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Animal African trypanosomosis (AAT), a disease affecting livestock, is caused by parasites of the Trypanosoma genus (mainly T. vivax and T. congolense). AAT is widespread in Sub-Saharan Africa, where it continues to impose a heavy socio-economic burden as it renders development of sustainable livestock rearing very strenuous. Active case-finding and the identification of infected animals prior to initiation of drug treatment requires the availability of sensitive and specific diagnostic tests. In this paper, we describe the development of two heterologous sandwich assay formats (ELISA and LFA) for T. congolense detection through the use of Nanobodies (Nbs). The immunisation of an alpaca with a secretome mix from two T. congolense strains resulted in the identification of a Nb pair (Nb44/Nb42) that specifically targets the glycolytic enzyme pyruvate kinase. We demonstrate that the Nb44/Nb42 ELISA and LFA can be employed to detect parasitaemia in plasma samples from experimentally infected mice and cattle and, additionally, that they can serve as ‘test-of-cure’ tools. Altogether, the findings in this paper present the development and evaluation of the first Nb-based antigen detection LFA to identify active T. congolense infections.

Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTA(TM) filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. Significance and Impact of the Study Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTA(TM) technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.

Studying the epidemiology of schistosomiasis-the most prevalent gastropod-borne human disease and an economic burden for the livestock industry-relies on adequate monitoring tools. Here we describe a molecular assay for detecting human and animal African schistosome species in their planorbid gastropod host (xenomonitoring) using a two-step approach. First, schistosome infections are detected and discriminated from other trematode infections using a multiplex polymerase chain reaction (PCR) that includes a trematode-specific marker (in 18S rDNA), a Schistosoma genus-specific marker (in internal transcribed spacer 2 [ITS2]) and a general gastropod marker (in 18S rDNA) as an internal control. Upon Schistosoma sp. detection, a second multiplex PCR is performed to discriminate among Schistosoma haematobium, Schistosoma mansoni, Schistosoma mattheei and Schistosoma bovis/Schistosoma curassoni/Schistosoma guineensis using markers of differential lengths in the cytochrome c oxidase subunit 1 (COX1) gene. The specificity of these assays was validated with adult worms, naturally infected gastropods and human urine and stool samples. Sensitivity was tested on experimentally infected snail specimens that were sacrificed 10 and 40 days post-infection in order to mimic a natural prepatent and mature infection, respectively. The assay provides a diagnostic tool to support the xenomonitoring of planorbid gastropods for trematode infections in a One Health context, with a focus on the transmission monitoring of schistosomiasis. ispartof: TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE vol:113 issue:11 pages:722-729 ispartof: location:England status: published

Background To improve agricultural practices and the food/feed security, plant breeding techniques were developed, including transgenesis commonly using Agrobacterium tumefaciens or biolistic technologies. To guarantee the traceability of GMO in food/feed chain and the consumer's freedom of choice, regulatory frameworks were established in many countries around the world, such as in Europe. Their implementations, including detection systems usually based on qPCR, are becoming complex and expensive regarding the number of analysis to perform. Moreover, the dispersion of publicly available information about developed GMO prevents to accurately estimate the efficiency of the standard detection system applied to unauthorized GMO. Scope and approach To illustrate this problem, the case of rice, one of the leading staple crops, was investigated. An overview of worldwide developed biotech rice generated by transgenesis was thus conducted, based on 1067 peer-reviewed publications, and analysed regarding inter alia their expressed genes of interest and the corresponding traits, their transformation processes and the elements composing their transgenic cassettes. From this work, the power and weakness of the standard detection system, notably used by the European enforcement laboratories, are evaluated. To strengthen this system, especially with unauthorized GMO, additional strategies are suggested. Moreover, given the growing interest for biotech rice produced by new plant breeding techniques, related challenges for their detection are discussed. Key findings and conclusions According to all collected information, suitable detection strategies, combining qPCR to additional technologies (e.g., DNA walking and NGS), are proposed to cover most of inventoried biotech rice. The present approach, including the data centralization to subsequently suggest appropriated detection strategies, can be extended to biotech events from different species.

Background Foodborne Neglected Zoonotic Helminths (FNZH) are parasites of both economic and public health importance. They include Taenia solium, Echinococcus granulosus sensu lato, Echinococcus multilocularis and Foodborne trematodes (FBT). FNZH are earmarked for major interventions for control, elimination and eradication. This systematic review highlights the progress towards development of rapid tests for the diagnosis of FNZH since 2010 when they were listed as neglected tropical diseases. Methodology A systematic search was conducted in three databases, World of Science, Embase and PubMed using the same search phrase. The search produced 480 hits. Three studies from back referencing were included. Only 22 of these met the inclusion criteria. Data was extracted from these and presented qualitatively. Results Twenty-five rapid diagnostic tests were found to have been developed since 2010, eight for diagnosis of T. solium infections, eight for echinococcosis and nine for FBT infections. The rapid tests for diagnosing T. solium infections included six antibody detecting and two antigen detecting tests. They constitute a combination among them, with some tests providing qualitative, others quantitative results. Similarly, seven out of the eight rapid tests developed for Echinococcus infections were antibody detecting tests save for one loop mediated isothermal amplification test. All of them were qualitative tests. For FBT infections, nine rapid tests were described; two antibody and one nucleic acid detecting test for diagnosis of Fascioliasis; three nucleic acid detecting tests for Opisthorchiasis; one antibody detecting test for Paragonimiasis; and for Clonorchiasis, one antibody and one nucleic acid detecting test. The FBT infection rapid tests were all qualitative in nature. Most of these tests have not undergone field evaluation in endemic areas where they will be used most. Conclusion This review describes the development and evaluation of rapid diagnostic tests, while highlighting the need for in depth validations of the tools to determine how well they can perform in endemic areas.

West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.

BACKGROUND: Microscopy and rapid diagnosis tests have a limited sensitivity in diagnosis of malaria by Plasmodium ovale. The LAMP kit (LoopAMP®) can be used in the field without special equipment and could have an important role in malaria control programmes in endemic areas and for malaria diagnosis in returned travellers. The performance of the Pan primer of the kit in detecting malaria by P. ovale was compared with the results of standard nPCR in samples of patients returning from P. ovale endemic areas. METHODS: Plasmodium ovale positive samples (29, tested by PCR and/or microscopy) and malaria negative specimens (398, tested by microscopy and PCR) were collected in different hospitals of Europe from June 2014 to March 2016 and frozen at -20 °C. Boil and spin method was used to extract DNA from all samples and amplification was performed with LoopAMP® MALARIA kit (Eiken Chemical, Japan) in an automated turbidimeter (Eiken 500). The results of LAMP read by turbidimetry and with the naked eye were compared. RESULTS: The kit showed a sensitivity of 100% and a specificity of 97.24% with positive and negative predictive values of 72.5 and 100%, respectively. Naked eyed readings were in accordance with turbidimetry readings (sensitivity, 92.5%, specificity, 98.96% and positive and negative predictive values, respectively, 90.24 and 99.22%). The limit of detection of LAMP assay for P. ovale was between 0.8 and 2 parasites/µl. CONCLUSIONS: The Pan primer of the Malaria kit LoopAMP® can detect P. ovale at very low-levels and showed a predictive negative value of 100%. This tool can be useful in malaria control and elimination programmes and in returned travellers from P. ovale endemic areas. Naked eye readings are equivalent to automated turbidimeter readings in specimens obtained with EDTA. This work received financial support from FIND with funds from the German Federal Ministry of Education and Research (BMBF) through the KfW Entwicklungsbank. Sí