A novel colorimetric assay for early differentiation of mucocutaneous and cutaneous leishmaniasis via species-specific identification

Mucocutaneous leishmaniasis (MCL) is a severe and debilitating progression of cutaneous leishmaniasis (CL) that occurs when the disease spreads to involve mucosal tissues. Contrasting CL, which can often be treated with local therapies, MCL requires aggressive systemic treatment, strict adherence to a 30-day regimen and regular monitoring to prevent recurrence. These requirements highlight the critical need for accurate and rapid early diagnosis to guide effective treatment strategies. However, differentiating between the Leishmania species responsible for MCL and CL remains a significant challenge, particularly in resource-limited settings. To address this gap, this study introduces a novel colorimetric assay that integrates the Spin-Tube platform with Dynamic Chemical Labeling (DCL) technology for species-specific identification of Leishmania parasites. This approach targets single nucleotide fingerprints (SNFs) within the conserved hsp70 gene, allowing precise differentiation between species associated with MCL and CL. The assay employs single-plex PCR followed by DCL-based detection of SNFs, providing rapid and visually interpretable results to facilitate species differentiation. The assay demonstrated remarkable sensitivity, with a detection limit of 1 copy of parasite DNA per μL and performed effectively even under resource-limited conditions. It was used to identify ten MCL patients, with the results confirmed through DNA sequencing. Its simplicity and rapid turnaround could make it an ideal diagnostic solution for endemic regions. By providing accurate early differentiation between CL and MCL, this assay enables the implementation of personalised treatment plans, minimising unnecessary exposure to toxic therapies and reducing the risk of irreversible mucosal damage for affected patients.