‘Bands’ parameter causes problems in Listeria typing assessment
The Pulsed Field Gel Electrophoresis (PFGE) gel quality is highly dependent on laboratory procedures, said the agency as it warned against taking shortcuts.
Improvement measures need to be taken to improve the quality of the ‘bands’ parameter and ensure onwards inter-laboratory comparison of PFGE profiles, it said.
Findings come as part of the third round of the External Quality Assessment (EQA) scheme for the typing of Listeria monocytogenes.
The EQA covers the PFGE method, conventional serological typing and PCR-based molecular typing.
A total of 22 laboratories registered for the EQA-3 with 20 laboratories completing it and two opting out without submitting results. 18 produced PFGE results and 16 participated in the serotyping exercise.
Eight of these 16 laboratories performed conventional phenotypic serotyping, while 13 performed molecular PCR-based serotyping.
PFGE gel results
The majority (67%) of the laboratories were able to produce a PFGE gel of sufficiently high quality to allow for comparison with profiles obtained by other laboratories.
Comparability primarily relies on the use of correct running conditions, distinct bands and a good quality image acquisition.
Gels were normalised and interpreted using the specialised software BioNumerics (BN) software.
Listeriosis is a relatively rare but serious foodborne disease, with 1,763 confirmed human cases reported in the EU in 2013.
Compared to other foodborne infections under EU surveillance, listeriosis caused the most severe human disease, with 99% of the cases hospitalised
Fourteen laboratories completed the gel analysis and generally did so with high quality (93%) and in accordance with the guidelines.
When evaluating the scores of the seven parameters, it is clear the quality of the bands on the gels is the major challenge for the participating laboratories, said ECDC.
“This third EQA for PFGE typing of Listeria demonstrates that the majority of participating laboratories were able to produce good results. However, an increase in gels being graded ‘Poor’ and not suitable for inter-laboratory comparison was seen since EQA-2.
“This decrease in quality only highlights that PFGE is a highly person-dependent method, and a method that requires many parameters to be correct.”
Six laboratories scored ‘Poor’ in one or more parameters, and were not able to produce gels of sufficiently high quality to ensure inter-laboratory comparisons.
“The parameter ‘Bands’ was especially a problem to the participants, and 22% of gels run with enzyme ApaI and 12% of the gels run with AscI obtained a score of 1 (‘Poor’) in this parameter.
“Therefore it is stressed that plugs should be cut thinly, that the cutting enzyme is used with the correct enzyme buffer and the running buffer is fresh and of the right concentration.
“Furthermore, the overexposure of a gel can lead to single bands looking very ‘fat’ and therefore being assigned as double band.”
Of the eight who performed the conventional serotyping of L. monocytogenes half were able to correctly serotype all eleven EQA test strains.
Three participants failed to report the correct result of one strain and one participant failed with five strains, only submitting 55% correct results.
Eleven of the 13 participants in the molecular serotype of L. mono were able to correctly serotype all EQA test strains, which is an improvement from last year’s eight participants which submitted 100% correct results. One participant only had 45% correctly submitted results.
Regarding results of the conventional serotyping and the molecular serotyping, incorrect results could primarily be attributed to one laboratory for each method, said ECDC.
“The quality of the molecular (PCR-based) serotyping performed by the participants was very high and 84% of the participants scored 100% correct results. The quality of the conventional serotyping was somewhat lower with only 50% of the participants obtaining 100% correct results.
“However, the majority (7/8) of participants obtained a good score ≥90%. Compared to the PCR method the conventional phenotypic serotyping is much more expensive, laborious and slow and furthermore it requires experienced personnel.”
Test strains were chosen to cover the most prevalent serotypes present in isolates causing human disease.
The average percentage of correctly typed strains for conventional serotyping was 91%, an increase from EQA-2 mainly attributed to one difficult strain in the previous EQA.
Molecular surveillance system implemented as part of TESSy, relies on the European Food-and Waterborne Diseases and Zoonoses network (FWD-Net) laboratories to produce comparable typing results.
Molecular typing method used for EU-wide surveillance is PFGE. Phenotypic serotyping is included in TESSy and PCR-based serotyping has also been since 2012.