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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - EbolaMoDRAD (Ebola Virus: Modern Approaches for developing bedside Rapid Diagnostics – Sofia ref.: 115843)

Teaser

The overall aim of EbolaMoDRAD was to develop and deliver rapid and bedside diagnostic tool(s) that will significantly increase our capacity to handle the Ebola Virus Disease (EVD) in West Africa and also future outbreaks. To achieve this, the following specific objectives...

Summary

The overall aim of EbolaMoDRAD was to develop and deliver rapid and bedside diagnostic tool(s) that will significantly increase our capacity to handle the Ebola Virus Disease (EVD) in West Africa and also future outbreaks. To achieve this, the following specific objectives were set out:
-to develop a biosafe detection method (diagnostic tool) for use at points of care in EVD endemic countries;
-to validate the diagnostic tools first in BSL4 and reference laboratories and then in the field;
-to implement a strong capacity building programme in West Africa with focus on rapid diagnostic, biosafety measure and outbreak management;
-to disseminate widely the project and its results to public health bodies, NGO, soutbreak management teams and local hospitals in West Africa.
Within this period, successful progress was made to achieve this objective. By using Ebola blood samples (from NHP), we could confirm our earlier data on our recently developed inactivation vacuum tube for Ebola virus. We also succeeded in pushing our isothermal assays towards two final versions that could be used in the diagnostic laboratory or field setting in future outbreaks. We have identified conventional real-time PCR assays that are most suitable as Ebola diagnostics and we are now looking to make these into formats that can be deployed in the field. We developed over 100 cartridges which contain the reagents and a silicon chip in order to diagnose Ebola infection. In addition, we developed and validated immunochromatographic flow tests (LFD) with a sensitivity of 89% and specificity of 98% by using clinical samples in Africa and Europe. We are now exploring possibilities to bring this assay to the marke and which can be used in future outbreaks.

Work performed

In 2016 our work on inactivating Ebola directly in blood tubes was published (J Clin Microbiol. 2016 Oct;54(10):2521-9). This novel inactivation of Ebola virus allows the samples to be handled outside of the high containment laboratory, which means easier and more rapid diagnoses. The isothermal assays have all advanced towards final versions that could be used in the diagnostic laboratory or field setting. Considerable progress has been made in the development of a single linear RCA assay. The RPA assays have undergone significant development. Triplex lateral flow RPA molecular diagnostics have been produced and are suitable for use in an outbreak setting and these are being tested in Dakar. The Ebola LAMP assay has been evaluated with clinical samples from two institutes. Two independent partners have tested and identified conventional real-time PCR assays that are most suitable as Ebola diagnostics and are now looking to make these into formats that can be deployed in the field. Clonit have developed over 100 cartridges which contain the reagents and a silicon chip to diagnose Ebola. We have expressed, purified all Zaire strain EBOV proteins and also Antibodies against them. The functional pAbs have been distributed to EbolaMoDRAD partners. We have successfully synthesized 386 Scan peptides covering all 6 Ebolavirus proteins. They have been used together with recombinant proteins for the production/printing of a 2nd generation 16-well antigen microarray. After the production of a 3rd generation as the final prototype, it was used for epitope mapping of IgG positive convalescent human sera of individuals previously infected with EBOV. First immunochromatographic flow tests (LFD) were developed and validated. It showed a sensitivity of 89% and specificity of 98% in further field trials with a total number of 250 tests. A SOP for rapid bedside inactivation of EBOV for serology testing using 10% Triton X-100 was developed and was presented at different meetings. The validation activities were carried out in the laboratories in Europe and on the field, using samples gathered in the project’s “disseminated” BioBank. Dissemination of knowledge produced by the project has provided scientists, public health staff and health workers in the field with enhanced skills and SOPs to enable them to implement the rapid diagnostic techniques for future outbreaks. The outcome of the second workshop “Mobile Laboratories” was published in a peer review journal.

Final results

We developed several molecular and serological tools for biosafe, point-of-care diagnostics, such as an inactivation vaccum tube, LFD assay for antigen and IgM detection for Ebolavirus as well as RPA for molecular testing. The successful assays were validated either at BSL4-laboratories in Europe and/or in endemic regions in Africa. The outcome of EbolaMODRAD has been communicated and disseminated through extensive workshops, linking to international organizations (WHO, MSF) and/or peer review publications.
EbolaMoDRAD has investigated the influence of the extraction reagents from the direct inactivation blood tubes on the real-time PCR and also development of these reagents into freeze-dried reagents that can be easily stored for extended periods of time. Regarding the isothermal diagnostic development, the direct novel RNA detection approach has a strong potential to be patented for use in molecular diagnostics. This will serve as a method for RNA-based detection, opening new doors for diagnostic approaches. We also developed strips capable of detecting the RPA-based Ebola diagnostic. These strips offer the potential of instrument-free molecular amplification with a simple lateral flow read-out. This type of diagnostic could sit well alongside established conventional serological diagnostic test strips. The development of the Clonit complete sample to answer system has significantly progressed. Extraction of clinical samples for molecular testing still remains an Achilles heel for many molecular diagnostics as these systems are both column and centrifuge-based, or rely on very expensive robotic machinery. AJ Innuscreen has made great advances in the field of extraction, particularly with a bespoke solution of automated nucleic acid extraction. We have shown that the production of recombinant EBOV proteins enables the development of highly specific serological assays for analysing immune responses against EBOV. The assay can be developed for diagnosing acute infections and analysing EBOV vaccine-induced responses as well as analysing the seroprevalence against EBOV in larger population studies. LFA antigen and IgM detection has been developed and partly validated, these tools together will contribute to a better rapid field diagnostic. All developed diagnostic tools for a rapid diagnosis of Ebola infection developed were validated, representing one of the successes of the project. Collaborations between several partners beyond the objective of this project were established, which may lead to new initiatives for development of diagnostic tools for neglected pathogens.

EbolaMoDRAD publications: http://www.ebolamodrad.eu/index.php/press-and-publications
Project coordinator: Ali Mirazimi, FoHM
Contact details: Ali.Mirazimi@folkhalsomyndigheten.se

Website & more info

More info: http://www.ebolamodrad.eu/.