Multiplex Polymerase Chain Reaction for Detection of Leishmania Sp. in Sandflies of the Paraná River Islands, Southern Brazil.

Abstract

Leishmaniases are classified as tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL). Brazil is among the countries with the highest number of TL and VL cases. This study was undertaken to standardize the multiplex polymerase chain reaction (PCR) for the detection of the genus Leishmania in sandflies of endemic regions, on islands in the Upper Paraná River, northwestern Paraná. The sandflies were collected on 10 islands, from November 2012 to November 2014, with Falcão light traps, identified and conserved in tubes containing isopropanol, for subsequent DNA extraction. Two pairs of primers were used for multiplex PCR: A1/A2 and 5Llcac/3Llcac. Nyssomyia neivai was the predominant species of the collected specimens. A total of 3870 samples of female sandflies were analyzed and submitted to multiplex PCR, for the validation of the technique. All pools showed the 220 bp fragment for sandfly DNA detection, but no ∼120 bp fragment of Leishmania DNA was found. Although no natural infection of Ny. neivai by Leishmania was found in this study, the interaction of sandflies with Leishmania and its natural reservoirs continues in these Paraná River islands, despite the low diversity of the sandfly fauna. Some of these islands have permanent residents and are frequented by tourists.