Monitoring of antimicrobial resistance in Campylobacter EURL AR activities in framework of the new EU regulation Lina Cavaco

Transcription

1 Monitoring of antimicrobial resistance in Campylobacter EURL AR activities in framework of the new EU regulation Lina Cavaco 1 DTU Food, Technical University of Denmark

2 Outline EURL-AR presentation and tasks Tasks related to new monitoring Brief overview Campylobacter in the monitoring Sampling AST and panel of antimicrobials to be tested Ring trials for Campylobacter AST in EURL-AR network Results of most recent Campylobacter AST trial (2013) Challenges 2 DTU Food, Technical University of Denmark

3 The tasks of the EURL-AR Scientific advice and support to the Commission Co-ordination of National Reference Laboratories and provision of technical support Creation of the network of NRL s Workshops Dissemination of knowledge and information Collection of information on activities at the NRL s Ring trials, comparative testing and quality assurance Confirmatory testing Evaluation and development of analytic methods 3 DTU Food, Technical University of Denmark

4 The Tasks of the EURL-AR Co-ordination of National Reference Laboratories and provision of technical support Creation of the network of NRL s The address list Workshops One annual workshop next April 2015 Training courses aimed at the specific methods to apply in the monitoring (2013 was training on AST incl. Campylobacter, this year was focused on selective isolation of presumptive ESBL-, AmpC or carbapenemase producing E. coli Site visits Dissemination of knowledge and information Home page: Newsletters E-learning site ( ongoing preparation) Collection of information on activities at the NRL s Questionnaires 4 DTU Food, Technical University of Denmark

5 The tasks of the EURL-AR (The ring trial system - EQAS) AST of eight Salmonella, Campylobacter, E.coli, Staphylococcus and Enterococcus strains MRSA isolation identification and spa typing Genotyping (gene EQAS) New one on the way, awaiting approval of WP 2015 Supply labs with original ref. strains E. coli ATCC 25922, C jejuni ATCC 33560, S. aureus ATCC and E. faecalis ATCC Results and comments submitted through a web-based data entry program, using individual passwords Instant individual evaluation reports with suggestions for corrective actions Yearly over-all evaluation reports on the web 5 DTU Food, Technical University of Denmark

6 Work of EURL-AR and the monitoring according to EU 652/2013 Participation in the EFSA Workgroups Scientific advice to the EU Commission and participation on the Preparation and discussion of the contents and methods used in the regulation Facilitating training Facilitating ring trials Development of methods and confirmatory testing Thus, the activities aim at implementing, from an analytical point of view, the provisions of monitoring of antimicrobial resistance set down the EU regulation 652/ DTU Food, Technical University of Denmark

7 Campylobacter monitoring acc EC 652/2013 Sampling (a) In the years 2014, 2016, 2018 and 2020 for laying hens, broilers and fresh meat thereof, and fattening turkeys. (b) In the years 2015, 2017 and 2019, for pigs, bovines under one year of age, pig meat and bovine meat. Sample nrs 170 isolates for AST for each combination of bacterial species and type of sample of animal population or food category listed in point 1(a), (b), (c), (e) and (f). in Member States with a production of less than tonnes -85 isolates is minimum In Member States with low bacterial prevalence or low number of epidemiological units, all available isolates at the end of the monitoring period shall be included in the antimicrobial susceptibility testing. Disclaimer: for exact information and further info on sampling design and representative sampling - consult the regulation and the EFSA technical specifications for the sampling procedures! 7 DTU Food, Technical University of Denmark

8 8 DTU Food, Technical University of Denmark

9 Campylobacter monitoring Testing ID Campylobacter C. jejuni /C. coli Recommended PCR method for speciation under: pylobacter_november_2013.pdf Acc: Denis, C. Soumet, K. Rivoal, G. Ermel, D. Blivet, G. Salvat and P. Colin. Development of a m-pcr assay for simultaneous identification of Campylobacter jejuni and C. coli. Letters in Applied Microbiology 1999, 29, AST MIC testing Antimicrobial panel and ranges acc EFSA technical specifications not changed from previous reccomendations QC and storage of isolates (for 5 years) Data collection- isolate-based data - EFSA 9 DTU Food, Technical University of Denmark

10 Choice of antimicrobials and ranges Insert reasons from EFSA 10 DTU Food, Technical University of Denmark

11 Rationale for antimicrobials to be tested Macrolides (erythromycin, clarithromycin, azithromycin) are considered first choice drugs for therapy of human infections with Campylobacter spp. Acquired resistance is caused by single point mutations in the ribosomal DNA resulting in cross-resistance between the different macrolides. Macrolides are commonly used in food-animals and resistance to macrolides is reported, predominantly in C. coli. Erythromycin is commonly used in the test panels as representative of the macrolides. (Fluoro)quinolones (ciprofloxacin, nalidixic acid) Resistance to quinolones is due to mutation in the gyrase gene resulting in resistance. Both nalidixic acid and ciprofloxacin are used in existing monitoring programmes as indicators of acquired resistance to quinolones in Campylobacter spp. 12 DTU Food, Technical University of Denmark

12 Rationale for antimicrobials to be tested (cont) Tetracyclines are considered alternative choices for treatment of Campylobacteriosis. But acquired resistance does occur. Therefore inclusion of this class is relevant. Resistance in Campylobacter spp. is encoded by tet(o), a gene that expresses a ribosomal protection protein. Streptomycin Resistance to aminoglycosides in Campylobacter spp. is encoded by APH and AAD-genes. Although cross resistance between different aminoglycosides is incomplete, streptomycin resistance can be used as indicator for acquired genes. 13 DTU Food, Technical University of Denmark

13 The tasks of the EURL-AR Ring trials, comparative testing and quality assurance Ring trials, comparative testing and quality assurance Proficiency tests -include one annual ring trial on AST of Campylobacter Confirmatory testing Confirmation of results and characterization of monitoring isolates Evaluation and development of analytic methods Establishing reference strain collections for relevant research and requests Methods for detection of relevant resistances- very focused on the needs in monitoring. 14 DTU Food, Technical University of Denmark

14 Focus of the EURL-AR EQAS s Area: Antimicrobial Resistance Microorganisms: Campylobacter Salmonella E. coli Enterococci Staphylococci (MRSA, Genotyping and ESBL/AmpC) Methods for phenotypic antimicrobial susceptibility testing (AST): Microbroth / Agar dilution (MIC-test) 15 DTU Food, Technical University of Denmark

15 Objectives of the EQAS To have laboratories evaluate their performance of antimicrobial susceptibility testing (AST) To assess the quality of AST in European reference laboratories And improve the quality of monitoring data to EFSA To identify challenges within the methods To evaluate the effect of training courses 16 DTU Food, Technical University of Denmark

16 Quality Assurance of the EQAS Accredited methods (relevant for the EQAS): MIC testing according to EN/ISO Provider of EQAS (serotyping and AR) according to ILAC G13 (International Laboratory Accreditation Cooperation) International standards/guidelines ISO (International Organization for Standardization) CLSI (Clinical and Laboratory Standards Institute) EUCAST (The European Committee on Antimicrobial Susceptibility Testing 17 DTU Food, Technical University of Denmark

17 18 Campy EQAS AST- Participation countries 30 sets of Campylobacter results 18 DTU Food, Technical University of Denmark

18 19 Comparison to former EQASs 19 DTU Food, Technical University of Denmark

19 20 Campylobacter results strains Test strain AST in % correct total C-8.1 (C. coli) C-8.2 (C. coli) C-8.3 (C. jejuni) C-8.4 (C. jejuni) C-8.5 (C. jejuni) C-8.6 (C. coli) C-8.7 (C. coli) C-8.8 (C. coli) Only 3 incorrect identifications out of 240 identifications reported > 98.8 % correct 20 DTU Food, Technical University of Denmark

20 21 Campylobacter results antimicrobials Overall percentage AST correct: 96.5% Antimicrobial % of correct AST Ciprofloxacin 96.6 Erythromycin 97.5 Gentamicin 99.6 Nalidixic acid 93.2 Streptomycin 96.6 Tetracycline DTU Food, Technical University of Denmark

21 Most frequent problems Campylobacter Standardization and harmonization of methods Sometimes growth issues on MIC plates (EURL-Campy contacted a few times) Issues with acquisition or supplementation of media Less problems with AST results now that disk diffusion results are not accepted (formerly some labs encountered problems due to the method used New challenges with new monitoring not yet observed 22 DTU Food, Technical University of Denmark

22 Thank you!! From the EURL-AR team and our network 23 DTU Food, Technical University of Denmark