Defined Fecal Microbiota Transplantation for Clostridium Difficile Diarrhea
Defined Fecal Microbiota Transplantation for Clostridium Difficile Diarrhea
To confirm and extend the work of Trede and Rask-Madsen (Lancet 1989;1:1156-1160) that
 administration of a defined fecal microbiota will lead to rapid and sustained resolution of
 C. difficile associated chronic relapsing diarrhea.

 The current rationale behind FMT for CDI is that introduction of microbes from a healthy
 donor allows restoration of a normal microbial community in the diseased host with consequent
 suppression of C. difficile colonization and disease pathogenesis. The first modern use of
 FMT was reported in a 1958 case series of 4 patients with pseudomembranous enterocolitis. The
 first case of confirmed CDI treated with FMT was reported in 1983; treatment was curative.
 Until 1989, retention enemas were the most common technique for FMT. Alternative methods for
 delivering FMT have included fecal infusion via duodenal tube (1991), rectal tube (1994), and
 colonoscopy (1998). FMT for recurrent CDI has been used successfully whether administered by
 nasogastric tube, rectal administration by colonoscopy, rectal tube including
 self-administration at home by enema. FMT has proven to be remarkably effective and
 remarkably safe without any significant problems (see below and attached reviews and
 meta-analyses).

 Increasing, interest is emerging regarding the changes in the intestinal microbiota
 associated with CDI. In 2008 Chang et al. constructed small (< 200 sequences per subject) 16S
 rRNA gene libraries from the stools of 4 patients with first-time CDI and 3 patients with
 recurrent CDI. Based on 16S rRNA gene classification, they found that the fecal microbiomes
 of patients with an initial episode of CDI were similar at the phylum level to healthy
 subjects (i.e., the majority of sequences belonged to dominant fecal phyla Bacteroidetes and
 Firmicutes), while a major reduction or loss of Bacteroidetes was observed in patients with
 recurrent CDI. The loss of the Bacteroidetes was accompanied by the expansion of other phyla,
 including Proteobacteria and Verrucomicrobia, which are normally minor constituents of the
 fecal microbiota. Khoruts et al. (2010) compared the microbiota of a patient with recurrent
 CDI before and after FMT by using terminal-restriction fragment length polymorphism and
 clone-based 16S rRNA gene sequencing. Before transplantation, the patient's microbiota were
 deficient in members of Bacteroides and instead were composed of atypical fecal genera such
 as Veillonella, Clostridium, Lactobacillus, Streptococcus, and unclassified bacteria similar
 to Erysipelothrix. Two weeks after infusion of donor fecal suspension, the bacterial
 composition of her feces approached normal and was dominated by Bacteroides sp. strains.

 In 1989, Tvede and Rask-Madsen used a combination of nine normal fecal organisms to treat 6
 patients with chronic relapsing C. difficile diarrhea. These investigators cultivated 10
 strains of bacteria, including Enterococcus (Streptococcus) faecalis (1108-2), Clostridium
 innocuum (A27-24), Clostridium ramosum (A3I-3), Bacteroides ovatus (A40-4), Bacteroides
 vulgatus (A33-14), Bacteroides thetaiotaomicron (A33-12), Escherichia coli (1109), E. coli
 (1108-1), Clostridium bifermentans (A27-6), and Blautia producta (Peptostreptococcus
 productus) (1108-2) in broth for 48 h to a concentration of approximately 10 to the 9th power
 bacteria/mL. Two mL from each bacterial culture were admixed with 180 mL saline that had been
 pretreated in an anaerobic chamber for 24 h; the bacterial suspension was then instilled
 rectally. This procedure was followed promptly by a decline of C. difficile to undetectable
 levels by culture and the loss of detectable toxin from the stools. Normal bowel function was
 restored within 24 hours, and abdominal symptoms disappeared. Stool cultures and toxin assays
 for C. difficile remained negative during a year of follow-up. It is especially important to
 note that feces from none of the 6 patients contained Bacteroides sp.
This study will initially enroll 12 subjects. Based on the relatively large experience with
 FMT reported to date, we anticipate a prompt and sustained response as determined by a
 cessation of fever, leukocytosis and diarrhea and a loss of abdominal discomfort in >8 (80%)
 of the subjects. If such a response is not observed in >3 of the first 6 patients treated, we
 will reevaluate the composition of the FMT mixture and/or consider whether more than one
 inoculum should be given. If the 80% or greater success rate is achieved, clinical follow-up
 and studies of the microbiome will continue for a total of 12 months. During that time, we
 would plan to obtain new approvals to test other methods of inoculum delivery (e.g., via NG
 tube or oral administration in a buffered solution). In contrast to the use of raw stool of
 unknown and variable composition, the inoculum is defined and grown under anerobic conditions
 with defined media. Pre-administration oft therapy the inoculum will be examined by gram
 strain and then will be cultured to ensure that it contains the orgamisms that were used. The
 chance of a contaminant is thought to be very unlikely and contamination with an unknown
 pathogen essentially impossible. As an additional safety measure the inoculum is given into
 the gastrointestinal tract which is designed for food and other non-sterile materials.

 We anticipate a prompt and sustained response, as determined by a cessation of fever,
 leukocytosis and diarrhea and a loss of abdominal discomfort in >8 (80%) of the subjects. If
 such a response is not observed in >3 of the first 6 patients treated, we will reevaluate the
 composition of the FMT mixture and/or consider whether more than one inoculum should be
 given. If the 80% success rate is achieved, clinical follow-up and studies of the microbiome
 will continue for a total of 12 months.

 Inclusion Criteria:

 Only VA patients will be eligible for the study if they have had a confirmed diagnosis of CDI
 that has been treated for 10-14 days with recommended doses of metronidazole or vancomycin
 and has either failed to respond, or has responded and relapsed within 4 weeks of the end of
 treatment. The diagnosis will be regarded as confirmed by the presence of diarrhea (>3
 unformed stools in a 24-hour period for 2 successive days) and abdominal discomfort. The
 presence of fever, leukocytosis, and a serum albumin <3 gm/dL will be recorded but will not
 be necessary for the diagnosis. Patients will be included after they have given informed
 consent and signed the appropriate consent form that has been approved by the Baylor IRB.

 Procedure: Patients with proven recurrent CDI at the MEDVAMC will be asked to participate.
 The health records of all VA patients entered will be flagged that they are enrolled in a
 reseach study. We presume that the strains described by Tvede and Rask-Madsen were clinical
 isolates as there is no record of these strains in later publications and they are not
 available from any of the public strain repositories such as ATCC, DSMZ, or CCUG. In place of
 these specific strains, we will use the following fully sequenced type strains, originally
 isolated from human feces, and purchased from ATCC: Bacteroides ovatus ATCC 8483, Bacteroides
 vulgatus ATCC 8482, and Bacteroides thetaiotaomicron ATCC 29148. Primary cultures will be
 grown according to GLP procedures (SOP attached) on pre-reduced anaerobic blood agar ANABA
 (Remel, Lenexa, KS), then broth cultures of each strain will be anaerobically cultivated at
 37°C for 48 hours in pre-reduced chopped meat medium that will be made in house. Bacteria
 will be collected by centrifugation at 1,600 x g for 10 minutes and resuspended in 5 mL
 sterile pre-reduced 0.9% saline, using Macfarlane standards to estimate bacterial
 concentrations to target a final concentration of 10 to the power 8 to 9 cfu/mL. Aliquots
 (0.1 mL) will be removed for serial dilution and plating and anaerobic incubation at 37°C on
 pre-reduced ANABA plates to determine actual bacterial concentrations. Two mL of each of the
 three saline suspensions will be combined with 194 mL sterile pre-reduced 0.9% saline to form
 the infusion cocktail. The 200 mL bacterial suspension will then be administered into the
 small intestine using an Olympus ultra-slim gastro-duodenoscope. The small bowel route is
 chosen to avoid the potentially deleterious effects of gastric acid on survival of the
 inoculum and to prevent or reduce reflux of the volume administered into the stomach.
 Subjects will be then followed for up to 12 months.

 Transplant procedure: Patients will be pretreated with 4 days of oral vancomycin (125 mg q 6
 hr) to reduce the C. difficile load, with the final dose being given at 6 PM on the evening
 before the procedure (approximately 14-16 hours before FMT). On the evening before FMT and
 again on the morning of the procedure, patients will receive 20 mg of omeprazole by mouth. On
 the morning of the procedure, a "super-slim" 5.5 mm diameter gastroscope will be passed per
 mouth into the patient's small intestine with an attempt to place the tip at or beyond the
 ligament of Treitz. Two hundred mL of the bacterial suspension will be instilled into the
 small intestine via a catheter introduced through the biopsy channel of the endoscope and the
 flushed with 25 mL of sterile pre-reduced 0.9% saline. After removal of the endoscope, after
 recovery, patients will be allowed to resume a normal diet and physical activities.

 Subjects will be seen daily when in the hospital (and contacted daily by phone after they are
 discharged) for 14 days. Subjects will then be contacted at 30 days and monthly for 3 months,
 then every 3 months for one year. Symptoms will be recorded at each contact using a standard
 questionnaire. Laboratory examinations including CBC with differential, basic metabolic panel
 and albumin will be obtained at 3 days, at 10 days and at 30 plus or minus 7 days.

 The results of clinical outcome will be combined with evaluation of the effect of the therapy
 on the fecal microbiome. Stools sample will be collected regularly up to 1 year or until
 proven relapse. Stools samples and rectal swabs will be otained daily during the initial
 study period until discharge from hospital or 14 days and then stool samples at 30 days and
 at 3 month intervals for one year. After discharge from hospital samples will be sent to the
 laboratory using cold packs by Federal Express. This should allow us to examine in detail the
 effect of the therapy on the C. difficile as well as on the fate of the administered bacteria
 (for which the entire genome has been sequenced and published). for details see attachments).
Inclusion Criteria:

 Only VA patients will be eligible for the study if they have had a confirmed diagnosis of
 CDI that has been treated for 10-14 days with recommended doses of metronidazole or
 vancomycin and has either failed to respond, or has responded and relapsed within 4 weeks
 of the end of treatment. The diagnosis will be regarded as confirmed by the presence of
 diarrhea (>3 unformed stools in a 24-hour period for 2 successive days) and abdominal
 discomfort. The presence of fever, leukocytosis, and a serum albumin <3 gm/dL will be
 recorded but will not be necessary for the diagnosis. Patients will be included after they
 have given informed consent and signed the appropriate consent form that has been approved
 by the Baylor IRB.

 Exclusion Criteria:

 Exclusion criteria include: treatment with major immunosuppressive agents including
 prednisone >10 mg/day (or its equivalent), calcineurin inhibitors, mammalian target of
 rapamycin (mTOR) inhibitors, lymphocyte-depleting biological agents, anti-tumor necrosis
 factor agents, and others; chemotherapeutic antineoplastic agents; decompensated liver
 cirrhosis; serum creatinine >4 or need for hemodialysis; presence of an active malignancy
 other than superifical skin cancer (eg, basal cell); HIV/acquired immune deficiency
 syndrome; recent bone marrow transplant, or other cause of severe immunodeficiency;
 requirement for concurrent antimicrobial therapy; contraindication for ultra-slim endoscopy
 including severe chronic heart or lung disease; a chronic bedridden state; and any other
 condition suggesting that life span will not be >1 yr.

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