Laboratory and field evaluation of LAMP assays for canine visceral leishmaniasis diagnosis.

Abstract

Visceral leishmaniasis (VL) caused by Leishmania infantum remains a major public health concern in endemic regions and has shown geographic expansion in recent decades. Accurate and accessible diagnostics are essential for detecting infection in canine reservoirs that sustain transmission. Loop-mediated isothermal amplification (LAMP) represents a promising alternative to PCR-based techniques, combining diagnostic accuracy with lower cost and operational simplicity. Five LAMP assays targeting kinetoplast DNA (kDNA), internal transcribed spacers (ITS1 and ITS2), and the K26 gene were evaluated and compared with nested PCR targeting ITS1 (nPCR-ITS1). Most assays demonstrated species-level specificity for L. infantum, with the exception of the ITS2-targeting assay, which showed cross-amplification with Leishmania amazonensis. The kDNA-, ITS1-, and K26-targeting assays showed analytical sensitivity comparable to or higher than nPCR-ITS1. Considering its analytical performance and shared molecular target with the reference method, the ITS1-targeting assay (LAMP-P34) was selected for field evaluation. In canine samples from endemic and non-endemic areas, LAMP-P34 showed 80.4% sensitivity (37/46) and 100% specificity (0/58), using a reference panel based on nPCR-ITS1 positivity in lymph node or bone marrow samples to define infection and combined negative serology and molecular results to define non-infected dogs. Agreement with nPCR-ITS1 was almost perfect (κ = 0.887; 116/122, 95.1%). The high concordance with nPCR-ITS1 together with a simplified workflow underscores the potential of LAMP-P34 to expand access to molecular diagnosis and strengthen surveillance and control strategies in endemic areas.