Hrt Williams

National survey of practice of faecal microbiota transplantation for Clostridium difficile infection in the UK

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Ethnic variation in the frequency of IBD related polymorphisms in IRGM, ATG16L1 and IL23R

Introduction Recent genome wide association studies have identified a number of key single nucleotide polymorphisms (SNPs) that contribute to the susceptibility of inflammatory bowel disease (IBD). SNPs in the autophagy-related 16-like 1 gene (ATG16L1; rs2241880) and immunity-related GTPase family M gene (IRGM; rs13361189 and rs4958847) have been shown to confer susceptibility to Crohns Disease (CD) in European/North American populations. In addition, a SNP in the interleukin 23 Receptor (IL23R, rs11209026) has been shown to confer protection against both CD and ulcerative colitis (UC) in Caucasian patients. These findings are in contrast to studies from East Asia that have shown no association of these SNPs with inflammatory bowel disease (IBD). The aim was to investigate the prevalence of IBD related SNPs in ATG16L1, IRGM and IL23R in a cohort of South Asian IBD patients and controls. The results were compared with a cohort of white Northern European IBD patients to determine if there is interethnic variation in the minor allele frequency for the disease cohorts. Methods IBD patients were recruited from the IBD clinics of five hospitals in North West London. Indian Asian patients with all four grandparents originating from South Asia and a confirmed diagnosis of IBD were included in the study. Patients and controls were genotyped using pyrosequencing and the results compared using the χ2 test. Results 216 Indian Asian IBD patients (60 CD: 156 UC), 203 healthy Indian Asian controls and 146 white Northern European IBD patients (98 CD: 48UC) were recruited. All SNPs were in Hardy Weinberg equilibrium. There was no significant difference in the minor allele frequency (MAF) between Indian Asian IBD patients and ethnically matched controls for any of the 4 SNPs. However, the MAF for SNPs in IRGM and IL23R was significantly different between Indian Asian and European CD cohorts (IRGM rs13361189: 0.23 vs 0.11, p=0.004; IRGM rs4958847, 0.28 vs 0.15 p=0.005; IL23R 0.8 vs 6.1, p=0.02). Conclusion The polymorphisms of ATG16L1, IRGM and IL23R that are associated with IBD in Western populations show no association with IBD in Indian Asians. These findings are similar to a Japanese study that showed these SNPs were not associated with CD in this ethnic group; however, one cannot exclude the possibility that an analysis of a larger sample size in the Indian Asian population might unveil such an association. The SNP frequencies in the South Asian IBD cohorts were significantly different to the European disease controls for SNPs in IRGM and IL23R showing that there are clear interethnic variations in risk allele frequencies.

How helpful are serological markers in differentiating crohn's disease from ulcerative colitis in indian asian inflammatory bowel disease patients?

Introduction Atypical perinuclear antineutrophil cytoplasmic antibody (pANCA), anti-Saccharomyces cerevisiae antibody (ASCA) and more recently Outer Membrane Porin C (Omp-C) have been extensively studied in European and North American IBD populations. There is emerging evidence that the combination of these autoantibodies may increase the accuracy of diagnosis in IBD, however it is not known how reliable these markers are in IBD patients of different ethnicities. The aims of this study were to first determine the prevalence of ASCA, Omp-C and atypical pANCA in Indian Asian IBD patients and controls. The second aim was to determine the ability of ASCA and atypical pANCA to discriminate between CD and UC in Indian Asians. Methods A total of 191 Indian Asian IBD patients (UC 139, CD 52) and 36 healthy ethnically matched controls were included in the study. Sera were analysed for the presence of ASCA IgA and IgG and Omp-C antibodies using a commercially available ELISA (Quanta Lite; INOVA Diagnostics). To test for atypical pANCA, indirect immunofluorescence was performed on sera diluted 1/10 and tested on ethanol fixed neutrophil substrate (INOVA diagnostics) using FITC-conjugated rabbit antihuman IgG. Sensitivity and specificity were estimated for each serological marker and the relationship between disease phenotype and serological positivity was tested using the χ2 test. Results Accuracy of the serological markers ASCA (IgA or IgG isotypes) and Omp-C for differentiation of healthy controls from those with CD showed a sensitivity of 50% and 54% but a higher specificity (75% and 78%). The sensitivity and specificity of pANCA was calculated for differentiating UC from controls as 48% and 97% respectively. The combination of ASCA-/pANCA+ resulted in better diagnostic accuracy for differentiating CD from UC (sensitivity 32% and specificity 92%) than either test alone. There was no significant association with atypical pANCA and UC phenotype (p=0.58). Both ASCA and Omp-C positivity correlated with small bowel involvement in CD patients (p=0.01 and p=0.04 respectively). Conclusion The low sensitivity of ASCA, Omp-C and pANCA limits their use for IBD screening in Indian Asian populations but the high specificity of combining ASCA and ANCA can be helpful in differentiating between UC and CD in this ethnic group. The reported sensitivities and specificities of ASCA, Omp-C and pANCA in Indian Asians are similar to those observed in Caucasian populations. In addition, the association of ASCA and Omp-C with CD phenotype has also been reported in Caucasian CD patients suggesting similar pathological mechanisms in both ethnic groups.

OC-063 Gut microbiota-host bile acid metabolism interactions in clostridium difficile infection: the explanation for the efficacy of faecal microbiota transplantation?

Introduction Faecal microbiota transplantation (FMT) effectively treats recurrent Clostridium difficile infection (CDI), yet the mechanisms underlying its efficacy are poorly-defined. In vitro, conjugated primary bile salts (i.e. taurocholic acid) promote the germination of C. difficile, whilst secondary bile salts (i.e. deoxycholic acid) inhibit vegetative growth of the organism. As gut microbiota-derived enzymes (i.e. bile salt hydrolases (BSH)) are responsible for bile acid metabolism in vivo, we hypothesised that the efficacy of FMT may reflect transfer of BSH-producing bacteria, with restoration of a gut bile acid profile that inhibit germination/vegetative growth of C. difficile. Method Faecal samples were collected from patients with recurrent CDI pre-FMT (n=26), at 8–12 weeks after successful FMT, and also from stool donors (n=17). Bacterial DNA was used for microbial profiling (via 16S rRNA gene sequencing) and for qPCR of BSH genes. Liquid chromatography-mass spectrometry was used for bile acid profiling. BSH enzyme activity was established using a plate-based precipitation assay. Results Microbial and bile acid profiles from pre-FMT patients were markedly different to those found in the post-FMT and donor groups (p<0.001, PERMANOVA); qPCR confirmed enrichment of BSH-producing organisms post-FMT. Taurocholic acid levels were elevated (and deoxycholic acid levels reduced) pre-FMT compared to donors and post-FMT (p<0.001, Wilcoxon-Mann-Whitney test). By Spearman’s rank, abundance of BSH-producing bacteria negatively correlated with taurocholic acid and positively correlated with deoxycholic acid levels (Figure 1), with p<0.05 for this correlation for levels of both bile acids with Bacteroides vulgatus, Blautia obeum, Dorea longicatena, and Eubacterium rectale. Stool BSH activity was negligible pre-FMT, but was significantly increased post-FMT (p<0.002, Wilcoxon-Mann-Whitney). Abstract OC-063 Figure 1 Heatmap demonstrating the correlation between abundance of BSH-secreting organisms and gut levels of taurocholic and deoxycholic acids (Spearman’s rank). Conclusion The gut microbiota is enriched with BSH-producing bacterial species post-FMT for CDI, and these organisms are present within the gut microbiota of donors. The increased relative abundance of BSH-producing organisms post-FMT was negatively correlated with gut taurocholic acid levels, positively correlated with deoxycholic acid levels, and associated with increased BSH activity. These data collectively support a hypothesis of transfer of BSH-producing organisms during FMT linked to reconstitution of a gut bile acid profile unfavourable to the germination and growth of C. difficile. Disclosure of Interest None Declared

OC-040 National Survey of Practice of Faecal Microbiota Transplantation for Clostridium Difficile Infection in the United Kingdom

Introduction The National Institute of Health and Care Excellence recommend the use of faecal microbiota transplantation (FMT) for recurrent or refractory Clostridium difficile infection (CDI), as studies have shown it to be a highly effective therapy with a primary cure rate of over 90%.1 We aimed to conduct a National survey to explore current practice of FMT and the challenges faced by hospitals in setting up this novel treatment strategy. Methods UK gastroenterologists, microbiologists and infectious disease physicians were invited to take part in a National survey by completing an online questionnaire over a five month period from October 2015. Results A total of 255 responses were obtained, of which 219 were evaluable. These came from 124 microbiologists/infectious disease clinicians and 95 gastroenterologists. The survey covered 130 independent sites: 112 acute NHS Trusts in England, 9 hospitals in Scotland and 9 hospitals in Wales. Only 28% (36/130) had performed FMT for refractory or recurrent CDI, of which 58% (21/36) of sites had experience of performing FMT for over 1 year, but only 19% (7/36) had treated at least 10 patients. 67% (24/36) made FMT on site while 33% (12/36) obtained FMT from elsewhere to administer at their hospital. Apart from one site that used FMT for refractory ulcerative colitis there were no other indications for its use. Of the 94 independent sites that did not perform FMT for refractory or recurrent CDI, 45% (42/94) believed that they were unable to do it due to lack of facilities, 38% (36/94) did not know where to start, however only 5% (5/94) felt reluctant to do it because of its perceived unpleasantness. Of those sites not performing the procedure, 70% (66/94) suggested that they would be keen to have support in setting up an FMT service for CDI locally. Only 29% (27/94) of the sites that did not perform FMT had referred their patients elsewhere; primarily to Glasgow, Birmingham and Exeter. Conclusion In the largest National survey done to date exploring the practice of FMT in UK, we have shown that only a quarter of responding sites performed FMT for recurrent or refractory CDI. There are significant challenges faced by hospitals in setting up this service. However, most welcomed support due to unfamiliarity with the perceived logistical hurdles. A central quality controlled and regulated FMT preparation, delivery and support service for the UK may be an efficient model to ensure continued and safe access to this novel treatment strategy for patients in the NHS. Reference 1 https://www.nice.org.uk/guidance/ipg485 Disclosure of Interest None Declared

PWE-094 Understanding The Efficacy of Faecal Microbiota Transplantation in Clostridium Difficile Infection: Re-Establishment of Gut Microbiota with The Ability to Degrade Bile?

Introduction Faecal microbiota transplantation (FMT) has recently emerged as a highly-effective therapy for recurrent/ refractory Clostridium difficile (recently re-named Peptoclostridium difficile) infection (CDI); however, the specific mechanisms underlying the efficacy of FMT remain largely unclear. Given that different bile salt metabolites differentially affect C. difficile’s ability to germinate and grow both in vitro and in vivo, we hypothesised that CDI is characterised by perturbed bile acid metabolism, and that FMT may exert its efficacy through re-establishment of gut microbiota that restore this process to normal. Methods Stool samples were collected from healthy volunteer donors participating in an FMT programme, whilst serial stool samples were collected from a patient successfully treated with FMT for refractory CDI both pre- and post-transplantation. Samples were assayed for structure of the gut microbiota using 16 S rRNA gene sequencing, and for bile acid profiling via liquid chromatography mass spectrometry (LC-MS). Presence of bile salt hydrolases (responsible for deconjugation of glycine- and taurine-conjugated primary bile acids within the gut) was assessed via PCR of bacterial DNA extracted from stool. Results A 61 year-old man with refractory CDI was treated with FMT. He demonstrated a modest improvement in diarrhoea after a first FMT, but an immediate, complete and sustained resolution of symptoms after a second FMT from a different donor (performed two weeks after the first). 16 S rRNA gene sequencing demonstrated a pattern of faecal bacterial communities that closely resembled that of the healthy donors by one week after the second FMT. Faecal LC-MS analysis revealed the patient’s gut bile acid profile pre-FMT to be enriched sixfold in taurocholic acid (a potent trigger for C. difficile spore germination in vitro). Post-FMT, the patient’s gut bile acid profile resembled that of healthy donors, with loss of taurocholate and enrichment of secondary bile acids (which are recognised in vitro as inhibitors of C. difficile growth). PCR of bacterial DNA extracted from faeces displayed no detectable BSH genes in the recipient either pre-FMT or by one week following the first FMT, but BSH presence was confirmed in the recipient by one week following the second FMT, as well as in both donors. Conclusion FMT may restore bile-degrading members of the gut microbiota, and consequently restore a normal bile acid metabolism to the gut that protects against C. difficile germination. Disclosure of Interest None Declared

PTH-069 Effect of co-morbidities on urinary metabolic profiling in the characterisation of patients with inflammatory bowel disease

Introduction Several studies have successfully used metabolic profiling (metabonomics) of urine to distinguish patients with IBD from healthy controls,1many discriminatory metabolites identified relate to microbial or host-microbial co-metabolism, supporting the concept of gut dysbiosis in IBD pathogenesis. An individual’s metabolic phenotype may be influenced by factors including comorbidities such as diabetes mellitus, and previous studies have been restricted to IBD patients with no significant comorbidities which does not accurately reflect clinical practice. In order to assess the potential of metabonomics in a clinically relevant population, this study analysed urinary metabolic profiles of IBD patients with and without comorbidities. Method Nuclear magnetic resonance spectroscopy was used to acquire urinary metabolic data from 51 IBD patients with at least one significant comorbidity (including diabetes mellitus, asthma and ischaemic heart disease), 46 patients with IBD alone, and 54 healthy controls. Groups were matched for age, sex, race, BMI and IBD diagnosis. As a preliminary analysis, resonances specific for metabolites influenced by gut microbes based on prior observations were integrated and analysed using appropriate univariate statistics. Results Univariate analysis showed that hippurate excretion was significantly lower in patients with IBD and comorbidities compared to healthy controls (p = 0.01), as well as patients with IBD alone (p = 0.04) confirming results of other published studies. In addition, trimethylamine (TMA) levels were relatively increased in patients with IBD and comorbidities (p = 0.03) and IBD alone (p = 0.07) compared to healthy controls, although trimethylamine-N-oxide (TMAO) levels showed no significant difference due to IBD or comorbid status. Conclusion In this study urinary hippurate was significantly lower in IBD patients, regardless of other comorbid diagnoses or treatments, which is consistent with previous studies. Hippurate is not a specific marker for IBD but rather related to gut microbiome metabolic dysfunction as it has also been observed when comparing the urine metabolic profile of lean and obese people and in intestinal parasitic infections. The relative increase in TMA has not been previously reported in humans, but correlates with a study that shows increasing TMA with progression of IBD in the IL10 knock-out mouse model.2Future work will include multivariate analysis of this dataset to elucidate metabolic phenotypes associated with complex comorbidities in IBD. Disclosure of interest None Declared. References Williams HR et al.Am J Gastroenterol. 2009;104(7):1894 Murdoch T et al. Anal. Chem. 2008;80:5524–5531

OC-011 Exhaled volatile organic compound breath analysis in inflammatory bowel disease

Introduction Distinguishing between Crohn’s disease (CD) and ulcerative colitis (UC) is important for determining management and prognosis. Selected ion flow tube mass spectrometry (SIFT-MS) may be used to analyse volatile organic compounds (VOCs) in exhaled breath: these may be altered in disease states, and distinguishing breath VOC profiles can be identified.1A recent paediatric study used SIFT-MS to distinguish IBD patients from healthy controls (HC).2The aim of this pilot study was to identify, quantify and analyse VOCs present in the breath of adult IBD patients and controls, potentially providing insights into disease pathogenesis and complementing current diagnostic algorithms.Abstract OC-011 Table 1 Sensitivity (%) Specificity (%) Q2value CD vs HC 94.4 94.4 0.78 UC vs HC 90.5 94.4 0.66 CD vs UC 88.9 90.0 0.69 Method SIFT-MS breath profiling of 56 individuals (20 UC, 18 CD and 18 healthy controls) was undertaken. Multivariate analysis included principal components analysis (PCA) and partial least squares discriminant analysis with orthogonal signal correction (OSC-PLS-DA). Receiver Operator Characteristic (ROC) analysis was performed for each comparative analysis using statistically significant VOCs. Results OSC-PLS-DA modelling was able to distinguish both CD and UC from healthy controls and from one other with good sensitivity and specificity. ROC analysis using combinations of statistically significant VOCs (dimethyl sulphide, hydrogen sulphide, hydrogen cyanide, ammonia, butanal and nonanal) gave integrated areas under the curve (AUC) of 0.86 (CD vs HC), 0.74 (UC vs HC) and 0.83 (CD vs UC). Conclusion SIFT-MS breath profiling was able to distinguish IBD patients from controls, as well as separate UC from CD, using both multivariate and univariate statistical techniques. The specific VOCs characterising the breath in IBD relate to bacterial dysbiosis (sulphur compounds and ammonia) and oxidative stress (aldehydes)3– both mechanisms implicated in disease pathogenesis. Disclosure of interest None Declared.Abstract OC-011 Figure 1 References Popov TA. Human exhaled breath analysis. Ann Allergy Asthma Immunol. 2011;106(6):451–6 Patel N, Alkhouri N, Eng K, et al. Metabolomic analysis of breath volatile organic compounds reveals unique breathprints in children with inflammatory bowel disease: a pilot study. Aliment Pharmacol Ther. 2014;40(5):498–507 Bos LD, Sterk PJ, Schultz MJ. Volatile metabolites of pathogens: a systematic review. PLoS Pathog. 2013;9(5):e1003311

The prevalence of abnormal hepatic biochemistry and hepatobiliary morbidity in a cohort of patients with inflammatory bowel disease

Introduction There is a recognised association between Inflammatory Bowel Disease (IBD) and both abnormal hepatic biochemistry and hepatobiliary morbidity. Hepatic steatosis, primary sclerosing cholangitis (PSC), cholelithiasis and drug reactions have all been linked, but the nature of the association and risk factors are unclear. The aims of this study were: (1) to assess the prevalence of, and risk factors for abnormal biochemistry in a cohort of patients with IBD; (2) to determine the prevalence of hepatobiliary morbidity in those patients with abnormal liver function tests (LFTs). Methods Ethical approval was obtained to take demographic and clinical data retrospectively from the IBD database of a secondary/tertiary referral centre, entered prospectively from January 2006 to September 2009. IBD diagnosis was based on endoscopic, radiological and histological criteria and disease phenotyping was according to the Montreal classification. Abnormal hepatic biochemistry was defined as elevation above the laboratory upper limit of normal for ALT (>40 IU/l) and/or ALP (>130 IU/l) and/or Bilirubin (>17 mmol/l) at time of entry into the database. Fishers Exact and Mann-Whitney U tests were used. Results Of 493 patients in the database, 370 (75%) had hepatic biochemistry available. Of these, the prevalence of abnormal hepatic biochemistry was 17.8% and 10.8% had abnormal ALT. African-Caribbean patients had a high prevalence of abnormal hepatic biochemistry (11/23, 48%, p=0.001), in contrast to South Asian patients (5/49, 10%, p=0.16) and White British patients (24/152, 15.8%, p=0.4). Overall, there was no association with age, sex, diagnosis (Crohns or Ulcerative Colitis), disease phenotype or use of Azathioprine or Infliximab. Hepatobiliary morbidity was identified in 18/64 (28%) of those with abnormal LFTs. 6 patients (1.6%) had PSC, of whom 5 had raised ALP. Conclusion More than one in six patients with IBD in this cohort had abnormal hepatic biochemistry and of these, over a quarter had identifiable hepatobiliary morbidity. Surprisingly, the prevalence of abnormal LFTs was higher than expected in African-Caribbean patients and low in South Asians. IBD type and phenotype was not associated with abnormal liver biochemistry. Confirmation and further evaluation of these findings should be sought prospectively in an independent cohort.

PTU-047 Tolerability of thiopurines in a UK South Asian inflammatory bowel disease population

Introduction The thiopurine drugs, azathioprine and mercaptopurine are commonly used in the treatment of both Crohns disease (CD) and ulcerative colitis (UC). In general, they are well tolerated and safe. However, side effects, serious enough to warrant drug withdrawal, are reported in up to 24% of patients.1 Common reasons for thiopurine withdrawal include gastric intolerance, leucopaenia and deranged liver function tests. Previous studies in the UK have concentrated on Caucasian IBD populations; there are no previous studies of the tolerability of thiopurine drugs in the UK South Asian IBD population. Methods The aim of this study was to determine the frequency of thiopurine withdrawal in a UK South Asian IBD cohort. The medical notes of South Asian inflammatory bowel disease patients attending clinic in five North West London hospitals between 2007 and 2009 were reviewed. Any patients treated with thiopurines were noted and patients withdrawing from treatment within 6 months of starting treatment were recorded. Results Three hundred and seventy-one South Asian IBD patients were identified. One hundred and fifty-one patients (41%) were started on a thiopurine. In 34.4% of cases (52/151) South Asian patients stopped first-line immunosuppression with a thiopurine within 6 months of starting. In five cases the reason for drug withdrawal was not documented. In the remaining 47 cases, the reasons reported for stopping the drug included gastric intolerance (17), flu-like symptoms (3), pancreatitis (5), hepatitis (3), rash (3), myelotoxicity (3) and non-specific symptoms (including headache, itching, increased urinary frequency and paraesthesia) (13). Conclusion This study has revealed a high level of thiopurine withdrawal in a cohort of South Asian IBD patients. Although TPMT levels were not available for all South Asian patients in our study, previous research has shown that TPMT polymorphisms are of a similar frequency in British Caucasians and South Asians. A high proportion of South Asian patients stopped thiopurine treatment for non-specific symptoms (13/47; 28%). A possible reason to explain this could be difficulty in communication with non-English speaking patients, in particular a lack of drug information leaflets translated into other languages. This may mean that patients do not know what to expect when starting thiopurines. Clinicians need to be aware of this and information leaflets on thiopurines should be translated for ethnic minorities who are unable to read English.