Ahmed Abd El Wahed who is based at Georg-August University, Goettingen, Germany shares his experiences with development and use of field diagnostics that can fit in a suitcase. This work received support from the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Geneva, Switzerland. He explains the concept here …
Laboratory diagnosis mainly depends on nucleic acid detection by real-time polymerase chain reaction (PCR), which is available in central laboratories and has a turnaround time of more than two hours. Since real-time RT-PCR assays are not suitable for on-site screening, samples collected from local hospitals, treatment center or at the site of an outbreak have to be sent to laboratories over long distances for testing.
In developing countries, the necessary equipment for diagnosis is only available in few central laboratories, which are not accessible and of limited capacity to test large numbers of incoming samples. Moreover, transport conditions of samples are inadequate and therefore lead to unreliable results.
For decentralizing of the molecular diagnostics, there is a need for a simple molecular point-of-need test. We have developed a mobile suitcase laboratory (62+49+30 cm) containing all reagents and equipment for the detection of the nucleic acid of infectious agents using the recombinase polymerase amplification (RPA) technology. The RPA assay run at a constant temperature (42°C) for a maximum of 15 minutes.
Moreover, all reagents are cold-chain independent and the mobile laboratory is operated by a solar power battery. The nucleic acid extraction is performed by a magnetic bead based method, in which a simple fast lysis protocol is applied. In case of highly infectious agents such as Ebola virus, the inactivation step was performed in a glovebox.
The suitcase lab is designed to detect almost 30 pathogens such as Dengue, Zika, Chikungunya, Ebola, Coronaviruses as well as Tuberculosis, Leptospira, Salmonella typhi and paratyphi, malaria, avian influenza, and Leishmania.
The suitcase lab featured in a presentation at the 65th Annual Meeting of the American Society of Tropical Medicine in Atlanta under the title of “Novel Extraction Protocol and Recombinase Polymerase Amplification Assay for Detection of Leishmania Donovani In 30 Minutes.” Authors/presenters included Dinesh Mondal, Prakash Ghosh, Md. Anik Ashfaq Khan, Faria Hossain, Susanne Böhlken-Fascher, Greg Matlashewski, Axel Kroeger, Piero Olliaro, and Ahmed Abd El Wahed.
Their work highlighted Leishmania donovani (LD), a protozoan parasite transmitted to humans by sand flies, which causes Visceral Leishmaniasis (VL). Currently, diagnosis is based on presence of anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings.
In this study, we report on the development and testing of a novel extraction protocol in combination with recombinase polymerase amplification (RPA) assay for the detection of LD. The LD RPA assay detected equivalent to one LD genomic DNA. The RPA assay was performed at constant temperature (42°C) and the total assay runtime including the extraction procedure was 30 minutes.
The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100% agreement.
To allow the use of the assay at field settings, a mobile suitcase laboratory (56+45.5+26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method, in which a simple fast lysis protocol was applied.