Study Design

The complete methods are included in the Supplementary Appendix, which along with the research protocol is available with the full text of this article at NEJM.org.

In this open-label, randomized, controlled trial, we compared three treatment regimens: the infusion of donor feces preceded by an abbreviated regimen of vancomycin and bowel lavage, a standard vancomycin regimen, and a standard vancomycin regimen with bowel lavage.

The study was conducted at the Academic Medical Center in Amsterdam. Patients who had been admitted to referring hospitals were visited by the study physicians, who performed the randomization. All participants provided written informed consent. A data and safety monitoring board monitored the trial on an ongoing basis. The research protocol was approved by the ethics committee at the Academic Medical Center. The first and last two authors vouch for the accuracy and completeness of the reported data and for the fidelity of the report to the study protocol.

Study Population

Included in the study were patients who were at least 18 years of age and who had a life expectancy of at least 3 months and a relapse of C. difficile infection after at least one course of adequate antibiotic therapy (≥10 days of vancomycin at a dose of ≥125 mg four times per day or ≥10 days of metronidazole at a dose of 500 mg three times per day). C. difficile infection was defined as diarrhea (≥3 loose or watery stools per day for at least 2 consecutive days or ≥8 loose stools in 48 hours) and a positive stool test for C. difficile toxin. Available isolates were characterized by polymerase-chain-reaction (PCR) ribotyping.19

Exclusion criteria were prolonged compromised immunity because of recent chemotherapy, the presence of human immunodeficiency virus (HIV) infection with a CD4 count of less than 240, or prolonged use of prednisolone at a dose of at least 60 mg per day; pregnancy; use of antibiotics other than for treatment of C. difficile infection at baseline; admission to an intensive care unit; or need for vasopressor medication.

Treatments

Patients received an abbreviated regimen of vancomycin (500 mg orally four times per day for 4 or 5 days), followed by bowel lavage with 4 liters of macrogol solution (Klean-Prep) on the last day of antibiotic treatment and the infusion of a suspension of donor feces through a nasoduodenal tube the next day; a standard vancomycin regimen (500 mg orally four times per day for 14 days); or a standard vancomycin regimen with bowel lavage on day 4 or 5. Patients in whom recurrent C. difficile infection developed after the first donor-feces infusion were given a second infusion with feces from a different donor. Patients in whom antibiotic therapy failed were offered treatment with donor feces off protocol.

Infusion of Donor Feces

Donors (<60 years of age) were volunteers who were initially screened using a questionnaire addressing risk factors for potentially transmissible diseases. Donor feces were screened for parasites (including Blastocystis hominis and Dientamoeba fragilis), C. difficile, and enteropathogenic bacteria. Blood was screened for antibodies to HIV; human T-cell lymphotropic virus types 1 and 2; hepatitis A, B, and C; cytomegalovirus; Epstein–Barr virus; Treponema pallidum; Strongyloides stercoralis; and Entamoeba histolytica. A donor pool was created, and screening was repeated every 4 months. Before donation, another questionnaire was used to screen for recent illnesses.

Feces were collected by the donor on the day of infusion and immediately transported to the hospital. Feces were diluted with 500 ml of sterile saline (0.9%). This solution was stirred, and the supernatant strained and poured in a sterile bottle. Within 6 hours after collection of feces by the donor, the solution was infused through a nasoduodenal tube (2 to 3 minutes per 50 ml). The tube was removed 30 minutes after the infusion, and patients were monitored for 2 hours. For patients who had been admitted at referring hospitals, the donor-feces solution was produced at the study center and immediately transported and infused by a study physician.

Outcomes

The primary end point was cure without relapse within 10 weeks after the initiation of therapy. For patients in the infusion group who required a second infusion of donor feces, follow-up was extended to 10 weeks after the second infusion. The secondary end point was cure without relapse after 5 weeks. Cure was defined as an absence of diarrhea or persistent diarrhea that could be explained by other causes with three consecutive negative stool tests for C. difficile toxin. Relapse was defined as diarrhea with a positive stool test for C. difficile toxin. An adjudication committee whose members were unaware of study-group assignments decided which patients were cured.

Patients kept a stool diary and were questioned about stool frequency and consistency, medication use, and adverse effects on days 7, 14, 21, 35, and 70 after the initiation of vancomycin. Stool tests for C. difficile toxin were performed in a central laboratory (Premier Toxins A&B, Meridian Bioscience) on days 14, 21, 35, and 70 and whenever diarrhea occurred.

Analysis of Fecal Microbiota

We analyzed the fecal microbiota for bacterial diversity by extracting DNA from samples from patients before and after donor-feces infusion and from the respective donor samples.20 We then characterized 16S ribosomal RNA gene amplicons using the Human Intestinal Tract Chip (HITChip), a phylogenetic microarray, as described previously.21 We estimated the diversity of the bacterial communities before and after donor-feces infusion using Simpson's Reciprocal Index of diversity,22 on a scale ranging from 1 to 250, with higher values indicating greater diversity.

Statistical Analysis

The objective was to determine the superiority of donor-feces infusion, as compared with vancomycin, both without and with bowel lavage. A cure rate of 90% for donor-feces infusion13,14 and of 60% for antibiotic therapy2,6 was assumed. Per group, 38 patients were needed to achieve a power of 80% to detect a difference between groups with a one-sided level of significance of 0.025. To account for dropouts, we planned to enroll 40 patients per group. All analyses were performed on a modified intention-to-treat basis with the exclusion of one patient who required high-dose prednisolone treatment after randomization but before the study treatment was initiated. Differences in cure rates were assessed with Fisher's exact probability test. Since the trial was terminated early according to the Haybittle–Peto rule (i.e., P<0.001 for the primary end point), rate ratios for the primary end point (overall cure) were calculated with their exact 99.9% confidence interval.

On the basis of Simpson's Reciprocal Index of diversity,22 the statistical significance of a change in microbiota diversity was assessed with the use of a paired-samples Student t-test. A principal component analysis was performed on profiles derived from the HITChip phylogenetic microarray.21 Wilcoxon signed-rank tests were performed with the application of the Benjamini–Hochberg approach to determine microbial groups that were significantly different in matched pairs of fecal samples obtained from patients before and after infusion.23