1.1 Introduction

The ongoing coronavirus (COVID-19) pandemic has caused a global health crisis, which has demonstrated the critical importance of rapid and reliable pathogen detection. Similarly, in the field of plant health, the recent emergence of several pests in the EPPO region, such as Xylella fastidiosa and tomato brown rugose fruit virus, required rapid responses from diagnostic laboratories and other stakeholders to allow national plant protection organisations to take appropriate measures based on a reliable diagnosis.

The movement of pests has considerably increased in the past century because of increased and diversified international trade. Climate change also has an impact on plant health, for examplethe expansion of pest distribution (IPPC 2021). In addition, the rapid growth of the human population and the resulting increased demand for food are putting enormous pressure on agro-ecosystems and the stakeholders involved in food production (Rodrigues et al. 2017). To avoid losses and achieve sustainable plant-based food production, prevention or limitation of damage caused by plant pests through the use of timely and coordinated measures is essential. The rapid and accurate detection of plant pest is a foundation of successful pest control and will become more important in the future in the context of further reductions in the use of chemical pesticides.

Guidelines for plant pest diagnosis are provided in the International Standard for Phytosanitary Measures 27 ‘diagnostic protocols for regulated pests’ developed by the International Plant Protection Convention (IPPC). IPPC Standards are recognised by the World Trade organization as reference Standards for international trade. In addition, guidelines on diagnostic are also developed by Regional Plant Protection Organisations (RPPOs). The European and Mediterranean Plant Protection Organization (EPPO) is the RPPO for Europe,Footnote 1 and has established a work programme on diagnostics since the 1990s, and developed a number of horizontal and pest specific diagnostic Standards (series PM 7 of EPPO Standards). Additional requirements may be specified by other national bodies involved in plant health.

A variety of tests are used to diagnose pathogens. These tests may be developed by commercial companies or research institutions. The reliability of diagnostic tests depends on the intended use of the tests, their performance characteristics and associated uncertainty obtained from validation studies and the experience of the laboratories (EPPO PM 7/76 2018a). Validation, i.e., the process by which laboratories ensure that a test is fit for purpose based on the evaluation of its performance characteristics, consists of several steps described in Fig. 1.1, each of which needs to be carefully planned and executed. The most important part of the validation process is the determination of the performance characteristics of a test. Performance characteristics that are frequently used to characterise tests include: analytical sensitivity, analytical specificity (inclusivity and exclusivity), selectivity, repeatability and reproducibility (EPPO PM 7/98(5) 2021a). Guidance for the evaluation of these performance characteristics has been developed by EPPO and is included in the Standard PM 7/98(5) (2021a). The performance characteristics of a test can be determined within one laboratory (i.e., ‘in-house’) during a validation study (referred to as intralaboratory studies) or by several laboratories in interlaboratory comparisons. Guidance on the organisation of interlaboratory comparisons is provided in the EPPO Standard PM 7/122(1) (2014). There are different types of interlaboratory comparisons, but this book focuses on the organisation of test performance studies which aim at evaluating the performance of (a) test(s) by two or more laboratories using defined samples. Overall, validation is a demanding process in terms of time and resources (expertise, money). In plant health, given the large number of pests, matrices and methods and the combinations of these, data on the performance of diagnostic tests is not always available and validation of tests is mainly performed by laboratories based on their need to be accredited ISO/IEC 17025 (2005) to perform their activities.

Fig. 1.1
figure 1

The validation process in Plant Health. (Adapted from EPPO PM 7/98 (4) (2019))

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The need for a reliable diagnosis in plant health has been recognised by the European Commission and was supported by the funding of the VALITEST Project (www.valitest.eu, 2018–2021; grant agreement N° 773,139) through the EU Horizon 2020 research and innovation programme. The most important aims of the VALITEST Project were to: (1) provide more complete and precise descriptions of the performances of diagnostic tests; (2) stimulate, optimise and strengthen the interactions between stakeholders in plant health to promote better diagnostics; and (3) lay the foundations for structuring the quality and commercial offers of plant health diagnostics tools. Fulfilling these goals was ensured by the creation of a multidisciplinary consortium that brought together leading EU public, private, academic and industrial organisations and other stakeholders from the plant health and diagnostics sectors.

One of the core activities of the Project was the production of validation data for existing tests through the organisation of 12 TPS. In total the performance of 83 tests covering 11 pests recognised as a priority was evaluated during two rounds of TPS (Trontin et al. 2021). As the provision of validation data for selected tests is time consuming and requires a significant investment of human and financial resources, one of the outcomes of VALITEST was also the development of a guidance documents to assist in the organisation of TPS, and to improve the diagnostic procedures and the validation framework.

The purpose of this book is to provide practical and technical guidance for the organisation of TPS for plant pests based on the experience gained by the TPS organisers through the organisation of TPS in the framework of VALITEST. The major aspects and challenges faced during the preparation, organisation and reporting of TPS are identified and can be used by organisers of future TPS not only in the field of plant pest detection, but also in other areas of microbiology. They are mainly illustrated using the case study of a TPS organised on tomato spotted wilt orthotospovirus in the framework of VALITEST but this example can be easily adapted to the specifics of different tests for which validation is required. Practical templates developed in the framework of VALITEST for specific steps of TPS organisation are also given in the Appendices.

1.2 Common Terms Used in This Book (EPPO PM 7/76 (5) (2018a))

  • Accuracy - the ability of a test to detect true positives and true negatives, as (true positives + true negatives)/total population of negatives.

  • Analytical sensitivity - the smallest amount of the target that can be detected reliably, and sometimes referred to as the ‘limit of detection’. Further details on the procedures to determine analytical sensitivity are given in EPPO PM 7/98 “Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity”.

  • Analytical specificity (including inclusivity and exclusivity; see below) - further details on the procedures to determine analytical specificity (inclusivity, exclusivity) are given in PM 7/98 “Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity”.

  • Diagnostic sensitivity - the proportion of infected or infested samples that test positive, as compared with results from alternative tests (or a combination of tests).

  • Diagnostic specificity - the proportion of uninfected or uninfested samples that test negative (i.e., true negatives) compared with results from alternative tests (or a combination of tests).

  • Exclusivity - performance of a test with regards to cross-reactions with a range of non-targets (e.g., closely related organisms, contaminants).

  • Inclusivity - the performance of a test with a range of target organisms that covers genetic diversity, different geographic origins or hosts.

  • Interlaboratory comparison - organisation, performance and evaluation of measurements or tests on the same or similar items by two or more laboratories in accordance with predetermined conditions (i.e., proficiency testing, test performance studies).

  • Matrix - type of material (e.g., leaves of tomato, pepper seeds …).

  • Methods - methods include bioassay methods, biochemical methods, fingerprint methods, isolation/extraction methods, molecular methods, morphological and morphometric methods, pathogenicity assessments and serological methods.

  • Repeatability - the level of agreement between replicates of a sample tested under the same conditions.

  • Reproducibility - the ability of a test to provide consistent results when applied to aliquots of the same sample tested under different conditions (e.g., different times, operators, equipment, locations).

  • Robustness of a test - the extent to which altered test conditions (e.g., temperature, volume, reagents) affect the established test performance values (e.g., analytical sensitivity, analytical specificity).

  • Selectivity - the extent to which variations in the matrix affect the test performance (i.e., matrix effects).

  • Test - the application of a method to a specific pest and a specific matrix.

  • Test performance study (ring tests, collaborative trials) - evaluation of the performance of one or more tests by two or more laboratories using defined samples (evaluation of a test).

  • Validation - process carried out to provide objective evidence that a test is suitable for the circumstances of its use (ISO/IEC 17025 2005).