title: Development of a recombinase polymerase amplification assay for the detection of pathogenic Leptospira.
abstract: Detection of leptospires based on DNA amplification techniques is essential for the early diagnosis of leptospirosis when anti-Leptospira antibodies are below the detection limit of most serological tests. In middle and low income countries where leptospirosis is endemic, routine implementation of real-time PCR is financially and technically challenging due to the requirement of expensive thermocycler equipment. In this study we report the development and evaluation of a novel isothermal recombinase polymerase amplification assay (RPA) for detection of pathogenic Leptospira based on TwistAmp chemistry. RPA enabled the detection of less than two genome copies per reaction. Retrospective evaluation revealed a high diagnostic accuracy (sensitivity and specificity of 94.7% and 97.7%, respectively) compared to culturing as the reference standard. RPA presents a powerful tool for the early diagnosis of leptospirosis in humans and in animals. Furthermore, it enables the detection of the causative agent in reservoirs and environment, and as such is a valuable adjunct to current tools for surveillance and early outbreak warning.
authors.affiliation: KIT Biomedical Research, WHO/FAO/OIE and National Leptospirosis Reference Centre, Royal Tropical Institute, Meibergdreef 39, Amsterdam 1105, The Netherlands. a.ahmed@kit.nl.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Ahmed
authors.affiliation: KIT Biomedical Research, WHO/FAO/OIE and National Leptospirosis Reference Centre, Royal Tropical Institute, Meibergdreef 39, Amsterdam 1105, The Netherlands. H.v.d.Linden@kit.nl.
authors.forename: Hans
authors.initials: H
authors.lastname: van der Linden
authors.affiliation: KIT Biomedical Research, WHO/FAO/OIE and National Leptospirosis Reference Centre, Royal Tropical Institute, Meibergdreef 39, Amsterdam 1105, The Netherlands. r.hartskeerl@kit.nl.
authors.forename: Rudy A
authors.initials: RA
authors.lastname: Hartskeerl
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D006801:Humans; D007919:Leptospira / Q000235:genetics / Q000302:isolation & purification*; D007922:Leptospirosis / Q000175:diagnosis*; D021141:Nucleic Acid Amplification Techniques; D045522:Recombinases / Q000378:metabolism; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D045522:Recombinases; D004259:DNA-Directed DNA Polymerase