EURL-Salmonella. interlaboratory comparison study on the detection of Salmonella in samples from the primary production XVI (2013)

Transcription

1 EURL-Salmonella interlaboratory comparison study on the detection of Salmonella in samples from the primary production XVI (2013) Boot socks with environmental material laying hen flock Angelina Kuijpers

2 Study is combined EURL-Salmonella / CEN mandate The data are differently treated for: CEN mandate: testing the performance of the method (validation of annex D of ISO 6579) Kirsten EURL-Salmonella: performance of the NRLs for the prescribed method (annex D of ISO 6579) This presentation Angelina

3 Time table Week (2013) Dates Subject February Mailing of the protocol, SOP and instructions for the web based test report to the NRLs by February Sending the link and the password for the electronic results form to the participants by March Mailing of the parcels to the NRLs as Biological Substance Cat. B (UN3373) by door-to-door courier service Preparation of the media by the NRLs March Performance of the study 13 Before 30 March Deadline for completing the electronic submission of results: 29 March 2013 (23:59). After this deadline the electronic submission form will be closed.

4 EURL-Salmonella Detection study differences with earlier studies: No use of reference material (capsules or lenticule discs) Artificially contamination of samples at laboratory EURL-Salmonella before sending to NRLs Use of webbased testreport

5 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella QUESTIONS Boot socks with or without the addition of fluid (Peptone Saline Solution, BPW or Water) Influence on -backgroundflora in the faeces -Salmonella Salmonella serovars. : S. Enteritidis (SE) or S. Typhimurium (STM)? Artificially contamination: Level of contamination - reference material (lenticules) - culture (dilution) Stability of samples during - storage (5 ºC) - transport (10-15 ºC)

6 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella EXPERIMENT (I): Boot socks in a plastic bag Addition : -15 ml of BPW, Peptone Saline Solution, Water or No fluid -10 gram of environmental material (mainly faeces from laying hen flock) CFU/boot sock of SE or STM or CFU/boot sock of STM or Blanc Storage of boot socks (5 each) at 5 ºC, 10 ºC and 15 ºC during 0, 7, 14 and 21 days

7 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella EXPERIMENT (II) Test of boot socks during storage: -presence of Salmonella according ISO 6579 annex D (MSRV) -level of background flora (Enterobacteriacea and aerobic bacteria:according to ISO & ISO 4833) Test at the start of the experiment -Inoculation level of culture STM or SE, counted on XLD

8 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella RESULTS & CONCLUSIONS (I) Salmonella Typhimurium is more stable than S. Enteritidis -STM (5 CFU) all 5 samples positive after 15 days of storage at 5 ºC -SE (5 CFU) no positive results after 7 days of storage at 5 ºC The addition of fluid to the boot socks gives a more stable sample -Background flora of the faeces is 1 log higher after 14 days of storage with fluid -Few more Salmonella positive samples after 21 days of storage with fluid No difference between type of fluid: Peptone Saline Solution, BPW or water STM low and high level contaminated samples are stable during storage at 5 ºC for 14 days -all 5 samples low level STM (5 CFU/boot sock) and high level STM (55 CFU/ boot sock) were tested positive after 14 days at 5 ºC

9 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella RESULTS & CONCLUSIONS (II) Samples were less stable after storage 7 days 10 ºC -boot socks without the addition of faeces : STM (13 and 55 CFU/boot sock) 21 days of storage at 10 ºC all 5 samples positive -boot socks with faeces: STM (55 CFU/boot sock) for 14 days at 15 ºC scored 3/5 positives STM (13 CFU/boot sock) for 13 days at 10 ºC scored 4/5 positives Artificially contamination with a diluted culture of STM -A diluted culture of STM added to the boot socks with faeces gave sufficient stable samples for use in the study - pre-artificially contaminated samples mimic better routine samples than mixture of matrix and reference materials

10 Pre-test : stability and homogeneity of samples Boot socks with chicken faeces and Salmonella CONCLUSIONS Sample used for the study: -One pair of boot socks in a plastic bag -15 ml of Peptone Saline Solution -10 gram of environmental material (faeces form laying hen flock) Artificially contaminated with a diluted culture of -STM low level (10-15 CFU/boot sock) -STM high level ( CFU/boot sock) -Blanc

11 Preparation of Boot socks

12 Study design Control (n=6) boot socks No matrix added Test samples (n=24) boot socks with 10 g environmental material S. Typhimurium low (STM9) 2 8 S. Typhimurium high (STM81) 2 8 Blank (BL) 2 8

13 Method study Annex D of ISO 6579 (prescribed) -selecitive enrichment on MSRV -Isolation medium XLD & 2nd agar for choice

14 Participants 36 Laboratories 28 NRLs from 27 EU MSs 4 NRLs from EU candidate MS - Croatia, Serbia, Bosnia and Herzegovina & FYROM 3 NRLs from European Free Trade Associations (EFTA) - Norway, Switzerland & Iceland 1 NRL from a third country on request of DG-Sanco : - Israel

15 Level of contamination Date of testing Low level STM CFU/sample High level STM CFU/sample 1 March 2013 Inoculum of boot sock 11 March 2013 after storage at 5 C MPN of boot socks with environmental material & inoculated with STM (95 % confidence limit) (1.1-10) (53-490) Performance of the study 11 March MPN: Most Probable Number

16 Transport of study material Cooling devices & Temperature logger Transport as Biological Substances Cat. B UN3373 by TNT door-to-door Transport time : - 29 parcels 1 day - 7 parcels 2 days DELAY Arrival at the NRL- non EU, because of problems at: - 1x customs 3 days extra - 1x airport 1 day extra - 3x parcel sent to institute EU-MS (extra transport to non-eu-ms) Temperature during transport & storage at the NRL majority<5 C except 1x delay at customs 2 days at 16 C extra transport from the airport (1x) or another country (2x) to NRL between 5-10 C

17 Material & Methods FOLLOW as much as possible YOUR ROUTINE PROCEDURE Addition of BPW 225 ml to plastic bag with boot socks (BPW at least room temperature) Mix gently knead or shake.. (Pulsifier not advisable) Incubation BPW at 37ºC for 18 ± 2 h Selective enrichment on MSRV Isolation on XLD and 2 nd plate for choice

18 Background flora environmental material of laying hen flock Date Enterobacteriaceae CFU/g Aerobic bacteria CFU/g 22 January *10 4 2* March 2013 after storage at 5 C 1*10 4 1*10 7

19 Accreditation according ISO/IEC participants 3 NRLs (EU-MS) accreditation planned in 2013/2014 Accredited method: 33 NRLs Annex D of ISO NRLs also ISO 6579 According to EC Regulations (EC No 882/2004), each NRL should have been accredited for their relevant work field before 31 December 2009 (EC No 2076/2005).

20 Good performance Minimum result Positive % No. of positive samples / total No. of samples Control Samples (boot socks without matrix) STM high 100 % 2/2 STM low 50 % 1/2 Blank 0 % 0/2 Samples (boot socks with Environmental material) STM high 80 % 7/8 STM low 60 % 5/8 Blank 15% at max 1/8

21 Specificity, sensitivity & accuracy (%) Control samples: boot socks without matrix The NRLs scored an excellent result for the control samples!! Specificity MSRV/X Blank 100 Sensitivity STM low 99 STM high 100 Accuracy All samples 99.5 X = plating out medium with the highest number of positive isolations

22 Specificity, sensitivity & accuracy (%) Samples: boot socks with chicken faeces Specificity MSRV/X Blank 99.7 Sensitivity STM low 94.4 STM high 98.3 Accuracy All samples 95.8 X = plating out medium with the highest number of positive isolations No difference between isolation media XLD or other medium

23 STM high : MSRV/x Number of positive isolations Lab code

24 STM low : MSRV/x Number of positive isolations Lab code

25 RESULTS: performance of the NRLs Control boot socks without chicken faeces: excellent - All NRLs scored correctly blank and STM controls - 1 NRL scored 1/2 STM low negative Samples : boot socks with environmental material/chicken faeces - 1 NRL scored 1/8 blank boot socks false positive - 3 NRLs scored 1/8 STM high negative - 2 NRLs scored 1/8 STM low and 1/8 STM high negative - 10 NRLs scored 1/8 STM low negative - 2 NRLS scored 2/8 STM low negative 19 from the 36 participants tested all boot socks with faeces contaminated with STM positive. All NRLs scored within the lines of GOOD PERFORMANCE!!

26 Increase in positive results after additional 24 h of incubation MSRV 24/48 h % positive results Increase additional incubation Blank 0/0.3% 0.3 % (1) STM low 91/94 % 3 %(9) STM high 96/98% 2 %(6) Laboratory 15 found 50 % more STM samples positive after 48 h of incubation (24h: 8 pos; 48h: 16 pos)

27 Conclusions (1) All participants achieved the level of good performance The laboratories scored excelent results for the control samples The Sensitivity and Accuracy rates for the artificially contaminated boot socks was high 96 % 48 hours of incubation of MSRV gave overall 2-3 % more positive results.

28 Conclusions (2) The samples used in this study boot socks with environmental material (chicken faeces) artificially contaminated with a diluted culture of Salmonella (STM) : - mimic well routine samples - gave good results in this study - were easier to test by the participants.but more work for the EURL: within a few days preparation of 1200 samples Webbased report - easier for the EURL to analyse the results hopefully also easier for the participants?

29 THANKS for your participation & cooperation in this study Angelina Kuijpers