Fecal Microbiota Transplant in Children With and Without IBD
Children, under 18 years of age, with and without IBD, underwent FMT as clinically indicated for the treatment of recurrent CDI. All of the patients required the following: (i) at least three recurrences of CDI, each with at least three episodes of diarrhoea per day; (ii) C. difficile positive for each recurrence on laboratory testing either by C. difficile toxin polymerase chain reaction (PCR) or enzyme immunoassay (ELISA) of C. difficile toxins and (iii) had symptoms that resolved or improved with CDI antibiotic treatment. Related donors were used for the FMT; all donors completed a health screening questionnaire, had a history and physical examination by their primary medical doctor and had blood and stool screened for potential pathogens following previously published protocols.
Faecal microbiota transplantation recipients stopped antibiotics to treat CDI 48 h prior to the FMT procedure. Donors were given a mild laxative on the day prior to the procedure (one capful of Miralax or 200 mg of Colace) and donor stool was collected within 12 h of FMT. Up to 100 g of donor stool (range 38–100 g, median 100 g, mean 92 g) was vortexed with 400 mL of nonbacteriostatic saline and filtered. Prepared stool was then delivered to recipient via colonoscopy, primarily in the caecum, with a small amount of stool delivered through the rest of the colon as colonoscope was withdrawn. Recipients remained lying flat for 2 hours after the FMT procedure and received loperamide to aid retention of stool; children <43 kg received 2 mg loperamide after FMT and 1 mg before discharge and those >43 kg received 4 mg after FMT and 2 mg prior to discharge.
Baseline information for FMT recipients was collected including age, gender, race, medications, antibiotic use, gastric acid suppression use, probiotic use, whether the patient had IBD, and if so IBD phenotype, location and IBD medications. Donor information recorded included age, gender, race, relationship with recipient, medications, recent antibiotic use (with exclusion of those who had received antibiotics within the last 3 months) and medical history. Follow-up clinic visits and/or phone calls were conducted at 1–4 days, 2–10 weeks, 10–20 weeks and 6 months after FMT. Adverse effects were screened for at each time point including fever, chills, malaise, fatigue, anorexia, abdominal pain, diarrhoea, constipation, nausea and vomiting.
Stool was collected from both donor and recipient prior to FMT, and from recipients at 2–10 weeks, 10–20 weeks and 6 months after FMT; the samples were immediately frozen at −80 °C until analysis. DNA extraction was performed on faecal samples using the MoBio PowerSoil DNA Isolation Kit (Carlsbad, CA, USA). Golay barcoded primers were used to amplify the v4 hypervariable region of the 16S rRNA gene. Sequencing was performed on the Illumina MiSeq platform using 2 × 150 bp paired-end reads. Paired-end reads were joined with FastqJoin from EA-utils, and only reads that matched in the overlapping region at ≤6% error were kept. De-multiplexed sequences were filtered for quality and clustered into operational taxonomical units using QIIME 1.8 software. Taxonomy was assigned using the open reference method and the Greengenes 16S database (version gg_13_8). QIIME was used to assess richness (via the observed species metric), to generate taxa plots, and to calculate UniFrac distances and generate unweighted principal coordinate analysis (PCoA) plots, visualised using EMPeror software. All alpha and beta diversity analyses were performed with samples rarefied to a sequencing depth of 8000. Two-sample nonparametric t-tests are used to calculate P-values for alpha diversity analyses. Post-FMT statistics were calculated using the 6 month timepoint. Differentially abundant taxa were identified using Linear Discriminant Analysis Effect Size (LefSe) software. Default settings were used to determine significance thresholds (i.e. alpha % 0.05 for the Kruskal–Wallis test among classes and R 2.0 for the logarithmic linear discriminant analysis score).
Polymerase chain reaction of the toxin B gene was performed on recipient stool samples at 10–20 weeks and 6 months after FMT using the commercial BD GenOhm C. difficile assay (BD Diagnostics, Inc, Sparks, MD, USA) directly on stool as per the manufacturer's instructions, which has been shown to have high sensitivity and specificity for detection of C. difficile from frozen paediatric stool samples.
For a subset of stool samples where the genus Clostridium was identified on 16SrRNA sequencing, selective anaerobic culture of stool samples for C. difficile was performed following previously described methods.
Institutional Review Board approval was obtained for this study at both the Johns Hopkins Hospital and INOVA Fairfax hospital.
Materials and Methods
Children, under 18 years of age, with and without IBD, underwent FMT as clinically indicated for the treatment of recurrent CDI. All of the patients required the following: (i) at least three recurrences of CDI, each with at least three episodes of diarrhoea per day; (ii) C. difficile positive for each recurrence on laboratory testing either by C. difficile toxin polymerase chain reaction (PCR) or enzyme immunoassay (ELISA) of C. difficile toxins and (iii) had symptoms that resolved or improved with CDI antibiotic treatment. Related donors were used for the FMT; all donors completed a health screening questionnaire, had a history and physical examination by their primary medical doctor and had blood and stool screened for potential pathogens following previously published protocols.
Faecal microbiota transplantation recipients stopped antibiotics to treat CDI 48 h prior to the FMT procedure. Donors were given a mild laxative on the day prior to the procedure (one capful of Miralax or 200 mg of Colace) and donor stool was collected within 12 h of FMT. Up to 100 g of donor stool (range 38–100 g, median 100 g, mean 92 g) was vortexed with 400 mL of nonbacteriostatic saline and filtered. Prepared stool was then delivered to recipient via colonoscopy, primarily in the caecum, with a small amount of stool delivered through the rest of the colon as colonoscope was withdrawn. Recipients remained lying flat for 2 hours after the FMT procedure and received loperamide to aid retention of stool; children <43 kg received 2 mg loperamide after FMT and 1 mg before discharge and those >43 kg received 4 mg after FMT and 2 mg prior to discharge.
Baseline information for FMT recipients was collected including age, gender, race, medications, antibiotic use, gastric acid suppression use, probiotic use, whether the patient had IBD, and if so IBD phenotype, location and IBD medications. Donor information recorded included age, gender, race, relationship with recipient, medications, recent antibiotic use (with exclusion of those who had received antibiotics within the last 3 months) and medical history. Follow-up clinic visits and/or phone calls were conducted at 1–4 days, 2–10 weeks, 10–20 weeks and 6 months after FMT. Adverse effects were screened for at each time point including fever, chills, malaise, fatigue, anorexia, abdominal pain, diarrhoea, constipation, nausea and vomiting.
Stool was collected from both donor and recipient prior to FMT, and from recipients at 2–10 weeks, 10–20 weeks and 6 months after FMT; the samples were immediately frozen at −80 °C until analysis. DNA extraction was performed on faecal samples using the MoBio PowerSoil DNA Isolation Kit (Carlsbad, CA, USA). Golay barcoded primers were used to amplify the v4 hypervariable region of the 16S rRNA gene. Sequencing was performed on the Illumina MiSeq platform using 2 × 150 bp paired-end reads. Paired-end reads were joined with FastqJoin from EA-utils, and only reads that matched in the overlapping region at ≤6% error were kept. De-multiplexed sequences were filtered for quality and clustered into operational taxonomical units using QIIME 1.8 software. Taxonomy was assigned using the open reference method and the Greengenes 16S database (version gg_13_8). QIIME was used to assess richness (via the observed species metric), to generate taxa plots, and to calculate UniFrac distances and generate unweighted principal coordinate analysis (PCoA) plots, visualised using EMPeror software. All alpha and beta diversity analyses were performed with samples rarefied to a sequencing depth of 8000. Two-sample nonparametric t-tests are used to calculate P-values for alpha diversity analyses. Post-FMT statistics were calculated using the 6 month timepoint. Differentially abundant taxa were identified using Linear Discriminant Analysis Effect Size (LefSe) software. Default settings were used to determine significance thresholds (i.e. alpha % 0.05 for the Kruskal–Wallis test among classes and R 2.0 for the logarithmic linear discriminant analysis score).
Polymerase chain reaction of the toxin B gene was performed on recipient stool samples at 10–20 weeks and 6 months after FMT using the commercial BD GenOhm C. difficile assay (BD Diagnostics, Inc, Sparks, MD, USA) directly on stool as per the manufacturer's instructions, which has been shown to have high sensitivity and specificity for detection of C. difficile from frozen paediatric stool samples.
For a subset of stool samples where the genus Clostridium was identified on 16SrRNA sequencing, selective anaerobic culture of stool samples for C. difficile was performed following previously described methods.
Institutional Review Board approval was obtained for this study at both the Johns Hopkins Hospital and INOVA Fairfax hospital.
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