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Methods
DOI 10.1007/s12161-010-9154-4
B. Lombard (*)
AFSSA-LERQAP (French Food Safety AgencyLaboratory
for Study and Research on Food Quality and Food Processing),
23 avenue du Gnral de Gaulle,
94706 Maisons-Alfort, France
e-mail: b.lombard@afssa.fr
A. Leclercq
WHO Collaborative Center for Foodborne Listeriosis,
National Reference Center for Listeria, Institut Pasteur,
28 rue du Docteur Roux,
75724 Paris cedex 15, France
e-mail: alexlec@pasteur.fr
Introduction
Reference methods in food microbiology, for the analysis of
food, animal feeding stuffs, samples from primary production and food processing and handling, are standardised at
the international level by International Organization for
Standardization (ISO) and at the European level by
European Committee for Standardization (CEN) (Lombard
et al. 1996; Lombard 2004; Leclercq 2002; Leclercq et al.
1999; Lombard and Leclercq 2010). The standardisation
process consists of establishing reference documents in
agreement with all parties and countries concerned. These
reference methods are mostly based on conventional
microbiology, enabling the major characteristic of a
standard to be met: the largest consensus and the broadest
applicability in food microbiology worldwide, including
developed countries themselves. Application of reference
methods requires skilled staff and is labour-intensive and
time-consuming, due to the time needed by microorganisms
to grow in or on culture media.
Innovative methods in food microbiology, so called
rapid methods, have been developed to achieve results
within a shorter time and sometimes with higher performance than classical methods based on conventional
microbiology. They can be classified into three main types:
(1) solid culture media other than those prescribed in the
standard reference methods, mostly based on chromogenic
substrates so as to enable easier visual recognition of the
target bacteria on the plates, (2) immunoenzymatic kits,
such as ELISA, and (3) molecular-based methods, such as
hybridisation probes or PCR, either conventional or real-
easy to carry out and/or automation, analytical performances at least as good as those of the reference method. In
other words, an alternative method is designed and intended
to be used instead of the corresponding reference method.
The term alternative method refers to the combination of
product, equipment and complete operating procedure,
from sample preparation to final result. It includes all the
elements necessary for implementing the alternative method: equipment, culture media, software for analysis of
results, etc.
In general, validation of a method consists of confirming, by examination and provision of objective evidence,
that the particular requirements for a specific intended use
are fulfilled (Leclercq 2002). For a specific intended use, a
laboratory has to reply to the question: why am I analysing
the samples and for what purpose are the resulting data to
be used? For objective evidence, laboratories need to have
or refer to validation studies. For confirmation, laboratories
have to compare information obtained from validation
studies with customer's requirements.
In order to have a common reference protocol to conduct
the validation of these innovative methods, CEN has
developed a standard on the validation of alternative
methods in food microbiology, EN ISO 16140 (Anonymous
2003), a standard adopted in parallel by ISO. According to
the standard on general requirements and guidance for
microbiological examinations (EN ISO 7218 (Anonymous
2007) Clause 14.2), the validation of alternative methods
shall be performed according to EN ISO 16140.
This paper will mainly present the protocol for the
validation of alternative methods according to the EN ISO
16140 Standard, then will give the revision status of this
standard and introduce validation/certification systems, as
well as their relation with laboratory accreditation and
regulatory aspects, and the use of validated methods.
Part 1 on terminology
Part 2 on validation of proprietary methods
Part 3 on in-house method validation, distinguishing in
particular the case where the method to be validated is
compared or not to a RM, and the case of cultivable/
non-cultivable microorganisms (e.g. viruses)
Part 4 on method verification, i.e. how a laboratory can
verify that it is correctly implementing a validated
method, in particular in the context of laboratory
accreditation
Official Methods scheme of AOAC International, Performance Tested Methods scheme of AOAC Research Institute
in the USA and NordVal of NMKL in the European Nordic
countries. The Official Methods scheme and NordVal
follow a technical protocol similar but not identical to that
of EN ISO 16140. In contrast, the technical protocol of the
Performance Tested Methods scheme does not include an
inter-laboratory study. In addition, the organisations
concerned do not operate as certification bodies, lacking
in particular the control of the manufacturer's quality
assurance.
In the future, official reference laboratories, such as
Community or National Reference Laboratories in Europe,
may become new actors in the field of validation of AMs,
depending on possible new regulatory duties given by their
prescribers, the Directorate-General for Health and Consumers (DG SANCO) of the European Commission, or
national competent authorities in charge of official food
control.
Another point is that certification bodies contract with
expert laboratories to conduct the two parts of the
validation study described in EN ISO 16140: the methods
comparison study and the inter-laboratory study. The two
certification schemes (AFNOR Validation and MicroVal),
as well as NordVal, require that the expert laboratory be
accredited according to EN ISO 17025 for the relevant RM,
whereas this is not the case with AOAC. However, such an
accreditation does not appear to cover an expert laboratory's
entire field of competence necessary to implement the
experimental designs, calculations and interpretation of
the different phases of the validation study. Such a
competence includes (1) the organisation of the interlaboratory study, which is particularly difficult in food
microbiology, given the living nature of the bacteria, and
(2) the quality assurance of the expert laboratory's strain
collection (traceability, characterisation), a key element to
conduct inclusivity and exclusivity testing, as well as to
perform artificial contamination. It is true that a single
reference document for validation of AMs, the EN ISO
16140, is crucial, but the guarantee of its correct application
with reliable results is no less important. It would require in
the future the development of the accreditation of expert
laboratories for the application of EN ISO 16140, in the
same way that proficiency testing (PT) scheme providers
can be accredited according to EN ISO 17043 (Anonymous
2010) to organise these PT schemes.
Conclusion
In numerous cases, laboratories performing routine food
microbiological analyses do not use the RMs based on
conventional microbiology but increasingly often choose
proprietary methods, based on innovative technologies,
which provide results in a shorter amount of time, are
easier to perform, require fewer resources in terms of
skilled staff, and are more automated. The validation of
these proprietary methods according to EN ISO 16140, and
their subsequent certification, give assurance to the laboratories and their customers (in the food industry, food
distribution, public control authorities) that the results
obtained with these methods are valid and at least as good,
if not better, than those obtained with the corresponding
RMs, the gold standards.
In certain cases, laboratories may also use in-house
methods that they have developed. The revised EN ISO
16140 will provide a tool to the laboratories to validate
these methods and to give confidence in their reliability,
both to their customers and to accreditation bodies.
The revised EN ISO 16140 will also define what method
verification in food microbiology is, guiding the laborato-
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