Résumé
ABSTRACT Cutaneous leishmaniasis (CL) is the most prevalent form of leishmaniasis, with up to 1 million new cases reported annually across more than 90 endemic countries. In Morocco, CL is caused by three Leishmania species: Leishmania major , L. tropica , and L. infantum . Sensitive and specific parasite detection and accurate species identification are essential for proper diagnosis, treatment decisions, epidemiological studies, and effective disease surveillance. Commonly used PCR-based diagnostic methods require multi-step procedures, such as sequential amplification (Noyes nested PCR) or enzymatic digestion (ITS1-RFLP), and are not sufficiently resolutive for precise species identification. In this study, we introduce TIM (for T ropica , I nfantum , M ajor ), a novel, flexible, and straightforward molecular diagnostic tool based on standard PCR procedures. TIM targets specific motifs within kinetoplast DNA minicircle sequences, allowing for Leishmania detection in a single multiplex reaction. It can be adapted with a preliminary nested amplification step to enhance sensitivity or with species-specific primers for accurate species-level identification. When applied to CL clinical samples from Morocco, TIM demonstrated higher sensitivity than established methods. Notably, TIM also enabled the identification of mixed interspecific profiles directly from clinical samples, which may indicate either co-infections with distinct species or the presence of hybrid genotypes. With a streamlined protocol requiring only six primers, TIM is rapid, flexible, and well suited for both routine diagnostics and large-scale epidemiological investigations. IMPORTANCE This study introduces TIM, a novel molecular diagnostic assay developed to enhance the detection and species identification of Leishmania parasites from clinical samples in Morocco. TIM relies on simple PCR procedures, and it is tailored to the detection of three species that are endemic to the region: L. major , L. tropica , and L. infantum . TIM demonstrated superior sensitivity and improved species-level resolution when compared to the diagnostic methods commonly used in Morocco. Notably, TIM also enables the direct detection of mixed interspecific profiles from cutaneous clinical samples. Thanks to its versatility and simplicity, TIM offers a practical tool to significantly strengthen leishmaniasis surveillance, prevention, and control strategies in Morocco and surrounding regions.