Detection and Identification of Leishmania DNA within Naturally Infected Sand Flies by Seminested PCR on Minicircle Kinetoplastic DNA

Abstract

A seminested PCR assay was developed in order to amplify the kinetoplast minicircle of Leishmania species from individual sand flies. The kinetoplast minicircle is an ideal target because it is present in 10,000 copies per cell and its sequence is known for most Leishmania species. The two-step PCR is carried out in a single tube using three primers, which were designed within the conserved area of the minicircle and contain conserved sequence blocks. The assay was able to detect as few as 3 parasites per individual sand fly and to amplify minicircle DNA from at least eight Leishmania species. This technique permits the processing of a large number of samples synchronously, as required for epidemiological studies, in order to study infection rates in sand fly populations and to identify potential insect vectors. Comparison of the sequences obtained from sand flies and mammal hosts will be crucial for developing hypotheses about the transmission cycles of Leishmania spp. in areas of endemicity.

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

“…An important limitation of this study is that both ELISA for diagnosis of asymptomatic infection in humans and genus specific PCR assay for detecting infection in sandflies may not be the most valid techniques to employ. Consequently, prevalence estimates of infection by L. chagasi might not be accurate (Aransay et al 2000, Romero et al 2009). However, we were less concerned in providing exact prevalence estimates than with showing autochthonous transmission in the area by identifying L. chagasi infection in all important elements of the transmission chain.…”

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

“…Such studies have been conducted either by dissecting individual insects and detecting the parasites under a microscope, or polymerase chain reaction (PCR)-based techniques to detect Leishmania DNA in sand flies (Kato et al, 2005). The former is laborious and timeconsuming due to the low Leishmania infection rate in sand flies in most endemic areas (Aransay et al, 2000;Kato et al, 2005). The latter method has been proved to have both high sensitivity and specificity; however, the PCR methods require specialized instruments and take several hours, which make their use in resource-limited countries and under field conditions unfeasible.…”

Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

“…Actualmente, la detección de parásitos del género Leishmania en los insectos incluye métodos que van desde la búsqueda directa de los flagelados en el tracto digestivo del insecto hasta técnicas moleculares que usan principalmente K ADN (12,(26)(27)(28)(29)(30). La técnica de reacción en cadena de la polimerasa (PCR), por su alta sensibilidad y especificidad, ha facilitado adelantar estudios ecoepidemiológicos en áreas de alta endemicidad, suministrando datos más precisos en periodos de tiempo más cortos (17,29).…”

“…La PCR como una de estas técnicas con capacidad de producir cientos de copias de ADN se ha utilizado en estudios entomológicos con diferentes fines (11,12).…”