EFSA Scientific Report (2004) 18, 1-13 on the Evaluation of Seven New Rapid post mortem BSE Tests

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Scientific Report of the European Food Safety Authority on the Evaluation of Seven New Rapid post mortem BSE Tests Question N° EFSA-Q-2003-084 Adopted on 16 November 2004

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. At present 5 rapid BSE test kits are approved by the EC for the post mortem testing of slaughtered cattle 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 the third European evaluation exercise for newly developed rapid post mortem and live animal TSE/BSE tests. This call for the expression of interest remained open until 4th April 2003. The laboratory evaluation of selected rapid post mortem BSE tests was divided in two phases: a phase I laboratory evaluation and a field trial. The laboratory evaluation was organised, carried out and analysed by the European Commission’s Institute of Reference Materials and Measurements (IRMM) and the results were assessed by EFSA’s Working Group on TSE Testing. Only those candidates that had successfully passed the phase I laboratory evaluation were allowed to enter into the field trial. This EFSA Scientific Report provides synopses and conclusions on the results from the phase I laboratory evaluation of 10 tests and from 7 finalized field trials. Based on an overall assessment on the application dossiers, the phase I laboratory evaluation, the field trials and the approval of the package inserts, the experts of EFSA´s Working Group on TSE Testing express their favourable opinion on 7 new tests and recommend these test for approval by the European Commission in the framework of regulation (EC) No 999/2001.

Key words: BSE, Bovine Spongiform Encephalopathy, TSE, Transmissible Spongiform Encephalopathy, post mortem, rapid BSE test, evaluation, field trial, Regulation (EC) No 999/2001.

Background According to EU legislation all slaughtered cattle over the age of 30 months have to be tested using one of the EC approved1 rapid BSE tests. In addition, a defined number of fallen stock over 24 months of age as well as all emergency slaughtered cattle over 24 months of age have to be tested for BSE with one of the approved rapid test. At present, test kits of five manufacturers (Prionics Check Western, Enfer, Bio-Rad TeSeE, Prionics Check LIA and InPro CDI-5) are approved by the EC and listed in the Annex X to Regulation (EC) No 999/2001, as amended by Regulation (EC) No 1053/20032. The EC carried out a first evaluation of rapid post mortem BSE tests in 1999. Four tests were evaluated on brain tissue from clinical BSE cases. Three of these tests, Prionics Check Western, Enfer, Bio-Rad TeSeE, performed satisfactorily and were later approved pursuant to Regulation (EC) No 999/2001. The results were reported by the EC Directorate-General (DG) XXIV in their document on “the Evaluation of Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines, 8 July 1999”. A second laboratory evaluation by IRMM in 2002 covered five post mortem rapid tests. This evaluation was split into two phases, a phase I laboratory evaluation with a reduced samples size, and a second phase field trial to assess the performance of the tests under field condition. Two tests (Prionics Check LIA and InPro CDI-5) passed the field trial and were approved pursuant to Regulation (EC) No 999/2001 in 2003. The results of the laboratory evaluation were reported by IRMM in their document on “the Evaluation of Five Rapid Tests for the Diagnosis of Transmissible Spongiform Encephalopathy in Bovines” (2nd study), 27 March 2002.The design of the field trial was based on the opinion of the SSC on “Design of a Field Trial for the Evaluation of New Rapid BSE post mortem Tests” (22 February 2002). 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” (6 March 2003). Following a call for expression of interest in the Official Journal of the European Union (No C15) on 22 January 2003, twelve parties indicated their interest in participating in the third European evaluation exercise for their newly developed rapid post mortem TSE/BSE tests.

Terms of references EFSA was requested by the EC to take on the responsibility for the scientific aspects of the evaluation of new rapid TSE/BSE tests. Besides the evaluation of the technical dossiers of newly developed tests and the results of a phase I laboratory evaluation the EFSA Working Group on TSE Testing was also asked to assess the field trial results of the submitted rapid BSE post mortem and to provide a recommendation these tests.

Evaluation of new rapid post mortem tests for the diagnosis of BSE The evaluation of the new BSE tests covered: 1. Assessment of the application dossier 2. Phase I laboratory evaluation3 3. Field trial4 4. Package insert 1

As laid down in Annex 10, chapter C to Regulation 999/2001 OJ L 152, 20.6.2003, p.8. 3, 4 Reports with detailed results of phase I laboratory evaluation and field trials: www.irmm.jrc.be 2

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1. Assessment of the application dossiers The dossiers were assessed based on pre-defined criteria covering the soundness of the underlying scientific test principle, available experimental evidence on sensitivity, specificity and reproducibility, practicability of testing procedures and stage of development of the test. Based on the assessment by EFSA’s Working Group on TSE Testing, the following 10 tests were offered to enter into the phase I laboratory evaluation: CediTect BSE Test; CEDI Diagnostics, the Netherlands FRELISA BSE; Fujirebio, Japan IDEXX HerdChek BSE Antigen Test Kit, EIA; IDEXX Laboratories Inc., USA Institut Pourquier Speed’it BSE; Institut Pourquier, France PDL Rapid BSE Test; Prion Developmental Laboratories Inc., USA Prionics Check PrioSTRIP; Prionics A.G., Switzerland Priontype post mortem; Labor Diagnostik GmbH Leipzig, Germany Roboscreen Beta Prion BSE EIA Test Kit; Roboscreen GmbH, Germany Roche Applied Science PrionScreen; Roche Diagnostics GmbH, Germany Test for the Diagnosis of TSE in Ruminants; Universitätsklinikum Hamburg-Eppendorf, Germany♦ _________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Enfer TSE Kit version 2.0, automated sample preparation∗; Enfer Scientific Ltd., Ireland ∗ Additionally, the EC requested to include the already approved Enfer TSE Kit version 2.0 in the evaluation since the test developer had developed and proposed an automated sample preparation. Such a major modification requires a full re-evaluation of the test as laid down in regulation (EC) 999/2001. ♦

The test developer never subjected the test to the phase I laboratory evaluation.

2. Phase I laboratory evaluation The phase I laboratory evaluation was organised, carried out and analysed by the Institute IRMM. The protocol on the organisation can be found in Annex 1 of the EC DG-JRC Global Report on the laboratory phase I: “The evaluation of 10 rapid post mortem tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines”. This report provides all 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. The provision of suitable positive samples was ensured by the Veterinary Laboratory Agency’s (VLA) TSE archive, New Haw, United Kingdom, and the Agence Française de Sécurité Sanitaire des Aliments (AFSSA) in Lyon, France. These samples were originally not collected according to IRMM’s technical specifications but all were in a good condition especially regarding the conserved morphology, size, weight and freshness. These samples allowed an unambiguous sampling following a well established permutation scheme to produce equivalent sample sets to all participants. This sampling scheme aimed at avoiding a possible discrimination of any test and guaranteed a fair distribution of sub-samples. 2.1. Parameters The laboratory phase I was designed to assess the following parameters:

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a) Diagnostic sensitivity: may be defined as the proportion of known clinically affected reference animals that test positive in the assay. Therefore 50 true positive samples were included in the exercise. The tissue was supplied in frozen form. b) Diagnostic specificity: is defined as the proportion of uninfected reference animals that test negative in a reference test. The diagnostic specificity was evaluated by testing 150 samples of known negative tissue. The tissue was supplied in frozen form. c) Detection limit: The test detection limit is defined as the smallest detectable amount of an analyte. Because of the nature of the assay, this determination may be relative. Each participant was supplied with minced central nervous tissue from BSE positive animals. This positive tissue was diluted on site in test specific buffer and in freshly prepared homogenate made up from negative central nervous tissue to produce dilutions of 1:5, 1:50, 1:100 and 1:200. Aliquots of each dilution were coded by Commission staff present on site and was tested in duplicate on at least three different plates. d) Repeatability: Each sample was tested in duplicate on the same microtiter plate or gel even if the protocol foresaw only a single analysis. The freshly prepared dilution series homogenates were tested in duplicate on up to three different microtiter plates or gels. 2.2. Summary of results phase I laboratory evaluation Table 1, depicted from the IRMM-Report on the phase I laboratory evaluation presents a summary of diagnostic sensitivity, diagnostic specificity and detection limits of 10 tests. Sensitivity in % Specificity in % (95 % confidence interval) (95 % confidence interval) CediTect BSE Test PDL Rapid BSE Test FRELISA BSE IDEXX HerdChek BSE Antigen Test Kit, EIA Institut Pourquier Speed’it BSE Priontype post mortem Prionics Check PrioSTRIP Roboscreen Beta Prion BSE EIA Test Kit Roche Applied Science PrionScreen Enfer TSE Kit v2.0, (autom. sample prep.)

Detection limit∗

100 (94 %) 96 (87.4 %) 100 (94 %) 100 (94 %)

100 (98 %) 100 (98 %) 100 (98 %) 100 (98 %)

≥ 1:200 1:10 ≥ 1:200 ≥ 1:200

100 (94 %)

100 (98 %)

1:64

100 (94 %) 100 (94 %) 100 (94 %)

100 (98 %) 100 (98 %) 100 (98 %)

1:25 1:100 ≥ 1:200

100 (94 %)

99.3 (96.2 %)

100 (94 %)

100 (98 %)

1:100 ≥ 1:200

∗ Numbers correspond to the dilution level where > 80 % of all replicates were still classified positive. Some tests could have higher detection limits.

Nine of the 10 tests demonstrated a 100 % diagnostic sensitivity. The PDL Rapid BSE Test (Prion Developmental Laboratories Inc., USA) did not correctly classify 3 true positive samples in an initial analysis. These samples were repeatedly tested in duplicate starting from the remaining homogenates and starting with new sub-samples from the remaining tissue slices, respectively. Only one of the 3 samples was now tested positive in duplicate on the new tissue slice, indicating a possible sampling effect. Tests starting from the remaining homogenates were still negative for all 3 samples. http://www.efsa.eu.int

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Nine tests showed a 100 % diagnostic specificity. One negative sample out of 3 “initial reactives” analysed by Roche´s PrionScreen test was also positive in retesting and thus was classified positive according to the test developer’s data assessment criteria. The positive pool for the analytical sensitivity testing was initially prepared using brainstem tissue from six different BSE positive animals. The testing of the dilution series for the assessment of an analytical sensitivity resulted for most of the tests in detection limits of 1:100 to 1:200, keeping in mind that some tests could produce signals on even higher dilutions. All samples were analysed in duplicate and most tests showed a good repeatability. 2.3. Conclusions on the results of the phase I laboratory evaluation Based on the results of the phase I laboratory evaluation EFSA´s Working Group on TSE Testing agreed on 9 tests to enter the field trial (CediTect BSE Test; Enfer TSE Kit version 2.0, automated sample preparation; FRELISA BSE; IDEXX HerdChek BSE Antigen Test Kit, EIA; Institut Pourquier Speed’it BSE; Prionics Check PrioSTRIP; Priontype post mortem; Roboscreen Beta Prion BSE EIA Test Kit; Roche Applied Science PrionScreen). No favorable opinion was given on the phase I laboratory evaluation results of the PDL Rapid BSE Test as two true positive samples were classified negative. This weakness of the diagnostic sensitivity testing is confirmed by the detection limit. Therefore the test was not allowed to enter the field trial. The judging on those two false negative test results becomes even more crucial considering that none of the positive samples included in the test panel can be declared as a weak positive, i.e. originating from an animal that is consistently resulting in low signals with most or all of the tests. 3. Field Trial The purpose of the field trial is to demonstrate a non-inferiority of new tests as compared to already approved tests. The field trial was performed according to a former opinion of the SSC on a “Design of a field trial for the evaluation of new rapid BSE post mortem tests” (22 February 2002) which was amended by EFSA’s Working Group on TSE Testing (EFSA Scientific Report, 2004, 1, 1-10 “on the design of a field trial protocol for the evaluation of new rapid BSE post mortem tests”). This field trial protocol defines that the performance of any new rapid BSE test should statistically not be inferior to the performance of currently approved tests. At present (November 2004) 7 test developers have finalised the field trial. Thus at the moment the assessment on the field trial results of 7 tests can be provided based on 7 EC DGJRC Field Trial Reports 2004 prepared by the IRMM on the performance of the following rapid post mortem BSE tests: CediTect BSE Test, Enfer TSE Kit version 2.0, automated sample preparation, IDEXX HerdChek BSE Antigen Test Kit, EIA, Institut Pourquier Speed’it BSE, Prionics Check PrioSTRIP, Roboscreen Beta Prion BSE EIA Test Kit, Roche Applied Science PrionScreen. 3.1. Parameters The field trial protocol was designed to assess the following parameters:

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a) Diagnostic sensitivity relative to reference tests: 200 true positive samples that were tested positive using a reference test, b) Diagnostic specificity relative to reference tests: 10,000 consecutive samples from healthy slaughtered cattle that were tested negative using a reference test, c) Performance of the new tests on poor quality negative samples: 200 poor quality negative samples that were tested negative using a reference test. The field trial should enable also the assessment of parameters like robustness, applicability in different laboratories and the expected rate of false initial results under high throughput field conditions. Additionally the conduction of the field trial provides the opportunity of necessary cut-off adjustment before market introduction. The conditions for field trial according to the protocol (EFSA Scientific Report, 2004, 1, 1-10 on the design of a field trial protocol for the evaluation of new rapid post mortem tests) were as following: a) Each new test should be compared to at least two reference tests as regards the sensitivity (200 true positive samples) and the specificity (10,000 negative samples). The maximum number of samples for each of the sensitivity and specificity testing analyzed by one of the reference tests should not exceed 70 %. b) The sensitivity testing (200 true positive samples) should be performed in National Reference Laboratories (NRLs) of one or more Member States (or Switzerland). Derogating from the general rule that sensitivity testing should be carried out in one or more NRL(s), certain other laboratories may take part in the study if the respective NRL does not conduct the study itself and if the responsible NRL agrees. c) The true positive samples can be provided by the National Reference Laboratories (NRL). They should be well documented (origin and age of the sample, e.g. subpopulation; condition of the sample, e.g. autolysis etc.; brain region used; storage conditions; duration of storage). d) In order to avoid test results being discrepant between the new rapid test and the approved test due to an uneven distribution of prion proteins the samples should be made homogeneous. The homogenisation treatment must follow an available protocol that has no or minimal influence on subsequent steps. Other equivalent homogenisation protocols can be used if their use is justified to IRMM. (Companies would have to show that their homogenisation protocol does not discriminate the reference test). e) The specificity testing (10,000 negative samples) should be performed with the agreement of the NRL in experienced high throughput routine laboratories. The samples should be prepared according to the company’s protocol. Fresh material immediately adjacent to the usual sampling region can be used for the comparison of the tests. Laboratories of at least two Member States (or one Member State and Switzerland) should be involved at the discretion of the company. The maximum proportion of samples tested in a single laboratory should not exceed 70 % of all samples. At least two batches of the test kits should be included. http://www.efsa.eu.int

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f) At least two batches of the new tests have to be included for the estimation of the sensitivity and the specificity. g) Raw data on the testing of the 200 true positive samples have to be submitted to IRMM at least on a weekly basis. Raw data on the testing of the 10,000 negative samples from routine slaughter and the raw data from the on the testing of the 200 poor quality samples have to be communicated to IRMM on a daily basis. h) Description of the test procedure: it was the duty of the test developer to provide clear, stringent and detailed descriptions of the test procedures used during the field trial. The test procedures used or the clarified test procedures if clarification was necessary would be defined as part of the approval. Later changes in the test procedure would invalidate the approval. 3.2. Summary of results Tables 2-8, depicted from the respective IRMM reports, present summarised results on the diagnostic sensitivity, diagnostic specificity and on the poor quality negative samples. The results of tests are listed in an alphabetic order of the test kit’s name: Table 2: CediTect BSE Test; CEDI Diagnostics, the Netherlands, (PVDF filter chemiluminescence ELISA for the detection of Proteinase K resistant PrP using a monoclonal antibody) Sample (n)

Initial reactives

Correctly identified

%

Specificity

4057 6047

0 0

4057 6047

Total Sensitivity

10104 143 60

0 143 *57

10104 143 *59

100

203 210

*200

*202

99.5

0

210

210

0

210

Total Poor quality samples Total

95 % CI limit, one sided poisson

Reference test Bio-Rad TeSeE Prionics Check Western

99.97 Bio-Rad TeSeE Prionics Check Western 97.7 Bio-Rad TeSeE

100

98.6

* Three samples were not identified as positive in the first test although their values were clearly above the background of negative samples. Two of them tested positive in a repeat test. These two samples would have been classified ‘initial reactive’ in the first test and positive in the retest if the new method of the RLUcut-off and Cut-off plate calculation had been applied as proposed by the test manufacturer retrospectively and agreed by the EFSA Working Group.

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Table 3: Enfer TSE Kit version 2.0 (automated sample preperation) Enfer Scientific Ltd., Ireland, (Chemiluminescence ELISA detecting Proteinase K resistant PrPSc using a polyclonal antibody and an enhanced chemiluminescent reagent) Sample (n)

Initial reactives

Correctly identified

7301 3191

8 53

7301 3191

10492

61

10492

Sensitivity

200

200

200

Total Poor quality samples

200

200

200

200

0

200

Total

200

0

200

Specificity Total

%

95 % CI limit, one sided poisson

Reference test Bio-Rad TeSeE Prionics Check Western

100

99.97 Bio-Rad TeSeE Prionics Check Western

100

98.5 Bio-Rad TeSeE Prionics Check Western

100

98.5

Table 4: IDEXX HerdChek BSE Antigen Test Kit, EIA; IDEXX Laboratories Inc., USA (Antigen-capture enzyme-linked immunoassay for detection of PrPSc using a monoclonal antibody, no Proteinase K treatment) Sample (n) Specificity Total

Initial reactives

♦

7177 3188

∇

10365

∇

Correctly identified

3 1 4

%

95 % CI limit, one sided poisson

7177 3187

Reference test Bio-Rad TeSeE Prionics Check LIA

∇ ∇

10364 99.99

99.95 Bio-Rad TeSeE Prionics Check Western

Sensitivity

200

200

200

Total Poor quality samples Total

200 200

200 0

200 200

100

200

0

200

100

98.5 Bio-Rad TeSeE 98.5

♦

This number includes a total of 287 samples from fallen stock (LDA 50, St. Lo, France) that were analysed in the frame of the specificity testing. ∇ One sample was repeatedly tested positive but remained negative when tested with reference tests by the French NRL and by the VLA Weybridge. According to the field trial this result was justified as false positive.

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Table 5: Institut Pourquier Speed’it BSE; Institut Pourquier, France (Chemiluminescence ELISA detecting Proteinase K resistant PrPSc using a monoclonal antibody) Sample (n)

Initial reactives

Correctly identified

5092 1996

6 10

5092 1996

3000

3

3000

10088

*19

10088

Sensitivity

200

200

200

Total Poor quality samples

200 200

0

200

Total

200

0

200

Specificity

Total

♦

200

♦

200

%

95 % CI limit, one sided poisson

Reference test Enfer TSE Prionics Check LIA Prionics Check Western

100

99.97 Bio-Rad TeSeE Prionics Check Western

100

98.5 Bio-Rad TeSeE Prionics Check Western

100

98.5

* Eleven samples were classified initially reactive and eight as ‘doubtful’ (grey zone). All samples were re-tested according to the test kit manual and correctly classified negative. ♦ In the first set of 'VLA panel' samples tested at the French NRL four samples were classified negative; one sample was 'doubtful' in the initial test. The initially 'doubtful' sample was re-tested according to the test kit manual. It was positive in duplicate measurements starting from the original sample preparation. All four initially negative samples were positive in duplicate when re-tested from the original 50 % tissue, 50 % water homogenate according to the field trial protocol. A re-test that started with the initially produced homogenates ended in three negative and one 'doubtful' (grey zone) classifications. The French NRL informed IRMM that pieces of tissue were found in the original sample preparations indicating a considerable heterogeneity of the preparations. This finding was considered by the EFSA Working Group and it was decided to test another set of 200 'VLA panel' samples with test at VLA Newcastle. All of the new aliquots gave initial reactive (positive) readings.

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Table 6: Prionics Check PrioSTRIP; Prionics A.G., Switzerland (Lateral flow immunoassay – PrioSTRIP - using two different monoclonal antibodies to detect Proteinase K resistant PrP). Sample (n)

Initial reactives

Correctly identified

4102 3879

0 4

4102 3879

940

0

940

2019 10940 75

0

2019

*4

*10940

75

75

126

126

126

201 223

201 ∇ 2

201 223

1 224

♦

0 223

%

95 % CI limit, one sided poisson

Bio-Rad TeSeE Prionics Check Western Prionics Check LIA Enfer TSE

Specificity

Total

100

99.97 Prionics Check Western Prionics Check LIA

Sensitivity Total Poor quality samples Total

∇♦

1 3

Reference test

100

98.5 Prionics Check Western Enfer TSE

99.6

97.9

* Two initial reactive results only in automated reading, one in visual reading and one in both automated and visual reading. ∇ Initial reactive in visual and automated reading. ♦ Initial reactive found in both in automated reading and still positive in one well in a retest (automated reading) and thus considered as a false positive. The sample was reported to be autolysed and derived from fallen stock. The sample was originally reported as specificity sample but afterwards reported as autolysed sample from fallen stock. Table 7: Roboscreen Beta Prion BSE EIA Test Kit; Roboscreen GmbH, Germany (Classical two-sided immunoassay using two different monoclonal antibodies directed against bovine Proteinase K resistant PrP). Sample (n)

Initial reactives

Correctly identified

%

3044 3385

0 4

3044 3385

4007 10436 200

3 7 200

4007 10436 200

Total Poor quality samples

200 200

200 0

200 200

100

Total

200

0

200

100

Specificity Total

95 % CI limit, one sided poisson

Bio-Rad TeSeE Prionics Check Western Enfer TSE 100

99.97 Prionics Check Western Bio-Rad TeSeE

Sensitivity

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Reference test

98.5 Prionics Check Western Bio-Rad TeSeE 98.5

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Table 8: Roche Applied Science PrionScreen; Roche Diagnostics GmbH, Germany (ELISA for the detection of PrPSc in streptavidin-coated microplates after Proteinase K digestion). Sample (n) Specificity Total

Initial reactives

Correctly identified

%

5033 5040

2 0

5033 5040

10073 200

2 0

10073 200

100

200 200

200 *54

200 199

100

200 200

*54

199

99.5

200

0

95 % CI limit, one sided poisson

Bio-Rad TeSeE Prionics Check Western 99.97 Prionics Check Western Bio-Rad TeSeE

Sensitivity Total Poor quality samples A “macerates” * Total Poor quality samples B “tissues” * Total

Reference test

98.5 Prionics Check Western Bio-Rad TeSeE 97.7

0

Bio-Rad TeSeE

200

100

98.5

* The VLA panel of poor quality negative samples (‘macerates’ 50 % tissue - 50 % water) initially produced a rate of 54 (~ 28 %) initial reactives. The test developer argued that this might be due to the sample preparation and not to limitations of the test. This was supported by the fact that the analysis of 200 autolysed tissue samples did not reveal any problems of the test with respect to autolysed material. Roche also provided evidence that the results obtained with the 200 positive samples, originally prepared in the same way as the poor quality macerates, were not biased by the effects occurred during the analysis of the poor quality macerates. 3.3. Conclusions on the results of the field trials EFSA’s Working Group on TSE Testing agreed that these 7 tests (CediTect BSE Test; Enfer TSE Kit version 2.0, automated sample preparation; IDEXX HerdChek BSE Antigen Test Kit, EIA; Institut Pourquier Speed’it BSE; Prionics Check PrioSTRIP; Roboscreen Beta Prion BSE EIA Test Kit, Roche Applied Science PrionScreen) have fulfilled the requirements of the field trial. It was concluded that their performance is not inferior to already approved tests. 4. Package inserts The package inserts of these 7 tests were accepted 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 Based on an overall assessment on 7 new rapid post mortem BSE tests, covering the application dossier, a phase I laboratory evaluation with 50 positive samples and 150 negative samples, a field trial with 200 positive samples, 200 poor quality negative samples and more than 10,000 samples from healthy slaughtered animals and the approval of package inserts, the experts of EFSA´s Working Group on TSE Testing express their favourable opinion and http://www.efsa.eu.int

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recommend the following tests for approval by the European Commission in the framework of regulation (EC) No 999/2001: - CediTect BSE Test - Enfer TSE Kit version 2.0, automated sample preparation - IDEXX HerdChek BSE Antigen Test Kit, EIA - Institut Pourquier Speed´it BSE - Prionics Check PrioSTRIP - Roboscreen Beta Prion BSE EIA Test Kit - Roche Applied Science PrionScreen

Documents provided to EFSA Letter with the ref. D(2003)CB/MG/ac/310087 from the European Commission – Health & Consumer Protection Directorate-General, requesting to take on the responsibility for the scientific aspects of the evaluation of rapid TSE/BSE tests. Applications from interested parties: Following the call of expression of interest on 22 January 2003, twelve organisations submitted their application and dossiers. EC DG-JRC Reports of the Laboratory Phase I on the performance of rapid post mortem BSE tests: CediTect BSE Test (13 Feb 2004), Enfer TSE Kit version 2.0, automated sample preparation (15 April 2004), IDEXX HerdChek BSE Antigen Test Kit, EIA (15 April 2004), Institut Pourquier LIA BSE (15 April 2004), Prionics Check PrioSTRIP (13 Feb 2004), Roboscreen BSE post mortem Kit (13 Feb 2004), Roche Applied Science PrionScreen (15 April 2004); Philipp, W., Van Iwaarden, P., Goll, M., Kollmorgen, N., Schimmel, H., and Vodrazka, P., (2004), Institute for Reference Materials and Measurements (IRMM). EC DG-JRC Global Report on the Laboratory Phase I: “The evaluation of 10 rapid post mortem tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines”; Philipp, W., Van Iwaarden, P., Goll, M., Kollmorgen, N., Schimmel, H., Vodrazka, P., (08 Oct 2004), Institute for Reference Materials and Measurements (IRMM). EC DG-JRC Field Trial Reports 2004 on the performance of rapid post mortem BSE tests: CediTect BSE Test (06 Sept 2004), Enfer TSE Kit version 2.0, automated sample preparation (06 Sept 2004), IDEXX HerdChek BSE Antigen Test Kit, EIA (06 Sept 2004), Institut Pourquier LIA BSE (06 Sept 2004), Prionics Check PrioSTRIP (30 June 2004), Roboscreen BSE post mortem Kit (06 Sept 2004), Roche Applied Science PrionScreen (6 Sept 2004, amendment on 9 Nov 2004); Philipp, W. and Vodrazka, P., Institute for Reference Materials and Measurements (IRMM). EC DG-JRC Global Field Trial Report 2004: the field trial of 7 rapid post mortem tests for the diagnosis of Bovine Spongiform Encephalopathy in Bovines; Philipp, W. and Vodrazka, P., Institute for Reference Materials and Measurements (IRMM).

References 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 22.01.2003, pp. 13-14.

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EC 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 1999. EC DG-JRC Report on the “Evaluation of five rapid tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines (2nd study) on 27 March 2002, IRMM. www.irmm.jrc.be EC DG-JRC Global Report on the Laboratory Phase I: “The evaluation of 10 rapid post mortem tests for the diagnosis of Transmissible Spongiform Encephalopathy in bovines”; Philipp, W., Van Iwaarden, P., Goll, M., Kollmorgen, N., Schimmel, H., Vodrazka, P., (08 Oct 2004), Institute for Reference Materials and Measurements (IRMM). EC DG-JRC Global Field Trial Report 2004: the field trial of 7 rapid post mortem tests for the diagnosis of Bovine Spongiform Encephalopathy in Bovines; Philipp, W. and Vodrazka, P., Institute for Reference Materials and Measurements (IRMM). EC (European Community), 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 31.05.2001, pp. 1-40. EC (European Community), 2003. 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 20.06.2003, pp. 8-9. EFSA Scientific Report (2004) 1, 1-10 on the “Design of a field trial for the evaluation of new rapid BSE post mortem tests”. SSC Opinion on 22 February 2002. Opinion of the Scientific Steering Committee on “Design of a field trial for the evaluation of new rapid BSE post mortem tests”. SSC Opinion on 6 March 2003. Opinion of the Scientific Steering Committee on “The field trial evaluation of two new rapid BSE post mortem tests.”

EFSA Scientific Expert Group members Concepcion Gomez-Tejedor, Martin H. Groschup, James Hope, Peter Lind, Johannes Löwer (chairman), Jean-Yves Madec, Danny Matthews, 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 and AFSSA Lyon are acknowledged for their support. 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. http://www.efsa.eu.int

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