Abstract
Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with a broad spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Systemic Stx toxemia is considered to be central to the genesis of HUS. Distinct methods have been used to evaluate anti-Stx response for immunodiagnostic or epidemiological analysis of HUS cases. The development of enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay to detect the presence of specific antibodies to Stx has introduced important advantages for serodiagnosis of HUS. However, application of these methods for seroepidemiological studies in Argentina has been limited. The aim of this work was to develop an ELISA to detect antibodies against the B subunit of Stx2, and a WB to evaluate antibodies against both subunits of Stx2 and Stx1, in order to analyze the pertinence and effectiveness of these techniques in the Argentinean population. We studied 72 normal healthy children (NHC) and 105 HUS patients of the urban pediatric population from the surrounding area of Buenos Aires city. Using the WB method we detected 67% of plasma from NHC reactive for Stx2, but only 8% for Stx1. These results are in agreement with the broad circulation of Stx2-expressing STEC in Argentina and the endemic behavior of HUS in this country. Moreover, the simultaneous evaluation by the two methods allowed us to differentiate acute HUS patients from NHC with a great specificity and accuracy, in order to confirm the HUS etiology when pathogenic bacteria were not isolated from stools.
Highlights
Verocytotoxin-producing Escherichia coli (E. coli), referred to as Shiga toxin (Stx) -producing E. coli (STEC), infection is associated with a spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) [1,2,3]
The objectives of the present study were 1) to develop a standard antibody enzyme-linked immunosorbent assay (ELISA) to detect anti-Stx2 B subunit, and a western blot (WB) assay against the whole Stx2 and Stx1 proteins; 2) to correlate the results from anti-Stx2B ELISA with those from anti-Stx2 WB and to validate them to be used in our population; and 3) to study the presence of antibodies against Stx2 A and B subunits in normal healthy children and HUS patients from Argentina
The intensity of the reactivity was variable, the strongest signals were detected in HUS or HUSrecovered patients (HUSrec) plasma
Summary
Verocytotoxin-producing Escherichia coli (E. coli), referred to as Shiga toxin (Stx) -producing E. coli (STEC), infection is associated with a spectrum of clinical manifestations that include diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) [1,2,3]. Stxs are a family of protein toxins that share a structure of polypeptide subunits consisting of an enzymatically active A subunit (approx 32 kDa) that is linked to a pentamer of B (binding) subunits (approx 7,5 kDa) [5]. Among the Stxs produced by human STEC isolates, Stx and Stx2c show the highest association with HUS [8]. Stx is serologically distinct from Stx (and Stx2c) and these toxins do not show cross-neutralization by homologous antisera in Vero cell monolayers [7,9]. Stx is completely neutralized by anti-Stx2c antiserum, whereas Stx2c is only partially neutralized by Stx antiserum [10]
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