Fecal Sample Collection Research Articles

Plasma calprotectin – preanalytical stability and interference from hemolysis

As an activation product of neutrophil granulocytes, calprotectin has been widely used in fecal samples for diagnosis and monitoring of patients with inflammatory bowel disease. However, fecal sample collection is cumbersome, and pre-analytical sources of error are plentiful. Therefore, plasma calprotectin is being investigated as a promising new biomarker. To utilize any biomarker, pre-analytical factors such as stability and susceptibility to interference from hemolysis must be established. We present precision estimates, stability results as well as interference study on plasma calprotectin in EDTA-plasma using the Thermo Fischer Phadia 250 EliATM Calprotectin immunoassay. Precision was estimated by the use of patient pools as well as internal quality controls provided by the manufacturer. Coefficients of variance were below 6.9% for patient samples. Calprotectin was stable in EDTA plasma after storage at 5–8 °C for up to 4 days, as well as after long-term storage at −20 °C. Susceptibility to interference from hemolysis was high, especially for low concentrations of calprotectin (<50 ng/mL) where hemoglobin levels above 0.02 mmol/L lead to false increase in calprotectin concentrations of up to 100%.

Circadian rhythms in metabolic organs and the microbiota during acute fasting in mice.

The circadian clock regulates metabolism in anticipation of regular changes in the environment. It is found throughout the body, including in key metabolic organs such as the liver, adipose tissues, and intestine, where the timing of the clock is set largely by nutrient signaling. However, the circadian clocks of these tissues during the fasted state have not been completely characterized. Moreover, the sufficiency of a functioning host clock to produce diurnal rhythms in the composition of the microbiome in fasted animals has not been explored. To this end, mice were fasted 24 h prior to collection of key metabolic tissues and fecal samples for the analysis of circadian clock gene expression and microbiome composition. Rhythm characteristics were determined using CircaCompare software. We identify tissue‐specific changes to circadian clock rhythms upon fasting, particularly in the brown adipose tissue, and for the first time demonstrate the rhythmicity of the microbiome in fasted animals.

Trans-anal irrigation in patients with multiple sclerosis: Efficacy in treating disease-related bowel dysfunctions and impact on the gut microbiota: A monocentric prospective study.

BackgroundConstipation and faecal incontinence are not so uncommon in patients with multiple sclerosis, impairing quality of life. The gut microbiota is altered in multiple sclerosis patients and likely contributes to disease pathogenesis. Trans-anal irrigation has been proven to allow treatment of neurogenic bowel dysfunction and may affect gut microbiota.ObjectivesThe primary outcome was trans-anal irrigation effectiveness on constipation and faecal incontinence. The secondary outcome was gut microbiota profiling compared to healthy subjects and during trans-anal irrigation adoption.MethodsWe conducted a prospective cohort study on multiple sclerosis patients, screened with Patient Assessment of Constipation Quality of Life questionnaire before undergoing constipation and faecal incontinence scoring, abdomen X-ray for intestinal transit time, compilation of food and evacuation diaries and faecal sample collection for gut microbiota analysis before and after 4 weeks of trans-anal irrigation.Results and ConclusionsEighty patients were screened of which nearly half had intestinal symptoms. The included population (n = 37) was predominantly composed of women with significantly longer disease duration, higher mean age and disability than the excluded one (p < 0.05). Twelve patients completed the trans-anal irrigation phase, which led to significant improvement of bowel dysfunction symptom-related quality of life, increase in gut microbiota diversity and reduction of the proportions of pro-inflammatory taxa (p < 0.05). Trans-anal irrigation was safe, satisfactory and could help counteract multiple sclerosis-related dysbiosis.

A Comparative Study of Mini-FLOTAC With Traditional Coprological Techniques in the Analysis of Cetacean Fecal Samples.

Free-ranging cetaceans are considered sentinels for the marine ecosystem's health. New and non-invasive methods have been set up for the collection of fecal samples from free-ranging big whales at sea, permitting to gain an excellent epidemiological picture of parasitic infections in wild populations. To select the best protocol to be used for copromicroscopic examination in cetaceans stool samples, we evaluated the sensitivity of two commonly used techniques, i.e., a sedimentation-floatation method and the Mini-FLOTAC through validation by helminth isolation from the digestive tract. For this aim, gastrointestinal content and fecal samples were collected during necropsy from 44 cetaceans, including bottlenose dolphins (Tursiops truncatus), striped dolphins (Stenella coeruleoalba), sperm whales (Physeter macrocephalus), Risso's dolphins (Grampus griseus), Cuvier's beaked whales (Ziphius cavirostris), pilot whale (Globicephala melas), and fin whales (Balaenoptera physalus). Helminths were recovered through washing and filtering of the gastrointestinal contents and morphologically identified. Copromicroscopic examinations were performed on formalin-preserved fecal samples, using a sodium nitrate, sodium thiosulphate, and sucrose solution (s.g. = 1.450) for both methods. Helminths belonging to 9 taxa (i.e., the trematodes Synthesium tursionis, Synthesium delamurei, Campula palliata, Braunina cordiformis, Pholeter gastrophilus, the nematode Anisakis sp., cestodes of the family Tetrabothriidae and the acanthocephalan Bolbosoma sp.) were isolated. Eggs referable to the same taxa, with the exception of cestodes, were found in copromicroscopic analyses. Sensitivity of the Mini-FLOTAC method appeared higher or equal for all taxa, proving superior to the sedimentation-flotation method for the detection of all except Anisakis sp. The concordance of the two tests indeed revealed a moderate to perfect agreement (kappa values 0.42–1). Not excluding the limitations inherent to the techniques themselves, explanations for false-negative results at copromicroscopy could be linked to parasite-related factors, including prepatent infections, low parasitic burdens, or intermittent egg shedding. Notwithstanding these limitations, this study evidenced that the Mini-FLOTAC protocol approximates more accurately the composition of the gastrointestinal helminthic community of cetaceans from copromicroscopic examination, providing at the same time a quantitative estimation.

A Wipe-Based Stool Collection and Preservation Kit for Microbiome Community Profiling.

While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and a dissolvable wipe used with DESS (dimethyl sulfoxide, ethylenediaminetetraacetic acid, sodium chloride) solution. These samples were sequenced and analyzed for taxonomic abundance metrics, bacterial metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R2 of 0.96 for species across all kingdoms, as well as exhibiting a maintenance of Shannon diversity (3.1-3.3) and species richness (151-159) compared to frozen samples. Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. These data support that the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.

The seventh survey of the Tromsø Study (Tromsø7) 2015-2016: study design, data collection, attendance, and prevalence of risk factors and disease in a multipurpose population-based health survey.

Aims:The Tromsø Study is an ongoing population-based health study in Tromsø, Norway, initiated in 1974. The purpose of the seventh survey (Tromsø7) 2015–2016 was to advance the population risk factor surveillance and to collect new types of data. We present the study design, data collection, attendance, and prevalence of risk factors and disease.Methods:All inhabitants in Tromsø municipality, Norway, aged 40 years and older (N=32,591) were invited to a health screening including extensive questionnaires, face-to-face interviews, biological sampling (blood, urine, saliva, nasal/throat swabs, faeces), measurements (anthropometry, blood pressure, pulse, pulse oximetry) and clinical examinations (pain sensitivity, echocardiography, cognitive, physical, and lung function, accelerometer measurements, eye examinations, carotid ultrasound, electrocardiography, dual-energy X-ray absorptiometry, and heart, lung and carotid auscultation). New research areas in this round were dental and oral health examinations, collection of faecal samples for studies of normal bacterial flora and antibiotic resistance, and 24-hour urine samples for examination of sodium and iodine intakes.Results:Attendance was 65% (N=21,083), and was higher in women, age group 50–79 years, previous attenders, and Norwegian-born individuals. Cardiovascular risk factor levels and prevalence of chronic obstructive lung disease decreased since the last survey, while the prevalence of obesity and diabetes increased.Conclusions:Attendance was stable from the sixth survey. Interaction with participants might be key to maintain participation. Favourable trends in risk factors continue, except for a continued increase in obesity. Both new data collection technology and traditional physical examinations will be crucial for the impact of future population studies.

Standards for Collection, Preservation, and Transportation of Fecal Samples in TCM Clinical Trials.

BackgroundUnlike chemical drugs with a single or a few kinds of active compounds, traditional Chinese medicines (TCMs)uses herbal formulas composed of numerous kinds of chemical constituents. Therefore, TCM clinical trials require unique and stricter standards for collecting, preserving, and transporting fecal samples than those used for chemical drugs. Unfortunately, there are no special standards for processing fecal samples in TCM clinical trials.MethodsWe invited interdisciplinary experts within TCM clinical trials and gut microbiome research to help formulate this standard. After more than a year’s in-depth discussion and amendments, we achieved a standard via expert interviews, literature research, questionnaire surveys, and public opinion solicitation. This standard has been reviewed and approved by the Standards Office of China of the Association of Chinese medicine.ResultsWe established a sample information processing method prior to TCM clinical sample collection, which is adapted to the unique features of TCM. The method formulates detailed processing requirements for TCM information in addition to the factors that may disturb the gut microbiome. We also constructed a set of methods for collecting, preserving, and transporting fecal samples that meet the characteristics of TCM. These methods formulate detailed operating specifications on the collection approaches, storage conditions, transportation requirements, and management of fecal samples.ConclusionsThis standard guides the information processing prior to sample collection and the standard operating procedures for the collection, preservation, and transportation of fecal samples in TCM clinical trials, which also can be used as a reference by clinicians and researchers in modern medicines.

Altered Gut Microbiota as an Auxiliary Diagnostic Indicator for Patients With Fracture-Related Infection.

Preoperative diagnosis of fracture-related infection (FRI) is difficult for patients without obvious signs of infection. However, specific profiles of gut microbiota may be used as a potential diagnostic tool for FRI as suggested by a previous study. The fecal microbiome was compared between 20 FRI patients (FRI group), 18 fracture healed patients (FH group), and 12 healthy controls (HC group) included after collection of fecal samples and evaluation. The α and β diversity indices were used to characterize the fecal microbiome. Dysbiosis indexes were constructed based on the characteristic high-dimensional biomarkers identified in the fecal microbiota from the three groups by linear discriminant analysis and generalized linear model analysis to quantify the dysbiosis of fecal microbiota. The effectiveness of α and β diversity indices and dysbiosis indexes was assessed in distinguishing the fecal microbiome among the three groups. The influences of serum inflammatory factors on gut microbiota were also addressed. The α diversity indices were significantly different between the three groups, the highest in HC group and the lowest in FRI group (P < 0.05). The β diversity indices showed significant phylogenetic dissimilarity of gut microbiome composition among the three groups (P < 0.001). The dysbiosis indexes were significantly higher in FRI group than in FH and HC groups (P < 0.001). The area under Receiver operating characteristic curve showed the characteristics of gut microbiota and the gut microbiota was found as effective in distinguishing the three groups. The dysbiosis in the FRI patients was associated with systemic inflammatory factors. In addition, significant differences in the gut microbiota were not observed between the FRI patients versus without sinus tract or pus before operation. Since FRI patients, with or without sinus tract or pus, have a characteristic profile of gut microbiota, their gut microbiota may be used as an auxiliary diagnostic tool for suspected FRI.

PSI-4 Effects of Sorghum Inclusion in Lamb Diets on Apparent Total Tract Digestibility

Abstract The objective of this experiment was to evaluate the effect of grain sorghum inclusion on the apparent total tract digestibility of lamb diets. This experiment utilized 24 Rambouillet lambs (46.1 ± 3.90 kg) that were previously used in a growth experiment. Animals were housed in 1 of 24 pens with 1 lamb per pen and 6 pens per treatment. Lambs were randomly assigned 1 of the 4 treatment diets prior to the growth experiment and remained on the same treatment for over the 10-d experimental period. Treatments included 1) Control (0% sorghum inclusion); 2) Sorg10 (10% corn substituted for sorghum); 3) Sorg20 (20% corn substituted for sorghum); and 4) Sorg30 (30% corn substituted for corn). Diets were ground using hammermill and titanium dioxide was added to the ground diet at 0.4% as an indigestible marker. Lambs were fed these diets for 7-d prior to fecal sample collection to allow for steady-state marker concentrations. Fecal grab samples were collected every 6 hours over the final 3 d and were composited by animal to represent each 2-hour period of a 24-hour cycle. Feed and composited fecal samples were analyzed for crude fat, crude protein, ADF, NDF, and ash. Data were analyzed using the LM function in R with a fixed effect of treatment. Significance was defined at P &amp;lt; 0.05 and means were separated using Tukey’s HSD where appropriate. Sorghum inclusion did not affect (P &amp;gt; 0.16) crude protein, crude fat, or acid detergent fiber digestibility. Neutral detergent fiber digestibility was reduced (P = 0.02) for animals consuming Sorg20 and Sorg30 compared with the control, and Sorg10 was intermediate. Organic matter digestibility tended to decrease (P = 0.08) with increasing sorghum inclusion. In conclusion, replacing corn with sorghum at levels up to 30% had minimal impact on nutrient digestibility in growing lambs.

Gut microbiota and voluntary alcohol consumption

Alcohol is part of the usual diet of millions of individuals worldwide. However, not all individuals who drink alcohol experience the same effects, nor will everyone develop an alcohol use disorder. Here we propose that the intestinal microbiota (IMB) helps explain the different consumption patterns of alcohol among individuals. 507 humans participated in this study and alcohol consumption and IMB composition were analyzed. On the other hand, in 80 adult male Wistar rats, behavioral tests, alcohol intoxication, fecal transplantation, administration of antibiotics and collection of fecal samples were performed. For identification and relative quantification of bacterial taxa was used the bacterial 16 S ribosomal RNA gene. In humans, we found that heavy episodic drinking is associated with a specific stool type phenotype (type 1, according to Bristol Stool Scale; p < 0.05) and with an increase in the abundance of Actinobacteria (p < 0.05). Next, using rats, we demonstrate that the transfer of IMB from alcohol-intoxicated animals causes an increase in voluntary alcohol consumption in transplant-recipient animals (p < 0.001). The relative quantification data indicate that the genus Porphyromonas could be associated with the effect on voluntary alcohol consumption. We also show that gut microbiota depletion by antibiotics administration causes a reduction in alcohol consumption (p < 0.001) and altered the relative abundance of relevant phyla such as Firmicutes, Bacteroidetes or Cyanobacteria (p < 0.05), among others. Benjamini–Hochberg false discovery rate (FDR) correction was performed for multiple comparisons. These studies reveal some of the consequences of alcohol on the IMB and provide evidence that manipulation of IMB may alter voluntary alcohol consumption.

Identifying individual ungulates from fecal DNA: a comparison of field collection methods to maximize efficiency, ease, and success

Non-invasive genetic sampling can facilitate the identification of individual animals across a landscape, with applications to management and conservation. Fecal material is a readily available source of DNA, and various methods exist for collecting fecal samples for DNA preservation. In particular, swab methods offer considerable promise, but their utility in real-world field contexts remains relatively untested. We systematically compared multiple genetic fecal sampling methods across all stages of data collection and analysis, including sampling in the field, DNA extraction in the lab, and identification of individuals using microsatellite genotyping. We collected 112 fecal samples from black-tailed deer (Odocoileus hemionus columbianus) in the field in Mendocino County, California, across a range of sample conditions of unknown age. We systematically compared the efficiency, ease, and genotyping success of three methods for field collection and storage of ungulate fecal samples: whole pellets in ethanol, whole dry pellets in paper envelopes, and cotton swabs in buffer. Storage method, sample condition, and their interaction predicted genotyping success in the top binomial GLMMs. We found that swabbing pellets resulted in the greatest percentage of individually identifiable genotypes (81%, compared to 60% for dry samples and 56% for ethanol), despite lower DNA concentrations. While swabbing pellets requires a greater time investment in the field, the samples are easier and safer to store and transport, and subsequent labwork is more efficient as compared to whole-pellet collection methods. We, therefore, recommend the swab method for most contexts. We provide additional recommendations and field protocols based on subsequent collection of 2284 swab samples for a larger monitoring study of the deer population, given that this large number of samples spanned a range of sample conditions and time spent in storage.

Prevalence and Factors Associated with Cryptosporidium Infection in Calves in and around Nekemte Town, East Wollega Zone of Ethiopia.

Purpose Cryptosporidiosis, caused by a protozoan parasite of the genus Cryptosporidium, is a zoonotic disease that affects young animals and humans. The study was conducted from October 2020 to May 2021, with the objectives of estimating the prevalence of Cryptosporidium infection in calves and assessing the associated risk factors in Nekemte town, Ethiopia. Methods A cross-sectional study was adopted during the study period. Initially, 35 dairy farms were selected using a systematic study approach from which a total of 384 calves under the age of 12 months (75 calves from intensive, 51 from semi-intensive, and 258 from extensive dairy farms) were selected purposively for fecal sample collection. Fecal samples were collected directly from the rectum of each calf and tested for Cryptosporidium oocysts using Sheather's flotation and a modified Ziehl–Neelsen staining procedure. The data were analyzed using STATA statistical software version 13. Results Cryptosporidium infection was found in 53 of the 384 calves investigated, with an overall prevalence of 13.8% (95% CI = 8.1–17.6). Logistic regression analysis on the risk factors showed that types of farm, age, body condition scores, fecal consistency, types of feed, source of water, and farm hygiene were found to have a statistically significant influence on the shedding of Cryptosporidium oocysts by the calves (p < 0.05). Calves under the age of six months had a higher likelihood (OR = 2.7, 95% CI = 1.0–4.2, p < 0.001) of shedding the oocysts than the calves of 6 to 12 months of age. It was also confirmed that calves with poor body condition scores had a higher likelihood (OR = 2.6, 95% CI = 1.2–3.7, p=0.024) of shedding the Cryptosporidium oocysts than other ones. The likelihood of shedding a Cryptosporidium oocyst by the diarrheic calves was higher than by the nondiarrheic ones (OR = 3.1, p < 0.001). The infection was 2.6 times highest (16.8%, 31/185, 95% CI = 14.5–19.1, OR = 2.6, p=0.028) in calves feeding on grass alone, followed by milk and grass (15%) and milk (4.2%). Similarly, it was 2.2 times higher (18.5%, OR = 2.2, and p=0.002) in calves drinking river water than in calves drinking tap water. Moreover, calves kept under poor hygiene were more likely to shed Cryptosporidium oocysts (OR = 2.8, p=0.001) than calves kept under good hygiene. Conclusion In conclusion, cryptosporidiosis is one of the health problems of calves in and around Nekemte town, East Wollega zone. Our findings confirmed that risk factors such as types of farm, age, body condition scores, fecal consistency, types of feed, water source, and farm hygienic status were found to have a significant effect on the occurrence of Cryptosporidium infection in the calves. While the molecular-based study is required to be carried out aiming at species identification and the epidemiology of the parasite, particular attention should be paid to the control of the disease in the study area.

Evaluating live microbiota biobanking using an ex vivo microbiome assay and metaproteomics

ABSTRACT Biobanking of live microbiota is becoming indispensable for mechanistic and clinical investigations of drug–microbiome interactions and fecal microbiota transplantation. However, there is a lack of methods to rapidly and systematically evaluate whether the biobanked microbiota maintains their cultivability and functional activity. In this study, we use a rapid ex vivo microbiome assay and metaproteomics to evaluate the cultivability and the functional responses of biobanked microbiota to treatment with a prebiotic (fructo-oligosaccharide, FOS). Our results indicate that the microbiota cultivability and their functional responses to FOS treatment were well maintained by freezing in a deoxygenated glycerol buffer at −80°C for 12 months. We also demonstrate that the fecal microbiota is functionally stable for 48 hours on ice in a deoxygenated glycerol buffer, allowing off-site fecal sample collection and shipping to laboratory for live microbiota biobanking. This study provides a method for rapid evaluation of the cultivability of biobanked live microbiota. Our results show minimal detrimental influences of long-term freezing in deoxygenated glycerol buffer on the cultivability of fecal microbiota.

Comparison of Fecal Sample Collection Methods for Microbial Analysis Embedded within Colorectal Cancer Screening Programs.

Colorectal cancer screening programs with fecal sample collection may provide a platform for population-based gut microbiome disease research. We investigated sample collection and storage method impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods [i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries] among 19 healthy volunteers. Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha diversity metrics, and the first principal coordinates of the beta diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4°C or 30°C, and after screening for the presence of occult blood in the stool. In addition, accuracy was estimated for samples frozen immediately compared to samples with no solution (i.e., the putative gold standard). When compared with the putative gold standard, we observed significant variation for all collection methods. However, interindividual variability was much higher than the variability introduced by the collection method. Stability ICCs were high (≥0.75) for FIT tubes that underwent colorectal cancer screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30°C (range, 0.41-0.90) and room temperature (range, 0.06-0.94). Paper-based collection cards and different types of FIT are acceptable tools for microbiome measurements. Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs.

INTESTINAL EPITHELIAL CELL-SPECIFIC CFTR KNOCKOUT MICE EXHIBIT FECAL MICROBIAL DYSBIOSIS

Abstract BACKGROUND Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF) disease. Fecal microbial dysbiosis has the potential of inducing intestinal inflammation and has been reported in people with CF as well as CF mouse models. CF mice consuming an osmotic polyethylene glycol (PEG) laxative in drinking water to prevent intestinal obstruction have low levels of intestinal inflammation and low intestinal bacterial loads. In contrast, CF mice consuming a nutritionally complete liquid diet to prevent intestinal impaction exhibit intestinal inflammation and high intestinal bacterial loads (R. C. De Lisle, et al., Am J Physiol 293, G577-G584, 2007) To investigate the development of dysbiosis in a CFTR-deficient intestine, we transitioned adult intestinal epithelial cell-specific knockout and wild-type (WT) mice from life-long consumption of PEG laxative to a nutritionally-complete liquid diet for a two week period before collection of fecal samples. METHODS Fecal samples collected from B6.Cg-Tg(Vil1-cre)-Cftrf10/f10 intestinal-specific Cftr knockout mice (iCftr KO) and their WT sex-matched littermates were DNA extracted and prepared for 16S rRNA sequencing and evaluation of the microbiome. Microbial community composition, richness and diversity indices, and relative abundance of bacterial species of interest were evaluated. RESULTS 16S rRNA sequencing and metagenomic analysis revealed significant differences in microbial composition with intestinal-specific loss of CFTR as compared to WT, as well as a significant decrease in Shannon diversity and Chao-1 richness indices. A significant deficit of taxa was seen in the intestinal-specific CF mice, as compared to WT, which corresponded with a significant increase in bacterial species that potentially serve as provocateurs of intestinal inflammation including Helicobacter typhlonius, Clostridium perfringens, Ruminococcus gnavus, and Tyzerella spp. CONCLUSIONS As compared to WT, the transition from PEG laxative to a nutritionally-complete liquid diet resulted in overt differences in the fecal microbiota composition in iCftr KO mice which included enrichment of potential bacterial pathobionts capable of stimulating intestinal inflammation. These studies suggest that the CFTR-deficient intestinal epithelium provides an environment selective for potential bacterial pathobionts. The paradigm of the study may prove useful for investigating the development of dysbiosis and corresponding changes in intestinal mucosal immunity.

Neuroimmunological investigations of cerebrospinal fluid in patients with recent onset depression \u2013 a study protocol

BackgroundA proinflammatory response has been suggested to be involved in the pathophysiology of depression in a subgroup of patients. However, comprehensive largescale studies on neuroimmunological investigations of the cerebrospinal fluid (CSF) are lacking and no largescale longitudinal CSF studies comparing patients with depression to healthy controls currently exist.MethodsA longitudinal case-control study including at least 100 patients with first time depression (ICD-10: F32) within the past year with ongoing symptoms and at least 100 sex and age matched healthy controls with collection of CSF, blood, and fecal samples. All individuals will be evaluated by neurological examination including neurological soft signs, interviewed for psychopathology assessment and have symptomatology evaluated by relevant rating scales. Level of functioning and quality of life will be evaluated by a panel of interview questions and rating scales, and cognitive function assessed by a relevant test battery. In addition, a large number of potential confounders will be registered (BMI, smoking status, current medication etc.). Primary outcomes: CSF white cell count, CSF/serum albumin ratio, CSF total protein levels, IgG index, CSF levels of IL-6 and IL-8, and the prevalence of any CNS-reactive autoantibody in CSF and/or blood. Secondary outcomes: exploratory analyses of a wide range of neuroimmunological markers and specific autoantibodies. Power calculations are computed for all primary outcomes based on previous CSF studies including patients with depression and healthy controls.DiscussionThis study will represent the hitherto largest investigation of CSF in patients with recent onset depression compared to healthy controls. We expect to elucidate neuroimmunological alterations in individuals with depression and characterize an immunological profile paving the way for the development of effective treatments based on biomarkers.Trial registrationThe study is approved by The Regional Committee on Health Research Ethics (Capital Region, j.no: H-16030985) and The Danish Data Protection Agency (j.no: RHP-2016-020, I-Suite no.: 04945).

Impact of Multi-metal Mixture Administration Through Drinking Water on the Gut Bacterial Microflora and Oxidative Stress Parameters in Male and Female Mice

Background: Heavy metal containing wastes reaches to the food chain either directly or indirectly. These ingested toxic elements manifest direct impact on the gut ecosystem and its overall functioning. The present study explores the alteration in mice gut bacteria on exposure to mixture of toxic heavy metals through drinking water. Methods: Twelve experimental groups of Swiss albino male and female mice were exposed to the metal mixture of varying concentrations. Profiling of gut bacterial flora was done by periodical collection of fecal samples via culture-based technique. Redox status of all experimental animals was analyzed in blood samples collected on the day 30. Results: In comparison to the controls, nearly a 10-fold decline in colony forming units/ml was observed at higher modal concentrations (50× &amp; 100×) at the end of 15 days, but 100-fold reduced bacterial count was recorded following 30 days of dosing. Sex specific significant alteration in the bacteria count and diversity was also observed. Overall experimental results showed a heavy metal dose-dependent decline in bacterial count and loss in diversity. Disturbance in the oxidative stress markers was recorded in response to high dose of metal mixture. In group receiving 100× dose, malondialdehyde levels were increased in the erythrocytes (P&lt;0.05), and all of the other antioxidant parameters were decreased (P&lt;0.05), except for reduced glutathione in both male and female mice. Conclusion: The present work is the first report on the multiple heavy metals induced gut microbiota alterations and its correlation to oxidative stress.

Fecal Calprotectin Is Increased in Stroke.

Background: While there have been major advances in unveiling the mechanisms comprising the ischemic cascade of CNS, stroke continues to be a significant burden. There is a need to extend the focus toward peripheral changes, and the brain–gut axis has recently gained much attention. Our study aimed to evaluate gut inflammation and its association with blood variables in stroke using fecal calprotectin (FC). Methods: Fecal samples were obtained from 27 stroke patients and 27 control subjects. FC was quantitatively measured using a commercial ELISA. Laboratory data on the fecal sample collection were also collected, including CBC, ESR, glucose, creatinine, total protein, albumin, transaminases, and CRP. Results: There was a significant increase in FC levels in stroke patients compared to the controls. Furthermore, FC in stroke patients was negatively correlated with the Glasgow Coma Scale. Moreover, FC in stroke patients was positively correlated with CRP and negatively correlated with lymphocyte count and albumin. Conclusions: Our findings show that increased FC is associated with consciousness and systemic response in stroke and warrants further studies to elucidate the usefulness of FC in the management of stroke.

Clinical health issues, reproductive hormones, and metabolic hormones associated with gut microbiome structure in African and Asian elephants

BackgroundThe gut microbiome is important to immune health, metabolism, and hormone regulation. Understanding host–microbiome relationships in captive animals may lead to mediating long term health issues common in captive animals. For instance, zoo managed African elephants (Loxodonta africana) and Asian elephants (Elephas maximus) experience low reproductive rates, high body condition, and gastrointestinal (GI) issues. We leveraged an extensive collection of fecal samples and health records from the Elephant Welfare Study conducted across North American zoos in 2012 to examine the link between gut microbiota and clinical health issues, reproductive hormones, and metabolic hormones in captive elephants. We quantified gut microbiomes of 69 African and 48 Asian elephants from across 50 zoos using Illumina sequencing of the 16S rRNA bacterial gene.ResultsElephant species differed in microbiome structure, with African elephants having lower bacterial richness and dissimilar bacterial composition from Asian elephants. In both species, bacterial composition was strongly influenced by zoo facility. Bacterial richness was lower in African elephants with recent GI issues, and richness was positively correlated with metabolic hormone total triiodothyronine (total T3) in Asian elephants. We found species-specific associations between gut microbiome composition and hormones: Asian elephant gut microbiome composition was linked to total T3 and free thyroxine (free T4), while fecal glucocorticoid metabolites (FGM) were linked to African elephant gut microbiome composition. We identified many relationships between bacterial relative abundances and hormone concentrations, including Prevotella spp., Treponema spp., and Akkermansia spp.ConclusionsWe present a comprehensive assessment of relationships between the gut microbiome, host species, environment, clinical health issues, and the endocrine system in captive elephants. Our results highlight the combined significance of host species-specific regulation and environmental effects on the gut microbiome between two elephant species and across 50 zoo facilities. We provide evidence of clinical health issues, reproductive hormones, and metabolic hormones associated with the gut microbiome structure of captive elephants. Our findings establish the groundwork for future studies to investigate bacterial function or develop tools (e.g., prebiotics, probiotics, dietary manipulations) suitable for conservation and zoo management.

Comparison of fecal and oral collection methods for studies of the human microbiota in two Iranian cohorts

BackgroundTo initiate fecal and oral collections in prospective cohort studies for microbial analyses, it is essential to understand how field conditions and geographic differences may impact microbial communities. This study aimed to investigate the impact of fecal and oral sample collection methods and room temperature storage on collection samples for studies of the human microbiota.ResultsWe collected fecal and oral samples from participants in two Iranian cohorts located in rural Yazd (n = 46) and urban Gonbad (n = 38) and investigated room temperature stability over 4 days of fecal (RNAlater and fecal occult blood test [FOBT] cards) and comparability of fecal and oral (OMNIgene ORAL kits and Scope mouthwash) collection methods. We calculated interclass correlation coefficients (ICCs) based on 3 alpha and 4 beta diversity metrics and the relative abundance of 3 phyla. After 4 days at room temperature, fecal stability ICCs and ICCs for Scope mouthwash were generally high for all microbial metrics. Similarly, the fecal comparability ICCs for RNAlater and FOBT cards were high, ranging from 0.63 (95% CI: 0.46, 0.75) for the relative abundance of Firmicutes to 0.93 (95% CI: 0.89, 0.96) for unweighted Unifrac. Comparability ICCs for OMNIgene ORAL and Scope mouthwash were lower than fecal ICCs, ranging from 0.55 (95% CI: 0.36, 0.70) for the Shannon index to 0.79 (95% CI: 0.69, 0.86) for Bray-Curtis. Overall, RNAlater, FOBT cards and Scope mouthwash were stable up to 4 days at room temperature. Samples collected using FOBT cards were generally comparable to RNAlater while the OMNIgene ORAL were less similar to Scope mouthwash.ConclusionsAs microbiome measures for feces samples collected using RNAlater, FOBT cards and oral samples collected using Scope mouthwash were stable over four days at room temperature, these would be most appropriate for microbial analyses in these populations. However, one collection method should be consistently since each method may induce some differences.