CRISPR-LAMP test advances Leishmania detection

Tegumentary leishmaniasis is a parasitic disease found across the Americas that causes skin and mucosal lesions and can lead to disfigurement and stigma. Early and accurate diagnosis matters for treatment and control, but the most sensitive tests today—PCR-based molecular diagnostics—need specialized laboratories and trained staff that are often absent in rural, low-resource areas. To address that gap, Vanessa Adaui and colleagues set out to adapt a cutting-edge molecular approach so it could be more practical outside advanced labs. They combined the highly specific CRISPR-Cas12a system, previously used with PCR preamplification, with loop-mediated isothermal amplification (LAMP), a simpler method that amplifies DNA at a single temperature. The team focused on two commonly used multi-copy genetic targets: 18S rDNA for broad, pan-Leishmania detection and kinetoplast DNA (kDNA) minicircles for detecting the Leishmania ( Viannia ) subgenus. Their goal was to create assays that produce clear readouts by fluorescence or on lateral flow strips and that could be simplified further for use near the point of care in endemic regions.

The researchers optimized LAMP-coupled CRISPR-Cas12a assays and tested them with both fluorescence-based and lateral flow readouts. In analytical tests using Leishmania braziliensis M2904 genomic DNA, the assays detected as little as 0.2 genome equivalents per reaction. The kDNA-targeting assay reliably detected all tested species of the Leishmania ( Viannia ) subgenus, while the 18S rDNA assay showed pan- Leishmania detection capability. Importantly, there was no cross-reactivity with DNA from other protozoan pathogens Trypanosoma cruzi and Plasmodium falciparum, the bacterial pathogen Mycobacterium tuberculosis, or with human DNA. The team then applied the assays to 90 clinical skin lesion samples from the Cusco region of Peru and compared results to kDNA real-time PCR. With a fluorescence readout, the LAMP-CRISPR assays achieved a sensitivity of 90.9% for kDNA and 72.7% for 18S rDNA, both with 100% specificity. Lateral flow strip results matched fluorescence detection in 18 tested samples, with a single discrepancy in the 18S assay linked to low parasite load.

These findings suggest the LAMP-CRISPR-Cas12a approach could become a new generation of molecular tools for leishmaniasis diagnosis and surveillance. Because LAMP operates at a constant temperature and lateral flow strips provide simple visual readouts, the assays are amenable to further simplification and optimization for low-resource settings where conventional PCR is impractical. The use of multi-copy targets like 18S rDNA and kDNA minicircles supports sensitive detection across species important to human disease, and the lack of cross-reactivity with other pathogens and human DNA indicates strong specificity. While the work reported here was performed under laboratory conditions, the combination of sensitivity, specificity, and adaptable readouts positions these tests to support earlier diagnosis, better patient management, and improved surveillance efforts in endemic areas. The authors note the potential for these tools to contribute to One Health strategies for disease control, linking human, animal, and environmental health considerations.