EU-RL- Campylobacter Proficiency test no 6, 2010 Swab ring test. Eva Olsson Engvall, EU-RL Campylobacter

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1 EU-RL- Campylobacter Proficiency test no 6, 2010 Swab ring test Eva Olsson Engvall, EU-RL Campylobacter

2 At the workshop in 2009 we agreed on a ring test that would include Identification of Campylobacter species in swab samples with pure cultures of: Reference strains, clinical isolates Isolates submitted by the NRLs to the CRL from the EU baseline survey (BS) in 2008 Other organisms than Campylobacter, but Campylobacter related organisms, aka CROs Methods for identification: any method for analysis could be applied

3 The EU-RL tested many strains by phenoand genotyping and 10 were selected Phenotyping: Basically as described in ISO :2006 for identification of thermophilic Campylobacter spp: Microscopic morphology and motility, growth at 25 C (mae) and at 41.5 C (ae), biochemical tests: oxidase, catalase, hippurate hydrolysis, indoxyl acetate and production of H 2 S on TSI agar

4 PCRs: PCR based assays and mass spec -Denis et al 1999, identification of C. jejuni and C. coli -Wang et al 2002, identification of C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus -Fermér and Engvall 1999, identification of C. jejuni, C. coli, C. lari, and C. upsaliensis -Marshall et al 1999, identification of Campylobacter, Arcobacter, and Helicobacter -Lawson et al 1997, identification of C. upsaliensis and C. helveticus Matrix assisted laser desorption time of flight mass spectrometry, MALDI-TOF

5 Description of the 10 strains Sample no Identity of strain Origin Characteristics & Comments 1 C. jejuni Broiler Hippurate negative. C. jejuni according to PCRs 2 C. lari Broiler Phenotypic reactions agree with C. lari, e.g. hippurate and indoxyl acetate negative 3 C. hyointestinalis Cattle Phenotypic reactions agree with C. hyointestinalis, e.g. TSI positive 4 C. avium Broiler Hippurate positive, but not C. jejuni according to PCRs. Slow growth 5 C. jejuni Broiler Phenotypic reactions agree with C. jejuni, e.g. hippurate positive To be continued

6 continued description of the 10 strains Sample no Identity of strain Origin Characteristics & Comments 6 Arcobacter butzleri Broiler Could be mistaken for Campylobacter sp. Phenotypic reactions agree with A. butzleri, e.g. growth in aerobic atmosphere. PCR facilitates identification 7 C. jejuni Broiler Hippurate negative. C. jejuni according to PCRs 8 Helicobacter canadensis Broiler Could be mistaken for Campylobacter sp. Slow and sparse growth. PCR facilitates identification 9 C. upsaliensis Dog Phenotypic reactions agree with C. upsaliensis, e.g. weakly positive or negative for catalase, slow growth. PCR facilitates identification 10 C. coli Broiler Weakly hippurate positive. C. coli according to PCRs

7 Shipment The strains were sent as live cultures on swabs in Amies transport medium with charcoal. The swabs were thoroughly packed and shipped by regular air mail in padded envelopes The transport time varied, in some cases it took 5 days and the test was re-sent by courier to 5 labs

8 Participation in the Swab ring test 33 NRLs participated, 30 NRLs in the 27 MSs and 3 non- EU countries (CH, NO, MK) The identifications were done by a combination of phenotyping and PCR (at 18 NRLs), only phenotyping (at 9 NRLs), only PCR (at 6 NRLs). In addition to standard pheno/pcr methods: three NRLs sequenced 16S rrna, 3 used latex agglutination, 2 used API- Campy and in 1 NRL mass spectrometry technique by Maldi-Tof was used.

9 35 30 Results (sample 4 deleted) Camp sp C. jejuni uncorrect no growth C. lari C. hyoint not Camp sp C. upsal C. coli

10 Sample No Content Max no Your result Your result No of NRLs with of points Species ID No of points 2 points 1 points 0 points No growth 1 Hippurate negative C. jejuni C. lari C. hyointestinalis C. avium deleted from ring test Hippurate positive C. jejuni Not Campylobacter spp /Arcobacter Hippurate negative C. jejuni Not Campylobacter spp / Helicobacter C. upsaliensis C. coli Total no available points 16

11 Comments and Conclusions Difficult test - but also challenging Courier delivery should be used Majority of NRLs correctly identified C. jejuni, C. coli and C. lari strains as Campylobacter spp Difficulties to grow some strains own initiatives rewarding! More reliable identification by PCR or a combination of phenotyping and PCRs compared to only phenotyping

12 Thank you for your collaboration! Comments? Another similar test next year? Thank you for your attention and collaboration!

Voluntary PCR ring test. Boel Harbom CRL - Campylobacter

Voluntary PCR ring test. Boel Harbom CRL - Campylobacter Voluntary PCR ring test Boel Harbom CRL - Campylobacter Voluntary PCR ring test September 2008 At the CRL- Campylobacter workshop in 2007 it was agreed that the CRL would organise a voluntary PCR ring