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A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Studying microbiome-disease associations likely requires microbial data to be pooled from multiple studies. Our findings suggest collection methods that are most optimal to be used standardly across future WGSS microbiome studies. Few previous studies have assessed stability and “gold-standard” concordance of fecal sample collection methods for whole-genome shotgun metagenomic sequencing (WGSS), an increasingly popular method for studying the gut microbiome. We used WGSS data to investigate ambient temperature stability and putative gold-standard concordance of microbial profiles in fecal samples collected and stored using fecal occult blood test (FOBT) cards, fecal immunochemical test (FIT) tubes, 95% ethanol, or RNAlater. Among 15 Mayo Clinic employees, for each collection method, we calculated intraclass correlation coefficients (ICCs) to estimate stability of fecal microbial profiles after storage for 4 days at ambient temperature and concordance with immediately frozen, no-solution samples (i.e., the putative gold standard). ICCs were estimated for multiple metrics, including relative abundances of select phyla, species, KEGG k-genes (representing any coding sequence that had >70% identity and >70% query coverage with respect to a known KEGG ortholog), KEGG modules, and KEGG pathways; species and k-gene alpha diversity; and Bray-Curtis and Jaccard species beta diversity. ICCs for microbial profile stability were excellent (≥90%) for fecal samples collected via most of the collection methods, except those preserved in 95% ethanol. Concordance with the immediately frozen, no-solution samples varied for all collection methods, but the number of observed species and the beta diversity metrics tended to have higher concordance than other metrics. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT cards, FIT tubes, and RNAlater are acceptable choices for fecal sample collection methods in future WGSS studies. IMPORTANCE A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Studying microbiome-disease associations likely requires microbial data to be pooled from multiple studies. Our findings suggest collection methods that are most optimal to be used standardly across future WGSS microbiome studies.

Unlike 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. We 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 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. We 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. This 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.

The critical role of gut microbiota in human health and disease has been increasingly illustrated over the past decades, with a significant amount of research demonstrating an unmet need for self-monitor of the fecal microbial composition in an easily-accessible, rapid-time manner. In this study, we employed a tool for Smartphone Microbiome Evaluation and Analysis in Rapid-time (SMEAR) that uses images of fecal smears to predict microbial compositional characteristics in a human cohort. A subset of human fecal samples was randomly retrieved from the second wave of data collection in the Healthy Life in an Urban Setting (HELIUS) study cohort. Per sample, 16S rRNA gene sequencing data was generated in addition to an image of a fecal smear, spread on a standard A4 paper. Metagenomics-paired pictures were used to validate a computer vision-based technology to classify whether the sample is of low or high relative abundance of the 50 most abundant genera, and α-diversity (Shannon-index). In total, 888 fecal samples were used as an application of the SMEAR technology. SMEAR gave accurate predictions whether a fecal sample is of low or high relative abundance of Sporobacter , Oscillibacter and Intestinimonas (very good performance, AUC > 0.8, p-value < 0.001, for all models), as well as Neglecta , Megasphaera , Lachnospira , Methanobrevibacter , Harryflintia , Roseburia , and Dialister (good performance, AUC > 0.75, p-value < 0.001, for all models). Likewise, SMEAR could classify whether a fecal sample was of low or high α-diversity (AUC = 0.83, p-value < 0.001). Our study demonstrates that SMEAR robustly predicts microbial composition and diversity from digital images of fecal smears in a human cohort. These findings establish SMEAR as a new benchmark for rapid, cost-effective microbiome diagnostics and pave the way for its direct application in research settings and clinical validation.

Pelvic floor disorders are highly prevalent among reproductive-age women in sub-Saharan Africa and commonly lead to urinary incontinence, pelvic organ prolapse, bowel dysfunction, sexual problems, and reduced quality of life. Pelvic floor muscle exercise, defined as repeated contraction and relaxation of the pelvic floor muscles, is an effective preventive and non-surgical treatment of pelvic floor disorders. However, despite the high burden of pelvic floor disorders in Ethiopia, the significance and role of pelvic floor muscle exercise have not yet been studied in a study setting. This study aimed to assess the Pelvic Floor Muscle Exercise Practice and Its Determinants among Postpartum Women in Gurage Zone, Central Ethiopia. A community-based cross-sectional study was employed from May 12 to June 12, 2023. A 422 postpartum women were selected using a systematic random sampling technique. A pre-tested and structured questionnaire was used for data collection. Data were entered into Epidata 3.1 and exported into SPSS version 26 software for analysis. Both bivariable and multivariable binary logistic regressions were performed. Variables with a P-value < 0.05 at a 95% confidence interval were considered statistically significant. In this study,420 study participants were included with a response rate of 99.5%. The proportion of women practicing pelvic floor exercises was 12.14% (95% CI: 6.3, 18.7). Higher education (AOR = 1.40; 95% CI: 1.27, 4.31), ANC visits (AOR = 4.31;95%CI:1.36, 9.57), women with urinary incontinence (AOR = 6.47;95%CI:3.96, 11.54), and women’s knowledge (AOR = 6.31;95%CI:3.59, 12.23) were determinants of pelvic floor muscle exercise practice. The present study showed that 87.86% of postpartum mothers lacked proper pelvic floor muscle exercise practices. Thus, encouraging women to attend ANC visits as recommended, providing awareness through health education, and offering counseling on pelvic floor muscle exercises can increase their practice and help reduce pelvic floor disorders.

Operative vaginal delivery is the use of forceps or vacuum devices to assist the eligible laboring mother to avoid poor birth outcomes. It is associated with increased maternal, neonatal morbidity and perinatal complications if it is not used appropriately. Instrumental delivery use needs health care providers' skills, knowledge, and decision-making ability for good maternal outcomes. This study aimed to assess immediate unfavorable birth outcomes and associated factors of operative vaginal delivery among women delivered in East Gojjam Zone Public Hospitals, North West Ethiopia. The study design was institution based cross-sectional and consecutive sampling procedure was used to select 313 mothers in the study, from March 1, 2019, to April 30, 2019. We used Epi data version 3.1 for data entry and SPSS version 25 software for cleaning and analysis. A Bivariable logistic regression analysis was used to identify the association between each outcome variable and each factor. Again, a multivariable logistic regression analysis was employed to identify factors associated with each outcome variable, and variables with a p-value less than 0.05 were taken as significant variables. The overall unfavorable maternal outcomes of operative vaginal delivery were found to be 32.9% [95% CI: 27.8, 38.3]. No formal education (AOR = 8.36; 95% CI: 1.01, 69.2), rural residence (AOR: 11.77; 95% CI: 2.02, 68.41), male sex of the neonate (AOR: 2.87; 95% CI: 1.08, 7.61) and zero station during instrumental application (AOR: 6.93; 95% CI: 1.75, 27.5) were factors associated with unfavorable maternal outcomes. The study also showed that the magnitude of unfavorable neonatal outcomes was 34.8% (95% CI: 29.7, 40.3). Vaginal first-degree tear (AOR = 0.03, 95% CI: 0.001, 0.951) and blood transfusion (AOR = 7.38, 95% CI: 1.18-46.15) was statistically significant factors associated with unfavorable neonatal outcomes. The overall unfavorable maternal and neonatal outcomes of operative vaginal delivery were high compared with some other studies done in Ethiopia.

ObjectivesTo assess psychological maladjustment in adolescents with functional constipation.Study designWe conducted a cross-sectional survey in five schools. Adolescents aged between 13 and 18 years were included in the study. Validated questionnaires were used to collect bowel habits and demographic data, health-related quality of life (HRQoL) and psychological maladjustment. Rome III criteria were used to diagnose constipation.Results1697 adolescents were recruited (boys 779 (45.9%), mean age 15.06 years and SD 1.6 years). Prevalence of constipation was 6.7%, of whom 52 were boys (45.6%) and 62 were girls (54.4%). 38 adolescents (33.3%) with constipation and 230 controls (14.5%) had significant psychological maladjustment. Among seven different personality dimensions used to assess psychological maladjustment, children with constipation had significantly more deficits than controls in hostility and aggression (14.2 vs 12.6 in controls (mean difference 1.54, 95% CI (0.89 to 2.19) p<0.001), negative self-esteem (12.0 vs 10.5 in controls, mean difference 1.54 95% CI (0.96 to 2.06) p<0.001), negative self-adequacy (11.9 vs 9.8 controls, mean difference 2.07 95% CI (1.46 to 2.67) p<0.001), emotional unresponsiveness (12.9 vs 11.5 controls, mean difference 1.44 95% CI (0.84 to 2.04) p<0.001), emotional instability (17.1 vs 15.6, mean difference 1.53 95% CI (0.86 to 2.2) p<0.001) and negative world view (12.1 vs 10.2 controls, mean difference 1.91 95% CI (1.24 to 2.59) p<0.001). The total HRQoL of adolescents with constipation was lower than controls (70.6 vs 79.0 mean difference 9.48 95% CI (1.4 to 6.7) p<0.05).ConclusionA significant proportion of children with constipation are suffering from psychological maladjustment.

Background The effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods. Results Fourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods. Conclusion Our results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.

Wild animal diets are challenging to quantify, and the various methods for doing so have strengths and weaknesses. Combining multiple methods can allow ecologists to assess their level of confidence in particular results, increase sample size, and investigate diet over varying time scales. The biases of traditional gut content–based methods are mostly well understood. Newer methods may have important biases that can only be worked out through comparison to established ones. We collected data on the diet of wild Plains Hog-nosed Snakes (Heterodon nasicus) using multiple, fundamentally dissimilar methods, combined analytically using a Bayesian framework to describe an ontogenetic dietary shift. Gut contents were the most straightforward, but yielded a small sample size that fell below any reasonable threshold for making generalizations. Stable isotopes indicated an obvious ontogenetic dietary shift, but were labor-intensive, and conclusions are limited by multiple methodological caveats including similarity among prey groups, maternal carryover effects, and uncertainty in trophic enrichment factors. Fecal environmental DNA (eDNA) was intermediate in terms of effort, yielding results congruent with the other two methods, but the interpretation of which would likely have been confounded by contaminants had we not used all three methods in tandem. Several apparent artifacts are discussed. There are some reassuring similarities among methods. There are also several differences. The most complete picture uses data from all methods taken together. Future studies should attempt to compare the biases, expense, and potential drawbacks of these and other methods in greater detail.

Background The microbial community analysis of stools requires optimised and standardised protocols for their collection, homogenisation, microbial disruption and nucleic acid extraction. Here we examined whether different layers of the stool are equally representative of the microbiome. We also studied the effect of stool water content, which typically increases in diarrhoeic samples, and of a microbial disruption method on DNA integrity and, therefore, on providing an unbiased microbial composition analysis. Results We collected faecal samples from healthy subjects and performed microbial composition analysis by pyrosequencing the V4 region of the 16S rRNA gene. To examine the effect of stool structure, we compared the inner and outer layers of the samples (N = 8). Both layers presented minor differences in microbial composition and abundance at the species level. These differences did not significantly bias the microbial community specific to an individual. To evaluate the effect of stool water content and bead-beating, we used various volumes of a water-based salt solution and beads of distinct weights before nucleic acid extraction (N = 4). The different proportions of water did not affect the UniFrac-based clustering of samples from the same subject However, the use or omission of a bead-beating step produced different proportions of Gram-positive and Gram-negative bacteria and significant changes in the UniFrac-based clustering of the samples. Conclusion The degree of hydration and homogenisation of faecal samples do not significantly alter their microbial community composition. However, the use of bead-beating is critical for the proper detection of Gram-positive bacteria such as Blautia and Bifidobacterium .

This study assessed the efficiency of sewage treatment plants (STPs) in removing sterols based on chemical analyses of both influents and effluents. Samples from 3s and three tertiary plants were collected and analyzed by gas chromatography mass spectrometry for 23 individual sterols including mestranol, norethindrone, equol, estrone, equilin, norgestrel, 17α-ethinylestradiol, 17α-estradiol, 17β-estradiol, estriol, dihydrocholesterol (cholestanol), coprostanol, epicoprostanol, cholesterol, desmosterol, campesterol, stigmasterol, β-sitosterol, coprostanone, cholestanone, epicholestanol, stigmastanol, and 24-ethylcoprostanol. The percentage of sterols remaining in effluent samples (compared to influent samples) ranged from 0% to 80% and varied among sterol compounds and with STP location and treatment type. Differences in the efficiency of sterol removal for secondary and tertiary STPs were statistically significant. Although the concentration of sterol compounds differed between influents and effluents, sterol abundances remained the same. The most abundant sterol detected was cholesterol, followed by the fecal sterol coprostanol, and the plant sterols 24-ethylcoprostanol and β-sitosterol. For three STPs, the hormone estrone was detected in effluents at concentrations of 0.03–0.05 μg L −1 . Ten sterol ratios specific for human fecal contamination and eight sterol ratios for differentiating among multiple sources of fecal contamination were calculated and showed that 12 ratios for influent and nine ratios for effluent were successful for human fecal source tracking. Based on sterol ratio values in this study, new criteria for identification of human fecal contamination were suggested.