Molecular Detection of Leishmania in Wild Caught Sand Flies of Larroussius Subgenus in Iran: Combined Use of Internal and External Morphological Characters as a Mean to Differentiate Morphologically Similar Females.

OBJECTIVE: Phlebotomine sand flies are the primary vectors of leishmaniasis; visceral form of disease is transmitted mainly by species belonging to Larroussius and Adlerius subgenus. Species identification of some females in Larroussius subgenus is not easy due to the high similarity. Accurate species identification enables control operations to target only main vectors and increase understanding of ecological needs, bionomic characteristics, and behavioral traits. The purpose of current study was to use two approaches based on internal and external morphological characters to identify the wild caught of female specimens belonged to Larroussius subgenus and investigate Leishmania infection. METHODS: Totally, 128 specimens belonging to Larroussius' subgenus were collected from a VL focus in northwestern Iran, species differentiation was done by two approaches proposed in literature: (1) characters of pharyngeal armature, number of Spermathecal segment, length of spermathecal neck, palpal and ascoid formulae; (2) blindly on the basis of the shape of the base of spermathecal duct. Their potential infection to Leishmania was investigated by kDNA-Nested-PCR. RESULTS: The results of species identification were consistent based on two methods. Among three identified species, Phlebotomus perfiliewi was found to be the dominant species, followed by Ph. neglectus and Ph. tobbi. Two specimens of Ph. perfiliewi were found to be infected by Leishmania infantum, which emphasizes the role of this species in VL transmission in study area. CONCLUSION: It is suggested that combination of the characters used here be considered for species identification of female of Larroussius subgenus to take advantage of maximum characters, especially where species present sympatrically.

Comparison of Three Commonly Used Genetic Markers for Detection of Leishmania Major: An Experimental Study.

BACKGROUND: Leishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome. METHOD: In the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions. RESULTS: The lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes). CONCLUSION: In conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites.