Development of a recombinase polymerase amplification lateral flow assay for the detection of active Trypanosoma evansi infections

Abstract

Background

Animal trypanosomosis caused by Trypanosoma evansi is known as “surra” and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings.

Methodology/Principle findings

An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool.

Conclusions/Significance

Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF ( TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.

Author summary

Neglected tropical diseases (NTDs) affecting humans and/or domestic animals severely impair the socio-economic development of endemic areas. One of these diseases, animal trypanosomosis, affects livestock and is caused by the parasites of the Trypanosoma genus. The most widespread causative agent of animal trypanosomosis is T. evansi, which is found in large parts of the world (Africa, Asia, South America, Middle East, and the Mediterranean). Proper control and treatment of the disease requires the availability of reliable and sensitive diagnostic tools. DNA-based detection techniques are powerful and versatile in the sense that they can be tailored to achieve a high specificity and usually allow the reliable detection of low amounts of parasite genetic material. However, many DNA-based methodologies (such as PCR) require trained staff and well-equipped laboratories, which is why the research community has actively investigated in developing amplification strategies that are simple, fast, cost-effective and are suitable for use in minimally equipped laboratories and field settings. In this paper, we describe the development of a diagnostic test under a dipstick format for the specific detection of T. evansi, based on a DNA amplification principle (Recombinase Polymerase Amplification aka RPA) that meets the above-mentioned criteria.