PFGE typing finds inter-lab comparison possible but reveals quality concerns
The European Center for Disease Prevention and Control (ECDC) said 85% of participating laboratories were able to produce at least one PFGE gel sufficient to allow inter-laboratory comparison but overall quality varied considerably.
Findings come from the fourth round of the External Quality Assessment (EQA) scheme for the typing of L. monocytogenes organised for labs in the Food- and Waterborne Diseases and Zoonoses Network (FWD-Net).
Pulsed Field Gel Electrophoresis (PFGE) is the gold-standard for typing of L. monocytogenes and used for EU-wide surveillance usually performed with two enzymes (ApaI and AscI).
Serotyping analysis
Conventional phenotypic and molecular serotyping are included in the European Surveillance System (TESSy).
Twenty-three laboratories completed the exercise and the majority (70%) completed the full EQA scheme. In total, 20 participated in the PFGE part and 19 in the serotyping part.
Conventional serotyping results were provided by nine laboratories (47%) and molecular serotyping by 16 participants. Six did both serotyping methods.
It assessed the serotype determination by conventional antigen-based typing of somatic ‘O’ antigens and flagellar ‘H’ antigens and/or PCR-based molecular serotyping.
Quality of conventional serotyping increased from EQA-3, mainly due to the absence of one difficult strain included in the previous EQA.
Conventional serotyping is more expensive, laborious, and slow, and requires experienced personnel in comparison to molecular serotyping.
The performance of the molecular serotyping corresponds to that of EQA-3, where 85% of the participants correctly serotyped all eleven test-strains.
In the serotyping part of the EQA, a trend towards substituting the conventional serotyping with molecular has been seen, with an increase of participation from seven to 16 participants in 2015.
ECDC said 78% and 81% of participants correctly serotyped all test strains by conventional and molecular methods, respectively.
PFGE and software
Compared with the first scheme in 2012 a reduction in the percentage performing PFGE has been seen from 94% to 87%.
Parameters were image and running conditions, cell suspension, bands, lanes, restriction, gel background and DNA degradation.
ECDC said in the longer term, whole genome sequence (WGS)-based methods will take over from both of the current methods.
Four laboratories scored poor for the PFGE gel part, according to the TIFF Quality Grading Guidelines 2016, where seven parameters are used for grading given scores between one and four.
One was a first time participant and the other three improved general performance from the last time but still produced profiles of insufficient quality for inter-laboratory comparison.
Fourteen laboratories analysed the gels and were able to produce XML-export files according to protocol, including five parameters scored 1-3. Re-submission of results was necessary for four participants.
BioNumerics (BN) is software developed for PFGE gel analysis. For most participants (71%), the analysis of gels were of a fair [2] to excellent [3] quality.
Only five analyses (from four participants) obtained the score poor [1], meaning these analysis could not produce PFGE profiles for inter-laboratory comparison.
