Bacteriology
Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

https://doi.org/10.1016/j.diagmicrobio.2008.03.017Get rights and content

Abstract

In a study conducted in João Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them.

Introduction

Diarrheal diseases remain as an important public health concern. Diarrheagenic Escherichia coli (DEC) is among the most important bacterial enteric pathogens, particularly in developing countries (Franzolin et al., 2005; Nataro and Kaper, 1998). E. coli is a clonal species, and the characterization of lipopolysaccharide O antigen and the flagellar H antigen on the cell surface allows the grouping of pathogenic clones within this species. DEC is classified into 6 categories: enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adhering E. coli (DAEC) (Nataro and Kaper, 1998). EPEC has been divided into typical EPEC (t-EPEC) and atypical EPEC (a-EPEC) (Kaper, 1996). The identification of DEC strains requires that these organisms be differentiated from nonpathogenic members of the microbiota. Serotyping is the gold standard method for the determination of H and O antigens; however, it is insufficient to identify a strain as diarrheagenic. Thus, other analyses are required for the identification of DEC as the HEp-2 adherence assay (Scaletsky et al., 2002a) and the detection of nucleic acid biomarkers.

EPEC has been a leading cause of childhood diarrhea in developing countries, but its frequency in industrialized areas has been decreasing (Nataro and Kaper, 1998, Trabulsi et al., 2002). Recent studies have also shown a clear declining in the incidence of EPEC strains in developing countries (Okeke et al., 2000, Rodrigues et al., 2004, Zamboni et al., 2004). The main mechanism of EPEC pathogenesis is a lesion called attaching and effacing (A/E), which is characterized by intimate adherence of the bacteria to the intestinal epithelium (Trabulsi et al., 2002). The eae gene, in the pathogenicity island, the locus of enterocyte effacement, and the bfpA gene, in the plasmid, and the EPEC adherence factor (EAF) have been used to classify this group of bacteria into typical and atypical strains (Kaper, 1996). The EPEC category harboring the EAF plasmid is classified as t-EPEC and strains of the A/E genotype, and those not harboring the EAF plasmid (bfpA) is classified as a-EPEC (Trabulsi et al., 2002). Many studies have assessed the epidemiologic significance of a-EPEC in gastrointestinal illness (Hellard et al., 2001, Nguyen et al., 2006, Robins-Browne et al., 2004). Various lines of evidences suggest that a-EPEC is closer to EHEC (Trabulsi et al., 2002). This pathotype causes diarrhea and hemolytic uremic syndrome, as well as harbors eae and Shiga toxin genes (stx1 and/or stx2) (Nataro and Kaper, 1998).

DEC bacteria, other than EPEC and EHEC, have been characterized by their ability to produce distinctive patterns of adherence in cultured epithelial cells (Nataro and Kaper, 1998). These adherence tests have demonstrated that certain adherent non-EPEC E. coli strains can also generate diarrhea (Nataro and Kaper, 1998). EAEC has emerged as a significant diarrheal pathogen in multiple population groups, especially in children with persistent diarrhea living in developing countries (Nguyen et al., 2006). EAEC was found to have an association with acute diarrhea in children in some reports (Cohen et al., 2005, Nguyen et al., 2006, Okeke and Nataro, 2001, Scaletsky et al., 2002a) but not in others (Almeida et al., 1998, Cravioto et al., 1991, Gioppo et al., 2000, Gomes et al., 1998, Keskimaki et al., 2001, Oberhelman et al., 1998, Scaletsky et al., 2002b, Souza et al., 2002). The aggregative adherence (AA) phenotype of EAEC is encoded in 55- to 65-MDa plasmids, collectively called pAA. A DNA fragment from a noncharacterized region of this plasmid works as a specific EAEC probe, which distinguishes EAEC strains from other E. coli bacteria (Harrington et al., 2006).

The epidemiologic significance of each E. coli category in childhood diarrhea varies depending on different geographic locations. Very few studies have investigated the etiology of acute childhood diarrhea in the northeast of Brazil (Fang et al., 1995, Guerrant et al., 1983, Scaletsky et al., 2002b). Many diarrhea studies were conducted predominantly in urban centers within the southeast of Brazil (Almeida et al., 1998, Borges et al., 1991, Gomes et al., 1989, Gomes et al., 1991, Gomes et al., 1998, Rodrigues et al., 2004, Scaletsky et al., 2002a, Souza et al., 2002).

The Brazilian Northeast region has the highest rates of childhood mortality in the country (Barreto and Carmo, 2007, Silva Jr, 2004), and acute diarrhea is an important death cause (7.8 per 100 000 infant) (Silva, 2004). Therefore, acute diarrhea represents 1 of the important childhood ailments urging for treatment and prevention in this region of Brazil. The Pediatric Hospital Arlinda Marques in João Pessoa, state of Paraíba, has an average of 7456 children per month, 80% of them with diarrhea. In this study, we used microbiologic assays to perform the most comprehensive etiologic assessment ever made in João Pessoa, evaluating infants with diarrhea requiring medical assistance. We investigated enteropathogens detected in stools of infants with diarrheal disease compared with those of age-matched controls.

Section snippets

Study design

Our study was conducted at the Pediatric Complex Arlinda Marques, a hospital assisting children from a poor section of urban João Pessoa, a coastal city in Paraiba, a northeast state of, Brazil. This city has a population of 700 000 inhabitants and a tropical climate with an average temperature of 26.6 °C. Typically, it has 2 distinctive seasons: i) the rainy season, from April through September, and ii) the dry season, from October through March. For our study, we selected 290 infants <24

Prevalence of enteropathogens

A wide range of bacterial pathogens was detected during this study (Table 2), all of which were significantly associated with diarrhea (P < 0.01). Pathogens were detected in the stools of 62.7% of the cases and 12.4% of the controls. EAEC was the pathogen most frequently detected in the cases (25%), followed by EPEC (11%), ETEC (10%), Salmonella (8%), and Shigella sp (4%). t-EPEC and a-EPEC were isolated in 2% and 9% of the cases, respectively.

EAEC was the most frequent enteropathogen in the

Discussion

In this report, we present data from a case–control study conducted during a 12-month period to determine the role of various categories of DEC in childhood diarrhea in Joao Pessoa, northeast of Brazil. We included only infants younger than 2 years old once acute diarrhea is more prevalent and has the direst consequences in this age range.

Stools of case and control groups were processed exactly the same way, both logistically and methodologically. The prevalence of enteropathogens was 62.7% in

Acknowledgments

The authors are grateful to Mônica Vieira for the colony hybridization assays. Staff members of the Arlinda Marques Pediatric Hospital and of the Center for Tropical Medicine at the Federal University of Paraíba, Paraíba, Brazil, kindly provided assistance and support. The authors also thank the parents who gave permission for their children to participate in this study. This work was granted by the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP. Mr. Antônio Fernandes Filho and Ms.

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