Update on activities in ISO and CEN. Kirsten Mooijman
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1 Update on activities in ISO and CEN Kirsten Mooijman
2 Relevant groups in ISO and CEN ISO/TC34/SC9: ISO: International Standardisation Organisation TC34: Technical Committee 34 on Food products SC9: Subcommittee 9: Microbiology CEN/TC275/WG6: CEN: European Committee for Standardisation TC275: Technical Committee 275 for Food analysis Horizontal methods WG6: Working Group 6 for Microbiology of the food chain Annual meetings of SC9 and WG6: 9-13 May 2016, Paris, France 2
3 EN ISO Detection of Salmonella FprEN/FDIS (Final Draft International Standard) voting 12/11/ /01/2016. Outcome: positive with editorial and few technical comments FDIS is final stage before publication technical comments can not be taken into account Still some of the technical comments were important, especially to change Annex F for preparation of dairy products from normative to informative to make sure that future improvements in sample preparations in ISO can be taken into account for dairy products Until April 2016: written consultation of ISO (and CEN) members to agree with last changes: outcome positive Publication final ISO in summer/fall 2016??? 3
4 PCR monophasic Salmonella Typhimurium In 2014 decided to start standardisation of PCR method for identification of monophasic Salmonella Typhimurium Technical part done in CEN-TAG3 (molecular methods), p.l. Burkhard Malorny (Germany), in cooperation with ISO-WG10 (serotyping Salmonella), p.l. Kirsten Mooijman 2015: collection of methods, conclusions: Focus on monostm lacking the second phase (1,4,[5],12:i:-); Standardise >1 protocol: Gel-based PCR, Real time PCR, gelbased with Real time reagents; Determine performance characteristics with one set of strains. Early 2016: Inventory among NRLs for interest and strains (Kirsten) Start drafting protocols (Burkhard) 4
5 PCR monostm results inventory Positive replies of in total 23 laboratories: Belgium (2x), Cyprus, Denmark, Germany, Estonia, Finland, France (2x), Greece, Italy, Latvia, Luxembourg, Netherlands, Poland (2x), Romania, Slovak Republic, Spain, Switzerland, United Kingdom (2x), USA 14 laboratories interested to review the draft protocols 21 laboratories interested to participate in interlaboratory study for determining the performance characteristics. Important that participating laboratories are able to perform gel based PCR as well as real-time PCR; 21 laboratories indicated to have interesting strains (few strains up to >100) for the interlaboratory study, including strains with deviating behavior in a PCR assay. 5
6 PCR monostm next steps Selection of strains (EURL and NRL Germany) Collection of selected strains (EURL) Test draft protocols with set of strains (EURL and NRL Germany) Amend draft protocols when needed (CEN-TAG3) As soon as technical work is finished, movement from CEN-TAG3 to ISO-WG10 Call for PCR experts for ISO-WG10 (by secretariat ISO SC9) Launch New Work Item Proposal (NWIP) in ISO for a new part 4 of ISO 6579 (ISO/TS ) with tentative title: Polymerase chain reaction (PCR) for identification of monophasic Salmonella Typhimurium (1,4,[5],12:i:-) Organisation of an interlaboratory study for determining performance characteristics as soon as the final draft is available 6
7 Harmonisation of incubation temperature (I) In 2014 at meetings ISO/TC34/SC9 and CEN/TC275/WG6 agreed to use broader temperature range for incubation of non-selective media: C instead of 37 C ± 1 C (harmonisation with US). At lab of Adria, France (Daniele Sohier) experiments performed to test influence of incubation temperature on growth of Salmonella and several Enterobacteriaceae species. Conclusions: no impact of incubation at 35 C or at 37 C on growth of Salmonella spp. some impact on the growth of some (other) Enterobacteriaceae species In 2015 proposed to set up a protocol to test with members of ISO/CEN the possible broader temperature range for incubation of selective media. Especially test the influence on the growth of Enterobacteriacea. 7
8 Harmonisation of incubation temperature (II) Protocol concerns culturing of routine samples, preferably with high amount of background flora (e.g. raw products, pps samples) and if possible with Salmonella, in MKTTn at 37 C ± 1 C and at 35 C ± 1 C. Incubation of Plating-out from MKTTn-37 C also at 37 C and plating-out from MKTTn-35 C also at 35 C. Protocol drafted by Daniele Sohier (France) and Kirsten Mooijman (EURL-Salmonella) and tested in their labs. Improved protocol and reporting form recently sent to members ISO and CEN, asked to perform experiments until January NRLs-Salmonella interested to perform some experiments as well? 8
9 CEN mandate M/381 (history) Aim: to have standardised and validated methods that are referred to in legislation in order to support the EU food policy Project started in 2007 Concerned international standardisation and validation of 15 microbiological methods, including validation of method for detection of Salmonella in samples from primary production stage (pps) Performance characteristics for detection of Salmonella in pps samples determined from EURL-Salmonella interlaboratory studies of 2008 (chicken faeces), 2012 (pig faeces) and 2013 (boot socks combined EURL/CEN-mandate study) 9
10 Finalisation CEN mandate M/381 CEN mandate project ends in June By then all 15 EN/ISO standards including the performance characteristics have to be published Raw data of all studies have to remain available for possible future recalculations will probably be stored (per study) at DG-Sante and CEN Agreed at meeting of CEN to publish all validation studies in a special issue of the International Journal of food Microbiology All participants in validation studies shall be mentioned in the Acknowledgements and shall agree on publication of the validation study/studies 10
11 CEN-TAG9 Pre-enrichment step Task group raised in 2012 in trying to come to an optimal preenrichment medium for detection of several (Gram neg) pathogenic bacteria, to resuscitate stressed or damaged cells. Experiments have been performed with replacement of peptone by chemically defined products (poor growth of many strains) and by testing different strains in BPW prepared from different batches of peptones (variable results). Start development of a protocol to determine performance characteristics for the pre-enrichment step (e.g.in BPW) Also impact of modifications on sample preparation protocols will be considered for the pre-enrichment step, e.g. soaking of dry samples, pooling of test portions, different dilution factors, prewarming of BPW, alternative BPW formula for acidifying food items 11
12 Revision of ISO 6887 series: Microbiology of the food chain Preparation of test samples FDIS voting will soon be launched of: Part 1: General rules for the preparation of the initial suspension and decimal dilutions (including info on pooling of samples and verification protocol for pooling) Part 2: Specific rules for the preparation of meat and meat products Part 3: Specific rules for the preparation of fish and fishery products Part 4: Specific rules for the preparation of miscellaneous products (e.g. animal feed, eggs, cocoa products, acidic products) Revision (probably) will soon start of: Part 5: Specific rules for the preparation of milk and milk products 12
13 ISO series Method validation Publication approximately summer 2016: Part 1: Vocabulary Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method 2nd Working Draft (April 2016): Part 3: Protocol for the verification of (validated) reference and validated alternative methods implemented in a single laboratory CD voting until mid April 2016: Part 4: Protocol for in-house (single) laboratory method validation Part 5: Protocol for factorial interlaboratory method validation Part 6: Protocol for the validation of alternative methods for microbiological confirmation and typing 13
14 Other subjects of possible interest (I) Revision of EN ISO General requirements and guidance for microbiological examinations Update with information/reference to new techniques Reduce calculation section (refer to Excel calculators) Reduce equipment section Revision of ISO/TS Specific requirements and guidance for proficiency testing by interlaboratory comparison: To make document full standard (instead of Technical Specification), as TS is not recognised in some countries Take into account new information on statistical aspects for PT schemes To include PT schemes for viruses, parasites, primary production, yeasts and moulds, molecular methods 14
15 Other subjects of possible interest (II) ISO 11133:2014 Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media Preparation of an amendment for correction of errors and to clarify some aspects Preparation of amendment on confirmation media and reagents Revision of document after finalisation of the 2 amendments Development of an international standard, in (likely) three parts on whole genome sequencing (project leader USA): 1. Wet laboratory sequencing and analysis of sequence data 2. Validation of data and methods 3. Metadata and sequence repository 15
16 Other subjects of possible interest (III) Proposal at last meeting of ISO-SC9 to start standardisation of a method for molecular (PCR) serotyping of Salmonella. SC9-secr will launch an enquiry (at SC9 members) on the need to standardise a method. With this enquiry a method proposal by US, including validation data, will be included. Opinion NRLs? Next annual meeting of ISO/TC34/SC9 and CEN/TC275/WG6 on June 2016 in Tokyo (or nearby), Japan 16
17 Thank you Questions? 17
Update on activities in ISO and CEN. Kirsten Mooijman
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