This report (EQA-6) presents the results of the sixth round of the external quality assessment scheme for typing of verocytotoxin-producing E. coli (VTEC) funded by ECDC. The EQA-6 was carried out from January to April 2015 and included the following methods: pulsed-field gel electrophoresis (PFGE), O:H serotyping, detection of virulence genes (eae, vtx1, vtx2 and ehxA), subtyping of the vtx genes, phenotypic detection of verocytotoxin/Shiga toxin production (VT/Stx), fermentation of sorbitol, production of Beta-glucuronidase, enterohaemolysin, and extended BetaBeta-lactamase (ESBL). A total of 29 laboratories participated in at least one part of the EQA-6. Twenty-two laboratories (76%) reported PFGE results, 17 laboratories (59%) participated in full O:H serotyping of all strains (26 laboratories submitted O group results for at least one strain and 17 laboratories submitted H-types for at least one strain). Genotypic detection of eae, vtx1 and vtx2 was performed by 24-26 laboratories (an average of 83-90%), 19 (66%) for ehxA, and 22 (76%) participated in subtyping of vtx genes. Sixteen laboratories who participated in phenotypic detection; 7 (25%) for VCA (Vero cell assay), 24 (83%) for fermentation of sorbitol, 15 (55%) for Beta-glucuronidase, 15 (52%) for enterohaemolysin and 16 (55%) for ESBL. Twenty-two laboratories participated in the PFGE part of the EQA-6, and 16 (73%) were able to produce a PFGE gel of sufficiently high quality to allow comparison with profiles obtained by other laboratories. The subsequent normalisation and interpretation of the profiles were performed using the specialised software suite BN. Fifteen laboratories completed the gel analysis, and 87% performed fair to good, in accordance with the guidelines. Of the 17 participants, an average of 78% (range 59-100%) could correctly determine the O:H serotype of the strains (some laboratories only typed a selection of the ten test strains). The more common serotypes obtained better typing results: O157:H7 serotype was typed correctly (100%) by all 17 participants, while both O41:H26 and O174:H21 were typed correctly by 10 laboratories (59%). The results for the genotypic detection of virulence genes were generally very good: eae (97%), vtx1 (99%), vtx2 (98%) and ehxA (98%). False positive results were reported once for vtx1 and vtx2 and false negative results were submitted four times for vtx2. For vtx subtyping, eight false negative results were received for vtx2; six laboratories failed to detect the vtx2d gene. The virulence genes aggR and aaiC were reported by 19 and 16 participants, respectively. The one aaiC positive test strain was correctly determined by two participants while 14 participants failed to detect the gene. Whole genome sequencing (WGS) of this strain revealed a new variant of aaiC, not previously described. The primers and probe for either conventional PCR or Real Time PCR (RT PCR) used by the 14 participants with negative aaiC results are presumed to be unable to anneal to the new aaiC gene variant. One of the test strains harboured the aggR gene which was correctly reported by all laboratories. The percentage for correct results for phenotypic characterisation was 100% for VCA, 99% for ESBL production, 89% haemolysin production, 96% for Beta-glucuronidase production and 98% for sorbitol fermentation.
