The epidemiological and clinical characteristics and nutritional development of infants with acute diarrhoea, in north-eastern Brazil.

In order to assess the epidemiological and clinical characteristics and changing nutritional status of infants suffering from acute diarrhoea, 103 infants with such diarrhoea and the same number of age-matched controls were investigated at the Universidade Federal do Rio Grande do Norte's Paediatric Hospital, in north-eastern Brazil. Each child with diarrhoea was given oral rehydration or, in the severe cases, intravenous rehydration. Each subject was checked for enteropathogens and his or her weight, height and weight-for-height, weight-for-age and height-for-age Z-scores were evaluated immediately after any clinical dehydration had been corrected and 30 days later. In the infants aged <6 months, a diet that included foods other than breast milk (odds ratio=9.41), including one in which breast milk was supplemented with other foods (odds ratio=4.69), was found to be statistically associated with diarrhoea. The enteropathogens found most commonly in the children with diarrhoea were rotavirus (36.9%), enteropathogenic Escherichia coli (11.6%) and Shigella (11.6%). Just four (5.2%) of the 77 cases with adequate follow-up showed persistent diarrhoea. At presentation or as soon as any clinical dehydration had been corrected, the infants with diarrhoea had significantly lower weights and weight-for-height and weight-for-age Z-scores than the controls. Thirty days later, however, the weight-for-height and weight-for-age Z-scores of the cases had increased significantly, to the point when they were not significantly different from the baseline values for the controls. The negative consequences of diarrhoea on weight-for-height and weight-for-age Z-scores and the recovery of these parameters after 30 days with rehydration reflect the acute but reversible influence of diarrhoea on infant nutritional status.

A randomized double-blind clinical trial of the effect of non-absorbable oral polymyxin on infants with severe infectious diarrhea.

The present study evaluated the effect of non-absorbable oral polymyxin on the duodenal microflora and clinical outcome of infants with severe infectious diarrhea. Polymyxin was chosen because classic enteropathogenic Escherichia coli was more sensitive to this antibiotic. Twenty-five infants were randomly assigned to a 7-day treatment with oral polymyxin (2.5 mg/kg in 4 daily doses) or placebo. Duodenal and stool cultures were performed before and after the treatment. Five patients were excluded during the study because of introduction of parental antibiotic therapy due to clinical sepsis (N = 3) or rapid clinical improvement (N = 2). In the polymyxin group, small bowel bacterial overgrowth occurred in 61.5% of the cases (8/13) before treatment and in 76.9% (10/13) after treatment. In the placebo group these values were 71.4% (5/7) and 57.1% (4/7), respectively. By the 7th day, clinical cure was observed in 84.6% of the cases (11/13) in the polymyxin group and in 71.4% (5/7) in the placebo group (P = 0.587). Considering all 25 patients included in the study, clinical cure occurred on the 7th day in 12/14 cases (85.7%) in the polymyxin group and 6/11 cases (54.5%) in the placebo group (P = 0.102). Clinical sepsis occurred in 3/11 (27.3%) of the patients in the placebo group and in none (0/14) in the polymyxin group (P = 0.071). Oral polymyxin was not effective in reducing bacterial overgrowth or in improving the clinical outcome of infants hospitalized with severe infectious diarrhea. Taking into account the small sample size, the rate of cure on the 7th day and the rate of clinical sepsis, further studies with greater number of patients are necessary to evaluate these questions.

A randomized double-blind clinical trial of the effect of non-absorbable oral polymyxin on infants with severe infectious diarrhea

The present study evaluated the effect of non-absorbable oral polymyxin on the duodenal microflora and clinical outcome of infants with severe infectious diarrhea. Polymyxin was chosen because classic enteropathogenic Escherichia coli was more sensitive to this antibiotic. Twenty-five infants were randomly assigned to a 7-day treatment with oral polymyxin (2.5 mg/kg in 4 daily doses) or placebo. Duodenal and stool cultures were performed before and after the treatment. Five patients were excluded during the study because of introduction of parental antibiotic therapy due to clinical sepsis (N = 3) or rapid clinical improvement (N = 2). In the polymyxin group, small bowel bacterial overgrowth occurred in 61.5 percent of the cases (8/13) before treatment and in 76.9 percent (10/13) after treatment. In the placebo group these values were 71.4 percent (5/7) and 57.1 percent (4/7), respectively. By the 7th day, clinical cure was observed in 84.6 percent of the cases (11/13) in the polymyxin group and in 71.4 percent (5/7) in the placebo group (P = 0.587). Considering all 25 patients included in the study, clinical cure occurred on the 7th day in 12/14 cases (85.7 percent) in the polymyxin group and 6/11 cases (54.5 percent) in the placebo group (P = 0.102). Clinical sepsis occurred in 3/11 (27.3 percent) of the patients in the placebo group and in none (0/14) in the polymyxin group (P = 0.071). Oral polymyxin was not effective in reducing bacterial overgrowth or in improving the clinical outcome of infants hospitalized with severe infectious diarrhea. Taking into account the small sample size, the rate of cure on the 7th day and the rate of clinical sepsis, further studies with greater number of patients are necessary to evaluate these questions.

Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp.

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.

Assessment of gliadin in supposedly gluten-free foods prepared and purchased by celiac patients.

BACKGROUND: The present study was designed to evaluate the presence of gliadin in homemade foods prepared by patients with celiac disease and/or their relatives, as well as in processed products consumed by such patients in São Paulo, Brazil, by enzyme immunoassay (EIA) and Western blot (WB) analysis. METHODS: One hundred ninety samples were analyzed: 108 homemade foods prepared in homes of patients with celiac disease, 81 processed products, and 1 positive control of homemade food. All samples were analyzed by EIA based on monoclonal antibodies to heat stable omega-gliadins and related prolamins from wheat, rye, and barley. Samples were also analyzed using the WB technique. RESULTS: Only one (0.9%) of 108 homemade foods contained detectable amounts of gliadin, as determined by EIA. Twelve of 81 processed products contained gliadin by EIA, as follows: 5 of 61 without gluten listed in the ingredients, 2 of 11 malt extracts, 1 of 2 wheat starches, 1 of 2 types of beer, and all 3 positive control products. Gliadin content of these products was between 4 and 10 mg of gliadin/100 g of product, except for the wheat starch sample (28 mg of gliadin/100 g) and all 3 samples with gluten (>4000 mg of gliadin/100 g). The positive control of homemade food contained 152 mg of gliadin/100 g. One hundred three of 190 samples were analyzed by WB, and 21 of these were gliadin positive. A comparison of results obtained by EIA and WB showed no statistical differences between the methods. CONCLUSIONS: The greater part of the foods prepared in homes of patients with celiac disease and most processed products supposed to be gluten-free did not contain gliadin. Therefore, celiac patients adequately prepare gluten-free homemade food and have the expertise to purchase processed gluten-free food in São Paulo, Brazil.

[Assessment of gliadin in pharmaceutical products - important information to the orientation of celiac disease patients]. / Pesquisa de gliadina em medicamentos--informação relevante para a orientação de pacientes com doença celíaca.

BACKGROUND: Some drugs might contain gliadin which can be dangerous for celiac disease patients. OBJECTIVE: Detect gliadin in pharmaceutical products commonly used in Brazil. METHODS: We analyzed 78 pharmaceutical products selected aleatory from a list of 180 products most frequently sold at Brazilian community pharmacies. The analyzed samples were analgesics (n = 9), anthelmintics (n = 3), antacids (n = 8), antibiotics (n = 13), anticholesteremics (n = 1), anticonvulsants (n = 2), antidepressants (n = 2), antiemetics (n = 3), antihypertensives (n = 3), antihistaminics (n = 3), anti-inflammatories (n = 7), antipyretics (n = 2), bronchodilators (n = 1), laxatives (n = 1), oral contraceptives (n = 5) and vitamins (n = 10). The samples were analyzed by enzyme immunoassay based on monoclonal antibodies omega-gliadins, the elected technique according to the Codex Alimentarius Commission WHO/FAO. All samples were analyzed in duplicate. The sensitivity of this test is 4 mg of gliadin/100 g of product. RESULTS: Only one (1.3%) out of 78 pharmaceutical products contained detectable amounts of gliadin (5.5 mg/100 g). The active ingredient of this drug is ranitidine. According to the Codex Alimentarius Commission WHO/FAO the intake of 10 mg of gliadin/day should not be exceeded by celiac disease patients. Considering the amount of gliadin in each capsule of ranitidine, the ingested quantity would be lower than the maximum allowed for celiac patients. CONCLUSIONS: In this study gliadin was not detected in pharmaceutical products in harmful amount for celiac disease patients.

Studies of the small bowel surface by scanning electron microscopy in infants with persistent diarrhea.

We describe the ultrastructural abnormalities of the small bowel surface in 16 infants with persistent diarrhea. The age range of the patients was 2 to 10 months, mean 4.8 months. All patients had diarrhea lasting 14 or more days. Bacterial overgrowth of the colonic microflora in the jejunal secretion, at concentrations above 10(4) colonies/ml, was present in 11 (68.7%) patients. The stool culture was positive for an enteropathogenic agent in 8 (50.0%) patients: for EPEC O111 in 2, EPEC O119 in 1, EAEC in 1, and Shigella flexneri in 1; mixed infections due to EPEC O111 and EAEC in 1 patient, EPEC O119 and EAEC in 1 and EPEC O55, EPEC O111, EAEC and Shigella sonnei in 1. Morphological abnormalities in the small bowel mucosa were observed in all 16 patients, varying in intensity from moderate 9 (56.3%) to severe 7 (43.7%). The scanning electron microscopic study of small bowel biopsies from these subjects showed several surface abnormalities. At low magnification (100X) most of the villi showed mild to moderate stunting, but on several occasions there was subtotal villus atrophy. At higher magnification (7,500X) photomicrographs showed derangement of the enterocytes; on several occasions the cell borders were not clearly defined and very often microvilli were decreased in number and height; in some areas there was a total disappearance of the microvilli. In half of the patients a mucus-fibrinoid pseudomembrane was seen partially coating the enterocytes, a finding that provides additional information on the pathophysiology of persistent diarrhea.

Studies of the small bowel surface by scanning electron microscopy in infants with persistent diarrhea

We describe the ultrastructural abnormalities of the small bowel surface in 16 infants with persistent diarrhea. The age range of the patients was 2 to 10 months, mean 4.8 months. All patients had diarrhea lasting 14 or more days. Bacterial overgrowth of the colonic microflora in the jejunal secretion, at concentrations above 10(4) colonies/ml, was present in 11 (68.7 percent) patients. The stool culture was positive for an enteropathogenic agent in 8 (50.0 percent) patients: for EPEC O111 in 2, EPEC O119 in 1, EAEC in 1, and Shigella flexneri in 1; mixed infections due to EPEC O111 and EAEC in 1 patient, EPEC O119 and EAEC in 1 and EPEC O55, EPEC O111, EAEC and Shigella sonnei in 1. Morphological abnormalities in the small bowel mucosa were observed in all 16 patients, varying in intensity from moderate 9 (56.3 percent) to severe 7 (43.7 percent). The scanning electron microscopic study of small bowel biopsies from these subjects showed several surface abnormalities. At low magnification (100X) most of the villi showed mild to moderate stunting, but on several occasions there was subtotal villus atrophy. At higher magnification (7,500X) photomicrographs showed derangement of the enterocytes; on several occasions the cell borders were not clearly defined and very often microvilli were decreased in number and height; in some areas there was a total disappearance of the microvilli. In half of the patients a mucus-fibrinoid pseudomembrane was seen partially coating the enterocytes, a finding that provides additional information on the pathophysiology of persistent diarrhea

The gut at war: the consequences of enteropathogenic Escherichia coli infection as a factor of diarrhea and malnutrition.

Diarrheal disease is still the most prevalent and important public health problem in developing countries, despite advances in knowledge, understanding, and management that have occurred over recent years. Diarrhea is the leading cause of death in children under 5 years of age. The impact of diarrheal diseases is more severe in the earliest periods of life, when taking into account both the numbers of episodes per year and hospital admission rates. This narrative review focuses on one of the major driving forces that attack the host, namely the enteropathogenic Escherichia coli (EPEC) and the consequences that generate malnutrition in an early phase of life. EPEC serotypes form dense microcolonies on the surface of tissue-culture cells in a pattern known as localized adherence (LA). When EPEC strains adhere to epithelial cells in vitro or in vivo they cause characteristic changes known as Attaching and Effacement (A/E) lesions. Surface abnormalities of the small intestinal mucosa shown by scanning electron microscopy in infants with persistent diarrhea, although non-specific, are intense enough to justify the severity of the clinical aspects displayed in a very young phase in life. Decrease in number and height of microvilli, blunting of borders of enterocytes, loss of the glycocalyx, shortening of villi and presence of a mucus pseudomembrane coating the mucosal surface were the abnormalities observed in the majority of patients. These ultrastructural derangements may be due to an association of the enteric enteropathogenic agent that triggers the diarrheic process and the onset of food intolerance responsible for perpetuation of diarrhea. An aggressive therapeutic approach based on appropriate nutritional support, especially the utilization of human milk and/or lactose-free protein hydrolyzate-based formulas and the adequate correction of the fecal losses, is required to allow complete recovery from the damage caused by this devastating enteropathogenic agent.

[Association of patterns of Escherichia coli adherence to HEp-2 cells with acute and persistent diarrhea]. / Associação de padrões de adesão de Escherichia coli às células HEp-2 com diarréia aguda e persistente.

In Brazil diarrhea is the cause of approximately 15% of death among infants. Enteropathogenic E coli is the most important bacterial agent causing acute diarrhea, which is defined as less than 14 days of duration. About 30% of these cases may evolve to persistent diarrhea, defined as lasting more than 14 days. In this work it was carried out a case-control study including 34 children under 2 years of age, and admitted to hospital facilities in São Paulo for rehydration therapy. Thirty-four age matched children hospitalized in the same facilities, and presenting no gastrointestinal symptoms were included as controls. Stool samples were analyzed for the presence of bacterial pathogens (diarrheagenic E coli, Shigella, Salmonella, Yersinia, and Campylobacter), protoparasytes, rotavirus, and enteric adenovirus. The E coli strains isolated were analyzed for their ability to adhere to HEp-2 cultured cells, in a 3 h adhesion assay. Search for homology with DNA probes for localized adherence (EAF, eaeA probes), AA (enteroagregative adherence) (AA probe), and diffuse adherence (F1845, AIDA-I probes) was carried out by the colony hybridization method. Twenty-four of the cases were acute diarrhea and 10 persistent diarrhea. Strains with localized adherence were associated with acute and persistent diarrhea. About 23.5% of E coli were associated with typical Enteropathogenic E coli strains (EAF+, eaeA+). Enteroaggregative E coli (EAggEC) (AA+) was isolated only from cases and in similar frequency for acute and persistent diarrhea. Diffusely adherent E coli (DAEC) which did not hybridize with the diffuse adherence probes were isolated among cases and controls. E coli eaeA+ with localized-like adherence was isolated from cases in a frequency three times higher than in controls, suggesting that it may really have a pathogenic potential.

A localized adherence-like pattern as a second pattern of adherence of classic enteropathogenic Escherichia coli to HEp-2 cells that is associated with infantile diarrhea.

Escherichia coli strains that cause nonbloody diarrhea in infants are known to present three distinct patterns of adherence to epithelial cells, namely, localized (LA), diffuse (DA), and aggregative (AA) adherence. Strains with LA (typical Enteropathogenic Escherichia coli [EPEC]) are well recognized as a cause of secretory diarrhea, but the role of strains with DA (DAEC) is controversial, and strains with AA (EAEC) have been more frequently related to persistent diarrhea whereas its relationship with acute diarrhea is not well defined. To determine the relationship of the different types of E. coli adherence patterns with acute diarrhea (lasting less than 14 days) and persistent diarrhea (lasting more than 14 days) in São Paulo, Brazil, we studied stool specimens from 40 infants under 1 year of age with diarrhea and 40 age-matched control infants without any gastrointestinal symptoms. Twenty-eight (35.0%) of eighty cases yielded adherent E. coli (HEp-2 cells). Strains with localized and aggregative adherence were associated with acute and persistent diarrhea. A total of 11.2% of the adherent strains were typical EPEC serotypes and hybridized with the enteroadherence factor probe; 5.0% were EAEC and hybridized with the EAEC probe. DAEC strains were isolated from 10.0% of patients and 7.5% of controls and did not hybridize with the two probes used (daaC and AIDA-I). Strains with a localized adherence-like pattern (atypical EPEC) were found significantly more frequently (P = 0.028) in cultures from children with diarrhea (17.5%) than in controls (2.5%).

Phenotypic and genetic features of Escherichia coli strains showing simultaneous expression of localized and diffuse adherence.

We have previously shown that some Escherichia coli strains isolated from children with diarrhea present the so-called 'localized and diffused adherence (LA/DA) pattern' in which both localized adherence (LA) and diffused adherence (DA) are expressed simultaneously. In the present study, we show that the LA adherence of these strains is genetically and phenotypically similar to that so far described for enteropathogenic E. coli (EPEC) as determined by DNA hybridization and electron microscopy. On the other hand, the DA is encoded by genes not homologous to the DAEC or AIDA-I DNA probes. In addition, the LA/DA strains are able to invade eukaryotic cells both in vitro and in vivo. In the rabbit ileal loop assay their invasion capacity goes beyond the enterocyte and reaches the muscularis mucosae as determined by transmission electron microscopy. These findings suggest that the LA/DA adherence pattern may be linked to a new E. coli virulence category which in the case of the strains studied may be associated to other virulence traits that enable them to more deeply invade the intestinal mucosa.

[Classic enteropathogenic Escherichia coli]. / Escherichia coli enteropatogênica clássica (EPEC).

Enteropathogenic Escherichia coli. (EPEC) are gram negative bacteria of great importance in the etiology of diarrhea in children under two years of age living in developing countries. It was first reported in 1946. Its pathogenic mechanism was unknown until 1970 when several papers were published in the attempt to find out their physiophatologic mechanism. These bacteria interfere on the small bowel resulting in parcial or sub total villous atrophy. This injury will revert when the infection subsides.

Culture supernatant of Shiga toxin-producing Escherichia coli strains provoke fluid accumulation in rabbit ileal loops.

Production of Shiga toxin (Stx) in Escherichia coli strains belonging to serogroups O26, O111, and O157 was evaluated in the rabbit ileal loop assay and results were compared to those using tissue culture assays and DNA hybridization with specific probes for Stx1 and Stx2. All 14 Shiga toxin-producing E. coli strains tested provoked fluid accumulation in the rabbit intestinal loop. Eleven strains hybridized with Stx1 probe, one strain with Stx2 and two strains with both probes. Filtered culture supernatants of all E. coli strains presented cytotoxic effects in both HeLa and Vero cells. In this study, we found a strong association between the production of Stx and its effect in an animal model. This is the first description of high-level Stx-producing E. coli O111ac isolated in Brazil.

Attaching and effacing enteropathogenic Escherichia coli O18ab invades epithelial cells and causes persistent diarrhea.

A case of persistent diarrhea following Escherichia coli O18ab gastroenteritis is reported. Electron microscopy of a biopsy of the small intestine showed effacement of the brush border, attachment of bacteria to the epithelial cells with pedestal formation, and bacteria within the enterocytes. The bacterial isolate was an enteropathogenic E. coli isolate which did not contain the adherence factor (EAF) but possessed the attaching-effacing eae gene, was able to invade HeLa cells in a gentamicin invasion assay, and also invaded rabbit intestinal cells. Results suggest that E. coli organisms of the O18ab serotype may cause diarrhea by an as yet unknown pathogenic mechanism, involving attaching to and effacing of enterocytes followed by invasion of the epithelial cells.

Clonal structure and virulence factors in strains of Escherichia coli of the classic serogroup O55.

Virulence properties and genetic variation as determined by multilocus enzyme electrophoresis were studied in 70 strains of Escherichia coli 055, a common serogroup of enteropathogenic E. coli (EPEC), a major cause of infantile diarrhea in developing countries. Nearly 40% of the strains were originally isolated in Brazil and represented serotypes 055:H6, 055:H7, and 055:H51 and nonmotile (055:H-) strains. The analysis of electrophoretic variants of 20 enzymes defined seven distinct electrophoretic types (ETs). ET 1 was represented by 41% of the strains, including strains which usually hybridized with DNA probes for the intimin gene (eaeA), the EPEC adherence plasmid (EAF), and the gene for the pilin subunit of the bundle-forming pilus (bfpA). The ET 1 strains were also typically serotype 055:H6, displayed localized adherence (LA) in tissue culture assays, and were positive in the fluorescent-actin staining test for intimate cell adherence. These same characteristics were observed in the closely related ETs 2 to 4, which clustered in the same branch as ET 1. No known virulence marker could be identified in ET 6. ET 5 included 23 strains, all of which carried the eaeA gene but otherwise displayed a striking array of distinct virulence traits. This ET was represented by 055:H7 strains with phenotypes as diverse as the simultaneous expression of LA and diffuse adherence and the ability to form a newly described adherence pattern, called LA-like adherence. The results suggest that ET 5 marks a special pathogenic clone with a propensity to acquire virulence factors which may facilitate the emergence of new pathogenic strains.

Plasmid coding for drug resistance and invasion of epithelial cells in enteropathogenic Escherichia coli 0111:H.

Enteropathogenic Escherichia coli (EPEC) can adhere to, invade and multiply in human epithelial cells. To define the elements required for bacterial invasion, we isolated from an 0111:H- EPEC a 6.6 kb plasmid that is capable of conferring to an avirulent, non-adherent E. coli K12 strain (DK1) the capacity to invade epithelial cells. With this system a dissociation was possible between bacterial invasion and adherence to epithelial cells. Bacteria containing this plasmid synthesise a protein of 32 kDa (pl 4.93) which seemed to be required for cell invasion. The results provide a new basis for strategies to prevent EPEC infections.