Insaf BEL HADJ ALI ¹ Yusr Saadi ¹ Imen Khammeri ² Hejer Souguir ¹ Emna Harigua ¹ Hamed Chouaieb ² Ahmed Sahbi Chakroun ¹ Meryem LEMRANI ³ Aicha Kallel ⁴ Kalthoum Kallel ⁴ Nabil Haddad ⁵ Oussayma El-Dbouni ⁶ Rhea Coler ^7,8,9 Steven Reed ¹⁰ Akila Fathallah- Mili ² Ikram GUIZANI ^1*

• ¹ University Tunis El Manar, Laboratory of Molecular Epidemiology and Experimental Pathology, Pasteur Institute of Tunis, Tunis, Tunisia
• ² Parasitology Department, Faculty of Medicine Ibn el Jazzar, University Hospital Farhat Hached, University of Sousse, Sousse, Tunisia
• ³ Laboratory of Parasitology and Vector-Borne-Diseases, Institut Pasteur du Maroc, Casablanca, Morocco
• ⁴ Parasitology department, La Rabta University Hospital, Tunis, Tunisia
• ⁵ Laboratory of Immunology and Vector Borne Diseases, Faculty of public health, Lebanese University, Beirut, Lebanon
• ⁶ Infectious Diseases division, Rafic Hariri University Hospital, Beirut, Lebanon
• ⁷ Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, Washington, United States
• ⁸ Department of Pediatrics, School of Medicine, University of Washington, Seattle, Washington, United States
• ⁹ Department of Global Health, University of Washington, Seattle, WA, United States
• ¹⁰ HDT Bio, Seattle, WA, United States

Cutaneous leishmaniases (CL), a wide range of cutaneous diseases caused by diverse species of Leishmania genus parasites, are among most neglected infectious diseases. While it is not fatal, CL are highly morbid with disfiguring lesions, which could be chronic, leaving life-long unsightly scars; they are combined with psychological distress and social stigma. Efficiency of treatment highly depends on the infecting Leishmania species. Diagnosis is mainly based on microscopic direct examination (DE) of Giemsa-stained smears needing experienced microscopists. It can be laborious and time consuming when the parasite load is low. DE is poorly sensitive and does not identify Leishmania species. So far, only DNA assays accurately identify the species. Despite their wide use for generic detection, PCR methods also require equipment and additional steps to identify causal Leishmania species. L. major is hyperendemic in many countries in Africa, Middle East and Asia, where other species co-occur with different endemicity levels according to the situations. This complicates disease management and treatment, notably as leishmaniases distribution and epidemiology remain poorly understood. Here, we aimed for a simple and rapid molecular diagnostic test to detect and identify L. major, a predominant CL causal species, which could be prone to become a control tool at point of care, in endemic areas, using isothermal Recombinase DNA Amplification (Recombinase Polymerase Amplification, RPA/ or Recombinase Aided Amplification, RAA) coupled to detection by lateral flow (LF) chromatography on a PCRD cassette. To develop an L. major species-specific RPA-LF assay, computational analysis 70 Leishmania DNA targets, identified through bibliography and databases searches, selected 5 targets. We designed and tested 7 primers-pair/probe sets to specifically amplify L. major DNAs. First, the primers were tested for species specificity and sensitivity using basic RPA chemistry. Then, to develop RPA coupled LF detection, we shifted to the nfo chemistry; This way, we retained one set for further investigation, which confirmed it is L. major speciesspecific. Tested on 86 human cutaneous samples, this selected set was able to detect 100% of L. major infections in confirmed CL patients. We did not observe any cross reactivity with lesions due to L. infantum or L. tropica.