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

• Published in: PLoS neglected tropical diseases 2020-02, Vol.14 (2), p.e0008044-e0008044
• Main Authors: Li, Zeng, Pinto Torres, Joar Esteban, Goossens, Julie, Stijlemans, Benoit, Sterckx, Yann G-J, Magez, Stefan
• Format: Article
• Language: English
• Subjects: Agarose; Agglutination tests; Amplification; Analytical methods; Analytical techniques; Animals; Assaying; Beef cattle; Biochemistry; Biology and Life Sciences; Deoxyribonucleic acid; Detection; Diagnostic software; Diagnostic systems; Diseases; DNA; DNA, Protozoan - genetics; Domestic animals; Electrophoresis; Gel electrophoresis; Gels; Genes; Genetic research; Glycoproteins; Health aspects; House mouse; Immunology; Infection; Infection control; Infections; Laboratories; Medicine and Health Sciences; Methods; Mice; Mice, Inbred C57BL; Nucleic Acid Amplification Techniques - methods; Nucleic acids; Nucleotide sequence; Parasites; PCR; Polymerase chain reaction; Polymerase Chain Reaction - methods; Protocols; Recombinase; Recombinases - metabolism; Research and Analysis Methods; Ribosomal DNA; Sensitivity; Sensitivity and Specificity; Setting (Literature); Software; Strains; Supervision; Surra; Technology; Time; Tropical climate; Tropical diseases; Tropical environment; Trypanosoma - genetics; Trypanosoma - isolation & purification; Trypanosoma evansi; Trypanosomiasis - diagnosis; User requirements
• ISSN: 1935-2735; 1935-2727; 1935-2735
• DOI: 10.1371/journal.pntd.0008044

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. 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. 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.