Twelfth external quality assessment scheme for typing of Shiga toxin-producing Escherichia coli

This report presents the results of the 12th round of the external quality assessment (EQA-12) scheme for typing of Shiga toxin-producing Escherichia coli (STEC). This EQA was organised for national public health reference laboratories (NPHRLs) providing data to the Food- and Waterborne Diseases and Zoonoses Network (FWD-Net) managed by the European Centre for Disease Prevention and Control (ECDC). Since 2012, the unit of Foodborne Infections at Statens Serum Institut (SSI) in Denmark has arranged the EQA under a framework contract with ECDC. EQA-12 contained serotyping, detection of virulence genes, and molecular typing-based cluster analysis.

Twenty-six laboratories participated in the EQA-12 scheme, with 25 (96%) performing the serotyping part, 25 (96%) determining the virulence profile, and 23 (88%) engaging in cluster identification using WGS data analysed by different approaches. In O:H serotyping, an average score of 97% was achieved by participants. The performance in detecting the virulence genes was also high: 97% for stx1 and 98% for stx2, 98% for aggR and esta genes, and 96% for eae gene. The average score of laboratories that correctly performed the stx subtyping were 95% for stx1, 94% for stx2, and 93% combined stx1 and stx2. In general, the performance of the cluster detection was high, with 15/23 (65%) laboratories correctly identifying the cluster of closely related strains.

Human STEC infection is a zoonotic disease. For 2022, 8 565 confirmed cases of STEC infection were reported by 29 EU/EEA countries. Twenty-three countries reported at least one confirmed STEC case and three countries reported zero cases (Bulgaria, Cyprus and Lithuania). In 2022, the EU notification rate was 2.1 per 100 000 population. For 2022, there was an increase of 8.8% in the annual notification rate reported compared with year 2021 (1.9 cases per 100 000 population). In 2022, the six most frequently reported serogroups were O157 (21.3%), O26 (19.4%), O103 (6.6%), O146 (5.5%), O145 (4.4%), and O91 (2.9%) [1].

Since 2007, ECDC has been responsible for the EU-wide surveillance of STEC, including facilitating the detection and investigation of food-borne outbreaks. Surveillance data, including basic typing parameters and molecular typing data for the isolated pathogen, are reported by Member States to The European Surveillance System (TESSy). The surveillance system relies on the capacity of NPHRLs in FWD-Net providing data to produce comparable typing results. To ensure that the EQA is linked to the development of surveillance methods used by NPHRLs, a molecular typing-based cluster analysis using whole genome sequencing (WGS)-derived data has been included since EQA-8.

The objectives of the EQAs are to assess the quality and comparability of typing data reported by NPHRLs participating in FWD-Net. Test strains for the EQA were selected to cover strains currently relevant to public health in Europe and represent a broad range of clinically relevant types of STEC. Twelve test strains were selected for serotyping/virulence profile determination and molecular typing-based cluster analyses. Additional eight strains (sequences) were included for the molecular typing-based cluster analysis. Twenty-six laboratories registered and 26 completed the exercise, comparable to EQA-11.

The full O:H serotyping was performed by 85% (22/26) of participating laboratories, with an average score
of 97%. In general, the more common European serotypes generated the highest scores compared to the less common serotypes, such as O8:H4, O80:H2, and O154:H31, which proved more difficult to identify, particularly if participants used phenotypic methods. Notably, not all laboratories demonstrated the capacity to determine all O groups and H types, and the participation in H typing was lower (22/26) compared to the O grouping (25/26) but higher than H typing in EQA-11 (19/26) most likely reflecting a shift towards WGS-based methods. The shift towards WGS was also seen in the reported O-grouping results: 68% (17/25) used WGS-based methods, which is higher than EQA-11 (60%), EQA-10 (52%), EQA-9 (50%), and EQA-8 (26%).

The quality of the virulence profile determination results was generally good, with high average scores of 96%, 97%, and 98% for eae, stx1, and stx2, respectively, similar to previous EQAs.

In EQA-12, two other diarrhoeagenic E. coli (DEC) pathotypes were included, EAEC strain3 (aggR gene) and ETEC Strain11 (esta gene) testing the participating laboratories in their abilities to detect STEC hybrid strains. The detection performance of the aggR gene was higher (23/24, 98%) than in both EQA-11 (95%) and EQA-10 (94%). Similar to aggR, the performance for esta was also higher (98%) than EQA-11 (89%). This variance in performance was attributed to four laboratories that couldn't identify the gene in strain11. All laboratories, except one, utilised a WGS-based method.

Of the 26 laboratories participating in the EQA-12, 23 (88%) performed molecular typing-based cluster analysis using WGS data analysed by different approaches. Notably, all laboratories used WGS in both EQA-12 and EQA-11 and none chose PFGE, a decrease from EQA-10 (2 laboratories) and EQA-9 (8 laboratories). The purpose of the cluster analysis part of the EQA was to assess the NPHRL’s ability to identify a cluster of genetically closely related strains, i.e. to correctly categorise the cluster test strains regardless of the method used. The focus is on the result, not a specific procedure.

Fifteen participants (65%) correctly identified the cluster of closely related ST301 strains defined by precategorisationfrom the EQA provider among the 12 test strains and eight test strains (genomic sequences).

In this EQA, participants were free to choose their preferred analytical method for the WGS-based cluster identification. An allele-based method was most frequently used; 87% (20/23) used core genome MultiLlocus Sequence Type (cgMLST) compared to 13% (3/23) using single nucleotide polymorphism (SNP) for the reported cluster analysis as the main analysis.

In general, for cgMLST the reported results from the participants were at a comparable level despite using various analysis and different allelic calling methods.

For inter-laboratory comparability and communication about cluster definitions, cgMLST using a standard scheme (e.g. Enterobase) gives a very high degree of homogeneity in the results, while the use of non-standardised SNP analysis may be more challenging. There are two main challenges: difficulty in comparing SNP with cgMLST results, and variations between SNP analyses in general, as demonstrated in this EQA, which makes the comparison and communication of the results between laboratories difficult. The latter was reflected in the reported results, as all three of the laboratories that used SNP-based analysis did not identify the pre-determined cluster.

The participants assessed additional genomes, some of which were modified by the EQA provider to provide a realistic view of various quality issues. Notably, only 48% (11/23) of the participants reported quality issues with the modified sequence containing 8% contamination with Shigella sonnei . In contrast, all participants (100%) correctly identified the poor quality of strain18, a non-cluster sequence with reduced coverage and removal of genes. Assessing both contamination with a different species and poor quality is crucial before conducting WGS analysis.

A feedback survey was sent to assess the STEC EQA scheme. The questionnaire contained both questions related to accreditation and information on the individual report; 58% (15/26) responded. Overall, the survey revealed an appreciation for QC assessment but highlighted the need for the EQA provider to optimise analyses also to IonTorrent data in addition to the standardised Illumina data. Streamlining the reporting form, especially for virulence gene determination, was suggested, along with exploring sending isolates for multiple EQAs simultaneously during sequencing runs. All of the responders appreciated the format, but some listed recommendation for improvements.