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DAFM NRL Newsletter Volume 6, Issue 1.

NRL Contacts

Antimicrobial Resistance

Zoonoses (Salmonella)

Ms R Slowey

Listeria

Staphylococci

Milk & Milk Products

Ms B Hickey

E. coli (VTEC)

Dr M Gutierrez

TSE’s

Dr M McElroy

Residues/Chemical Elements

Dr C Mannion

Pesticide Residues

Dr J Garvey

Campylobacter, TB & Johnes Disease

Dr J Egan

Animal Proteins

Dr J Choiseul

Exotic Animal Diseases

Drs R O’Neill & E Ryan

Parasite

Dr W Byrne

Contagious equine metritis

Dr Eoin Ryan & Mr J Moriarty

Volume 6, Issue 1 2015

BACKWESTON

DAFM LABORATORIES

BACKWESTON

Newsletter

Activities of National Reference Laboratories Introduction In 2006, following the designation of a number of additional European Union Reference Laboratories (EURL’s), Member States were required under Article 33 of Regulation 882 / 2004 to designate one or more National Reference Laboratory (NRL) for each EURL. The Departments of Health and Children and Agriculture, Food and the Marine (DAFM), as the Irish Competent Authorities, assigned these NRL functions to a number of laboratories including those within the Backweston Laboratory Campus. The DAFM laboratories also host a number of NRL established under EU or national legislation focusing specifically on animal health.

In this issue:

Report on 9th

Annual Workshop of the EURL for E. coli (2014) p. 2

Report on 9th

Annual Workshop of the EURL for Listeria monocytogenes (2015) p.7 Report on the Annual Workshop of the EURL for Antimicrobial Resistance (2015) p. 11

Report Annual Workshop on EURL Residues Berlin (2014) p. 13 Report on 19

th Workshop for the Control of Antimicrobial Residues in

Food from Animal Origin LC-MS/MS Method for Aminoglycosides in Meat and Milk (2014) p. 15 Report on the Annual Workshop of the EURL for Chemical Elements in Food of Animal Origin (2014) p. 20 Report on the 8

th and 9

th Annual Workshop of the EURL for Animal

Proteins in Feedingstuffs (2014 and 2015) p. 24


Abbreviations PT- Proficiency Test MS- Member States NGS- Next Generation Sequencing WGS- Whole Genome Sequencing FBO- Food Business Operator

NRL E. coli Report on 9th Annual Workshop of the EURL for E. coli, Rome, October 20- 21st, 2014 NRL Represenataive: Montserrat Gutierrez (VPHRL, Backweston Campus)

The 9

th EURL workshop provided an opportunity to

discuss the results in proficiency testing organised by the EURL and to disseminate information in the topic of Verotoxigenic E. coli (VTEC) including the progress in the development of molecular typing database. Valentina RIZZI (EFSA) gave an update on the annual reporting of VTEC in the EU and on EFSA activities for molecular typing data collection for food and animal isolates. In 2012 data on VTEC from food and from animals was reported by 21 and 11 M, respectively. Although different methods were used, ISO16654 was most frequently used; therefore the data was solely for O157 in many cases. For fresh bovine meat (carcass swabs or fresh meat) 4,603 results samples from 9 countries, were received, showing a prevalence of 1.3% of VTEC and 0.1% of O157 (fluctuations among countries: lowest 0%, highest 5.5%). The number of samples per country was varied, ranged from 25 to 1,923. Regarding animal samples, a total of 7,843 VTEC results were received from 7 countries (rectal swabs, hide swabs and faeces), showing a prevalence of 7.4% of VTEC and 1.8% of O157. The number of samples per country varied, ranged from 56 to 1,553, and the prevalence was also very variable, from 1.7% to 35.7%. Human pathogenic VTEC strains were detected by the reporting MS from fresh bovine meat occasionally and at low levels. The human pathogenic VTEC serogroups isolated from bovine meat and cattle samples included VTEC O157, O26, O91, O103 and O145. A total of 41 outbreaks of human pathogenic E. coli were reported, 12 were supported by strong-evidence (9 of them O157 and one O113). Of

these, 6 were linked to bovine meat and meat products. The 10 outbreaks reported by Ireland were water-borne. Regarding the progress in the molecular database for foodborne pathogens, the following is a scheme of the joint project. There will be one database at EFSA for food, feed and animal isolates, and another database at ECDC, which will contain all the EFSA data plus the human data. This latter database will be interrogated when looking at links between human and non-human sources.

Ms Rizzi informed the audience of the completion of the task of the working group to define the structure of the data collection system, the integration with the human data and the common nomenclature and data dictionaries for human, food, feed and animal isolates. A technical report will be published by end of the year. EFSA also launched three procurements to produce SOPs for molecular testing of Salmonella, Listeria and VTEC isolates and interpretation of molecular typing data. The reports for these are under final approval and will be published on the EFSA website. Although the EFSA database is not operative yet, data will have to be provided at result-based level according to the Standard Sample Description version 2. If one country has several data providers collecting data, the country shall coordinate internally to guarantee the uniqueness of the identifiers for each submitted PFGE image, result, isolate and sample taken at the national level. The access to joint EFSA-ECDC database depends on the type of data (sensitive and non-sensitive) and the users. A Collaboration Agreement will be signed by all parties to define data ownership, access, use, publication, procedures and confidentiality. A Steering Committee, with ECDC and EFSA, the relevant EURLs and ECDC’s curators will be set up to monitor and evaluate the pilot phase, identify needs for revision of the data collection system


and develop standard operating procedures for scientific data analyses. Ivo Van WALLE (ECDC) indicated that the molecular database at ECDC is being populated in a voluntary basis with human data from National Public Health Reference Laboratories since 2013. There are 11 countries contributing for VTEC, with 558 cases since 2013, 11 countries for L. monocytogenes and 572 cases, and 18 countries for Salmonella and 5,369 cases. A comparison of these human isolates from different countries has permitted the detection of multi-country clusters for Salmonella (n=95) and L. monocytogenes (n=26) but none for VTEC, which in his opinion raised questions about the suitability of the current molecular surveillance for VTEC.

PFGE is the main typing method, with MLVA also in use for S. typhimurium and plans to incorporate MLVA for S. enteritidis. The database is however designed to incorporate new typing methods and pathogens and to assist transition to whole genome sequencing as a typing method an expert group called FWDNEXT has been set up. Alfredo CAPRIOLI (EURL) informed the audience that a questionnaire distributed in the preceeding January by the EURL to 35 NRLs showed that 27 availed of PFGE, 8 of MLVA for O157 and 7 of NGS. Approx. 6,000 strains are generated each year, although it remarked that their geographical origin was not homogeneous. Of these, 1,761 O157 strains were identified as available. Twenty-eight NRLs have taken part in PTs on PFGE which demonstrated capacity for this typing procedure among NRLs. The EURL continue to provide training on typing, BioNumerics and plans to have a training event in 2015 on NGS. Stefano MORABITO (EURL) gave a presentation entitled “Next Generation Sequencing (NGS): International initiatives and application in surveillance and outbreak investigations”. This

technology is emerging due to the falling costs of carrying out sequencing with new systems such as Illumina and Ion Torrent Sequencing. Examples of use of NGS are projects such as the Foodborne Pathogens Genome Project and more recently the Global Microbial Identifier. Interest spans to organisations such as CDC, EFSA and even ISO has a proposal for standardisation of WGS for food microbial typing. Flemming SCHEUTZ (WHO-International Escherichia and Klebsiella Centre) presented real applications of WGS to VTEC surveillance and outbreak investigation. User-friendly WGS tools (MLST, SNPs, prediction of bacterial species, plasmid typing among others) are available from http://www.genomicepidemiology.org/, an organisation located at DTU and lead by Frank Aarestrup, also head of the EURL Antimicrobial Resistance. His group has applied WGS to type for vtx and eae genes, including their multiple variants and many other virulence factors. When applied to outbreak investigation the results were not only equivalent to traditional methods but also faster. A most challenging project is underway for serotyping O and H antigens by WGS, looking at the wzx and wzy (O-antigen flippase and polymerase) and the wzm and wzt (ABC transporter) genes for O serotyping and fliC (flagellin) and flkA, flnA, flmA, fllA genes for the H serotyping. Results obtained prove WGS is able to serotype most of strains, and has revealed some interesting facts of uncommon strains showing unusual genes combinations.

The application to typing other bacteria was also presented. In particular in L. monocytogenes real-time typing of human isolates from 2014 is carried by WGS alone, with an MLST protocol that looks at 25 loci instead than the conventional 7, and that has proven its usefulness in the Danish outbreak related to the meat product called ”rullepølse”.

http://www.genomicepidemiology.org/

http://www.dtu.dk/English/Service/Phonebook.aspx?lg=showcommon&id=39147&type=person




Valeria MICHELACCI (EURL) presented the results of a genome-based phylogenetic analysis carried out in O26 strains. Since the 90’s O26 strains vtx2 positive MLST type ST29 are emerging as causing disease to humans with 53 cases in Italy in the period 2011 to 2013. A group of human (n=21) and non-human (n=10) O26 strains were analysed by MLST, SNPs based phylogenetic tree and virulence genes. The results indicate that ST29 isolates are a subset circulating mostly in humans and the presenter postulated that the reservoir is unknown but probably not cattle.

Rosangela TOZZOLI (EURL) presented the results of a collaborative study for the validation of the ISO 16654:2001 and a study for ISO/TS 13136:2012 validation in sprouts. The equivalence study between ISO 16654 and the NMKL method No 164 in milk was agreed in Cairo in 2007, mandate M/381. It was carried out in 2012, with 15 participating laboratories, each analysing 24 samples with either no inoculum, low level (10-50 cfu/ml) or high level inoculum (100-500 cfu/ml). Outcome of the study was a sensitivity of 100% and a specificity of 94.4% (the specificity at less than 100% is due to cross-contamination). The sprouts collaborative study was similarly designed and conducted at the end of 2013, with samples inoculated only with O157 serotype. Laboratories were requested to carry out the preliminary enrichment followed by ISO 16654 and ISO 13136. The results were better for 13136 than 16654:

ISO 16654 ISO 13136

Specificity 99.1% 100%

Sensitivity low inoculum

75.89% 96%

Sensitivity high inoculum

96.42% 99.04%

Detection limit LOD 50%

8.141 Not given

The results of the EU-RL PT12 on the detection of VTEC and other pathogenic E. coli in sprouts were presented. Thirty-four NRLs representing 28 MS and the NRLs of Norway, Switzerland, Argentina and Egypt took part. Three samples were distributed: blank, spiked with VTEC and spiked with EIEC (ipaH). The evaluation of the laboratories

was based on a penalty points system for incorrect results as follows: RT-PCR screening step where virulence genes were incorrectly identified

4 points for each incorrect/missing result on vtx genes

2 points for each incorrect result on other virulence genes

1 point for missing results on other virulence genes

Isolation step:

4 points for the lack of isolation of VTEC strains

2 points for the lack of isolation of other patho-groups

Performance in the overall procedure: 8 points considered as the threshold for under-performance (but score higher than 8 without errors on vtx genes or VTEC isolation was still considered as satisfactory). Ten laboratories underperformed, although for 8 of them the points were assigned for tests not carried out (missing results). The results of the EU-RL PT13 on the identification and typing of VTEC were presented. For this PT, laboratories received 7 strains to carry out: I) amplification of the main virulence genes (vtx1 group, vtx2 group, eae) and of genes that represent markers of the enteroaggregative adhesion, aaiC and aggR; II) serogrouping of at least the strains that belongs to the following 13 serogroups: O26, O45, O55, O91, O103, O104, O111, O113, O121, O128, O145, O146, O157, which were selected on the basis of their prevalence in human infections in Europe; III) sub-typing of VT-coding genes (vtx1a, vtx1c, and vtx1d and from vtx2a to vtx2g); and IV) determination of the PFGE profile. Thirty-four NRLs representing 28 MS and NRLs of Norway, Switzerland, Turkey, Argentina and Russia participated. The evaluation used the penalty points system. PCR detection of virulence genes:

4 points for each incorrect or missing result on vtx genes

2 points for each incorrect result on other virulence genes

1 point for missing results on other virulence genes

Serogroup identification

4 points for incorrect identification of the ”top5” serogroups

2 points for incorrect identification of the other 8 serogroups

Subtypes of the vtx genes:


1 point for each incorrect or missing results

A sum of 4 points was considered as the threshold for under-performance in each of the sections. The evaluation demonstrated 7 laboratories underperforming on the detection of virulence genes, 3 on the serogroup identification and 11 on the subtyping of vtx genes. 24 out of 38 laboratories provided a false positive result for the vtx2c gene in strain 2, which was positive for vtx2d, the combination of the 2 subtypes is apparently very unusual and the high rate of false positive results seems to be related to insufficient temperature in PCR conditions. Flemming SCHEUTZ (WHO-International Escherichia and Klebsiella Centre) presented the results of the ECDC-FWD 5th External Quality Assessment (EQA) on the identification and typing of VTEC. Twenty-nine laboratories received 10 strains and were asked to carry out detection of virulence genes and vtx subtyping, serotyping, PFGE and phenotypic testing for production of Verocytotoxin/Shiga toxins, ESBL, β-glucuronidase, enterohaemolysin and fermentation of sorbitol. Compared to the 4

th EQA, there was, in general, an

increase in the participation and success rate of the molecular analyses and a decrease in the participation of the phenotypic analyses. Anna Aspan (National Veterinary Institute of Sweden-SE) presented some of the work they have carried out. She presented the following diagram attempting to put some figures on the weight of different sources of VTEC in humans:

They investigated differences in virulence between different variants of VTEC O157:H7 with results published (Ref: Molecular Typing of Escherichia coli O157:H7 Isolates from Swedish Cattle and Human Cases: Population Dynamics and Virulence. J Clin Microbiol. 2014; 52: 3906-12). Another

research contribution has been to prove that environmental sampling consisting of dust, overshoe, and pooled pat samples, is better than pooled, individual faecal sampling for determining the cattle herd status in naturally infected dairy herds (Ref: Environmental sampling for evaluating verotoxigenic Escherichia coli O157: H7 status in dairy cattle herds. J Vet Diagn Invest. 2013; 25: 189-98). Ms Aspan gave an overview of a slaughterhouse prevalence study sampling faeces and ear samples and highlighted the finding of positive samples that were negative to PCR screening (isolation was attempted in all samples regardless of PCR result).

Her research also included vaccination of calves with a Canadian vaccine, however 3 calves got very sick and died after vaccination and the study was abandoned. Lisa BYRNE, (Centre for Infectious Disease Surveillance and Control (Colindale), UK), presented results of 4 years (2009-2012) of enhanced surveillance in VTEC in England. The summary of cases is as follows:

Most of cases were O157 (98.8%), but she commented that it is only since December 2012 that implementation of GI PCR at frontline laboratories has taken place (as a result and in 2014 to date, 90 non-O157 isolates versus 27 O157 isolates have been obtained). Characterisation of O157 for isolates from the period 2009-2012 showed 36 different phage-types, with domination


of PT 8 and PT 21/28 (~30% each). Two-thirds of all strains carried VT2 genes, 33% encoded VT1 + VT2 while VT1 only strains were rare (n=20). Overall incidence: 1.80 per/100,000 person years. 61% bloody diarrhoea, 34.3% were hospitalised and 5.5% of VTEC cases (n=202) developed Haemolytic Uraemic Syndrome (HUS). Incidence was four times higher in rural vs urban areas. MLVA use has permitted the identification in recent years of 5 times more clusters, it is reckoned that in 2013 278 (43%) cases are part of clusters that they would not necessarily have been detected through traditional means before. Camilla SEKSE (Norwegian Veterinary Institute, NO) presented some research on E. coli O145. Since atypical Enteropathogenic (stx-, eae+) E. coli O145 isolates are prevalent among sheep in Norway, some studies were conducted to compare these with human pathogenic O145 strains. Ovine E. coli O145 were of same serotype as most of the human O145 isolates: O145:H25 and O145:H28. Human and ovine isolates were also similar with respect to their profile to 21 virulence genes and molecular genotype (PFGE and MLVA) and although all ovine O145 isolates were lacking the stx genes, they may be considered potentially human pathogenic either as a cause of diarrhoea by itself or as precursors for STEC. Ms Seske also alerted the audience of the lack of specificity of the O145 real-time PCR primers by Perelle et al. 2004 that are from the ihp1 gene. These are the ones cited in the ISO 13136:2012. She found superior results using the Fratamico et al. 2009 primers for the wzy gene. Perelle primers seemed to be specific to O145:H28 serotype and missed 37 out of 112 isolates. For that reason the Norwegian NRL has replaced Perelle primers with Fratamico primers. Martial PLANTADY (DG SANTE) presented the latest on the EU draft guidance document on the application of Article 14 of Regulation (EC) No 178/2002 as regards food contaminated by STEC. EFSA scientific opinion of VTEC seropathotype and criteria regarding pathogenicity assessment of 2013 concluded that due to the plasticity of STEC genome it is difficult to designate individual serotypes as pathogens and it proposed a molecular approach that is summarised in the following table:

The Commission wanted to guide most specifically for cases that fall in group II, and are proposing to take into account the type of food, the consumption habits and the traceability information. Moreover they are guiding with the aim of having the same rules for import and domestic production. The decision follows this diagram:

Peter Feng (Food and Drug Administration (FDA), US) of the Centre for Food Safety and Applied Nutrition, Maryland, gave a very comprehensive talk on the complexities related to making health risk decisions on results obtained from testing for VTEC in fresh produce. He is a STEC Microbiological Expert so he participates in outbreak investigations, contributes to sampling programs and acts as court witness if industry challenges results. As there are many stx producing bacteria but not all have same human pathogenicity, he explains that he gathers all information on the serotype and the stx and eae subtypes to determine the risk of the produce before deciding if a recall is needed or not. When deciding the outcome of the product it is important to have a speedy result and this is why he has adopted the use of an array to check the presence of a multitude of genes. Unfortunately the decision of what constitutes risk is not under agreement. At the CFIA Conference in Ottawa in 2010 two proposals were made for categorising strains as risky: 1) Virulence plus serotype: stx2 (but not 2e, 2f or 2g), eae, and certain O types (the “big” ones which differ in US and EU) 2) Virulence only, with the following profiles stx2a and/or 2c+eae stx2d alone


stx1a+eae (some serotypes) When 132 strains from the survey of fresh produce were checked against the above criteria only a small subset fitted in the different profiles, 1 to 15 on each, however there were some others that did not fit the criteria and were considered EHEC. The presenter who is also advisor in surveys in fresh produce remarked also the high prevalence of VTEC in spinach compared to other products. Finally he presented a report from the American Academy of Microbiology to which he contributed called E. coli: good, bad and deadly: http://academy.asm.org/images/stories/documents/EColi.pdf Stefano MORABITO (EURL) gave a presentation on Enteroaggregative and other unusual E. coli. Enteroaggregative Haemorrhagic E. coli (EAHEC), like the strain causing the German sprout outbreak, were first seen in 1992 and are nowadays considered an emerging pathogroup. The German outbreak triggered a much higher interest in the potential of “other” E. coli as human pathogens. All the Diarrheagenic E. coli groups have been observed as producing Vtx “into the wild” (excluding EIEC, so far, but including ExPEC). Even other bacterial species have been described (E. cloacae, E. albertii) as vtx positive. Lytic phages are responsible for vtx genes spread to E. coli strains. Whole genome comparison of Vtx-phages from EAHEC strains demonstrated that at least 2 vtx-phages were able to infect EaggEC recipients. In some cases these new pathogenic groups are stable and have augmented virulence potential (EAHEC). In some cases they might be spread with food although for unusual VTEC food this seems to be an accidental vehicle for human-borne disease. Food contamination with human-borne E. coli occurs most probably due to food handling. Morabito concluded that although the detection of such unusual VTEC in food appears to be not useful in the routine controls, the E. coli Network, in the framework of Regulation 882/2004, must be prepared to apply the methods for their detection in support to the epidemiological investigation, in case an outbreak or sporadic cases linked to food occur.

NRL Listeria monocytogenes

Report on 9th Annual Workshop of the EURL for Listeria monocytogenes, ANSES, Paris, March 25- 27th , 2015

NRL Representative:

Bernadette Hickey (DSL, Backweston Campus)

Participants from all MS and Norway attended the meeting. Martial Plantady, DG Sante was present on 26 and 27

th March. A wide range of topics were

discussed; including the effect of pooling on the detection of Listeria monocytogenes (Lm) and the effect of the quantity of sample used for enumeration on the results,the applicability of the draft ISO 11290-1and 2 for the detection of new species. Typing method including WGS were discussed The meeting was briefed on the progress of the establishment of the database for Listeria monocytogenes PFGE profiles from non-human sources. An update on EFSA activities was also provided. The topics of the agenda are divided into sections- General, Epidemiological context, Detection / Enumeration, Characterisation epidemiosurveillance and Microbiological Shelf Life Studies.

General and Regulatory Update Bertrand Lombard (EU-RL Lm) gave an overview of the reorganisation of EURL activities within ANSES. Changes have been made to the structure and personnel within the EURL’s in ANSES. Martial Plantady (DG SANTE) gave a short presentation on a possible amendment on the microcriteria for Listeria monocytogenes in RTE able to support the growth of Lm, Category 1.2. When the FBO is unable to demonstrate that the RTE product will not exceed 100cfu/g at the end of the shelf life then the criteria of absent in 25g before it leaves the control of FBO applies. There is no micro-criteria applicable when the product is on the market and at the end of the shelf life. Member states are applying different rules, some MS apply absent across the food chain, others apply 100cfu/g at the end of shelf life. This is leading to a distortion of markets and an un harmonised approach. The Codex has absent in 25g or another validated limit but based on information provided by the FBO. DG SANTE has a draft discussion paper that they will present to the

http://academy.asm.org/images/stories/documents/EColi.pdf

http://academy.asm.org/images/stories/documents/EColi.pdf


Micro criteria working group on 28/04/2015. They will also send out questionnaires to MS to gather information on current practices applied. NRL’s asked Martial Plantady to consider other changes to the microcriteria also, such as including vulnerable populations in Category 1.1 and changing the foot note 8 where it states that “products with a shelf life of less than five days are automatically considered as belonging to “Category 1.3 unable to support the growth of Listeria monocytogenes. Epidemiological Context Valention Rizzi (EFSA) spoke on the rise of reported invasive listerosis. The cases continue to rise and it is of great concern as the infection is acquired from consumption of contaminated RTE foods. Despite the rise in listerosis, Listeria monocytogenes was seldom detected above the levels set in the Regulation (EC) 2073/2005. RTE fishery products, mainly smoked fish are most frequently associated with non compliances with 4.6 % of single samples and 19.9 % of batches non compliant. There were 12 food borne outbreaks in 2013.The food vehicles implicated were ; crustaceans, shellfish and molluscs and products thereof (3 outbreaks of which crab meat was implicated in 2 outbreaks), cheese, meat and meat products, pig meat and products thereof, vegetables and juices and products thereof ( mixed salad) Johanna TAKKINEN (ECDC, Stockholm) presented data on human listerosis. Only invasive listerosis is reported. ECDC examined all the data from 2006 -2013 on listerosis and can conclude that 99.5% of the human listerosis infections were acquired within the EU. There were 1763 cases of listerosis in 2013, an increase of 8.6% on 2012. In 2012 there were 1642 confirmed cases of human listerosis resulting in a 10.5% increase on 2011. The EU notification rate is 0.44 per 100,000 of the population. There is a significant slowly increasing trend. The highest rate of fatality is in the male population aged over 75. However the risk is higher when over 45. There appears to be a higher incidence in the summer months. There is concern over the increasing level of listerosis as the population is aging. Jens Kirk Andersen (DTU, DE) gave an interesting presentation on the outbreak that occurred in Denmark in 2014. Denmark has the highest rate of

listerosis in the EU over the last number of years. 44 cases of listerosis were reported, 16 deaths occurred giving it a 36% fatality rate. The product responsible was Rullepølser, a Danish spiced meat. The Danish authorities were fortunate to be in a position to identify the contaminated food due to a pilot Whole Genome Sequencing (WGS )project. The Statens Serum Institute (SSI) in Copenhagen are responsible for the typing Lm isolates from humans in Denmark and also for the curation of human isolates for Listeria monocytogenes for ECDC. In December 2013 they commenced whole genome sequencing (WGS) on human isolates. In the spring of 2014 they added food isolates from official control samples. On the 28

th April an official

control samples was taken of Rullepølse and on 6

th May 2014 it was confirmed as positive for Lm.

Product was recalled. On 27th

May the authorities permitted production to recommence. In June, SSI identified 5 possible identical strains indicating an outbreak. On 3

rd July, SSI found 2/5 strains from

patients to be identical on WGS. On 16th

July the isolate from the Rullepølse food was tested and found to be identical. On the 8

th August the

Authorities sampled 10 batches (n=5) of product and some environmental samples at the FBO. 29 isolates were found to be identical by WGS to the strains from the patients. Twenty-eight of the isolates were from food and 1 from the environment. Of the 28 products samples found positive for Lm, only 4 had a count >10cfu/g when enumerated (when taken from the production plant and not tested at the end of the shelf life) and only 1 count exceed 100cfu/g. They believe the contamination occurred at the plant and was on the surface of the product. This product was then sold to other FBO’s who cut it and extended the shelf life. Following the outbreak there was a workshop with authorities, industry, universities, consumer representatives and International experts. There was an evaluation of all procedures in place. Working groups were established with expertise from controlling authorities, knowledge in food production, vulnerable population. The Danish have a set of recommendations. There should be sharing of data and isolates from self-checking, development of plans for intelligent sampling, trend analysis should be used in self checking, there should be access to the data from self checking, There are concerns about vulnerable groups and their understanding of shelf life and storing of foods


Suzanne Thisted Lambertz (SVI, Sweden) gave a presentation on a food outbreak that is occurring in Sweden for a year. In 2013, 9 cases of listerosis presented and an investigation team was formed in January 2014. The PFGE profiles were compared to the profiles from food isolates in the Swedish database. There was no exact match but 1 similar match for salmon in 2010. They compared the profiles to the EURL Lm database and found 3 matches, cheese, fresh cut meat and deli products – sausages from foods tested in 2013. The Swedish authorities thought they found the source in cold meat from an FBO in Sweden and the production line was closed. However the same pattern is presenting in listerosis in humans now making the link with the production facility uncertain. Detection/ Enumeration Damien MICHELON (EU - RL) presented the results of the inter-laboratory trial on the detection of Listeria monocytogenes in iceberg salad conducted in 2014. 34 NRL’s participated and the performance of the network of NRL’s were satisfactory. Lena BARRE (EU-RL) presented results of trials carried out on subsampling test portions. The distribution of microorganisms is heterogenous in solid matrices. The subsampling of the test portion has a large effect on the measurement of uncertainty (MU). EN ISO 6887-2-5 on the preparation of test samples for microbiological analysis does not give details on how to sub sample the test portion. In 2013 the EU-RL compared the results obtained when 100g test portions and a 10g test portions were analysed. Better results and a lower level of measurement of uncertainty were obtained when 100g test portion was taken. The use of larger test portions increases the reliability of the enumeration test results. There are technical difficulties to using 100g test portions in routine laboratories. The ISO working group on the revision of the EN 6887 series did not accept 100g but will add a note stating that

“Using larger test portions will increase the reliability of enumeration test results, particularly for heterogeneous contamination in matrices.”

The EURL want to evaluate the effect of using 25g rather than 10 g in the enumeration of Lm. They have asked for the assistance of the NRLs’ in

performing this study on naturally contaminated products.

Damien MICHELON (EU–RL) spoke on work undertaken to look for effective methods to inoculate samples to simulate a naturally occurred contamination (structure, physico-chemical parameter, microflora…). Inoculation of samples is required for preparation of PT trial, validation work, shelf life studies etc. They have carried out a bibliographical study on inoculation methods. They examined 2 methods of inoculating samples, addition of inoculum with a pipette to each individual sample portion (one by one –OBE) and addition of the inoculum to a sample matrix, blended (BC) and then subsampled. Better results we obtained when inoculum was added and blended. The OBE method was better to preserve the structure of the food but the BC method was less labour intensive and was more reproducible and the results were less varied.

Nathalie GNANOU–BESSE (EU- RL) briefed the meeting a study to evaluate the loss in the sensitivity of the method to Detect Lm when pre enriched samples are pooled.

EN ISO DIS 6887-1; “Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - General rules”, permits the use of pooling for qualitative test procedures when the procedure has been validated. Two types of pooling are proposed in EN ISO DIS 6887, dry pooling and wet pooling. In dry pooling when n= 5 then 125 g (5 x 25g) is weighed and 1125mls of enrichment broth is added. In wet pooling each 25g sample is enriched separately and then combined in the selective broths. A protocol on how to carry out the validation is listed in Annex D of EN ISO DIS 6887-1. The EURL Lm developed and validated a model for the growth of Lm in half fraser broth and can then calculate the loss in sensitivity when samples are pooled. They calculated a 10% loss in sensitivity. They have not carried out a study on dry pooling and do not intend to do so as they know that loss in sensitivity is higher in dry pooling and they do not consider that this is acceptable.

Lena BARRE (EU-RL) gave a presentation on the applicability of the draft EN ISO 11290-1 and 2 on the detection and enumeration of Listeria monocytogenes and Listeria species. The previous


versions of the methods were for the Detection and enumeration of Listeria monocytogenes but it was agreed at ISO that the method should also include Listeria spp. However new Listeria spp .are being identified all the time and there are now 17 Listeria spp. The EURL undertook a study to assess the suitability of the draft methods for the detection and enumeration to the new Listeria spp. The results are preliminary and more work needs to be undertaken. There was difficulty in detecting the following; L. cornellensis, L. grandensis, L. rocourtiae and L. weihenstephanensis after pre enrichment and enrichment. Issues were encountered with the Voges Proskauer test proposed for identifying Listeria spp.

The gene prs specific for Listeria species was not detected in 9 out of the 13 strains.

Shelf Life Studies Annie BEAUFORT (EU – RL) informed the meeting that the revision of the EURL Lm Technical Guidance Document for conducting shelf-life studies on Listeria monocytogenes in ready-to-eat foods (2014) was completed and accepted by SCoFCAH in 2014 . In 2014 a working group was established to develop a CEN ISO standard for food challenge tests.

Anne Laure LAURDEUX (EU- RL) presented the results of a study using an air brush to apply surface contamination on product for challenge studies.

Helen BERGIS (EU- RL) informed the meeting that they had obtained permission to evaluate the temperature data collected from retail during the baseline study to see the temperature distribution in the MS. More than 10,000 temperature measurements were collected during the survey. The temperature of the retail cabinet and the surface temperature were recorded. Characterisation and Epidemiology Damien MICHELON (EU – RL) gave an overview of the results obtained by the NRL’s in the Proficiency trials for typing and characterisation. The PT covered typing by conventional agglutination, molecular and characterisation by PFGE. 11 strains were despatched to the NRL’s. 32 NRL’s registered to participate with different numbers of NRL’s participating in the different typing procedures. 12 NRL’s performed all 3

methods. The issues that gave rise to the main deviations were discussed. Benjamin FELIX (EU – RL) and Valentina RIZZI ( EFSA) presented progress on the development of the European Reference Laboratory Listeria monocytogenes database (EURLLmDB). EFSA published a vision paper on molecular testing of pathogens in December 2012. The EU RL LM database will move to EFSA where all the databases for non- human isolates for pathogens will reside. A lot of progress has taken place over the year in conjunction with the EURL’s for Salmonella and E.coli on harmonisation of protocols for analysis, interpretation, evaluation of profiles and maintenance of database. Work is on -going between EURL, DG SANTE, EFSA and ECDC on harmonisation and implementation of the databases. The EFSA database will be piloted during 2015. The call for participants will be made to the competent authorities in Member states. The EURL have developed software and scripts to allow them to act as a coordinator for other laboratories in France. The network of French laboratories can submit data to the EURL for Listeria. Eva Moller NIELSEN (SSI Denmark) and Charlotta LOFSTROM (DTU Denmark) gave a presentation on Whole Genome Sequencing (WGS) for outbreak detection and investigation. WGS permits the typing of isolates in real time and this is the best way to detect clusters and outbreaks. The SSI commenced using WGS in December 2013 on their isolates obtained from humans and operated a pilot study on food isolates from official controls in 2014. Prior to WGS SSI were using different techniques and different sera to identify multiple pathogens. They were unable to carry out some of the characterisation in real time as they needed sufficient isolates of the same pathogen to run a technique. WGS permits the laboratory to have harmonised protocols for the pathogens once the DNA is extracted instead of different techniques. There are some drawbacks to WGS at present. Currently there are no harmonised protocols on analysis, validation and the interpretation of data and its relatedness in epidemiology. The pilot study will undertake studies to evaluate the possible sources of errors with WGS such as contributions from sequencing errors, different sequencing platforms and different data analysis pipelines


NRL Anti-microbial Resistance

Report on the EURL AR Workshop, Copenhagen, Denmark, April 23-24th, 2015

NRL representative:

Rosemarie Slowey (Bacteriology, Backweston)

Rosa PERAN (EU Commission) gave an update on the EU Action Plan to combat Antimicrobial Resistance (AMR). This was a 5 year action plan, written in 2011, which comprised 12 actions in 7 areas. Actions 2 and 3 relate to the prudent use of veterinary antimicrobials and in 2014 the Commission adopted a proposal on veterinary medicines. The revised veterinary medicines legislation will deal with the prudent use of antimicrobials in the veterinary sector, the preservation of critically important antimicrobials and the strengthening of the regulation of the use of medicated feed for animals. A proposal on the prevention of bacterial infections in animals has been adopted by the Commission (Action 5). Scientific advice on the development of new antimicrobials for veterinary medicine has been published (Action 7). There have been international and bilateral talks on the problem of Antimicrobial Resistance (Action 8). Surveillance systems on AMR have been strengthened and a report on the monitoring of Veterinary Antimicrobial Consumption was published in 2014. Decision 2013/652/EU has also come into force, which allows for harmonised monitoring of antimicrobial resistance in the agricultural sector (Action 10). Communication, Education and Training (Action 12) in the area of AMR is on-going and has included EURL training of NRL staff.

Pierre Alexandre BELOEIL (EFSA) spoke on enhanced cooperation between agencies, which has seen ECDC and EFSA publish joint summary reports, ECDC, EFSA and the EMA publish the JIACRA report and EFSA and the EMA working together on a new EC mandate on alternatives to Antimicrobial use in agriculture. In preparation for the reporting of data generated by testing done under Decision 2013/652/EU, training has been given to relevant personnel in MS on data reporting, guidance documents have been published and data models updated. It is anticipated that the 2014 report will be as concise as possible, with some data appearing in

appendices, and that it will allow comparison with human data and year- on – year comparison of results for MS. There will continue to be a focus on Multi- drug resistance and co-resistance to critical antimicrobials. The use of aggregated data and inferred phenotypes was proposed and discussed at the meeting.

Gunnar KAHLMETER (EUCAST) explained how ECOFFs are set for bacteria. He explained the importance of sample size, including data from investigators from several geographical regions and the removal of results that came from testing with truncated MIC distributions. The latter is a factor which affects data from the new standardised plates, which limits the use of the data gathered by EFSA for setting the missing ECOFFs; the dilution ranges in the standardised plates are insufficient to allow a complete MIC distribution to be plotted.

Cristina GARCIA- GREALIS (NRL, BE) reported her findings on the use of TBX agar in E coli isolation as part of 2013/652/EU; there were fewer “false positive” colonies using the TBX compared to Mc Conkey and a confirmatory step was not necessary.

Therese WESTRELL (ECDC) gave an update from the Food Waterborne Disease Network. In 2013, isolates approximately 19% and 15% of laboratory confirmed cases of Salmonella and Campylobacter, respectively, underwent Antimicrobial Susceptibility Testing. There is no compulsory testing of human- derived isolates and the methods used in laboratories are not as standardised as on the veterinary side. In 2014, it is anticipated that the FWD Network will undertake external QA in association with Statens Serum Institute. The 2013 ECDC/ EFSA summary report was written with the aim of allowing comparison of human and animal data. Cases that were recorded as travel- associated were dropped from the datasets. The AST results for S. typhimurium, S. enteritidis, S. infantis, and S. derby and C. coli and C. jejuni were considered separately in the report. The limitations for the 2014 report include the fact that some countries do not document patients’ travel histories, that not all report all drug/ bug combinations and that the veterinary testing panel is more extensive than that used on the human side.

Frank AARESTRUP (EURL) reviewed the use of WGS (Whole Genome Sequencing) as a tool in the monitoring of resistance genes. Traditional AST


requires comprehensive standardisation of methods and has been found to have an inaccuracy rate of about 9%. WGS has the potential to simplify the workflow in the lab and to provide information that previously would have required multiple tests. Next Generation Sequencing (NGS) appears to be equal/ superior to phenotypic testing in terms of accuracy. It has the potential to give a complete picture of clones and resistance genes that are circulating.

Jean PATEL (CLSI) gave an update on ISO and CLSI standardisation of resistance genes. It is anticipated that future versions of the M100 standard will include guidelines on the interpretation of genotyping results. It is hoped that in 2016 interpretative criteria for the identification of bacteria and fungi by DNA/ target sequence will be published. The second edition of the “guidelines on nucleic acid sequencing methods in diagnostic laboratory medicine” has been published. When setting standards for new methods, such as NGS, a method should be selected that produces reproducible results and detects biological changes. The results produced by testing should allow early and clear treatment decisions to be made and should generate a set of resistance data that would guide future empiric therapy. NGS methods can range from high resolution (short and long reads with de novo assembly) to low resolution (targeted genomic sequencing with reference assembly). Elements of the testing that need to be standardised include which data are used to make decisions, the level of correlation needed to recommend interpretation and the development of QC parameters. In order to set standards in this area a method would have to be selected, QC parameters set for it, a pathogen/ matrix combination selected to work on and interpretative guidelines set for it.

Danilo Wo Fo LONG (WHO) described renewed global efforts that have been taking place aimed at increasing awareness of AMR. The Global AMR Action plan is based on the principles of improving awareness, strengthening knowledge and the evidence base for AMR, decreasing the incidence of infection, optimising the use of Antimicrobials and developing the economic case for sustainable development. AMR surveillance in the human sector is expanding in Europe with the work of EARSNET and in Asia and Eastern Europe through the CAESAR network. Antimicrobial use surveillance is also being strengthened.

Henk VAN ORMEL (FAO) explained how his organisations aims which centre on the development of sustainable agriculture and the elimination of hunger could not be achieved without finding solutions to Antimicrobial Resistance. He outlined how increasing demand for animal protein worldwide will put pressure on agricultural production systems. He described the routine use of antimicrobials in the US production systems and how in poorer regions of the world, the selling of antimicrobials is completely unregulated. However, Tuberculosis is a huge problem in developing countries and MDR has emerged in spite of TB antimicrobials not being used in agriculture. The FAO has been involved in initiatives such as the development of laboratory capacity, public education and CPD schemes in developing countries.

Pierre Alexandre BELOEIL (EFSA) gave an overview of the JIACRA report (Joint Report on the Integrated Analysis of the consumption of Antimicrobials agents and the occurrence of antimicrobial resistance in bacteria from humans and food). The report looked at consumption using a population correction unit compared to summary indicators of resistance and the total tonnes of active substance compared to the estimated biomass of humans/ animals. The latter analysis was done, animal consumption of antimicrobials was less than human consumption in 15/26 countries (including Ireland), similar in 3/26 countries and greater in 8/26 countries. There was no association between the use of 3

rd/

4th

generation Cephalolporins in animals and resistance to these drugs in bacteria of human origin. However, the consumption of Fluoroquinolones, Macrolides and Tetracyclines in the agricultural sector was positively associated with resistance in bacteria from humans. The report concluded that the epidemiology of resistance is complex and several factors as well as antimicrobial consumption are involved.


NRL Residues

Report Annual Workshop on EURL Residues, Berlin, 16th-18th June, 2014

NRL Representative:

Lynda Harman (VPHRL, Backweston Campus)

This workshop was presented by Joachim Polzer, Head of EURL for Residues, Berlin. He was assisted by scientists, technical assistants and the workshop secretary from EURL Berlin. The workshop was attended by 30 participants from EU National Reference Laboratories, 2 external guest speakers and 1 European Commission representative. The workshop was split into two parts; the theoretical part was for two days and the practical part for the final day. Joachim POLZER and Petra GOWIK (EURL) presented an outline of the 2014 workshop program. The results of the surveys from the 2013 workshop were synopsised including suggestions made for topics to be included in the 2014 workshop. An overview of EURL activities for the past few years including training, preparation of standards and proficiency testing was presented. Of particular interest was a website FIS-VL.bund.de. This website FIS-VL is an Information System for Consumer Protection and Food Safety. FIS-VL is accessible for all members of authorities at federal, state and regional level, meeting tasks in the area of consumer protection and food safety and is a web-based platform supporting information exchange and collaboration between members (to become a member, registration is via a sign-up procedure). Also a draft standard “Performance Characteristics for Multi Residues Methods (MRMs) for Veterinary Drugs” has been sent to Codex Alimentarius Commission for adoption at the meeting in July 2014. Frank SWARTENBROUX (DG SANTE) spoke about recent and future developments in EU legislation related to residues. The main points of news from the Commission were a proposal of a new food and animal health regulation, reference points for action and allowed substances and zero tolerance. The proposal of a new food and animal health regulation will probably be adopted in 2016 and will replace 96/23/EC (the annexes will remain valid until replaced). For RPAs there will be follow-up discussions with an EU working group in autumn with a view to working out procedural

aspects and this should lead to new legislation to replace 2005/34/EC. For allowed substances and zero tolerance residues of allowed substances these should be judged based on risk assessment rather than analytical capability. Zero tolerance should be restricted to residues of non-allowed substances. Future work is hoped to include a revision of Commission Decision 2002/657/EC to include validation of multi-compound/multi-class methods and new technologies, cascade MRLs and sampling muscle versus injection sites. Wolfgang RADECK (EURL) presented the first validation results of a multi-residue screening method of group B compounds using LC-Q-TOF. Due to economic pressure and increasing numbers of analytes to be monitored multi-residue and multi-class methods have become the focus in recent years. The aim of this work was to complete a comprehensive validation of a multi-residue screening method. The matrices used were muscle and liver of bovine, porcine, ovine, equine and poultry samples covering more than 200 regulated analytes at their levels of interest. The validation study included 47 anthelmintics, 35 coccidiostats, 41 NSAIDs, 103 antibiotics and 18 sedatives. Two different extractions were used depending on sample type and analyte. The experimental design for validation was 16 runs; each run contained 10 samples (8 spiked, 1 matrix blank and 1 reagent blank). The spiking levels were 2, 5, 25, 50, 100, 150, 200μg/kg. The data was acquired qualitatively using mass and retention time in three ways, auto MS/MS for unknown screening, targeted MS/MS using a spectra library and all ion MS/MS using a spectra library. It was also acquired quantitatively which has been extremely time-consuming and has not been completed yet. The developed and validated method now allows screening of muscle samples for over 180 analytes. More work will be done to develop the quantitation method. Frank HAMANN (EURL) spoke about antiparasitics and their groups according to Council Directive 96/23/EC. He also gave more detail about the previously mentioned internet platform FIS-VL. He went step-by-step through the sign-up procedure available and encouraged participants to sign up. Manfred STOYKE (EURL) spoke about the NRCP and news regarding NSAIDs. According to regulation (EU) 37/2010 NSAIDs are authorised for food-producing animals and most have an MRL. The non-compliant results for this group have varied from 14 to 56 samples per year since 2004.


The most frequently found analytes were phenylbutazone, diclofenac, ibuprofen, tolfenamic acid, metamizole and meloxicam. This shows a probability of misuse of these drugs in food-producing animals. There is also possible contamination with drugs frequently used in human medicine via feed or drinking water in the production of food of animal origin. To select the matrix analysed we must look at the importance for human consumption, (e.g. frequency of consumption or children’s food) and have targeted sensitive detection of banned veterinary drugs. In most member states milk is analysed for NSAIDs including pharmaceuticals used in human medicine (e.g. ibuprofen). The average number of analytes is eight per member state. The minimum should be phenylbutazone, flunixin, diclofenac, metamizole, tolfenamic acid, carprofen, ibuprofen and naproxen. Peter FURST (EFSA) spoke about establishing RPAs, the EFSA CONTAM panel approach. A veterinary medicinal product (VMP) may not be placed on the market in a member state unless a marketing authorization has been issued by the competent authorities of that member state or by the centralised procedure. A VMP may only be authorized if residues of the pharmacologically active substance in foodstuffs produced from treated animals will be harmless to consumers, in accordance with regulation (EU) 470/2009. These substances are listed in table 1 “allowed substances” in regulation (EU) 37/2010. The use of other non-authorised pharmacologically active substances is not allowed, a specific list of non-allowed substances is listed in table 2 “prohibited substances” in regulation (EU) 37/2010. Substances which are not classified as allowed substances need a reference point for action established to ensure the functioning of controls of food of animal origin. Food of animal origin containing residues of such substances at or above the RPA is considered not to comply with community legislation. In current practice the RPAs take into account the lowest residue concentration that can be quantified with a validated analytical method and are based on minimum required performance limits (MRPLs). Currently no consideration is given to the toxicological profile of non-allowed substances when establishing RPAs. RPAs for chloramphenicol, nitrofuran metabolites, medroxyprogesterone acetate and malachite/leuco-malachite green are laid down in Annex II of Commission Decision 2002/657/EC. The new approach proposed by the

EFSA CONTAM panel takes into account both analytical and toxicological considerations. The aim is to define an analytical concentration for a non-allowed substance that can be determined by official control laboratories and is low enough not to pose a health concern for consumers. It is important to note that this EFSA guidance does not replace a full risk assessment. The proposed step-wise approach considers many factors such as analytical capability, toxicity, pharmacological activity and also includes the establishment of a reasonably achievable lowest limit of quantitation (RALLOQ), a toxicological screening value (TSV) and a toxicologically based limit of quantitation (TBLOQ). Finally the CONTAM panel considered whether RPAs should be set for different matrices based on consumption patterns and tissue distribution characteristics of pharmacologically active substances. Setting values for all possible matrix/substance combinations was considered to be impractical so the panel concluded that the RPAs should be matrix independent and should take into account the overall intake of food of animal origin. Renat SEMILOV (Russian State Centre for Quality and Standardization of Veterinary Drugs and Feed, RU) gave an overview of their activities in the national residue monitoring program of Russia. This centre operates as a screening and confirmatory laboratory for the monitoring program for control of residues of veterinary drugs and environmental conditions in Russia. They have developed over 50 methods to detect toxic metals, mycotoxins, PCBs, pesticides, hormones, antibiotics, coccidiostats, nitroimidazoles, dyes and NSAIDs and currently analyse for over 200 compounds. They also organise proficiency tests for regional laboratories and participate in international proficiency tests. Manfred STOYKE (EURL) presented the evaluation of the 2013 proficiency test for coccidiostats in poultry muscle, liver and egg. The aim of this proficiency test was to promote the residue analysis of coccidiostats in different poultry matrices. The minimum required analytes are diclazuril and nicarbazin (in egg), lasalocid, maduramycin, monensin, narasin, robenidine and salinomycin. Analytes recommended for detection are amprolium, decoquinate, clopidol, halofuginone, laidlomycin propionate, nequinate, semduramycin and toltrazuril. The summary of 2012 non-compliant coccidiostat samples showed that more than 70% of non-compliant samples


were either poultry or egg so these matrices were chosen for the 2013 proficiency test. The proficiency test samples were prepared in EURL Berlin and their homogeneity and stability were analysed. The proficiency test participants included 26 NRLs, 2 official laboratories, 9 routine field laboratories and 9 other laboratories from the rest of the world. 45 laboratories returned confirmatory results while 1 laboratory did screening only. The most frequently analysed compounds were salinomycin, narasin, monensin, robenidine, maduramycin, diclazuril, nicarbazin and lasalocid and only 27 laboratories analysed for toltrazuril sulfone. There were a relatively high percentage of false negative results (34%). All laboratories used LC-MS/MS for analysis which showed a very low probability of false positive findings. Beatrice GIFFEI (EURL) spoke about an investigation into the influence of phospholipids on veterinary drug analysis. Phospholipids can be problematic in residue analysis as they can cause ion suppression or enhancement which may affect quantitation and lead to false positive or false negative results. Their effects were studied and analysed by LC-MS/MS using existing EURL methods for NSAIDs and coccidiostats. The conventional SPE used for NSAIDs was ISOLUTE®C18 (EC) and for coccidiostats was Oasis HLB®. The alternative clean-ups for selective phospholipids removal investigated in this study were HybridSPE® phospholipid from Sigma-Aldrich and Phree™ Phospholipid removal from Phenomenex.

NRL the Control of Antimicrobial Residues

Report on 19th Workshop for the Control of Antimicrobial Residues in Food from Animal Origin LC-MS/MS Method for Aminoglycosides in Meat and Milk, Fougères, France October 2-3rd, 2014

NRL Representative:

Jean Kane (VPHRL, Backweston Campus)

Pascal SANDERS (EURL) welcomed participants to the annual EU-RL workshop. The 2-day workshop was attended by 33 participants, 28 from EU member states, 2 from candidate countries, 2 from EFTA countries and 1 representative of the European Commission. Eric Verdon (EURL) re-iterated the welcome and addressed some workshop logistical and administrative issues.

Eric VERDON (EURL) gave an overview of EU-RL activity for the period 2013-2014. This included: Method development, evaluation and validation of microbiological/immunological methods and physicochemical methods for the analysis of antibiotics in various matrices; Proficiency testing studies; Visits to NRLs; Training sessions for NRLs network; Specific support/expert study visits and other representative activities. The most recent EU-RL publications on antibiotic residues were listed. Feedback from EU-RL Workshop 2013 was reviewed and the response to the questionnaire issued to NRLs for 2014-2015 activities was analysed. The presentation concluded with an outline of the 2-day workshop programme.

Frank SWARTENBROUX (DG SANTE) presented news from the EU Commission. There is a proposal for a new food and animal health regulation. The proposal is expected to be adopted in 2016 however progress depends on the position adopted by the incoming European Parliament. The new regulation repeals Directive 96/23/EC but annexes will remain valid until they are replaced. A generic opinion on Reference Points for Action (RPAs) has been published however issues regarding chloramphenicol and nitrofurans are on-going. Discussion will continue at the next residue working group meeting.

Eric VERDON and Valerie GAUDIN (EURL) presented a short overview of the control for aminoglycosides in the 2014 EU National Residue


Monitoring Plans (NRMPs). NRMPs were sorted by species/products (red meat, poultry meat, milk, eggs, aquaculture and honey). The review included details of aminoglycosides analysed, screening/confirmation methods used, analytical limits proposed and the number of member states analysing for aminoglycosides. Aminoglycosides account for about 2% of antibiotic usage. Streptomycin, neomycin and gentamicin are the most commonly used. There are fewer methods for milk, eggs and aquaculture than red meat, poultry meat and honey. CCα and CCβ values vary greatly depending on method, matrix and species.

Valerie GAUDIN (EURL) evaluated biological methods for the screening of aminoglycosides. Screening methods should be wide spectrum, sensitive, rapid, cheap, easy to use and have high sample through-put. Methods discussed were Receptor tests, Elisa, Radioimmunoassay, Charm test, Ampoule/microplate, Plate tests and commercially available Biosensors. Advantages and disadvantages of the methods were outlined. The presentation concluded with an update on new developments in Biosensors.

Murielle GAUGAIN (EURL) presented an analysis of aminoglycosides in veterinary medicine. Aspects covered were chemical and physical properties, pharmacology, therapeutic indications, European regulations and laboratory strategy. Aminoglycosides are white powders, highly soluble in water and stable in the presence of heat, acid and base. They are highly basic and hydrophilic compounds making their extraction from matrices and chromatography difficult. A study involving the analysis of muscle and kidney samples from two pigs treated with gentamicin indicated that aminoglycosides have a high affinity for kidney making it the target matrix. Aminoglycosides are among the most toxic antibiotics. They are a group for which antimicrobial resistance is growing rapidly hence some aminoglycosides are reserved for human medicine only. European regulations have set maximum residue limits for aminoglycosides however apramycin and paromomycin are not permitted for use in animals producing milk for human consumption.

Michele LAURENTIE (EURL) gave a presentation on aminoglycoside pharmacokinetics and residue depletion. Residue depletion depends on the pharmacokinetic process which consists of four phases: Absorption, Distribution, Metabolism and Excretion (ADME). Aminoglycoside residue depletion studies show high levels in kidney, the

second target tissue is liver with low levels in muscle and fat. Factors influencing residue depletion are formulation used, route of administration and physiological conditions such as the age and health of the animal. Tables detailing aminoglycoside residue depletion, MRLs for aminoglycosides and withdrawal times for aminoglycosides were presented. Apramycin and paromomycin are not permitted for use in animals producing milk for human consumption. Kanamycin, spectinomycin and paromomycin are not permitted for use in animals producing eggs.

Patricia MUNOZ (CNA-AECOSAN, ES) outlined the development of a method for the analysis of aminoglycosides in eggs by UHPLC-MS/MS. The method focused on non-authorised aminoglycosides. The selection of extraction mixture, SPE, UHPLC and MS conditions were described. The optimum pH for the extraction of aminoglycosides is pH 1 to 2. Compounds are polar and have poor solubility in organic solvents. They also adhere to glass which should be avoided in the extraction process. Weak cation exchange (WCX) is the SPE of choice. Direct injection of the SPE eluate gave better chromatography and more reproducible results. Best resolution, peak shapes and responses were obtained with 20 mM HFBA in the mobile phase. Critical stages of the procedure include pH adjustment, LC gradient optimization (organic/aqueous percentage and curve profile at each step) and fine adjustment of HFBA in the mobile phase. The method was validated as a qualitative/confirmatory method in accordance with Decision 2002/657/EC. Further work is required to improve the chromatic separation of gentamycin C2 and gentamycin C2a. Quantitative validation in milk is in progress with promising results.

Marie-Pierre CHOTARD (EURL) presented a method for the detection and dosage of aminoglycoside residues in kidney by LC-MS/MS and application to muscle and milk. Standard preparation and storage, sample preparation, extraction procedure, LC-MS/MS method, quantification and decision making were outlined. The adsorption of aminoglycosides on glassware was again stressed and the preparation of aminoglycoside solutions in polymethylpentene (PMP) or polypropylene (PP) flasks is recommended. The method is divided into three steps: liquid/liquid extraction after acidification, ultracentrifugation and injection in the LC/MS-MS system. To optimize chromatographic conditions


and stability aminoglycosides are injected in 5% TCA (not water). Two internal standards are used for quantification, tobramycin and dihydrostreptomycin. The response function is quadratic (y=a+bx+cx

2) for spectinomycin and

linear (y=ax+b) for all the other aminoglycosides analysed. The method was validated in accordance with Decision 2002/657/EC. The experimental plan, trueness and precision data, analytical limits and results of stability in kidney were presented. The method was slightly adapted and validated for muscle and milk. Adaptions include working standard concentration, internal standard concentration and injection volume. Tables detailing validation results for muscle and milk and stability studies in both matrices were presented.

Murielle GAUGAIN (EURL) discussed ‘Aminoglycosides in Muscle - Solid Phase Extraction (SPE) and Matrix Effects’. Matrix effects in pork, turkey, chicken, and beef muscle after liquid/liquid extraction were assessed by comparing responses obtained for matrix-matched standards to that of reagent standards. Tabulated results were presented. Solid phase extraction tests were carried out on strong cation exchange, weak cation exchange and polymeric cartridges from various suppliers. Plexa polymeric cartridges from Agilent showed promising results. SupelMIP cartridges for aminoglycosides prepared by Supelco are currently under test.

Murielle GAUGAIN (EURL) presented stability study designs for standard solutions, matrix samples and a statistical approach. For standard solutions the mean response of 6 injections of stored solution (Ci) is compared with the mean response of 6 injections of freshly prepared solution (Co). Injections of fresh and stored solution are alternatively injected into the LC-MS/MS to eliminate effects of equipment. For matrix samples the mean concentration of 3 matrix samples at each term of the stability study (Ci) is compared with the initial concentration (Co). Stability criteria for standards and matrix is 80% < Ci/Co *100 < 120%. The statistical approach outlined the calculation of independent analyses required to detect the selected target percentage difference.

Murielle GAUGAIN (EURL) described specific cases regarding kidney and muscle control of aminoglycosides. Remote access to the instrument was possible and chromatograms and spectra were displayed. The first case was a muscle sample from the national plan which was suspected to

contain antibiotics when screened by a microbiological test. Further screening by LC-MS/MS indicated the suspect presence of neomycin and dihydrostreptomycin (DHS). The sample was analysed by the ‘Confirmation of Aminoglycosides by LC-MS/MS’ method. Criteria for a valid batch of analysis were outlined: presence of the internal standard in all samples; presence of 2 chromatographic peaks (1 for each transition) in the ½MRL level spiked sample with a signal to noise ratio greater than 3; CCα < CCα max and R

2 > 0.97. Neomycin and DHS levels less than

their respective CCα values were confirmed present in the sample. The sample was deemed compliant. Discussion included specificity problems between neomycin and paromomycin and chromatographic separation of the two is recommended. The second case was a kidney sample from a pig treated with gentamicin. The sample was diluted by 1/20

th with negative

material. The levels of gentamicin C1a, gentamicin C1 and gentamicin C2 in the standard were calculated by applying the percentages from the certificate of analysis of the gentamicin standard. The sum of gentamicin C1a, gentamicin C1 and gentamicin C2 in the sample was then calculated using the sum MRL calculation outlined in SANTE/2004/2726-rev-4 Dec. 2008 Guidelines for the implementation of Decision 2002/657/EC.

Regine FUSELIER (EURL) presented an overview of 3 proficiency tests for banned substances: carbadox in pig muscle, dye residues in prawns and chloramphenicol in urine and pig muscle. A PT for carbadox in pig muscle was organised in 2013. Pigs were treated with carbadox, depletion and homogeneity studies were carried out and samples of incurred material were sent to 18 participants for analysis. All 18 participants used LC-MS/MS technologies. 11 participants applied methods for the analysis of both QCA and DCBX and 7 analysed for QCA only. For QCA 11 of 18 participants used validated methods. For DCBX 3 of 11 participants used validated methods. Raw data results were presented as the standard deviation of the consensus assigned values were too large to evaluate proficiency according to z-scores (ISO 17043). Participants could still evaluate their results by comparison with the assigned value. A PT for dye residues in prawns was organised in 2013. The aim of the PT was the evaluation of the participant’s proficiency to detect, confirm and quantify dye residues and their leucobase in prawns. Prawns were treated with malachite green, crystal violet and brilliant green.


Quantification, homogeneity and stability studies were carried out and incurred samples were sent to 24 participants for analysis. All results were submitted before the deadline, 23 of the 24 participants used LC-MS/MS technologies. Three participants obtained false positive results in the blank material. Z-scores were calculated for material 2 (LMG) only. Two participants obtained questionable z-scores and one participant obtained an unsatisfactory z-score. For the other materials/results raw data was again presented as the standard deviation of the consensus assigned values were too large to evaluate proficiency according to z-scores. A PT for chloramphenicol residues in pig muscle and urine was organised in 2014. Incurred material was obtained from animals treated with chloramphenicol palmitate and blank material was collected from animals before treatment. Samples were analysed to verify the target concentration and to verify the absence of chloramphenicol in the blank materials. Samples were sent to 45 participants but 2 participants did not return results. There were a large number of false positive results reported for the blank materials. Statistical evaluation of material 3 showed good results with all z-scores below 2. There was no statistical evaluation of material 4 due to a stability issue. Results for materials 5 and 6 were fairly scattered with one z-score > 5.

Detlef BOHM (BVL, DE) outlined a PT for multi-antibiotics in pig muscle. Three 3kg samples of pig muscle were homogenised. Two samples (A and B) were spiked at 1.5MRL with 4 antibiotics selected from 8 antibiotic groups. Sample C was used as a blank sample. Portions of 30g were deep-frozen and shipped with dry ice to 17 participants within 24 hours. Homogeneity studies were carried out on 12 samples randomly chosen and analysed in duplicate. Stability studies indicated that all substances were stable for 5 weeks at -25⁰C and with the exception of penicillin G for 3 days at 4 ⁰C. On evaluation of the participants results no substances were detected in the blank sample, there were two false negative results (penicillin G in sample A and tiamulin in sample B) and one false positive result (doxycycline in sample B). 14 laboratories investigated all substances. 89.8% of results were satisfactory, 5.1% questionable and 5.1% unsatisfactory.

Regine FUSELIER (EURL) presented news on PTs in progress at EU-RL Anses-Fougeres. A PT for multi-antibiotics in milk from various species is on-going. Samples for screening and confirmation will be

dispatched in December 2014 (or possibly January 2015) to NRLs in charge of antibiotic residue control in milk. Samples should be analysed within one month to avoid stability issues. Results will be evaluated for false positive/negative results and the assigned values will be calculated. The final report will be issued in March 2015. PTs for nitrofurans (matrix not specified) and multi-antibiotics in pig muscle are planned for 2015.

Sophie MOMPELAT (EURL) presented ‘Fate of Ceftiofur in Poultry and a metabolomics approach’. Ceftiofur is a 3

rd generation cephalosporin

administered by subcutaneous injection or direct injection in eggs to prevent early mortality in one day old chicks. A possible outcome of use is the emergence of extended-spectrum cephalosporin (ESCs) resistant E. coli and Salmonella. The main aim of the study was to find a relevant analytical strategy to monitor ceftiofur in poultry. Targeted and non-targeted approaches were explored and results and conclusions presented. There is no apparent persistence of ceftiofur in muscle, plasma or kidney. Further matrices should be explored ( feather), for possible longterm persistence of ceftiofur. Increased knowledge of the identity, specificity and persistence of ceftiofur markers is required.

George KAKLAMANOS (RIKILT, NL) described a recent case study in the Netherlands regarding the detection of nitrofurans in farm animals. Nitrofurans are synthetic broad-spectrum antimicrobial agents. Due to their genotoxic and carcinogenic potential they have been banned for use in most countries. Detection of nitrofurans in meat products relies on the determination of their corresponding metabolites. The metabolites accumulate in tissue where they are stable and can be analysed long after administration. ELISA screening methods and LC-MS/MS screening and confirmatory methods have been developed for nitrofurans in various matrices including eggs, liver, muscle etc. However, there is limited availability of developed methods for the detection of nitrofurans at farm level in matrices such as urine and serum. In the case study outlined urine, meat and feed samples were found positive for furazolidone. The feed producer was thought to be the source of the nitrofurans as additional cases of positive samples later in the year were linked to the same feed producer. Feed raw materials from the feed producer have been analysed for nitrofurans but the source of the contamination is still not clear. An outline of the


methods applied to feed, meat, urine and serum was described. The presentation concluded with a general discussion on nitrofuran analysis by EU member states.

Anton KAUFMANN (Kantonales Labor, CH) presented ‘HRMS based confirmation of positive findings equivalent to CD 2002/657/EC’. The presenter stated that the possibility of false positive or false negative findings when following Commission Decision (CD) 2002/657/EC is rare but possible and even likely in some situations. The confirmation of sebutylacine in tarragon was presented as an example. A sample was confirmed positive by LC-MS/MS based on CD 2002/657/EC criteria but was shown to be negative when additional voluntary tests were performed. Experiments to compare the likelihood of false positive/negative results when applying high resolution mass spectrometry (HRMS) criterion versus CD 2002/657/EC criteria for triple quadropole (QqQ) systems indicated that false positives and false negatives are less likely by using HRMS criterion. In the case of QqQ, ion ratio deviation is the main cause of false negative results. Ion ratio can be affected by signal suppression and distorted by matrix peaks. When applying CD 2002/657/EC criteria, a deviating ion ratio deems a suspected peak as a false detect even if the concentration is above the CCβ. Commission Decision 2002/657/EC uses ion ratio as ‘killer criterion’ as it over rules CCβ which is a statistically solid concept. CD 2002/657/EC was written when QqQ instruments were not as sensitive, baselines were flat and no interfering compounds were visible. QqQ sensitivity has improved but selectivity has remained stagnant. The presenter concluded: CD 2002/657/EC was an innovative document when it was written but now needs major revision; QqQ instruments are not sufficiently selective for the sensitivity they provide; HRMS based confirmation meets or exceeds QqQ performance; and ion ratio are dangerous when they are used as ‘killer criterion’.

Celia Suarez PANTALEON (CER Groupe Belgium) described a flow cytometry immunoassay method for the detection of antibiotics in porcine tissue (including aminoglycosides). Due to the wide range of veterinary antimicrobial agents available there was a need for a broad range screening method for antibiotic residues in meat. Challenges included the number of antibiotics, their different physicochemical behaviour and sensitivity requirements (dependent on EU regulatory limits).

The ‘Bead Plex’ kit was developed as a screening multi-residue flow cytometry-based immunoassay able to detect the most relevant members of 10 antibiotic families in porcine tissue at or below their regulatory limits. In-house validation following CRL guidelines for screening methods demonstrated that the prototype kit could be a candidate to be implemented into national control plans for the screening of antibiotics in porcine meat. The kit is rapid, specific, robust, user friendly and capable of high sample through-put. Identification is at family level which reduces the costs of confirmatory analysis. The next step is to organise an inter-laboratory study to compare results of the Bead Plex kit with those of reference physicochemical methods.

Eric VERDON (EURL) demonstrated the navigation of the EU-RL website Anses-Fougeres. The purpose of the website is to create non-stop access to EU-RL information. Users of the site include EU National Reference Laboratories, the European Commission, National Laboratories of Candidate Countries and Laboratories of Third Countries. The restricted area is for authorised members only and different levels of access apply. The current website may be accessed at http://crl.fougeres.anses.fr/ until the end of 2014 however there are new websites under construction for each of the Anses EU-RLs. A transitional period will operate in 2015 where both old and new sites will be available but from 2015 onwards the EU-RL Anses Fougeres website will be http://anses.fr/fr... .

Delegates at the 19th

Annual Workshop of the EURL for Antimicrobial and Dye Residues in Food, ANSES, Fougères, October 2014.

http://crl.fougeres.anses.fr/

http://anses.fr/fr


NRL Chemical Elements in Food of Animal Origin

Report on the Annual Workshop of the EURL for Chemical Elements in Food of Animal Origin, Rome, Italy, 17th October, 2014

NRL Representative:

Marion Barrett (VPHRL, Backweston Campus)

Laura CIARALLI (EURL) opened the 2014 workshop by welcoming all participants, including the invited guest speakers. The workshop was attended by 45 participants which included 31 representatives of EU National Reference Laboratories from 27 member states, 1 representative from a laboratory outside the EURL-CEFAO network, 1 external expert, 7 staff from the EURL-CEFAO at the ISS in Rome and 5 invited speakers, including a representative from the European Commission. This workshop covered a range of relevant topics including developments in EU legislation, current and future proficiency testing, method development for heavy metals and toxic elements in cheese, stabilisation of digestion solutions for the analysis of mercury (Hg) in food and training on LoD and LoQ.

Frank SWARTENBROUX (European Commission) commenced the meeting by giving an overview of recent and future developments in EU legislation relating to 96/23/EC for residue control. There is currently a proposal for a new Food and Animal Health Regulation involving measures relating to controls and monitoring. This proposal would lead to the repeal of 96/23/EC, of which the annexes would remain valid until they are replaced. The progress of this new regulation depends on the position of the incoming European Parliament, with expected adoption in 2016 and possible application in 2017.

For Cadmium (Cd), there is to be a review of the existing maximum levels (MLs) with the setting of additional ones, although it is not possible to lower MLs for major contributors such as cereals, vegetables and potatoes. There is to be a progressive implementation of mitigation measures such as a decrease of phosphates in fertiliser, as it is rich in Cadmium. Member states (MSs) are to monitor progress and send data to the European Food Safety Authority (EFSA), and the situation is to be reassessed by the end of

2018. New MLs for Cadmium are proposed for food for infants and young children from 2015, as EFSA has recommended a reduction in exposure for this consumer group. It is intended to have differentiated MLs for liquid vs. powder and cow’s milk vs. soy based products. Also proposed are new MLs for cocoa and chocolate from 2019, with differentiated MLs depending on the cocoa solids content of the product. A fine-tuning of MLs for fish and shellfish of various groups is also proposed.

For Arsenic (As), the major contributors to dietary exposure are cereals, fish and food supplements such as Hijiki seaweed. Currently the discussion on MLs for rice and its derived commodities (i.e. rice cakes) is in its final stage. However, differentiated MLs may be required for white vs. brown rice and for rice destined for food products for infants and young children.

For Lead (Pb), discussions on legislative changes for MLs are also in the final stage and proposals are ready. Protection from exposure has been increased and extended to lower MLs for infant formula (differentiated for liquid vs. powder forms) and lower MLs for ready-to-eat and other food and drinks, such as follow-on formulas, aimed at this vulnerable age group. There is also a fine-tuning of MLs proposed for lead in vegetables, a lowering of MLs for fruit juices and wine and a lowering of MLs for cephalopods to the normal fish level.

For Mercury (Hg), the EFSA risk-benefit analysis of fish consumption related to methylmercury is expected at the end of 2014 and discussions are expected to start early in 2015. There is an EFSA mandate on data collection and annual reporting which involves a definition data model and adjustment of standard sample description (SSD). A pilot study to test data submission is to be carried out in 2015/2016 with 4 or more member states, with the first annual data collection at sample level from 2016 to be published as an annual report in 2017.

Piotr ROBOUCH (EC-JRC-IRMM) gave a presentation on how to determine LoD (limit of detection) and LoQ (limit of quantification). The LoD is derived from the smallest measure that can be detected with reasonable certainty for a given analytical procedure. The LoQ is the lowest concentration determined with satisfactory measurement. The Critical Measurement Value is the hidden parameter, which is the lowest signal


that can be detected with reasonable certainty. Some typical experimental examples were examined as to the effect of these parameters on uncertainties for data results. The working hypothesis assumes a linear response at low concentrations and that samples are undergoing the full analytical procedure. The signal to noise distribution, slope and critical value are important for both LoD and LoQ calculation. Issues were discussed including normal distributions, equivalence, standard deviation of the blank, paired observation and calibration. Different scenarios of calculation were explored using the ISO approach and the Quick Din Method. A comprehensive list of examples and relevant literature was given, including the website www.eurachem.org which has relevant free downloads available for LoD and LoQ calculations and on which the Eurachem Method Validation Guide for 2014 will soon be available.

Martijn VAN DER LEE (RIKILT, NL) spoke on method development and results for detection of heavy metals and toxic elements in cheese, specifically Arsenic (As), Lead (Pb), Cadmium (Cd), Chromium (Cr), Nickel (Ni) and Mercury (Hg). There are many difficulties associated with analysing for chemical elements in cheese such as the high fat, salt and protein content, non-homogeneity and low concentration levels. The fat content in particular is relevant to the weight of the sample amount chosen for analysis; a low sample weight affects the LoD, while too high a weight will have too high a fat content. Difficulties also arise on how to spike cheese, due to the non-homogeneity of the sample. To circumvent this problem, milk was also obtained for this experiment, spiked, and made into cheese. Three dairy products were sourced from the ISS in Rome; ripe cheese, Primo cheese and milk from previous PTs. FAPAS milk powder was also used as a baseline reference. The rind of the cheese was removed and the cheese grated. On average, 0.3-0.5g of cheese was used, and 2-5ml of milk. Microwave-assisted digestion was used with nitric acid added to make a final sample volume of 50ml, and total digestion was achieved. Measurements were taken by ICPMS and GFAAS. For Cd, it was found that the method of standard addition improves results and good recoveries were achieved with a higher percentage recovery for the cheese samples for both methods. For Pb, both methods also revealed similar results with the spiked samples giving higher than expected recoveries than their unspiked counterparts. Milk

and milk powder were not analysed for Cr or Ni, and the results for cheese were similar for both methods with GFAAS (with method of standard addition) giving similar results to ICPMS. GFAAS with external calibration gave poor results for both these elements. For As, some levels in the samples were too low to measure, while spiking in cheese analysed by ICPMS was found to increase recovery. In conclusion, many difficulties were observed and the LoD in GFAAS was not sufficient at times. Interference was seen in ICPMS with recoveries too high in milk and spiked cheese and recoveries too low in cheese. It is proposed that ICPMS in DRC (dynamic reaction cell) mode using oxygen might be an option to use in the future.

Gerhard LIFTINGER (IAGES, OS) discussed the stabilisation of digestion solutions for analysing mercury in food. Losses of mercury in such solutions can be associated with adsorption on the walls of the vessel, evaporation after reduction and ‘inerting’ in complexes or amalgams. References are different and sometimes even contradictory concerning Nitric acid (HN03), Potassium Dichromate (K2Cr2O7) and Hydrochloric acid (HCl) as agents used for stability. For the experiment, these solutions and also Potassium Permanganate (KMnO4) and Gold (Au) solution were each investigated as to their ability to stabilise Hg in digest solutions, and were added to solutions with different concentrations of Hg. These were stored in the dark at room temperature in Sarstedt tubes and analysed on a range of different days against freshly made calibration solutions. The instrument used was a flow injection CV-AAS, with 0.2% NaBH4 as reductant and 3% HCl as carrier. It was found that nitric acid alone was not suitable for the stabilisation of mercury solutions. When HCl was added to the nitric acid it was found that it was suitable for stabilisation from a concentration of 2ml/l for at least five weeks. In contrast, Potassium Dichromate at higher concentrations suppressed the signal and was stable only for a few days at 0.005% while also being toxic. The use of gold can only be partly recommended and some signal suppression was also detected over the ten-day experiment, so a further study would be required. Potassium permanganate cannot be recommended at all as with increasing concentration the signal was reduced, and it is believed that Manganese Oxide (MnO2) is formed which absorbs the mercury. Longer-term studies are required as most of these chemical

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experiments only took place over a period of several weeks.

Jens JØrgen SLOTH (Technical University of Denmark, DK) presented an update on the analysis of lipid-soluble arsenic compounds (arsenolipids) in marine oils. He gave a rundown on the history of analytical progress in the identification of arsenolipids. While arsenic itself was first documented in marine oils in the 1960s/1970s, the first arsenolipid was only identified in marine algae in the late 1980s (Morita & Shibata, 1988). From then until 2004, research focused mainly on the hydrolysis products. In 2007 intact arsenolipids were analysed by LC-ICPMS by Sloth & Amlund who found four main arsenolipids and some minor ones. In more recent years, research has discovered many new types of arsenolipid compounds (Arsenic-containing fatty acids, Arsenic-containing hydrocarbons, etc) and 53 different compounds have been identified so far. There are analytical challenges in the speciation analysis and methods such as HPLC-ICPMS and qTOF-MS are being used. There is also a question of cellular toxicity with arsenic-containing hydrocarbons, whose cytotoxicity was found in one study to be comparable to that of arsenite. The biosynthesis route is still unclear as is the biological role of arsenolipids. Future food safety control on arsenic would appear to be more complex than has previously been thought and advanced analytical techniques will be required.

Francesco CUBADDA (Department of Food Safety and Veterinary Public Health, IT) spoke on the analytical determination of inorganic arsenic in food; from research to legislation. Arsenic occurs in the environment in different organic and inorganic forms, with the organic forms including 15 arsenosugars, 50 arsenolipids and 10 other organoarsenic species. Inorganic arsenic (iAs) is far more toxic than organic and is a human carcinogen, thus the need arises to discriminate between the different arsenic compounds in food. The main foods of concern regarding iAs are rice, wheat and shellfish, which generally contain low arsenic levels but of which iAs is a much higher proportion of the total arsenic content. Opinions vary on a legal maximum level but it is agreed there is no ‘safe’ level, and there is a concern about exposure in infants and young children.

Speciation analysis is used to appropriately assess the risk of dietary arsenic exposure and to distinguish between the inorganic and organic forms; this typically involves HPLC-ICPMS or SPE &

HG-AAS. Due to the naturally-occurring nature of arsenocompounds, however, speciation is proving far more complex than originally thought. There is a need for more speciation data for different foods, increased risk assessments and validated methods for various food matrices. Also, some matrices are more challenging for analysis than others, especially shellfish and edible algae (seaweeds). These contain organoarsenical compounds that make the determination of total vs iAs content more complex. Following the new risk assessments of arsenic in food in 2009, the process leading to the introduction of legal limits for iAs in foodstuffs has commenced. An amendment of EC Regulation 1881/2006 as regards maximum limits of iAs is expected by the end of this year; products being regulated could include various types of rice, rice products and milled rice destined for use in the production of baby foods. A future maximum level may be set at 0.2-0.3µg/kg of iAs. This would be the first time ever a maximum limit has been introduced in EU legislation for an element species.

Laura CIARALLI (EURL) discussed the planning of PTs for 2015. As of 1

st January 2015, MLs for Cd in

infant formulae and follow-on formulae (both liquid and powdered) will come into effect according to EU Regulation 488/2014, amending EU Regulation 1881/2006. Consequently, the 22

nd

PT in April will be on powdered infant formula based on animal protein. This matrix is quite complex from an analytical point of view, so the proposal of this PT is considered to be useful to the network. The outcome can also be used to compare the performance of the network with results previously obtained in PTs on milk. The elements considered will be As, Cd, Pb and Mo (Molybdenum). Mo was included due to an EFSA Scientific Opinion (2013) that detailed the unavailability of data on its intake and health outcomes. The 23

rd PT will be on freeze-dried

freshwater fish in July, and will examine the elements Cd, Pb, Total As and Hg. This is a matrix of great interest and a PT on fish has not been carried out since 2010. The priority is to verify the stability of the network’s performance taking into account the advent of new staff in laboratories and new and different analytical techniques.

Laura CIARALLI and Andrea COLABUCCI (EURL) gave a presentation on EURL-CEFAO follow-up actions. EURLs have been requested by the EU Commission this year to enhance collaboration with the NRL network and to strictly follow the


protocol set down for performance evaluation. Therefore the EURL-CEFAO enhanced the actions in place to comply with the Commission’s advice. The tasks of the NRLs are set down in Reg EC 882/2004, including participation in PTs and the annual workshop organised by the EURL and taking advice and recommendations given by the EURL into consideration. Various issues and deviations from the tasks were discussed, such as underperformance or lack of participation in PTs. The EURL provides assistance and investigation to laboratories experiencing these issues, and emphasis is given to resolving issues for element/matrix combinations where MLs are set in legislation. Where possible, extra exercises are organised for underperforming laboratories and close assessment of the results is carried out by the EURL. If problems persist, the Commission must officially be informed and supplied with a detailed report of the main findings and corrective actions taken. The achievement of a good collaboration between the EURL and the NRLs is essential for high quality and the uniformity of analytical results.

Marina PATRIARCA (NRL for Heavy Metals in Food, IT) gave a talk on ISO 13528 and beyond. This presentation was based on training material presented at Eurachem Workshops on Proficiency Testing in Istanbul and Berlin, October 2014, by Marina Patriarca, Piotr Robouch and Michael Koch. The scope of ISO 13528 includes analysing data obtained from PT schemes, using this data to design new PT schemes and providing recommendations on the interpretation of PT data by participants, accreditation bodies and other interested parties. Essentially, it provides harmonised guidance for PT providers regarding statistical design issues and evaluation of performance over time. The revised version of this standard is out next year and will fundamentally be the same, with some new ideas and alignment with existing standards that have come into being since its original publication. Terminology has been aligned with the International Vocabulary of Metrology (VIM), some new symbols are included and the scope is extended. There is extra information on the design of PT schemes where there are small numbers of participants and on methods of determining the assigned value. The Eurachem website (www.eurachem.org) has a wide range of useful information regarding PT schemes and corrective actions for problems that the NRL laboratories may find extremely useful.

Anna Chiara TURCO (EURL) gave the final talk of the day on the outcome of the 20

th PT. Frozen

kidney was chosen for this exercise as the last PT on this matrix was in 2011 and offal is included in most of the National Residue Control Plans for EU member states. The analytes chosen for analysis were Cd, Pb, Cu & Hg. Kidney samples were spiked with these elements, apart from Cu, which was to be measured at the natural level present in the tissue (approx. 4mg/kg). The aim of the exercise was to give the NRLs a tool to check the performance of their methods and to assess sample compliance. After the production of the kidney material at the ISS, AA techniques were used to check homogeneity. Repeatability was found to be very good and the stability criteria were met on PT test items. All (28) laboratories of the NRL network took part in the exercise and submitted results. For Cd, 5 laboratories submitted results produced by two different techniques and the data from one other laboratory was excluded in the statistical evalution as it was higher than the upper limit of 50% of the median. The distribution proved normal when this outlier was removed. No statistical difference was found between the analytical techniques. For Pb, all NRLs submitted results, with two data points determined by F-AAS and one other outlier datum excluded from the evaluation. The distribution was then unimodal and met all criteria. For Cu, 28 NRLs submitted data and no data were excluded. Distribution was normal with a slight positive skew, which was thought to be produced by results from laboratories with less experience in ICP-MS. For Hg, 29 NRLs submitted results. One clearly aberrant datum and two data higher than the upper limit of 50% of the median were excluded. The results had a normal unimodal distribution, standard deviations were comparable and the mean was robust. ICP-MS proved comparable to HG-VGM with no differences found. Performance on the whole was better for analysis of Cd and Pb than for the analysis of Cu and Hg. The performance of the network was satisfactory in terms of data dispersion for Cd, Pb and Hg, but not for Cu, while an improvement in performance has been noted for Hg.

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NRL Animal Proteins in Feedingstuffs

Report on the 8th and 9th Annual Workshop of the EURL for Animal Proteins in Feedingstuffs, Riga 21-22nd May, 2014 and Backweston, 21- 23 April 2015

NRL representative:

James Choiseul (2014) and Ruairi Colbert (2015) (FFGPD, Backweston Campus)

This meeting is held annually to inform the National Reference Laboratories for Animal Protein (NRL-AP) network of developments in the area of feedingstuff analysis and to discuss issues such as new or revised scientific protocols. The 8

th

meeting was attended by c. 50 delegates from 27 countries and the 9

th meeting was attended by c.

60 delegates from 26 countries.

8th

Conference Report

Guilbert BERBEN (EURL) presented the activities of the EURL for 2013, addressing each work area set out in the founding regulation. Progress in developing the FiSH (fluorescent in situ hybridisation) method of AP detection was described. As a method that requires visualisation of florescent particles, the main issue is difficulty in preparing sample material which is sufficiently transparent to allow this. The EURL is currently validating the porcine PCR assay in-house, and plans an inter-laboratory validation for autumn 2014. A number of minor changes to the mandatory SOPs were outlined. These will be shown in the next versions.

Pascal VEYS (EURL) presented the results of the 2014 PT. Overall performance was good, especially considering that the difficulty of the tests have increased gradually since inception. According to an EU scale the EURL PT has shifted from a grade 1 PT (easy) to a grade 3 PT (difficult). In this PT the Irish NRL regretfully inverted the sample numbers when returning samples following analysis. This is noted in the final report. The Irish NRL has raised a non-conformance to address this error. Overall some issues were identified regarding the identification of feather meal with 21 out of 27 labs failing to detect this material. The EURL noted that it is important to adhere absolutely to the procedure and that NRL’s should ensure that

Official Labs do not deviate from the official method.

Leo VAN RAAMSDONK (RIKILT, NL) made a presentation concentrated on a number of inconsistencies in the official SOP which makes its implementation difficult. These were presented to the EURL for review. Additionally an updated version of the Aries decision support system was shown, and delegates were asked to consider if this system should be maintained or discontinued.

James CHOISEUL (NRL, IE) reported on a number of experiments conducted by the Irish NRL which demonstrated that if sample material is ground before testing, as required by legislation, it increases significantly the number of spicules present in the sample and therefore can result in a sample that should be reported as negative (i.e. less than 5 spicules detected) being instead positive. The EURL proposed to organise a wider trial to investigate the significance of this finding.

Angus WEIR (VLA, UK) gave a presentation detailing the decline in BSE cases in the UK since the introduction of the feed ban but also the development of a new form of BSE (Barb BSE) which though occurring in very low numbers, is associated with areas of the UK (e.g. Scotland) which were outside the main BSE epidemic zone.

Martial PLANTARDY (DG SANTE) detailed the impact that the new controls regulation will have on the NRL network. The main changes highlighted were:

The inclusion of ‘other official activities’ so that the EU will be able to expand the scope of activities within sectorial specific roles without reference to the European Parliament.

Cost based mandatory fees for most official controls

Enforced administrative assistance and co-operation

A new integrated information management system, IMSOC, incorporating current systems such as TRACE and RASFF.

For EURL’s, new responsibilities will include the requirement to maintain lists of NRLS’s, to compile information on research activities and to provide official training courses for Official Labs.

Paschal VEYS (EURL) made a presentation of an investigation into alleged contamination of


aquafeed with animal protein In June 2013, non-ruminant AP was reintroduced as an ingredient in aquafeed. Subsequently, industry data suggested that up to 60% of aquafeed was contaminated with ruminant DNA. In order to investigate this, the analysed fish meal samples at from the market and at place of production. The results of this analysis is that fishmeal samples are not contaminated by ruminant AP, but that the porcine blood added to the fishmeal can be frequently contaminated by ruminant AP.

Jutta ZAGON (Federal Institute for Risk Assessment, DE) gave a presentation on the application of aptameres in the detection of bovine thrombin. Aptameres are single stranded DNA fragments which behave like antibodies. They can be generated by means of a process called Systemic Evolution of Ligands by Exponential Enrichment (SELEX). Using a novel diagnostic technique called ELONA, similar to the well-known ELISA method, they can be used to identify material of animal origin. Initial tests have shown this method to be sensitive, reproducible and easy to apply for differentiating between bovine and porcine material. Development continues.

9th

Conference Report

Guilbert BERBEN (EURL) presented a summary of the EURL-AP activities in 2014. The EURL-AP will seek accreditation for proficiency testing (ISO 15189) in the future. Discussion on the availability of ‘pure’ reference material took place. The EURL-AP will source material from EFPRA and will make this material available to the NRL-AP network.

Janka MATRAI (JRC-IRMM) gave a presentation on the production of Porcine pDNA Calibrants. It was stressed that Calibrants should only undergo 5 freeze thaw cycles.

Olivier FUMIÈRE (EURL) presented information on the recent Porcine PCR assay validation study for the detection of Porcine DNA in feed. After reviewing the results of the study, the method was officially declared valid for the detection of pig PAPs in feed at the level of 0.1% (w/w). The final report describing the in-house validation as well as the inter-laboratory study will be published soon. The NRLs already have access to the method and an implementation test will be organized at the end of 2015.

Lucie CARROUÉE (DG Sante) made a presentation beginning with EFSA’s recent reports and opinions, specifically:

BSE risk in bovine intestines and mesentery was commented and will lead to an amendment to the list of MRS;

The scientific opinion on the scrapie situation within the EU after 10 years of surveillance;

Protocol for further lab investigation of atypical BSE cases. Comments on the revision of the feed ban were explained to the NRLs with an eye to the future relaxation for pig and poultry. It appeared that only 31 RASFF alerts were posted over the period February 2013 –April 2015. DG Sante reminded that the strict following of the SOP for method selection and implementation is a prerequisite. A revision of the obligation of bilateral agreement before export of non-ruminant PAP is under discussion. In this respect it is also important that GTH detection performs excellently in the EU. DG Sante requested the EURL-AP to see how a GTH proficiency test could be organised. The proposed revision of the SRM measures is presented according to the OIE standards and the increased number of Member States with a negligible risk status. Finally an update of the BSE surveillance was presented. The number of classical BSE cases still diminished while the number of atypical cases seems to have reached a basal level. A GTH PT will be organised for 2016.

Guilbert BERBEN (EURL) gave presentation of the IAG-RIKILT 2014.11 report. The report questioned the revised method for microscopy and PCR. Following discussions with RIKILT, EURL-AP and the commission it was agreed that a revised report would be published.

Paschal VEYS (EURL) proposed that the EURL-AP will carry out a collaborative study on the effect of grinding feed sample on bone fragment numbers. The Irish NRL have demonstrated that grinding increases the bone spicule numbers in a sample. They presented this at the Riga workshop. Veys also presented the 2014 EURL-AP PT results for microscopy. 78% of NRLs had excellent results. A third of NRL’s were able to detect blood. The 2015 PT test will combine microscopy and PCR.

Guilbert BERBEN (EURL) updated NRL’s on the work programme for the EURL-AP of 2015. There will be expert training in Microscopy in the


Autumn and this will be EU funded. Basic training will still be available but there will be a charge for the NRL. An opened discussion followed which as was chaired by Fumière (PCR) & Veys (Microscopy). Topics included the following:

PCR method:

Positive results near cut-off values but below Ct-values of a 0.1% control sample;

Uncertainty of the method;

Legitimacy of PCR on fish feed with blood products;

Internal validation of the method towards accreditation bodies.

Microscopy method:

Possibility of a SOP guiding the successive determinations,

Relevance of flotate observation with stereomicroscope,

How to deal with particles of undefined nature?

Amount of material on slides.

Marie Claire LECRENIER (EURL) presented ‘Mass spectrometry for the detection of bloodmeal/products’ gelatine analysis using different analytical methods such as PCR, infrared spectroscopy and mass spectrometry (in collaboration with York University). Initial results were presented.

Gilbert BERBEN (EURL) summarised the conclusions of the workshop as follows:

1. The JRC-IRMM provided advice on using the PCR Porcine Calibrants.

2. PCR method for pig target is validated. Extraction will be considered during the implementation test in autumn.

3. Upcoming PCR target: poultry. 4. Do not perform PCR if feed is not

intended for fish. 5. SOP operational scheme was modified

regarding aquafeed known to contained blood products:

6. Need for clarification on number of determinations in microscopy.

7. Grinding study will be organised by EURL-AP on a voluntary basis.

8. The amount of sediment put on slides could be studied.

9. Next PT will be a joint Microscopy – PCR test.

10. GTH PT planned for 2016. It is asked to NRLs to send information about their lab able to perform the test.

11. Some NRLs didn’t provide their data for the new database. If not received within a short period, their data will disappear because by switching from the old to the new database, the old database will be completely erased and only the information put in the new database will be taken into consideration.

12. The possibility to produce extraction reference material will be studied with JRC-IRMM.

13. Next workshop will be held in Belgium for the 10th anniversary of the EURL-AP.

Delegates at the 9th

Annual Workshop of the EURL for Animal Proteins in Feedingstuffs, Backweston, April 2015

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