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ABSTRACTEarlier 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 Leishmaniain 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 Leishmaniadetection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PDqPCR 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.

ABSTRACTWe present an algorithm based on three PCR assays for Leishmania(Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.

We present an algorithm based on three PCR assays for Leishmania (Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.