title: Real-time PCR assay for detection and quantification of Leishmania (Viannia) organisms in skin and mucosal lesions: exploratory study of parasite load and clinical parameters.
abstract: Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (<3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.
authors.affiliation: Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru. jara.portocarrero.m@upch.pe
authors.forename: Marlene
authors.initials: M
authors.lastname: Jara
authors.forename: Vanessa
authors.initials: V
authors.lastname: Adaui
authors.forename: Braulio M
authors.initials: BM
authors.lastname: Valencia
authors.forename: Dalila
authors.initials: D
authors.lastname: Martinez
authors.forename: Milena
authors.initials: M
authors.lastname: Alba
authors.forename: Carlos
authors.initials: C
authors.lastname: Castrillon
authors.forename: Maria
authors.initials: M
authors.lastname: Cruz
authors.forename: Israel
authors.initials: I
authors.lastname: Cruz
authors.forename: Gert
authors.initials: G
authors.lastname: Van der Auwera
authors.forename: Alejandro
authors.initials: A
authors.lastname: Llanos-Cuentas
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Dujardin
authors.forename: Jorge
authors.initials: J
authors.lastname: Arevalo
mesh_terms: D000328:Adult; D018105:DNA, Kinetoplast / Q000032:analysis / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D005260:Female; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000469:parasitology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009092:Mucous Membrane / Q000469:parasitology*; D059208:Parasite Load / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012867:Skin / Q000469:parasitology*
chemical_list: D018105:DNA, Kinetoplast; D016054:DNA, Protozoan
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