High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh.

BACKGROUND: Diarrheal disease is a leading cause of morbidity and mortality globally, especially in low- and middle-income countries. High-throughput and low-cost approaches to identify etiologic agents are needed to guide public health mitigation. Nanoliter-qPCR (nl-qPCR) is an attractive alternative to more expensive methods yet is nascent in application and without a proof-of-concept among hospitalized patients. METHODS: A census-based study was conducted among diarrheal patients admitted at two government hospitals in rural Bangladesh during a diarrheal outbreak period. DNA was extracted from stool samples and assayed by nl-qPCR for common bacterial, protozoan, and helminth enteropathogens as the primary outcome. RESULTS: A total of 961 patients were enrolled; stool samples were collected from 827 patients. Enteropathogens were detected in 69% of patient samples; More than one enteropathogen was detected in 32%. Enteropathogens most commonly detected were enteroaggregative Escherichia coli (26.0%), Shiga toxin-producing E.coli (18.3%), enterotoxigenic E. coli (15.5% heat stable toxin positive, 2.2% heat labile toxin positive), Shigella spp. (14.8%), and Vibrio cholerae (9.0%). Geospatial analysis revealed that the median number of pathogens per patient and the proportion of cases presenting with severe dehydration were greatest amongst patients residing closest to the study hospitals." CONCLUSIONS: This study demonstrates a proof-of-concept for nl-qPCR as a high-throughput low-cost method for enteropathogen detection among hospitalized patients

Is detection of enteropathogens and human or animal faecal markers in the environment associated with subsequent child enteric infections and growth: an individual participant data meta-analysis.

BACKGROUND: Quantifying contributions of environmental faecal contamination to child diarrhoea and growth faltering can illuminate causal mechanisms behind modest health benefits in recent water, sanitation, and hygiene (WASH) trials. We aimed to assess associations between environmental detection of enteropathogens and human or animal microbial source tracking markers (MSTM) and subsequent child health outcomes. METHODS: In this individual participant data meta-analysis we searched we searched PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus for WASH intervention studies with a prospective design and concurrent control that measured enteropathogens or MSTM in environmental samples, or both, and subsequently measured enteric infections, diarrhoea, or height-for-age Z-scores (HAZ) in children younger than 5 years. We excluded studies that only measured faecal indicator bacteria. The initial search was done on Jan 19, 2021, and updated on March 22, 2023. One reviewer (AM) screened abstracts, and two independent reviewers (AM and RT) examined the full texts of short-listed articles. All included studies include at least one author that also contributed as an author to the present Article. Our primary outcomes were the 7-day prevalence of caregiver-reported diarrhoea and HAZ in children. For specific enteropathogens in the environment, primary outcomes also included subsequent child infection with the same pathogen ascertained by stool testing. We estimated associations using covariate-adjusted regressions and pooled estimates across studies. FINDINGS: Data from nine published reports from five interventions studies, which included 8603 children (4302 girls and 4301 boys), were included in the meta-analysis. Environmental pathogen detection was associated with increased infection prevalence with the same pathogen and lower HAZ (ΔHAZ -0·09 [95% CI -0·17 to -0·01]) but not diarrhoea (prevalence ratio 1·22 [95% CI 0·95 to 1·58]), except during wet seasons. Detection of MSTM was not associated with diarrhoea (no pooled estimate) or HAZ (ΔHAZ -0·01 [-0·13 to 0·11] for human markers and ΔHAZ -0·02 [-0·24 to 0·21] for animal markers). Soil, children's hands, and stored drinking water were major transmission pathways. INTERPRETATION: Our findings support a causal chain from pathogens in the environment to infection to growth faltering, indicating that the lack of WASH intervention effects on child growth might stem from insufficient reductions in environmental pathogen prevalence. Studies measuring enteropathogens in the environment should subsequently measure the same pathogens in stool to further examine theories of change between WASH, faecal contamination, and health. Given that environmental pathogen detection was predictive of infection, programmes targeting specific pathogens (eg, vaccinations and elimination efforts) can environmentally monitor the pathogens of interest for population-level surveillance instead of collecting individual biospecimens. FUNDING: The Bill & Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office

Introducing riskCommunicator: An R package to obtain interpretable effect estimates for public health

Common statistical modeling methods do not necessarily produce the most relevant or interpretable effect estimates to communicate risk. Overreliance on the odds ratio and relative effect measures limit the potential impact of epidemiologic and public health research. We created a straightforward R package, called riskCommunicator, to facilitate the presentation of a variety of effect measures, including risk differences and ratios, number needed to treat, incidence rate differences and ratios, and mean differences. The riskCommunicator package uses g-computation with parametric regression models and bootstrapping for confidence intervals to estimate effect measures in time-fixed data. We demonstrate the utility of the package using data from the Framingham Heart Study to estimate the effect of prevalent diabetes on the 24-year risk of cardiovascular disease or death. The package promotes the communication of public-health relevant effects and is accessible to a broad range of epidemiologists and health researchers with little to no expertise in causal inference methods or advanced coding

Shared bacterial communities between soil, stored drinking water, and hands in rural Bangladeshi households.

Understanding household-level transmission pathways of fecal pathogens can provide insight for developing effective strategies to reduce diarrheal illness in low- and middle-income countries. We applied whole bacterial community analysis to investigate pathways of bacterial transmission in 50 rural Bangladeshi households. SourceTracker was used to quantify the shared microbial community in household reservoirs (stored drinking water, soil, and hands) and estimate the percentage of fecal-associated bacteria from child and mothers' feces in these reservoirs. Among the reservoirs studied, most bacterial transfer occurred between mothers' and children's hands and between mothers' hands and stored water. The relative percentage of human fecal-associated bacteria in all household reservoirs was low. We also quantified the number of identical amplicon sequence variants within and between individual households to assess bacterial community exchange in the domestic environment. Intra-household sharing of bacteria between mothers' and children's hands and between hands and soil was significantly greater than inter-household sharing

Shared bacterial communities between soil, stored drinking water, and hands in rural Bangladeshi households.

Understanding household-level transmission pathways of fecal pathogens can provide insight for developing effective strategies to reduce diarrheal illness in low- and middle-income countries. We applied whole bacterial community analysis to investigate pathways of bacterial transmission in 50 rural Bangladeshi households. SourceTracker was used to quantify the shared microbial community in household reservoirs (stored drinking water, soil, and hands) and estimate the percentage of fecal-associated bacteria from child and mothers' feces in these reservoirs. Among the reservoirs studied, most bacterial transfer occurred between mothers' and children's hands and between mothers' hands and stored water. The relative percentage of human fecal-associated bacteria in all household reservoirs was low. We also quantified the number of identical amplicon sequence variants within and between individual households to assess bacterial community exchange in the domestic environment. Intra-household sharing of bacteria between mothers' and children's hands and between hands and soil was significantly greater than inter-household sharing

Human metabolome variation along the upper intestinal tract

Most processing of the human diet occurs in the small intestine. Metabolites in the small intestine originate from host secretions, plus the ingested exposome1 and microbial transformations. Here we probe the spatiotemporal variation of upper intestinal luminal contents during routine daily digestion in 15 healthy male and female participants. For this, we use a non-invasive, ingestible sampling device to collect and analyse 274 intestinal samples and 60 corresponding stool homogenates by combining five mass spectrometry assays2,3 and 16S rRNA sequencing. We identify 1,909 metabolites, including sulfonolipids and fatty acid esters of hydroxy fatty acids (FAHFA) lipids. We observe that stool and intestinal metabolomes differ dramatically. Food metabolites display trends in dietary biomarkers, unexpected increases in dicarboxylic acids along the intestinal tract and a positive association between luminal keto acids and fruit intake. Diet-derived and microbially linked metabolites account for the largest inter-individual differences. Notably, two individuals who had taken antibiotics within 6 months before sampling show large variation in levels of bioactive FAHFAs and sulfonolipids and other microbially related metabolites. From inter-individual variation, we identify Blautia species as a candidate to be involved in FAHFA metabolism. In conclusion, non-invasive, in vivo sampling of the human small intestine and ascending colon under physiological conditions reveals links between diet, host and microbial metabolism

High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh. S1 Dataset

Data collected from 961 patients with results of enteropathogen detection by Nanoliter-qPCR. This dataset supports the PLOS ONE paper, "High-throughput low-cost nl-qPCR for enteropathogen detection: A proof-of-concept among hospitalized patients in Bangladesh.

Identification of Widespread Antibiotic Exposure in Patients with Cholera Correlates with Clinically Relevant Microbiota Changes

Background: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. Methods: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. Results: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P =. 040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P <. 001). Conclusion: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study

Profiling the human intestinal environment under physiological conditions

The spatiotemporal structure of the human microbiome1,2, proteome3 and metabolome4,5 reflects and determines regional intestinal physiology and may have implications for disease6. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals7. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. Collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease