Title : Preliminary evaluation of loop-mediated isothermal amplification for detecting Leishmania infantum in canine conjunctival swabs
Abstract:
Canine visceral leishmaniasis is a major parasitic disease of public health importance in endemic areas, but diagnosis can be challenging because clinical manifestations are variable and conventional methods may differ in sensitivity according to the biological specimen and analytical approach. This study aimed to investigate the feasibility of loop-mediated isothermal amplification (LAMP) for the detection of Leishmania infantum in conjunctival swabs collected from dogs with suspected canine visceral leishmaniasis, contributing to the development of a practical molecular diagnostic workflow. Biological samples were obtained from naturally exposed dogs and evaluated using conventional parasitological and molecular approaches, while LAMP reactions targeting the K26 gene were optimized at 65 °C. DNA extracted from conjunctival swabs using a commercial kit and a Direct Boil protocol was evaluated, including serial DNA dilutions to assess inhibition and analytical performance. Preliminary data from the study population showed heterogeneous results among diagnostic approaches: direct examination was positive in 11 of 37 samples, while conventional polymerase chain reaction (PCR) using RV1/RV2 was positive in 30 of 32 samples, PCR from kit-extracted conjunctival swabs was positive in 43 of 44 samples, and PCR from Direct Boil extracts was positive in 36 of 50 samples. During LAMP optimization, diluted DNA preparations produced more consistent amplification than highly concentrated extracts, indicating that matrix-related inhibition may affect reaction performance. These findings support further optimization and validation of conjunctival-swab LAMP as a rapid molecular tool for canine visceral leishmaniasis, with potential application in veterinary diagnosis and One Health surveillance.
Keywords: canine visceral leishmaniasis, LAMP, Leishmania infantum, conjunctival swab, molecular diagnosis, One Health
