Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).Neste trabalho avaliamos o uso da reação em cadeia da polimerase (PCR) na investigação da presença de parasitas Leishmania (Kinetoplastida:Trypanosomatidae) em flebotomíneos individuais. Para isso, foram utilizados três pares flebotomíneo/parasita: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis e Lutzomyia migonei/Leishmania braziliensis, todos eles incriminados na transmissão de leishmaniose cutânea ou visceral. O DNA total a ser amplificado foi extraído de flebotomíneos inteiros, contendo ou não o parasita, sem dissecção prévia do trato digestivo ou combinação de indivíduos. Conteúdos do trato digestivo de flebotomíneos, em especial sangue de camundongo, não interferiram na reação de amplificação. Dez parasitas Leishmania sp. por flebotomíneo foram suficientes para detecção com iniciadores gênero-específicos. Com a utilização de iniciadores para os complexos L. braziliensis e L. mexicana, respectivamente, foi possível discriminar entre L. braziliensis e L. amazonensis, em flebotomíneos infectados experimentalmente (L. migonei)

Ecoepidemiological aspects of visceral leishmaniasis in an endemic area in the Steel Valley in Brazil: An ecological approach with spatial analysis.

Leishmaniases are a group of infectious diseases transmitted by phlebotomine sand flies, and their distribution depends on the presence of vectors, parasites, reservoirs and susceptible hosts in the same environment. In the last decades, visceral leishmaniasis (VL) has become urbanized and reached economically important cities in countries within the transmission zone. Our study was conducted in one of those cities-Ipatinga-in the state of Minas Gerais, Brazil, where the first autochthonous case of VL dates back to 2011. Since no data regarding the epidemiological triad of VL (etiological agent/vector/domestic reservoir) were available for this city, we characterized the local entomological fauna, identified the presence of specific Leishmania DNA in the captured phlebotomine sand flies, and assessed the incidence of canine and human VL. For the entomological survey, we set twenty light traps in ten districts of the city with reports of human and canine VL. The insect captures were performed monthly, during one year, starting in March 2015. A total of 1501 specimens of phlebotomine sand flies belonging to 16 distinct species were captured, with predominance (61.9%) of Lutzomyia longipalpis. Leishmania infantum DNA was detected in L. longipalpis and in Evandromyia cortelezzii test samples. A total of 9,136 dogs were examined, 1,355 of which (14.8%) were serologically positive for VL. The cases were georeferenced and the data were plotted in thematic maps, along with human cases of VL registered by the local Department of Health, during the study period. Our results confirm that the VL transmission cycle is active in Ipatinga, with the presence of vectors carrying Leishmania DNA, canine and human cases of the disease. Spatial analysis allowed for the observation of a positive relationship between canine and human cases of VL and the identification of areas with high priority for control actions in the city. The mapping of high-risk areas, together with an epidemiological study in urban areas, is fundamental to improve the efficacy of the Program for Surveillance and Control of VL (PSCVL) in Brazil