Rapid isothermal molecular tests to discriminate between Leishmania braziliensis and Leishmania infantum infections in dogs.

Abstract

BACKGROUND: We standardized two recombinase polymerase amplification (RPA) assays coupled with lateral flow (LF) strips for the detection of Leishmania braziliensis and Leishmania infantum kinetoplast DNA (kDNA). METHODS: The RPA-LF assays were tested at different temperatures and reaction times, using DNA from cultured L. braziliensis and L. infantum. The L. infantum RPA-LF was also tested using clinical samples (bone marrow and skin) from infected and uninfected dogs. RESULTS: The detection limits (analytical sensitivity) of the assays were 0.04&#xa0;pg/&#x3bc;l and 0.04&#xa0;ng/&#x3bc;l for L. braziliensis and L. infantum kDNA, respectively. Using clinical samples, the L. infantum RPA-LF successfully detected the parasite kDNA in bone marrow (21/30; 70.0%) and skin samples (23/30, 76.6%) from naturally infected dogs. We found an almost perfect agreement (kappa&#x2009;=&#x2009;0.807) between RPA-LF for L. infantum and our reference quantitative real-time polymerase chain reaction (qPCR), considering clinical samples with a quantification cycle (C)&#x2009;<&#x2009;30, whereas the agreement with samples with a C&#x2009;>&#x2009;30 (lower parasite loads) was moderate (kappa&#x2009;=&#x2009;0.440). CONCLUSIONS: The RPA-LF assays developed here may be promising diagnostic tools for point-of-care diagnosis of L. infantum and L. braziliensis infection in dogs, particularly in remote rural areas lacking laboratory infrastructure.