title: A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis.
abstract: Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists' attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA ( approximately 120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.
authors.affiliation: Centre for Applied Entomology and Parasitology, Institute of Science and Technology in Medicine, Keele University, Staffordshire ST5 5BG, UK.
authors.forename: Shalindra
authors.initials: S
authors.lastname: Ranasinghe
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Rogers
authors.forename: James G C
authors.initials: JG
authors.lastname: Hamilton
authors.forename: Paul A
authors.initials: PA
authors.lastname: Bates
authors.forename: Rhayza D C
authors.initials: RD
authors.lastname: Maingon
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis; D005260:Female; D007303:Insect Vectors; D007891:Leishmania / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000635:transmission; D011576:Psychodidae / Q000469:parasitology*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan