clinical trials also has been applied unevenly to commercial entities.24 In addition, the enforcement discretion policy, perhaps unintentionally, has opened the possibility of establishing nonprofit stool banks, such as OpenBiome at the Massachusetts Institute of Technology, which is able to ship standardized fecal microbiota across state lines with only limited regulatory oversight.24 The FDA, charged with the incredibly difficult task of protecting patients today and in the future, as well as guiding the development of better therapeutics, continues to adjust its policies to accommodate the new paradigms intrinsic to this field. As a drug, live microbiota is unparalleled in its compositional and metabolic complexity. Moreover, its composition is intrinsically highly dynamic and responsive to external factors such as diet.25 This complexity may prove too unsettling for the regulators and may require paradigm shifts as it once did for other cellular therapies. Functionally, gut microbiota fit the description of an organ, and given its tight co-evolutionary linkage to its specific host species, it can be considered a human organ composed of microbial cells.10,26 Therefore, it may be reasonable to borrow elements of regulation for microbiota products from those applied to tissue transplantation.27 Most importantly, therapeutics development in this area should be guided by the best science. Undoubtedly, that will require reworking of longestablished paradigms of the germ theory of disease and acceptance of a new conceptual framework that is grounded in microbial ecology and the growing understanding of microbiota-host interactions. Ultimately, we all—physicians, scientists, developers, and regulators—need to be informed by continued research, basic and clinical, to allow establishment of this promising new class of therapeutics into mainstream medicine.
References 1. Hamilton MJ, Weingarden AR, Unno T, et al. High-throughput DNA sequence analysis reveals stable engraftment of gut microbiota following transplantation of previously frozen fecal bacteria. Gut Microbes 2013;4:125–135. 2. Shahinas D, Silverman M, Sittler T, et al. Toward an understanding of changes in diversity associated with fecal
Clinical Gastroenterology and Hepatology Vol.
-,
No.
-
microbiome transplantation based on 16S rRNA gene deep sequencing. MBio 2012;3. 3. van Nood E, Vrieze A, Nieuwdorp M, et al. Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med 2013;368:407–415. 4. Weingarden AR, Chen C, Bobr A, et al. Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection. Am J Physiol Gastrointest Liver Physiol 2014;306:G310–G319. 5. Fuentes S, van Nood E, Tims S, et al. Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection. ISME J 2014;8:1621–1633. 6. Shankar V, Hamilton MJ, Khoruts A, et al. Species and genus level resolution analysis of gut microbiota in Clostridium difficile patients following fecal microbiota transplantation. Microbiome 2014;2:13. 7. Gough E, Shaikh H, Manges AR. Systematic review of intestinal microbiota transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection. Clin Infect Dis 2011;53: 994–1002. 8. Kassam Z, Lee CH, Yuan Y, et al. Fecal microbiota transplantation for Clostridium difficile infection: systematic review and meta-analysis. Am J Gastroenterol 2013;108: 500–508. 9. Surawicz CM, Brandt LJ, Binion DG, et al. Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am J Gastroenterol 2013;108:478–498; quiz 499. 10. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science 2005;307:1915–1920. 11. Aroniadis OC, Brandt LJ. Fecal microbiota transplantation: past, present and future. Curr Opin Gastroenterol 2013;29:79–84. 12. Smits LP, Bouter KE, de Vos WM, et al. Therapeutic potential of fecal microbiota transplantation. Gastroenterology 2013; 145:946–953. 13. Starkey JM, MacPherson JL, Bolgiano DC, et al. Markers for transfusion-transmitted disease in different groups of blood donors. JAMA 1989;262:3452–3454. 14. Hamilton MJ, Weingarden AR, Sadowsky MJ, et al. Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection. Am J Gastroenterol 2012;107:761–767. 15. Bakken JS, Borody T, Brandt LJ, et al. Treating Clostridium difficile infection with fecal microbiota transplantation. Clin Gastroenterol Hepatol 2011;9:1044–1049. 16. Youngster I, Russell GH, Pindar C, et al. Oral, capsulized, frozen fecal microbiota transplantation for relapsing Clostridium difficile infection. JAMA 2014;312:1772–1778. 17. Guarino A, Giannattasio A. New molecular approaches in the diagnosis of acute diarrhea: advantages for clinicians and researchers. Curr Opin Gastroenterol 2011;27:24–29. 18. Lamendella R, VerBerkmoes N, Jansson JK. ‘Omics’ of the mammalian gut–new insights into function. Curr Opin Biotechnol 2012;23:491–500. 19. Lawley TD, Clare S, Walker AW, et al. Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice. PLoS Pathog 2012;8:e1002995. 20. Petrof EO, Gloor GB, Vanner SJ, et al. Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut. Microbiome 2013;1:3.
-
2014
FMT for Mainstream Medicine
5
21. Buffie CG, Bucci V, Stein RR, et al. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 2014 Epub ahead of print.
26. Ochman H, Worobey M, Kuo CH, et al. Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol 2010;8:e1000546.
22. Olle B. Medicines from microbiota. Nat Biotechnol 2013; 31:309–315.
27. Smith MB, Kelly C, Alm EJ. Policy: how to regulate faecal transplants. Nature 2014;506:290–291.
23. Guidance for Industry: Enforcement Policy Regarding Investigational New Drug Requirements for Use of Fecal Microbiota for Transplantation to Treat Clostridium difficile Infection Not Responsive to Standard Therapies. Available from: http://www. fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory Information/Guidances/Vaccines/ucm361379.htm. 24. Ratner M. Fecal transplantation poses dilemma for FDA. Nat Biotechnol 2014;32:401–402. 25. David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2014; 505:559–563.
Reprint requests Address requests for reprints to: Alexander Khoruts, MD, Department of Medicine, Division of Gastroenterology, University of Minnesota, 2101 6th Street SE, Medical Biosciences Building, Room 3-184, Minneapolis, Minnesota 55414. e-mail: [email protected]; fax: (612) 625-2199. Conflicts of interest The authors disclose the following: Alexander Khoruts, Michael Sadowsky, and Matthew Hamilton have received funding from CIPAC, LLC, to perform research on frozen fecal microbiota; and Michael Sadowsky and Matthew Hamilton have provided consulting services for CIPAC.
