Stool Collection Research Articles

Frequency of Testing for Clostridioides difficile in Adults Hospitalized with Diarrhea in Louisville, Kentucky

Background: Although Clostridioides difficile infections (CDIs) are associated with significant morbidity and mortality, CDI disease burden may be underestimated if a high proportion of inpatients with diarrhea do not have stool specimens collected for CDI diagnostic testing. The objective of this study was to define the frequency of stool specimen collection and testing for CDI in adult hospitalized patients with diarrhea. Methods: A cross-sectional study was conducted in all 9 adult hospitals (total, 3,532 beds) in Louisville (adult aged ≥18 years; population 599,276) to identify patients with diarrhea and to observe the frequency of stool specimen collection for CDI diagnosis. For 7 consecutive days in December 2018, each ward was visited to identify new onset diarrhea (>3 loose stools in 24 hours) among Louisville adults: first via electronic medical record (EMR) review, then by nurse interviews, and finally by interviewing patients. For patients with diarrhea, research staff reviewed EMRs to determine whether a stool specimen was collected for CDI diagnosis, and they interviewed nurses about potential noninfectious causes of diarrhea. Results: Among 2,565 hospitalized adults (with 14,042 patient days), research staff identified 167 patients (47% men; median age, 64 years) with new onset diarrhea, 1.2 diarrhea cases per 100 patient days. Patients with diarrhea were initially ascertained by EMR review (50%), nurse interviews (42%) or patient interviews (8%); all cases identified by patient interviews were identified by nurses the following day (but many cases identified by nurses were never identified by EMR review). Nurses indicated that 67 cases had a potential noninfectious cause of diarrhea (eg, laxatives, feeding tube, colostomy, liquid diet, etc). Stool specimens were collected by hospital staff for CDI testing from 53 of 167 patients (32%) with diarrhea; 10 of 67 patients (15%) with diarrhea for whom nurses reported potential noninfectious causes of diarrhea (laxative use, enteric feeding, or gastric survey) in the past 24 hours; and 43 of 100 patients (43%) with diarrhea with no reported potential noninfectious causes of diarrhea. Stool collection frequency was similar on weekdays and weekends. Conclusions: The low frequency of CDI diagnostic testing of hospitalized patients with diarrhea indicates that CDI may be underdiagnosed in these hospitals and suggests, given that only 32% of patients with diarrhea had a stool specimen collected, that the CDI disease burden may be 3 times larger than currently appreciated. New-onset diarrhea occurred in >1% of patients each day; the most effective method for identifying patients with diarrhea was via nurse interviews.Funding: Pfizer Vaccines supported this study.Disclosures: Frederick Angulo, Kimbal D. Ford, Joann Zamparo, Elisa Gonzalez, Sharon Gray, David Swerdlow, and Catia Ferreira all report salary from Pfizer.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy

ObjectiveAltered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear.DesignCross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients...

Abstract A26: Pilot trial to examine the effect of ginger on the gut microbiome: The Minnesota Cancer Clinical Trials Network

Abstract Experimental and epidemiologic evidence shows that the use of ginger root may reduce the incidence of colorectal cancer (CRC), but the mechanism through which ginger exerts its effect is unclear. Recently, an imbalanced gut microbiome was linked to inflammation, immune response, and CRC risk. Thus, we hypothesize that ginger intake may decrease CRC risk via changing the gut microbiome. The goals of this study are (1) assess whether ginger intake shifts the distribution of the gut microbiome from proinflammatory, CRC-predisposing bacteria and towards anti-inflammatory, CRC-protective bacteria and (2) evaluate the feasibility of conducting a large randomized clinical trial (RCT) leveraging the recently established Minnesota Cancer Clinical Trials Network (MNCCTN). We will conduct a pilot double-blind RCT, called “GINGER,” of 95-100 subjects aged 50-75 years old who were diagnosed with colorectal adenoma within the last 5 years (ClinicalTrials.gov NCT03268655). The subjects will be randomized to receive either 2,000 mg of ginger extract per day or matching placebo (1:1) for 6 weeks; the treatment phase will be followed by a 6-week washout. Fecal samples to characterize the gut microbiome will be collected at baseline, midpoint (week 6), and study end. Microbiome composition will be characterized by sequencing microbial 16S ribosomal RNA genes. In addition, pre- and postexposure urine samples will be collected to examine the correlation between bacteria and urinary metabolite of prostaglandin E2, which is an inflammatory biomarker and a putative biomarker for CRC. The methodology for “GINGER” was informed by an RCT recently completed by our group that involved aspirin as the intervention. In that RCT, which included 50 subjects and 5 stool collections within 12 weeks, 49 subjects completed the entire study and all stool collections. Adherence rates for the aspirin measured by pill count was at least 90%. Thus, these pilot data support the feasibility of the GINGER study. To implement the “GINGER,” we are using the resources of the MNCCTN, established in 2017. The MNCCTN, funded by Minnesota legislature and led by Masonic Cancer Center, University of Minnesota, will bring the “GINGER” and subsequent cancer clinical trials to communities throughout Minnesota, thus reducing the patients’ burden of travel and related costs. The network partners with Minnesota’s five largest health care providers (Essentia Health, Sanford Health, Mayo Clinic Cancer Center, Metro-Minnesota Community Oncology Research Consortium, and Fairview Health System) to provide infrastructure for conducting cancer trials at 18 sites outside the Twin-Cities metro area and Rochester, Minnesota. The “GINGER”—the first study conducted within the network—will begin recruitment in 3 sites by the end of November and in 6 sites by the end of 2018. Based on our previous experience, we expect to recruit 24-25 (25%) subjects in 3 months, 50%, in 6 months and all subjects by the end of 2019. Citation Format: Anna Prizment, Timothy R. Church, Dorothy Hatsukami, Robert Madoff, Christopher Staley, Robert J. Straka, Allison Iwan, Jenn Stromberg, Ya-Feng Wen, Cheryl Stibbe, Marie Rahne. Pilot trial to examine the effect of ginger on the gut microbiome: The Minnesota Cancer Clinical Trials Network [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A26.

Abstract A25: Collecting gut microbiome in the 10,000 Families Study

Abstract The 10,000 Families Study (10KFS) is a family-based prospective cohort study, the goal of which is to collect information about health-related outcomes, including cancer, in multigeneration families. Since 2017, we have been conducting a pilot feasibility study to prepare for the implementation of 10KFS. At baseline, eligible participants answer a questionnaire about demographics and lifestyle factors and attend a health fair during which trained staff measure their blood pressure, vision, hearing, and several other characteristics, as well as collect blood, urine, and saliva. Also, during the heath fair participants receive easy-to-use kits that enable them to collect stool samples in the convenience of their home. The method for stool collection to characterize gut microbiome in this study was chosen based on a small pilot of three methods: (1) Omnigene Gut (DNA Genotek), (2) iSWAB-Microbiome Collection Kit (Mawi Corporation), and (3) a homemade kit including a BD culture swab and vial with RNAlater preservative. The second method (iSWAB) was unanimously chosen by the volunteers as most acceptable due to the ease of stool collection. The acceptability and reduction of the participant’s burden are of primary importance in this study, since an adult participant often collects stool samples from one or more of their children. The quality of DNA assessed by spectrophotometry (based on 260/280 and 260/230 ratios) was similar for the iSWAB and the Omnigene kits (gold standard), and the quantity using the iSWAB kit was sufficient to characterize gut microbiome. Thus, in our study, stool samples are collected by participants using iSWAB kits and then returned by mail within four days of collection to the central laboratory biorepository at the University of Minnesota – Advanced Research and Diagnostic Laboratory, where they are immediately frozen and stored at -80°C. To date, stool samples from 62 participants aged 0 to over 90 years have been returned (out of 139 kits that were given to participants). This collection method meets the requirements necessary for stool collection in large population studies: (1) it is fast and easy for participants to use; (2) a preservative in the tube allows stool samples to be kept without freezing for up to 8 weeks while maintaining intact and viable bacteria; (3) samples use little space during shipment and storage; and (4) the method is cost efficient. Citation Format: Anna Prizment, Deanna Gabrielson, DeAnn Lazovich, Sharon Minnerath, Heather H. Nelson, Jenny N. Poynter, Cavan Reilly, Michelle Roesler, Logan G. Spector, Bharat Thyagarajan. Collecting gut microbiome in the 10,000 Families Study [abstract]. In: Proceedings of the AACR Special Conference on Modernizing Population Sciences in the Digital Age; 2019 Feb 19-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(9 Suppl):Abstract nr A25.

Galactoligosaccharide and a prebiotic blend improve colonic health and immunity of adult dogs

This study aimed to evaluate the effects of two prebiotics in different concentrations on nutrient digestibility, fermentative products and immunological variables in adult dogs. Twenty-four adult dogs were randomly divided into six blocks according to their metabolic body weights (BW0.75); within these groups, dogs were randomized to four treatments: control without prebiotics (CO); inclusion of 0.5% prebiotic blend Yes-Golf (B1); inclusion of 1.0% galactooligosaccharide (GOS); and inclusion of 1.0% prebiotic blend Yes-Golf (B2). The experiment lasted 30 days, with 20 days adaptation and 10 days stool and blood collection. Results were analyzed for normality and means were separated by ANOVA and adjusted by the Tukey test at the significance level of 5.0%. Prebiotic supplementation had no effect on apparent digestibility coefficients (ADC), total stool production and fecal scores (p > 0.05). Prebiotics evaluated also did not alter fecal pH, nor the concentrations of ammonia, lactic acid, short chain fatty acids (SCFA) and most fecal branched chain fatty acids (BCFA) (p > 0.05). The addition of GOS decreased the concentration of iso-valeric acid (p = 0.0423). Regarding immunological variables, concentrations of fecal IgA were not influenced by the treatments. Treatments GOS and B2 increased the total number of polymorphonuclear cells, as well as the oxidative burst in relation to treatments B1 and CO (p < 0.0001). Treatment B2 improved the rate of S. aureus phagocytosis in relation to CO (p = 0.0111), and both the GOS and B2 treatments had a better index for E. coli phagocytosis than the CO treatment (p = 0.0067). In conclusion, there was indication that both prebiotics GOS and B2 at 1.0% inclusion improved the immunity of healthy dogs.

Association Between the Gut Microbiota and Blood Pressure in a Population Cohort of 6953 Individuals.

BackgroundSeveral small‐scale animal studies have suggested that gut microbiota and blood pressure (BP) are linked. However, results from human studies remain scarce and conflicting. We wanted to elucidate the multivariable‐adjusted association between gut metagenome and BP in a large, representative, well‐phenotyped population sample. We performed a focused analysis to examine the previously reported inverse associations between sodium intake and Lactobacillus abundance and between Lactobacillus abundance and BP.Methods and ResultsWe studied a population sample of 6953 Finns aged 25 to 74 years (mean age, 49.2±12.9 years; 54.9% women). The participants underwent a health examination, which included BP measurement, stool collection, and 24‐hour urine sampling (N=829). Gut microbiota was analyzed using shallow shotgun metagenome sequencing. In age‐ and sex‐adjusted models, the α (within‐sample) and β (between‐sample) diversities of taxonomic composition were strongly related to BP indexes (P<0.001 for most). In multivariable‐adjusted models, β diversity was only associated with diastolic BP (P=0.032). However, we observed significant, mainly positive, associations between BP indexes and 45 microbial genera (P<0.05), of which 27 belong to the phylum Firmicutes. Interestingly, we found mostly negative associations between 19 distinct Lactobacillus species and BP indexes (P<0.05). Of these, greater abundance of the known probiotic Lactobacillus paracasei was associated with lower mean arterial pressure and lower dietary sodium intake (P<0.001 for both).ConclusionsAlthough the associations between overall gut taxonomic composition and BP are weak, individuals with hypertension demonstrate changes in several genera. We demonstrate strong negative associations of certain Lactobacillus species with sodium intake and BP, highlighting the need for experimental studies.

Evaluation of Changes in Gut Microbiota in Patients with Crohn's Disease after Anti-Tnfα Treatment: Prospective Multicenter Observational Study.

Background: Crohn’s disease is believed to result from the interaction between genetic susceptibility, environmental factors and gut microbiota, leading to an aberrant immune response. The objectives of this study are to evaluate the qualitative and quantitative changes in the microbiota of patients with Crohn’s disease after six months of anti-tumor-necrosis factor (anti-TNFα) (infliximab or adalimumab) treatment and to determine whether these changes lead to the recovery of normal microbiota when compared to a control group of healthy subjects. In addition, we will evaluate the potential role of the Faecalibacterium prausnitzii/Escherichia coli and Faecalibacterium prausnitzii/Clostridium coccoides ratios as indicators of therapeutic response to anti-TNFα drugs. Methods/Design: This prospective multicenter observational study will comprise a total of 88 subjects: 44 patients with Crohn’s disease scheduled to start anti-TNFα treatment as described in the drug specifications to control the disease and 44 healthy individuals who share the same lifestyle and eating habits. The presence of inflammatory activity will be determined by the Harvey-Bradshaw index, analytical parameters in blood, including C-reactive protein, and fecal calprotectin levels at commencement of the study, at three months and at six months, allowing the classification of patients into responders and non-responders. Microbiota composition and the quantitative relationship between Faecalibacterium prausnitzii and Escherichia coli and between Faecalibacterium prausnitzii and Clostridium coccoides group as indicators of dysbiosis will be studied at inclusion and six months after initiation of treatment using ultra sequencing with Illumina technology and comparative bioinformatics analysis for the former relationship, and digital droplet PCR using stool samples for the latter. Upon inclusion, patients will complete a survey of dietary intake for the three days prior to stool collection, which will be repeated six months later in a second collection to minimize dietary bias. Discussion: In this study, massive sequencing, a reliable new tool, will be applied to identify early biomarkers of response to anti-TNF treatment in patients with Crohn’s disease to improve clinical management of these patients, reduce morbidity rates and improve efficiency.

"Your Finger or Mine?"-Patient Preferences in the Collection of Fecal Occult Blood Testing in the Emergency Department.

There are a number of presenting Emergency Department complaints that may necessitate the testing of stool for the presence of blood. A provider-performed digital rectal examination is frequently performed solely for the collection of stool for a fecal occult blood testing (FOBT). With increased emphasis on patient autonomy and shared decision-making, it is appropriate to consider patient preferences with regard to who performs the invasive collection of stool. Our objective was to determine patient preference in regard to provider versus self-collection of stool sample for use in FOBT and identify patient demographics associated with those more likely to self-collect. Finally, we examined specimen adequacy and patient satisfaction by open-ended comment. We conducted a 1-year, prospective, non-randomized, observational trial with a convenience sample of 100 patients. After enrollment, study participants were offered the choice of self-collection versus provider collection. Stool collection proceeded according to participant preference. Simple descriptive statistics were used to analyze the primary outcome. The secondary proportional univariate outcomes were evaluated with chi-square testing (alpha level-0.05). We used a multivariable logistic regression model to evaluate the association of independent variables with the likelihood to perform self-collection. A total of 100 patients were enrolled over 1 year, with 51% choosing to collect their own sample. Younger age was the only statistically significant predictor for self-collection (p<0.001); furthermore, for every year of increasing age, patients were 3% less likely to choose self-collection. All self-collected samples were adequate for testing. In univariate analyses, patients younger than 35 were more likely to perform self-collection (66%) compared with those over 35 (36%). Those over 64 self-collected only 24% of the time. Neither sex nor race were associated with collection method preference. When offered the choice between self-directed or provider-performed stool sample collection, more than half of participants chose to obtain their own sample; furthermore, age was the only statistically significant predictor of preference for collection method. All patients who self-collected were able to provide an adequate sample. Our evidence suggests the offer of FOBT self-collection is a feasible method to enhance patient autonomy and improve therapeutic alliances.

Stereotactic ablative body radiotherapy (SABR) combined with immunotherapy (L19-IL2) versus standard of care in stage IV NSCLC patients, ImmunoSABR: a multicentre, randomised controlled open-label phase II trial

BackgroundAbout 50% of non-small cell lung cancer (NSCLC) patients have metastatic disease at initial diagnosis, which limits their treatment options and, consequently, the 5-year survival rate (15%). Immune checkpoint inhibitors (ICI), either alone or in combination with chemotherapy, have become standard of care (SOC) for most good performance status patients. However, most patients will not obtain long-term benefit and new treatment strategies are therefore needed. We previously demonstrated clinical safety of the tumour-selective immunocytokine L19-IL2, consisting of the anti-ED-B scFv L19 antibody coupled to IL2, combined with stereotactic ablative radiotherapy (SABR).MethodsThis investigator-initiated, multicentric, randomised controlled open-label phase II clinical trial will test the hypothesis that the combination of SABR and L19-IL2 increases progression free survival (PFS) in patients with limited metastatic NSCLC. One hundred twenty-six patients will be stratified according to their metastatic load (oligo-metastatic: ≤5 or poly-metastatic: 6 to 10) and randomised to the experimental-arm (E-arm) or the control-arm (C-arm). The C-arm will receive SOC, according to the local protocol. E-arm oligo-metastatic patients will receive SABR to all lesions followed by L19-IL2 therapy; radiotherapy for poly-metastatic patients consists of irradiation of one (symptomatic) to a maximum of 5 lesions (including ICI in both arms if this is the SOC). The accrual period will be 2.5-years, starting after the first centre is initiated and active. Primary endpoint is PFS at 1.5-years based on blinded radiological review, and secondary endpoints are overall survival, toxicity, quality of life and abscopal response. Associative biomarker studies, immune monitoring, CT-based radiomics, stool collection, iRECIST and tumour growth rate will be performed.DiscussionThe combination of SABR with or without ICI and the immunocytokine L19-IL2 will be tested as 1st, 2nd or 3rd line treatment in stage IV NSCLC patients in 14 centres located in 6 countries. This bimodal and trimodal treatment approach is based on the direct cytotoxic effect of radiotherapy, the tumour selective immunocytokine L19-IL2, the abscopal effect observed distant from the irradiated metastatic site(s) and the memory effect. The first results are expected end 2023.Trial registrationImmunoSABR Protocol Code: NL67629.068.18; EudraCT: 2018–002583-11; Clinicaltrials.gov: NCT03705403; ISRCTN ID: ISRCTN49817477; Date of registration: 03-April-2019.

Rectal Swabs as an Alternative Sample Collection Method to Bulk Stool for the Real-Time PCR Detection of Giardia duodenalis.

.Though bulk stool remains the gold standard specimen type for enteropathogen diagnosis, rectal swabs may offer comparable sensitivity with greater ease of collection for select pathogens. This study sought to evaluate the validity and reproducibility of rectal swabs as a sample collection method for the molecular diagnosis of Giardia duodenalis. Paired rectal swab and bulk stool samples were collected from 86 children ages 0–4 years living in southwest Niger, with duplicate samples collected among a subset of 50 children. Infection was detected using a previously validated real-time PCR diagnostic targeting the small subunit ribosomal RNA gene. Giardia duodenalis was detected in 65.5% (55/84) of bulk stool samples and 44.0% (37/84) of swab samples. The kappa evaluating test agreement was 0.81 (95% CI: 0.54–1.00) among duplicate stool samples (N = 49) and 0.75 (95% CI: 0.47–1.00) among duplicate rectal swabs (N = 48). Diagnostic sensitivity was 93% (95% CI: 84–98) by bulk stool and 63% (95% CI: 49–75) by rectal swabs. When restricting to the lowest three quartiles of bulk stool quantitation cycle values (an indication of relatively high parasite load), sensitivity by rectal swabs increased to 78.0% (95% CI: 64–89, P < 0.0001). These findings suggest that rectal swabs provide less sensitive and reproducible results than bulk stool for the real-time PCR diagnosis of G. duodenalis. However, their fair sensitivity for higher parasite loads suggests that swabs may be a useful tool for detecting higher burden infections when stool collection is excessively expensive or logistically challenging.

The Gut Microbiome and Diabetes Status in the Multiethnic Cohort

Abstract Objectives As features of the gut microbiome may promote the development of type 2 diabetes (T2D), we examined the hypothesis that gut microbiome composition differs by glycemic/diabetes status within a subset of the Multiethnic Cohort. We also estimated the association of lipopolysaccharide-binding protein (LBP) as a measure of circulating bacterial endotoxin with T2D. This outer membrane component of gram-negative bacteria may affect glucose metabolism. Methods In 2013–16, cohort members from 5 ethnic groups completed clinic visits, questionnaires, and stool collections. Participants with self-reported T2D and/or taking medication were considered T2D cases. Those with fasting glucose &amp;gt;125 and 100–125 mg/dL were classified as undiagnosed (UT2D) and prediabetes (PT2D). We characterized the gut microbiome through 16S rRNA gene sequencing (V1-V3). Plasma LBP was measured by ELISA. Linear regression was applied to estimate associations of gut microbiome community structure and LBP with T2D status adjusting for relevant confounders. Results Among 1756 participants (59.9–77.4 years), 315 (18%) were T2D, 158 (9%) UT2D, 518 (29%) PT2D, and 765 (44%) normoglycemic (NG). The Shannon diversity index was lower (6.30, 6.25, 6.28, 6.18; P = 0.02) and LBP was higher (26.0, 26.6, 28.6, 28.2 µg/mL; P = 0.0009) in T2D than NG participants. Of 10 phyla, Actinobacteria and Firmicutes were inversely associated with T2D status (P = 0.004). Six of 161 genera were significantly related to T2D status after Bonferroni adjustment: the abundance of Clostridium sensu stricto 1, Lachnospira, Lachnospiraceae NC2004, and Peptostreptococcaceae was lower, while Lachnospiraceae uncultured and Escherichia-Shigella were more abundant among T2D than NG participants. In general, those with PT2D and UT2D had values closer to NG than T2D individuals. Conclusions Participants with T2D showed a lower abundance of bacteria capable of fermenting plant polysaccharides and higher levels of gram-negative endotoxin-producing bacteria indicating that a less favorable pattern of gut microbiome community structure may contribute to T2D through endotoxin binding to toll-like receptors via LBP and activation of the NFkB pathway associated with chronic systemic inflammation. Funding Sources NIH grants P01CA169530, U01CA164973, P30CA071789, #UL1TR000130, R01HL140335.

Gut microbial diversity, inflammation, and oxidative stress are associated with tacrolimus dosing requirements early after heart transplantation.

IntroductionEffective tacrolimus (TAC) dosing is hampered by complex pharmacokinetics and significant patient variability. The gut microbiome, a key mediator of endotoxemia, inflammation and oxidative stress in advanced heart failure (HF) patients, is a possible contributor to interindividual variations in drug efficacy. The effect of alterations in the gut microbiome on TAC dosing requirements after heart transplant (HT) has not been explored.MethodsWe enrolled 24 patients (mean age = 55.8 ±2.3 years) within 3 months post-HT. Biomarkers of endotoxemia ((lipopolysaccharide (LPS)), inflammation (tumor necrosis factor-α (TNF-α)) and oxidative stress (8,12-iso-Isoprostane F-2alpha-VI) were measured in 16 blood samples. 22 stool samples were analyzed using 16S rRNA sequencing. TAC dose and serum trough level were measured at the time of stool and blood collection. TAC doses were reported in mg/kg/day and as level-to-dose (L/D) ratio, and categorized as ≤ vs. > median.ResultsThe median TAC dose was 0.1 mg/kg/day and L/D ratio was 100.01. Above the median daily weight-based TAC dose was associated with higher gut microbial alpha diversity (p = 0.03); similarly, TNF-α and 8,12-iso-Isoprostane F-2alpha-VI levels were lower and LPS levels were higher in the above median TAC group, although these findings were only marginally statistically significant and dependent on BMI adjustment. We observed n = 37 taxa to be significantly enriched among patients with > median TAC dose (all FDR<0.05), several of which are potential short-chain fatty acid producers with anti-inflammatory properties, including taxa from the family Subdoligranulum.ConclusionsOur pilot study observed gut microbial alpha diversity to be increased while inflammation and oxidative stress were reduced among patients requiring higher TAC doses early after HT.

Early Gut Microbiota Changes in Preterm Infants with Bronchopulmonary Dysplasia: A Pilot Case-Control Study.

Bronchopulmonary dysplasia (BPD) is a complex chronic lung disease that primarily affects premature or critically ill infants. This pilot study investigated early changes in gut microbiota composition in BPD patients and explored the potential risk factors associated with these changes. Preterm infants admitted to our neonatal intensive care unit with a gestational age of 26 to 32 weeks were prospectively surveyed and eligible for stool collection on days 7 and 28 of postnatal age between February 2016 and June 2017. A 16S rRNA sequencing approach was applied to compare the gut microbiota composition between the BPD group and controls. Multiple linear regression analysis was used to identify the predictor variables. Eight subjects in the BPD group and 10 subjects in the preterm group were analyzed during the observation period. Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes were the four dominant bacteria phyla of intestinal microflora. A significantly lower diversity of gut microbiota was observed in the BPD group compared with the preterm group on day 28 (number of observed operational taxonomic units, p = 0.034; abundance-based coverage estimator, p = 0.022; Shannon index, p = 0.028). Multiple linear regression analysis revealed that high Neonatal Therapeutic Intervention Scoring System score (≧19) at 24 hours was statistically significant in predicting the proportion of aerobic with facultative anaerobic bacteria on day 28 (p = 0.002). Infants with BPD are prone to develop gut dysbiosis in early life. A higher severity of illness and treatment intensity may indicate a higher risk of disrupting an anaerobic environment in the gut during the first month of life. · BPD patients are prone to develop gut dysbiosis.. · Lower diversity of gut microbiota.. · Higher risk of disrupting anaerobic environment..

Predicted Metabolic Pathway Distributions in Stool Bacteria in Very-Low-Birth-Weight Infants: Potential Relationships with NICU Faltered Growth.

Many very-low-birth-weight (VLBW) infants experience growth faltering in early life despite adequate nutrition. Early growth patterns can affect later neurodevelopmental and anthropometric potentials. The role of the dysbiotic gut microbiome in VLBW infant growth is unknown. Eighty-four VLBW infants were followed for six weeks after birth with weekly stool collection. DNA was extracted from samples and the V4 region of the 16S rRNA gene was sequenced with Illumina MiSeq. A similar microbiota database from full-term infants was used for comparing gut microbiome and predicted metabolic pathways. The class Gammaproteobacteria increased or remained consistent over time in VLBW infants. Out of 228 metabolic pathways that were significantly different between term and VLBW infants, 133 pathways were significantly lower in VLBW infants. Major metabolic differences in their gut microbiome included pathways involved in decreased glycan biosynthesis and metabolism, reduced biosynthetic capacity, interrupted amino acid metabolism, changes that could result in increased infection susceptibility, and many other system deficiencies. Our study reveals poor postnatal growth in a VLBW cohort who had dysbiotic gut microbiota and differences in predicted metabolic pathways compared to term infants. The gut microbiota in VLBW infants likely plays an important role in postnatal growth.

Gender Differences in Neurodegeneration, Neuroinflammation and Na+-Ca2+ Exchangers in the Female A53T Transgenic Mouse Model of Parkinson's Disease.

Twelve-month-old male mice expressing the human A53T variant of α-synuclein (A53T) develop dopamine neuron degeneration, neuroinflammation, and motor deficits, along with dysfunctions of the mitochondrial Na+-Ca2+ exchanger (NCX) isoforms 1 (NCX1) and 3 (NCX3) in the nigrostriatal system. Since gender is thought to play a role in the etiology of Parkinson’s disease (PD), we characterized neurochemical and behavioral alterations in 12-month-old female A53T transgenic mice. We investigated the presence of dopaminergic degeneration, astrogliosis and microgliosis using immunohistochemistry for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (IBA-1) in both the substantia nigra pars compacta (SNc) and striatum. In the same regions, we also evaluated the co-localization of NCX1 in cells positive for IBA-1 and the co-localization of NCX3 in TH-positive neurons and fibers. Furthermore, in both male and female mice, we performed motor (beam walking and pole tests) and memory [novel object recognition (NOR) and spontaneous alternation] tasks, together with tests to evaluate peripheral deficits (olfactory and stool collection tests). Female A53T transgenic mice displayed degeneration of nigral dopaminergic neurons, but neither microgliosis nor astrogliosis in the SNc and striatum. Moreover, female A53T transgenic mice displayed co-localization between NCX1 and IBA-1 positive cells in the striatum but not SNc, whereas NCX3 did not co-localize with either TH-positive terminals or neuronal bodies in the nigrostriatal system. Furthermore, female A53T transgenic mice showed increased crossing time in the beam walking test, but no impairments in the pole or memory tests, and in tests that evaluated peripheral deficits, whereas male A53T transgenic mice displayed motor, memory and peripheral deficits. Immunohistochemical and behavioral results obtained here in the female mice differ from those previously observed in males, and suggest a dissimilar influence of NCX1 and NCX3 on dopaminergic function in female and male A53T transgenic mice, strengthening the validity of these mice as a model for studying the etiological factors of PD.

Evaluation of a ketogenic diet for improvement of neurological recovery in individuals with acute spinal cord injury: study protocol for a randomized controlled trial

BackgroundTherapies that significantly improve the neurological and functional recovery of individuals with spinal cord injury (SCI) are still urgently needed. The ketogenic diet (KD) has been shown to improve forelimb motor function in an SCI rat model, likely by reducing inflammation and cell death in the spinal cord. Furthermore, our recent pilot study in patients with SCI showed that, compared with a standard hospital diet (SD), 5 weeks of KD started during acute care improved upper extremity motor function and reduced serum levels of a neuroinflammatory blood protein. The primary goals of the current study are to: 1) show the safety and feasibility of administering a KD during acute care for SCI; 2) determine if consuming 5 weeks of a KD significantly improves motor and sensory functions, functional independence and glycemic control; and 3) quantify serum biomarkers that are linked to improvements in neurological recovery and functional independence via targeted proteomics.Methods/designIn a single-masked, longitudinal, randomized, parallel-controlled study, a total of 60 eligible, acutely traumatic spinal cord injured (cervical 5 to thoracic 12) participants ranging in age from 18 to 60 years with American Spinal Injury Association impairment scale (AIS) grades A–C (AIS-A, sensorimotor complete; AIS-B, sensory incomplete/motor complete; and AIS-C, nonfunctional motor incomplete) are being enrolled. Neurological and functional examinations, resting energy expenditure, blood, urine, and stool collections, and protein analyses related to neurological recovery will be performed within 72 h of injury (baseline measure) and repeated after 5 weeks of KD or SD (discharge measure). We anticipate a completion rate of 80% with a total of 48 participants.DiscussionIntervention with a more neuroprotective diet during acute care of SCI can be implemented anywhere in the world at low cost and without major regulatory hurdles. Better functional recovery will lead to a better quality of life and long-term health outcomes in individuals with SCI. While this study targets SCI, if successful it has the potential to improve neurological outcomes for individuals with various traumatic injuries.Trial registrationNCT03509571 Registered on April 28, 2018.

Effects of incremental exercise and dietary tryptophan supplementation on the amino acid metabolism, serotonin status, stool quality, fecal metabolites, and body composition of mid-distance training sled dogs.

Exercise improves the health of dogs; however, the extreme exertion experienced by sled dogs may lead to variable metabolic and fecal characteristics. Nutritional interventions, such as dietary tryptophan (Trp), may reduce the prevalence of these exercise-induced disturbances. Sporting diets tend to have high crude protein concentrations in contrast to adult maintenance diets and this results in less Trp relative to other amino acids (AA). Therefore, sporting dogs represent an ideal cohort to assess the effects of supplemental Trp. The objective was to evaluate the effects of supplemental dietary Trp and an incremental training regimen on AA and serotonin status, fecal scores and metabolites, and body composition in client-owned Siberian huskies. Sixteen dogs (nine females and seven males) were used, with a mean age of 4.8 ± 2.5 yr and body weight (BW) of 24.3 ± 4.3 kg. Dogs were blocked for sex, age, and BW and randomly allocated into two groups with eight fed a dry extruded control diet (Ctl) and eight fed Ctl supplemented with Trp to reach a Trp:large-neutral AA (LNAA) ratio of 0.075:1 (treatment, Trt). The exercise regimen was designed to increase in distance each week, but weather played a role in setting the daily distance. Each week BW was recorded and food allotments were adjusted to maintain initial BW. Pre and post-exercise blood samples were taken every 3 wk, dogs then received a meal followed by 1, 2, and 4 h post meal blood collections (serum AA, serotonin). Stool collection and scoring occurred each week and body composition was measured on weeks -1 and 11. Serotonin, AA, fecal metabolite, and body composition data were analyzed using PROC MIXED of SAS with dog as a random effect and week and Trt as fixed effects. Stool score data were analyzed using PROC FREQ to compare stool score and Trt, and PROC CORR was used to analyze associations between fecal score, temperature, humidity, and run distance. Dogs on Trt had greater fasted Trp compared with baseline, greater post-meal Trp and serotonin compared with baseline, greater post-meal Trp compared with fasted, and greater post-meal Trp and serotonin compared with Ctl (P < 0.05). Fecal data indicated that Trp improved stool scores (P < 0.05) yet had no effect on fecal metabolites. An overall increase in lean and decrease in fat mass was found (P < 0.05), but Trt had no effect on body composition. Optimization of the dietary Trp:LNAA ratio may help to improve GI health without compromising performance in actively training sled dogs.

Abstract B13: Development of a low-cost method for collecting fecal samples in clinical trials

Abstract Although immune checkpoint inhibitors (ICIs) have shown promise in treating various cancers, fewer than half of patients with most tumor types experience a durable response. Thus, there is a need for biomarkers to better predict outcomes. Recent studies suggest that the presence of a handful of microbial species and greater alpha-diversity in the gut may serve as a biomarker for and might facilitate ICI responses. However, the specific bacteria, or bacterial communities, that associate with improved ICI responses vary across study populations, and the factors that contribute to these discrepant findings remain elusive. Thus, a standardized method by which biospecimens may be collected, transported, and stored for gut microbiome studies in the context of ICI therapy is needed. In this study, we evaluated a method for shipping fecal samples using a low-cost (&amp;lt;$3.00) ThinPrep Pap Test® methanol-based preservative kit. Under an IRB-approved protocol, we recruited patients with melanoma, Merkel cell carcinoma, endometrial cancer, and non-small cell lung cancer who had experienced &amp;gt;1 year of durable tumor response after ICI therapy. Patients were provided with stool collection kits and either asked to collect fecal samples at home within 48 hours of their clinic visit and store at 4°C (fresh) or asked to place stool in preservative and ship at ambient temperature to our laboratory (fixed). For fresh samples, a portion of each sample was frozen and another portion placed into preservative as a paired control. DNA was extracted using Zymo Quick-DNA™ Fecal/Soil Microbe kit. For fixed samples, methanol was removed by evaporation prior to DNA extraction. Microbial composition was analyzed with 16S rRNA amplicon sequencing with V1-V2 primers with 150bp paired-end sequencing using an Illumina platform. Among n=10 samples collected in clinic, fixed portions demonstrated decreased alpha-diversity and a uniform shift in beta-diversity compared to the paired frozen portions. In all fixed samples, Faecalibacterium and Roseburia relative abundance decreased with a corresponding increase in Bacteroides. Among a second set of n=11 samples that were placed in preservative by patients and then shipped to our laboratory, we analyzed the effects of shipping by comparing fresh stool samples and shipped fixed samples collected by the same patient within one month. The data revealed similar trends, suggesting that fixation, rather than shipping, drives the overall effect. We are currently verifying the relative abundance of specific bacterial species in both sets of samples using quantitative RT-PCR. Our data show that methanol-based preservation must be optimized prior to clinical utilization for accurate assessment. If optimized, we have identified a low-cost method to collect fecal samples that could be adopted in community practices and low-income areas. Ongoing work from our group includes optimization of processing procedures, ratio of fecal matter to preservative, and storage conditions. Citation Format: Kimberly E. Peloza, Joell J. Gills, Fyza Y. Shaikh, James R. White, Sara Glass, Carisse Lansiquot, Courtney Stevens, William Assan, William H. Sharfman, Dung T. Le, Jarushka Naidoo, Evan J. Lipson, Drew M. Pardoll, Cynthia L. Sears. Development of a low-cost method for collecting fecal samples in clinical trials [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B13.

The Effectiveness of Phytopharmaca of Ethanol Extract Sambiloto Leaf as an Alternative of Control to Coccidiosis

Sambiloto can increase phagocytic and lymphocytic cells, so that it can treat coccidia and can become coccidiostat (sulfaquinoxalin). Utilization of medicinal plants for the purpose of prevention and treatment of parasites for the prevention and treatment of diseases in animals. This study aims to is to analyze, the effectiveness, and evaluate the Extract Sambiloto Leaves ( ESL) as anticoccidial, against Eimiria spp . The material of this research is extract ethanol of sambiloto leaf (ESL) in capsules, 20 male Etawah Crossbreed Goats aged 3-4 months. The obtained data were analyzed with one-way ANOVA,and followed with LSD test. This study used experimental with post test only control group design with 5 treatment groups and 4 repetitions. Group T1(Negative Control: Curmint vit Plus:500mg ), T2 (Positive Control, without anticoccidial), T3( ESL caps: 750 mg ), T4 (ESL caps:1125 mg ); and T5 (ESL caps: 1500 mg). Stool collection two (2) times, (Pre research) before treatment and after treatment. Acounting of oocysts using the Floatation Mc Master methode. The results showed a significant difference (P <0.05) between group. Groups T1, T2, T3, T4 and T5 showed coccidial infestation was decreased in the number of oocysta (Oocysta per Gram = OPG) - respectively T1: 71.62% (beginning 2960-end 840), T2: 38.78% (3816-2323), T3: 51.96% (3632 - 1636), T4: 75.15% (3928-976), and T5: 68.91% (2856-888). The results showed that T1 groups no significant T4 and T5 (P>0.05), T1,T4 and T5 were significantly different (P <0.05) with T2, and T3. Conclusion, Ethanol extract of Sambiloto Leaves (ESL) is effective as control of phytopharmaceutical alternative for coccidiosis based on dose EDS = 1125 mg with the highest reduction OPG amount of 75.15% in Etawah Crossbreed Goats.

The effect of legume supplementation on the gut microbiota in rural Malawian infants aged 6 to 12 months

Common bean and cowpea contain about 25% protein and 25% fiber, and are recommended as complementary foods in sub-Saharan Africa. The objective of this study was to determine if a daily legume supplement given to Malawian infants aged 6 to 12 mo alters the 16S configuration of the fecal microbiota as read out by amplicon sequence variants (ASVs). This study was conducted within the context of a randomized, double-blind, controlled clinical trial to assess whether cowpea or common bean supplementation reduced intestinal permeability or increased linear growth. There were 2 village clusters in which the study was conducted. Fresh stool collections were flash frozen from 236 infants at ≤6 time points. The stools were sequenced using Earth Microbiome project protocols and data were processed using Qiime and Qiita, open-source, validated software packages. α-diversity was measured using the Faith's test. The 16S configuration was characterized by determining the weighted UniFrac distances of the ASVs and comparing them using permutational multivariate ANOVA. Among the 1249 samples analyzed, the α-diversity of the fecal microbiome was unchanged among subjects after initiation of legume supplementation. Neither cowpea nor common bean altered the overall 16S configuration at any age. The 16S configuration differed between children with adequate and poor linear growth aged from 6 to 9mo, but no specific ASVs differed in relative abundance. The 16S configuration differed between children with normal and abnormal intestinal permeability at 9mo, but no specific ASVs differed in relative abundance. Among categorical characteristics of the population associated with different 16S configurations, village cluster was most pronounced. Legume supplementation in breastfed, rural African infants did not affect the structure of the gut microbial communities until the children were aged 9mo. This trial was registered at clinicaltrials.gov as NCT02472262.