Although real-time reverse transcription RT-PCR is currently used as the standard method for EBOV molecular diagnosis, it has some limitations in terms of cost, equipment, and turn-around time. This project team successfully implemented isothermal amplifictaion (Recombinase Polymerase Amplification (RPA)) in a mobile suitcase laboratory for EBOV point-of-care (POC) detection at Ebola treatment centers (ETC) during the large Ebola virus disease (EVD) outbreak in West-Africa 2014-2015 We successfully demonstrated that the use of RPA helped avoid saturating ETC capacities with suspect cases and that easily trained teams can reliably deploy this fast method. In the Ebola outbreak in Guinea up to 90% of patients tested negative but were rarely diagnosed for differentials. Malaria and other diseases went under-diagnosed, which most likely lead to more deaths from these infectious agents. Another important aspect is that, the fear of the patients of having Ebola, while they are suffering from other disease must be overcome by providing the correct diagnosis of the type of the pathogen timely. Two mobile laboratories are currently deployed in DRC and stationed at stationed at Bikoro, Mbandaka and Itipo providing diagnostics for EBOV. In our team RPA assays have been developed for the detection of Plasmodium falciparum, Yellow Fever virus (YFV), Dengue virus (DENV) and Salmonella typhi. Currently we are preparing dried primer sets to be used with RPA reagents for these teams. The RPA assay is six times faster than conventional real-time PCR while yielding the same analytical sensitivity and specificity. In addition, the RPA format (equipment size, cold-chain independent reagents) makes rapid on-site testing feasible and affordable. We developed a mobile laboratory, which consists of a hard plastic glovebox, a Diagnostics-in-a-Suitcase (DiaS), and a solar panel and power pack set. The disassembled glovebox is kept in a metal box (80 × 60 × 41 cm) with other necessary materials (disinfectant solution, extraction kits, filter tips, racks, vortex, heat block, autoclavable plastic bags and personal protective equipment (PPE). The total weight is 28 kg for the box and 16 kg for the DiaS. Sample inactivation and RNA extraction using the SE kit so far were done in the glovebox. This allowed handling of hazard group 4 samples. The RT-RPA assay was performed in the DiaS containing the ESEQuant TS2 device with integrated touchscreen to operate the device and display the results without the need of a laptop, which is not easy to disinfect.
