title: Viability and burden of Leishmania in extralesional sites during human dermal leishmaniasis.
abstract: BACKGROUND
  The clinical and epidemiological significance of Leishmania DNA in extralesional sites is obscured by uncertainty of whether the DNA derives from viable parasites. To examine dissemination of Leishmania during active disease and the potential participation of human infection in transmission, Leishmania 7SLRNA was exploited to establish viability and estimate parasite burden in extralesional sites of dermal leishmaniasis patients.
  
  
  METHODS
  The feasibility of discriminating parasite viability by PCR of Leishmania 7SLRNA was evaluated in relation with luciferase activity of luc transfected intracellular amastigotes in dose-response assays of Glucantime cytotoxicity. Monocytes, tonsil swabs, aspirates of normal skin and lesions of 28 cutaneous and 2 mucocutaneous leishmaniasis patients were screened by kDNA amplification/Southern blot. Positive samples were analyzed by quantitative PCR of Leishmania 7SLRNA genes and transcripts.
  
  
  RESULTS
  7SLRNA amplification coincided with luciferase activity, confirming discrimination of parasite viability. Of 22 patients presenting kDNA in extralesional samples, Leishmania 7SLRNA genes or transcripts were detected in one or more kDNA positive samples in 100% and 73% of patients, respectively. Gene and transcript copy number amplified from extralesional tissues were comparable to lesions. 7SLRNA transcripts were detected in 13/19 (68%) monocyte samples, 5/12 (42%) tonsil swabs, 4/11 (36%) normal skin aspirates, and 22/25 (88%) lesions; genes were quantifiable in 15/19 (79%) monocyte samples, 12/13 (92%) tonsil swabs, 8/11 (73%) normal skin aspirates.
  
  
  CONCLUSIONS
  Viable parasites are present in extralesional sites, including blood monocytes, tonsils and normal skin of dermal leishmaniasis patients. Leishmania 7SLRNA is an informative target for clinical and epidemiologic investigations of human leishmaniasis.
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Cali, Colombia.
authors.forename: Ibeth
authors.initials: I
authors.lastname: Romero
authors.forename: Jair
authors.initials: J
authors.lastname: Téllez
authors.forename: Yazmín
authors.initials: Y
authors.lastname: Suárez
authors.forename: Maria
authors.initials: M
authors.lastname: Cardona
authors.forename: Roger
authors.initials: R
authors.lastname: Figueroa
authors.forename: Adrian
authors.initials: A
authors.lastname: Zelazny
authors.forename: Nancy
authors.initials: N
authors.lastname: Gore Saravia
mesh_terms: D002470:Cell Survival; D017930:Genes, Reporter; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification* / Q000502:physiology*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D050296:Microbial Viability; D009000:Monocytes / Q000469:parasitology; D014066:Palatine Tonsil / Q000469:parasitology; D010274:Parasitology / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D016053:RNA, Protozoan / Q000096:biosynthesis / Q000235:genetics; D020733:RNA, Small Cytoplasmic / Q000096:biosynthesis / Q000235:genetics; D018271:Signal Recognition Particle / Q000096:biosynthesis / Q000235:genetics; D012867:Skin / Q000469:parasitology
chemical_list: C115990:7SL RNA; D016053:RNA, Protozoan; D020733:RNA, Small Cytoplasmic; D018271:Signal Recognition Particle; D008156:Luciferases