Validation of an immunochromatographic assay for the diagnosis of diarrheagenic Escherichia coli

Abstract

Acute diarrheal infections are still identified as a significant cause of morbidity and mortality, especially in developing countries. Annually approximately 1.5 million diarrhea-related deaths are estimated and about 2 billion cases of diarrheal diseases, mostly concentrated in impoverished areas of the world. Among the pathogens causing diarrhea diarrheagenic Escherichia coli are responsible for 30-40% of episodes of acute diarrhea in these countries. In this context, the diagnosis is an important tool to minimize and control events. Among the diarrheagenic E. coli, the categories of epidemiological relevance are: enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and E. coli producing the Shiga toxin (STEC) and its subgroup enterohemorrhagic E. coli (EHEC). Unfortunately, EAEC does not present a common virulence factor amongst all isolates, the other three pathotypes produce the virulence factors that serve as targets for its diagnosis, EPEC and EHEC express intimin and secreted protein (Esps), for ETEC the main virulence factors are the heat-stable toxins (ST) and the heat-labile (LT) and the most potent cytotoxins Shiga (Stx1 and/or Stx2) are secreted by STEC and EHEC. Comparing detection methods, the immunoassay has several advantages for rapid tests, including high specificity, sensitivity, and easy sample preparation. Thus, the polyclonal and monoclonal antibodies (MAb) obtention allowed the standardizations of a fast and low-cost capture method for ETEC, EPEC and STEC/EHEC detection through its virulence factors in bacterial isolates. All antibodies were obtained at the Bacteriology Laboratory: monoclonal and polyclonal antibodies anti-ST, anti-LT and anti-Stx1, anti-Stx2 and anti-EspB. After collection, purification and characterization all antibodies were employed for the development of the immunochromatographic test (IC). Basically, the test strips composed of three types of membrane: fiber cellulose [sample pad and absorbing pad (Millipore®)], fiberglass [conjugate pad (Millipore®)] and nitrocellulose [HiFlow nitrocellulose (Millipore ®)]. Once prepared, the tape allows the sample present in the sample pad migrate and react with MAb conjugated to colloidal gold. The complex migrating below the tape and is captured by the polyclonal antibodies previously immobilized, creating a colored red line. The reaction is considered positive when the two lines test or positive controls are present. Monoclonal antibodies were conjugated to colloidal gold for evaluation of different concentrations of polyclonal antibodies. When these conditions were established, the detection limit using target antigens were evaluated and presented an immunochromatographic positive assay reaction after 15 minutes. These results lead us to the objective of this project that consists in determining the sensitivity, specificity and efficiency of this immunochromatographic assay using isolates characterized as diarrheagenic E. coli to define the sensitivity and isolates from microbiota and other Enterobacterial to define the specificity of the assay.