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Leishmania tropica is one of the main causative agents of cutaneous leishmaniasis (CL). Population structures of L. tropica appear to be genetically highly diverse. However, the relationship between L. tropica strains genomic diversity, protein coding gene evolution and biogeography are still poorly understood. In this study, we sequenced the genomes of three new clinical L. tropica isolates, two derived from a recent outbreak of CL in camps hosting Syrian refugees in Lebanon and one historical isolate from Azerbaijan to further refine comparative genome analyses. In silico multilocus microsatellite typing (MLMT) was performed to integrate the current diversity of genome sequence data in the wider available MLMT genetic population framework. Single nucleotide polymorphism (SNPs), gene copy number variations (CNVs) and chromosome ploidy were investigated across the available 18 L. tropica genomes with a main focus on protein coding genes. MLMT divided the strains in three populations that broadly correlated with their geographical distribution but not populations defined by SNPs. Unique SNPs profiles divided the 18 strains into five populations based on principal component analysis. Gene ontology enrichment analysis of the protein coding genes with population specific SNPs profiles revealed various biological processes, including iron acquisition, sterols synthesis and drug resistance. This study further highlights the complex links between L. tropica important genomic heterogeneity and the parasite broad geographic distribution. Unique sequence features in protein coding genes identified in distinct populations reveal potential novel markers that could be exploited for the development of more accurate typing schemes to further improve our knowledge of the evolution and epidemiology of the parasite as well as highlighting protein variants of potential functional importance underlying L. tropica specific biology., Competing Interests: The authors have declared that no competing interests exist.

Background: Cutaneous leishmaniasis (CL) remains a prioritized neglected tropical disease. CL novel presentations call for updating its features., Methods: A multiregional cohort of 396 patients with confirmed CL was reviewed. Lesion's clinical stage and eruption type were assigned. Disease was considered as extensive if numerous (≥5), large (>3 cm), disfiguring, threatening vital sensory organs, and/or older than 12 months. Microscopically, Ackerman's inflammatory pattern, Ridley's pattern (RP), and parasitic index (PI) were recorded. Microscopic variables pertaining to the organisms, epidermis, and host's inflammatory response were also assessed. All cases were confirmed and speciated molecularly., Results: In our region, 71.8% of cases showed extensive disease with 15.7% exceeding 12 months duration. Leishmania tropica accounted for 91.3% of cases while Leishmania major constituted 8.7% and presented solely as dry lesions. The dominant inflammatory composite consisted of plasma cells, lymphocytes, and histiocytes. Granulomatous inflammation was present in 55.5%. Most cases showed interface changes (72.7%), spongiosis (75.3%), and marked epidermal hyperplasia (63.9%). Transepidermal elimination of organisms was present in 29.2% of cases. None of traditional classification patterns (clinical stage, microscopic pattern, and RP) showed the predicted linear correlation with lesion age. High and low PI levels correlated with early and healing microscopic patterns, respectively, but did not correlate with the corresponding RPs. PI was bimodal with peaks at 3-6 and 9-12 months., Conclusion: Cutaneous leishmaniasis is an evolving disease defying the traditional prediction classifications. Our study sets the ground for adopting updated clinical courses, microscopic presentation, and species mapping., (© 2019 The International Society of Dermatology.)

Cutaneous Leishmaniasis (CL) is a parasitic infection classified by the WHO as one of the most uncontrolled spreading neglected diseases. Syria is endemic for Leishmania tropica and Leishmania major, causing CL in the Eastern Mediterranean. The large-scale displacement of Syrian refugees exacerbated the spread of CL into neighboring countries. Therapeutic interventions against CL include local, systemic and physical treatments. The high risk for drug-resistance to current treatments stresses the need for new therapies. Imiquimod is an immunomodulatory drug with a tested efficacy against L. major species. Yet, Imiquimod efficacy against L. tropica and the molecular mechanisms dictating its potency are still underexplored. In this study, we characterized the effect of Imiquimod against L. tropica and L. major, and characterized the molecular mechanisms dictating its anti-leishmanial efficacy against both strains. We also investigated the potency and molecular mechanisms of an Imiquimod analog, EAPB0503, against these two strains. We have tested the effect of Imiquimod and EAPB0503 on macrophages infected with either L. major, L. tropica strains, or patient-derived freshly isolated L. tropica parasites. The anti-amastigote activity of either drugs was assessed by quantitative real time PCR (RT-PCR) using kinetoplast specific primers, confocal microscopy using the Glycoprotein 63 (Gp63) Leishmania amastigote antibody or by histology staining. The mechanism of action of either drugs on the canonical nuclear factor kappa- B (NF-κB) pathway was determined by western blot, and confocal microscopy. The immune production of cytokines upon treatment of infected macrophages with either drugs was assessed by ELISA. Both drugs reduced amastigote replication. EAPB0503 proved more potent, particularly on the wild type L. tropica amastigotes. Toll-Like Receptor-7 was upregulated, mainly by Imiquimod, and to a lesser extent by EAPB0503. Both drugs activated the NF-κB canonical pathway triggering an immune response and i-NOS upregulation in infected macrophages. Our findings establish Imiquimod as a strong candidate for treating L. tropica and show the higher potency of its analog EAPB0503 against CL., Competing Interests: The authors of this study have consented to its submission. The authors have declared that no competing interests exist.

Visceral leishmaniasis, a fatal disease if not treated, is caused by Leishmania parasites. This disease might be overlooked in the Middle East because of limited awareness and low incidence. We report 5 patients who died of visceral leishmaniasis in Lebanon and make recommendations to improve faster diagnosis and treatment.