Gluten-related immunogenic peptides in stool as means to monitor compliance with gluten-free diet in children with newly dia gnosed coeliac disease

Objectives and study: To compare the values of gluten-related immunogenic peptides (GIP) in stool and anti-tissue transglutaminase IgA antibodies (anti-tTG IgA) in blood in children newly diagnosed with coeliac disease (CD). Methods: All children (2–15 y) newly diagnosed with CD between May 2018 and May 2020 at our clinic who complied with the inclusion criteria were invited to join the prospective study. During workup for CD, a stool sample to measure GIP was taken together with a blood sample to measure anti-tTG IgA. All newly diagnosed children were invited 4 months later for a check-up. Children and their caregivers were asked about known non-compliance with the gluten-free diet (GFD), a blood sample was taken to measure the anti-tTG IgA, and a stool sample was collected to measure GIP. Blood was evaluated for anti-tTG IgA by ELISA, and the stool was tested by quantitative Sandwich ELISA designed to detect and quantify GIP using the G12 antibody. Values of GIP and anti-tTG IgA were compared in terms of their relation to the upper limit of normal (ULN) of the particular method. Results: 29 children (18 girls) were enrolled in the study. The values of GIP in stool at the time of diagnosis were above the ULN (0.15 µg/g) in all children. Average 4.21, median 3.29, standard deviation (SD) 3.7. After the four months, all but three (89.7%) had values of GIP in the reference range. Average 0.29, median 0.12, SD 0.73. Similarly, anti-tTG IgA values were above the ULN (9.9 U/mL) at the time of diagnosis in all children. Average 164, median 195, SD 49. Although the anti-tTG IgA levels were lower at check-up in all but one child, only 10 (34.5%) showed values within the normal range, with an average of 27.9, median 12.0, and SD 38.9. All children declared strict adherence to GFD. Discussion: Using the GIP concentration in stool, adherence to GFD in our cohort of children is very good, better than that described in literature. Conclusion: Measuring GIP in stool could prove a more sensitive indicator of adherence to GFD in the early months after the diagnosis of CD when anti-tTG IgA are still elevated above the ULN due to their well-described gradual decrease after GFD initiation.

Metagenomic Analysis of the Pediatric-Onset Multiple Sclerosis Gut Microbiome

Background and Objectives:Little is known of the functional potential of the gut microbiome in pediatric-onset multiple sclerosis (MS). We performed metagenomic analyses using stool samples from individuals with pediatric-onset MS and unaffected controls.Methods:Persons ≤21 years old enrolled in the Canadian Pediatric Demyelinating Disease Network providing a stool sample were eligible. Twenty MS patients (McDonald criteria) with symptom onset <18 years were matched to 20 controls by sex, age (±3 years), stool consistency, and race. Microbial taxonomy and functional potentials were estimated from stool sample-derived metagenomic reads and compared by disease status (MS vs controls) and disease-modifying drug (DMD) exposure using alpha-diversity, relative abundance, and prevalence using Wilcoxon rank-sum, ALDEx2 and Fisher’s exact tests, respectively.Results:Individuals with MS were aged 13.6 years (mean) at symptom onset and 8 were DMD naïve. Mean ages at stool sample were 16.1 and 15.4 years for MS and control participants, respectively; 80% were girls. Alpha-diversity of enzymes and proteins did not differ by disease or DMD status (p>0.20), but metabolic pathways, gene annotations and microbial taxonomy did. Individuals with MS (vs controls) exhibited higher methanogenesis prevalence (odds ratio=10, p=0.044), and Methanobrevibacter abundance (log2 fold-change[LFC]=1.7, p=0.0014), but lower homolactic fermentation abundance (LFC=-0.48, p=0.039). Differences by DMD status included lower phosphate butyryltransferase for DMD-naïve vs exposed MS patients (LFC=-1.0, p=0.033).Discussion:The gut microbiome’s functional potential and taxonomy differed between individuals with pediatric-onset MS versus controls, including higher prevalence of a methane-producing pathway from Archaea and depletion of the lactate fermentation pathway. DMD exposure was associated with butyrate-producing enzyme enrichment. Together these findings indicate that the gut microbiome of individuals with MS may have a disturbed functional potential.

Relationship Functioning and Gut Microbiota Composition Among Older Adult Couples: Feasibility of Data Collection

An emerging area of research extends work on couple functioning and physical health to gut health, a critical marker of general health and known to diminish with age. As a foray into this area, we conducted a pilot study to determine feasibility of data collection (questionnaires and a stool sample) among older adult couples. Participants were recruited from the community using a variety of methods including social media. Among 41 persons responding with interest across recruitment sources, 32 were contacted for screening. Inclusion criteria were: age 60+, marriage or cohabiting partnership, and English speaking/understanding. Exclusion criteria were a gastrointestinal disorder, receiving enteric nutrition, use of antibiotics (past month), cancer treatment (past 6 months), and a +COVID-19 diagnosis (past 2 months). Among 31 eligible couples, 30 consented. All 60 participants completed questionnaires and provided a stool sample using DNAgenotek’s OMR-200 collection kit, chosen for its ease and because samples can be stored at room temperature for 60 days. Sample characteristics were: M (SD) age = 66.57 (4.78); 53.3% female; 91.7% White; 1.7% Latinx; and 78.3% college-educated. 2 couples were same-sex. 43% reported at least one health condition and 25% reported use of a proton pump inhibitor (which can affect the gut microbiome), though none daily. Relational well-being was moderate-high on average per measures of dyadic adjustment and intimacy. Despite original plans to recruit couples in-person from a retirement community, remote operations were feasible via online assessment and study-coordinated shipping, a necessary yet fruitful shift due to the SARS-CoV-2 pandemic.

The Impact of a Mediterranean Diet on the Gut Microbiome in Healthy Human Subjects: A Pilot Study

<b><i>Background and Aims:</i></b> Despite the reported salutary benefits of a Mediterranean diet (MD) on a wide variety of health conditions, the specific microbial changes associated with an MD within the gastrointestinal (GI) tract are not well studied. Specifically, although population and survey-based studies have shown microbial changes, there are no published data on how an MD alters the gut flora in a controlled setting. <b><i>Methods:</i></b> We recruited 10 healthy subjects, each of whom gave a stool sample at baseline and then was provided with prepared meals of a “typical” American diet; after 2 weeks, a second stool sample was collected. All subjects were then provided with prepared meals based on the MD for another 2 weeks, followed by a final stool sample collection. Stool samples were batch analyzed with DNA extraction, and sequencing libraries were generated. Measures of bacterial diversity, species richness, and enterotypes were performed. <b><i>Results:</i></b> All ten subjects tolerated the diets well. Bacterial diversity increased with an MD, as measured by alpha diversity via the Simpson index. Furthermore, there were significant differences in 5 bacterial genera between the 2 diets. <b><i>Conclusion:</i></b> This small pilot study of controlled diets demonstrates that the MD can rapidly alter the gut microbiome in healthy subjects at the level of global microbial diversity and individual genera. These data confirm the findings of previous observational studies and establish the feasibility of conducting longer term studies on the impact of the MD on the flora of the GI tract and its relationship to digestive diseases.

761. Host Intestinal Defenses Against Clostridioides difficile Infection in Chemotherapy Patients

Background Clostridioides difficile infection (CDI) is a common complication in patients undergoing cancer treatment with cytotoxic chemotherapy. Exposure to antibiotics or chemotherapy disrupts the microbiome by killing protective intestinal flora which consequently promotes C. difficile spore germination and disease. The host defense against CDI includes colonization resistance conferred by the healthy microbiome and innate defenses provided by intestinal epithelial cells. One protective factor secreted by Paneth cells of the intestinal epithelium is lysozyme, an enzyme that degrades the cell walls of Gram-positive bacteria such as C. difficile. We hypothesized that chemotherapy-induced mucosal barrier injury and the resultant death of Paneth cells leads to decreased production of lysozyme. We thus sought to examine changes in lysozyme concentration in stools of chemotherapy patients. Methods We collected stool samples from six patients undergoing cancer treatment at four different time points. The first stool sample corresponded to the day prior to the start of chemotherapy (day zero). We then performed ELISA assays to determine the lysozyme concentration for each stool sample. Results On day zero, the lysozyme levels (n=6) averaged 268.1 ± 131.7 ng/mL. Over the course of chemotherapy, the lysozyme levels decreased 78.70 ± 24.19% from the starting value. The lowest values were observed around days 5 through 11 for most patients, coinciding with when they were most neutropenic around day 11. One of the patients developed CDI on day 5 and experienced more fluctuating lysozyme levels thereafter. On the day that the patient developed CDI, lysozyme was measured as 6.63 ng/mL. Throughout treatment, 3/6 patients showed recovery of lysozyme production with white blood cell recovery. Conclusion Our data indicate that chemotherapy causes decreased concentrations of lysozyme in stool. Low lysozyme levels could in part account for the increased susceptibility to CDI during chemotherapy. Future experiments will include bioinformatics analyses to determine how the microbiome changes in response to chemotherapy. Together, these experiments will inform our approach to determining patient susceptibility to chemotherapy-associated CDI. Disclosures All Authors: No reported disclosures

Identification and Prevalence of Digestive Tract Endoparasites of Goats in Ujungpangkah, Gresik District

This study aims to determine the prevalence and species of endoparasite that infect the digestive tract of goats in Ujungpangkah Sub-District, Gresik District. This study was conducted in February-March 2021 with 100 samples of goat’s stool. Sample examination was conducted in the laboratory of the Division of Veterinary Parasitology, Faculty of Veterinary Medicine, Universitas Airlangga. In fecal examination found four types of endoparasites, which were: Eimeria sp. 62%, Blastocystis sp. 5%, Haemonchus sp. 4%, Strongyloides sp. 2% and mixed infection prevalence was 3%. This study showed a prevalence of 76% digestive tract endoparasite. The Chi-Square test showed significant differences (p <0.05) between groups of goats aged under one year (57%) and over one year (43%).

Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract

Investigation of the impact of stool collection methods on the metabolomics analysis/profiles of infant fecal samples

Metabolomic studies are important to understand microbial metabolism and interaction between the host and the gut microbiome. Although there have been efforts to standardize sample processing in metabolomic studies, infant samples are mostly disregarded. In birth cohort studies, the use of diaper liners is prevalent and its impact on fecal metabolic profile remains untested. In this study, we compared metabolite profiles of fecal samples collected as solid stool and those collected from stool saturated liner. One infant's stool sample was collected in triplicate for solid stool and stool saturated liner. Comprehensive metabolomics analysis of the fecal samples was performed using NMR, UPLC and DI-MS. The total number, identities and concentrations of the metabolites were determined and compared between stool sample collection methods (stool vs. liner). The number and identity of metabolites did not differ between collection methods for NMR and DI-MS when excluding metabolites with a coefficient of variation (CV) > 40%. NMR analysis demonstrated lowest bias between collection methods, and lowest technical precision between triplicates of the same method followed by DI-MS then UPLC. Concentrations of many metabolites from stool and stool saturated liner differed significantly as revealed by Bland-Altman plots and t-tests. Overall, a mean bias of 10.2% in the Bland-Altman analysis was acceptable for some metabolites confirming mutual agreement but not for others with a wide range of bias (-97-117%). Consequently, stool and stool-saturated liner could be used interchangeably only for some select metabolite classes e.g. amino acids. Differences between the metabolomic profiles of solid stool samples and stool saturated liner samples for some important molecules e.g., ethanol, fumarate, short chain fatty acids and bile acids, indicate the need for standardization in stool collection method for metabolomic studies performed in infants.