IMPORTANCE OF STANDARDISATION AND VALIDATION OF MOLECULAR DIAGNOSTIC TECHNIQUES FOR AQUATIC ANIMAL PATHOGENS AND DISEASES FOR INTERNATIONAL TRADE AND TRANS-BOUNDARY MOVEMENT OF AQUATIC ANIMALS AND ANIMAL PRODUCTS - BARRY J. HILL

Fish Diseases Commission, OIE

The Office International des Epizooties (OIE) is an inter-governmental organisation established in 1924 in order to promote world animal health. One of its main activities is to provide guidelines and standards for health regulations applicable to international trade in living animals and their products.

The Sanitary and Phytosanitary (SPS) Agreement under the auspices of the World Trade Organisation (WTO) states that animal health requirements established by countries importing live animals or their products should be based on international standards, guidelines and recommendations, primarily those developed by the OIE. The Agreement states that animal health measures which conform to the recommendations of OIE will be presumed to be consistent with the SPS Agreement. Thus, OIE recommendations should, until further notice, provide the basis for all national animal health regulations with respect to international trade. However, any country may introduce health conditions for imports of animals which provide a higher level of health protection than would be achieved by strict application of the relevant OIE standards, on condition that it can provide scientific justification (usually based upon a full risk assessment). The fundamental principle involved is that health certification should be a means of facilitating international trade and should not be used to restrict it by requiring unjustified health conditions.

The OIE standards applicable to international trade in aquatic animals are laid out in the International Aquatic Animal Health Code and in the Diagnostic Manual for Aquatic Animal Diseases. Laboratories involved in the diagnosis and/or health certification for aquatic animal diseases are recommended to be fully conversant with the technical procedures and protocols in the OIE Diagnostic Manual. The Manual provides a uniform approach to the detection of the diseases and pathogens listed in the OIE Code and standardised requirements for health certification in connection with international trade in aquatic animals and their products. The OIE takes the view that the only dependable current approach to the diagnosis of aquatic animal diseases lies in the specific identification of the pathogens using laboratory methods. Health certification programmes aim to determine, from the results provided by standardised laboratory procedures performed with samples selected according to defined rules, the health status of aquatic animal stocks from a particular production site or a geographical zone or an entire country. Thus, the Manual provides a general basis for health surveillance/control programmes, as well as setting out standard diagnostic methods which can be applied in any competent laboratory involved in diagnosis of aquatic animal diseases.

The International Aquatic Animal Health Code covers a list of diseases notifiable to the OIE, and a list of other significant diseases in aquatic animals, which are currently:

Therefore, a total of 14 diseases of fish, 6 diseases of molluscs and 6 diseases of crustaceans are listed by OIE.

In the current version of the Manual (1997 Ed.), the more sophisticated methods involving molecular biological techniques such as DNA probes and the polymerase chain reaction (PCR) are recommended for only a few of the diseases. For the others, such techniques have yet to be adapted for routine diagnostic purposes and remain largely a research tool. Molecular biological tests for diagnosis of clinical outbreaks or for identification of pathogens isolated during health surveillance programmes are described for 5 of the fish diseases (epizootic haematopoietic necrosis, bacterial kidney disease, piscirickettsiosis, Gyrodactylosis and viral encephalopathy and retinopathy), and 2 of the diseases of crustacea (nuclear polyhedrosis baculoviroses and white spot disease). For none of the listed diseases is a molecular biology technique recommended for "screening" of the aquatic animal population to demonstrate it is free of infection (i.e. for health certification purposes).

The task of compiling the Manual is assigned to the OIE Fish Diseases Commission (FDC), who will continually revise and update it as existing laboratory techniques are improved and more rapid, sensitive and/or specific methods are developed. It is intended to publish a new edition approximately every 3 years. The aim of the FDC is to include only those test procedures which have become standardised through widespread use over several years, or those which are so precisely described and reproducible that they can effectively be carried out to the same standard in any laboratory with the necessary facilities. For most of the diseases, a choice of test methods is given for identification of the specific pathogen, but for screening aquatic animal populations to demonstrate freedom from a particular pathogen, a single prescribed method is given.

The next revision of the Manual is currently underway and a new edition will appear in the year 2000. Each chapter will be sent to another leading expert on the disease or pathogen in question for an independent review prior to submission to the FDC for its approval. When the FDC is satisfied with all the disease chapters, the OIE will submit them to the national delegates (usually the Chief Veterinary Officer, or equivalent, of each country) of the 152 Member Countries for national expert opinion on them. The FDC will then take into account the views and suggested amendments arising from this wide consultation and, if necessary, will refer these to the relevant author of the chapter for any final revisions to be incorporated. Formal acceptance of all the methods is by the agreement of the national delegates at their next annual meeting (held at OIE, Paris, in May each year). On publication some months later, the methods described in the Diagnostic Manual are regarded by the WTO as the only international standard methods for the purpose of health certification requirements attached to international trade in aquatic animals and their products.

A noticeable change in the next edition will be the introduction of molecular diagnostic techniques for several more of the listed diseases, particularly those of molluscs and crustacea. In most cases, the chapter for each disease has been written by the nominated OIE expert at the designated OIE Reference Laboratory for that particular disease. The expert has been appointed on the basis of their recognised high level of expertise for the disease(s) in question. In some cases, the molecular diagnostic technique described has been developed at the Reference Laboratory itself, but in others the method has been developed elsewhere and described in the scientific literature. Whatever the origins of the molecular diagnostic methods, it is important that they have been standardised by the laboratory which developed them and preferably validated (see below) in comparison with the existing conventional methods. Ideally, each molecular diagnostic method should have been previously published in the peer-reviewed scientific literature and should have been subjected to independent trials in other laboratories such that it has received scientific and international acceptance.

The next edition of the Diagnostic Manual will also include chapters on the principles of validation of diagnostic assays for infectious diseases and on good laboratory practice, quality control and quality assurance, based on those in the OIE Manual of Standards for Diagnostic Tests and Vaccines. According to the OIE Standards Commission, validation of a new test "is a complex procedure which includes the development, evaluation and standardisation of the test method; determining the repeatability; determining diagnostic and relative specificity and sensitivity; determining reproducibility; and doing the statistical analysis of the results". Validation is performed after the test method has been established and includes the establishment of the cut-off values for negative, positive and suspected-positive samples with diagnostic sensitivity and specificity being the primary parameters estimated in the test validation. It is important to establish that the sensitivity is at least as good as other standardised methods already in use and is of sufficient specificity to avoid false positive results. A validated test should consistently provide results that identify animals as positive or negative for a particular pathogen or a specific antigen/genomic sequence characteristic of that pathogen.

It is important to use only standardised and, as far as possible, validated test methods for diagnosing aquatic animal diseases accurately. This is particularly important for health certification of animals intended for international trade when the importing country has a requirement that animals are free of particular diseases. This is to ensure that there is a "level playing field" in disease-free certification, regardless of the country of origin. Clearly, there will not be a level playing field if the same tests used in different countries have different levels of reliability and give markedly different degrees of certainty for the absence of specific infections. To facilitate international trade in aquatic animals, it is imperative that specific disease-free certification of a farm, zone or country provides the same level of confidence irrespective of the exporting country. For this purpose, molecular diagnostic tests should be applied only if they have been standardised and validated. In the Asian Region, there is increasing use of molecular-based tests for screening of shrimp populations for serious viral diseases. If these tests are to generate meaningful data on disease distribution and, in particular, the absence of diseases from particular locations, testing standards will need to improve and provide consistent and comparable results and a common level of certainty.