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

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 pg/μl and 0.04 ng/μl 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 = 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 (Cq) < 30, whereas the agreement with samples with a Cq > 30 (lower parasite loads) was moderate (kappa = 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.