Humoral immune response to Shiga Toxin 2 (Stx2) among Brazilian urban children with hemolytic uremic syndrome and healthy controls

Guirro et al. BMC Infectious Diseases 2014, 14:320 http://www.biomedcentral.com/1471-2334/14/320 RESEARCH ARTICLE Open Access Humoral immune response to Shiga Toxin 2 (Stx2) among Brazilian urban children with hemolytic uremic syndrome and healthy controls Mirian Guirro1, Roxane Maria Fontes Piazza2, Renato Lopes de Souza3 and Beatriz Ernestina Cabilio Guth1* Abstract Background: Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. Stx is essential in the pathogenesis of HUS, which has been mostly related to Stx2-producing isolates. Very limited data exist on the immune response to STEC in the Brazilian population. In this study, the prevalence of immunoglobulin G (IgG) antibodies to Stx2 was investigated in sera of children diagnosed with HUS and of healthy children in the city of São Paulo, Brazil. Methods: IgG-antibody reactivity to Stx2 was determined by immunoblotting (WB) and enzyme-linked immunosorbent assay (ELISA) in sera from 13 children with HUS aged 8 months to 6 years and 54 healthy urban children aged 5 months to 7 years. Results: A positive immune response to the A and B subunits of Stx2 was observed in 46.1% HUS patients and in 16.6% healthy individuals by WB. All HUS patients and 62.9% healthy children showed IgG antibodies to the Stx2 A subunit. The frequency of antibodies to both subunits or only to the A subunit of Stx2 was significantly higher in HUS patients than controls (p < 0.05). Also, the mean OD value obtained by ELISA was higher in that group. Considering children’s age, the frequency of reactivity to either the A subunit or both subunits of Stx2 was considerably higher in HUS children up to three years old compared to controls in the same age range. Moreover, in almost 37% of healthy children, no immune response to Stx2 was detected independently of the child’s age. Conclusions: The seroepidemiolgy of anti-Stx2 antibodies was described for the first time in healthy children and children with HUS in Brazil. The percentage of individuals showing antibodies against Stx2 was higher among HUS patients than controls, and in spite of the low number of notified HUS cases, STEC strains are circulating in our settings. In addition, the results obtained also corroborated previous data on the increased sensitivity and specificity of WB compared to toxin-based enzyme immunoassays. Background Shiga toxin-producing Escherichia coli (STEC) infection can induce hemolytic uremic syndrome (HUS), a thrombotic microangiopathy characterized by acute renal failure, thrombocytopenia and hemolytic anemia. O157:H7 is the most prominent STEC serotype implicated in serious outbreaks and sporadic cases of HUS. However, in the last decade, a wide range of non-O157 STEC serotypes have shown a significant etiological role in the illness [1,2]. Worldwide, there is substantial geographic variation * Correspondence: 1 Department of Microbiology, Immunology, Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil Full list of author information is available at the end of the article in the prevalence of STEC serotypes as well as in the incidence of HUS. In Brazil, human STEC infections have been linked mostly to sporadic cases of nonbloody diarrhea associated mainly with non-O157 strains [3,4]. However, HUS cases associated with O157 as well as non-O157 STEC infections have more recently been described in São Paulo State [5-8]. The most important virulence property of STEC is its ability to produce Shiga toxins (Stx), central in the pathogenesis of HUS [9]. Stx consist of one enzymatically active A subunit (32 kDa) linked to a pentamer of B subunits (7.5 kDa), and are produced during mucosal colonization and delivered to the circulation [10]. There is increasing evidence demonstrating the damage caused to vascular © 2014 Guirro et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guirro et al. BMC Infectious Diseases 2014, 14:320 http://www.biomedcentral.com/1471-2334/14/320 endothelial cells in various organs and tissues, including kidneys and gastrointestinal tract [11,12]. The toxin family contains two major groups that are serologically distinct, called Stx1 and Stx2. The latter has multiple subtypes or variants in a range of combinations [13]. Among the Stx produced by human STEC isolates, Stx2 and Stx2c show the highest association with severe cases of HUS [14-16]. Protective immunity to STEC infection is likely to result from the interplay between antibodies that inhibit colonization of the bowel and those that neutralize Stx [17]. Experimental and clinical findings suggest that Stx antibodies can develop a role in the protective immune response as well as contributing to HUS resistance [17-19]. However, several epidemiological analyses have demonstrated that approximately 75% of HUS cases occur in children less than 5 years old, suggesting that the disease may be associated with the absence of preexisting immunity in the pediatric population. Neutralization assay in cell cultures was the earliest approach for detecting antibodies to Stx in human serum. However, some studies found nonspecific neutralizing activity in this assay due to a lipoprotein component of the serum [20]. To overcome these limitations, other assays such as enzymelinked immunosorbent assay (ELISA) and Western blot (WB) have been employed [17,20-22]. Information about the nature and measurement of the immune response directed against Stx in Brazil is limited. Palmeira et al. [23] investigated the ability of sera obtained from healthy adults to neutralize Stx2. Thus, seroepidemiological data regarding anti-Stx2 antibodies in the Brazilian population and the use of serodiagnosis could help to explain the epidemiological profile of the illness. Therefore, the aim of the present study was to investigate the prevalence of IgG antibodies to Stx2 in sera of children diagnosed with HUS and of healthy children (control population) in the city of São Paulo, SP, Brazil, using the accurate and sensitive techniques WB and ELISA, outlining the first report on the analysis of Stx antibodies in children in Brazil. Methods Human serum samples – patients and controls Sera were obtained from 13 patients aged 8 months to 6 years, presenting with typical symptoms of HUS. All patients developed HUS after gastroenteritis, with diarrheal prodromes for a median of 6.4 days [8]. The samples were collected in pediatric in (...truncated)