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 Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE Question N EFSA-Q Adopted on 26 September 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 had been conducted by the European Commission. In the absence of such data, 5 rapid post mortem TSE tests which performed 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 European Commission s Institute of Reference Materials and Measurements (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, 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 (Nor98) 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 also evaluated 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 On 17 May 2005, the EFSA TSE Testing WG adopted its Scientific Report on the Evaluation of Rapid post mortem TSE (EFSA, 2005) on six post mortem tests. In July 2005, EFSA has received IRMM s reports on the laboratory evaluation of three additional post mortem TSE tests intended for small ruminants. The assessment consists of the application dossiers, on results of the laboratory evaluation and of the package inserts of the three tests. 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 recommends two tests (IDEXX Herdchek, InPro CDI-5) for approval by the European Commission to be used in the field to assess the prevalence of classical scrapie and BSE in brainstem samples of sheep. Both tests are also recommended for the detection of atypical scrapie (Nor98) using cerebrum or cerebellum samples. In addition the IDEXX Herdchek test can be recommended also for brainstem samples to detect atypical scrapie (Nor98). The Fuijrebio FRELISA post mortem Test, (Fujirebio Inc.) is not recommended for approval on small ruminant tissue. In respect to goats the EFSA Working Group on TSE Testing recommended on the basis of current limited scientific knowledge that, in terms of testing, this species should be equally treated as sheep. 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 16

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). Page 3 of 16

4 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. 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 Nor98 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 Nor98 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). Page 4 of 16

5 2. Evaluation of rapid post mortem tests for the diagnosis of TSE in small ruminants During January to June 2004, 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 (Nor98) 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 reducing the minimum number of positive samples from 300 to 200. The IRMM report and its 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. On 17 May 2005, the EFSA TSE Testing WG adopted its Scientific Report on the Evaluation of Rapid post mortem TSE (EFSA, 2005): Bio-Rad TeSeE, Bio-Rad TeSeE sheep/goat, Enfer TSE Test, v.2.0 (automated sample preparation), Institut Pourquier Scrapie ELISA Test, Prionics Check LIA SR, Prionics Check Western SR. At this time the laboratory evaluation of the 3 tests listed below was not finalised: - Fujirebio FRELISA post mortem Test, Fujirebio Inc. Japan - IDEXX Herdchek BSE post mortem Test, IDEXX Laboratories Inc., USA - InPro CDI-5 rapid post mortem BSE Test, (provisionally EU approved test for small ruminants; in the meantime the InPro CDI-5 test was licensed by Beckman Coulter, USA.) IRMM finalised the laboratory evaluation in July 2005 and forwarded the results to EFSA (EC, June 2005; EC, July 2005a; EC, July 2005b) This EFSA Scientific Report provides an overview on the evaluation of the three tests and the conclusions and recommendations of the EFSA TSE Testing WG. Page 5 of 16

6 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 2.1 Assessment of the application dossier The EFSA Scientific Report on the Evaluation of Rapid post mortem TSE Tests intended for Small Ruminants, adopted on 17 May 2005, provides detailed information on the assessment of the application dossier and on the selection of tests for the laboratory evaluation (EFSA, 2005). The Fujirebio FRELISA post mortem Test, the IDEXX Herdchek BSE post mortem Test and the InPro CDI-5 rapid post mortem BSE Test were accepted for the laboratory evaluation. 2.2 Laboratory evaluation The laboratory evaluation was organised, carried out and analysed by IRMM. The published IRMM reports on the evaluation of rapid post mortem tests for the diagnosis of TSE in sheep provide details on the organisation and execution 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, EC June 2004) The following parameters were examined in the evaluation: 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 (Nor98) on cerebrum samples from 3 different sheep including the determination of the analytical sensitivity. f) Detection of BSE in brainstem samples of 3 experimentally infected sheep including the determination of the analytical sensitivity. 1 IRMM Reports on the evaluation of rapid post mortem tests in the EU Scrapie evaluation: Page 6 of 16

7 2.2.2 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 Nor98 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 s WG on TSE Testing recommended that rapid tests intended for small ruminants should be evaluated also 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/brainstem 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 brainstem. Seven of these lymph nodes from six different animals were negative in all four of previously evaluated tests applied to them (EFSA, 2005). 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 investigation was carried out by the Friedrich-Loeffler-Institut, Insel-Riems, Germany, using the L42 mab. Two of six tested samples gave an IHC-PrP positive lymphoid follicle implying, operationally at least, that the others could be regarded as negatives and that the calculated diagnostic sensitivities for these four tests were likely to be minimum estimates as well as for this evaluation. Page 7 of 16

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 / Nor98 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. About one thousand negative samples from each tissue (brainstem, lymphnodes, spleen) were used to assess the diagnostic specificity. It should be pointed out that for the evaluation of the three tests (Fujirebio FRELISA, IDEXX Herdchek, InPro CDI-5) comparable sample sets with aliquots from the same tissue homogenates from classical scrapie and BSE in sheep samples were used as for the evaluation of the first six recommended small ruminant tests (EFSA, 2005; EC, June 2004 and EC, April 2005). However this does not apply to the three cerebrum Nor98 samples. Although the three tests were applied to cerebrum samples from the same sheep as the first six recommended tests, the tissue for these three tests was not sourced from exactly the same site of the cerebrum as for the evaluation of the first six tests. The results of the three tests on the Page 8 of 16

9 Nor98 samples therefore should not be directly compared with the results of the first six tests previously published in the EFSA Scientific Report (EFSA, 2005). However the three tests were applied to comparable aliquots of tissue homogenate from Nor98 cerebrum samples. Therefore the test performance of the three tests on Nor98 samples can be compared to each other Results of the Evaluation The diagnostic sensitivities and specificities of three tests evaluated for their performance in identifying evidence of a prion protein disorder or TSE in sheep brainstem or cerebrum, lymph nodes or spleen are summarised in Table 2. These data are taken from the corresponding IRMM reports. Table 2: Diagnostic sensitivity and specificity of the 3 tests for different tissues Test Fujirebio FRELISA IDEXX Herdchek InPro CDI-5 Brainstem Specificity % 100 (99.7)* 100 (99.7) 99.9 (99.5) Sensitivity % 97.6 (95.2) 100 (98.8) 100 (98.8) Lymph nodes Specificity % Sensitivity % not done 99.2 ( ) 94.2 ( ) not done Spleen Specificity % Sensitivity % not done 99.5 ( ) 94.2 ( ) not done Cerebrum of 3 Nor98 cases 1:1 (1) Dilution not detected detected 1:200 (1) 1:500 (1) Brainstem of 3 experimentally BSE infected sheep Dilution detected 1:10 (2) 1:50 (1) 1:500 (2) 1:1 (1) 1:25 (2) 1:25 (1) 1:50 (1) * 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. The IDEXX Herdchek BSE post mortem Test and the InPro CDI-5 rapid post mortem BSE Test were tested only on two brainstem samples of BSE infected sheep. Page 9 of 16

10 Diagnostic specificity was 100 % for the the Fujirebio FRELISA and the IDEXX Herdchek and 99.9 % for the InPro CDI-5 on true negative brainstem samples from sheep. While the IDEXX Herdchek and the InPro CDI-5 test gave diagnostic sensitivity of 100 %, the Fujirebio FRELISA could detect only 97.6 % (240 of 246) true positive brainstem samples from clinical, confirmed cases of classical scrapie in sheep. According to the instructions of the Fujirebio s package insert, 6 true positive samples were classified negative and were considered false negative. Three out of these samples were also positive in a duplicate retest using the remaining tissue. The IRMM report concluded, based on the results of retesting false negative samples that the false negative test results were most probably not due to a lack of the test s diagnostic sensitivity. However following a request by EFSA for more details on the false negative test results, EFSA has received the information from the IRMM that the six cases were scattered over 5 different plates on 3 dates, which does not constitute the degree of "clustering" indicative of a singular technical error. The SSC Opinion on A programme for the evaluation of rapid post mortem tests to detect TSE in small ruminants Evaluation of Rapid post mortem Tests to Detect TSE in Small Ruminants (SSC, 2002b) states that a test should detect ideally 100% of all positive specimens as positive. In the absence of an EC evaluated and approved rapid post mortem test for small ruminants, the EFSA Working Group applied the criterion that a test has to demonstrate, with a probability of 95 %, that its sensitivity is not below 98 % (99 %) of the sensitivity of other recommended tests, if a sample size comprises at least 138 (258) samples that are positive by one of the approved tests. This criterion is defined by the SSC Opinion (2002a) and the EFSA Scientific Report (2005) to compare the diagnostic sensitivity of evaluated BSE post mortem tests to already approved tests The detection limits of each test relative to its cut-off point were determined for the three tests applied to brainstem samples from classical scrapie cases. They were ranked accordingly by analytical sensitivity as follows: IDEXX Herdchek test (detection limit at 1:2000 and 1:4000 for samples from UK and Cyprus) > InPro CDI-5 test (detection limit at 1:500 for samples from UK and Cyprus) > Fujirebio FRELISA test (detection limit at 1:100 for UK samples and 1:200 for samples from Cyprus) All three tests correctly identified, as TSE positive brainstem samples, clinical cases of experimental BSE in sheep. The BSE in sheep samples are identified as PG0948/03, PG0168/04 and PG1578/04. Only the Fujirebio FRELISA test was evaluated on all 3 BSE in sheep samples, the IDEXX Herdchek test on the samples PG0168/04 and PG1578/04 and the InPro CDI-5 test on the samples PG0948/03 and PG0168/04. The application of the three 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. Only the sample PG168/04 was used for a dilution series for all three tests. The detection limits on this sample were evaluated at 1:500 for the IDEXX Herdchek test, 1:50 for the InPro CDI-5 test and 1:10 for the Fujirebio FRELISA test. Detection limits on sample PG1578/04 were at 1:500 for the IDEXX Herdchek test and at 1:50 for the Fujirebio FRELISA test; detection limits on sample PG 948/04 were at 1:25 for the InPro CDI-5 test and at 1:10 for the Fujirebio FRELISA test. Page 10 of 16

11 The three tests were also applied to dilution series of three atypical scrapie Nor98 samples. The detection limits were at 1:500, 1:200 and 1:1 for the IDEXX Herdchek test, twice at 1:25 and once at 1:1 for the InPro CDI-5 test. The Fuijrebio FRELISA gave only negative results on the three Nor98 samples When applied to the panel of samples from mesenteric lymph node and from spleen tissue, the IDEXX Herdchek was on samples of both tissues about 94 % sensitive and about 99 % specific. The Fujirebio FRELISA test and the InPro CDI-5 test were not applied to lymphatic tissue. 2.3 Package inserts The package inserts of recommended 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. Conclusions The EFSA TSE Testing WG commented on the AFSSA Note and Opinion in its Scientific Report on the Evaluation of Rapid post mortem TSE adopted on 17 May 2005 (EFSA, 2005). For the conclusions on the test results the previously published EFSA Scientific Report on six evaluated TSE tests for small ruminants was considered (EFSA, 2005). The Experts of the EFSA TSE Working Group conclude: 1) Two tests (IDEXX Herdchek, InPro CDI-5) performed satisfactorily in terms of diagnostic sensitivity and diagnostic specificity when applied to brainstem from clinical, confirmed cases of classical scrapie in sheep. There are differences in the analytical sensitivities with the IDEXX Herdchek test showing the highest analytical sensitivity in that it was able to detect positive, non-autolysed brainstem samples up to a dilution of 1/4000 comparable with the corresponding results of the previously evaluated Bio-Rad TeSeE sheep/goat test. 2) The EFSA Working Group discussed potential reasons for the failure of the Fuijrebio FRELISA test on true positive samples. The false negative test results could have been due to a lack of sensitivity, lack of robustness, inappropriate instructions of the package inserts more in particular in respect to the homogenization process or due to a mishandling, applying not accurately the instructions of the package insert. Also a combination of these potential factors can not be excluded. Page 11 of 16

12 Apart from the unexpected (false negative) results, identified after decoding of the samples, no handling errors or mismatches were indicated. If mishandling had been occurred this should have been identified and indicated before the samples were decoded. This does not apply for the evaluation of the Fuijrebio FRELISA test. The EFSA Working Group could not accept the retesting of the false negative test results and therefore concluded that the Fuijrebio FRELISA test did not perform satisfactorily in terms of diagnostic sensitivity when applied to brainstem from clinical, confirmed cases of classical scrapie in sheep. 3) All three tests were able to detect PrP Sc in three brainstem samples of experimental BSE in sheep. The IDEXX Herdchek test gave the best analytical sensitivity in detecting the relatively protease-resistant PrP of experimental BSE in sheep cases at a dilution of 1:500 of the two tested samples. 4) While the IDEXX Herdchek test and the InPro CDI-5 test were able to detect all three Nor98 cases, the Fuijrebio FRELISA test gave only negative results on these samples. The IDEXX Herdchek test showed a analytical sensitivity on two samples up to the dilution step of 1:200 and 1:500, but like the InPro CDI-5 test tested positive only in the undiluted sample (1:1) of one of the three Nor98 cases. The results indicate that one of the three cerebrum samples had only a very low PrPsc concentration rather than a weak analytical sensitivity of the IDEXX Herdchek test and InPro CDI-5 test. 5) Only the IDEXX Herdchek test was evaluated against lymph nodes and spleen tissue and was able to detect PrP Sc with ~ 99 % diagnostic specificity and ~ 94 % 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 reports provide regulatory bodies with a rationale basis for the selection of tests for their various needs. For the recommendations on the three tests evaluated here, the previously published EFSA Scientific Report on six TSE tests evaluated for small ruminants was considered (EFSA, 2005). The EFSA WG notes that only two tests (IDEXX Herdchek, InPro CDI-5) performed satisfactorily in terms of diagnostic sensitivity when applied to brainstem 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. The IDEXX Herdchek test and the InPro CDI-5 test were able to detect all three atypical scrapie (Nor98) cases, the Fuijrebio FRELISA test failed on these samples. 1) Two tests, the IDEXX Herdchek and the InPro CDI-5, therefore can be recommended to be used in the field to assess the prevalence of classical scrapie and BSE in sheep in brainstem samples. Page 12 of 16

13 2) The Fuijrebio FRELISA post mortem Test, (Fujirebio) is not recommended for approval on small ruminant tissue. 3) The two tests (IDEXX Herdchek, InPro CDI-5) might also be used for the detection of atypical scrapie (Nor98) 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 only the IDEXX Herdchek test can be recommended also for brainstem (obex) samples to detect atypical scrapie (Nor98). 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 brainstem 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. 4) If the intention is to extend the use of tests to lymph node or to spleen samples, the IDEXX Herdcheck test could be suitable to detect PrP sc in these tissues. 5) 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 (communicated by the TSE Reference Lab). 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, the EFSA Working Group on TSE Testing recommends that, in terms of testing, goats should be equally treated as sheep. 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 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- Page 13 of 16

14 Lopez, M., Goll, M., LeGuern, L., Schimmel, H. and 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. EC, (June 2005). DG-JRC Report on the Fujirebio FRELISA rapid post mortem BSE test in the EU scrapie test evaluation Philipp, W.J., Van Iwaarden, P., Kollmorgon, N. and Vodrazka, P., (10 June 2005). Institute for Reference Materials and Measurements (IRMM) Reference Materials Unit, Geel, Belgium. EC, (July 2005a). DG-JRC Report on the Beckman Coulter s InPro CDI TM rapid post mortem test (version 2) in the EU scrapie test evaluation Philipp, W.J., Kollmorgon, N., Van Iwaarden, P. and Vodrazka, P., (04 July 2005). Institute for Reference Materials and Measurements (IRMM) Reference Materials Unit, Geel, Belgium. EC, (July 2005b). DG-JRC Report on the IDEXX HerdChek rapid post mortem test (version 2) in the EU scrapie test evaluation Philipp, W.J., Van Iwaarden, P.,Kollmorgon, N., Schimmel, H. & Vodrazka, P., (05 July 2004). Institute for Reference Materials and Measurements (IRMM) Reference Materials Unit, Geel, Belgium. Acknowledgement The chairman and the members of the Scientific Expert Working Group are acknowledged for their valuable contribution to this mandate. EFSA Scientific Expert Group members are: Concepcion Gomez-Tejedor, Martin H. Groschup, James Hope, Peter Lind, Johannes Löwer (chairman), Jean-Yves Madec, Kath Webster, Emmanuel Vanopdenbosch, Angus Wear. The VLA Newcastle is acknowledged for their support. The TSE Archive, VLA, UK is thanked for the supply of brainstem from experimentally BSE infected sheep approved by Independent Archive Advisory Group (IAAG, UK) and Dr. S. Benestad, National Veterinary Institute, Norway, for the supply of Nor98 samples. We further acknowledge the support of Wolfgang Philipp and his colleagues of the IRMM for the organisation of the evaluation and the preparation of the reports and to Koen Van Dyck (DG SANCO) for clarifications on procedural issues. Page 14 of 16

15 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, Nor98. 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 Nor98 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 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 Page 15 of 16

16 EFSA (2005). Scientific Report of the European Food Safety Authority on the Evaluation of Rapid post mortem TSE. EFSA Journal 31, 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 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 Nor98 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 Nor98. 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, Page 16 of 16