FECAL MICROBIOTES TRANSPLANTATION TECHNOLOGIES: MEDICAL, BIOTECHNOLOGICAL AND REGULATORY ASPECTS

of successful enema transplantation of suspended feces to four patients with pseudomembranous colitis. The Clostridium difficile role in the developme nt of this disease was discovered Fecal microbiota transplantation (FMT) is a treatment method based on donor’s fecal solution injection into the patient’s gastrointestinal tract. FMT is effectively used in the treatment of recurrent Clostridium difficile infection. There is also growing interest in the therapeutic application of the method to treat metabolic, autoimmune and other disorders that was not previously associated with intestinal microbiota. Despite the promising results of FMT use, the organizational and legal matters and that of the safety FMT application have not yet been resolved in the European and Ukrainian medical community. The purpose of this review was to summarize information on the FMT application and the regulatory aspects of its use. in accordance with the applicable legal provisions. The analysis of the practical instructions provisions of for FMT applying in clinical practice was carried out, and the bioethical problems associated with the FMT use were investigated.

The important role of the gastrointestinal tract microbiota in maintaining normal physiological processes in the human body has been generally accepted fact for a long time. The idea of a direct link between human health and the activity of intestinal microorganisms was formulated by Ilya Mechnikov. A microbiota, or microbiome, is a population of microorganisms (ov erwhelmingly of bacteria) that coexist symbiotically with humans. The human intestinal microbiota includes about 10 14 microorganisms of almost 1000 different species, most of which are representatives of two phyla: Bacteroidetes and Firmicutes [1].
Today, th ere are numerous data that indicate th e role of one or another dys biotic disorders in the microbiome of the gastrointestina l tract in the development of obesity, Crohn's disease, ulcerative colitis, multiple scl erosis, and diabetes mellitus [2][3][4][5]. Poor effi cacy and overuse of antibacterial drugs contribute to the search for new treat-ments. Therefore, there is a growing interest in fecal microbiota transplantation. The essence is to transfer the microbiota of a healthy person feces to the gastrointestinal tract of a sick reci pient in order to restore the normal microbiota [6,7].
Prerequisites for the development of fecal microbiota transplantation. Although the FMT use is a little-known practice, it is not an innovative technology. It is first mentioned in me dical tr eatises of the IV century AD, found in China, which described a case of successful treatment of food poisoning. This technique was also used in veterinary medicine (treatment of colitis accompanied by diarrhea in horses).
The first publication on the FMT use to treat pseudomembranous colitis was published in 1958. Then Ben Eisman described a case of successful enema transplantation of suspended feces to four patients with pseudomembranous colitis. The Clostridium difficile role in the developme nt of this disease was discovered UDC 579. 61 [12].
We consider the features of FMT obtained from the European consensus on FMT in clinical practice [13].
Donor selection. The provisions of the European consensus provide for th orough screening of fecal material donors. To reduce possible risks, the age of the donor should not exceed 60 years. Healthy donors are usually selected from family members.
At the beginning of the process, potential donors should undergo a medical interview to rule out possible risk factors.
Key diseases / conditions to be tested in the selection of potential donors for pre-screening: Infectious diseases: • a history of HIV, hepatitis B virus or hepatitis C virus, syphilis, human T-lymphotropic virus type I or II, malaria, trypanosomiasis, tuberculosis presence; • known systemic infection not controlled at the time of donation; • use of il licit drugs; • in comprehensible sexual behavior (anonymous sexual intercourse; presence of a history of sexually transmitted diseases); • previously undergone organ / tissue transplantation as a recipient; • taking blood products (<12 months); • tattooing, piercing, acupuncture (<6 months); • recent treatment in unsatisfactory hygienic conditions; • risk of transmitting diseases caused by prions; • recent parasitosis or infectious diseases caused by rotavirus, Giardia lamblia and other microorganisms that affect the gastrointestinal tract; • recent stay in tropical countries, countries at high risk of infectious diseases or travelers' diarrhea (<6 months); • recent vaccination with alive attenuated virus if there is a risk of transmission (<6 months); • healthcare professionals (to eliminate the risk of transmission of multidrug-resistant organisms); • individual work with animals (to eliminate the risk of zoonotic infections transmission).
 Diseases of the gastrointestinal tract, metabolic and neurological diseases: • history of irritable bowel syndrome, inflammatory bowel disease, functional chronic constipation, celiac disease, other chronic diseases of the gastrointestinal tract; • history of chronic systemic autoimmune diseases associated with the gastrointestinal tract; • history or high risk of gastrointestinal cancer or polyposis; • recent diarrhea or hematochezia; • h i s t o r y o f n e u r o l o g i c a l o r neurodegenerative diseases; • history of mental disorders; • overweight or obesity (body mass index> 25 kg/m 2 ).
 Drugs that may affect the composition of the intestinal microbiota: • R e c e n t u s e o f a n t i b i o t i c s , immunosuppressants, chemotherapy (<3 months); • Prolonged therapy with proton pump inhibitors.
Selected donors for FMT must pass the analysis of blood and feces no later than 4 weeks before the donation of fecal material ( Table 1).
Preparation of material. The European consensus on the transplantation of fecal microbiota clearly regulates the amount of fecal material, the optimal time of delivery to the laboratory, the requirements for the premises in which feces are treated. A suspension of feces which can be prepared from fresh or frozen feces is used for FMT. The optimal method of fecal material preparation is uncertain. Randomized trials indicate that fresh and frozen material are equally effective in the treatment of C. difficile infections [14,15].
There is considerable heterogeneity in studies on the material for FMT preparation, which makes it difficult to draw a firm conclusion. However, it has been shown that infusions with water are more effective than with physiological saline (98.5% vs. 86%) [16].
Fresh fecal material must be processed within 6 hours of receipt. Approximately 50 g of donor fecal material is mixed with 150 ml of sterile sodium chloride solution. The mixture is filtered to remove large particles which may hinder the administration [13,17].
The use of frozen material contributes to the standardization of the FMT process and the development of fecal sample banks. Frozen fecal material should be stored at -80 C. When using the material in FMT, fecal suspension is thawed in a water bath at 37 C, mixed with physiological saline. The infusion should be performed within 6 hours after thawing [13].
Means of fecal material delivery. The means of fecal material delivery used today are conventionally divided into upper (oral capsules), middle (by esophagogastroduode-noscopy or nasogastric, nasojejunal, nasoduodenal catheter) and lower (by colonoscopy or retention enema). The literature discusses the effectiveness of different methods of fecal suspensions administration.
The main advantages of colonoscopy are the ability to visualize the process [18; 19], reliable delivery to the affected parts of the intestine [20], providing a larger volume of material, which will increase the success rate of the procedure [16]. But colonoscopy is an expensive, relatively risky invasive procedure.
The use of enemas is less invasive, relatively cheaper and easier way to perform. However, there is a problem of material retention, which may require multiple infusions to achieve clinical efficacy [21].
The use of medium means of delivery is a faster and cheaper way, better tolerated than colonoscopy [22]. However, smaller volumes of material are used, which is associated with lower clinical efficacy of this method. Wang and coauthors [7] note that the frequency of side effects was higher when using medium delivery methods compared to the lower ones (43.6% vs. 17.7%).
The capsules are minimally invasive and convenient. In addition, the capsules are more

Analysis of feces in specific cases
 Vibrio cholera and Listeria monocytogenes;  Antigens and / or acid staining for Isospora and Microsporidia;  Calprotectin;  Fecal antigen Helicobacter pylori;  Rotavirus aesthetically pleasing because patients prefer this method of delivery over others [23].
To date, in clinical practice there is no strong evidence of the most optimal means of fecal material delivery. The decision to choose a method should depend on the individual clinical situation. Schematic diagram of the FMT process is shown in Fig. 1.
Monitorin g. After FMT performing, experts r ecommend careful monitoring of patients. The length of the period during which the patient should be monitored has not been establish ed. If FMT procedure is successful and the clinical symptoms of the disease begin to decrea se progressively, European experts do not recommend retesting C. difficile in the feces, as toxins of this bacterium can be stored in the feces for several weeks [12,24].
Fecal microbiota transplantation in the treatment of infections caused by C. difficile. At present, the idea of the FMT effectiveness in the treatment of recurrent infection caused by C. difficile has been established. The efficiency indicator of treatment when using FMT is approximately 90%. At the same time, the efficiency indicator of long-term antimicrobial therapy is 2030% [25]. Numerous stu dies also demonstrate the FMT e fficiency in the treatment of recurrent C. difficile infection in elder ly patients, patie nts with comorbidities or weakened immunity [2628].
So, van Nood and coauthors [29] conducted a randomized open-labelled controlled clini cal trial in which patients with recurrent C. difficile infection were randomly assigned to one of three groups: 1) initial treatment with vancomycin (orally, by 500 mg 4 times a day for 4 days) followed by bowel lavage and infusion of donor suspended feces solution through a catheter; 2) standard course of therapy with vancomycin (orally, by 500 mg 4 times a day for 14 days); 3) standard course of therapy with vancomycin with bowel lavage. The endpoint was initially identified -the persistent disappearance of diarrhea (withi n 10 weeks of follow-up) caused by the bacterial infection.
In 13 of 16 patients in the first group there was a disappearance of diarrhea after the first administr ation of the drug. Three patients in this g roup were re-infused (from another donor), i n these circumstances, diarrhea disappeared in two of them. Disappearance of diarrhea was observed only in 4 of 13 patients of the second group, in 3 of 13 -in the third group.
Thus, FMT was more effective than the vancomycin use in the treatment of recurrent C. difficile infection.
Ethan Gough and coauthors in the review [16] showed that in 317 patients in the 27 studies reviewed, FMT showed efficacy in treating the disease in 92% of cases. Efficacy varied depending on the route of administration, the volume of drug administered, and the treatment before infusion.
Therefore, FMT is now the recommended treatment for the third recurrence of C. difficile infection. The acute form of the infection and the first recurrence are still treated with antibiotics. However, given the effectiveness and low frequency of complications, more FMT is predicted to be used in the first recurrences of the disease in the future [30].
Fecal microbiota transplantation in the treatment of inflammatory bowel disease. In addition to the treatment of C. difficile infections, FMT is considered a potential method of treating inflammatory bowel disease.
Encouraging results have been obtained with the use of FMT for the treatment of ulcerative colitis. In their study Ahmet Uygun and coauthors [31] demonstrated the FMT effectiveness in the treatment of ulcerative colitis. After the procedure, 21 of 30 patients showed a clinical response to treatment, 13 of 30 patients achieved remission by the twelfth week, 9 patients had no clinical response by the twelfth week. In 2012, a retrospective review of 62 patients with ulcerative colitis who underwent FMT over a 24-year period was conducted. In this study, the frequency of a positive FMT result was reported to be 91.9%, with 67.7% achieving complete clinical remission after FMT; 24.2% achieved partial remission, and only 8% did not have a positive response to FMT [32].
Available data on the FMT use in the Crohn's disease treatment are limited to descriptions of a series of studies on small groups or an isolated cases description. Vermeire and coauthors [33] reported no significant clinical or endoscopic improvement after 8 weeks in four Crohn's disease patients who underwent FMT through a nasojejunal catheter three times over a 2-day period. Temporary changes in the recipient's microbiota were observed in all patients (weeks 2-4), but the microbial composition of the intestine returned to baseline after eight weeks. These results, along with other studies, suggest that Crohn's disease has increased resistance to FMT compared with ulcerative colitis [34].
Fecal microbiota transplantation in the treatment of irritable bowel syndrome. FMT is also considered as a possible method of treating irritable bowel syndrome.
Many studies have shown that microbial changes in the intestine (reduced biodiversity and increased numbers of Bacteroidetes) are associated with the development of irritable bowel syndrome [35,36]. If the pathogenicity model of C. difficile infections is applied to irritable bowel syndrome, FMT may also be effective in this group of patients. Indeed, positive results have been reported in the treatment of irritable bowel syndrome with a predominance of diarrhea. Pinn and coauthors [37] informed of 13 patients with refractory irritable bowel syndrome, 70% of whom showed improvement in symptoms after FMT, including symptoms such as abdominal pain (72%), dyspepsia (67%), and bloating (50%). A randomized, double-blind, placebo-controlled study [38] showed that FMT alters the composition of the intestinal microbiota in patients with irritable bowel syndrome. However, placebo-treated patients had greater symptom relief compared with the FMT group. Therefore, more research is needed to better understand the effects of FMT on the patients with irritable bowel syndrome health and the subsequent wider use of FMT in treatment.
Fecal microbiota transplantation in the treatment of autoimmune diseases. There are publications that indicate a link between the functioning of the immune system and the microbiota [39,40], between changes in the intestinal microbiota and the development of autoimmune disorders, including idiopathic thrombocytopenic purpura, systemic lupus erythematosus, arthritis, Sjögren's syndrome, and Hashimoto's thyroiditis [41].
Borody and coauthors [42] reported the sudden disappearance of idiopathic thrombocytopenic purpura in a patient with prolonged ulcerative colitis who underwent FMT, which led to prolonged normalization of platelet counts and decreased ulcerative colitis activity.
There are also examples of treatment for multiple sclerosis. Thus, a clinical case is indicative -a 30-year-old man was observed for multiple sclerosis and trigeminal neuralgia. To treat constipation, the patient underwent five FMT procedures by rectal infusion, which resulted in the complete disappearance of the problem. At the same time, there was a decrease in the severity of neurological deficits caused by multiple sclerosis, including the restoration of the ability to walk independently. Initially, this was considered remission, but 15 years after FMT, the pa tient did not have period s of exacerbation [43].
In addition, FMT is trying to be used in the complex treatment of metabolic syndrome [4446], as well as for the correction of autism in children with complex therapy [47]. The evidence base for the treatment of these pathologies is still insufficient, but there are encouraging results.
Regulatory aspects. In July 2013, the US Food and Drug Administration (FDA) officially confirmed that it would regulate the use of human fecal-derived drugs in clinical practice. Due to the growing interest to FMT, the question of such a need has been raised in the United States and other countries for the past few years [48]. The European Medicines Agency does not currently regulate intestinal microbiota transplantation. The only conditions that the doctor must follow are to obtain the informed patient consent and to follow the donor selection protocol. Similar requirements are set in Australia [49].
Consider in more detail the regulation in the United States. A fecal microbiota transplantate used t o treat, alleviate, or prevent a disease falls within the United States acting drug definition. In the United States, a requirement was introduced in 2013 that physicians using FMTs must submit to the FDA an application for a new investigational drug (Investigative New Drug (IND) application), which is equivalent to filing an application for a clinical trial, with a detailed clinical research protocol. The IND submitted to the FDA, in addition to standard information, should include information on procedures restricting the transmission of pathogens to the subjects involved; intestinal microbiota donors screening procedures; description of pharmaceutical and biological testing methods of the fecal transplantate properties; description of the procedure for fecal microbiota introduction; the amount of administered product (dose) and frequency of administration; a description of the donor material obtaining process and its storage (e.g., fresh / frozen); description of the method of fecal transplantate preparation; characteristics of physicochemical and biological properties of the pharmaceutical substance [50].
Canada has made great strides in the development of FMT, so it is worth considering the position of the Canadian regulator on this new technology. Health Canada has classified FMT materials as "new biological drugs". As part of the documents submission for consideration to this agency, FMT protocols are considered by the Directorate of Biological and Gene Therapy. Examination of documents is fairly standard, including an on-site inspection to determine whether the rules and guidelines of good manufacturing practice are followed at the production site. In addition, Health Canada requires that donors should be screened for infections included in organ transplant screening protocols, as well as intestinal infections. Validated bacterial mixtures extracted from human fecal material should be monitored for contamination and cultural stability [6].
Economic aspects. C. difficile infection has a significant financial burden on the health care system and requires the development and implementation of more cost-effective treatments [51,52]. In the United States, the financial cost of hospital treatment of patients with C. difficile infection in 2015 was 6.3 billion US dollars [52]. In Europe, financial costs are estimated at about 3 billion euros [53].
Economic analyzes comparing treatment with FMT and antibiotics showed the potential cost-effectiveness of FMT in the treatment of recurrent infection caused by C. difficile.
So, in their work G. Konijeti and coauthors [54] compared four strategies for treating the recurrent infection caused by C. difficile: treatment with metronidazole; using vancomycin; using fidaxomicin; FMT. According to their estimates, the use of FMT by colonoscopy was the most cost-effective treatment strategy with an incremental costeffectiveness ratio (ICER) of $17.016.
Lauren Lapointe-Shaw and coauthors [55] analyzed the cost-effectiveness of six treatment strategies for recurrent C. difficile infection: metronidazole treatment; vancomycin treatment; fidaxomicin treatment; FMT with an enema treatment; FMT using a nasogastric catheter treatment; FMT by colonoscopy treatment. It should be noted that FMT by means of a colonoscopy showed the greatest efficiency and was less expensive, in comparison with the considered alternatives. According to all model parameters, an 87 % probability was determined that FMT by colonoscopy is the most profitable strategy.
Zainab I Abdali and coauthors [56] conducted an analysis using a decision model that represents the cost for an additional year of life adjusted for its quality (QALY). This analysis showed that the FMT use is a less expensive and more effective treatment compared to fidaxomicin and vancomycin. The incremental cost-effectiveness ratio was 242.514 pounds sterling / QALY. Therefore, based on economic analyzes, it can be argued that FMT is a more cost-effective way to treat recurrent C. difficile infection compared to antibiotic therapy.
Bioethical aspects of FMT use. Ethical aspects in the field of FMT can be summarized as follows [57]: 1. Problems related to donor selection. 2. Safety issues and the risk-benefit balance of the procedure.
3. Problems of informed consent.

Problems of commercialization.
Problems with donor's selection. To date, a number of different donors screening protocols for FMT have been published. However, there are some inconsistencies between the protocols regarding the requirements for certain tests or the frequency of their conduct. In addition to the uncertainties associated with the screening procedure, there are uncertainties related to the donor's profile -it has not been established whether children and pregnant women can become donors, or whether the donor's religious background related to a special diet should be taken into account. From the donor's point of view, the issues of confidentiality, information on the duration of screening and the possibility of refusal are also important [57]. In 2019, two cases of infection with multidrug-resistant organisms were registered, one of which resulted in the patient's death. These cases have led the FDA to revise its recommendations by introducing additional screening for multidrug-resistant organisms. Modern protocols are insufficient and do not take into account the possibility of microbiota perturbation by currently unknown mechanisms [58].
Safety issues and the risk-benefit balance of the procedure. Modern research, in most cases, demonstrates the safety of FMT. However, some patients have side effects -constipation, fever, increased levels of C-reactive protein. Risks should also include exacerbation of an existing disease, known and unknown infections, and so on. The follow-up period for patients who have undergone FMT is not long enough to identify long-term side effects. Another risk is the use of FMT in children. There is a critical period in infancy and early childhood, during which manipulations with the intestinal microbiota have the greatest impact on health and the brain, which can affect the overall development of the child [59,60]. The possibility of such side effects also raises ethical questions about patients' informed consent regarding the possibility of side effects in the future.
Problems of informed consent. From the recipient's point of view, the informed consent process should fully inform him of the main objectives, possible benefits and risks of the study. This aspect is the most ethically complex in the case of a child's treatment, as legal decisions are made by guardi ans who must consider both the risks and the benefits to the other person. From the donor's point of view, it is important to provide complete information on the procedure [58].
Problems of commercialization. The problem of FMT commercialization raises questions of ownership of the material, availability of data and biological material, as well as the consequences of the direct sale of material to the consumer [61]. The commercialized and widespread use of FMT raises the question of the risk-benefit balance, as unpredictable long-term side effects of FMT use are possible.
Some authors also highlight such a possible ethical issue as family relationships, as family members may become potential secondary recipients of the altered microbiota [57].
To date, the FMT effectiveness in the treatment of recurrent C. difficile-associated infections has been proven. The FMT use is also considered as a possible treatment for inflammatory bowel disease, irritable bowel syndrome, autoimmune and metabolic diseases. However, data on the FMT efficacy in these conditions are limited to isolated clinical cases and small studies, so further randomized controlled trials are needed. It is also important to regulate organizational, legal, technical and economic issues related to the widespread use of FMT. A necessary priority is to develop effective regulation that will protect patients and donors, prevent abuse of treatment. In addition, the issue of FMT use safety is important, so it is necessary to further determine the consequences of FMT long-term use.
This study did not receive any financial support from a government, community or commercial organization.