PROFICIENCY TESTS NO 19 AND EURL-Campylobacter National Veterinary Institute
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1 PROFICIENCY TESTS NO 19 AND EURL-Campylobacter National Veterinary Institute
2 NO OF NRLS PARTICIPATING IN THE PROFICIENCY TESTS 2017 PT PT PT PT PT PT PT PT 7 Enumeration PT PT PT PT PT PT PT PT 7 Detection and species identification
3 CAMPYLOBACTER-FREE MATRICES The chicken meat (PT19) and caecum samples (PT20) originated from a producer that according to the surveillance program had not had any Campylobacter for more than one year The broilers had been slaughtered at a slaughter house with a very low level of Campylobacter-positive flocks (0% Nov 2016 May 2017) Meat and ceacal material were tested negative for presence of Campylobacter
4 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00 04:00 08:00 12:00 16:00 20:00 00:00 T-log measurments Temperature 25,0 Kl. 10 PACKING 20,0 EURL-Camp 15,0 10,0 5,0 0,0-5,0 12/03/ /03/ /03/ /03/ /03/ /03/ /03/ ,0 Only PT 19 Date/Time
5 PT 19 ENUMERATION (DETECTION AND SPECIES IDENTIFICATION)
6 PROFICIENCY TEST NO 19 The objective was to assess the performance of the NRLs to enumerate, detect and species identify Campylobacter in minced chicken meat. The proficiency test involved: Detection and enumeration by direct culture on selective agar plates Subculture of suspected Campylobacter colonies on non selective agar plates Confirmation Voluntary detection after enrichment Voluntary species identification by biochemical or molecular methods
7 PT 19: BASIC OUTLINE SOP based on ISO :2006 Participants received minced chicken meat and 10 vials with freeze-dried sample (with or without Campylobacter) Homogenize and make a serial dilution Count colonies Tests for confirmation of Campylobacter spp. Calculate log cfu/g
8 PT 19: PROCEDURE Culture media; mccd agar Incubation time and temperatures; according to ISO 10272, Part 1: Detection method and Part 2: Colony-count technique Confirmation tests; morphology, motility, growth at 25ºC (microaerobic) and oxidase, and/or by molecular methods Species identification; Identification of Campylobacter species with biochemical tests and/or molecular methods, e.g. PCR, MALDI-TOF.
9 PT 19: STABILITY OF THE TEST The vials were tested for homogeneity and stability at The National Food Agency We also did enumerations in triplicates, with and without minced chicken meat
10 PT 19: NO OF DAYS FOR TRANSPORT OF PT 19 TO THE NRLS Arrival No of NRLs Start of analysis No of NRLs 14 th of March th of March 6 15 th of March 5 15 th of March 9 16 th of March 5 20 th of March 6 21 th of March 5 22 th of March 2 28 th of March 1 29 th of March 1 3 rd of April 1 The samples were distributed from the EURL on the 13 th of March. The 10 vials and the minced chicken meat were placed in a plastic container which was placed in a cool insulation box.
11 DESCRIPTION OF THE 10 VIALS IN PT 19 Sample Species Batch number No 1 Campylobacter coli + Escherichia coli Campylobacter jejuni Campylobacter coli Escherichia coli Campylobacter jejuni Negative - 7 Campylobacter jejuni Campylobacter coli Campylobacter lari Campylobacter jejuni 259
12 cfu/g PT 19: RESULTS OF ENUMERATION False pos: 1 neg: 1 neg: 1
13 HOW WAS PERFORMANCE CALCULATED? The Median Absolute Deviation (MAD) have been used to calculate performance Results within participants median ±2 MAD = 2 points Results between ±2 MAD and ±2,58 MAD = 1 points Results outside ±2,58 MAD = 0 points The maximum score (2 points for all samples with Campylobacter) was 16 points Calculate the score for each participant Grade Scoring limits for each performance grade Excellent % Good % Acceptable % Needs improvement % Poor 62.5%
14 PERFORMANCE PT % Excellent 90% Good 80% Acceptable 70% Needs improvement 60% Poor 50% 40% 30% 20% 10% 0% 27 28*
15 PERFORMANCE PT % Excellent 90% Good 80% Acceptable 70% Needs improvement 60% Poor 50% 40% 30% 20% 10% 0% 27 28*
16 PT 19: PERFORMANCE IN RELATION TO START OF ANALYSIS Day No of NRLs Performance Excellent Good Acceptable Need improvement Poor 14 th of March th of March th of March th of March th of March th of March th of March th of March rd of April 1 1
17 PERFORMANCE IN ENUMERATION OVER TIME 100 Excellent/Good Good Acceptable Needs improvement Poor
18 C. jejuni C. coli C. lari Camp spp. Other / No growth PT 19: SPECIES IDENTIFICATION Content of sample (vial) 1. C. coli + E. coli C. jejuni C. coli E. coli C. jejuni Negative C. jejuni C. coli C. lari C. jejuni 31
19 PERFORMANCE IN SENSITIVITY OF DETECTION AND IDENTIFICATION OF CAMPYLOBACTER IN PT 19 VOLUNTARY PARTS DETECTION CAMPYLOBACTER IDENTIFICATION CAMPYLOBACTER SPP. Good 3% Excellent 100% Excellent 97%
20 PERFORMANCE IN DETECTION (SE) OVER TIME 100 Excellent/Good Good Acceptable Needs improvement Poor
21 PERFORMANCE IN IDENTIFICATION (SE) OVER TIME 100 Excellent/Good Good Acceptable Needs improvement Poor
22 PT 20 DETECTION AND SPECIES IDENTIFICATION OF CAMPYLOBACTER
23 PROFICIENCY TEST NO 20 The objective was to assess the performance of the NRLs to detect and species identify Campylobacter in caecum samples. The proficiency test involved: Detection after enrichment Subculture of suspected Campylobacter colonies on non selective agar plates Confirmation Species identification by biochemical or molecular methods
24 PT 20: BASIC OUTLINE SOP based on ISO :2006 Participants received swab samples in transport media (with or without Campylobacter) Enrichment culture in Bolton broth Innoculate agar plates Tests for confirmation of Campylobacter spp. Tests for identification of Campylobacter spp.
25 PT 20: PROCEDURE Culture media; Bolton broth and mccda (and other selective media) Incubation time and temperatures; according to ISO 10272, Part 1: Detection method Confirmation tests; morphology, motility, growth at 25ºC (microaerobic) and oxidase, and/or by molecular methods Species identification; Identification of Campylobacter species with biochemical tests and/or molecular methods, e.g. PCR, MALDI-TOF.
26 PT 20: PREPARATION OF THE TEST Swab samples were prepared to resemble samples taken in transport crates for broilers Overnight cultures were prepared Caeca were cut and placed in a stomacher bag and mixed with BPW and Cary Blair transport medium Aliquots of BPW + caecum suspension, Cary Blair and 5 ml of the overnight culture were poured into each stomacher bag along with 2 swabs
27 PT 20: STRAINS TESTED Campylobacter: clinical strains of C. jejuni, C. coli and C. lari (the species that are most likely to be found in chicken feces) Non-Campylobacter: Clinical strains of Proteus, Candida and E. coli ESBL
28 PT 20: STABILITY OF THE TEST Pre-tests (October-March): For each strain that was tested, 12 replicates of swab samples were prepared Viable count of sample prior to enrichment Samples were kept at 8 degrees Samples were analysed in triplicates on day 1, 4, 5 and 6. Analysis: according to SOP (direct cultivation on CCDA) and serial dilutions to determine viable count
29 PT 20: BACTERIA ADDED TO THE SWAB SAMPLES Sample No. Bacterial species Biochemical properties Level 11 Campylobacter lari high 12 Campylobacter coli low 13 Campylobacter jejuni Hippurate + low 14 Campylobacter lari high 15 Escherichia coli 16 Campylobacter coli high 17 Escherichia coli 18 Proteus mirabilis 19 Campylobacter coli low 20 Campylobacter jejuni Hippurate high 21 Campylobacter jejuni Hippurate low 22 Proteus mirabilis 23 Candida spp.* 24 Campylobacter coli high 25 Campylobacter lari low 26 Campylobacter jejuni Hippurate low 27 Candida spp.* 28 Campylobacter lari high
30
31 PT 20: CORRECT REPORTED RESULTS PER SAMPLE IN DETECTION AND SPECIES IDENTIFICATION Number of NRLs Correct Campylobacter detection Correct species identification Sample No. C. lari C. Jejuni hipp Low (20 high)
32 PT 20: CORRECT REPORTED RESULTS PER LAB IN DETECTION AND SPECIES IDENTIFICATION Number of correct reported samples 18 Correct Campylobacter detection Correct species identification Lab ID
33 PERFORMANCE SE AND SP IN DETECTION OF CAMPYLOBACTER DETECTION CAMPYLOBACTER DETECTION NON CAMPYLOBACTER Acceptable 3% Acceptable 9% Good 15% Excellent 82% Excellent 91%
34 ACCURACY IN DETECTING POSITIVE AND NEGATIVE CAMPYLOBACTER SAMPLES Good 12% Acceptable 9% Excellent 79%
35 ACCURACY - COMPARISON WITH PREVIOUS PTS 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Excellent Good Acceptable Need improvement Poor
36 PERFORMANCE SE SPECIES IDENTIFICATION IN PT 20 Good 6% Acceptable 9% Needs improvement 3% Exellent 82%
37 IDENTIFICATION COMPARISON WITH PREVIOUS PTS 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Excellent Good Acceptable Needs improvement Poor
38 NUMBER OF CORRECT SPECIES IDENTIFICATIONS IN SAMPLES WITH CAMPYLOBACTER BY DIFFERENT METHODS Method for species identification Biochemical tests only Correct Sp id in all samples analysed Total 2 3 PCR assays only MALDI-TOF only PCR and biochemical tests PCR and MALDI-TOF MALDI-TOF and biochemical tests Biochemical tests, PCR and MALDI-TOF
39 CONCLUSIONS - PT19 AND PT20
40 FUTURE PTS PT21 AND PT22 PT21- Enumeration: Questback: dilutions Matrix chicken skin PT22- Detection and Species Id: The goal is to use freeze dried material for also PT22 Matrix? Date for sending next PT: 5th of March?
41 THANK YOU FOR YOUR PARTICIPATION AND FOR PROVIDING INFORMATION IN THE QUESTBACK REPORTS!
42 Hippurate hydrolysis C. jejuni C. coli C. lari Mixed C. jejuni and C. coli Campylobacter spp. but unable to identify species Growth of other, not Campylobacter No growth at all No species identification done PT 20: REPORTED SPECIES IDENTIFICATION Sample No. Bacterial species 11 Campylobacter lari Campylobacter coli Campylobacter jejuni Campylobacter lari Escherichia coli Campylobacter coli Escherichia coli Proteus mirabilis Campylobacter coli Campylobacter jejuni Campylobacter jejuni Proteus mirabilis Candida spp Campylobacter coli Campylobacter lari Campylobacter jejuni Candida spp Campylobacter lari
43 COMMENTS OR QUESTIONS ON PT20 Direct plating vs enrichment Bolton broth vs Preston broth
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