Detection and Identification of Leishmania Species Within Naturally Infected Sand Flies in the Andean Areas of Ecuador by a Polymerase Chain Reaction

Abstract

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.

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

“…Wild sand flies were collected in March, July and August 2012 in Andean areas of Ecuador; Alausi, Chanchan and Huigra (Province of Chimborazo), where Andean-type cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana is endemic (Kato et al, 2005.…”

“…Conventional PCR was performed with previously described primers specific for Leishmania minicircle kinetoplast DNA (mkDNA) (Kato et al, 2005(Kato et al, , 2007. The primer sequences used for amplification were 5´ -CTRGGGGTTGGTGTAAAATAG-3´ (L.MC-1S) and 5´ -TWTGAACGGGRTTTCTG-3´ (L.MC-1R) (Kato et al, 2005).…”

“…The primer sequences used for amplification were 5´ -CTRGGGGTTGGTGTAAAATAG-3´ (L.MC-1S) and 5´ -TWTGAACGGGRTTTCTG-3´ (L.MC-1R) (Kato et al, 2005). The reaction was carried out in a volume of 15 µl using a pair primers (0.4 µM each) and 2 x Ampli Taq Gold PCR Master Mix (Applied Biosystems, NJ, USA).…”

“…Estimation of Leishmania infection rates in the vector could serve as an indicator of a change in transmission intensity at a given endemic area. 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).…”

“…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.…”