First international proficiency testing for laboratory performance on Xylella fastidiosa detection

A proficiency test (PT) to evaluate the performance of laboratories involved in molecular and serological detection of X. fastidiosa was carried out in early 2017; 35 laboratories from EU/non- EU Countries tested 4 different methods to purify DNA, conventional and qPCR assays, and 2 ELISA tests. The number of resultant positive agreement/negative agreement/positive deviation/negative deviation was used to determine the laboratory performance (i.e. accuracy 100%). The overall results showed that all laboratories were able to correctly diagnose X. fastidiosa in the blind samples containing the highest X. fastidiosa concentrations, whereas the performance of several laboratories was negatively affected by the lack of detection in the samples with the lowest concentrations, both through molecular and serological tests. Accuracy level of 100% (laboratory conformed to the PT) was successfully recovered in the majority of the laboratories performing qPCR and PCR assays on DNA purified using at least 2 of the 4 tested protocols. The use of automated platform ensured higher laboratory performance. As expected, results of the ELISA tests generated lower performance values in the majority of the laboratories, due to the lack of detection of positive samples containing the lowest the bacterial concentration. This study provides a good overview on the laboratory performance for the diagnostics currently used in the EPPO countries and indicate useful improvements that laboratories can adopt to achieve a better performance

Spectral Imaging with QUBIC: building frequency maps from Time-Ordered-Data using Bolometric Interferometry

The search for relics from the inflation era in the form of B-mode polarization of the CMB is a major challenge in cosmology. The main obstacle appears to come from the complexity of Galactic foregrounds that need to be removed. Multi-frequency observations are key to mitigating their contamination and mapping primordial fluctuations. We present "Spectral-Imaging", a method to reconstruct sub-frequency maps of the CMB polarization within the instrument's physical bandwidth, a unique feature of Bolometric Interferometry that could be crucial for foreground mitigation as it provides an increased spectral resolution. Our technique uses the frequency evolution of the shape of the Bolometric Interferometer's synthesized beam to reconstruct frequency information from the time domain data. We reconstruct sub-frequency maps using an inverse problem approach based on detailed modeling of the instrument acquisition. We use external data to regularize the convergence of the estimator and account for bandpass mismatch and varying angular resolution. The reconstructed maps are unbiased and allow exploiting the spectral-imaging capacity of QUBIC. Using end-to-end simulations of the QUBIC instrument, we perform a cross-spectra analysis to extract a forecast on the tensor-to-scalar ratio constraint of σ(r)=0.0225 after component separation

Spectral Imaging with QUBIC: building astrophysical components from Time-Ordered-Data using Bolometric Interferometry

The detection of B-modes in the CMB polarization pattern is a major issue in modern cosmology and must therefore be handled with analytical methods that produce reliable results. We describe a method that uses the frequency dependency of the QUBIC synthesized beam to perform component separation at the map-making stage, to obtain more precise results. We aim to demonstrate the feasibility of component separation during the map-making stage in time domain space. This new technique leads to a more accurate description of the data and reduces the biases in cosmological analysis. The method uses a library for highly parallel computation which facilitates the programming and permits the description of experiments as easily manipulated operators. These operators can be combined to obtain a joint analysis using several experiments leading to maximized precision. The results show that the method works well and permits end-to-end analysis for the CMB experiments, and in particular, for QUBIC. The method includes astrophysical foregrounds, and also systematic effects like gain variation in the detectors. We developed a software pipeline that produces uncertainties on tensor-to-scalar ratio at the level of σ(r)∼0.023 using only QUBIC simulated data

First report of Xanthomonas citri pv. citri pathotype A causing Asiatic citrus canker in Martinique, France

Asiatic canker, caused by Xanthomonas citri pv. citri, is a major threat to worldwide citriculture. Three pathotypes differing in host range and hypersensitive reactions toward citrus species have been defined. Whereas pathotypes Aw and A* have a restricted host range, X. citri pv. citri pathotype A infects a broader range including most commercial citrus species and hybrids and can cause important economic losses in tropical and subtropical areas. Pathotype A strains, especially those assigned to lineage 1, were implicated in the major geographical expansion of X. citri pv. citri during the 20th century from their native area, Asia (Pruvost et al. 2014). X. citri pv. citri is listed as a quarantine pathogen in the European Union (EU) – Directive 2000/29/EC annex II A1. Martinique (France) is an outermost region of the EU in the eastern Caribbean Sea. Canker lesions were first observed at Morne Rouge, Martinique in June 2014 on grapefruit (Citrus paradisi), mandarin (C. reticulata), Tahiti lime (C. latifolia), and Valencia and Washington Navel oranges (C. sinensis). Official diagnostics, including bacterial isolations on YPGA or KC semiselective medium (Pruvost et al. 2005), PCR-based identification with 4/7 primers (Hartung et al. 1993), and pathogenicity tests, were performed following the EPPO standard PM7/44 (www.eppo.int) and identified isolates as X. citri pv. citri. Three strains isolated in Martinique in 2014 from grapefruit or Tahiti lime were further characterized (LL074-4, LL077-2, and LL079). Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (Almeida et al. 2010; Bui Thi Ngoc et al. 2010) identified Martinique strains as X. citri pv. citri with 100% sequence identity to the type strain LMG 9322. Using MLVA-31 targeting 31 minisatellites, Martinique strains were assigned to lineage 1 composed of pathotype A strains (Pruvost et al. 2014). All strains were inoculated by a detached leaf assay onto Mexican lime SRA 140 (C. aurantifolia), sweet orange Washington Navel SRA 102, and grapefruit Henderson SRA 336 (Bui Thi Ngoc et al. 2010). All inoculated leaves produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like colonies were reisolated from lesions that had developed. Boiled suspensions were assayed by PCR with 4/7 primers and produced the expected amplicon, fulfilling Koch's postulates. No lesions developed on the negative control consisting of sterile 0.01M tris buffer pH 7.2. This is the first report of X. citri pv. citri in Martinique and to our knowledge in the Caribbean region. Surprisingly, all strains collected to date in Martinique grew on YPGA supplemented with 300 mg liter−1 copper sulfate even when no extensive copper spray programs have been used, suggesting that copper-resistant strains may have been introduced. Disease is contained by tree removal and burning and the situation is presently under apparent control although positive trees were sporadically detected in 2015 in backyards or small orchards at Le Lorrain and Saint Pierre. An extensive surveillance program is currently implemented in Martinique for quarantine pathogens of citrus. (Texte intégral

What did we achieve with VALITEST an EU project on validation in plant pest diagnostics?

peer reviewedEnsuring the reliability of diagnostic activities is an essential cornerstone of Plant Health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories although companies commercializing diagnostic kits also produce validation data for their products. Due to the high number of pest , matrix and method combinations and given the significant resources required to validate tests, it is essential that validation data are shared with the entire diagnostic community and produced in a harmonized way to facilitate their use by different stakeholders. Indeed, the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic laboratories and also involve National Plant Protection Organizations. The VALITEST EU project (2018-2021) was established to tackle all these issues. New validation data for tests targeting important pests for the EPPO region were produced. Guidelines to improve and harmonize the validation framework were developed. Sharing of validation data and experience was ensured through the development of new or existing databases, the organization of training courses and the dissemination of the project outputs in scientific publications and Standards. Finally, the involvement of researchers, diagnosticians, policy makers, inspectors, industries etc. and the establishment of the European Plant Diagnostic Industry Association were important actions to strengthen the interactions between Plant Health stakeholders

Comparative and collaborative studies for the validation of a nested PCR for the detection of Xanthomonas axonopodis pv. dieffenbachiae from Anthurium samples

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N-PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N-PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90*6, 88*7 and 47*2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92*5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N-PCR assay. The N-PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N-PCR had undeniable advantages compared to the reference method (less labour-intensive and less time-consuming). In addition, post-test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N-PCR assay has since been included in a revised version of the EPPO detection protocol. © 2013 British Society for Plant Pathology

Minimum performance parameters to select tests for validation and selection of laboratories for TPS

The aim of deliverable 1.1. is to prepare criteria to select tests for validation and to select laboratories for TPS (test performance study). Criteria for selection of tests for the TPS for each pest have been set (see Tables 7-12). These criteria have been divided in five groups: 1) validation data, 2) applicability, 3) protocols, 4) chemicals and 5) equipment. For selection of participants for the TPS selection criteria have also been set (see Table 13). Amongst the most important criteria for selection for participants of TPS are technical expertise for the pest group and the method, authorization to work with the specific pest and that the participating laboratory has quality assurance in place. These criteria enable evaluation of whether participants are proficient to perform the tests, have the necessary equipment and a permit to work with viable regulated organism. The scope of the testing for specific pests was set and common rules for each selection process was defined

List of criteria the reference materials have to meet for use in validation studies

A list of general minimum criteria was developed to be used in preparation of reference materials to be used in interlaboratory testing, including validations through test performance studies. The list was based on previously identified criteria in standards including ISO standards, EPPO guidelines, deliverables of relevant projects, related fields, and own experience of project partners. Several additional criteria were proposed: ‘availability’, ‘purity’ and ‘commutability’. These criteria result from a series of discussions, taking into account previous work and relevant international standards. Where relevant, each criterion was first defined as a series of levels from the highest to lowest ranking with the lowest ranking considered to be the minimum. Depending on the intended use, the reference material may need to fulfil higher levels of selected criteria or some criteria may not be relevant at all. The criteria were tested by the organizers of the TPS in round 1 of validation within VALITEST project which include bacteria, viruses, nematodes and fungi. The systematic and structured approach of describing RMs was found to be useful in promoting transparent descriptions of the RMs used and comparability of TPSs, as well as a step toward implementing FAIR data principles. The criteria defined here are applicable, potentially with minor modification, beyond the scope of this deliverable and project to other pests and other uses of RM

First international proficiency testing for laboratory performance on Xylella fastidiosa

A proficiency test (PT) to evaluate the performance of laboratories involved in molecular and serological detection of Xf was carried out in early 2017. Thirty-five laboratories from EU/non-EU Countries tested 4 different methods to purify DNA, conventional and qPCR assays, and 2 ELISA tests. The number of resultant positive agreement/negative agreement/positive deviation/negative deviation was used to determine the laboratory performance (i.e. accuracy 100%). The overall results showed that all laboratories were able to correctly diagnose Xf in the blind samples containing the highest Xf concentrations, whereas the performance of several laboratories was negatively affected by the lack of detection in the samples with the lowest concentrations, both through molecular and serological tests. Accuracy level of 100% (laboratory conformed to the PT) was successfully recovered in the majority of the laboratories performing qPCR assays on DNA purified using at least 2 of the 4 tested protocols. The use of automated platform ensured higher laboratory performance. As expected, results of the ELISA tests generated lower performance values in the majority of the laboratories, due to the lack of detection of positive samples containing the lowest the bacterial concentration. This study provides a good overview on the laboratory performance for the diagnostics currently used in the EPPO countries and indicate useful improvements that laboratories can adopt to achieve a better performanc