Development of rapid field diagnostics for identification, control and management of haemorhagic fever outbreaks
The VHF Diagnostics project aims at developing and validating diagnostic tools for early viral haemorrhagic fever outbreak detection and control. A frontline line-assay carrying antigens of all African haemorrhagic fever viruses is to be developed and validated for widespread use in local hospitals. Line assays are third-generation (3G) immunoblots using purified recombinant proteins sprayed onto membranes irrespective of their molecular weight. This means that highly concentrated protein can be arranged onto membranes in the manner of a barcode.
There are no problems with contaminating extra bands, or with spacing of target molecules. This assay is easy to use and the results are easy to interpret. It is also cheap to produce and has a long shelf life. This tool will help to improve outbreak detection in Africa. The project includes a long-term field trial in local hospitals. A mobile integrated fluorescent real-time-PCR (F-RT-PCR) for outbreak investigation teams is also to be developed. It will consist of a panel of F-RTPCRs covering all haemorrhagic fever viruses, a robust simple nucleic acid extraction protocol and ready-to-use lyophilised PCR mixes. This tool will help outbreak investigation teams to improve their ability to manage and control haemorrhagic fever outbreaks.
A consortium of three European laboratories and four African laboratories will provide the expertise for the development, validation and adaptation to filed use of both tools.[+] Read More
Control of viral hemorrhagic fever (VHF) outbreaks critically depends on early detection, and an early alert, to allow, define, and deliver an appropriate response. To improve the early detection of cases, adequate tools have to be developed to allow early detection in the basic (field) conditions of local hospitals. Once the outbreak is identified, case management also needs on-site tools, such as viral genome detection, to contain the spread of the outbreak by carefully identifying and monitoring viraemic patients able to transmit the virus.
The general objective of VHF Diagnostics is to make adequate tools available to identify VHF outbreaks on-site at an early stage, and to support and complement the control of an outbreak. To reach this general objective, the consortium will develop:
Both assays will cover Ebola-Virus (EBOV), Marburg Virus (MRGV), Crimean-Congo-Virus (CCHFV) Lassa virus (LASV), Rift Valley Fever Virus (RVFV), Yellow Fever virus (YFV) and Dengue virus 1-4 (DENV). The F-RT-PCR will additionally cover the most important viral differentials Influenza A virus (FLUAV) and Influenza B virus (FLUBV).
LA: To develop LA, purified recombinant proteins will be expressed in the in vitro RTS-500 system (Roche) and sprayed onto immunoblot strips in the manner of a barcode. The LA will be designed for VHF circulating in Africa. Validation of the LA will be achieved by using available sera in the consortium of laboratories, which will be centralised in a repository for VHF diagnostics development. F-RT-PCR: The consortium will validate existing F-RT-PCRs for field use (EBOV, MBGV 12, CCHFV 13, RVFV 14, DENV 15, FLUAV, FLUBV16).
Additionally, F-RT-PCRs not yet described for LASV and YFV will be designed and validated for field use. To assess the sensitivity of each assay, the partners will generate RNAstandards for each aetiological agent derived from sections of the respective genomes. The specificity of the assays will be evaluated with recent isolates of each aetiological agent and patient and/or rodent samples provided by the collaborating laboratories. They will adapt the extraction of nucleic acids from blood samples to field conditions. The partners will also develop lyophilised ready-to-use PCR mixes for each aetiological agent to allow field PCR without the need for refrigeration facilities.
LA: The partners expect to be able to produce the envisioned line assay and test its applicability in local hospitals in Mali and Guinea. They hope to prove that an easy-touse frontline test indeed is a tool to reduce alert time in the case of an outbreak. The consortium hopes to detect either a YFV, a RVFV or a LASV outbreak during the evaluation period.
F-RT-PCR: The partners expect to develop an integrated toolbox for mobile outbreak investigation teams that will enable them to perform initial differential diagnostics and follow up patients during the containment of the outbreak. This will contain a field-evaluated set of lyophylised PCR mixes for VHFV plus FluA and B virus detection in combination with a field-evaluated simple extraction protocol. If successful, it may be possible to produce the LA assay for the African market.
LA: A fully functional and validated VHF-line assay can be used in regional hospitals and by outbreak investigation teams.
F-RT.-PCR: A fully functional and validated set of VHF F-RT-PCRs could increase the efficiency of outbreak investigation teams.