REPORT TO THE EUROPEAN COMMISSION ON THE INTERLABORATORY COMPARISON TEST n 2011-ILC-01

Transcription

1 Organisation des Comparaisons Interlaboratoires Fiche d enregistrement Date de création : 17/03/10 Date de la révision : Date d archivage : Laboratoire LGP, La Tremblade Edition n 1 Réf. : E.CI.1.07 REPORT TO THE EUROPEAN COMMISSION ON THE INTERLABORATORY COMPARISON TEST n 2011-ILC-01 Provider of the Interlaboratory Comparison (ILC) test ILC test number Objective Content of the test Targeted diseases Material preparation information Time schedule Ifremer, Genetic and Pathology Laboratory, Community Reference Laboratory for Mollusc Diseases, Av. de Mus de Loup, La Tremblade, France 2011-ILC-01 To test the competency of participants regarding the detection of OsHV-1 by Real Time PCR in Pacific oyster (Crassostrea gigas) tissue 24 samples of tissue from Pacific oyster (Crassostrea gigas) One plasmidic DNA suspension corresponding to 10 5 viral copies/µl to prepare the standard curve and which should be used as positive control. Infection with OsHV-1 Samples were prepared by aliquoting ethanol fixed mantle suspensions from Pacific oysters. These samples had previously been checked by Real Time PCR. Reference results have been established based on these results (Table 1). Date of announcement 17/05/2011 Dead line for inscription 27/05/2011 Date of starting 31/05/2011 Date of the end 31/08/2011 Number of participants Details of the 15 participating laboratories are given in Table 2. Samples were sent by express mail using Chronopost company. Date of sample sending, package tracing codes and dates of sample reception were recorded for each Sample sending participant. The samples were packaged in order to ensure their protection. Pipettes (2µl, 20µl, 200µl and 1000 µl) and thermocycler for real-time PCR are necessary to perform the test. For more details consider section 3 of the following Required equipment document OsHV-1 detection and quantification by Real Time Polymerase Chain Reaction available on the EURL website Recommendation for test performance It was recommended to treat and test samples according to the document OsHV-1 detection and quantification by Real Time Polymerase Chain Reaction It was also recommended to test samples at once and to send results before the 31 st of August Recommendation for result restitution Expected results were qualitative : presence (+) or absence (-) It was recommended to indicate as well Ct values Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 1 /12

2 Results sending Once the test was performed results had to be reported using the electronic form available through the NRL section of the EURL website (follow the pages: NRL s section > Proficiency test > Results). For each participant, we established (Table 3, Figure 1): - the percentages of good answers corresponding to the number of results similar to the reference ones, For each sample, we established (Figure 2): - the percentages of good answers corresponding to the number of results similar to the reference ones, Results analysis Results synthesis We also established from the percentages of good answers of the 15 laboratories (Table 4): - the average - the minimum value - the first quartile - the median - the third quartile - the maximum value - the Kappa coefficient in order to appreciate the general agreement between participants After receiving results from participants, the EURL analysed results and sent a coded report informing each participant about their own results and global results. Decoded version of this report is only sent to the European Commission. Average 93,06% Minimum value 54,17% Results synthesis (All participants included) First quartile 95,83% Median 95,83% Third quartile 100% Maximum value 100% Kappa coefficient 0,76 Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 2 /12

3 Interpretation Maximum discrepancies in the results were observed for two samples: samples 2 and 7 (Figure 2). These samples were found negative after repeated PCR testing but gave positive results in three laboratories. Most of the errors came from an overdetection of the virus in some non infected samples which could be attributed to a lack of specificity of the assay in some laboratories conditions (laboratories A, D, G, F, B and C). Two laboratories (I and O) obtained negative results in infected samples. In the context of the laboratory I, looking at the Ct value, it seems that it is only an error of rewriting. In the context of the laboratory O, this under detection seems to be due to a lack of sensitivity of the PCR assay in the lab conditions. Three categories have been considered: no error is perfect one to three errors is almost perfect more than three errors requires investigation to find out the source of the problem. J K H No error: M Perfect E L N I A One to three errors: D Almost perfect G F B More than three errors: Investigation needed C O Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 3 /12

4 Conclusions Seven laboratories (J, K, H, M, E, L, N) out of the 15 participating ones presented perfect level for the detection of OsHV-1 by PCR. Six laboratories (I, A, D, G, F, B) presented one to three errors. Most of these errors seem to be due to a bad determination of the Ct cut-off value (above/bellow which a sample should be considered negative/positive). Considering the Ct values, in one case, the error seems to be due to a problem of contamination (sample 9, G) and in one case the error seems to be due to a rewriting mistake (sample 15, I). Two laboratories (C and O) presented more than three errors. For these laboratories, investigations should be carried out to understand the lack of sensitivity and specificity of the assay in their laboratory conditions. Contact persons Name ARZUL Isabelle JOLY Jean-Pierre ROBERT Maeva Address IFREMER, Av de Mus de Loup, La Tremblade FRANCE Tel iarzul@ifremer.fr jpjoly@ifremer.fr mrobert@ifremer.fr Date: the 17 th of October 2011 The coordinator: Isabelle ARZUL P.S. All the results are appended at the end of this report (Annex 1 and 2) Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 4 /12

5 Table 1 - Results of reference Sample number Host species OsHV-1 (+ or -) 1 Crassostrea gigas + 2 Crassostrea gigas 3 Crassostrea gigas + 4 Crassostrea gigas 5 Crassostrea gigas + 6 Crassostrea gigas 7 Crassostrea gigas 8 Crassostrea gigas + 9 Crassostrea gigas 10 Crassostrea gigas + 11 Crassostrea gigas 12 Crassostrea gigas 13 Crassostrea gigas 14 Crassostrea gigas 15 Crassostrea gigas + 16 Crassostrea gigas 17 Crassostrea gigas 18 Crassostrea gigas 19 Crassostrea gigas + 20 Crassostrea gigas + 21 Crassostrea gigas + 22 Crassostrea gigas + 23 Crassostrea gigas + 24 Crassostrea gigas + Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 5 /12

6 Table 2 - Participants details Institute Country Address Phone Central Veterinary Institute Veterinary Institute Instituto de Investigaciones Marinas FLI Insel Riems- Institute of Infectology CEFAS Istituto Zooprofilattico Sperimentale delle Venezie Institute for Diagnosis and Animal Health Agri-Food and Biosciences Institute-Belfast Marine Scotland - Science Marine Laboratory Institute Of Infectious And Parasitic Diseases Technical University of Denmark National Institute of Aquatic Resources National veterinary institute (SVA) Marine Institute IFREMER-LGP Institute of Marine Research The Netherlands Slovenia Spain Germany England UK Italy Romania Northern Ireland UK Scotland UK Greece Denmark Sweden Ireland France Norway Edelhertweg PH Lelystad Gerbiceva Ljubljana C/ Eduardo Cabello VIGO Südufer Greisfwald - Insel Riems Weymouth Laboratory Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB Via L. Da Vinci, Adria (Ro) Street Dr Staicovici 63, Sector Bucharest Stoney Road-Stormont- Belfast BT4 3SD UK PO Box , Victoria Road Aberdeen AB11 9DB 26th Oktobrioy No Thessaloniki Fish Diseases Laboratory Stigbøjlen Frederiksberg C Division of Fish Travvägen 20 S Uppsala Rinville Oranmore Co. Galway Av de Mus de Loup La Tremblade PO Box 1870 Nordnes NO Bergen (0) (0) (0) Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 6 /12

7 Table 3 and Figure 1- Results (%) by participant Lab. J K A H I M E D G F B L C O N Mean 100% 100% 96% 100% 96% 100% 100% 96% 96% 88% 96% 100% 75% 54,17% 100% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% J K A H I M E D G F B L C O N Report to the European Commission on the interlaboratory comparison test n 2010-ILC-01 Page 7 /12

8 Figure 2 - Results (% of good answers) per sample 100% 75% 50% 25% 0% Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 Sample 23 Sample 24 Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 8 /12

9 Table 4 Average, minimum value, first quartile, median, third quartile, maximum value and Kappa coefficient (general level of agreement regarding the scale of Landis and Koch [1977]) obtained by all the participating laboratories. Average (%) Minimum value (%) First quartile 1 (%) Median 2 (%) Third quartile 3 (%) Maximum value (%) Kappa value 4 93,06% 54,17% 95,83% 95,83% 100,00% 100,00% 0, % of participants obtained between mimimum value and first quartile of good answers. 2-50% of participants obtained between mimimum value and median of good answers % of participants obtained between mimimum value and third quartile of good answers. 4 - Scale of Landis and Koch (1977) 1 : < 0 poor agreement; slight agreement; fair agreement; moderate agreement; substantial agreement; almost perfect agreement; 1 perfect agreement 1 Landis J.R. & Koch G.G., The Measurement of Observer Agreement for Categorical Data, Biometrics 33: Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 9 /12

10 Annex 1- Results of each laboratory Sample Reference result J K A H I M E D G F B L C O N Reference results Good answers Wrong answers Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 10 /12

11 Sample Reference (Ct value) J K A H I M E D G F B L C O N 1 19,22 17,88 13,06 23,39 12,96 10,68 18,64 17,59 21,94 20,6 14,39 17,7 14,46 19,53 18,65 21, , , ,69 34, ,63 23,82 14,98 30,29 23,83 13,39 24,89 24,31 25,28 25,15 20,43 20,07 18,35 20,12 20,56 27, ,48 0 0, 37* ,5 35, ,38 19,56 15,92 21,8 18,55 16,74 18,12 19,95 23,18 22,7 15,41 21,61 17,55 21,7 0 21, , > , , ,2 30, , ,30 31, ,7 16,37 11,79 20,45 17,23 20,89 22,4 14,14 20,85 14,71 21, , , , ,5 35, ,83 16,69 12,26 20,66 15,36 12,76 19,33 17,57 20,05 19,5 14,94 19,99 14,07 20,8 0 23, , , , , * , , , , > ,82 21,17 13,49 27,99 15,95 9,4 19,52 17,72 20,88 19,7 16,63 18,57 15,26 20,8 0 22, , , , , , , > , ,82 23,25 11,57 23,68 16,91 12,44 18,39 15,47 19,89 18,7 14,92 20,18 14,97 21,5 0 20, ,62 22,35 12,54 22,26 16,07 12,15 21,09 15,78 22,88 21,7 13,82 19,96 14,28 20,7 0 20, ,19 18,3 0 25,59 21,18 13,3 20,16 18,03 23,93 20,7 16,09 20,43 17,47 22,3 19,29 23, ,62 20,28 12,49 20,41 21,4 15,48 22,99 19,09 22,52 21,2 16,78 19,08 14,04 21,9 0 21, ,73 23,58 12,98 23,3 19,04 15,19 21,7 20,8 23,71 23,2 17,85 21,23 16,72 21,6 0 24, ,34 18,83 12,16 23,54 17,04 11,2 21,41 17,86 23,09 19,6 13,81 18,66 14,43 23, ,84 Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 11 /12

12 Annex 2 - Ct values per sample: reference values in black and mean (± standard error) of the values obtained by the participants in red Ct value Mean Reference Sample Report to the European Commission on the interlaboratory comparison test n 2011-ILC-01 Page 12 /12