Influence of hydrometeorological risk factors on child diarrhea and enteropathogens in rural Bangladesh.

BACKGROUND: A number of studies have detected relationships between weather and diarrhea. Few have investigated associations with specific enteric pathogens. Understanding pathogen-specific relationships with weather is crucial to inform public health in low-resource settings that are especially vulnerable to climate change. OBJECTIVES: Our objectives were to identify weather and environmental risk factors associated with diarrhea and enteropathogen prevalence in young children in rural Bangladesh, a population with high diarrheal disease burden and vulnerability to weather shifts under climate change. METHODS: We matched temperature, precipitation, surface water, and humidity data to observational longitudinal data from a cluster-randomized trial that measured diarrhea and enteropathogen prevalence in children 6 months-5.5 years from 2012-2016. We fit generalized additive mixed models with cubic regression splines and restricted maximum likelihood estimation for smoothing parameters. RESULTS: Comparing weeks with 30°C versus 15°C average temperature, prevalence was 3.5% higher for diarrhea, 7.3% higher for Shiga toxin-producing Escherichia coli (STEC), 17.3% higher for enterotoxigenic E. coli (ETEC), and 8.0% higher for Cryptosporidium. Above-median weekly precipitation (median: 13mm; range: 0-396mm) was associated with 29% higher diarrhea (adjusted prevalence ratio 1.29, 95% CI 1.07, 1.55); higher Cryptosporidium, ETEC, STEC, Shigella, Campylobacter, Aeromonas, and adenovirus 40/41; and lower Giardia, sapovirus, and norovirus prevalence. Other associations were weak or null. DISCUSSION: Higher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Our findings emphasize the heterogeneity of the relationships between hydrometeorological variables and specific enteropathogens, which can be masked when looking at composite measures like all-cause diarrhea. Our results suggest that preventive interventions targeted to reduce enteropathogens just before and during the rainy season may more effectively reduce child diarrhea and enteric pathogen carriage in rural Bangladesh and in settings with similar meteorological characteristics, infrastructure, and enteropathogen transmission.

Burden, Clinical Characteristics, Risk Factors, and Seasonality of Adenovirus 40/41 Diarrhea in Children in Eight Low-Resource Settings.

Background: The application of molecular diagnostics has identified enteric group adenovirus serotypes 40 and 41 as important causes of diarrhea in children. However, many aspects of the epidemiology of adenovirus 40/41 diarrhea have not been described. Methods: We used data from the 8-site Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development Project birth cohort study to describe site- and age-specific incidence, risk factors, clinical characteristics, and seasonality. Results: The incidence of adenovirus 40/41 diarrhea was substantially higher by quantitative polymerase chain reaction than enzyme immunoassay and peaked at ∼30 episodes per 100 child-years in children aged 7-15 months, with substantial variation in incidence between sites. A significant burden was also seen in children 0-6 months of age, higher than other viral etiologies with the exception of rotavirus. Children with adenovirus 40/41 diarrhea were more likely to have a fever than children with norovirus, sapovirus, and astrovirus (adjusted odds ratio [aOR], 1.62; 95% CI, 1.16-2.26) but less likely than children with rotavirus (aOR, 0.66; 95% CI, 0.49-0.91). Exclusive breastfeeding was strongly protective against adenovirus 40/41 diarrhea (hazard ratio, 0.64; 95% CI, 0.48-0.85), but no other risk factors were identified. The seasonality of adenovirus 40/41 diarrhea varied substantially between sites and did not have clear associations with seasonal variations in temperature or rainfall. Conclusions: This study supports the situation of adenovirus 40/41 as a pathogen of substantial importance, especially in infants. Fever was a distinguishing characteristic in comparison to other nonrotavirus viral etiologies, and promotion of exclusive breastfeeding may reduce the high observed burden in the first 6 months of life.

MicroRNA Expression and Intestinal Permeability in Children Living in a Slum Area of Bangladesh.

Introduction: MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. Changes in miRNA expression have been reported in a number of intestinal diseases, in both tissue samples and readily accessible specimens like stools. Pathogenic infections, diet, toxins, and other environmental factors are believed to influence miRNA expression. However, modulation of miRNAs in humans is yet to be thoroughly investigated. In this study, we examined the expression levels of two human miRNAs (miRNA-122 and miRNA-21) in stool samples of a group of Bangladeshi children who had an altered/increased intestinal permeability (IIP). Methods: Stool samples were collected from children with IIP (L:M > 0.09) and normal intestinal permeability (NIP) (L:M ≤ 0.09). Quantitative PCR was performed to quantify the levels of miRNA-122 and miR-21 in stools. Commercial ELISA kits were used to measure gut inflammatory markers Calprotectin and REG1B. Serum samples were tested using Human Bio-Plex Pro Assays to quantify IL-1ß, IL-2, IL-5, IL-10, IL-13, IFN-γ, and TNF-α. Total nucleic acid extracted from stool specimens were used to determine gut pathogens using TaqMan Array Card (TAC) system real-time polymerase chain reaction. Results: The expression levels of miRNA-122 (fold change 11.6; p < 0.001, 95% CI: 6.14-11.01) and miR-21 (fold change 10; p < 0.001, 95% CI: 5.05-10.78) in stool were upregulated in children with IIP than in children with normal intestinal permeability (NIP). Significant correlations were observed between stool levels of miR-122 and miR-21 and the inflammatory cytokines IL-1ß, IL-2, IFN-γ, and TNF-α (p < 0.05). Children with IIP were frequently infected with rotavirus, Campylobacter jejuni, Bacteroides fragilis, adenovirus, norovirus, astrovirus, and various Escherichia coli strains (ETEC_STh, ETEC_STp, EAEC_aaiC, EAEC_aatA) (p < 0.001). miR-122 significantly correlated with the fecal inflammatory biomarkers REG1B (p = 0.015) and Calprotectin (p = 0.030), however miR-21 did not show any correlation with these fecal biomarkers.

HLA class I and II associations with common enteric pathogens in the first year of life.

BACKGROUND: genetic susceptibility to infection is mediated by numerous host factors, including the highly diverse, classical human leukocyte antigen (HLA) genes, which are critical genetic determinants of immunity. We systematically evaluated the effect of HLA alleles and haplotypes on susceptibility to 12 common enteric infections in children during the first year of life in an urban slum of Dhaka, Bangladesh. METHODS: a birth cohort of 601 Bangladeshi infants was prospectively monitored for diarrhoeal disease. Each diarrhoeal stool sample was analyzed for enteric pathogens by multiplex TaqMan Array Card (TAC). High resolution genotyping of HLA class I (A and B) and II (DRB1, DQA1, and DQB1) genes was performed by next-generation sequencing. We compared the frequency of HLA alleles and haplotypes between infected and uninfected children. FINDINGS: we identified six individual allele associations and one five-locus haplotype association. One allele was associated with protection: A*24:02 - EAEC. Five alleles were associated with increased risk: A*24:17 - typical EPEC, B*15:01 - astrovirus, B*38:02 - astrovirus, B*38:02 - Cryptosporidium and DQA1*01:01 - Cryptosporidium. A single five-locus haplotype was associated with protection: A*11:01~B*15:02~DRB1*12:02~DQA1*06:01~DQB1*03:01- adenovirus 40/41. INTERPRETATION: our findings suggest a role for HLA in susceptibility to early enteric infection for five pathogens. Understanding the genetic contribution of HLA in susceptibility has important implications in vaccine design and understanding regional differences in incidence of enteric infection. FUNDING: this research was supported by the National Institute of Health (NIH) and the Bill and Melinda Gates Foundation.

Clinical performance of the H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ assays, novel stool antigen tests for diagnosis of Helicobacter pylori.

Infection with Helicobacter pylori is a global health issue, and rapid and accurate testing is a key to diagnosis. We aimed to assess the performance of two novel enzyme immunoassays (EIA), the H. PYLORI QUIK CHEK™ and the H. PYLORI CHEK™ assays, for the detection of H. pylori antigen in stool. Patients from five geographically diverse sites across the USA, Germany, and in Bangladesh were tested for infection with Helicobacter pylori with the two novel stool antigen tests and two commercially available stool antigen assays. All patients provided a stool sample and underwent esophagogastroduodenoscopy for biopsy. Results were compared to a clinical diagnosis using a composite reference method consisting of histological analysis and rapid urease testing of the biopsy. A total of 271 patients, 68.2% female and mean age of 46 years, were included. The overall prevalence of H. pylori infection was 24.1%. The sensitivity of the H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ was 92% and 91%, respectively. The specificity of H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ was 91% and 100%, respectively. No significant cross-reactivity against other gut pathogens was observed. The H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ assays demonstrate excellent clinical performance compared the composite reference method.

Lewis Blood-group Antigens Are Associated With Altered Susceptibility to Shigellosis.

In a cohort of infants, we found that lack of the Lewis histo-blood group antigen was associated with increased susceptibility to shigellosis. Broadly inhibiting fucosylation in epithelial cells in vitro decreased invasion by Shigella flexneri. These results support a role for fucosylated glycans in susceptibility to shigellosis.

Fecal MicroRNAs as Potential Biomarkers for Screening and Diagnosis of Intestinal Diseases.

MicroRNAs (miRNAs) are a class of conserved endogenous, small non-coding RNA molecules with a length of 18-25 nucleotides that regulate gene expression by RNA interference processes, including mRNA chopping, mRNA deadenylation, and translation inhibition. miRNAs maintain the physiological functions of the intestine and are instrumental in gut pathogenesis. miRNAs play an important role in intercellular communication and are present in all body fluids, including stools with different composition and concentrations. However, under diseased conditions, miRNAs are aberrantly expressed and act as negative regulators of gene expression. The stable and differentially expressed miRNAs in stool enables miRNAs to be used as potential biomarkers for screening of various intestinal diseases. In this review, we summarize the expressed miRNA profile in stool and highlight miRNAs as biomarkers with potential clinical and diagnostic applications, and we aim to address the prospects for recent advanced techniques for screening miRNA in diagnosis and prognosis of intestinal disorders.

Effect of water, sanitation, handwashing and nutrition interventions on enteropathogens in children 14 months old: a cluster-randomized controlled trial in rural Bangladesh.

BACKGROUND: We evaluated the impact of low-cost water, sanitation, handwashing (WSH) and child nutrition interventions on enteropathogen carriage in the WASH Benefits cluster-randomized controlled trial in rural Bangladesh. METHODS: We analyzed 1411 routine fecal samples from children 14±2 months old in the WSH (n = 369), nutrition counseling plus lipid-based nutrient supplement (n = 353), nutrition plus WSH (n = 360), and control (n = 329) arms for 34 enteropathogens using quantitative PCR. Outcomes included the number of co-occurring pathogens; cumulative quantity of four stunting-associated pathogens; and prevalence and quantity of individual pathogens. Masked analysis was by intention-to-treat. RESULTS: 326 (99.1%) control children had one or more enteropathogens detected (mean 3.8±1.8). Children receiving WSH interventions had lower prevalence and quantity of individual viruses than controls (prevalence difference for norovirus: -11% [95% confidence interval [CI], -5 to -17%]; sapovirus: -9% [95%CI, -3 to -15%]; and adenovirus 40/41: -9% [95%CI, -2 to - 15%]). There was no difference in bacteria, parasites, or cumulative quantity of stunting-associated pathogens between controls and any intervention arm. CONCLUSIONS: WSH interventions were associated with fewer enteric viruses in children aged 14 months. Different strategies are needed to reduce enteric bacteria and parasites at this critical young age.

Duodenal Microbiota in Stunted Undernourished Children with Enteropathy.

BACKGROUND: Environmental enteric dysfunction (EED) is an enigmatic disorder of the small intestine that is postulated to play a role in childhood undernutrition, a pressing global health problem. Defining the incidence of this disorder, its pathophysiological features, and its contribution to impaired linear and ponderal growth has been hampered by the difficulty in directly sampling the small intestinal mucosa and microbial community (microbiota). METHODS: In this study, among 110 young children (mean age, 18 months) with linear growth stunting who were living in an urban slum in Dhaka, Bangladesh, and had not benefited from a nutritional intervention, we performed endoscopy in 80 children who had biopsy-confirmed EED and available plasma and duodenal samples. We quantified the levels of 4077 plasma proteins and 2619 proteins in duodenal biopsy samples obtained from these children. The levels of bacterial strains in microbiota recovered from duodenal aspirate from each child were determined with the use of culture-independent methods. In addition, we obtained 21 plasma samples and 27 fecal samples from age-matched healthy children living in the same area. Young germ-free mice that had been fed a Bangladeshi diet were colonized with bacterial strains cultured from the duodenal aspirates. RESULTS: Of the bacterial strains that were obtained from the children, the absolute levels of a shared group of 14 taxa (which are not typically classified as enteropathogens) were negatively correlated with linear growth (length-for-age z score, r = -0.49; P = 0.003) and positively correlated with duodenal proteins involved in immunoinflammatory responses. The representation of these 14 duodenal taxa in fecal microbiota was significantly different from that in samples obtained from healthy children (P<0.001 by permutational multivariate analysis of variance). Enteropathy of the small intestine developed in gnotobiotic mice that had been colonized with cultured duodenal strains obtained from children with EED. CONCLUSIONS: These results provide support for a causal relationship between growth stunting and components of the small intestinal microbiota and enteropathy and offer a rationale for developing therapies that target these microbial contributions to EED. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov number, NCT02812615.).

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

Protection From Natural Immunity Against Enteric Infections and Etiology-Specific Diarrhea in a Longitudinal Birth Cohort.

BACKGROUND: The degree of protection conferred by natural immunity is unknown for many enteropathogens, but it is important to support the development of enteric vaccines. METHODS: We used the Andersen-Gill extension of the Cox model to estimate the effects of previous infections on the incidence of subsequent subclinical infections and diarrhea in children under 2 using quantitative molecular diagnostics in the MAL-ED cohort. We used cross-pathogen negative control associations to correct bias due to confounding by unmeasured heterogeneity of exposure and susceptibility. RESULTS: Prior rotavirus infection was associated with a 50% lower hazard (calibrated hazard ratio [cHR], 0.50; 95% confidence interval [CI], 0.41-0.62) of subsequent rotavirus diarrhea. Strong protection was evident against Cryptosporidium diarrhea (cHR, 0.32; 95% CI, 0.20-0.51). There was also protection due to prior infections for norovirus GII (cHR against diarrhea, 0.67; 95% CI, 0.49-0.91), astrovirus (cHR, 0.62; 95% CI, 0.48-0.81), and Shigella (cHR, 0.79; 95% CI, 0.65-0.95). Minimal protection was observed for other bacteria, adenovirus 40/41, and sapovirus. CONCLUSIONS: Natural immunity was generally stronger for the enteric viruses than bacteria, potentially due to less antigenic diversity. Vaccines against major causes of diarrhea may be feasible but likely need to be more immunogenic than natural infection.

Serum Adipokines, Growth Factors, and Cytokines Are Independently Associated with Stunting in Bangladeshi Children.

Growth in young children is controlled through the release of several hormonal signals, which are affected by diet, infection, and other exposures. Stunting is clearly a growth disorder, yet limited evidence exists documenting the association of different growth biomarkers with child stunting. This study explored the association between different growth biomarkers and stunting in Bangladeshi children. A quasi-experimental study was conducted among 50 stunted (length-for-age Z-score (LAZ) < -2 SD) and 50 control (LAZ ≥ -2 SD) children, aged 12-18 months, residing in a Bangladeshi slum. The enrolled stunted children received an intervention package, which included food supplementation for three months, psychosocial stimulation for six months, and routine clinical care on community nutrition center at the study field site. The controls received routine clinical care only. All children were clinically screened over the study period. Length, weight, fasting blood and fecal biomarkers were measured. All biomarkers levels were similar in both groups except for oxyntomodulin at enrolment. Leptin (adjusted odds ratio, AOR: 4.0, p < 0.01), leptin-adiponectin ratio (AOR 5.07 × 108, p < 0.01), insulin-like growth factor-1 (IGF-1) (AOR 1.02, p < 0.05), and gamma interferon (IFN-γ) (AOR 0.92, p < 0.05) levels were independently associated with stunting at enrolment. Serum leptin, leptin-adiponectin ratio, interleukin-6 (IL-6), IL-10, tumor necrosis factor-alpha (TNF-α), and fecal alpha-1-antitrypsin (AAT) levels increased significantly (p < 0.001), while IFN-γ levels significantly decreased among stunted children after six months of intervention. Leptin, leptin-adiponectin ratio, IGF-1, and IFN-γ are independently associated with stunting in Bangladeshi children. This trial was registered at clinicaltrials.gov as NCT02839148.

Screening for coeliac disease in children and adults living in a slum of Dhaka, Bangladesh.

BACKGROUND AND OBJECTIVE: Serological screening with a confirmation through biopsy has improved the understanding of coeliac disease (CD) epidemiology worldwide. Prevalence of CD in Bangladesh is not yet explored and therefore, we aimed to assess the seroprevalence of CD in slum-dwelling malnourished children and adults in Dhaka. METHODS: Serum samples were collected from three different cohorts: stunted (length-for-age Z-scores (LAZ) <-2) and at risk of stunting children (LAZ <-1 to -2) and malnourished adults (body mass index <18.5 kg/m2). Samples from all the participants were assessed for anti-tissue transglutaminase antibody (tTG-IgA) and total serum IgA by ELISA. Positive tTG-IgA and randomly selected low IgA values were reconfirmed using anti-tTG-IgG and gliadin IgG ELISA. CD was diagnosed when second screening tests were found positive and the participants were further investigated by small bowel biopsy. RESULTS: A total of 818 participants (240 stunted, 272 at risk of stunting children and 306 malnourished adults) were enrolled in the study. Overall, anti-tTG-IgA was positive in 5/818 (0.6%; 95% CI 0.25% to 1.46%). Of the five positive cases, anti-tTG-IgG and gliadin IgG were found positive in only one participant. Duodenal biopsy of positive participant revealed characteristic lesions of CD. Randomly selected low IgA values were found negative in tTG-IgG and gliadin IgG for all the participants. No participant was found total IgA deficient. CONCLUSION: The incidence of coeliac autoimmunity is low in malnourished slum dwellers regardless of age in Bangladesh. It is important to investigate the nationwide prevalence to reveal the definite picture.

Evaluation of Real-time PCR for Diagnosis of Post-Kala-azar Dermal Leishmaniasis in Endemic Foci of Bangladesh.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) is a sequel to visceral leishmaniasis (VL), which is found in VL-endemic countries including Bangladesh. Because of these enigmatic cases, the success of the National Kala-azar Elimination Program is under threat. To date, diagnostic methods for PKDL cases in endemic regions have been limited to clinical examination and rK39 test or microscopy, and a suitable and accurate alternative method is needed. In this study, we investigated the application of real-time polymerase chain reaction (PCR) as a potential method for diagnosis of PKDL in comparison with microscopy. METHODS: Ninety-one suspected macular PKDL cases from Mymensingh district, Bangladesh, were enrolled in the study after diagnosis by clinical examination and an rK39 strip test. All of them responded after completion of the treatment with miltefosine. During enrollment, a skin biopsy was done for each patient, and both microscopy and real-time PCR were performed for detection and quantification of Leishmania donovan body (LDB) and LD DNA, respectively. RESULTS: Real-time PCR detected 83 cases among all suspected PKDL patients, with an encouraging sensitivity of 91.2% (83.4%-96.1%), whereas microscopy showed 50.6% (39.9%-61.2%) sensitivity. Among all suspected PKDL cases, 42 cases were positive in both microscopy and qPCR, whereas 41 cases were detected as positive through qPCR only. CONCLUSIONS: This study provides evidence that real-time PCR is a promising tool for diagnosis of PKDL in endemic regions. In addition to diagnosis, the quantitative ability of this method could be further exploited for after-treatment prognosis and cure assessment of PKDL cases.

Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study.

BACKGROUND: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING: Bill & Melinda Gates Foundation.

Examining the relationship between blood lead level and stunting, wasting and underweight- A cross-sectional study of children under 2 years-of-age in a Bangladeshi slum.

Elevated blood lead level (BLL) is known to cause cardiac, immune, and cognitive damage but had not been thoroughly studied in relation to stunting among children under two years of age. We primarily aimed to assess the relationship between elevated BLL, the accumulation of concerned amount of the metal lead in blood and stunting and secondarily-wasting and underweight amongst Bangladeshi children less than two years of age. For this cross-sectional study, BLL measurements, anthropometric data, and socioeconomic indicator information were collected and analyzed for 729 children under two years of age upon enrollment in the MAL-ED study conducted in a Bangladeshi slum area. Univariate, bivariate and multivariate analyses were carried out to observe the proportion and mean and contribution of elevated BLL and other relevant variables in explaining the occurrence of stunting. Of the enrolled subjects, 39.0% were stunted [length-for-age z score (LAZ<-2)], 50.3% were male, and 86.6% had an elevated BLL (≥5µg/dL). Mean BLL of stunted children was 8.47 ± 3·37 µg/dL and 8.10 ± 3·80 µg/dL for non-stunted children. Proportion of children with elevated BLL was not significantly different between the stunted and non-stunted groups (p>0.05). When adjusted for other variables, elevated BLL was found to be a significant predictor of stunting and underweight (p<0.05) but not wasting (p>0.05). Elevated BLL (p<0·01), child's gender and weight (p<0·001), maternal body mass index (BMI) (p<0.05) and severe household food insecurity (p<0·05) were all significantly associated with stunting in the multivariate model. Increased odds of stunting was also observed for increased BLL. The findings suggest that chronic lead poisoning is significantly associated with high level of stunting among child slum dwellers in Bangladesh. These findings strengthen the argument for improved lead reduction efforts in Bangladesh, where lead poisoning and stunting are both highly prevalent.

Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells.

The stimulation of angiotensin II (Ang II), the effector peptide of renin-angiotensin system, has been reported to increase the expression of vascular endothelial growth factor (VEGF) through the activation of the Ang II type 1 receptor (AT1R). In this study, we investigated whether hyperglycemia (HG, 33 mM glucose) in ARPE-19 cells could promote the expression of VEGF independently of Ang II through prorenin receptor (PRR), via an NADPH oxidase (Nox)-dependent mechanism. ARPE-19 cells were treated with the angiotensin converting enzyme (ACE) inhibitor perindopril to block the synthesis of Ang II. Treatment with HG induced VEGF expression in ARPE-19 cells, which was attenuated by pretreatment with the inhibitors of Nox, but not those of nitric oxide synthase, xanthine oxidase and mitochondrial O2 synthesis. In addition, Nox-derived [Formula: see text] and H2O2 signaling in the regulation of VEGF was determined by using both polyethylene glycol (PEG)-catalase (CAT) and PEG-superoxide dismutase (SOD). We demonstrated that small interfering RNA (siRNA)-mediated knockdown of PRR, Nox2 and Nox4 significantly reduced the HG-induced stimulation of VEGF. On the other hand, Nox4 overexpression significantly potentiated PRR-induced stimulation of VEGF under hyperglycemia in ARPE-19 cells. Furthermore, Nox4 was shown to be associated with enhanced activities of ERK1/2 and NF-κB (p65), indicating their involvement in PRR-induced activation of VEGF under HG in ARPE-19 cells. Our results support the hypothesis that Nox4-derived reactive oxygen species (ROS) signaling is implicated in the hyperglycemia-induced increase of VEGF expression through PRR in ARPE-19 cells. However, further work is needed to evaluate the role of PRR and Nox-s in HG-induced stimulation of VEGF in vivo.

Bangladesh Environmental Enteric Dysfunction (BEED) study: protocol for a community-based intervention study to validate non-invasive biomarkers of environmental enteric dysfunction.

INTRODUCTION: Environmental enteric dysfunction (EED) is a subacute inflammatory condition of the small intestinal mucosa with unclear aetiology that may account for more than 40% of all cases of stunting. Currently, there are no universally accepted protocols for the diagnosis, treatment and ultimately prevention of EED. The Bangladesh Environmental Enteric Dysfunction (BEED) study is designed to validate non-invasive biomarkers of EED with small intestinal biopsy, better understand disease pathogenesis and identify potential therapeutic targets for interventions designed to control EED and stunting. METHODS AND ANALYSIS: The BEED study is a community-based intervention where participants are recruited from three cohorts: stunted children aged 12-18 months (length for age Z-score (LAZ) <-2), at risk of stunting children aged 12-18 months (LAZ <-1 to -2) and malnourished adults aged 18-45 years (body mass index <18.5 kg/m2). After screening, participants eligible for study provide faecal, urine and plasma specimens to quantify the levels of candidate EED biomarkers before and after receiving a nutritional intervention. Participants who fail to respond to nutritional therapy are considered as the candidates for upper gastrointestinal endoscopy with biopsy. Histopathological scoring for EED will be performed on biopsies obtained from several locations within the proximal small intestine. Candidate EED biomarkers will be correlated with nutritional status, the results of histochemical and immunohistochemical analyses of epithelial and lamina propria cell populations, plus assessments of microbial community structure. ETHICS AND DISSEMINATION: Ethics approval was obtained in all participating institutes. Results of this study will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov ID: NCT02812615. Registered on 21 June 2016.

The MicroRNA-21 signaling pathway is involved in prorenin receptor (PRR) -induced VEGF expression in ARPE-19 cells under a hyperglycemic condition.

PURPOSE: MicroRNAs (miRNAs/miRs) are involved in a large number of biological functions and diseases, such as cancer, cardiovascular diseases, and diabetes. MiR-21 has been reported to target Sprouty homolog 1 (SPRY1), SMAD7, and PTEN. In this study, we examined the underlying role of miR-21 in the regulation of prorenin receptor (PRR)-mediated induction of vascular endothelial growth factor (VEGF) expression via targeting SMAD7, SPRY1, and PTEN in a hyperglycemic condition. METHODS: PRR-mediated induction of VEGF under a hyperglycemic condition (high glucose, 33mM) was studied by treating ARPE-19 cells with perindopril (10 µmol/l), which inhibits angiotensin II-mediated signaling. ARPE-19 cells exposed to normal glucose (NG, 5.5 mM) were considered as the control. To examine the role of miR-21 in the regulation of SPRY1, SMAD7, PTEN, and VEGF, ARPE-19 cells cultured in NG or high glucose were transfected with scramble negative control (Scr), a miR-21 mimic, or a miR-21 antagomir. To investigate the role of PRR and the small GTP-binding protein RAC1 in the regulation of miR-21, the expression of PRR and RAC1 was silenced by transfecting ARPE-19 cells with their corresponding siRNAs. RESULTS: Compared with the NG control, high glucose significantly induced the expression of PRR, VEGF, VEGFR2, and miR-21 but significantly suppressed the expression of SPRY1, SMAD7, and PTEN at the transcript and protein levels. In contrast, silencing the expression of PRR significantly abolished the high glucose-induced expression of VEGF, VEGFR2, and miR-21. Knockdown of RAC1 significantly attenuated the high glucose-induced expression of LOX, CTGF, and miR-21, suggesting that PRR and RAC1 are involved in the CTGF/LOX-mediated regulation of miR-21. Furthermore, high glucose dramatically increased the levels of pERK (p44), hypoxia-inducible factor (HIF-1α), and VEGF. However, this effect was antagonized by the miR-21 antagomir, indicative of the involvement of high glucose-induced miR-21 in the regulation of VEGF through ERK signaling. CONCLUSIONS: Our findings, for the first time, showed that the pleiotropic action of miR-21 induced the expression of pERK, HIF-1α, and VEGF in the high glucose condition by simultaneously targeting SPRY1, SMAD7, and PTEN in ARPE-19 cells. Therefore, miR-21 may serve as a potential therapeutic target for diabetes-induced retinal pathology.

Amoebic liver abscess in northern Sri Lanka: first report of immunological and molecular confirmation of aetiology.

BACKGROUND: Since 1985, amoebic liver abscess (ALA) has been a public health problem in northern Sri Lanka. Clinicians arrive at a diagnosis based on clinical and ultrasonographic findings, which cannot differentiate pyogenic liver abscess (PLA) from ALA. As the treatment and outcome of the ALA and PLA differs, determining the etiological agent is crucial. METHODS: All clinically diagnosed ALA patients admitted to the Teaching Hospital (TH) in Jaffna during the study period were included and the clinical features, haematological parameters, and ultrasound scanning findings were obtained. Aspirated pus, blood, and faecal samples from patients were also collected. Pus and faeces were examined microscopically for amoebae. Pus was cultured in Robinson's medium for amoebae, and MacConkey and blood agar for bacterial growth. ELISA kits were used for immunodiagnosis of Entamoeba histolytica infection. DNA was extracted from selected pus samples and amplified using nested PCR and the purified product was sequenced. RESULTS: From July 2012 to July 2015, 346 of 367 clinically diagnosed ALA patients admitted to Jaffna Teaching Hospital were enrolled in this study. Almost all patients (98.6%) were males with a history of heavy alcohol consumption (100%). The main clinical features were fever (100%), right hypochodric pain (100%), tender hepatomegaly (90%) and intercostal tenderness (60%). Most patients had leukocytosis (86.7%), elevated ESR (85.8%) and elevated alkaline phosphatase (72.3%). Most of the abscesses were in the right lobe (85.3%) and solitary (76.3%) in nature. Among the 221 (63.87%) drained abscesses, 93.2% were chocolate brown in colour with the mean volume of 41.22 ± 1.16 ml. Only four pus samples (2%) were positive for amoeba by culture and the rest of the pus and faecal samples were negative microscopically and by culture. Furthermore, all pus samples were negative for bacterial growth. Antibody against E. histolytica (99.7%) and the E. histolytica antigen were detected in the pus samples (100%). Moreover, PCR and sequencing confirmed these results. CONCLUSION: To our knowledge, this is the first report from Sri Lanka that provides immunological and molecular confirmation that Entamoeba histolytica is a common cause of liver abscesses in the region.