Distribution of Escherichia coli Pathotypes along an Urban-Rural Gradient in Ecuador.

Diarrheal diseases are a leading cause of mortality and morbidity in low- and middle-income countries. Diarrhea is associated with a wide array of etiological agents including bacterial, viral, and parasitic enteropathogens. Previous studies have captured between- but not within-country heterogeneities in enteropathogen prevalence and severity. We conducted a case-control study of diarrhea to understand how rates and outcomes of infection with diarrheagenic pathotypes of Escherichia coli vary across an urban-rural gradient in four sites in Ecuador. We found variability by site in enteropathogen prevalence and infection outcomes. Any pathogenic E. coli infection, coinfections, diffuse adherent E. coli (DAEC), enteroinvasive E. coli (EIEC), and rotavirus were significantly associated with acute diarrhea. DAEC was the most common pathotype overall and was more frequently associated with disease in urban areas. Enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) were more common in rural areas. ETEC was only associated with diarrhea in one site. Phylogenetic analysis revealed that associations with disease were not driven by any single clonal complex. Higher levels of antibiotic resistance were detected in rural areas. Enteropathogen prevalence, virulence, and antibiotic resistance patterns vary substantially by site within Ecuador. The variations in E. coli pathotype prevalence and virulence in this study have important implications for control strategies by context and demonstrate the importance of capturing within-country differences in enteropathogen disease dynamics.

Gut Microbiome Changes with Acute Diarrheal Disease in Urban Versus Rural Settings in Northern Ecuador.

Previous studies have reported lower fecal bacterial diversity in urban populations compared with those living in rural settings. However, most of these studies compare geographically distant populations from different countries and even continents. The extent of differences in the gut microbiome in adjacent rural versus urban populations, and the role of such differences, if any, during enteric infections remain poorly understood. To provide new insights into these issues, we sampled the gut microbiome of young children with and without acute diarrheal disease (ADD) living in rural and urban areas in northern Ecuador. Shotgun metagenomic analyses of non-ADD samples revealed small but significant differences in the abundance of microbial taxa, including a greater abundance of Prevotella and a lower abundance of Bacteroides and Alistipes in rural populations. Greater and more significant shifts in taxon abundance, metabolic pathway abundance, and diversity were observed between ADD and non-ADD status when comparing urban to rural sites (Welch's t-test, P < 0.05). Collectively our data show substantial functional, diversity, and taxonomic shifts in the gut microbiome of urban populations with ADD, supporting the idea that the microbiome of rural populations may be more resilient to ADD episodes.

First quantitative assessment of the adsorption of a fluorocarbon gas on phospholipid monolayers at the air/water interface.

HYPOTHESIS: Fluorocarbon gases introduced above monolayers of phospholipids at the air/water interface were recently found to promote the adsorption of diverse molecular compounds, with potential application in drug-loaded microbubble design. Quantitative determination of the fluorocarbon present in the monolayers is strongly needed for the development of such applications. We hypothesized that neutron reflectometry (NR) and ellipsometry experiments would allow quantification of the fluorocarbon trapped in the monolayers. EXPERIMENTS: We report the first quantitative determination of the extents of adsorption of perfluorohexane (F-hexane) on different phospholipid monolayers with respect to both their phase and isotopic form. To this aim, we applied an approach based on co-modeling the data obtained from NR and ellipsometry. FINDINGS: We found that F-hexane adsorbs strongly in monolayers of dipalmitoylphosphatidylcholine (DPPC) when they are both in the liquid expanded (LE) and liquid condensed (LC) phases, but to different extents according to the isotopic form of the phospholipid. Kinetic resolution of the interfacial composition from data on both isotopic contrasts (assuming chemical identicality) was therefore not possible using NR alone, so an alternative NR/ellipsometry co-modeling treatment was applied to data from each isotopic contrast. F-hexane adsorbs more abundantly on monolayers of hydrogenous DPPC than chain-deuterated DPPC when they are in the LE phase, whilst the opposite was observed when they monolayers are in the LC phase. The extents of adsorption of F-hexane in monolayers of dimyristoylphosphatidylcholine (DMPC, LE phase) and distearoylphosphatidylcholine (DSPC, LC phase) concurs with the strong dependence of those with phospholipids of different isotopic contrasts according to the monolayer phase. This new methodology can lead to advances in the novel characterization of fluorocarbons interacting with phospholipid monolayers of relevance to applications such as in the shells of fluorocarbon-stabilized medically-oriented microbubbles.

Synthesis and physicochemical evaluation of fluorinated lipopeptide precursors of ligands for microbubble targeting.

Ligand-targeted microbubbles are focusing interest for molecular imaging and delivery of chemotherapeutics. Lipid-peptide conjugates (lipopeptides) that feature alternating serine-glycine (SG) n segments rather than classical poly(oxyethylene) linkers between the lipid polar head and a targeting ligand were proposed for the liposome-mediated, selective delivery of anticancer drugs. Here, we report the synthesis of perfluoroalkylated lipopeptides (F-lipopeptides) bearing two hydrophobic chains (C n F2 n +1, n = 6, 7, 8, 1-3) grafted through a lysine moiety on a hydrophilic chain composed of a lysine-serine-serine (KSS) sequence followed by 5 SG sequences. These F-lipopeptides are precursors of targeting lipopeptide conjugates. A hydrocarbon counterpart with a C10H21 chain (4) was synthesized for comparison. The capacity for the F-lipopeptides to spontaneously adsorb at the air/water interface and form monolayers when combined with dipalmitoylphosphatidylcholine (DPPC) was investigated. The F-lipopeptides 1-3 demonstrated a markedly enhanced tendency to form monolayers at the air/water interface, with equilibrium surface pressures reaching ≈7-10 mN m-1 versus less than 1 mN m-1 only for their hydrocarbon analog 4. The F-lipopeptides penetrate in the DPPC monolayers in both liquid expanded (LE) and liquid condensed (LC) phases without interfacial film destabilization. By contrast, 4 provokes delipidation of the interfacial film. The incorporation of the F-lipopeptides 1-3 in microbubbles with a shell of DPPC and dipalmitoylphosphatidylethanolamine-PEG2000 decreased their mean diameter and increased their stability, the best results being obtained for the C8F17-bearing lipopeptide 3. By contrast, the hydrocarbon lipopeptide led to microbubbles with a larger mean diameter and a significantly lower stability.

Metagenomic Signatures of Gut Infections Caused by Different Escherichia coli Pathotypes.

Escherichia coli is a leading contributor to infectious diarrhea and child mortality worldwide, but it remains unknown how alterations in the gut microbiome vary for distinct E. coli pathotype infections and whether these signatures can be used for diagnostic purposes. Further, the majority of enteric diarrheal infections are not diagnosed with respect to their etiological agent(s) due to technical challenges. To address these issues, we devised a novel approach that combined traditional, isolate-based and molecular-biology techniques with metagenomics analysis of stool samples and epidemiological data. Application of this pipeline to children enrolled in a case-control study of diarrhea in Ecuador showed that, in about half of the cases where an E. coli pathotype was detected by culture and PCR, E. coli was likely not the causative agent based on the metagenome-derived low relative abundance, the level of clonality, and/or the virulence gene content. Our results also showed that diffuse adherent E. coli (DAEC), a pathotype that is generally underrepresented in previous studies of diarrhea and thus, thought not to be highly virulent, caused several small-scale diarrheal outbreaks across a rural to urban gradient in Ecuador. DAEC infections were uniquely accompanied by coelution of large amounts of human DNA and conferred significant shifts in the gut microbiome composition relative to controls or infections caused by other E. coli pathotypes. Our study shows that diarrheal infections can be efficiently diagnosed for their etiological agent and categorized based on their effects on the gut microbiome using metagenomic tools, which opens new possibilities for diagnostics and treatment.IMPORTANCEE. coli infectious diarrhea is an important contributor to child mortality worldwide. However, diagnosing and thus treating E. coli infections remain challenging due to technical and other reasons associated with the limitations of the traditional culture-based techniques and the requirement to apply Koch's postulates. In this study, we integrated traditional microbiology techniques with metagenomics and epidemiological data in order to identify cases of diarrhea where E. coli was most likely the causative disease agent and evaluate specific signatures in the disease-state gut microbiome that distinguish between diffuse adherent, enterotoxigenic, and enteropathogenic E. coli pathotypes. Therefore, our methodology and results should be highly relevant for diagnosing and treating diarrheal infections and have important applications in public health.

Locals get travellers' diarrhoea too: risk factors for diarrhoeal illness and pathogenic Escherichia coli infection across an urban-rural gradient in Ecuador.

OBJECTIVES: Diarrhoea is a common and well-studied cause of illness afflicting international travellers. However, traveller's diarrhoea can also result from travel between high and low disease transmission regions within a country, which is the focus of this study. METHODS: We recruited participants for a case-control study of diarrhoea at four sites along an urban-rural gradient in Northern Ecuador: Quito, Esmeraldas, Borbón and rural communities outside of Borbón. At each of these sites, approximately 100 subjects with diarrhoea (cases) were recruited from Ministry of Health clinics and were age-matched with subjects visiting the same clinics for other complaints (controls). RESULTS: Travellers to urban destinations had higher risk of diarrhoea and diarrhoeagenic Escherichia coli (DEC) infections. Travel to Quito was associated with diarrhoea (aOR = 2.01, 95% CI = 1.10-3.68) and travel to Guayaquil (another urban centre in Ecuador) was associated with Diffuse Adherent E. coli infection (OR = 2.09, 95% CI = 1.01-4.33). Compared to those not travelling, urban origins were also associated with greater risk of diarrhoea in Esmeraldas (aOR = 2.28, 95% CI = 1.20-4.41), and with higher risk of diarrhoeagenic E. coli infections in Quito (aOR = 2.61, 95% CI = 1.16-5.86), with >50% of travel from Quito and Esmeraldas specified to another urban destination. CONCLUSIONS: This study suggests that individuals travelling from lower-transmission regions (rural areas) to higher transmission regions (urban centres) within a single country are at a greater risk of acquiring a diarrhoea-related illness. Investments to improve water, sanitation and hygiene conditions in urban areas could have impacts on outlying rural areas within a given country.

Bile acid malabsorption in patients with chronic diarrhea and Crohn's disease.

INTRODUCTION AND AIM: Crohn's disease (CD) is a form of inflammatory bowel disease and is mainly characterized by diarrhea and abdominal pain. The aim of our study was to analyze the usefulness of performing a 75SeHCAT scan in CD patients with chronic diarrhea and suspected bile acid malabsorption (BAM). In addition, we aimed to determine whether there was a relationship with the clinical features of the disease and a previous bowel resection. PATIENTS AND METHODS: this was an observational cross-sectional study of 39 patients with a diagnosis of CD and chronic diarrhea. All cases underwent a 75SeHCAT scan for BAM diagnosis, after discarding disease activity. RESULTS: the study cohort included 19 females and 20 males. The median age was 44 years and the majority of patients were A2 L1 B1 according to the Montreal classification; 84.6% of patients had undergone a previous bowel resection. BAM was present in 97.4% of patients (100% and 83.3% of patients with and without previous surgery, respectively), which was severe in 92.1% of cases. Treatment with bile acid sequestrants was initiated and a favorable response was obtained in 72.2% of patients. The relationship between BAM degree (moderate or severe), bowel surgery and the response to bile acid sequestrant treatment was also analyzed but not statistically significant. CONCLUSION: BAM is a frequent cause of diarrhea in CD patients in endoscopic or radiological remission. This condition was present in all patients with a history of a bowel resection. A response to bile acid sequestrants treatment was observed in 73% of patients.

Is a second recombinant human thyrotropin stimulation test useful? The value of postsurgical undetectable stimulated thyroglobulin level at the time of remnant ablation on clinical outcome.

OBJECTIVE: The management of patients with differentiated thyroid cancer (DTC) has changed in recent years, and monitoring depends on the risk of persistent/recurrent disease. The objective was to assess the prognostic value of a single stimulated thyroglobulin (Tg) measured at the time of the first radioiodine therapy (Stim-Tg1), and the utility of a second stimulated Tg measurement performed 6-12 months later (Stim-Tg2). We also examined the role of neck ultrasound (US) in the early diagnosis of recurrence. DESIGN: This was a retrospective observational cohort study conducted in a tertiary referral hospital. Of 213 evaluated patients with DTC, 169 were finally included. METHODS: Measurement of Stim-Tg1, Stim-Tg2 and neck US. RESULTS: Stim-Tg1 was undetectable in 71 of 169 patients (42%). All of them (71/71) continued to have negative Stim-Tg2. Seventy of 71 had an excellent response to the first treatment. Sixty-eight of 71 had no evidence of disease after an average follow-up of 7·2 years. In patients with detectable Stim-Tg1 (98/169; 58%), Stim-Tg2 became negative in 40. The negative predictive value (NPV) of Stim-Tg1 was 0·96. The optimal Stim-Tg1 cut-off level for identifying persistence was 3·65 ng/ml. Recurrence was detected in 14 patients. Neck US was useful for identifying local recurrence (13/14; 92·85%). CONCLUSIONS: Stim-Tg1 is a reliable marker with a high NPV. A second stimulation test should be avoided in patients with negative Stim-Tg1. In patients with biochemical persistence, Stim-Tg2 is useful for confirming/ruling out final status. Neck US plays a valuable role in the early diagnosis of recurrence.

Thyroid remnant ablation success and disease outcome in stage III or IV differentiated thyroid carcinoma: recombinant human thyrotropin versus thyroid hormone withdrawal.

BACKGROUND: Most publications to date compare outcomes after post-surgical thyroid remnant ablation stimulated by recombinant human thyrotropin (rhTSH) versus thyroid hormone withholding/withdrawal (THW) in low-recurrence risk differentiated thyroid carcinoma (DTC) patients. We sought to perform this comparison in high-risk patients. METHODS: We retrospectively analyzed ~9-year single-center experience in 70 consecutive adults with initial UICC (Union for International Cancer Control) stage III/IV, M0 DTC undergoing rhTSH-aided (N.=54) or THW-aided (N.=16) high-activity ablation. Endpoints included ablation success and DTC outcome. Assessed ≥1 year post-ablation, ablation success comprised a) no visible scintigraphic thyroid bed uptake or pathological extra-thyroidal uptake; b) undetectable stimulated serum thyroglobulin (Tg) without interfering autoantibodies; c) both criteria. DTC outcome, determined at the latest visit, comprised either 1) "no evidence of disease" (NED): undetectable Tg, negative Tg autoantibodies, negative most recent whole-body scan, no suspicious findings clinically, on neck ultrasonography, or on other imaging; 2) persistent disease: failure to attain NED; or 3) recurrence: loss of NED. RESULTS: After the first ablative activity, ablation success by scintigraphic plus biochemical criteria was 64.8% in rhTSH patients, 56.3% in THW patients (P=NS). After 3.5-year versus 6.2-year median follow-up (P<0.05), DTC outcomes were NED, 85.2%, persistent disease, 13.0%, recurrence, 1.9%, in the rhTSH group and NED, 87.5%, persistent or recurrent disease, 6.3% each, in the THW group (P=NS). CONCLUSION: In patients with initial stage III/IV, M0 DTC, rhTSH-aided and THW-assisted ablation were associated with comparable remnant eradication or DTC cure rates.

Fluorometric enzymatic autoindicating biosensor for H2O2 determination based on modified catalase.

Our general aim is to develop reversible optical biosensors which can be used for continuous monitoring. In this paper we propose a biosensor for H(2)O(2) determination. The bioreceptor is catalase (Cat) previously linked to a Ruthenium O(2)-sensitive fluorophore (Cat-Ru). It is based on the reversible H(2)O(2) disproportionation into O(2) and H(2)O. First, the fluorescent-enzymatic system was optimized for batch measurements (linear response ranges from 1×10(-4) to, at least, 1×10(-3) M H(2)O(2)). Because of its reversibility, the same enzyme aliquot can be used for performing the whole calibration step (and the subsequent determination). Secondly, the optical sensor was prepared by Cat-Ru immobilization in a polyacrylamide film. The sensor permits H(2)O(2) determination in a similar concentration range as in batch mode and can be used during at least 1 month. A mathematical model has also been developed which permits the effect of the experimental parameters to predict. The model also explains the sensor behavior if different fluorophores are used, and shows that the analytical signal only slightly depends on the initial concentration of the O(2) in the sample. Finally an alternative sensor is presented based on a commercially available O(2) fluorescence sensor linked to catalase. This system gives an analytical behavior similar to that shown for the Cat-Ru sensor.

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems.

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.