Seventh external quality assessment scheme for Listeria monocytogenes typing

Human listeriosis is a relatively rare but serious foodborne disease with an EU notification rate of 0.47 cases per 100 000 population in 2018 [3]. The number of human listeriosis cases in the EU increased between 2008 and 2016. In 2017-2018, the level of reported cases was stable.

Since 2007, ECDC has been responsible for the EU-wide surveillance of listeriosis, including facilitating detection, and investigation of foodborne outbreaks. Surveillance data, including basic typing parameters for the isolated pathogen, are reported by European Union/European Economic Area (EU/EEA) countries to The European Surveillance System (TESSy), including molecular typing data. This molecular surveillance system relies on the capacity of laboratories to provide comparable data to FWD-Net. In order to ensure the EQA is linked to the development of surveillance methods used by NPHRLs in the EU/EEA, EQAs 5 to 7 featured a molecular typing-based cluster analysis using either pulsed-field gel electrophoresis (PFGE) and/or whole-genome sequencing (WGS)-derived data.

The objectives of the EQA are to assess the quality and comparability of typing data reported by NPHRLs participating in FWD-Net. Test isolates for the EQA were selected to cover isolates currently relevant for public health in the EU and represent a broad range of clinically relevant types for invasive listeriosis. Two sets of 11 test isolates were selected for serotyping and molecular typing-based cluster analysis. Eighteen laboratories signed up and 17 completed the exercise, representing a decrease in participation from 20 laboratories (15%) for EQA-5, but the same level of participation as for EQA-6. The majority of participants (12/17; 71%) completed the full EQA scheme.

In total, 14 (82%) participants participated in the serotyping part. Molecular serogrouping results were provided by 13 of 15 (93%) participants. Three participants performed both conventional serotyping and molecular serogrouping. The performance of molecular serogrouping was highest, with 100% correct results. For the conventional method, 75% of the participants correctly serotyped all test isolates. One new participant mistyped five of the 11 isolates. Since the first EQA in 2012, a trend towards substituting conventional serotyping with molecular serogrouping has been observed.

Of the 17 laboratories participating in EQA-7, 15 (88%) performed molecular typing-based cluster analysis using a method of their choice. 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 isolates, i.e. to correctly categorise the cluster test isolates regardless of the method used, not to follow a specific procedure.

The cluster of closely related isolates was pre-defined by the EQA provider using WGS-derived data. Therefore, as expected, the correct cluster delineation was difficult to obtain by the use of less discriminatory methods, e.g. PFGE. None of the three participants using PFGE did identify the correct cluster. Thirteen laboratories performed cluster analysis using WGS-derived data. Performance was high, with 100% of the participants correctly identifying the cluster of closely related isolates. An allele-based method was preferred since 84% (11/13) used core genome multilocus sequence type (cgMLST), compared with 16% (2/13) using single nucleotide polymorphism (SNP).

In EQA-7, the EQA provider introduced an additional part to the molecular typing-based cluster analysis: an assessment of four EQA provided genomes. This was designed to mimic an urgent outbreak situation, where sequence data may have been produced in other laboratories and the available sequences must be addressed despite, for example, possible poor quality. The majority of participants successfully identified the different characteristics in the modified genomes, and also correctly concluded one cluster isolate as being part of the cluster defined in the cluster analyses part of the EQA and one non-cluster genome as not being part of the cluster of closely related isolates.

In EQAs 5-7, participants were free to choose their preferred analytical method for the WGS-based cluster identification. The conclusion from EQA-5 was that cgMLST has higher consistency compared to SNP analysis. The conclusion was not as obvious in either EQA-6 or EQA-7, since only a few SNP analyses were reported in these schemes compared with six SNP analyses in EQA-5. One participant changed from SNP analysis to cgMLST and
identified the cluster correctly, unlike in EQA-6.