title: Detection of a broad range of Leishmania species and determination of parasite load of infected mouse by real-time PCR targeting the arginine permease gene AAP3.
abstract: Leishmaniasis is one of the world's most neglected infectious diseases, affecting around 12 million people and more than 350 million at risk of infection. The clinical picture varies from self-healing cutaneous lesions to severe visceral infections, but still no commercial vaccines for humans are available and the currently used drugs have unpleasant side effects. Here we report a real-time PCR assay targeting the arginine permease gene AAP3 that can be applied for all the nine different species of the Leishmania genus tested; 4 Old World species and 5 New World species, from both L. (Leishmania) and L. (Viannia) subgenera. No cross-reaction was seen with Trypanosoma cruzi, Trypanosoma brucei, human or mouse genomic DNA. The assay has a high sensitivity, with a limit of detection of 10fg DNA for L. (L.) major and L. (L.) donovani, and 100fg DNA for L. (V.) braziliensis, and can be used for both qualitative and quantitative purposes. This AAP3-Assay, run in duplex with a host specific gene-assay, was also successfully used for quantification of parasite load of footpads from L. (L.) major-infected mice. It can therefore be a valuable tool in applications like monitoring effects of drugs, the selection of vaccine candidates and in screening patients, including asymptomatic carriers.
authors.affiliation: National Centre for Tropical Infectious Diseases, Department of Medicine, Haukeland University Hospital, 5021 Bergen, Norway. Electronic address: marit.gjerde.tellevik@helse-bergen.no.
authors.forename: Marit Gjerde
authors.initials: MG
authors.lastname: Tellevik
authors.affiliation: Department of Clinical Science, University of Bergen, 5021 Bergen, Norway. Electronic address: Karl.Muller@k2.uib.no.
authors.forename: Karl Erik
authors.initials: KE
authors.lastname: Muller
authors.affiliation: Department of Clinical Science, University of Bergen, 5021 Bergen, Norway. Electronic address: Karen.Rebbestad@k2.uib.no.
authors.forename: Karen Rebbestad
authors.initials: KR
authors.lastname: Løkken
authors.affiliation: Department of Clinical Science, University of Bergen, 5021 Bergen, Norway. Electronic address: Audun.Nerland@k2.uib.no.
authors.forename: Audun Helge
authors.initials: AH
authors.lastname: Nerland
mesh_terms: D026923:Amino Acid Transport Systems, Basic / Q000235:genetics*; D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D007891:Leishmania / Q000145:classification* / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000469:parasitology*; D008807:Mice, Inbred BALB C; D059208:Parasite Load / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D026923:Amino Acid Transport Systems, Basic; D015800:Protozoan Proteins; C043909:arginine permease
keywords: AAP3; Arginine permease; Detection; Leishmania; Quantification; qPCR