Tenth external quality assessment scheme for Listeria monocytogenes typing in EU/EEA and EU enlargement countries, 2023

Human listeriosis is a relatively rare but serious food-borne disease with a European Union (EU) notification rate of 0.62 cases per 100 000 population in 2022 [3]. With an increase of 16% from 2021 to 2022 (2 365 and 2 738 cases, respectively) the number of cases in 2022 was even higher than before the COVID-19 pandemic (2019, 2 621 cases).

Since 2007, ECDC has been responsible for EU-wide surveillance of listeriosis, including facilitating the detection and investigation of food-borne outbreaks. Surveillance data, including certain basic typing parameters, are reported by  European Union/European Economic Area (EU/EEA) countries to The European Surveillance System (TESSy). Since 2012, the EQA scheme has covered molecular typing methods used for EU-wide surveillance.

EQA-10, conducted between May and November 2023, involved serotyping/grouping and molecular typing-based cluster analysis. The objective of this EQA was 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 that are currently pertinent for public health in Europe and to represent a broad range of clinically relevant types of invasive listeriosis. Seven test strains were selected for serotyping/grouping and molecular typing-based cluster analyses. An additional ten sequences were included for the molecular typing-based cluster analysis. Of the 23 laboratories that signed up, 21 completed the exercise. This represented an increase of one laboratory compared with EQA-9; however, the composition was different, as two laboratories from EQA-9 did not participate in EQA-10, two new laboratories joined EQA-10, and one laboratory from EQA-8 rejoined for EQA-10. Most participating laboratories (15/21; 71%) completed the full EQA scheme.

In total, 18 laboratories (86%) participated in the serotyping part of the exercise; all of them conducted PCR-based/WGS molecular serogrouping, and two laboratories (11%, 2/18) also conducted conventional antigen-based serotyping. On average, molecular serogrouping was performed well, with 95% correct results. For the conventional method, 100% of the participants correctly serotyped all seven test strains. One laboratory, participating for the first time, mistyped four of the seven strains in the molecular serogrouping, swapping two of the isolates. Since the first EQA in 2012, the trend has been towards replacing conventional serotyping with molecular serogrouping, showing strong performance.

Of the 21 laboratories participating in the EQA-10, 18 (86%) performed molecular typing-based cluster analysis using a method of their choice. The intent of the cluster analysis component of the EQA was to evaluate the NPHRLs’ capacity to identify a genetically closely related cluster. In other words, the goal was to accurately categorise the cluster test strains – regardless of the method used – rather than strictly be able to follow a specific procedure.

The cluster of seven closely related strains (three test strains and four strain sequences) was predefined by the EQA provider using data derived from whole genome sequencing (WGS). Therefore, as expected, the correct cluster delineation was not possible to obtain using less discriminatory methods (e.g. pulsed-field gel electrophoresis (PFGE)). Nevertheless, the three cluster strains were correctly identified by the participant that used PFGE, though they could not include the sequences provided in their analysis. Seventeen laboratories performed cluster analysis using WGS-derived data; only one used single-nucleotide polymorphism (SNP) as the main analysis. The submitted allelic differences (AD) clearly showed coherence despite the different approaches and schemes that the participants used. The most widely used core genome multilocus sequence type (cgMLST) scheme was Ruppitsch (9/16 laboratories), while the Pasteur scheme was less common (5/16). Most laboratories (11/16) reported 0–8 AD for the strains in the predefined cluster; however, not all included the strains with 7 or 8 AD in the reported cluster and others excluded the modified strain with low read R2 quality (strain13). When analysing the predefined cluster of the seven closely related strains, 47% (8/17) of the participants reported the same list of strains as the EQA provider. Two laboratories reported the cluster without strain13 and two laboratories employed a stringent cut-off, excluding borderline cluster strains. Furthermore, three of the laboratories that failed to identify the predefined cluster were suspected of submitting errors. Only two laboratories exhibited concerning errors: one laboratory interchanged strains and another included all sequence type 5 (ST5) in the cluster.

In general, most of the participants were able to identify the different characteristics and modifications of the EQAprovided sequences. All laboratories detected issues with the sequence that had a mix of sequence types and 94% of participants identified the quality control (QC) issues of low coverage. Different observations were made for both the strain with slightly reduced coverage and the strain with low read quality of R2.

A feedback survey was sent to the participants to assess their experience of EQA-10; 48% (10/21) of the participants responded. Of note, the QC evaluation of participant-sequenced data and the inclusion of low-quality data were considered useful by all respondents. Additionally, two respondents suggested uploading a TSV file instead of entering the data in a survey format to reduce typing errors and one suggested using an even bigger dataset to assess the overall cluster congruence between pipelines.