Disease surveillance methods used in the 8-site MAL-ED cohort study.

Describing the early life associations between infectious disease episodes and growth, cognitive development, and vaccine response in the first 2 years of life is one of the primary goals of the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. To collect high-resolution data during a critical early period of development, field staff visit each study participant at their house twice weekly from birth to 2 years of age to collect daily reported illness and treatment data from caregivers. Detailed infectious disease histories will not only allow us to relate the overall burden of infectious disease with the primary outcomes of the study, but will also allow us to describe the ages at which infectious diseases have the greatest effect on child health. In addition, twice-weekly visits allow for sample collection when diarrhea episodes are identified. This article describes the methods used to collect illness and treatment history data and discusses the a priori definitions of diarrhea and acute lower respiratory illness episodes.

Microbiologic methods utilized in the MAL-ED cohort study.

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.

Geography, population, demography, socioeconomic, anthropometry, and environmental status in the MAL-ED cohort and case-control study Sites in Fortaleza, Ceará, Brazil.

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort in the study's Fortaleza, Brazil, catchment area has a population of approximately 82 300 inhabitants. Most of the households (87%) have access to clean water, 98% have electricity, and 69% have access to improved toilet/sanitation. Most childbirths occur at the hospital, and the under-5 mortality rate is 20 per 1000 live births. The MAL-ED case-control study population, identified through the Institute for the Promotion of Nutrition and Human Development (IPREDE), serves 600 000 inhabitants from areas totaling about 42% of the city of Fortaleza. IPREDE receives referrals from throughout the state of Ceará for infant nutrition, and provides services including teaching activities and the training of graduate students and health professionals, while supporting research projects on child nutrition and health. In this article, we describe the geographic, demographic, socioeconomic, anthropometric, and environmental status of the MAL-ED cohort and case-control study populations in Fortaleza, Brazil.

High anti-Cryptosporidium parvum IgG seroprevalence in HIV-infected adults in Limpopo, South Africa.

A seroepidemiological study was performed to determine the seroprevalence of Cryptosporidium in human immunodeficiency virus (HIV)-infected adults and local university students in the Limpopo Province, South Africa. Using a custom anti-C. parvum immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), the seroprevalence of Cryptosporidium was found to be significantly higher (75.3%; 146 of 193) in HIV-infected individuals compared with student volunteers (32.8%; 19 of 58) (P < 0.001). A more recent diagnosis of HIV was associated with anti-C. parvum IgG seropositivity, as was lower weight among HIV-infected women. This is the first seroepidemiologic study of Cryptosporidium in rural South Africa, and it shows high endemicity among the HIV-infected population. In addition to raising the possibility of significant Cryptosporidium-related morbidities, this finding reveals that in Limpopo and perhaps in other low-income, rural populations, interrupting waterborne pathogen transmission will require strategies effective against environmentally hardy parasites such as Cryptosporidium.

Prolonged episodes of acute diarrhea reduce growth and increase risk of persistent diarrhea in children.

BACKGROUND & AIMS: Prolonged episodes of acute diarrhea (ProD; duration 7-13 days) or persistent diarrhea (PD; duration ≥14 days) are important causes of undernutrition, yet the epidemiology and nutritional impact of ProD are poorly understood. METHODS: We conducted a 10-year cohort study of 414 children from a Brazilian shantytown who were followed from birth; data were collected on diarrhea, enteric pathogens, and anthropometry. RESULTS: During 1276 child-years of observation, we recorded 3257 diarrheal episodes. ProD was twice as common as PD (12% and 5% of episodes, respectively); ProD and PD together accounted for 50% of all days with diarrhea. ProD was more common in infants whose mothers had not completed primary school (relative risk [RR], 2.1; 95% confidence interval: 1.02-2.78). Early weaning was associated with earlier onset of ProD (Spearman ρ = 0.309; P = .005). Infants with ProD were twice as likely to develop PD in later childhood (log rank, P = .002) compared with infants with only acute diarrhea (AD; duration <7 days), even after controlling for confounders. Children's growth was more severely stunted before their first episode of ProD, compared with AD (mean height-for-age Z score (HAZ) -0.81 vs -0.51, respectively, P < .05, unpaired t test). Following ProD, HAZ (ΔHAZ = -0.232) and weight-for-age (ΔWAZ = -0.26) significantly decreased (P < .005 in paired t tests). ProD was associated with Cryptosporidium and Shigella infections. CONCLUSIONS: ProD accounts for significant morbidity and identifies children at risk of a vicious cycle of diarrhea and malnutrition. Further studies are needed to address the recognition and control of ProD and its consequences in resource-limited settings and assess its role in PD pathogenesis.

Effects of vitamin A supplementation on intestinal barrier function, growth, total parasitic, and specific Giardia spp infections in Brazilian children: a prospective randomized, double-blind, placebo-controlled trial.

BACKGROUND: This study evaluates the effects of retinol on intestinal barrier function, growth, total parasites, and Giardia spp infections in children in northeastern Brazil. SUBJECTS AND METHODS: The study was a double-blind, randomized placebo-controlled trial (http://clinicaltrials.gov; register no. #NCT00133406) involving 79 children who received vitamin A 100,000-200,000 IU (n = 39) or placebo (n = 40) at enrollment, 4, and 8 months and were followed for 36 months. Intestinal barrier function was evaluated using the lactulose:mannitol ratio test. Stool lactoferrin was used as a marker for intestinal inflammation. RESULTS: The groups were similar with regard to age, sex, nutritional parameters (z scores), serum retinol concentrations, proportion of lactoferrin-positive stool samples, and intestinal barrier function. The lactulose:mannitol ratio did not change during the same time of follow-up (P > 0.05). The proportion of lactoferrin-positive samples evaluated at 1 month did not change between groups (P > 0.05). Total intestinal parasitic, specifically new, infections were significantly lower in the vitamin A treatment compared with control group; these were accounted for entirely by significantly fewer new Giardia infections in the vitamin A treatment group. The cumulative z scores for weight-for-length or height, length or height-for-age z scores, and weight-for-age did not change significantly with vitamin A intervention for 36 months of follow-up. CONCLUSIONS: These data showed that total parasitic infection and Giardia spp infections were significantly lower in the vitamin A treatment group when compared with the placebo group, suggesting that vitamin A improves the host's defenses against Giardia infections.

Faecal contamination of drinking water in a Brazilian shanty town: importance of household storage and new human faecal marker testing.

Worldwide, contaminated drinking water poses a major health threat, particularly to child development. Diarrhoea represents a large part of the water-related disease burden and enteric infections have been linked to nutritional and growth shortfalls as well as long-term physical and cognitive impairment in children. Previous studies detailed the frequency of infection and the consequences for child health in a shanty town in north-east Brazil. To determine the frequency of contaminated water, we measured faecal contamination in primary drinking water samples from 231 randomly selected households. Risk for contamination was compared across source and storage types. Nearly a third of the study households (70/231: 30.3%) had contaminated drinking water; the source with the highest frequency of contamination was well water (23/24: 95.8%). For tap water, the type of storage had a significant effect on the susceptibility to contamination (chi(2) = 12.090; p = 0.007). The observed pattern of contamination demonstrated the relative potential contributions of both source and storage. With evidence that supports the inclusion of source and storage in water quality surveys, this study, like others, suggests that contaminated drinking water in storage vessels may be an important factor for the documented diarrhoea disease burden in the Brazilian shanty town.

Detection of epithelial-cell injury, and quantification of infection, in the HCT-8 organoid model of cryptosporidiosis.

BACKGROUND: Intestinal cells grown in microgravity produce a three-dimensional tissue assembly, or "organoid," similar to the human intestinal mucosa, making it an ideal model for enteric infections such as cryptosporidiosis. METHODS: HCT-8 cells were grown in a reduced-gravity, low-shear, rotating-wall vessel (RWV) and were infected with Cryptosporidium parvum oocysts. Routine and electron microscopy (EM), immunolabeling with fluorescein-labeled Vicia villosa lectin and phycoerythrin-labeled monoclonal antibody to a 15-kD surface-membrane protein, and quantitative polymerase chain reaction (qPCR) using probes for 18s rRNA of C. parvum and HCT-8 cells were performed. RESULTS: The RWV allowed development of columnar epithelium-like structures. Higher magnification revealed well-developed brush borders at the apical side of the tissue. Incubation with C. parvum resulted in patchy disruption of the epithelium and, at the surface of several epithelial cells, in localized infection with the organism. EM revealed irregular stunting of microvilli, foci of indistinct tight junctions, and areas of loose paracellular spaces. qPCR showed a 1.85-log (i.e., 70-fold) progression of infection from 6 h to 48 h of incubation. CONCLUSION: The HCT-8 organoid displayed morphologic changes indicative of successful and quantifiable infection with C. parvum. The HCT-8 organoid-culture system may have application in interventional in vitro studies of cryptosporidiosis.

Angiotensin II subtype 1 receptor blockade inhibits Clostridium difficile toxin A-induced intestinal secretion in a rabbit model.

Angiotensin II (ANG II) has been described in the regulation of intestinal secretion and absorption via angiotensin subtype 1 (AT(1)) and AT(2) receptors, respectively, in rats. We investigated the role that ANG II plays in the rabbit ileal-loop model of Clostridium difficile infection. Expression of AT(1), the more abundant ANG II receptor, was demonstrated in ileal loops, and an AT(1) receptor blocker, losartan, inhibited hypersecretion induced by C. difficile toxin A (mean volume : length ratio, 0.27+/-0.06 vs. 0.60+/-0.06 mL/cm in controls). Losartan also decreased production of ANG II in the ileum (0.48+/-0.06 vs. 0.87+/-0.12 pg/mg in controls), raising the possibility that ANG II may participate in a positive feedback loop involving the hypersecretory response. Our findings suggest that ANG II plays a significant role in the pathogenesis of C. difficile toxin-induced diarrhea.

APOE4 protects the cognitive development in children with heavy diarrhea burdens in Northeast Brazil.

Polymorphisms in the apolipoprotein E (APOE) have constituted the major rationale to identify potential risk groups for developing late-onset Alzheimer's disease and help to predict recovery of cognitive function after brain injury. However, the APOE impact on cognitive development in children living in poor areas of the developing world, where we have discovered profound significant associations of early childhood diarrhea (at 0-2 y) with lasting impairments of growth, cognition, and school performance, is not known. Therefore, we conducted APOE genotyping in 72 Brazilian shantytown children under active surveillance since birth, using purified DNA extracted from buccal cell samples. We found a high frequency of APOE4 alleles (18% versus 9-11% expected) in children with lower diarrhea burdens. When we examined the children who experienced the heavier diarrhea burdens (greater than or equal to the median of seven illnesses in the first 2 y of life), those with APOE4 did significantly better in the coding subtest (p=0.01) when compared with APOE4-negative children with similar diarrhea burdens. Positive correlations between the APOE4 occurrence and coding scores remained, even after adjusting for family income, maternal education, and breast-feeding. Moreover, the APOE4-positive group, under heavy burdens of diarrhea, had preserved semantic fluency and the mean difference in fluency scores, p=0.025, a standardized coefficient for disproportional verbal fluency impairment. Our findings show that APOE4 is relatively common in favela children and suggest a protective role of the APOE4 allele in children with a history of heavy burdens of diarrhea in their first 2 y of life.

Laboratory diagnosis and susceptibility profile of Helicobacter pylori infection in the Philippines.

BACKGROUND: Helicobacter pylori diagnosis and susceptibility profile directs the applicability of recommended treatment regimens in our setting. To our knowledge, there is no published data on the culture and local susceptibility pattern of Helicobacter pylori in the Philippines. METHODS: 52 dyspeptic adult patients undergoing endoscopy from the Outpatient Gastroenterology clinic of the University of the Philippines-Philippine General Hospital underwent multiple gastric biopsy and specimens were submitted for gram stain, culture, antimicrobial sensitivity testing, rapid urease test and histology. Antimicrobial susceptibility testing was done by Epsilometer testing (Etest) method against metronidazole, clarithromycin, amoxicillin, and tetracycline. RESULTS: Sixty percent (60%) of the study population was positive for H. pylori infection (mean age of 44 years +/- 13), 70% were males. H. pylori culture showed a sensitivity of 45% (95% CI [29.5-62.1]), specificity of 98% (95%CI [81.5-100%]), positive likelihood ratio of 19.93 (95% CI [1.254-317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406-0.772]). All H. pylori strains isolated were sensitive to metronidazole, clarithromycin, amoxicillin and tetracycline. CONCLUSION: Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases.

Interleukin-8, tumor necrosis factor-alpha, and lactoferrin in immunocompetent hosts with experimental and Brazilian children with acquired cryptosporidiosis.

To examine the importance of intestinal inflammation in the diagnosis and pathogenesis of human cryptosporidiosis, stools of healthy adult volunteers before and after experimental infection were tested for fecal lactoferrin, interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha). Stool samples of Brazilian children with well-defined Cryptosporidium infection, with or without diarrhea, were also tested for IL-8 and TNF-alpha. Only one of the 14 volunteers challenged with Cryptosporidium had increased fecal lactoferrin. However, of 17 stool specimens from children with only Cryptosporidium infection from a previous study, 12 had mild to moderately elevated lactoferrin despite negative work-up for inflammatory enteritides. One of 10 adult volunteers who developed diarrhea with experimental cryptosporidiosis and three of 11 children with cryptosporidiosis and diarrhea had detectable fecal IL-8. The level of TNF-alpha was increased only in one of 14 volunteers and in none of the children. Although considered relatively non-inflammatory. cryptosporidiosis is often associated with mild inflammation, especially in children in an endemic area.

Age-specific Helicobacter pylori seropositivity rates of children in an impoverished urban area of northeast Brazil.

We determined age-specific Helicobacter pylori seropositivity rates of 166 children and 39 mothers in an urban shantytown in northeast Brazil. Seropositivity rates increased from 23.1% at 0 to 11 months of age to only 39.3% by 96 to 131 months of age and were 82.1% at maturity. We observed no correlation between the seropositivity of the mothers and the seropositivity of their children.

Association of vitamin A and zinc status with altered intestinal permeability: analyses of cohort data from northeastern Brazil.

To examine the association of intestinal barrier function with vitamin A deficiency and whether supplementation of micronutrients improves intestinal function and/or linear growth, height-for-age z-score (HAZ), concentrations of serum retinol and zinc, and intestinal permeability were determined in a cross-sectional sample of 75 children in northeastern Brazil. Effects of vitamin A and supplementation of zinc on intestinal permeability and growth were also determined comparing results before and after treatment in 20 children and age-matched controls. Lactulose:mannitol (L/M) permeability ratios inversely correlated with serum retinol concentrations (r = -0.55, p < 0.0005). Increased L/M permeability ratios with reduced concentrations of serum retinol were predominantly attributable to lower absorption of mannitol (r = 0.28, p = 0.02). L/M permeability ratios (p = 0.001) and HAZ scores (p = 0.007) improved with supplementation. It is concluded that impaired intestinal barrier function and linear growth shortfalls improve following supplementation of vitamin A and zinc in this setting.