Background Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP).

Methodology/Principal Findings LAMP-CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the Leishmania (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, or with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% for kDNA and 72.7% for 18S rDNA, both with 100% specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load.

Conclusions/Significance These new assays, being amenable to further simplification and optimization for their adoption in low-resource settings, hold promise as a new generation of accurate molecular tools for leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control.

The authors have declared no competing interest.

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This study used anonymized stored DNA samples that were assigned a code and had been isolated from different human skin ulcerative lesion specimen types, including biopsy, lancet scraping, cytological brush, swab, and filter paper. The respective patients had provided written informed consent for future research use of their specimens and clinical data. Patients with clinically suspected CL were recruited at the Hospital Nacional Adolfo Guevara Velasco (HNAGV) in Cusco, a region in Peru with endemic circulation of Leishmania (Viannia) parasites, most frequently L. braziliensis, L. guyanensis and L. lainsoni [57, 58], as part of another study between the HNAGV and UPCH (contract 095-2018-FONDECYT-BM-IADT-AV to JA, financed by CONCYTEC and The World Bank). The study protocol and informed consent (registration number 103155) were reviewed and approved by the ethics committee of the UPCH (IRB approval letter 063-05-19 dated 01/30/2019, latest renewed on 05/02/2023 with letter R-149-17-23).

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