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From: TSS (216-119-144-33.ipset24.wt.net)
Subject: EFSA Scientific Report Evaluation of BSE Tests for Live Cattle (PROTOCOL FOR CONSUMER PROTECTION ONLY)
Date: September 20, 2004 at 8:04 am PST

-------- Original Message --------
Subject: EFSA Scientific Report on the Design of a Field Trial Protocol for the Evaluation of BSE Tests for Live Cattle (provides a protocol for the purpose of consumer protection only.)
Date: Mon, 20 Sep 2004 10:00:08 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy
CC: CVM_BSE-L@LIST.NIH.GOV


EFSA Scientific Report on the Design of a Field Trial Protocol for the
Evaluation of BSE Tests for Live Cattle
Publication date: 17 September 2004

Adopted on 1 July 2004 (Question N° EFSA-Q-2003-084)

* 146 kB Report


* 90 kB Summary


Summary of the Report

The European Food Safety Authority 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 routine post mortem
testing of slaughtered cattle over 30 months of age in accordance with
the TSE Regulation (EC) No 999/2001. Following a 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 an EC evaluation exercise of their newly developed rapid BSE post
mortem and live animal tests.

It has been recognized that the availability of a rapid test for live
cattle would be a major advance in dealing with the problem of BSE and
TSE in general, but particularly with regard to epidemiological
screening. In the long term an accurate live animal test might offer the
possibility to reduce the number of culled animals after the detection
of one positive animal.

A rapid BSE test for live cattle could be approved for the purpose of
consumer protection, for epidemiological screening or for both. For the
purpose of consumer protection any new rapid BSE test including tests
for live animals should not be statistically inferior to that of the
currently approved post mortem tests.

This report provides a protocol for the design of a field trial protocol
for the evaluation of BSE tests for live cattle for the purpose of
consumer protection only.

http://www.efsa.eu.int/science/efsa_scientific_reports/bse_tse/612_en.html

EFSA Scientific Report (2004) 9, 1-8 on the Design of a Field Trial
Protocol for the
Evaluation of BSE Tests for Live Cattle.
Scientific Report of the European Food Safety Authority
on the Design of a Field Trial Protocol for the Evaluation of
BSE Tests for Live Cattle
Question N° EFSA-Q-2003-084
Adopted on 1 July 2004
Summary
The European Food Safety Authority 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 routine
post mortem testing of
slaughtered cattle over 30 months of age in accordance with the TSE
Regulation (EC) No
999/2001. Following a 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
an EC evaluation exercise of their newly developed rapid BSE post mortem
and live animal
tests.
It has been recognized that the availability of a rapid test for live
cattle would be a major
advance in dealing with the problem of BSE and TSE in general, but
particularly with regard
to epidemiological screening. In the long term an accurate live animal
test might offer the
possibility to reduce the number of culled animals after the detection
of one positive animal.
A rapid BSE test for live cattle could be approved for the purpose of
consumer protection, for
epidemiological screening or for both. For the purpose of consumer
protection any new rapid
BSE test including tests for live animals should not be statistically
inferior to that of the
currently approved post mortem tests.
This report provides a protocol for the design of a field trial protocol
for the evaluation of
BSE tests for live cattle for the purpose of consumer protection only.
Key words: BSE, Bovine Spongiform Encephalopathy, TSE, Transmissible
Spongiform
Encephalopathy, live animal test, evaluation, field trial, consumer
protection
Page 2 of 8
Background
According to EU legislation all slaughtered cattle over the age of 30
months have to be tested
using one of the approved1 rapid BSE tests. In addition, a certain
sample size of fallen stock
over 24 months of age as well as all emergency slaughtered cattle over
24 months of age have
to be subjected to an approved rapid test. Presently, test kits of five
manufacturers are in use
after having been evaluated and subsequently approved by the EC. In the
meantime, ten new
rapid tests have been developed and have taken part in an EC laboratory
evaluation exercise
according to a protocol defined by the European Commission. All of the
tests, which show the
potential to fulfil minimum parameter requirements, should be subjected
to an additional field
trial comparing the new rapid test with already approved tests.
Terms of Reference
EFSA was requested by the EC to take on the responsibility for the
scientific aspects of the
evaluation of rapid TSE/BSE tests.
Besides the evaluation of the technical dossiers of newly developed
tests and the results of a
first laboratory phase the EFSA Scientific Expert Working Group on TSE
Testing is also
asked to assess the results of field trials for submitted rapid BSE post
mortem and live animal
tests.
Design of a Field Trial Protocol for the Evaluation of Evaluation of BSE
Tests for Live Cattle
1. Introduction
Following a call for expression of interest the EU Commission received
20 proposals for new
tests following the publication in the O. J. C 15 of 22 January 2003 of
an open call for the
expression of interest to participate in a program for the evaluation of
tests for the diagnosis
of TSEs in ruminants. These include a series of new post mortem and live
animal BSE tests
and post mortem tests for small ruminants. Several parties indicated
their interest to
participate with their new rapid tests in an EC evaluation exercise. In
a pre-selection, one live
animal test, beside a number of post mortem tests, was selected for
participation in a
laboratory evaluation exercise conducted by the Institute of Reference
Materials and Methods
(IRMM). However, until now the only design for the evaluation of
diagnostic tests that has
been adopted is for post mortem tests (Opinion of the SSC on Design of
a field trial for the
evaluation of new rapid BSE post mortem tests adopted on 22 February 2002).
On 11 January 2002 the Scientific Steering Committee (SSC) recommended
that all the rapid
tests should undergo an evaluation under field conditions (field trial)
prior to approval. The
EFSA Scientific Report (2004) 1, 1-10 on the Design of a field trial
for the evaluation of new
rapid BSE post mortem tests provides an updated field trial protocol
based on this former
opinion of the SSC.
However, no design for a field trial was available for live animal
tests. In order to gather as
much experience in respect of BSE testing in live animals as possible,
an expert meeting was
held on 6th of August 2003, aiming to join the scientific and technical
experience available.
1 As laid down in Annex 10, chapter C to Regulation 999/2001
Page 3 of 8
2. Purpose
The Commission regulation 999/2001 foresees measures to prevent the
transmission of TSE
to humans, including the testing of animals before consumption, and the
use of laboratory
testing to give a full epidemiological picture of the situation as
regards TSE.
Live animal test could be applied for both purposes.
For the purpose of this evaluation the approach will be testing for
consumer protection and
not for epidemiological screening at this stage.
2.1. Testing for consumer protection
According to EU legislation all slaughtered cattle over the age of 30
months have to be tested
using one of the approved2 rapid BSE tests. In addition, fallen stock
over 24 months of age
as well as all emergency slaughtered cattle over 24 months of age have
to be subjected to an
approved rapid test. Presently, a number of new rapid tests have been
developed and have
taken part in an EC laboratory evaluation exercise according to a
protocol defined by the
European Commission. All of the test results showed the potential to
fulfil minimum
parameter requirements. In considering the evaluation report the SSC, in
agreement with the
IRMM, concluded that each of the new rapid tests should be subjected to
an additional field
trial comparing the new rapid test with already approved tests. The
performance of any new
rapid BSE test should not be statistically inferior to that of the
currently approved tests,
fulfilling the requirements as described in the Opinion of the SSC on
Design of a field trial
for the evaluation of new rapid BSE post mortem tests adopted on 22
February 2002). The
same requirements should be fulfilled for live animal tests used as an
alternative to a post
mortem test. The golden standard is the same for both post mortem tests
and live animals tests
for consumer protection i.e. samples from clinically affected animals,
confirmed BSE positive
by the reference tests.
2.2. Testing for epidemiological screening
Live animal BSE tests could also be applied to monitoring BSE in
epidemiosurveillance
programmes or certification of animals and herds. In that case, the test
performance
requirements regarding sensitivity and specificity may be different
(lower) to the
requirements when used as alternative for post mortem testing. In this
case the test
performance will be evaluated on the basis of samples from preclinically
sampled,
experimentally infected cattle from pathogenesis studies, rather than by
comparison with
approved post mortem tests on brain samples.
This document describes the design of such a field trial to evaluate the
live animal BSE tests
for its use for consumer protection, as developed by the working group
that met on 6 August
2003 and as modified at the plenary meeting on 18 September 2003.
The purpose of this field trial is to compare the sensitivity and
specificity of the new live
animal tests with approved post mortem BSE tests.
A live animal BSE test can be approved for purpose 2.1 (Consumer
protection), for both 2.1
and 2.2 (Consumer protection and epidemiological screening), or only for 2.2
(Epidemiological screening). It should be considered that if a live
animal test is approved for
consumer protection purposes (same performance as approved post mortem
tests) and in
addition also shows to be capable to detect infected animals earlier in
the incubation period, a
2 As laid down in Annex 10, chapter C to Regulation 999/2001
Page 4 of 8
confirmation by reference tests (histopathology, immunohistochemistry,
Western Blot, SAF)
or approved post mortem tests will not (always) be possible. The
consequences of this
situation should be carefully investigated, with regard to the measures
to be taken in the frame
of consumer protection.
The same is true for tests which are only approved for epidemiological
screening because
some of them could detect molecular changes or PrPSc in the earlier
stages which could be
absent at the later stages or clinical stage, thus making a confirmation
with approved post
mortem rapid tests or reference tests impossible. However, at present
there are no indications
for such a situation in case of BSE.
The working group recommends that a number of clinically healthy animals
that score
positive with an approved live animal BSE test should be kept alive and
clinically observed in
order to prove/disprove the results.
The evaluation concerns exclusively the use of live animal tests for BSE
testing and not for
other TSE, including sheep TSE.
3. Estimation of sensitivity relative to approved post mortem tests (test
purpose: consumer protection)
Sensitivity is the probability that a test recognizes confirmed positive
test specimens (or
animals) (true positives) as positive. Ideally, there should be no
false negative test results
(or animals), i.e. the sensitivity is 100 %.
It is important that a new live animal test for the purpose 2.1
(Consumer protection) should
not perform worse than an approved test, in this case an approved post
mortem rapid BSE test.
However, absolute equality can only be shown if all available and future
samples are tested by
both methods (in vivo and post mortem). Such an approach is impossible,
especially in the
case of live animal tests. Therefore, a sample size has to be selected
which will demonstrate
that, with a given probability, the sensitivity of the new rapid live
animal test when compared
to an approved post mortem test is not less than a given threshold
value. The key parameter
should be that all samples for the live animal test taken immediately
before slaughter of later
confirmed BSE cases must score positive. The sample size required also
depends on the
expected number of true positive results, which would be obtained using
the new live animal
rapid test (i.e. estimated prevalence of cases in the population).
Assuming that a new live
animal rapid test might be used in all Member States for a number of
years this expected
figure might be above 1000.
In order to demonstrate, with a probability of 95 %, that the
sensitivity of the new live animal
rapid test is not below 98 % (99 %) of the sensitivity of an approved
post mortem test, the
sample size has to comprise at least 138 (258) samples from animals in
the late stage of the
disease that are positive subsequently on brain material by one of the
approved post mortem
tests.
All of the remaining samples from animals in the end stage of BSE have
to be recognized by
the new live animal rapid test as positive. Any sample which tests
negative with the new live
animal rapid test must be re-tested in duplicate from the original
sample preparation. Both retest
results must be positive (2 out of 3 have to be positive).
Page 5 of 8
For practical reasons, it was decided that at least 200 samples
(including those that were
tested in the frame of the laboratory evaluation) from true positive BSE
animals at the end
stage of the disease should be tested by a new live animal rapid test,
which would ensure with
a 95 % probability that the sensitivity of the new rapid test is not
below 98.5 % compared
with the approved post mortem test.
The true positive samples can be provided by the National Reference
Laboratories (NRL) by
sampling (serum, urine, etc&) BSE suspected animals before slaughter,
which are afterwards
BSE confirmed. They should be well documented (nature, origin and age of
the source animal,
e.g. sub-population; condition of the sample, e.g. haemolysis etc.;
storage conditions; duration
of storage). A sufficient number of aliquots according to the test
requirements (e.g. serum,
tears, urine&) should be prepared for this purpose. At least one aliquot
must be archived.
The tests will be performed at least at two locations, including at
least one NRL. For
specificity testing the test should be performed in at least one NRL
besides the company.
NRL(s) will be chosen at the discretion of the company. The maximum
proportion of samples
tested in a single laboratory in a country must not exceed 70 % of all
samples. Derogating
from the general rule that sensitivity testing should be carried out in
at least one NRL besides
the company, certain state owned laboratories may take part in the study
if the NRL does not
conduct the study itself, if the state owned laboratory is in the
possession of a suitable number
of samples from confirmed BSE positive animals and if the responsible
NRL agrees.
At least two batches of the test kits, if appropriate, should be
included. It is the responsibility
of the company which intends to market the new rapid test to select a
NRL (or following the
derogation a state owned laboratory) as well as the approved tests used
for comparison. The
company should compensate the laboratories performing the comparisons
for all expenses.
Testing of the samples with the new assays should be executed by local
staff, or, at the
discretion of the NRL, by the test developers staff under supervision
of local NRL staff.
IRMM will be notified on the initiation of such studies (where the tests
will be carried out? on
how many samples? which approved test(s) has been chosen for comparison?
and when?) and
will collect and evaluate the data. The raw data will be communicated to
IRMM at least on a
weekly basis. The IRMM will provide a standardized data format.
4. Estimation of specificity relative to approved post mortem tests (test
purpose: consumer protection)
Specificity is the probability that a test recognizes truly negative
test specimens (or animals)
as negative. Ideally, there should be no false positive test results
(or animals), i.e. the
specificity is 100 %.
There is no way to prove exactly that a test is 100 % specific.
Therefore, a decision has to be
made on an acceptable value for specificity. As a specificity of 99.5 %
would still mean that
there are 500 false positive samples in 100,000 tested samples it was
decided that the
specificity should be between 99.95 % (50 false positive in 100,000
tested samples) and 99.99
% (10 false positive in 100,000 tested samples). To demonstrate such
specificity with a
probability of 95 % the number of samples to be tested should be between
5,988 and 29,950.
For practical reasons, it was decided that 10,000 consecutive samples
from confirmed non
BSE healthy slaughtered animals as well as animals with disorders
suspected of BSE, but
confirmed negative, should be used for the estimation of specificity
(see annex 1 of the
Opinion of the SSC on Design of a field trial for the evaluation of
new rapid BSE post
mortem tests adopted on 22 February 2002). The differential diagnostic
samples could
Page 6 of 8
include samples of animals with neurological disorders including
infectious causes
(parasitological, mycological, bacteriological, and virological), non
infectious causes
(traumatic, physical, chemical, metabolic and nutritional, congenital
and allergy) as well as
unknown causes (Saegerman et al., 2003). Based on the proposal of the
applicant the number
and nature of differential diagnosis samples will be determined by the
EFSA Scientific
Working Group on TSE testing. In order to evaluate a possible influence
of breed, an effort
should be made to include samples from the most common European breeds
in the test panel.
The tests should be performed with the agreement of the NRL in
experienced high throughput
routine laboratories. The samples should be prepared according to the
companys protocol.
Laboratories of at least two Member States (or one Member State and
Switzerland) will 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 if
appropriate should be included.
It is the responsibility of the company, which intends to market the new
rapid test to select the
laboratories. The company should compensate the laboratory performing
the comparisons for
all expenses. The NRL will be notified on the initiation of such
studies. It will take adequate
measures to avoid a disturbance of national statistics.
Discrepant results between the new live animal rapid test and the
approved post mortem test(s)
will be resolved by the responsible NRL and the EU Community Reference
Laboratory (CRL)
in Weybridge according to an algorithm agreed prior to the evaluation.
Results obtained in the
live animal test on samples according to the test requirements (e.g.
serum, tears, urine,&)
with confirmed positive results on brain sample will be excluded from
the calculation of
specificity.
IRMM will be notified on the initiation of such studies (where the tests
will be carried out? on
how many samples? which approved test(s) has been chosen for comparison?
and when?) and
will collect and evaluate the data. The raw data will be communicated to
IRMM on a daily
basis. The IRMM will provide a standardized data format.
5. Estimation of relative sensitivity and specificity for the detection of
preclinical cases (test purpose epidemiological screening)
After the establishment of proof of principle, samples originating from
pathogenesis studies
could be used to define the earliest point in the incubation period at
which tests are capable of
detecting infected cattle. However, as no approved reference preclinical
tests exists it has to
be taken into account that only a relative sensitivity and relative
specificity, in comparison
with the preclinical samples from the pathogenesis studies is feasible.
It should also be considered that if a live animal test is approved for
consumer protection
purposes (same performance as approved post mortem tests on clinically
affected animals)
and in addition is also shown to be capable of detecting infected
animals earlier in the
incubation period, a confirmation by reference tests (histopathology,
immunohistochemistry,
Western Blot, SAF) or approved post mortem tests will not (always) be
possible taking into
account that these tests as well as the approved rapid post mortem tests
are not validated for
the detection of preclinically affected animals but only for animals
that are known to be
clinically affected. The consequences of this situation should be
carefully investigated, with
regard to the measures to be taken in the frame of consumer protection.
Page 7 of 8
The same is true for tests which are only approved for epidemiological
screening because
some of them could detect molecular changes or PrPSc in the earlier
stages which could be
absent at the later stages or clinical stage, thus making a confirmation
with approved post
mortem rapid tests or reference tests impossible. However, if used only
for epidemiological
screening, a lower degree of specificity and sensitivity can be
acceptable. Because of all the
above mentioned uncertainties, the present protocol does not consider
the evaluation of live
animal tests for the purpose of epidemiological studies.
6. Influence of matrix on test performance
If the test is intended to be applicable to different matrices the test
should be evaluated on a
range of different samples from the same animal.
If the test is based on PrPSc detection, spike experiments at different
dilutions could be
considered. However, the spiking procedure has to be described in
detail, approved by the
company and validated.
For tests based on the detection of molecular changes or other
parameters (indirect detection)
only original experimental or field samples can be included, excluding
spiked samples.
The practicality of the sampling procedure according to the different
matrices should also be
evaluated as well as the variability of the quality of the samples which
could influence the
results or the applicability to only a part of the population (gender,
age, habitat, sedation
status,&).
The conditions for sampling, preparation and storage must be clearly
described by the
company in order to have guarantees for the best quality of the sample.
7. Description of the test procedure
A number of laboratories will be involved in the evaluation of the new
live animal rapid test.
It is essential that their results are comparable. It is therefore
necessary to have clear, stringent
and detailed descriptions of the test procedures. It is the duty of the
companies to provide
such descriptions and to ensure that they are understood in exactly the
same way in all
participating laboratories.
After the field trial is finished the laboratories involved should meet
in order to discuss
whether the written test procedures used during the field trials proved
to be accurate and fully
understandable. If necessary, test procedures may have to be clarified.
The clarified test procedures or if clarification is not necessary, the
test procedures used, will
then be defined as part of the approval. Later changes in the test
procedure will invalidate the
approval.
8. Evaluation of data
The data will be evaluated by the IRMM. The statistical analysis will be
designed to
demonstrate non-inferiority of the new live animal BSE test to the
already approved post
mortem rapid BSE tests for purpose 2.1 (Consumer protection), The
relative sensitivity and
specificity of the live animal BSE tests for purpose 2.2
(Epidemiological screening) is not
subject of this protocol.
9. Confidentiality
All data generated during the field trial must be kept confidential and
should not be made
accessible to third parties other than the test developer, the concerned
National Reference
Page 8 of 8
Laboratory, the testing laboratory, the European Food Safety Authority
and the European
Commission.
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.
References
EFSA Scientific Report (2004) 1, 1-10 on the Design of a field trial
for the evaluation of new
rapid BSE post mortem tests.
Enoe C., Georgiadis M. P., Johnson W. O. (2000). Estimation of
sensitivity and specificity of
diagnostic tests and disease prevalence when the true disease state is
unknown. Prev. Med.
Vet. 45: 61-81.
Gelfand, A., Smith, A. F. M. (1990). Sampling-based approaches to
calculating marginal
densities. J. Am. Statist. Assoc. 85: 398-409.
Hui, S. L., Walter, S. D. (1980). Estimating the error rates of
diagnostic tests. Biometrics 36
(1): 167-171.
Saegerman C., Claes L., Dewaele A., Desmecht D., Rollin F., Hamoir J.,
Gustin P., Czaplicky
G., Bughin J., Wullepit J., Laureyns J., Roels S., Berkvens D.,
Vanopdenbosch E., Thiry E.,
(2003). Differential diagnosis of neurologically expressed disorders in
Western European
cattle. Rev. sci. tech. Off. Int. Epiz. 22 (1): 83-102
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.
Walter, S. D., Irwig, L. M. (1988). Estimation of test error rates,
disease prevalence and
relative risk from misclassified data: a review. J. Clin. Epidemiol. 41
(9): 923-937.
EFSA Scientific Expert Group members
Thierry Baron, Concepcion Gomez-Tejedor, Martin H. Groschup, James Hope,
Peter Lind,
Johannes Löwer (chairman), Jean-Yves Madec, Danny Matthews, Gerard
Pascal, Vittorio
Silano, Martha Ulvund, Emmanuel Vanopdenbosch (rapporteur), Angus Wear.
Acknowledgement
The chairman and the members of the Scientific Expert Working Group are
acknowledged for
their valuable contribution to this mandate.

http://www.efsa.eu.int/science/efsa_scientific_reports/bse_tse/612/report09_bse02_tests_livecattle_en1.pdf

TSS





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