Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants

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1 EFSA Scientific Report (2005) 31, 1-17 on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants Question N EFSA-Q Adopted on 17 May 2005 Summary The European Food Safety Authority (EFSA) and its Scientific Expert Working Group on Transmissible Spongiform Encephalopathy (TSE) Testing were asked by the European Commission (EC) to take over the mandate of the former Scientific Steering Committee (SSC) for the scientific evaluation of rapid TSE/BSE (Bovine Spongiform Encephalopathy) tests. Until 2004 no evaluation of rapid TSE tests on material from small ruminants was conducted by the European Commission. In the absence of such data, 5 rapid post mortem TSE tests performing satisfactorily on bovine tissue were provisionally approved by the EC for the TSE monitoring of small ruminants in accordance with the TSE Regulation (EC) No 999/2001. Following an EC call for expression of interest in the Official Journal of the European Union (No C15) on 22 January 2003, several parties indicated their interest in participating in this third European evaluation exercise for newly developed rapid post mortem and live animal TSE/BSE tests. EFSA was asked to take over the scientific aspects of this evaluation and to assess the outcome of the IRMM evaluation of rapid TSE post mortem tests, taking also into account an opinion of the French Food Safety Agency (AFSSA), and to give recommendations on the approval of the tests. During January to June 2004, the European Commission s Institute of Reference Materials and Measurements (IRMM) carried out an evaluation of the diagnostic and analytical sensitivities, of the diagnostic specificity and repeatability of six rapid post mortem tests on samples from natural scrapie cases. Additionally the ability of these tests and their analytical sensitivity for the detection of atypical scrapie strain (Nor 98) in sheep tissue were evaluated. During August 2004, further brain samples from three clinical cases of sheep orally challenged with BSE-affected cattle brain homogenate were screened using each of the six rapid tests. In March 2005, in response to concerns of the EFSA WG on TSE Testing following confirmation of a case of BSE in a French goat, the six rapid tests were reevaluated against dilutions of brain homogenates from experimentally BSE infected sheep to provide analytical sensitivity for this material comparable to that previously obtained for scrapie.

2 The assessment comprised the assessment of the application dossiers, of the results obtained from the laboratory evaluation, organised, carried out and analysed by the IRMM, and of the package inserts. Based on an overall assessment on the application dossiers, the laboratory evaluation and the approval of the package inserts, the experts of EFSA s Working Group on TSE Testing express their opinion on 6 rapid post mortem TSE tests for small ruminants. Keywords: BSE, Bovine Spongiform Encephalopathy, TSE, Transmissible Spongiform Encephalopathy, post mortem, rapid BSE test, small ruminants, evaluation, Regulation (EC) No 999/2001, TSE monitoring. Page 2 of 17

3 Background According to Regulation (EC) No 999/2001 the prevention, control and eradication of certain transmissible spongiform encephalopathies (EC, 2001) in each Member State will be developed through an annual programme for monitoring BSE and scrapie that includes a screening procedure using rapid tests. Rapid tests shall be approved for that purpose in accordance with the comitology procedure (Commission proposal put for an opinion to the Standing Committee on the Food Chain and Animal Health) and listed in Annex X of Regulation (EC) No 999/2001. The list of rapid tests in Annex X to Regulation (EC) No 999/2001, as amended by Regulation (EC) No 1053/2003 (EC, 2003a), includes five tests: Prionics Check Western, Enfer, Bio-Rad TeSeE, Prionics Check LIA and InPro CDI-5. Regulation (EC) No 999/2001 does not specify the requirements for approving rapid tests for TSE monitoring. The policy so far has been that only tests which have performed satisfactorily in an independent evaluation have been approved for bovine animals. The Commission carried out a first evaluation of rapid post mortem TSE tests in Four tests were evaluated on brain tissue from clinical cases of BSE in cattle; three of these tests performed satisfactorily. Those three tests (Prionics Check Western, Enfer, Bio-Rad TeSeE) were later approved pursuant to Regulation (EC) No 999/2001. The results were reported by the European Commission DG XXIV in their document on the Evaluation of Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines (EC, 1999). A second laboratory evaluation by the Commission, again using brain tissue from clinical cases of BSE in cattle, covered five post mortem rapid tests and concluded in The sample size was smaller than in the first evaluation, and the Scientific Steering Committee (SSC) recommended that the tests should undergo a field trial with satisfactory results before they were approved. Two of the tests (Prionics Check LIA and InPro CDI-5) passed the field trial and were approved pursuant to Regulation (EC) No 999/2001 in The results of this laboratory evaluation were reported by the European Commission DG-JRC in their document on the Evaluation of Five Rapid Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines (2 nd study) (EC, 2002). The design of the field trial was based on the opinion of the SSC on the Design of a Field Trial for the Evaluation of New Rapid BSE post mortem Tests (SSC, 2002a). The results were reported in the Opinion of the Scientific Steering Committee on the Field Trial Evaluation of Two New Rapid BSE post mortem Tests (SSC, 2003). No evaluation of rapid TSE tests on material from small ruminants by the Commission was possible before February In the absence of such data, tests performing satisfactorily on bovine tissues were provisionally approved for small ruminants and used for active and passive surveillance for TSE in sheep and goats during (Bio-Rad TeSeE, Enfer Test, InPro CDI-5, Prionics Check Lia and Prionics Check Western). Terms of Reference The European Food Safety Authority is invited to assess the outcome of the IRMM evaluation of rapid TSE post mortem tests on tissues from small ruminants, taking also into account the opinion of AFSSA, and to give recommendations on the approval of the tests. Page 3 of 17

4 Assessment 1. TSE s in small ruminants Natural scrapie in small ruminants, in particular sheep, is a transmissible, progressive neurological disease with a range of clinical signs: weight loss, salivation, prorates and associated fleece loss and skin abrasions, nervousness and altered behaviour, ataxia of the hind limbs. In the past, the disease was confirmed by examination of the brain tissue for a triad of histopathological signs - vacuolation, loss of neurones and gliosis and, more recently, by immunhistochemical (IHC) or biochemical detection of abnormal prion protein (PrP Sc, the putative transmissible agent) in brain, spleen or lymphoid tissues. The classical case of scrapie the so-called typical scrapie defined above is associated with vacuolation, the accumulation of a relatively protease-resistant form of abnormal prion protein in the brainstem at the level of the obex and is usually, but not uniquely, found in animals carrying an ARQ or VRQ PrP allele (Hope, 1998). Evidence of natural infection can be found as early as two to four months of age in VRQ/VRQ lambs by IHC for abnormal PrP in tonsil biopsy samples (Schreuder et al., 1998) but, in general, the timing and spread of infection within a flock or individual sheep and the timing of disease depends inter alia on the route of exposure, breed and PrP genotype, and the type or strain of infecting agent (Van Keulen et al., 2000). In 2003, the molecular and histopathological spectrum of TSEs in sheep was extended by the discovery in Norway of an experimentally-transmissible, PrP-related, neurological sheep disease the Nor 98 sheep TSE phenotype - that was atypical of scrapie (Benestad et al., 2003). These findings were subsequently strengthened by the discovery of similar atypical scrapie cases in Germany and France (Buschmann et al., 2004a; Buschmann et al., 2004b; Madec et al., 2004). Other atypical TSE phenotypes have since been reported in Ireland (Onnasch et al., 2004), Portugal (Orge et al., 2004), Belgium (De Bosschere et al., 2004), Sweden, Finland, Spain and in the UK (Defra, unpublished). Atypical TSE has little or no vacuolation and abnormal PrP at the obex, but an intense cerebrum and cerebellar PrP Sc accumulation characterised by a smaller and less stable protease-resistant core of PrP Sc. This unusual distribution of histopathological lesions has already led to changes in the brain sampling protocols for sheep in several member states to facilitate a more comprehensive ascertainment and characterisation of these cases during scrapie surveillance. Atypical cases are more generally, but not uniquely, found in animals carrying an ARR, ARQ or AHQ PrP allele. For Nor 98 cases, this genotype correlation has been further refined recently to implicate another dimorphic codon in the PrP open reading frame, L141F (Moum et al., 2005). 2. Evaluation of rapid post mortem tests for the diagnosis of TSE in small ruminants During January to June 2004, the IRMM carried out an evaluation of the diagnostic and analytical sensitivities, of the diagnostic specificity and repeatability, and of an ability to detect an atypical strain of scrapie (Nor 98) in sheep tissues of six tests from four companies. The companies were: Bio-Rad (2 tests), Enfer Scientific Ltd., Institut Pourquier, Prionics (2 tests). This evaluation followed a protocol adopted from the opinion of the SSC on a Programme for the Evaluation of Rapid post mortem Tests to Detect TSE in Small Ruminants, 7-8 November 2002 (SSC, 2002b), amended by EFSA s TSE testing WG by reducing the number of positive samples from 300 to 200. The IRMM report and its Page 4 of 17

5 addendum on this evaluation are two of the supporting documents of this EFSA report provided to EFSA (EC, June 2004 and EC, April 2005). During August 2004, further brain samples from three clinical cases of sheep orally challenged with BSE-affected cattle brain homogenate were screened using each of the six rapid tests under the supervision of the VLA at the VLA Newcastle facilities, UK. In March 2005, in response to concerns of the EFSA TSE testing WG following confirmation of a case of BSE in a French goat, the six rapid tests were re-evaluated against dilutions of experimental BSE in sheep brain homogenates to provide analytical sensitivity for this material comparable to that previously obtained for scrapie. These data are provided as an addendum to the IRMM report in the supplementary documents of this Opinion. The French Food Safety Agency (AFSSA) has independently carried out an evaluation and issued an opinion on the performance of two rapid post mortem TSE tests on material from small ruminants. Based on that opinion the French government has addressed a note to the Commission asking it to reflect on the choice of the rapid test for the monitoring of TSE in small ruminants in The note and the opinion were considered by the EFSA WG on TSE Testing. The evaluation of the rapid TSE tests intended for small ruminant covered: 2.1. Assessment of the application dossier 2.2. Laboratory evaluation Package insert Assessment of the application dossier All companies of the provisionally approved tests for active and passive surveillance for TSE in sheep and goats during (Bio-Rad TeSeE, Enfer TSE Test, InPro CDI-5, Prionics Check Lia and Prionics Check Western) were invited to participate in this evaluation. The EFSA Working Group on TSE Testing agreed on the applications by Enfer Scientific Ltd. on a modification of the Enfer test with automated sample preparation (version 2.0) and by Prionics on the versions of the Prionics Check LIA and Prionics Check Western adapted to small ruminant tissue: Prionics Check LIA SR (small ruminants) and Prionics Check Western SR (small ruminants). Additionally all application dossiers submitted to EC call for expression of interest in the Official Journal of the European Union (No C15) on 22 January 2003 (EC, 2003b) were evaluated. The selection of tests was based on pre-defined criteria covering the soundness of the underlying scientific test principle, available experimental evidence on sensitivity, specificity and reproducibility on small ruminant tissue, practicability of testing procedures and stage of development of the test. Applying these criteria in the assessment of the dossiers, two applications were selected in July 2003 to participate for which EFSA has received a specifically prepared dossier for the small ruminant test evaluation: Bio-Rad TeSeE sheep/goat and the Institute Pourquier Scrapie ELISA test. 1 IRMM Report on the evaluation of rapid post mortem tests for the diagnosis of TSE in sheep: Page 5 of 17

6 In February 2005, two post mortem TSE tests which had been submitted to the above referred call of 2003 were agreed by the EFSA Working Group on TSE Testing to enter the laboratory evaluation: Idexx TSE post mortem test and Fujirebio FRELISA post mortem test. Each of the dossiers on the 4 additional tests submitted in 2003 (Bio-Rad TeSeE sheep/goat, Institut Pourquier Scrapie ELISA test, Idexx TSE post mortem test and Fujirebio FRELISA post mortem test) and agreed to enter the laboratory evaluation had provided experimental evidence on sensitivity and specificity testing on at least 100 small ruminant samples. In total 9 applications of BSE/TSE post mortem tests were refused for the evaluation on small ruminant tissue which provided no or not significant experimental evidence on specificity and sensitivity testing on small ruminant tissue to justify the forwarding to the large scale laboratory evaluation by the IRMM. In this respect EFSA refers also to the EC call for interests (OJ C15, 22 Jan 03) stating in bullet point 4. Validity of the call : The evaluation programme may be limited, cancelled or delayed if sample tissues or appropriate test material cannot be made available in due time or in sufficient amounts. The availability of atypical scrapie Nor 98 samples (at the time of sample collection) and especially the availability of samples from experimentally BSE infected sheep, of high scientific value, were very limited. The selection process resulted in finally 9 post mortem tests which were agreed by the EFSA Working Group on TSE Testing to enter the laboratory phase: - Bio-Rad TeSeE, Bio-Rad, France (provisionally EU approved test for small ruminants) - Bio-Rad TeSeE sheep/goat, Bio-Rad, France - Enfer TSE Test, version 2.0, automated sample preparation, Enfer Scientific Ltd., Ireland (version with manual sample processing provisionally EU approved test for small ruminants) - Fujirebio FRELISA post mortem Test, Japan - Idexx TSE post mortem Test, IDEXX Laboratories Inc., USA - InPro CDI-5, InPro Biotechnonology, USA (provisionally EU approved test for small ruminants) - Institut Pourquier Scrapie ELISA Test, France - Prionics Check LIA SR (small ruminants), Switzerland (provisionally EU approved test for small ruminants) - Prionics Check Western SR (small ruminants), Switzerland (provisionally EU approved test for small ruminants) At present the laboratory evaluation for 6 out of these 9 tests has been finalized Laboratory evaluation The laboratory evaluation was organised, carried out and analysed by the Institute IRMM. The published IRMM report on the evaluation of rapid post mortem tests for the diagnosis of TSE in sheep provides details on the organisation of the evaluation including the selection of positive and negative tissues, the sample preparation and testing procedures, the results and the comments by the participants Parameters (see IRMM report) The following parameters were examined in the evaluation: Page 6 of 17

7 a) Diagnostic sensitivity was defined as the ability to correctly identify infected tissues of true positive samples. b) Diagnostic specificity was defined as the ability to correctly identify non-infected tissues. c) Analytical sensitivity was defined as the ability to identify low concentration of PrP Sc (in a dilution series). d) Repeatability was determined by parallel analysis of two aliquots of the same tissue homogenate e) Detection of an atypical scrapie strain (Nor 98) on cerebrum samples from 3 different sheep including the determination of the analytical sensitivity. f) Detection of BSE in brain stem samples of 3 experimentally infected sheep including the determination of the analytical sensitivity Overview on the laboratory evaluation Consideration of the pathogenetic factors influencing the timing and expression of TSEs in small ruminants was critical for the test evaluation process. Tissues were sourced from natural clinical, confirmed cases from Cyprus, UK, Ireland, France and Norway (the atypical scrapie Nor 98 cases) occurring in a variety of breeds and PrP genotypes. The characteristics of these samples are summarised in Table 1 and further details of their selection, sample preparation and the testing procedures can be found in the IRMM report (p6-13, EC June 2004). The range of genotypes included in this evaluation corresponded to 93 % of those genotypes in which scrapie was confirmed in the EU15 member states during 2002 and The age range of the animals was 16 months to 6 years but, apart from the geographical spread of the cases, no attempt was made to diversify the range of natural scrapie strains included in the evaluation. During an initial review of the IRMM report, and in the light of a suspect and subsequently confirmed case of BSE in a French goat, the EFSA TSE testing WG recommended that the six rapid tests should re-evaluated against dilutions of experimental BSE in sheep brain homogenates to provide analytical sensitivity for this material comparable to that previously obtained for scrapie. Three midbrain/brain stem samples from orally-dosed sheep were provided by VLA for this purpose (Table 1; Bellworthy at al., 2005; Benestad et al., 2003). Ileal mesenteric lymph nodes and spleen samples were sourced from sheep that had been confirmed as scrapie cases by histology or immunocytochemical detection of abnormal PrP at the level of the brain stem. They were not tested directly for PrP Sc prior to the evaluation but only seven lymph nodes from six different animals were negative in all four tests applied to them and this triggered a retrospective investigation of sub-samples from these cases by IHC to determine if they contain no (detectable) PrP Sc or simply sub-optimal amounts of PrP Sc for rapid testing. This retrospective investigation was carried out by the Friedrich-Loeffler- Institut- (FLI), Insel-Riems, Germany. Page 7 of 17

8 Table 1: Characteristics of samples used in the TSE small ruminant evaluation Origin Cyprus France United Kingdom Tissue Specifically Tissue collected archive brainstem 121 mesenteric LN 121 spleen 121 brainstem 21 mesenteric LN 21 spleen 31 brainstem 67 mesenteric LN 85 spleen 72 brainstem/midbrain from sheep orallydosed with BSE (Bellworthy et al., 2005) 3 Prnp genotypes ARQ/ARQ VRQ/VRQ ARQ/VRQ ARQ/ARQ ARQ/VRQ ARQ/AHQ VRQ/VRQ ARH/ARH ARQ/ARH ARR/VRQ ARH/VRQ ARQ/ARQ ARQ/ARQ ARQ/ARQ Included breeds Chios (C) Friesland (F) C x F Manech Tete Rousse, Tete Noir Romney (1) and Suffolk (2) Ireland Norway brainstem 10 mesenteric LN 5 spleen 5 Cerebrum / Nor 98 3 ARQ/ARH ARQ/ARQ ARQ/VRQ ARQ/ARH ARQ/ARQ ARQ/VRQ Suffolk Suffolk X Norwegian white sheep Spel/Steigar Dala Negative control material was sourced from New Zealand from healthy adult sheep of mixed breed, but predominantly Romney, Suffolk and Perendale. The most frequent genotype in this sample was ARR/ARR Results of the Evaluation The diagnostic sensitivities and specificities of six tests evaluated for their performance in identifying evidence of a prion protein disorder or TSE in sheep brain stem or cerebrum, lymph nodes or spleen are summarised in Table 2. These data are taken from section of the IRMM report. All tests gave identical results for the duplicate samples. Page 8 of 17

9 Table 2: Diagnostic sensitivity and specificity of all tests for different tissues Test Brainstem Bio-Rad TeSeE Bio-Rad TeSeE, sheep/goat Enfer TSE Test v 2.0 Institut Pourquier Scrapie Test Prionics Check LIA Small Ruminant Prionics Check WB Small Ruminant Specificity % 100 (99.7)* 99.8 (99.4) 100 (99.7) 99.9 (99.5) 100 (99.8) 100 (99.8) Sensitivity % 99.6 (98.1) 100 (98.8) 100 (98.8) 100 (98.8) 100 (98.8) 100 (98.8) Lymph nodes Specificity % 100 (99.7) 100 (99.7) Sensitivity % 78.9 ( ) 94.2 ( ) aborted 97.8 ( ) 93.8 ( ) aborted 100 (99.7) 83.1 ( ) Spleen Specificity % 100 (99.7) 100 (99.7) Sensitivity % 85.1 ( ) Cerebrum of 3 Nor 98 cases Dilution detected 1:50 (1) 1:500 (2) 92.9 ( ) 1:50 (1) 1:500 (2) Brainstem of 3 experimentally BSE infected sheep Dilution detected 1:10 (1) 1:25 (1) 1:50 (1) 1:100 (3) not done not done not done not done 1:1 (3) 1:1 (1) 1:5 (2) Not detected 1:1 (2) 1:5 (1) 1:25 (2) 1:50 (1) 1:5 (2) 1:10 (1) 1:1 (3) 1:50(3) * Brackets: 95 % confidence intervals. Brackets: number detected out of a total of 3 samples tested, indicating the detection limit. Aborted: the evaluation was discontinued when a certain proportion of samples was tested and unsatisfactory results were obtained All six rapid tests applied to brain stem gave diagnostic sensitivities of % and diagnostic specificities of % When applied to the panel of mesenteric lymph node tissues, three tests (Bio-Rad TeSeE sheep and goat, Bio-Rad TeSeE and Prionics Check WB SR) gave 100 % specificity, one gave some possible false positives (Institut Pourquier Scrapie test, diagnostic specificity, 97.8 %) and two tests were withdrawn by the manufacturers because of unsatisfactory performance (Enfer TSE Test, v2.0 and Prionics Check LIA SR). The diagnostic sensitivities of the four tests which completed the evaluation on lymph nodes ranged from 78.9 to 94.2 % although only seven of the total 225 lymph node samples (196 animals) from TSE-confirmed sheep were negative in all four tests; all seven of these animals were of the ARQ/ARQ PrP genotype. Sufficient tissue was available from six of these sheep to allow IHC testing for abnormal prion protein using the L42 mab at FLI, Insel-Riems, Germany (Vorberg et al., 1999). Only one of these animals gave an IHC-PrP positive lymphoid follicle implying, Page 9 of 17

10 operationally at least, that the others should be regarded as negatives and that the calculated diagnostic sensitivities for these four tests are likely to be minimum estimates The detection limits of each test relative to its cut-off point (c/o) were determined for five of the six tests (excluding Prionics Check WB SR) applied to brain stem samples (Figure 1, which is a reproduction of Figure 57, p77, IRMM report, EC June 2004). They were ranked accordingly by analytical sensitivity as follows: Bio-Rad TeSeE sheep/goat > Bio-Rad TeSeE ~ Institut Pourquier Scrapie test > Enfer TSE Test, v2.0 * > Prionics Check LIA SR = Prionics Check Western SR. The Enfer TSE Test, v2.0 * showed a better sensitivity against UK samples than against Cyprus samples while with all four other tests the differential sensitivity was reversed. The Prionics Check WB SR test does not give a quantitative reading and so its data could not be processed in the same way. The detection limit of this test is about 1:100, similar to the Prionics Check LIA SR Mean response relative to cut-off 1 0,1 0,01 The results of the Prionics-Check Western SG are not included in the graph. The test identified positive dilution up to 1/100. 1:50 1:100 1:200 1:500 1:1000 1:2000 1:4000 negative Dilution Biorad TeSeE BioRad TeSeE SG Enfer TSE Institut Pourquier Speed'it scrapie Prionics-Check LIA SR cut-off Figure 1: Analytical sensitivity: serial dilution curves of the responses of five rapid tests relative to their cut-off points using brainstem tissue. The dilutions were done on positive brainstem samples from Cyprus. Further information on dilution series can be found in the IRMM evaluation report Three test manufacturers, Bio-Rad (2), Institut Pourquier and Prionics (1), allowed evaluation of their tests against dilution series of ileal mesenteric lymph node. Two tests, Bio- Rad TeSeE sheep/goat and Institut Pourquier Scrapie test, scored positive in this tissue at 1 :100 dilution (2/2 and 4/6, respectively), (Table 101, p 76, IRMM Report, EC June 2004) while the second Bio-Rad TeSeE test detected abnormal PrP in only 1 of 3 undiluted samples. The Prionics Check WB SR test did not score positive at 1:50 or greater dilutions and was not tested at 1:1. The two Bio-Rad tests were also able to detect abnormal PrP in undiluted spleen homogenate (Table 102, p76, IRMM Report, EC June, 2004). In a dilution series only the Bio-Rad TeSeE sheep/goat could detect the dilution of 1:10 (Table 52 and 53, p40, IRMM Report, EC June 2004). Page 10 of 17

11 We note that the original test material, 80 % homogenates of brain and 50 % homogenates of lymph nodes, may have contained large pieces of tissue of varying PrP Sc content and that this might theoretically compromise strict comparisons of analytical sensitivity especially in lymphoid tissues where the distribution of PrP Sc is more discrete and focally localized than in the brain. However, as pointed out by the IRMM research team, three of the four approved BSE tests detected dilutions of sheep brain stem in the same range, and with the same ranking, as reported from BSE test evaluations and proficiency testing on brain stem. This suggests that in practice sample heterogeneity did not affect the ranking of tests in terms of their analytical sensitivity All tests correctly identified, as TSE positive brain stem samples, three clinical cases of experimental BSE in sheep. The evaluation of the analytical sensitivity of these tests in detecting experimental BSE in sheep is provided in an addendum to the IRMM Report and summarized below in Figure 2. The BSE in sheep samples are identified as PG0948/03, PG0168/04 and PG1578/04. Bio-Rad TeSeE Bio-Rad TeSeE SG Enfer TSE Response relative to cut-off Response relative to cut-off :200 1:100 1:50 1:25 1:10 1:5 POS Response relative to cut-off Dilution of the positive homogenate 1:200 1:100 1:50 1:25 1:10 1:5 POS Dilution of the positive homogenate 1:200 1:100 1:50 1:25 1:10 1:5 POS Dilution of the positive homogenate Institut Pourquier Speed'it scrapie Prionics Prionics-Check LIA SR Response relative to cut-off Response relative to cut-off Cut-off _ Negative homogenate (individual readings) Dilutions of BSE in sheep homogenates (means of duplicate readings): PG0168 PG0948 PG1578 1:200 1:100 1:50 1:25 1:10 1:5 POS Dilution of the positive homogenate 1:200 1:100 1:50 1:25 1:10 1:5 POS Dilution of the positive homogenate Figure 2: Analytical sensitivity: serial dilution curves of the responses of five rapid tests relative to their cut-off points using brainstem tissue from clinical cases of BSE in orallydosed sheep (Bellworthy et al., 2005). All data points are means of two readings (duplicates, independent analyses of same homogenate). Dotted red lines represent cut-off values, with blue dashes representing individual readings of the negative sample on the Y-axis. The application of the rapid tests to these dilution series confirmed that all tests could detect a positive sample of 70 % aqueous tissue macerate but that they had differing analytical sensitivities. The six tests performed very differently on the three BSE in sheep samples. The Bio-Rad TeSeE sheep/goat had the greatest analytical sensitivity and was able to detect at Page 11 of 17

12 1:100 dilution (a 1:200 dilution was not tested with either of the Bio-Rad methods). The Bio- Rad TeSeE was able to detect 3 out of 3, at 1:10, 2 out of 3 at 1:25 and 1 out of 3 at 1:50 dilutions (PG168, PG1578 and PG1578 respectively). The Prionics Check WB SR is not included in this figure, but was able to detect all samples at 1:50 dilution. The Prionics Check LIA SR was able to detect all samples at 1:5 dilution and 1 out of 3 at 1:10 dilution (PG168). The Institut Pourquier Scrapie test was able to detect all samples at 1:25 dilutions and 1 out of 3 at 1:50 dilution (PG168). Enfer TSE Test, v2.0 detected all three neat samples (70 % tissue in water) and one out of 3 samples at 1:5 dilution (PG168) Package inserts The package inserts of these 6 tests were accepted by the WG after inclusion of requested amendments. These package inserts are part of the approval and changes to them should only be introduced after having notified to, and agreed by, the Community Reference Laboratory and the EC. The AFSSA Note and Opinion The AFSSA note presents a comparison of two ELISA tests, Bio-Rad TeSeE OV (now called Bio-Rad TeSeE sheep/goat) and Bio-Rad TeSeE, and the Prionics Check WB test for detection of a prion protein disorder or TSE in lymphoid tissue or spinal marrow (cord) from sheep during the pathogenesis of natural scrapie. If PrP immunohistochemistry (IHC) is used as the reference method for ascertainment of PrP abnormality or infection then the test performances can be summarized by their concordance indices (CI = [True +ve + True ve]/ [True +ve + True ve + False +ve + False ve]) as follows: for spinal cord, CI = 88 %, Prionics; 89 % Bio-Rad TeSeE; 94 % Bio-Rad TeSeE sheep/goat; for lymphoid tissues, CI = 93 %, Prionics; 97 % Bio-Rad TeSeE; 97 % Bio- Rad TeSeE sheep/goat. The AFSSA note also provides data indicating that during the pathogenesis of natural scrapie in their Langlade flock evidence of a prion protein abnormality, interpreted as a surrogate marker of infection, in lymphoid or spinal cord samples was first found using the Bio-Rad TeSeE sheep/goat test. Ranking of tests according to the earliest age of detection of a PrP Sc abnormality or TSE a measure of their relative analytical sensitivities - was: Bio-Rad TeSeE sheep/goat higher than Bio-Rad TeSeE higher than Prionics Check WB. The AFSSA Opinion concludes that for scientific reasons, that the active control program of TSEs in small ruminants to be led only using the test commercialized by the Bio-Rad Company, specifically the Bio-Rad TeSeE sheep/goat ELISA test. The AFSSA acknowledged the limitations of their study: a) only VRQ/VRQ sheep were tested and they have a reduced frequency in all French breeds, b) only animals naturally exposed to the Langlade type or strain of scrapie were examined, c) that their findings might not extend to other European strain isolates, and d) only a small number of animals were tested. They recommended the evaluation of test sensitivity and specificity in field conditions. Page 12 of 17

13 Conclusions The IRMM evaluation data extends the findings of AFSSA note and opinion covering more tests and more sheep tissue samples with wider genetic and geographic diversity. The EFSA WG noted that the Prionics Check WB in the AFSSA report is not the same test as the Prionics Check WB SR (small ruminants) assessed by the IRMM in their more recent evaluation but that these Prionics WB tests appear to rank similarly in terms of analytical sensitivity against the Bio-Rad tests. The IRMM study also evaluated, in a very limited number (n =3), the capability of these tests to detect PrP Sc of atypical scrapie (Nor 98) and experimental BSE in sheep. All tests performed satisfactorily in terms of diagnostic sensitivity and diagnostic specificity when applied to brain stem from clinical, confirmed cases of classical scrapie in sheep. There are differences in the analytical sensitivities with the Bio-Rad TeSeE sheep/goat test showing the highest analytical sensitivity in that it was able to detect a positive, non-autolysed brain stem sample at a dilution of 1/4000. With the exception of the Prionics Check LIA SR all other evaluated tests were able to detect the three atypical scrapie (Nor 98) cases when applied to undiluted cerebrum samples. However sample dilutions of all three Nor 98 samples were detected only by the two Bio-Rad tests (at a dilution of 1:50 or higher). All tests were able to detect PrP Sc in three brain stem samples of experimental BSE in sheep. The Bio-Rad TeSeE sheep/goat test gave the best analytical sensitivity in detecting the relatively protease-resistant PrP of experimental BSE in sheep cases (at a dilution of 1:100 in all three samples). While the Enfer TSE Test, v2.0 and Prionics Check LIA SR test gave no valid results when applied to lymph nodes, the other tests could detect PrP Sc in mesenteric lymph nodes of sheep with clinical scrapie although with a lower sensitivity as compared to brain stem samples. The highest diagnostic sensitivity (approximately 94 %) could be achieved using the Bio-Rad TeSeE sheep/goat and the Institut Pourquier Scrapie test. Only the two Bio-Rad tests were evaluated against spleen tissue and were able to detect PrP Sc with a 100 % diagnostic specificity and an % diagnostic sensitivity. Recommendations One pre-requisite of the comparison of TSE surveillance data across member states is that the screening (and confirmatory) tests are of similar diagnostic and analytical sensitivities. Tests have to be fit for purpose, and the selection of a test may change if the tissue, or purpose for which it is applied, is changed. The rigorous data provided in the IRMM report will provide regulatory bodies with a rationale basis for the selection of tests for their various needs. The EFSA WG notes that all tests performed satisfactorily in terms of diagnostic sensitivity when applied to brain stem from clinical, confirmed cases of classical scrapie in sheep. All tests were also able to detect PrP Sc in the three samples of experimental BSE in sheep provided by the VLA. All tests apart from the Prionics Check LIA SR were able to detect the three atypical scrapie (Nor 98) cases. Page 13 of 17

14 1, All tests therefore can be recommended to be used in the field to assess the prevalence of classical scrapie and BSE in sheep in brain stem samples. 2, With the exception of the Prionics Check LIA SR all tests might also be used for the detection of atypical scrapie (Nor 98) using cerebrum or cerebellum samples. If it is the goal to detect atypical cases in brainstem samples due to the low concentration of PrP Sc at this site (brainstem) only tests with high analytical sensitivity should be used. Therefore, in addition the two Bio-Rad tests can be recommended also for brainstem (obex) samples to detect atypical scrapie (Nor 98). According to current knowledge the distribution of PrP Sc in the brain of cases with atypical scrapie differs from the distribution in classical scrapie. While in classical scrapie a high concentration on PrP Sc is found in the brain stem its concentration is much lower in atypical scrapie where the highest concentration is found in cerebrum and cerebellum (Benestad et al., 2003; Buschmann et al., 2004a; Buschmann et al., 2004b). Therefore the EFSA Working Group on TSE Testing supports the legislative change (EC, 2005) that now requires the collection of cerebellum for surveillance purposes in order to reliably detect and characterize atypical cases. Based on the results with the limited number of cases used in this comparison at present tests with high analytical sensitivities for Nor 98 are the Bio-Rad TeSeE sheep/goat and Bio-Rad TeSeE. The recommendation above is supported by the experience in some Member States where the majority of atypical cases are detected using the Bio-Rad TeSeE. No data are available on the performance of these tests for the detection of BSE or atypical scrapie in goats. However atypical scrapie was recently detected in goats in France (Buschmann et al., 2004a) and Switzerland (TSE Reference Lab, not published). More research and published data would be necessary to substantiate the presence of atypical cases in goats. Nevertheless, on the basis of current limited scientific knowledge on the behaviour of BSE and atypical scrapie in goats, the EFSA Working Group on TSE Testing recommends that, in terms of testing, goats should be equally treated as sheep. If the intention is to extend the use of tests to lymph node samples, since the Enfer TSE Test, v2.0 and Prionics Check LIA SR tests did not give valid results when applied to lymph nodes, these tests can not be recommended for this purpose. If the intention is to extend the use of tests to spleen samples, tests not evaluated on this tissue can not be recommended for this purpose. Documents provided to EFSA Letter with the ref. D(2003)CB/MG/ac/ from the European Commission Health & Consumer Protection Directorate-General, requesting EFSA to take on the responsibility for the scientific aspects of the evaluation of rapid TSE/BSE tests. Letter with the ref. D(2003)KH/ip/ from the European Commission Health & Consumer Protection Directorate-General, requesting EFSA for an opinion on the evaluation of rapid TSE tests intended for small ruminants including the document Note from the French Government, dated 23 July 2003, and opinion of the French Food Safety Agency (AFSSA) of 19 May Page 14 of 17

15 Applications from interested parties: Following the call of expression of interest on 22 January 2003, twelve organisations submitted their application and dossiers. EC, (June 2004). DG-JRC Report: Evaluation of rapid post mortem tests for the diagnosis of TSE in sheep Philipp, W.J., Kollmorgon, N., Van Iwaarden, P., Charout-Got, J., Contreras- Lopez, M., Goll, M., LeGuern, L., Schimmel, H. & Vodrazka, P., (16 June 2004) Institute for Reference Materials and Measurements (IRMM) Reference Materials Unit, Geel, Belgium. EC, (April 2005). Addendum EC DG-JRC Report: Assessment of rapid scrapie tests with samples of experimental BSE infected sheep Wear, A. and Philipp, W., (April 2005); Veterinary Laboratories Agency (VLA) Newcastle, UK, and EC DG-JRC, Institute for Reference Materials and Measurements (IRMM), Reference Materials Unit, Geel, Belgium. References Bellworthy S.J., Hawkins S.A., Green R.B., Blamire I., Dexter G., Dexter I., Lockey R., Jeffrey M., Ryder S., Berthelin-Baker C., Simmons M.M., (2005). Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge. Vet Rec 156, Benestad S.L., Sarradin P., Thu B., Schonheit J., Tranulis M.A., Bratberg B., (2003). Cases of scrapie with unusual features in Norway and designation of a new type, Nor 98. Vet Rec 153, Buschmann A., Biacabe A.G., Ziegler U., Bencsik A., Madec J.Y., Erhardt G., Luhken G., Baron T., Groschup M.H., (2004a). Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. J Virol Methods 117, Buschmann, A., Luhken, G., Schultz, J., Erhardt, G., Groschup, M.H., (2004b). Neuronal accumulation of abnormal prion protein in sheep carrying a scrapie-resistant genotype (PrPARR/ARR). J Gen Virol 85, De Bosschere, H., Roels, S., Benestad, S.L., Vanopdenbosch, E., (2004). Scrapie case similar to Nor 98 diagnosed in Belgium via active surveillance. Vet Rec 155, EC, (1999). DG XXIV Consumer Policy and Consumer Health Protection Report on The evaluation of tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines on 8 July EC, (2001). Regulation No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain Transmissible Spongiform Encephalopathies. O. J. L 147 of , pp EC, (2002). DG-JRC Report on the Evaluation of five rapid tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines (2 nd study) on 27 March 2002, IRMM. EC, (2003a). Regulation No 1053/2003 of the European Parliament and of the Council of 19 June 2002 amending Regulation (EC) No 999/2001 as regards rapid tests. O. J. L 152 of , pp Page 15 of 17

16 EC, (2003b). Call for expression of an interest to participate in a programme for the evaluation of tests for the diagnosis of transmissible spongiform encephalopathies (TSE) in ruminants O. J. C15 of , pp EC, (2005). Regulation No 36/2005 of the European Parliament and of the Council of 12 January 2005 amending Annexes III and X to Regulation (EC) No 999/2001 as regards epidemio-surveillance for transmissible spongiform encephalopathies in bovine, ovine and caprine animals. O. J. L 010 of , pp Hope, J., (1998). Prion Protein-Related Diseases of Man and Animals. In Palmer, S.R., Soulsby, E.J.L., Simpson, D.I.H. (eds.) Zoonoses. Oxford University Press. pp Jeffrey, M., Martin, S., Gonzalez, L., Ryder, S.J., Bellworthy, S.J., Jackman, R. (2001). Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Path 125, Madec J.Y., Simon S., Lezmi S., Bencsik A., Grassi J., Baron T., (2004). Abnormal prion protein in genetically resistant sheep from a scrapie-infected flock. J Gen Virol 85, Moum T., Olsaker I., Hopp P., Moldal T., Valheim M., Moum T., Benestad S.L. (2005). Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor 98 cases. J Gen Virol 86, Onnasch, H., Gunn, H.M., Bradshaw, B.J., Benestad, S.L., Bassett, H.F. (2004). Two Irish cases of scrapie resembling Nor 98. Vet Rec 155, Orge L., Galo A., Machado C., Lima C., Ochoa C., Silva J., Ramos M., Simas J.P., (2004). Identification of putative atypical scrapie in sheep in Portugal. J Gen Virol 85, Schreuder, B.E.C., Van Keulen, L.J.M., Vromans, M.E.W., Langeveld, J.P.M., Smits, M., (1998). Tonsillar biopsy and PrP sc detection in the preclinical diagnosis of scrapie. Vet Rec 142, SSC, (2002a). Opinion of the Scientific Steering Committee on Design of a field trial for the evaluation of new rapid BSE post mortem tests adopted on 22 February SSC, (2002b). Opinion of the Scientific Steering Committee on A programme for the evaluation of rapid post mortem tests to detect TSE in small ruminants adopted on 7-9 November SSC, (2003). Opinion of the Scientific Steering Committee on The field trial evaluation of two new rapid BSE post mortem tests adopted on 6 March Van Keulen, L.J.M., Schreuder, B.E.C., Vromans, M.E.W., Langeveld, J.P.M. Smits, M.A., (2002). Pathogenesis of natural scrapie in sheep. Arch Virol Suppl 16, Vorberg I., Buschmann A., Harmeyer S., Saalmuller A., Pfaff E., Groschup M.H., (1999). A novel epitope for the specific detection of exogenous prion proteins in transgenic mice and transfected murine cell lines. Virology 255, Page 16 of 17

17 EFSA Scientific Expert Group members Concepcion Gomez-Tejedor, Martin H. Groschup, James Hope (rapporteur), Peter Lind, Johannes Löwer (chairman), Jean-Yves Madec, Kath Webster, Emmanuel Vanopdenbosch, Angus Wear. Acknowledgement The chairman and the members of the Scientific Expert Working Group are acknowledged for their valuable contribution to this mandate. The VLA Newcastle is acknowledged for their support. The TSE Archive, VLA, UK is thanked for the supply of brain stem from experimentally BSE infected sheep approved by Independent Archive Advisory Group (IAAG, UK). Thanks to Wolfgang Philipp and his colleagues of the IRMM for the organisation of the evaluation, the preparation of accurate, comprehensive and well structured reports, to Koen Van Dyck (DG SANCO) for clarifications on procedural issues. Page 17 of 17