Detection of Leishmania infantum in Lutzomyia longipalpis captured in Campo Grande, MS

Abstract

Leishmaniasis is a zoonotic disease caused by protozoa of the genus Leishmania (Ross, 1903) and is the focus of considerable attention in human and veterinary medicine. In the city of Campo Grande, MS, the causative agent of visceral leishmaniasis is Leishmania infantum (= L. chagasi) primary vector, comprising approximately 92.9% of the local sandfly population, is Lutzomyia longipalpis. The aim of this work was to compare real-time PCR with PCR as a tool for the detection of the kinetoplast DNA (kDNA) of L. infantum in sandflies. Sandflies of this species were caught, and a total of 38 samples with 1-4 individuals in each sample were obtained; these were distributed across 13 districts and divided between seven urban areas of the city of Campo Grande, MS. Three positive samples were found by PCR and, when using real-time PCR, this was able to detect the presence of this agent in 6 of the 13 districts sampled, which were all located on the outskirts of the city, where indicates the greater enzootic potential of these regions, as they are closer to natural forest reserves. We conclude that real-time PCR can be used for epidemiological studies of L. infantum.Keywords: PCR, real-time PCR, phlebotomines, epidemiology. ResumoA Leishmaniose é uma zoonose causada por protozoários do gênero Leishmania (Ross 1903), objetos de considerável atenção em medicina humana e veterinária. Na cidade de Campo Grande -MS, o agente etiológico da Leishmaniose Visceral é Leishmania infantum (= L. chagasi), e o principal vetor é a espécie Lutzomyia longipalpis, que representa cerca de 92,9% da população de flebotomíneos. Este trabalho teve como objetivo avaliar a PCR em tempo real como ferramenta para a detecção de kDNA de L. infantum em flebotomíneos, comparando-se com PCR convencional. Flebotomíneos dessa espécie foram capturados, somando 38 amostras de 1 a 4 espécimens cada, distribuídas em 13 bairros, divididos entre as 7 regiões urbanas da cidade de Campo Grande -MS, e armazenados a -70 °C até a extração de ADN e amplificação por PCR e PCR em tempo real. Das 38 amostras testadas, foram encontradas 3 amostras positivas pela PCR convencional e 11 pela PCR em Tempo Real. Na otimização da PCR em tempo real, a temperatura de dissociação do amplificado foi de 82, 89 °C. Neste estudo, utilizando-se a técnica da PCR em tempo real, foi possível detectar a presença desse agente em 6 dos 13 bairros amostrados, todos na periferia da cidade, indicando o maior potencial enzoótico dessas regiões, que têm maior proximidade com reservas de matas naturais. Conclui-se que a PCR em tempo real pode ser utilizada para estudo epidemiológico de L. infantum.

Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies

“…kDNA), which is present in high number of copies (~ 10,000 copies of minicircle molecules) per parasite [31]. In fact, other real-time PCR assays using the same target gene also reported very good analytical sensitivity [32,33].…”

Detection of Leishmania spp. in Bats from an Area of Brazil Endemic for Visceral Leishmaniasis

“…Thus, the oral route as a transmission mechanism for Leishmania in bats cannot be ruled out because (i) phlebotomines including L. longipalpis are very abundant in CG (De Oliveira et al., ; Silva et al., ; Cunha et al., ), (ii) bats belonging to the family Phyllostomidae show a generalist diet, opportunistically consuming arthropods (Gardner, ; Bernard, ; Zortéa, ), (iii) A. lituratus , A. planirostris and C. perspicillata , which were found to be infected by Leishmania in the present study, have already been reported supplementing their diet with arthropods in the study area (Martins et al., ) and (iv) bats and L. longipalpis have overlapping crepuscular and nocturnal activity patterns (Rebêlo, ; Feliciangeli et al., ). Furthermore, it has previously been shown that bats can become infected by trypanosomatid species when they ingest parasitized insects (Thomas et al., ; Ramírez et al., ).…”

“…In the city of CG, thousands of L. infantum seropositive dogs are killed annually, but the increasing incidence of HVL and CVL shows that the existing control measures are not effective (Cunha et al., ). Therefore, the classic epidemiological approach involving dogs–shadflies–humans should be reconsidered in this urban environment.…”

Contribution to the Knowledgment of Feeding Habbits of Lutzomyia (Lutzomyia) Longipalpis (Lutz & Neiva, 1912) in Association to Natural Infection by Leishmania (Leishmania) Infantum Chagasi (Cunha & Chagas, 1937)

“…Thus, if a sample showed a high parasitic load, this may be due to one or more infected phlebotomines [64]. In Camaçari (Salvador) was performed a compared study in diferents periods of captures from L. longipalpis, the results of parasite load was low and did not vary regardless of the season, despite the number of collected sand ies [65].…”