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The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Diarrhoeal disease attributable to enterotoxigenic Escherichia coli (ETEC) causes substantial morbidity and mortality predominantly in paediatric populations in low- and middle-income countries. In addition to acute illness, there is an increasing appreciation of the long-term consequences of enteric infections, including ETEC, on childhood growth and development. Provision of potable water and sanitation and appropriate clinical care for acute illness are critical to reduce the ETEC burden. However, these interventions are not always practical and may not achieve equitable and sustainable coverage. Vaccination may be the most cost-effective and equitable means of primary prevention; however, additional data are needed to accelerate the investment and guide the decision-making process for ETEC vaccines. First, to understand and quantify the ETEC disease burden, additional data are needed on the association between ETEC infection and physical and cognitive stunting as well as delayed educational attainment. Furthermore, the role of inappropriate or inadequate antibiotic treatment of ETEC-attributable diarrhoea may contribute to the development of antimicrobial resistance (AMR) and needs further elucidation. An ETEC vaccine that mitigates acute diarrhoeal illness and minimizes the longer-term disease manifestations could have significant public health impact and be a cost-effective countermeasure. Herein we review the ETEC vaccine pipeline, led by candidates compatible with the general parameters of the Preferred Product Characteristics (PPC) recently developed by the World Health Organization. Additionally, we have developed an ETEC Vaccine Development Strategy to provide a framework to underpin priority activities for researchers, funders and vaccine manufacturers, with the goal of addressing globally unmet data needs in the areas of research, product development, and policy, as well as commercialization and delivery. The strategy also aims to guide prioritization and co-ordination of the priority activities needed to minimize the timeline to licensure and use of ETEC vaccines, especially in in low- and middle-income countries, where they are most urgently needed.

Arthrospira platensis(commonly known as spirulina) is a photosynthetic cyanobacterium1. It is a highly nutritious food that has been consumed for decades in the US, and even longer by indigenous cultures2. Its widespread use as a safe food source and proven scalability have driven frequent attempts to convert it into a biomanufacturing platform. But these were repeatedly frustrated by spirulina’s genetic intractability. We report here efficient and versatile genetic engineering methodology for spirulina that allows stable expression of bioactive protein therapeutics at high levels. We further describe large-scale, indoor cultivation and downstream processing methods appropriate for the manufacturing of biopharmaceuticals in spirulina. The potential of the platform is illustrated by pre-clinical development and human testing of an orally delivered antibody therapeutic against campylobacter, a major cause of infant mortality in the developing world and a growing antibiotic resistance threat3,4. This integrated development and manufacturing platform blends the safety of food-based biotechnology with the ease of genetic manipulation, rapid growth rates and high productivity characteristic of microbial platforms. These features combine for exceptionally low-cost production of biopharmaceuticals to address medical needs that are unfeasible with current biotechnology platforms.

Background Clostridium difficile is the most common cause of hospital-acquired infections, responsible for >450000 infections annually in the USA. Probiotics provide a promising, well-tolerated adjunct therapy to standard C. difficile infection (CDI) treatment regimens, but there is a paucity of data regarding their effectiveness for the treatment of an initial CDI. Objectives We conducted a pilot randomized controlled trial of 33 participants from February 2013 to February 2015 to determine the feasibility and health outcomes of adjunct probiotic use in patients with an initial mild to moderate CDI. Methods The intervention was a 28 day, once-daily course of a four-strain oral probiotic capsule containing Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium lactis Bi-07 and B. lactis Bl-04. The control placebo was identical in taste and appearance. Registered at clinicaltrials.gov: trial registration number = NCT01680874. Results Probiotic adjunct therapy was associated with a significant improvement in diarrhoea outcomes. The primary duration of diarrhoea outcome (0.0 versus 1.0 days; P = 0.039) and two exploratory outcomes, total diarrhoea days (3.5 versus 12.0 days; P = 0.005) and rate of diarrhoea (0.1 versus 0.3 days of diarrhoea/stool diary days submitted; P = 0.009), all decreased in participants with probiotic use compared with placebo. There was no significant difference in the rate of CDI recurrence or functional improvement over time between treatment groups. Conclusions Probiotics are a promising adjunct therapy for treatment of an initial CDI and should be further explored in a larger randomized controlled trial.

Campylobacterinfections are among the leading bacterial causes of diarrhea and of ‘environmental enteropathy’ (EE) and growth failure worldwide. However, the lack of an inexpensive small animal model of enteric disease withCampylobacterhas been a major limitation for understanding its pathogenesis, interventions or vaccine development. We describe a robust standard mouse model that can exhibit reproducible bloody diarrhea or growth failure, depending on the zinc or protein deficient diet and on antibiotic alteration of normal microbiota prior to infection. Zinc deficiency and the use of antibiotics create a niche forCampylobacterinfection to establish by narrowing the metabolic flexibility of these mice for pathogen clearance and by promoting intestinal and systemic inflammation. Several biomarkers and intestinal pathology in this model also mimic those seen in human disease. This model provides a novel tool to testing specific hypotheses regarding disease pathogenesis as well as vaccine development that is currently in progress.Author SummaryCampylobacter jejunihas been identified as one of the leading causes of enteropathy and diarrhea. In developing countries, these repeated enteric infections often result in growth deficits and cognitive impairment. There is a lack of small animal models ofCampylobacterinfection. This is a major hurdle in understanding the pathogenesis ofCampylobacterinfection in order to lead to therapeutic treatments and vaccines. We have developed a highly reproducible mouse model ofCampylobacterinfection that has clinical outcomes that match those of malnourished children. We hope that these insights intoCampylobactersusceptibility will lead to the development of treatments against this major cause of diarrheal illness.

BACKGROUND: Environmental enteric dysfunction (EED) is widespread throughout the tropics and in children is associated with stunting and other adverse health outcomes. One of the hallmarks of EED is villus damage. In children with severe acute malnutrition (SAM) the severity of enteropathy is greater and short term mortality is high, but the metabolic consequences of enteropathy are unknown. Here, we characterize the urinary metabolic alterations associated with villus health, classic enteropathy biomarkers and anthropometric measurements in severely malnourished children in Zambia.METHODS/PRINCIPAL FINDINGS: We analysed 20 hospitalised children with acute malnutrition aged 6 to 23 months in Zambia. Small intestinal biopsies were assessed histologically (n = 15), anthropometric and gut function measurements were collected and the metabolic phenotypes were characterized by 1H nuclear magnetic resonance (NMR) spectroscopy. Endoscopy could not be performed on community controls children. Growth parameters were inversely correlated with enteropathy biomarkers (p = 0.011) and parameters of villus health were inversely correlated with translocation and permeability biomarkers (p = 0.000 and p = 0.015). Shorter villus height was associated with reduced abundance of metabolites related to gut microbial metabolism, energy and muscle metabolism (p = 0.034). Villus blunting was also related to increased sucrose excretion (p = 0.013).CONCLUSIONS/SIGNIFICANCE: Intestinal villus blunting is associated with several metabolic perturbations in hospitalized children with severe undernutrition. Such alterations include altered muscle metabolism, reinforcing the link between EED and growth faltering, and a disruption in the biochemical exchange between the gut microbiota and host. These findings extend our understanding on the downstream consequences of villus blunting and provide novel non-invasive biomarkers of enteropathy dysfunction. The major limitations of this study are the lack of comparative control group and gut microbiota characterization.

Background Clostridium difficile is a pathogen of rapidly increasing public health importance. An estimated quarter of a million Clostridium difficile infections (CDI) occur in the United States annually, at a resultant cost of 14,000 deaths and 1 billion dollars. Clostridium difficile related deaths have risen 400% over the last decade, and current standard antibiotic treatments are only 75 to 85% successful. Besides increasing the risk of antibiotic resistance and side effects, these treatments are very expensive. The most vulnerable population for Clostridium difficile is older adults, who make up approximately half of the cases, but account for 90% of the related deaths. Probiotics may have potential as adjunctive therapeutic agents for CDIs, however, current data is limited. Methods This pilot study is a single-site, randomized, placebo-controlled, double-blind, phase two clinical trial. The trial primarily evaluates the effect of four weeks of probiotic therapy in addition to standard of care on Clostridium difficile diarrhea duration and recurrence. Secondary outcomes include effect on fecal cytokines, fecal lactoferrin, and Clostridium difficile toxin density in stool, as well as patient functional status. Discussion This pilot study will determine the feasibility and effect size to conduct larger randomized controlled trials of probiotic interventions in patients with CDI, to determine the impact of probiotics on the symptoms of CDI. ClinicalTrials.gov Identifier: NCT01680874

There is strong evidence that exclusive breastfeeding (EBF) in the first 6 months of life reduces the risk of diseases in infancy and in later life.To understand the maternal reasoning that influences optimum infant feeding practices of caregivers in semirural communities of Limpopo province.Nested qualitative study among mothers in an ongoing birth cohort study was conducted; structured and semi-structured interviews were used to collect data. Data from 234 infants after 6 months of follow-up was included for quantitative analysis. Four focus discussion groups comprising 7 to 10 caregivers were used to obtain perception of mothers on breastfeeding. A semi-structured interview guide was used to stimulate discussions. Thematic content analyses were conducted to identify the main themes that influence breastfeeding practices of caregivers.Over 90% of the caregivers initiated breastfeeding after delivery. However, less than 1% of mothers practiced EBF by 3 months, and none of the children were exclusively breastfed for up to 6 months. All caregivers introduced non-breast milk liquids and solids by the second month of child's life. Common reasons for introducing non-breast milk foods included insufficiency of breast milk production, going back to work or school, and influence by elderly women (mothers/mothers-in-law) and church members.Exclusive breastfeeding was not practiced in this community due to cultural and religious beliefs and misinformation. The involvement of elderly women and church members in infant feeding education and promotion programs and the dissemination of breastfeeding information through mobile phones to younger mothers are recommended.

Molecular characterization of virulence and antimicrobial resistance profiles were determined for Shigella species isolated from children with diarrhea in Fortaleza, Brazil. Fecal specimens were collected along with socioeconomic and clinical data from children with moderate to severe diarrhea requiring emergency care. Shigella spp. were isolated by standard microbiological techniques, and we developed 4 multiplex polymerase chain reaction assays to detect 16 virulence-related genes (VRGs). Antimicrobial susceptibility tests were performed using disk diffusion assays. S. flexneri and S. sonnei were the predominant serogroups. S. flexneri was associated with low monthly incomes; more severe disease; higher number of VRGs; and presence of pic, set, and sepA genes. The SepA gene was associated with more intense abdominal pain. S. flexneri was correlated with resistance to ampicillin and chloramphenicol, whereas S. sonnei was associated with resistance to azithromycin. Strains harboring higher numbers of VRGs were associated with resistance to more antimicrobials. We highlight the correlation between presence of S. flexneri and sepA, and increased virulence and suggest a link to socioeconomic change in northeastern Brazil. Additionally, antimicrobial resistance was associated with serogroup specificity in Shigella spp. and increased bacterial VRGs.

ContextoA glutamina e a principal fonte de energia do enterocito e diarreia e perda de peso sao frequentes em pacientes infectados pelo HIV.ObjetivoDeterminar o efeito da alanil-glutamina sobre a permeabilidade e a absorcao intestinais nesses pacientes.MetodosEstudo duplo-cego, randomizado, controlado por placebo, utilizando doses isonitrogenicas de alanil-glutamina (24 g/dia) e de placebo (glicina, 25 g/dia) durante 10 dias. Antes e depois dessa suplementacao nutricional a excrecao urinaria de lactulose e manitol foi determinada por cromatografia liquida de alta performance.ResultadosQuarenta e seis pacientes com HIV/AIDS, sendo 36 do sexo masculino, com 37,28 ± 3 anos (media ± erro padrao) foram incluidos. Vinte e dois e 24 individuos foram tratados com alanil-glutamina e com glicina, respectivamente. Nos nove pacientes que relataram ter apresentado diarreia nos 14 dias anteriores ao inicio do estudo, a excrecao urinaria de manitol foi significativamente menor do que nos pacientes que nao referiram essa queixa [mediana (intervalo): 10,51 (3,01-19,75) vs 15,37 (3,93-46,73), P = 0,0281] e a razao lactulose/manitol foi significativamente mais elevada [mediana (intervalo): 0,04 (0,00-2,89) vs 0,02 (0,00-0,19), P = 0,0317]. Constatou-se tambem aumento significativo na excrecao urinaria de manitol no grupo tratado com alanil-glutamina [mediana (intervalo): 14,38 (8,25-23,98), antes vs 21,24 (6,27-32,99) apos o tratamento, n = 14, P = 0,0382].ConclusaoOs resultados do presente estudo sugerem que a integridade e a absorcao intestinais sao mais intensamente afetadas em pacientes com HIV/AIDS que tiveram diarreia recentemente. Adicionalmente, a suplementacao nutricional com alanil-glutamina associou-se a melhoria na absorcao intestinal.

Increased precipitation and temperature variability as well as extreme events related to climate change are predicted to affect the availability and quality of water globally. Already heavily burdened with diarrheal diseases due to poor access to water, sanitation and hygiene facilities, communities throughout the developing world lack the adaptive capacity to sufficiently respond to the additional adversity caused by climate change. Studies suggest that diarrhea rates are positively correlated with increased temperature, and show a complex relationship with precipitation. Although climate change will likely increase rates of diarrheal diseases on average, there is a poor mechanistic understanding of the underlying disease transmission processes and substantial uncertainty surrounding current estimates. This makes it difficult to recommend appropriate adaptation strategies. We review the relevant climate-related mechanisms behind transmission of diarrheal disease pathogens and argue that systems-based mechanistic approaches incorporating human, engineered and environmental components are urgently needed. We then review successful systems-based approaches used in other environmental health fields and detail one modeling framework to predict climate change impacts on diarrheal diseases and design adaptation strategies.

Enteroaggregative Escherichia coli (EAEC) is a common cause of infectious diarrhea, especially in children living in poor-resource countries. In this article, we present a SYBR Green-based real-time polymerase chain reaction (qPCR) method for quantitative detection of EAEC in DNA directly extracted from human stool samples. To test the proposed qPCR system, we examined specificity, sensitivity, repeatability, and also the degree of DNA extraction efficiency using EAEC strain 042 spiked into EAEC-free stool sample. The specificity of this assay was proved using six strains of EAEC, seven strains of other E. coli types, and one strain of Shigella. The detection limit of qPCR was 67 CFU/reaction. In naturally infected stool samples, we found EAEC in quantities varying from 6.7 × 10(5) to 2 × 10(9 ) CFU/g of feces. We could not detect any reduction after stool DNA extraction for the amounts of 10(7) and 10(6) CFU/mL of spiked EAEC. This qPCR assay is simple, rapid, reproducible, sensitive, specific, and allows rapid EAEC quantification to be used in a variety of further EAEC studies. This new quantitative method provides a relatively simple means to quantify EAEC, which will likely be key to understanding the pathophysiology and impact of EAEC infection.

This study determined the prevalence of Campylobacter jejuni/coli and its relation with nutritional status in children from Northeastern Brazil. This was a case-control study design. Stool samples were evaluated for hipO (C. jejuni), ask (C. coli), and cdtABC (C. jejuni's cytolethal distending toxin) genes. The nutritional status from these children was assessed by anthropometric measures and z-scores. C. jejuni and C. coli were detected in 9.6% (8/83) and 6.0% (5/83) in the diarrhea group and in 7.2% (6/83) and 1.2% (1/83) of the nondiarrhea group, respectively. Children with positive molecular detection of C. jejuni showed significantly lower z-scores than children without C. jejuni. The cdtABC operon was found in 57% of hipO(+) samples. C. jejuni/coli prevalence was similar in diarrhea and nondiarrhea groups. There was a significant association of C. jejuni infection with lower nutritional status.

Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enteropathogenic E coli or Salmonella). Viruses (such as noroviruses and rotaviruses) and protozoa (such as Cryptosporidium, Giardia, or Entamoeba histolytica) disrupt cell functions and cause short- or long-term disease. Much epidemiologic data about these pathogens have been collected from community- and hospital-acquired settings, as well as from patients with traveler's or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis, and treatment.

Campylobacter spp. were detected - using culture, ELISA, PCR, and qPCR - among children (0–36 months) with moderate to severe diarrhea in Northeastern Brazil. Our data showed that either the qPCR alone or PCR along with ELISA might be an alternative to culture to diagnose Campylobacter due to their enhanced sensitivity.

“Norwalk-like viruses” (NLVs) are a common cause of epidemic gastroenteritis in adults and children in developed countries. However, little is known about the role of NLVs in endemic pediatric gastroenteritis in developing countries. We sequenced Genogroup I and II NLV reverse transcription-polymerase chain reaction (RT-PCR) products from an 81-nucleotide region of the viral RNA polymerase gene to examine the molecular epidemiology of NLV infection in children younger than 5 years of age in Forteleza, Ceara, Brazil. NLV-positive PCR products were obtained from stool specimens collected over a 16-month period (1990–1991) from diarrhea cases and controls in a cohort of 120 children in an urban shantytown and from a study in the same city of hospitalized children with persistent diarrhea. Eight unique strains were detected in 15 specimens from 10 cohort children and in two hospital specimens. Nucleotide identity between the strains (5 Genogroup I, 3 Genogroup II) ranged from 63% to 88%. We designated these strains BraV1–8, for Brazil virus 1–8. The degree of genomic diversity of NLV strains we identified in this cohort during a short time period suggests multiple foci of infection within the community. Furthermore, sequence analysis of strains from two children with multiple symptomatic NLV infections indicates that infection with one strain was not protective against subsequent infection with a different strain in the same genogroup. These findings have implications for vaccine development and the prevention of pediatric gastroenteritis in developing countries. J. Med. Virol. 58:426–434, 1999. © 1999 Wiley-Liss, Inc.

While considerable progress has been made towards the Millennium Development Goals (MDGs) and childhood diarrheal diseases have reduced from 4.6 million to 0.8 million over the last three decades the number of diarrheal deaths remains unacceptably high. For diarrheal disease in particular coverage indicators for key preventive and curative interventions remain suboptimal suggesting that efforts to reduce diarrhea-related child deaths by two-thirds have stalled. This paper aims to identify research priorities using the Child Health and Nutrition Research Initiative’s (CHNRI’s) method for global childhood diarrhoeal disease over the next 15 years. Ten teams were established and over 150 experts participated generating and scoring 466 research questions. Despite a range of discovery-related research topics implementation research questions related to known interventions for childhood diarrheal diseases were ranked highly by most experts. In tandem with the Global Action Plan for Pneumonia and Diarrhea concerted efforts by a range of stakeholders in implementation research will be needed to equitably scale up already proven effective interventions. (excerpts)

Probiotics are derived from traditional fermented foods, from beneficial commensals or from the environment. They act through diverse mechanisms affecting the composition or function of the commensal microbiota and by altering host epithelial and immunological responses. Certain probiotic interventions have shown promise in selected clinical conditions where aberrant microbiota have been reported, such as atopic dermatitis, necrotising enterocolitis, pouchitis and possibly irritable bowel syndrome. However, no studies have been conducted that can causally link clinical improvements to probiotic-induced microbiota changes. Whether a disease-prone microbiota pattern can be remodelled to a more robust, resilient and disease-free state by probiotic administration remains a key unanswered question. Progress in this area will be facilitated by: optimising strain, dose and product formulations, including protective commensal species; matching these formulations with selectively responsive subpopulations; and identifying ways to manipulate diet to modify bacterial profiles and metabolism.

Enteroaggregative Escherichia coli (EAEC) is an important agent that causes endemic and epidemic diarrhoeal diseases worldwide. Several EAEC virulence-related genes (VRGs) have been described but their role in the clinical outcome of infection is not completely defined. This study investigated the prevalence of EAEC and potential associations of its VRGs with risk of or protection from diarrhoeal diseases in children from urban communities in north-eastern Brazil. The case–control study included 166 children, who had their stools evaluated for the EAEC diagnostic genes (aaiC and aatA) using PCR. Positive samples were further analysed by multiplex PCR and identified 18 VRGs. EAEC was found in the same proportion in both groups (41 %). The plasmid-borne gene encoding a hexosyltransferase homologue (capU) was the most frequently detected (89.6 %), followed by dispersin protein (aap, 58.2 %) and EAEC HilA homologue (eilA, 57.8 %). The AAF/III fimbrial subunit (agg3A) gene was observed at lower frequency (1.5 %). Plasmid-encoded toxin (pet) or AAF/II fimbrial subunit (aafA) was associated significantly with disease. AAF/IV fimbrial subunit (agg4A) or hypothetical plasmid-encoded haemolysin (orf61) was detected significantly more in controls than in children with diarrhoea. In addition, one set of genes in combination, aaiC and agg3/4C but lacking agg4A and orf61, was associated with diarrhoea cases; and another one, orf61 in the absence of pet and aafA, was correlated with control children. These data confirm a high prevalence, endemicity and heterogeneity of EAEC strains in the developing urban areas of north-eastern Brazil. Statistical correlation between cases and controls was seen with either isolated or combined sets of genes, suggesting that the pathophysiology of EAEC infection involves a complex and dynamic modulation of several VRGs.

Interest in global health (GH) among medical students worldwide is measurably increasing. There is a concomitant emphasis on emphasizing globally-relevant health professions education. Through a structured literature review, expert consensus recommendations, and contact with relevant professional organizations, we review the existing state of GH education in US medical schools for which data were available. Several recommendations from professional societies have been developed, along with a renewed emphasis on competencies in global health. The implementation of these recommendations was not observed as being uniform across medical schools, with variation noted in the presence of global health curricula. Recommendations for including GH in medical education are suggested, as well as ways to formalize GH curricula, while providing flexibility for innovation and adaptation.

Diarrhea in patients with acquired immunodeficiency syndrome (AIDS) may cause malabsorption of medications and failure of antiretroviral therapy (ART). We prospectively evaluated human immunodeficiency virus-1 (HIV-1)-infected patients with and without chronic diarrhea initiating ART in Haiti. We report mean plasma antiretroviral concentrations at 2 and 4 weeks. We measured plasma HIV-1 RNA levels at four points. Fifty-two HIV-1-infected patients (26 matched pairs) were enrolled. No differences in antiretroviral concentrations were detected. At week 24, 18/25 (72%) cases and 16/24 (68%) controls had undetectable plasma HIV-1 RNA levels (P = 0.69). Patients with plasma HIV-1 RNA levels > 50 copies/mL at week 24 had lower early efavirenz concentrations than patients with undetectable HIV-1 RNA (2,621 ng/mL versus 5,278 ng/mL; P = 0.02). Diarrhea at ART initiation does not influence plasma concentrations of the medications evaluated. Virologic outcome at Week 24 does correlate with efavirenz concentrations early in therapy but not with the presence of chronic diarrhea.

Infectious disease in the developing world continues to represent one of the greatest challenges facing humanity. Every year over a million children suffer and die from the sequela of enteric infections, and in 2008 was estimated almost 2.7 million (UNAIDS 2009 update) adults and children became infected with human immunodeficiency virus (HIV). While oral rehydration therapy for diarrhea and antiretrovirals (ARV) for HIV are critical, there is a place for adjunctive therapies to improve quality of life. The importance of the human microbiota in retaining health is now recognized, as is the concept of replenishing beneficial microbes through probiotic treatments. Studies have shown that probiotics can reduce the duration of diarrhea, improve gut barrier function, help prevent bacterial vaginosis (BV) and enhance immunity even in HIV-infected subjects. However, many issues remain before the extent of probiotic benefits can be verified, and their application to the developing world realized. This consensus report outlines the potential probiotic, and to a lesser extent prebiotic, applications in resource disadvantaged settings, and recommends steps that could bring tangible relief to millions of people. The challenges to both efficacy and effectiveness studies in these settings include a lack of infrastructure and funding for scientists, students and research projects in developing countries; making available clinically proven probiotic and prebiotic products at affordable prices; and undertaking appropriately designed clinical trials. We present a roadmap on how efficacy studies may be conducted in a resource disadvantaged setting among persons with chronic diarrhea and HIV. These examples and the translation of efficacy into effectiveness are described. © 2011 Landes Bioscience. Chemicals/CAS: glutamine, 56-85-9, 6899-04-3; retinol, 68-26-8, 82445-97-4; zinc, 14378-32-6, 7440-66-6

Toxin A (TxA) is able to induce most of the classical features of Clostridium difficile -associated disease in animal models. The objective of this study was to determine the effect of an inhibitor of adenosine deaminase, EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], on TxA-induced enteritis in C57BL6 mice and on the gene expression of adenosine receptors. EHNA (90 μmol/kg) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) 30 min prior to TxA (50 μg) or PBS injection into the ileal loop. A 2A adenosine receptor agonist (ATL313; 5 nM) was injected in the ileal loop immediately before TxA (50 μg) in mice pretreated with EHNA. The animals were euthanized 3 h later. The changes in the tissue were assessed by the evaluation of ileal loop weight/length and secretion volume/length ratios, histological analysis, myeloperoxidase assay (MPO), the local expression of inducible nitric oxide synthase (NOS2), pentraxin 3 (PTX3), NF-κB, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) by immunohistochemistry and/or quantitative reverse transcription-PCR (qRT-PCR). The gene expression profiles of A 1 , A 2A , A 2B , and A 3 adenosine receptors also were evaluated by qRT-PCR. Adenosine deaminase inhibition, by EHNA, reduced tissue injury, neutrophil infiltration, and the levels of proinflammatory cytokines (TNF-α and IL-1β) as well as the expression of NOS2, NF-κB, and PTX3 in the ileum of mice injected with TxA. ATL313 had no additional effect on EHNA action. TxA increased the gene expression of A 1 and A 2A adenosine receptors. Our findings show that the inhibition of adenosine deaminase by EHNA can prevent Clostridium difficile TxA-induced damage and inflammation possibly through the A 2A adenosine receptor, suggesting that the modulation of adenosine/adenosine deaminase represents an important tool in the management of C. difficile- induced disease.

Introduction Long-term care facilities (LTCF) residents have been estimated to have the highest incidence of diarrheal illness among adults living in the developed world. This study describes undiagnosed diarrhea, intestinal inflammation, and Clostridium difficile colonization in a LTC population and explores whether these are associated with functional decline, as defined by weight loss or a change in cognitive or ADL status. Methods An observational study of a convenience sampling of residents in a 180-bed LTCF was obtained; evaluation of stool and medical records was done. Stool specimens were evaluated for consistency, gross blood, inflammation (via quantitative fecal lactoferrin, IBD-SCAN), and C difficile (via PCR for gdh). SPSS and STATA were used and significance was set at P Results There were 46 stools collected; 13 of the subjects were male, 28 were older than 65 years, and 35 were prescribed 5 to 15 medications. Twenty-six of the 46 stools collected had elevated quantitative fecal lactoferrin levels. Although only 5 subjects were reported to have diarrhea (4 with elevated lactoferrin), 28 stool specimens were observed to be liquid or semi-solid (19 with elevated lactoferrin), and these liquid/ semisolid stools were significantly correlated with lactoferrin positivity (P = .017). In analysis of functional status, there was no statistically significant association between change in ADL (n = 17) or cognitive status (n = 5) and elevated lactoferrin. However, all 3 subjects who had significant weight loss had elevated lactoferrin, although the mean fecal lactoferrin was not statistically different from those without weight loss. Of the 2 samples with C difficile, both were liquid and, when compared with all other liquid stools (n = 22), the mean lactoferrin was statistically higher (134.1 versus 28.8 μg/mL, P = .008). These 2 subjects had neither weight loss nor change in cognitive status, but 1 had a change in ADL status. Discussion and conclusions Diarrhea in LTCF residents is underdiagnosed. Diarrhea and the presence of C difficile in the stool are associated with intestinal inflammation, as detected by fecal lactoferrin. With our small numbers, we were not able to identify a specific link; however, we were able to identify a correlation between weight loss and intestinal inflammation, but, with just 2 samples, not C difficile colonization. This relationship highlights the importance of larger studies to further examine the rate of diarrhea in LTCF; the effect of diarrhea and intestinal inflammation on weight loss; and the interaction of C difficile colonization with weight loss, malnutrition, and functional decline.

Highly-potent antiretroviral therapy is necessary to avoid viral replication in HIV patients; however, it can favor the appearance of resistance mutations. The mutations 41L, 67N, 70R, 210W, 215Y/F, 219E/Q, 44D and 118I are defined as nucleoside analogous mutations (NAMs), because they affect the efficacy of all nucleoside reverse transcriptase inhibitors (NRTI). The mutation most frequently associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) is 103N. 33W/F, 82A/F/L/T, 84V and 90M are called protease inhibitor resistance-associated mutations (PRAM), because they are associated with resistance to several protease inhibitors (PI). This study evaluated the development of resistance mutations and examine the susceptibility of HIV with these mutations to antiretrovirals in HIV-1 patients who have failed one or more therapy regimes. Analyses were made of 101 genotypic tests of patients with therapeutic failure to 2 or 3-drug regimens with NRTI, NNRTI or PI. We used the Stanford database to define the susceptibility profile of the viruses. The samples were divided into three treatment-failure groups: first (F), second (S) and multi-failure (MF) to antiretroviral regimens, and we correlated these groups with resistance profiles and principal mutations. There was a significant increase in resistance mutations V82A/F/L/T, I84V, L90M, M41L, K70R, L210W, T215Y/F and K219Q/E in MF. We also found significantly higher resistance to zidovudine, didanosine, stavudine and abacavir in MF. There was no increase in resistance to tenofovir (p=0.28) and lopinavir (p=0.079) in MF. A high degree of resistance to NNRTIs was observed in all groups. Increased resistance mutations will affect future therapeutic options for HIV patients in Brazil because it results in a significant increase in resistance to antiretroviral drugs.

All values reported as means±SD. P value was calculated by repeated measures ANOVA. Pair-wise comparisons were done using a significance criterion of 0.017 (0.05/3) in order to account for multiple comparisons. aP,0.017 when comparing the variable between "before IPAA" and "0-12 m after IPAA"; bP,0.017 when comparing the variable between "0-12 m after IPAA" and "12 m+ after IPAA"; cP,0.017 when comparing the variable between "before IPAA" and "12 m+ after IPAA"

Enteroaggregative Escherichia coli (EAEC) is a recognized cause of enteric disease in diverse clinical settings. Mucosal immunity towards EAEC infections is poorly understood. To better characterize immunoregulatory mechanisms underlying EAEC infections we constructed a computational model mimicking host responses to EAEC at the gut mucosa. Preliminary model calibration efforts demonstrated remarkable fitting to experimental data. Nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) has been targeted to modulate mucosal immune responses to EAEC. Wild type and conditional knockout mice lacking PPARγ in T cells were fed protein-deficient diets at weaning and challenged with 5x109cfu EAEC strain JM221. Quantitative RT-PCR data reveal that administration of GW9662, a potent PPARγ antagonist, for 7 days post infection or the deletion of PPARγ in T cells results in upregulation of pro-inflammatory cytokines including IL-6, TNF-α, and IL-1β when compared to non-treated infected animals. Histological analysis of colons divulges lower leukocyte infiltration and decreased mucosal thickness 14 days following EAEC infection after pharmacological blockade of PPARγ or deletion of PPARγ in T cells. These findings were accompanied by faster clearance of colonic EAEC in tissue-specific PPARγ null mice. In conclusion, we present a fully integrated approach to study host responses to EAEC that provides new insights on the pathogenesis and treatment of enteric infections.

Clostridium difficile is typically a harmless anaerobic bacterium but has recently re-emerged as a pathogen that can cause nosocomial diarrhea, colitis and death. To investigate the role of peroxisome proliferator-activated receptor (PPAR) γ in modulating immune responses to C. difficile we have performed several studies using a mouse model of antibiotic-induced C. difficile-associated disease (CDAD). The loss of PPARγ in T cells increased disease activity, body weight loss, and colonic inflammatory lesions after infection. It also resulted in upregulated IL-17 and MCP-1, and downregulated IL-10 expression, suggesting that a Th17 phenotype predominates during CDAD in mice lacking T cell PPARγ. Treatment of C. difficile-infected mice with PPAR γ agonists ameliorates disease severity and colonic lesions. RNA-seq results in colonic specimens indicated that three miRNAs were significantly overexpressed in infected mice: mmu-miR-146b, mmu-miR-1940, and mmu-miR-1298 (FDR P < 0.05). Real-time PCR results validated that infection results in overexpression of miR-146b, a molecule involved in regulating immunity and inflammation. Furthermore, NCOA4, a co-activator of PPARγ and target of miR-146b, was down-regulated in colons of infected mice. We provide novel evidence supporting a role for PPARγ in regulating C. difficile-related inflammation and immunopathology and explore the potential role of miRNA in modulating host responses to C. difficile.

Immunocompromised patients are susceptible to a variety of serious gastrointestinal infections. Among the most common or most severe are Mycobacterium avium-intracellulare, herpes simplex, candidiasis, cryptosporidiosis, and Salmonellosis. Some of these infections are difficult or impossible to treat successfully. The correct diagnosis often can only be established if it is first suspected and the appropriate studies are performed.