Methodological insights into fecal microbiota transplantation: Dissecting key approaches for success

Table 1 Clinical questionnaire to select potential donors

	Question
Medical history	Any history of
	Concurrent acute, medical illness?
	Any symptoms pertaining to gastrointestinal disease (nausea/ vomiting/ pain abdomen/ diarrhea/ blood in stool)?
	Chronic gastrointestinal (GI) disease (personal or family history), including functional GI disorders, inflammatory bowel disease, celiac disease or other chronic GI disease
	Any history of chronic illness (such as diabetes/ hypertension/ heart disease/ kidney disease/ liver disease/ HIV/malignancy)?
	Acute diarrhea (in the prospective donor or his/her contacts) in the past four weeks
	Typhus or other salmonellosis
	Tuberculosis (acute or past)
	Systemic autoimmune diseases, e.g., multiple sclerosis, connective tissue disorders
	Atopic diseases
	Neurological or psychiatric diseases [depression, chronic pain (fibromyalgia, chronic fatigue syndrome, etc.)]
	Obesity (body mass index > 30)
	Known food or medication allergy
	Cancer, including GI cancer or polyposis syndrome
Travel history	Any history of high-risk travel in the past six months
Social history	Any history of
	High risk sexual behavior
	Imprisonment
	Illicit drug use
	Body modifications such as tattooing, piercing, etc., in the past six months
	Occupational activity in a hospital or other health-care institution with patient contact; occupational activity in agriculture
Family history	Any history of premature colon cancer or early onset polyposis syndromes in first-degree family members
Treatment history	Any history of
	Recent hospitalization/discharge from long-term care facilities (past 6 months)
	Antibiotic treatment in the past three months
	Blood transfusions in the past five years
	Long-term drug treatment (e.g., with antibiotics, immunosuppressants, proton pump inhibitors, chemotherapy)
	Tissue/organ transplant
	Having been a participant in any clinical trial in the past six months
	Bowel resection or gastric reduction surgery

Table 2 Donor blood and stool testing

Blood testing
Complete blood count with differential
Blood sugar, renal and liver function tests
Hepatitis A, hepatitis B, hepatitis C and hepatitis E viruses
HIV-1 and HIV-2
Cytomegalovirus and Epstein-Barr Virus serology
Treponema pallidum (syphilis)
Nematodes (Strongyloides stercoralis)
Stool testing
Clostridioides difficile
Common enteric pathogens, including Salmonella, Shigella, Campylobacter, Shiga toxin-producing Escherichia coli, Yersinia and Vibrio cholerae
Antimicrobial-resistant bacteria and antimicrobial resistance gene testing
Norovirus, rotavirus, adenovirus
Giardia lamblia, Cryptosporidium spp., Isospora, Cyclospora and Microsporidia
Protozoa and helminths/ova and parasites (including Entamoeba histolytica, Blastocystis hominis and Dientamoeba fragilis)
Helicobacter pylori faecal antigen (for upper route of FMT delivery)

FMT: Fecal microbiota transplantation.

Table 3 Stool collection, processing and storage

Processing step	Description	Common variations/methods
Stool collection	Freshly passed stool is collected from a healthy, screened donor in a sterile container[17]	Anaerobic conditions: Certain protocols recommend oxygen-free collection and processing methods to preserve strict anaerobes[14,18,19]
		Pre-collection diet: Some studies suggest a high-fiber diet prior to donation to enhance microbial diversity[20,21]
Stool quantity	The amount of stool collected varies based on the protocol	Typically, 50-60 g of stool added to 250-300 mL of diluent[5,17]. A minimum of 25 g of feces is recommended for lower GI delivery, and 12.5 g for upper GI delivery[14,15]
Diluents/buffer solutions	Stool is diluted with a buffered solution to create a homogeneous slurry, and to a consistency that can be injected through the biopsy channel of scope	Commonly used diluents[5,22]:
		Normal saline (0.9% NaCl)-Most commonly used
		Sterile water (less preferred due to hypotonicity)
		Phosphate buffered saline
		Non-bacteriostatic saline (used to avoid antimicrobial preservatives)
		Milk or milk-based solutions (rarely)
Homogenization	Stool sample is mixed with the diluent to create a uniform suspension, ensuring even distribution of microbiota and removal of large particulate matter before filtration	Done by manual stirring with sterile spatula; laboratory blender. Performed under aerobic or anaerobic conditions[18,19]
Filtration	Large particles (undigested fiber, debris) are removed	Using[5]:
		Gauze filtration
		Stainless steel sieve (250-500 μm)
		Sterile mesh filters
Centrifugation	Further purification or concentration of microbiota; retain microbial rich supernatant	Low (3000-4000 × g), medium (5000-6000 × g) or high (up to 10000 × g) speed centrifugation; relative centrifugal force = 6000 × g for 15 minutes[23]. May be followed by re-suspension in diluent
Washing (optional step)	Component of microfiltration plus centrifugation, called washed microbiota transplantation	Integration of multiple filtration and washing steps, often performed using automated purification systems[16]; automated systems (e.g., GenFMTer) reduces host cells, debris, endotoxins (one-hour FMT protocol)[16]
Storage and handling	Stool preparation is handled under strict sterile conditions to prevent contamination. Can be used fresh or stored	Fresh use: Stored at 4 °C and used within 6 hours
		Frozen use: Stored at -80 °C with glycerol (10%) for long-term use (up to 2 years)

GI: Gastrointestinal; FMT: Fecal microbiota transplantation.

Table 4 Comparative analysis of different stool processing methods

Method	Pros	Cons	Recommended setting
Rough filtration	Simple, low cost	Low microbial purity, higher adverse events	Suitable for basic setups
Filtration plus centrifugation	Improved bacterial concentration	Moderate technical demands	Used in standard protocols
Microfiltration plus centrifugation	High microbial purity, fewer toxic reactions, delivers a precise dose of the enriched microbiota instead of using the weight of stool	Expensive, automated system e.g., GenFMTer needed	Ideal for advanced centers

Table 5 Comparison of common methods of fecal microbiota transplantation administration

	Enema	Capsule	Colonoscopy	Nasogastric, nasoduodenal or nasojejunal tube
Delivery	Distal colon	Small intestine/colon	Right colon/terminal ileum	Upper GI tract to stomach/duodenum/jejunum
Starting amount of feces	25-200 g	80-100 g	25-200 g	12.5-150 g
Final delivery volume per dose	300-500 mL	30-40 capsules	30-500 mL	30-500 mL
Pros	Well tolerated, no sedation, can be done at home	Non-invasive, faster, well-tolerated	Higher efficacy	Minimally invasive, avoids sedation risks
Cons	Retention difficulty, lower efficacy	Requires multiple doses, risk of gastric acid degradation; avoid FMT capsules if the patient has dysphagia, difficulty swallowing pills or gastroparesis	Invasive, requires sedation, risk of perforation	Risk of aspiration, patient discomfort

GI: Gastrointestinal; FMT: Fecal microbiota transplantation.

Table 6 Fecal microbiota transplantation: Current uses and future horizons

Established indication
Clostridioides difficile infection[7,11-14,48]
Investigational/emerging indications
Ulcerative colitis[45,49]	Primary sclerosing cholangitis[62,63]
Crohn’s Disease[47,50]	Intestinal pseudoobstruction[64]
Pouchitis[51]	Neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, multiple sclerosis)[65]
Irritable Bowel Syndrome[52]	Graft vs host disease[66]
Alcoholic hepatitis[53]	Human immunodeficiency virus[67]
Cirrhosis or hepatic encephalopathy[54]	Methicillin-resistant Staphylococcus aureus enterocolitis[68]
Obesity/metabolic syndrome[55]	Multidrug-resistant organisms[69]
Diabetes mellitus[56]	Post-stem cell transplant[70]
NAFLD/NASH[57]	Autologous FMT (preventive)[71,72]
Hepatitis B[58,59]	Autism spectrum disorders[73]
Pancreatitis[60]	Cancer[74]
Immune checkpoint inhibitor resistance[61]	Fecal incontinence[75]

FMT: Fecal microbiota transplantation; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis.

Table 7 Regulatory variations in fecal microbiota transplantation

Country	FMT classification
United States	Restricted use in CDI in line with FDA enforcement discretion policy[78]; investigational new drug approval used in context of clinical trials for other diseases
Belgium, Netherlands, Italy	Regulated as a tissue transplant, under European Union Tissue and Cells Directive
Australia	Regulated as a biological drug
United Kingdom, Germany, France, Ireland, Switzerland	Regulated as a medicinal drug -flexible use allowed

FMT: Fecal microbiota transplantation; CDI: Clostridioides difficile infection.