title: Real-time PCR to quantify Leishmania donovani in hamsters.
abstract: Visceral leishmaniasis, a vector-borne disease caused by Leishmania donovani and Leishmania infantum , currently affects 12 million individuals in 88 countries. In the present study, a real-time PCR (rt-PCR) assay has been optimized and validated against 2 other routine methods, i.e., microscopy and limiting dilution culture assay, to estimate parasite load in the liver of infected Syrian hamsters (Mesocricetus auratus). A set of specific primers amplified a 116-bp target template of the kinetoplastid DNA of L. donovani in a SYBR® Green-based rt-PCR assay. To assess the methods, we tested 2 anti-leishmanial compounds belonging to the class of arylimidamides, DB745 (2,5-bis[2-ethoxy-4-(2-pyridylimino)aminophenyl]furan) and DB766 (2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan) for efficacy in vivo in Syrian hamsters infected with L. donovani promastigotes. Parasite load was quantified in liver by all 3 methods and was found comparable. Of the 3 methods, rt-PCR was the fastest and most convenient, sensitive, and reproducible method.
authors.affiliation: Department of Global Health, University of South Florida, Tampa, Florida 33612, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Srivastava
authors.forename: J Mark
authors.initials: JM
authors.lastname: Sweat
authors.forename: Azliyati
authors.initials: A
authors.lastname: Azizan
authors.forename: Brian
authors.initials: B
authors.lastname: Vesely
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Kyle
mesh_terms: D000578:Amidines / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D006224:Cricetinae; D018105:DNA, Kinetoplast / Q000032:analysis / Q000302:isolation & purification; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D005663:Furans / Q000494:pharmacology / Q000627:therapeutic use; D007893:Leishmania donovani / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology*; D008099:Liver / Q000469:parasitology; D008297:Male; D008647:Mesocricetus; D051379:Mice; D008807:Mice, Inbred BALB C; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology / Q000627:therapeutic use; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C552046:2,5-bis(2-(2-propoxy)-4-(2-pyridylimino)aminophenyl)furan; C552045:2,5-bis(2-ethoxy-4-(2-pyridylimino)aminophenyl)furan; D000578:Amidines; D000981:Antiprotozoal Agents; D018105:DNA, Kinetoplast; D005663:Furans; D010767:Phosphorylcholine; C039128:miltefosine