The major challenge during isolation of EHEC from stool samples is the differentation of EHEC among commensal E. coli that usually form the majority of bacteria cultured. Additionally, the number of pathogens rapidly decreases during the development of HUS; therefore, an early sampling is crucial for a sufficient diagnosis.
As the Shiga toxin production is common in all EHEC, detection of Shiga toxin or the toxin encoding genes is nowadays the most frequently used diagnostic target. As EHEC frequently possess a virulence plasmide carrying a pathogenicity island with the locus of enterocyte effacement (LEE) and genes encoding for a type III secretion system, these genes can be detected via PCR or other molecular methods. Detection of such genes is highly indicative for an EHEC infection, however, all results have to be confirmed by cultivation of the respective pathogen. The EHEC isolate is subsequently used for further characterization and subtyping, which is essential for tracing transmissions and evolutionary analyses elucidating the emergence of certain clones.