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Evaluation of Methods for the Preservation of Human Fecal Samples, for Assessment of Microbiota Composition
probiotic and non-probiotic group did not significantly differ (p=0.808). Conclusion: Probiotic induced sepsis in patients with IBD on immunosupression is a very rare occurrence that is only documented in case reports. Given the elevated risk of CDI, pouchitis and other bowel pathology in this population, patients with IBD should be included in probiotic research going forward. References: 1. Meini S et al. Dec 2015;43(6):777-781.
Mo1909 DIRECT NUCLEIC ACID DETECTION OF DIARRHEAL PATHOGENS IN STOOL USING AN ANTIBODY-FREE LATERAL FLOW ASSAY Nicolaas H. Fourie, Sarah K. Abey, Eric Ferguson, Natnael Kenea, Ana F. Diallo, ChangHee Kim, Wendy A. Henderson Diarrheal disease remains a leading cause of childhood death in the developing world as well as being a significant health and financial burden, particularly to vulnerable populations such as the young, immunocompromised and elderly in developed countries. There is a need for rapid low-cost point-of-care detection of diarrhea causing pathogens to inform appropriate interventions and improve patient outcomes and mitigate financial burdens. We describe a gold nanoparticle lateral flow assay (LFA) for the direct detection of the nucleic acids of common diarrhea causing pathogens. The LFA used a sandwiched based nucleic acid capture design to produce a red color signal at detection dots and control dots for a given pathogen. The color signals are visible to the naked eye after 5 minutes. The paper based LFA was designed to be low-cost and suited for use in low resource environments. The LFA was optimized for the detection of rRNA and rDNA targets at concentrations between 1 and 100nM, although the lower detection limit is estimated to be around 0.5nM. No nucleic acid purification, washing or denaturing is necessary and stool samples are diluted with only water or PBS to facilitate flow. The tool has been optimized for the detection of the four most common diarrhea casing bacteria in the USA: EPEC, C. difficile, EAEC, and Campylobacter spp. Each target was validated using custom designed pathogen specific oligonucleotides which were tested for under various conditions. Once the assay and target sequences were optimized detection of the endogenous pathogen in stool was confirmed with PCR. The performance of the LFA is currently being tested against PCRbased gold standards in diarrhea pathogen detection in a large diarrhea cohort. The scope of testing, range of diarrheal pathogens and substrates will be expanded and optimized for. The LFA is rapid, sensitive, specific, easy to use and robust. It shows great potential as a tool to be deployed in the field, particularly in low resource environments where diarrhea burden is high and impacts are severe.
Mo1907 EVALUATION OF METHODS FOR THE PRESERVATION OF HUMAN FECAL SAMPLES, FOR ASSESSMENT OF MICROBIOTA COMPOSITION Tadasu Iizumi, Guillermo I. Perez Perez, Martin J. Blaser A major problem for clinical researchers is how to preserve human fecal samples for analysis of the gut microbiota. Often, fecal specimens have been stored or transported under ambient temperature before being frozen. To assess this problem, we selected four stabilizers: RNAlater (Qiagen), 100% ethanol, and the Stool Nucleic Collection and Preservation Tubes (Norgen) and OMNIgene Gut sample collection kit (DNAgenotek). Stool samples in the four different stabilizers were stored at 4 °C or at room temperature (RT) for 3 or 7 days. We extracted DNA from the fresh samples using either the MoBio PowerLyzer Power Soil DNA Isolation kit or Quiagen AllPrep DNA/RNA Universal kit. We found that the MoBio kit was superior to the Quiagen kit in the proportion of samples that were successfully amplified using universal 16S rRNA V4 region primers, and in their richness. There were 102 samples from 6 individuals (4 male / 2 female). We sequenced the extracted DNA using the Illumina MiSeq platform and performed data analysis using QIIME software (version Mac Qiime 1.9.1). After filtering procedures, sequences were clustered into operational taxonomic units (OTUs) using an open reference approach with UCLUST against the Greengenes Core set. Rarefaction analysis used whole phylogenetic diversity (PD) to assess α-diversity. Unweighted UniFrac distances were calculated to assess β-diversity, and visualized by principal coordinates analysis (PCoA); ANOVA was used to compare OTU and genus-level abundances. The MoBio extractions yielded a mean sampling depth of 10,053 ± 1,991. Assessment of αdiversity showed no significant differences in relation to stabilizer used, time of storage, or temperature. Differences in α-diversity mostly varied by subject. In analysis of β-diversity, the large differences in samples mainly were due to subject identity and not preservation method, time, or temperature. We next analyzed relative abundance at both the phylum and genus level, using fresh samples as the gold standard; abundances for each condition were almost identical at both taxonomic levels. We conclude from this study that commercial or inexpensive stool stabilizers were nearly identical in their utility at room temperature for up to seven days to preserve compositional characteristics of the fecal microbiota communities. Support: U01 AI122285-01 from NIAID
Mo1910 COMMON BACTERIAL AND VIRAL INFECTION IN INPATIENTS WITH IBD: RISK FACTORS AND IMPACT ON THE DISEASE OUTCOME Vaibhav Wadhwa, Rocio Lopez, Bo Shen Background: Patients with inflammatory bowel disease (IBD), particularly those with severe, refractory disease are more prone to infections. Infections in IBD patients are the common cause of poor disease outcome, increase morbidity and mortality. Our aim was to study the risk factors associated with the risk of three common infections namely - Clostridium difficile, Campylobacter and CMV in IBD inpatients as compared to diverticulitis patients. Methods: We performed a case control study using the National Inpatient Sample (NIS) database from 2013. The study group included all patients with Crohn's disease (CD) (ICD 9 Code: 555) & ulcerative colitis (UC) (ICD 9 Code: 556) and the control group included all patients with diverticulitis (ICD 9 Code: 562.11, 562.13). Both univariable and multivariable analyses were performed to assess the risk factors associated with C Diff (008.45), Campylobacter (008.45) and CMV (078.5). The case and control groups were compared using chi-square test for analysis. Continuous variables were compared using t-tests and categorical variables were compared using Rao-Scott chi-square tests. In addition, multivariable logistic regression analysis was performed to assess the association between disease group and cancer while adjusting for potential cofounders. A univariable analysis was performed to assess differences between subjects with UC & CD and those with diverticulitis; continuous variables were compared using t-tests and categorical variables were compared using Rao-Scott chi-square tests. All analyses were done using SAS (version 9.4, The SAS Institute, Cary, NC). Results: We analyzed more than 30 million hospitalizations in 2013, with 299,125 being IBD- related admissions and 299,765 being diverticulitis-related admissions. The rate of Campylobacter, C. diff and CMV infections were lowest in the diverticulitis group, followed by the CD group and highest in the UC group (Figure 1). After adjusting for all variables in the model, C diff infection was associated with sepsis (p<0.001) and dehydration (p<0.001) but not with bowel surgery or bowel perforation. Campylobacter infection was associated with increased risk of sepsis (p<0.001) and dehydration (p<0.001) but was shown to have decreased risk of bowel surgery (p<0.03). CMV infection in IBD patients was not shown to be associated with any adverse outcomes in the multivariate analysis. Conclusion: Campylobacter, C diff and CMV infections are more common in IBD patients than diverticulitis patients. These infections in IBD are associated with poor outcomes in IBD patients.
Mo1908 PROBIOTIC SEPSIS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE; IS IT SOMETHING TO WORRY ABOUT? Nicole T. Shen, Stephanie L. Gold, Yecheskel Schneider, Shirley A. Cohen-Mekelburg, Anna M. Maw, Carl V. Crawford Background: With expanding knowledge of the importance of fecal microbiota and its relationship to diseases such as inflammatory bowel disease (IBD) and C. dificile infections (CDI), probiotics are increasingly being prescribed. Traditionally, studies on probiotic efficacy and safety have excluded immunosupressed patients including those with IBD. This group has been considered high risk for probiotic bacteremia/fungemia because of rare case reports of translocation of probiotic across ulcerated intestinal mucosa, most commonly Lactobacillus followed by Saccharomyces.1 Case reports suggest risk factors for probiotic bacteremia include severe IBD or significant immunosuppression, suggesting the highest at risk population for probiotic gut translocation may be IBD patients on biologics. With the increased incidence of biologic use in the IBD population, studying the safety of probiotics in this patient population is critical. Methods: A single center chart review was conducted for patients with IBD on biologic therapy in the past 5 years. Baseline characteristics including gender, age, IBD type, duration of disease, prior steroid use and current biologic therapy were collected. Probiotic use including duration, type of probiotic, history of sepsis or systemic inflammatory response syndrome (SIRS) and hospitalizations while on the probiotic were recorded. Results: 204 patients with IBD on a biologic were isolated and 86(42.2%) were on a probiotic during the study period. The average age was 42 years, 88(43.1%) were male, 64.2% had Crohn's (CD), 34.8% had ulcerative colitis (UC), and the average disease duration was 14.28 years. 16.7% had an ileostomy. 67 of the patients were getting a bacterial probiotic and 21 were on a yeast based probiotic. The average Harvey Bradshaw Index was 3.5(median 4), and the average Partial Mayo Score was 2.43 (median 2). 72(35.2%) were on certolizumab, 33(16.1%) were on adalimumab, 70(34.3%) were on infliximab, 20(9.8%) were on vedolizumab, 1(0.5%) was on golimumab and 7(3.4%) were on ustekinumab. 77% of patients were additionally on oral steroids. No cases of probiotic sepsis or SIRS were encountered. 7(3.4%) of the patients on probiotics were hospitalized during their medication course; 1 for a liver transplant secondary to primary sclerosing cholangitis, 3 for dehydration, 1 for antibiotics for pyoderma gangrenosum and 1 for childbirth. The number of hospitalizations in the
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AGA Abstracts
AGA Abstracts
associated with significant post-prandial reductions in IL-12p70 (P=0.017), IL-1b (P=0.020), and ghrelin (P=0.017) compared to placebo subjects. The key findings of the present study, were that oral probiotic supplementation reduced responses that were consistent with "leaky gut syndrome" and transient reductions in chronic/metabolic disease risk.
Mo1909 DIRECT NUCLEIC ACID DETECTION OF DIARRHEAL PATHOGENS IN STOOL USING AN ANTIBODY-FREE LATERAL FLOW ASSAY Nicolaas H. Fourie, Sarah K. Abey, Eric Ferguson, Natnael Kenea, Ana F. Diallo, ChangHee Kim, Wendy A. Henderson Diarrheal disease remains a leading cause of childhood death in the developing world as well as being a significant health and financial burden, particularly to vulnerable populations such as the young, immunocompromised and elderly in developed countries. There is a need for rapid low-cost point-of-care detection of diarrhea causing pathogens to inform appropriate interventions and improve patient outcomes and mitigate financial burdens. We describe a gold nanoparticle lateral flow assay (LFA) for the direct detection of the nucleic acids of common diarrhea causing pathogens. The LFA used a sandwiched based nucleic acid capture design to produce a red color signal at detection dots and control dots for a given pathogen. The color signals are visible to the naked eye after 5 minutes. The paper based LFA was designed to be low-cost and suited for use in low resource environments. The LFA was optimized for the detection of rRNA and rDNA targets at concentrations between 1 and 100nM, although the lower detection limit is estimated to be around 0.5nM. No nucleic acid purification, washing or denaturing is necessary and stool samples are diluted with only water or PBS to facilitate flow. The tool has been optimized for the detection of the four most common diarrhea casing bacteria in the USA: EPEC, C. difficile, EAEC, and Campylobacter spp. Each target was validated using custom designed pathogen specific oligonucleotides which were tested for under various conditions. Once the assay and target sequences were optimized detection of the endogenous pathogen in stool was confirmed with PCR. The performance of the LFA is currently being tested against PCRbased gold standards in diarrhea pathogen detection in a large diarrhea cohort. The scope of testing, range of diarrheal pathogens and substrates will be expanded and optimized for. The LFA is rapid, sensitive, specific, easy to use and robust. It shows great potential as a tool to be deployed in the field, particularly in low resource environments where diarrhea burden is high and impacts are severe.
Mo1907 EVALUATION OF METHODS FOR THE PRESERVATION OF HUMAN FECAL SAMPLES, FOR ASSESSMENT OF MICROBIOTA COMPOSITION Tadasu Iizumi, Guillermo I. Perez Perez, Martin J. Blaser A major problem for clinical researchers is how to preserve human fecal samples for analysis of the gut microbiota. Often, fecal specimens have been stored or transported under ambient temperature before being frozen. To assess this problem, we selected four stabilizers: RNAlater (Qiagen), 100% ethanol, and the Stool Nucleic Collection and Preservation Tubes (Norgen) and OMNIgene Gut sample collection kit (DNAgenotek). Stool samples in the four different stabilizers were stored at 4 °C or at room temperature (RT) for 3 or 7 days. We extracted DNA from the fresh samples using either the MoBio PowerLyzer Power Soil DNA Isolation kit or Quiagen AllPrep DNA/RNA Universal kit. We found that the MoBio kit was superior to the Quiagen kit in the proportion of samples that were successfully amplified using universal 16S rRNA V4 region primers, and in their richness. There were 102 samples from 6 individuals (4 male / 2 female). We sequenced the extracted DNA using the Illumina MiSeq platform and performed data analysis using QIIME software (version Mac Qiime 1.9.1). After filtering procedures, sequences were clustered into operational taxonomic units (OTUs) using an open reference approach with UCLUST against the Greengenes Core set. Rarefaction analysis used whole phylogenetic diversity (PD) to assess α-diversity. Unweighted UniFrac distances were calculated to assess β-diversity, and visualized by principal coordinates analysis (PCoA); ANOVA was used to compare OTU and genus-level abundances. The MoBio extractions yielded a mean sampling depth of 10,053 ± 1,991. Assessment of αdiversity showed no significant differences in relation to stabilizer used, time of storage, or temperature. Differences in α-diversity mostly varied by subject. In analysis of β-diversity, the large differences in samples mainly were due to subject identity and not preservation method, time, or temperature. We next analyzed relative abundance at both the phylum and genus level, using fresh samples as the gold standard; abundances for each condition were almost identical at both taxonomic levels. We conclude from this study that commercial or inexpensive stool stabilizers were nearly identical in their utility at room temperature for up to seven days to preserve compositional characteristics of the fecal microbiota communities. Support: U01 AI122285-01 from NIAID
Mo1910 COMMON BACTERIAL AND VIRAL INFECTION IN INPATIENTS WITH IBD: RISK FACTORS AND IMPACT ON THE DISEASE OUTCOME Vaibhav Wadhwa, Rocio Lopez, Bo Shen Background: Patients with inflammatory bowel disease (IBD), particularly those with severe, refractory disease are more prone to infections. Infections in IBD patients are the common cause of poor disease outcome, increase morbidity and mortality. Our aim was to study the risk factors associated with the risk of three common infections namely - Clostridium difficile, Campylobacter and CMV in IBD inpatients as compared to diverticulitis patients. Methods: We performed a case control study using the National Inpatient Sample (NIS) database from 2013. The study group included all patients with Crohn's disease (CD) (ICD 9 Code: 555) & ulcerative colitis (UC) (ICD 9 Code: 556) and the control group included all patients with diverticulitis (ICD 9 Code: 562.11, 562.13). Both univariable and multivariable analyses were performed to assess the risk factors associated with C Diff (008.45), Campylobacter (008.45) and CMV (078.5). The case and control groups were compared using chi-square test for analysis. Continuous variables were compared using t-tests and categorical variables were compared using Rao-Scott chi-square tests. In addition, multivariable logistic regression analysis was performed to assess the association between disease group and cancer while adjusting for potential cofounders. A univariable analysis was performed to assess differences between subjects with UC & CD and those with diverticulitis; continuous variables were compared using t-tests and categorical variables were compared using Rao-Scott chi-square tests. All analyses were done using SAS (version 9.4, The SAS Institute, Cary, NC). Results: We analyzed more than 30 million hospitalizations in 2013, with 299,125 being IBD- related admissions and 299,765 being diverticulitis-related admissions. The rate of Campylobacter, C. diff and CMV infections were lowest in the diverticulitis group, followed by the CD group and highest in the UC group (Figure 1). After adjusting for all variables in the model, C diff infection was associated with sepsis (p<0.001) and dehydration (p<0.001) but not with bowel surgery or bowel perforation. Campylobacter infection was associated with increased risk of sepsis (p<0.001) and dehydration (p<0.001) but was shown to have decreased risk of bowel surgery (p<0.03). CMV infection in IBD patients was not shown to be associated with any adverse outcomes in the multivariate analysis. Conclusion: Campylobacter, C diff and CMV infections are more common in IBD patients than diverticulitis patients. These infections in IBD are associated with poor outcomes in IBD patients.
Mo1908 PROBIOTIC SEPSIS IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE; IS IT SOMETHING TO WORRY ABOUT? Nicole T. Shen, Stephanie L. Gold, Yecheskel Schneider, Shirley A. Cohen-Mekelburg, Anna M. Maw, Carl V. Crawford Background: With expanding knowledge of the importance of fecal microbiota and its relationship to diseases such as inflammatory bowel disease (IBD) and C. dificile infections (CDI), probiotics are increasingly being prescribed. Traditionally, studies on probiotic efficacy and safety have excluded immunosupressed patients including those with IBD. This group has been considered high risk for probiotic bacteremia/fungemia because of rare case reports of translocation of probiotic across ulcerated intestinal mucosa, most commonly Lactobacillus followed by Saccharomyces.1 Case reports suggest risk factors for probiotic bacteremia include severe IBD or significant immunosuppression, suggesting the highest at risk population for probiotic gut translocation may be IBD patients on biologics. With the increased incidence of biologic use in the IBD population, studying the safety of probiotics in this patient population is critical. Methods: A single center chart review was conducted for patients with IBD on biologic therapy in the past 5 years. Baseline characteristics including gender, age, IBD type, duration of disease, prior steroid use and current biologic therapy were collected. Probiotic use including duration, type of probiotic, history of sepsis or systemic inflammatory response syndrome (SIRS) and hospitalizations while on the probiotic were recorded. Results: 204 patients with IBD on a biologic were isolated and 86(42.2%) were on a probiotic during the study period. The average age was 42 years, 88(43.1%) were male, 64.2% had Crohn's (CD), 34.8% had ulcerative colitis (UC), and the average disease duration was 14.28 years. 16.7% had an ileostomy. 67 of the patients were getting a bacterial probiotic and 21 were on a yeast based probiotic. The average Harvey Bradshaw Index was 3.5(median 4), and the average Partial Mayo Score was 2.43 (median 2). 72(35.2%) were on certolizumab, 33(16.1%) were on adalimumab, 70(34.3%) were on infliximab, 20(9.8%) were on vedolizumab, 1(0.5%) was on golimumab and 7(3.4%) were on ustekinumab. 77% of patients were additionally on oral steroids. No cases of probiotic sepsis or SIRS were encountered. 7(3.4%) of the patients on probiotics were hospitalized during their medication course; 1 for a liver transplant secondary to primary sclerosing cholangitis, 3 for dehydration, 1 for antibiotics for pyoderma gangrenosum and 1 for childbirth. The number of hospitalizations in the
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AGA Abstracts
AGA Abstracts
associated with significant post-prandial reductions in IL-12p70 (P=0.017), IL-1b (P=0.020), and ghrelin (P=0.017) compared to placebo subjects. The key findings of the present study, were that oral probiotic supplementation reduced responses that were consistent with "leaky gut syndrome" and transient reductions in chronic/metabolic disease risk.