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.

Figures

Fig 1

Variability of kDNA targets in clinical samples and comparison of parasite loads estimated by the kDNA qPCR and G6PD qPCR assays. (A) Variations in kDNA minicircle numbers among Leishmania (Viannia) clinical isolates. The bars (right plot) show the ratio of kDNA to G6PD quantification results. (B) Comparison of sensitivities of the kDNA qPCR and G6PD qPCR assays for quantifying the parasite load in clinical samples. The histogram shows the distribution of the kDNA qPCR results with respect to those of G6PD qPCR (n = 87 values). (C) Correlation between parasite loads estimated by the kDNA qPCR and G6PD qPCR assays.

Fig 2

Flow diagram of the study procedures (diagnosis, parasite load determination, and species identification) performed on lesion biopsy specimens from patients with ATL. Superscript numbers indicate the following: (1) another specimen (scraping) available from the same lesion had a kDNA-positive qualitative PCR result; (2) these samples could not be quantified because they fell out of the limit of quantification; (3) mixed pattern for L. (V.) peruviana and L. (V.) braziliensis; (4) the species could not be assigned due to a lack of recognizable patterns common to reference Leishmania strains included in the species identification algorithm used herein, or the species could not be determined because of a very low parasite load or insufficient concentration of amplifiable genomic DNA.

Fig 3

Parasite load levels in human biopsy specimens. (A) Parasite load levels in clinical samples according to clinical manifestations. **, a significant difference was found in parasite loads between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions (P = 0.009). (B) Parasite load levels in CL lesions according to the infecting species. 1, the number of parasites per 106 human cells is indicated.