Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

Abstract

BackgroundCutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.Methodology/Principal FindingsWe applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4).Conclusion/SignificanceOur results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

“…Additionally, in the present study, swabs and biopsy samples had similar sensitivity values when the same chemistry was used for qPCR. These findings are in contrast to the higher accuracy of swab analysis in CL reported in previous studies (12,13,18).…”

“…Swabs and cytology brush samples of localized cutaneous leishmaniasis (CL) are more sensitive than lesion aspirate and biopsy samples (12,13). The utility of swabbased diagnosis in mucosal leishmaniasis (ML) has not been completely explored, as most studies have been conducted in highly controlled environments, usually using healthy controls for comparison (5).…”

Ability of Real-time PCR in diagnose Differentiation Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

“…Weigle and others showed that PCR sensitivity was higher than the conventional assays for the diagnosis of acute lesions while for chronic samples, the sensitivity of PCR was much higher than the conventional assays (27). and number or area of lesions, there was no difference in parasite load (30). Sirian and co-workers conducted a comparison between conventional, molecular, and immunohistochemical methods for CL detection and reported that immunohistochemical and molecular techniques were more sensitive (31)(32)(33).…”

Ability of Real-time PCR for Differential Diagnosis of Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology

“…Leishmania (Viannia) parasites. According to the parasite species, the patients' age, and number or area of lesions, there was no difference in parasite load (30). Sirian and co-workers conducted a comparison between conventional, molecular, and immunohistochemical methods for CL detection and reported that immunohistochemical and molecular techniques were more sensitive (31)(32)(33).…”

“…Nowadays, PCR-based testing of skin lesion biopsies is known as a sensitive and specific test for diagnosis and quantification of leishmaniasis (13)(14)(15)(16). The analysis of the load of leishmania parasites within the skin lesions would be important not only for diagnostic purposes, but also for an eventual follow-up of a patient's response to treatment (17). Accordingly, in the present study we applied a standardized qPCR assay to detect Leishmania.…”