European Union Reference Laboratory Research Articles

European union reference laboratory for feed additives: experience from 11 years organising proficiency testing

Feed additives are individually authorised within the European Union (EU) specifying the conditions of their use under real-world conditions. These criteria often include minimum or maximum level of their content in compound feed. In addition, feed business operators are obliged to indicate on the feed the level of the added content of the feed additive. While it is the task of the EU Member States official laboratories to perform control analyses to establish compliance with these provisions, the European Union Reference Laboratory for Feed Additives (EURL-FA) is organising since 2012 annual proficiency tests (PTs) for Member States’ National Reference Laboratories (NRLs). PTs organised by the EURL-FA are based on the principles that compound feed samples are prepared containing a known content of the target feed additive. These PT samples are then sent to the NRLs for analysis and the results reported back to the EURL-FA are subjected to statistical assessment to establish the laboratory specific proficiency to correctly analyse compound feed samples for the respective feed additive. In the current study, we looked at the outcome of this assessment across different PTs. The topics addressed were (1) the evaluation of the overall measurement capability of NRLs in respect to coccidiostats, trace elements and vitamin D3, (2) the challenge related to the preparation of homogenous test materials with particle-based feed additives, (3) a possible connection between the modified composition of preparations containing vitamin A and analytical results and (4) the impact of a training course on the proficiency of laboratories regarding carotenoid analysis.

Aluminium Determination in Foodstuffs by ICP-MS: Influence of Microwave Digestion Parameters on the Recovery

AbstractAluminium (Al) is the third most common element in the Earth’s crust and occurs naturally in drinking water and agricultural products, and humans are consequently exposed to the element from dietary sources. A tolerable weekly intake of 1 mg/kg has been established by the European Food Safety Authority (EFSA); however, no maximum levels for aluminium in foodstuffs have so far been established in the European Union (EU) legislation. Official food control requires validated methods for the determination of aluminium. Acid digestion assisted by microwaves is the main sample preparation technique used for the determination of aluminium, usually in combination with atomic spectrometry for quantification. In the present study, different parameters in the digestion step were investigated including test portion, digestion temperature, the reagent used and duration of the digestion to assess the aluminium extraction. The presented work is following up on an observation from a proficiency test (PT) on trace elements (including aluminium) in cocoa powder organised in 2020 by the European Union Reference Laboratory for metals and nitrogenous compounds in feed and food (EURL-MN), where the participant results for aluminium showed an unexpectedly large variation. In addition to the PT material, different certified reference materials were included in the present study, and the results highlighted that the temperature and reagent used are the most critical parameters to obtain a satisfactory sample digestion prior to aluminium determination. Based on the obtained results, it is recommended to digest food samples with a mix of ultrapure water and nitric acid for 25 min at a temperature of at least 240 °C with a mix of HNO3 and H2O to achieve satisfactory microwave-assisted digestion.

Migration of Melamine and Its Derivatives from Melamine/Bamboo/Wheat Straw-Made Tableware Purchased from Internet Markets or Retail Shops in China.

The ecofriendly and sustainable concept of bamboo- and wheat straw-made tableware has gained attention in recent years. However, it is necessary to note that these kinds of tableware are composed of melamine (MEL)-formaldehyde resin with the addition of bamboo fibers or wheat straw. This study aims to explore the potential migration of MEL and its derivatives from the tableware and conduct a risk assessment. The study involved 46 bowls or cups purchased from Internet markets or retail shops in China, whose raw materials included MEL, bamboo, and wheat straw. There were four pieces of glass- or ceramic-made tableware used as the control group. Migration testing was performed according to the test conditions selected from the European Union Reference Laboratory for Food Contact Materials. Considering the realistic worst-case scenario, we measured the concentrations of MEL and its derivatives in food simulants using ultra-performance liquid chromatography-tandem mass spectrometry and estimated the exposure risks for adults and 1-year-old infants. MEL and its derivatives could migrate from MEL-, bamboo-, and wheat straw-made tableware with varying concentrations. The total migration was ranked as follows: bamboo-made tableware > MEL-made tableware > wheat straw-made tableware > glass- or ceramic-made tableware (p < 0.001). The primary contributor to the total concentration for MEL- and bamboo-made tableware was MEL, whereas cyanuric acid (CYA) was the main contributor for wheat straw-made tableware. Based on the total concentration of MEL and its derivatives and the strictest TDI value, the proportions of the calculated hazard quotient ≥1 for MEL-, bamboo-, and wheat straw-made tableware in adults were 53.50%, 92.30%, and 1.90%; and the proportions in 1-year-old infants increased to 86.00%, 100.00%, and 7.40%. The utilization of MEL-, bamboo-, and wheat straw-made tableware could be regarded as a significant source of human exposure to MEL and its derivatives. It is advisable for both adults and infants to refrain from using tableware manufactured with MEL and bamboo fiber, as it may increase the susceptibility to MEL-related diseases.

Reflections on Feature Engineering and Design Using Causal Machine Learning (CML) for African Swine Fever (ASF) Diagnosis

Feature engineering is a crucial step in the process of machine learning, where raw data is transformed into meaningful features that can effectively represent the underlying patterns and relationships in the data. The goal is to improve the performance of machine learning models by providing them with more informative and meaningful input features. Automated feature engineering techniques, such as genetic algorithms, can also be used to automatically generate and optimise features. These methods search a space of potential features and select or create features based on their impact on the model's performance. Overall, feature engineering plays a crucial role in machine learning by enabling models to exploit the most relevant and informative aspects of the data, thereby improving their accuracy, robustness, and interpretability. This paper reports empirical studies aimed at demonstrating which types of technical features are best suited to establish relationships between ASF viruses and clinical symptoms to accurately diagnose ASF disease. Various machine learning models such as neural networks, decision trees, random forests, linear regression, and Bayesian regression accept ASF features and provide predictions. The experiment demonstrates the extent to which the machine learning model can establish correlations between ASF viruses and clinical symptoms by independently analysing the required feature. The focus is on establishing relationships between ASF viruses and clinical symptoms for diagnosis. Data from the European Union Reference Laboratory for African swine fever (ASF) was collected for the study. This paper provides essential information on ASF datasets based on the interpretation of results obtained by using appropriate samples and validated tests in combination with information from laboratory tests on ASF disease epidemiology, scenario, clinical signs, and lesions caused by different virulence. The study proposes to use causal ML to establish relationships between ASF viruses and symptoms to improve the accuracy of the ASF disease. In this study, the performance and validation of the models were measured using metrics such as R-squared, mean absolute error (MAE) and mean square error (MSE).

8.B. Workshop: Fit for purpose surveillance - strategies to implement effective surveillance systems in EU/EEA countries under the new Serious Cross Border Health Threats Regulation (2022/2371)

Abstract Background As part of the European Health Union package, the EU Regulation 2022/2371 on serious cross-border threats to health has entered into force on 26 December 2022. Chapter III (Articles 13-17) of this Regulation provide new provisions for Epidemiological surveillance, the Digital platform for surveillance, and EU reference laboratories. Methods Several Implementing Acts are currently in preparation, which will define in more detail the concrete implementation of these provisions, such as the list of communicable diseases, case definitions, procedures for the operation of the epidemiological network for epidemiological surveillance, the specifications of the digital platform for surveillance and its specific rules for functioning, as well as to designate EU reference laboratories. Via the EU4Health Programme, the European Commission is furthermore providing funding for Integrated Surveillance, Surveillance capacity building Results The workshop will inform on the current state of implementation, on the results of the preparatory works carried out so far as well as on an outlook to the short and longer term plans for the implementation of the regulation on serious cross-border threats to health. Furthermore, it will inform on a funding mechanism for the improvement of EU Surveillance Systems as one of the central lessons learnt from the COVID-19 pandemic and showcase a good national practice. Conclusions The COVID-19 pandemic has clearly illustrated that the creation and further development of sound, reliable, integrated and real-time digital surveillance systems are a very crucial component for preparedness against serious cross border health threats. The current work under the new regulation is a valuable opportunity for public health practitioners to improve surveillance at EU, national and regional level. The workshop will host panel presentations followed by a round-table discussion.

European Union Proficiency Tests for pesticide residues in cereals and feedstuff, from 2007 to 2022- Data collection experience

The European Union Reference Laboratory for pesticide residues in cereals and feeding stuff (EURL-CF) has, for 16 consecutive years since 2007, organized European Union Proficiency Tests (EUPTs) for analysis of multiple pesticide residues in various cereals and feedstuff commodities. The EUPTs are directed to National Reference Laboratories (NRLs), and official laboratories (OfLs) in European member (EU) states and in European Free Trade Association (EFTA) countries. The aim of the EUPTs is to gain information regarding the quality, accuracy and comparability of pesticide residue data in cereals and feedstuff reported by different EU and EFTA laboratories, to help evaluate and improve their performance. This article summarizes 16 years’ experience in processing and analyzing results for EUPTs for pesticide residues in cereals and feedstuff. It provides an overview of the performance and development of as many as 160 laboratories throughout the years. Results are highlighted in terms of the number of participants, analytical scope, z scores, false positive and false negative reported results, and classification of laboratories. The performance of laboratories in the EUPTs has improved over time. A notable increase was observed in NRLs and OfLs classified as category A, indicating satisfactory overall performance and scope coverage for the majority of participating laboratories.

Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Extended-spectrum β-lactamase- (ESBL) and AmpC- β-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three β-lactamase groups. The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

Results of the 2020 Genomic Proficiency Test for the network of European Union Reference Laboratory for Antimicrobial Resistance assessing whole-genome-sequencing capacities.

The global surveillance and outbreak investigation of antimicrobial resistance (AMR) is amidst a paradigm shift from traditional biology to bioinformatics. This is due to developments in whole-genome-sequencing (WGS) technologies, bioinformatics tools, and reduced costs. The increased use of WGS is accompanied by challenges such as standardization, quality control (QC), and data sharing. Thus, there is global need for inter-laboratory WGS proficiency test (PT) schemes to evaluate laboratories' capacity to produce reliable genomic data. Here, we present the results of the first iteration of the Genomic PT (GPT) organized by the Global Capacity Building Group at the Technical University of Denmark in 2020. Participating laboratories sequenced two isolates and corresponding DNA of Salmonella enterica, Escherichia coli and Campylobacter coli, using WGS methodologies routinely employed at their laboratories. The participants' ability to obtain consistently good-quality WGS data was assessed based on several QC WGS metrics. A total of 21 laboratories from 21 European countries submitted WGS and meta-data. Most delivered high-quality sequence data with only two laboratories identified as overall underperforming. The QC metrics, N50 and number of contigs, were identified as good indicators for high-sequencing quality. We propose QC thresholds for N50 greater than 20 000 and 25 000 for Campylobacter coli and Escherichia coli, respectively, and number of contigs >200 bp greater than 225, 265 and 100 for Salmonella enterica, Escherichia coli and Campylobacter coli, respectively. The GPT2020 results confirm the importance of systematic QC procedures, ensuring the submission of reliable WGS data for surveillance and outbreak investigation to meet the requirements of the paradigm shift in methodology.

European Union Reference Laboratories support the National food, feed and veterinary Reference Laboratories with rolling out whole genome sequencing in Europe.

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

Assessment of the Predictivity of DIO1-SK Assay to Investigate DIO1 Inhibition in Human Liver Microsomes

Introduction: The European Union Reference Laboratory for alternatives to animal testing (EU RL ECVAM) coordinates the validation of multiple thyroid in vitro assays aiming to set up an in vitro test battery. The validation consists of two independent parts: part 1 (Reproducibility assessment) in which the reproducibility of the method is evaluated by testing a small number of described inhibitors and part 2 (Predictivity assessment) where the predictivity of the method is investigated by testing a blinded set of substances. The deiodinases (DIO) are important regulators of thyroid hormone (TH) metabolism and potential targets for substance-induced thyroid disruption. We previously reported the part 1 results for the Sandell–Kolthoff reaction-based type 1 deiodinase (DIO1-SK) assay and an extensive description of the method. In this study, we report the results of the part 2 testing in the DIO1-SK assay. Methods: A set of 22 test substances consisting of known DIO inhibitors in vitro, as well as substances otherwise interfering with the TH system and substances with no such activities were tested in the standardized DIO1-SK assay. Experiments were performed on blinded substances, which were deblinded after experimental completion, statistically evaluated, and compared to literature data. Finally, an in vitro data interpretation procedure (DIP) was generated. Results and Discussion: Seven test substances produced a maximum DIO1 inhibition >90% and 11 test substances below 20%; they were regarded as inhibitors and noninhibiting substances, respectively. Two test substances, Ketoconazole and Silichristin, were found to be not applicable based on assay interference. Inhibition data were consistent with results of relevant in vitro and computational models. Using the variation of control data, an in vitro DIP was defined categorizing test substances (i) by efficacy using the maximum inhibition data and (ii) by potency using the IC50 data of the test substance.

Manganese uptake mediated by the NRAMP-type transporter MntH is required for acid tolerance in Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen that is characterized by its ability to withstand mild stresses (i.e. cold, acid, salt) often encountered in food products or food processing environments. In the previous phenotypic and genotypic characterization of a collection of L. monocytogenes strains, we have identified one strain 1381, originally obtained from EURL-lm, as acid sensitive (reduced survival at pH 2.3) and extremely acid intolerant (no growth at pH 4.9, which supports the growth of most strains). In this study, we investigated the cause of acid intolerance in strain 1381 by isolating and sequencing reversion mutants that were capable of growth at low pH (pH 4.8) to a similar extent as another strain (1380) from the same MLST clonal complex (CC2). Whole genome sequencing showed that a truncation in mntH, which encodes a homologue of an NRAMP (Natural Resistance-Associated Macrophage Protein) type Mn2+ transporter, is responsible for the acid intolerance phenotype observed in strain 1381. However, the mntH truncation alone was not sufficient to explain the acid sensitivity of strain 1381 at lethal pH values as strain 1381R1 (a mntH+ revertant) exhibited similar acid survival to its parental strain at pH 2.3. Further growth experiments demonstrated that Mn2+ (but not Fe2+, Zn2+, Cu2+, Ca2+, or Mg2+) supplementation fully rescues the growth of strain 1381 under low pH conditions, suggesting that a Mn2+ limitation is the likely cause of growth arrest in the mntH− background. Consistent with the important role of Mn2+ in the acid stress response was the finding that mntH and mntB (both encoding Mn2+ transporters) had higher transcription levels following exposure to mild acid stress (pH 5). Taken together, these results provide evidence that MntH-mediated Mn2+ uptake is essential for the growth of L. monocytogenes under low pH conditions. Moreover, since strain 1381 was recommended for conducting food challenge studies by the European Union Reference Laboratory, the use of this strain in evaluating the growth of L. monocytogenes in low pH environments where Mn2+ is scarce should be reconsidered. Furthermore, since it is unknown when strain 1381 acquired the mntH frameshift mutation, the ability of the strains used for challenge studies to grow under food-related stresses needs to be routinely validated.

Pyrrolizidine Alkaloids from Monofloral and Multifloral Italian Honey

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by plants as a self-defense against insects. After bioactivation in the liver, some PAs can cause acute or chronic toxicity in humans. The aim of this study was to determine the presence of PAs in 121 samples of monofloral and multifloral honey from three different Italian regions (Friuli-Venezia Giulia, Marche and Calabria) to meet the European Food Safety Authority (EFSA) suggestion. An in-house liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was validated according to European Union Reference Laboratory (EURL) performance criteria. This method allowed the detection and quantification of 35 PAs. Of the 121 honey samples, 38 (31%), mostly from Calabria, contained PAs. The total content of the PAs ranged from 0.9 µg/kg to 33.1 µg/kg. In particular, echimidine was the most prevalent PA. A rapid human exposure assessment to PAs in honey and a risk characterization was performed using the EFSA RACE tool. The assessment highlighted a potential health concern only for toddlers who frequently consume elevated quantities of honey. This study showed a low presence of PAs in Italian honey; however, the importance of continuously monitoring these compounds is stressed, along with the suggestion that the relevant authorities establish maximum limits to guarantee support for producers and consumer safety.

Trehalose transport occurs via TreB in Listeria monocytogenes and it influences biofilm development and acid resistance

Listeria monocytogenes is a pathogenic bacterium that can inhabit a diverse range of environmental niches. This is largely attributed to the high proportion of carbohydrate-specific phosphotransferase system (PTS) genes in its genome. Carbohydrates can be assimilated as sources of energy but additionally they can serve as niche-specific cues for L. monocytogenes to shape its global gene expression, in order to cope with anticipated stresses. To examine carbon source utilization among wild L. monocytogenes isolates and to understand underlying molecular mechanisms, a diverse collection of L. monocytogenes strains (n = 168) with whole genome sequence (WGS) data available was screened for the ability to grow in chemically defined media with different carbon sources. The majority of the strains grew in glucose, mannose, fructose, cellobiose, glycerol, trehalose, and sucrose. Maltose, lactose, and rhamnose supported slower growth while ribose did not support any growth. In contrast to other strains, strain1386, which belonged to clonal complex 5 (CC5), was unable to grow on trehalose as a sole carbon source. WGS data revealed that it carried a substitution (N352K) in a putative PTS EIIBC trehalose transporter, TreB, while this asparagine residue is conserved in other strains in this collection. Spontaneous mutants of strain 1386 that could grow in trehalose were found to harbour a reversion of the substitution in TreB. These results provide genetic evidence that TreB is responsible for trehalose uptake and that the N352 residue is essential for TreB activity. Moreover, reversion mutants also restored other unusual phenotypes that strain 1386 displayed, i.e. altered colony morphology, impaired biofilm development, and reduced acid resistance. Transcriptional analysis at stationary phase with buffered BHI media revealed that trehalose metabolism positively influences the transcription of genes encoding amino acid-based acid resistance mechanisms. In summary, our results demonstrated that N352 is key to the function of the sole trehalose transporter TreB in L. monocytogenes and suggest that trehalose metabolism alters physiology to favour biofilm development and acid stress resistance. Moreover, since strain 1386 is among the strains recommended by the European Union Reference Laboratory for conducting food challenge studies in order to determine whether or not L. monocytogenes can grow in food, these findings have important implications for food safety.

Statement on the risk assessment of maximum residue levels (MRLs) for oxamyl in view of consumer protection.

In accordance with Article 43 of Regulation (EC) No 396/2005, the European Commission requested EFSA to perform a risk assessment of the existing maximum residues levels (MRLs) for oxamyl considering the new toxicological reference values. Additionally, if needed to ensure adequate consumer protection, lower limits of quantification (LOQs) than those currently established in the legislation should be proposed. EFSA performed various consumer exposure calculation scenarios, considering the risk assessment values as available for the existing uses of oxamyl and the lowering of LOQs for several plant and animal commodities as suggested by the European Union Reference Laboratories for Pesticide Residues (EURLs). Based on the results of the consumer exposure assessment calculated considering the risk assessment values for crops with authorised oxamyl uses and the existing EU MRLs at the LOQ for remaining commodities (scenario 1), chronic consumer intake concerns were identified for 34 diets. Acute exposure concerns were identified for a wide range of crops, including crops with currently authorised oxamyl uses: bananas, potatoes, melons, cucumbers, carrots, watermelons, tomatoes, courgettes, parsnips, salsifies and aubergines/eggplants. Under exposure calculation scenario 3, which considered lowering of all MRLs to the lowest analytically achievable limits of quantification, EFSA concludes that chronic consumer exposure concerns can still not be excluded. Similarly, acute consumer exposure concerns were identified for 16 commodities, including crops with known authorised uses: potatoes, melons, watermelons and tomatoes, even though for these crops a lower LOQ as proposed by the EURLs were considered. Further refinements of the calculated exposure at the current stage were not possible by EFSA, but EFSA identified a list of commodities for which a lower LOQ than routinely achievable is expected to significantly reduce the consumer exposure and for which a risk management decision is required.

Co-occurrence of antibiotic and disinfectant resistance genes in extensively drug-resistant Escherichia coli isolated from broilers in Ilorin, North Central Nigeria

The occurrence of multidrug-resistant (MDR) bacteria in poultry poses the public health threat of zoonotic transmission to humans. Hence, this study assessed the occurrence of drug-resistant Escherichia coli in broilers in the largest live bird market in Kwara State, Nigeria in December 2020. Presumptive E. coli isolates were isolated using the European Union Reference Laboratory guideline of 2017 and confirmed via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Broth microdilution was performed on confirmed E. coli isolates to determine the minimum inhibitory concentration. Five extensively drug-resistant (XDR) isolates were selected for Illumina whole genome sequencing to predict the resistome, phylotype, sequence type, serotype, and diversity of mobile genetic elements in these isolates. Of the 181 broiler caecal samples, 73 E. coli isolates were obtained, of which 67 (82.0%) and 37 (50.6%) were determined as MDR (resistant to at least three classes of antibiotics) and XDR (resistant to at least five classes of antibiotics), respectively. Whole genome sequencing revealed diverse sequence types, phylogroups, and serotypes (ST165/B1 - O80:H19, ST115/A - Unknown: H7, ST901/B1 - O109:H4, ST4087/F - O117:H42, and ST8324/A - O127:H42). The XDR E. coli isolates encoded resistance to fluoroquinolones, fosfomycin, sulfamethoxazole, ampicillin and cephalosporins, trimethoprim, aminoglycosides, chloramphenicol, tetracycline, and macrolides. Mutations in the gyrA gene conferring resistance to fluoroquinolones were also detected. There was a positive correlation between phenotypic resistance patterns and the antibiotic resistance genes that were detected in the sequenced isolates. The XDR isolates also harbored two disinfectant resistance genes (qacE and sitABCD) that conferred resistance to hydrogen peroxide and quaternary ammonium compounds, respectively. The genome of the XDR isolates harbored several mobile genetic elements and virulence-associated genes, which were conserved in all sequenced XDR isolates. This is the first report of co-carriage of antibiotic resistance genes and disinfectant resistance genes in E. coli isolated from broilers in Ilorin, Nigeria. Our findings suggest that poultry are potential carriers of clonally diverse, pathogenic, MDR/XDR E. coli, which may have detrimental zoonotic potentials on human health.

6.C. The revised EU health security framework

Abstract As a response to the pandemic, the Commission's communication of 11 November 2020 proposed building blocks for a European Health Union (EHU) and advocated the strengthening of existing structures and mechanisms for better EU level protection, prevention, preparedness and response against human health hazards. It recommended a reinforced framework for cross-border cooperation against all health threats to protect better human lives and the internal market as well as to maintain the highest standards in the protection of human rights and civil liberties, and a strengthened EU role in international cooperation to prevent and control cross-border health threats and improve global health security. By upgrading the EU health security framework for cross-border health threats, the building blocks of the European Health Union bring greater overall impact while fully respecting the Member States’ competence in the area of health. Concretely, before the end of 2022, two pieces of legislation will have been adopted: the upgrading of Decision 1082/2013/EU and the strengthened mandate of the European Centre for Disease Prevention and Control (ECDC) and the European Medicines Agency (EMA) that go hand in hand. The new Regulation on serious cross-border threats to health, the overarching legislative piece of the EU Health Union Package, aims at a stronger and more comprehensive legal framework for the Union to better prepare and respond to serious cross-border threats to health and public health emergencies. It better governs actions at Member States and Union level on prevention, preparedness and response, surveillance, risk assessment, and early warning. Further, it ensures better arrangements for joint procurement at EU level. With its revised mandate, the ECDC will be able to issue recommendations to Member States regarding preparedness and response, host a new network of EU reference laboratories and establish an EU Health Task Force for crisis preparedness and rapid public health interventions in case of a major outbreak. The regulation reinforcing EMA's role in crisis preparedness and management of medicinal products and medical devices puts some of the structures and processes established during the COVID-19 pandemic on a permanent footing. EMA is now responsible for monitoring medicine shortages, as well as reporting shortages of critical medicines during a crisis. It will also coordinate responses of EU / EEA countries to shortages of critical medical devices and in-vitro diagnostics in crises. The EU Health Security Framework will link up to IHR (where EU is requesting to become a contracting party) and the pandemic treaty, and the parallel developments to improve pandemic preparedness and response in G7 and G20 framework (also new global health strategy). The revised legislation addresses the weaknesses evidenced by the COVID-19 pandemic and support actions that can be financed via the EU4Health programme and other EU funding instruments. DG SANTE and ECDC will present the new legislation; two Member States will share their views on the newly adopted legislation. HaDEA will present the funding opportunities through EU4Health to support the implementation of th e two new legislation. Speakers/Panellists Ingrid Keller European Commission, Luxembourg, Luxembourg Florina Telea European Commission, Brussels, Belgium

Technical specifications for a baseline survey on the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in pigs.

The European Commission requested scientific and technical assistance in the preparation of a EU‐wide baseline survey protocol for a European Union (EU) coordinated monitoring programme on the prevalence of methicillin‐resistant Staphylococcus Aureus (MRSA) in pigs. The objective of the survey is to estimate the MRSA prevalence in batches of fattening pigs at slaughter at both European and national levels, with a 95% level of confidence and a level of precision of 10% considering an expected prevalence of 50%. The survey protocol defines the target population, the sample size for the survey, sample collection requirements, the analytical methods (for isolation, identification, phenotypic susceptibility testing and further genotypic testing of MRSA isolates), the data reporting requirements and the plan of analysis. The samples are to be analysed according to the laboratory protocols available on the European Union Reference Laboratory (EURL‐AR) website. Generalised linear models will be used to estimate proportion (with 95% confidence intervals) of batches of slaughter pigs tested positive to MRSA. The necessary data to be reported by the EU Member States to support this baseline survey are presented in three data models. The results of the survey should be reported using the EFSA data collection framework.

Fate of Sulfonamides and Tetracyclines in Meat during Pan Cooking: Focus on the Thermodegradation of Sulfamethoxazole.

Although antimicrobials are generally found in trace amounts in meat, the human health risk they bear cannot be ignored. With the ultimate aim of making a better assessment of consumer exposure, this study explored the effects of pan cooking on sulfonamides and tetracyclines in meat. Screening of these antimicrobials in cooked meat was first performed by the European Union Reference Laboratory on the basis of HPLC-MS/MS analyses. A proof of concept approach using radiolabeling was then carried out on the most cooking-sensitive antimicrobial—sulfamethoxazole—to assess if a thermal degradation could explain the observed cooking losses. Degradation products were detected thanks to separation by HPLC and monitoring by online radioactivity detection. HPLC-Orbitrap HRMS analyses completed by 1D and 2D NMR experiments allowed the structural characterization of these degradation compounds. This study revealed that cooking could induce significant antimicrobial losses of up to 45% for sulfamethoxazole. Six potential degradation products of 14C-sulfamethoxazole were detected in cooked meat, and a thermal degradation pattern was proposed. This study highlights the importance of considering the cooking step in chemical risk assessment procedures and its impact on the level of chemical contaminants in meat and on the formation of potentially toxic breakdown compounds.

METHOD VALIDATION AND CALCULATION OF MEASUREMENT UNCERTAINTY FOR PESTICIDE RESIDUE ANALYSIS IN FRESH RED PEPPER

A multiple analysis method based on QuEChERS (fast, easy, inexpensive, effective, robust and safe) extraction method and liquid chromatography-tandem mass spectrometry (LC – MS/MS) was developed for 39 selected pesticides in fresh red pepper. This method has been validated according to the Sante guide published by the European Union Reference Laboratories (EURLs). The linearity study was conducted in the range of 1-80 µg/kg and in seven different concentrations. The correlation coefficients of the calibration curves were (R2)&gt; 0.992. Quantification limits (LOQs) ranged from 4 to 19 μg kg-1. The recovery values were between 70.84% and 108.0%. The repeatability values (RSDr), which were between 0.99% and 17.5%in terms of in-laboratory reproducibility values (RSDWR), were determined to be between 0.98% and 19.57%. Repeatability and trueness parameters were used to calculate measurement uncertainty. The extended uncertainty values were calculated as 20.88% to 49.94%. The method was successfully used to analyze 22 fresh red pepper samples. Nine different pesticide residues were identified in 12 samples. The amount of carbendazim in a sample was found to be above the legal limits of the EU and Turkey. A residue of carbofuran, which is banned in Turkey, was found in one sample.

Critical Implications of IVDR for Innovation in Diagnostics: Input From the BioMed Alliance Diagnostics Task Force.

Critical Implications of IVDR for Innovation in Diagnostics: Input From the BioMed Alliance Diagnostics Task Force.