Background: Cutaneous leishmaniasis (CL) is an infectious vector-borne disease caused by protozoa of the Leishmania genus that affects humans and animals. The distribution of parasites in the lesion is not uniform, and there are divergences in the literature about the choice of the better sampling site for diagnosis–inner or outer edge of the ulcerated skin lesion. In this context, determining the region of the lesion with the highest parasite density and, consequently, the appropriate site for collecting samples can define the success of the laboratory diagnosis. Hence, this study aims to comparatively evaluate the parasite load by qPCR, quantification of amastigotes forms in the direct exam, and the histopathological profile on the inner and outer edges of ulcerated CL lesions. Methods: Samples from ulcerated skin lesions from 39 patients with confirmed CL were examined. We performed scraping of the ulcer inner edge (base) and outer edge (raised border) and lesion biopsy for imprint and histopathological examination. Slides smears were stained by Giemsa and observed in optical microscopy, the material contained on the smears was used to determine parasite load by quantitative real-time PCR (qPCR) with primers directed to the Leishmania (Viannia) minicircle kinetoplast DNA. The histopathological exam was performed to evaluate cell profile, tissue alterations and semi-quantitative assessment of amastigote forms in inner and outer edges. Principal findings: Parasite loads were higher on the inner edge compared to the outer edge of the lesions, either by qPCR technique (P<0.001) and histopathological examination (P< 0.003). There was no significant difference in the parasite load between the imprint and scraping on the outer edge (P = 1.0000). Conclusion/Significance: The results suggest that clinical specimens from the inner edge of the ulcerated CL lesions are the most suitable for both molecular diagnosis and direct parasitological examination.

Cutaneous leishmaniasis (CL) is a vector-borne disease caused by Leishmania spp. Molecular methods such as conventional PCR (cPCR) and real-time PCR (qPCR) show high accuracy but remain restricted to reference centers. Loop-mediated isothermal amplification (LAMP) enables rapid DNA amplification under isothermal conditions. LAMP targeting 18S rDNA using WarmStart^® Colorimetric LAMP 2× Master Mix and Bst 2.0 DNA Polymerase with SYBR^® Green I (BstSg) was evaluated in swab samples (extracted by kit—SW-kit and direct boil—SW-DB) and specimens collected through biopsy from ulcerated lesions of patients attending a reference center in Rio de Janeiro, Brazil. The reference standard was defined as at least one positive parasitological or molecular (cPCR) test plus clinical confirmation. Analytical sensitivity reached 10 fg with WS and 10 pg with BstSg. The diagnostic accuracy of LAMP ranged from 51.8% to 78.6%, with sensitivity from 52.5% to 81% and specificity from 39.6% to 89.4%. LAMP, particularly when applied to swab samples with DNA extracted using the SW-kit protocol and amplified with BstSg, showed promising diagnostic accuracy and operational feasibility as a less invasive approach for CL diagnosis, eliminating the need for skin biopsy and reducing patient discomfort during sample collection.