Leishmaniases are neglected diseases caused by protozoans of the genus Leishmania that threaten millions of people worldwide. Cutaneous leishmaniasis (CL) caused by L. major is a typical zoonosis transmitted by phlebotomine sand flies and maintained in rodent reservoirs. The female sand fly was assumed to become infected by feeding on the skin lesion of the host, and the relative contribution of asymptomatic individuals to disease transmission was unknown. In this study, we infected 32 Meriones shawi, North African reservoirs, with a natural dose of L. major obtained from the gut of infected sand flies. Skin manifestations appeared in 90% of the animals, and xenodiagnosis with the proven vector Phlebotomus papatasi showed transmissibility in 67% of the rodents, and 45% were repeatedly infectious to sand flies. Notably, the analysis of 113 xenodiagnostic trials with 2189 sand flies showed no significant difference in the transmissibility of animals in the asymptomatic and symptomatic periods; asymptomatic animals were infectious several weeks before the appearance of skin lesions and several months after their healing. These results clearly confirm that skin lesions are not a prerequisite for vector infection in CL and that asymptomatic animals are an essential source of L. major infection. These data are important for modeling the epidemiology of CL caused by L. major.

Leishmania major is responsible for zoonotic cutaneous leishmaniasis. Therapy is mainly based on the use of antimony-based drugs; however, treatment failures and illness relapses were reported. Although studies were developed to understand mechanisms of drug resistance, the interactions of resistant parasites with their reservoir hosts and vectors remain poorly understood. Here we compared the development of two L. major MON-25 trivalent antimony-resistant lines, selected by a stepwise in vitro Sb(III)-drug pressure, to their wild-type parent line in the natural vector Phlebotomus papatasi. The intensity of infection, parasite location and morphological forms were compared by microscopy. Parasite growth curves and IC₅₀ values have been determined before and after the passage in Ph. papatasi. qPCR was used to assess the amplification rates of some antimony-resistance gene markers. In the digestive tract of sand flies, Sb(III)-resistant lines developed similar infection rates as the wild-type lines during the early-stage infections, but significant differences were observed during the late-stage of the infections. Thus, on day 7 p. i., resistant lines showed lower representation of heavy infections with colonization of the stomodeal valve and lower percentage of metacyclic promastigote forms in comparison to wild-type strains. Observed differences between both resistant lines suggest that the level of Sb(III)-resistance negatively correlates with the quality of the development in the vector. Nevertheless, both resistant lines developed mature infections with the presence of infective metacyclic forms in almost half of infected sandflies. The passage of parasites through the sand fly guts does not significantly influence their capacity to multiply in vitro. The IC₅₀ values and molecular analysis of antimony-resistance genes showed that the resistant phenotype of Sb(III)-resistant parasites is maintained after passage through the sand fly. Sb(III)-resistant lines of L. major MON-25 were able to produce mature infections in Ph. papatasi suggesting a possible circulation in the field using this vector.

Background: Drug development for leishmaniases treatment follows a very selective process in order to propose drug candidates that possess all the qualities that meet the strict specifications of the pharmaceutical industry. Drug resistance is a limiting factor that can impact the lifespan of a marketed drug. It is now essential that the risk of drug resistance be evaluated at the early stage of in vitro studies to discard a lead compound that could quickly generate drug resistance once available on the market. Objectives: This article aims to estimate the risk of drug resistance emergence for a promising drug candidate at the in vitro early stage of drug development. Methods: A sequential method is proposed to study some of the phenotypic characteristics and parameters of resistant parasites such as time to achieve maximal resistance during stepwise drug pressure, resistance amplitude, stability, fitness, and infectivity both in vitro and in vivo. Results: Some examples with drugs in clinical use and former drug candidates are given. Conclusions: This method providing an evaluation of the risk of drug resistance from an in vitro model of Leishmania donovani be extrapolated to other Leishmania species.

La leishmaniose cutanée zoonotique (LCZ) d’Algérie est une maladie négligée causée par le parasite Leishmania (L.) major MON-25 sauvage (WT), et transmise de l’animal à l’Homme par la piqûre d’un insecte vecteur compétent : Phlebotomus (Ph.) papatasi, lors d’un repas de sang pris sur le rongeur Meriones (M.) shawi, un réservoir naturel de la maladie. La LCZ constitue un problème majeur pour la santé publique de par l’augmentation alarmante du nombre d'isolats cliniques résistants de L. major apparus après des cas d’échecs thérapeutiques au Glucantime®. Le présent travail consiste à : 1) Mettre en place un élevage de mérions et de phlébotomes à partir d’individus capturés en zone d’endémie, 2) Sélectionner par pression médicamenteuse in vitro, des souches résistantes aux antimoniés trivalents (SbIII-R) et en étudier les caractéristiques phénotypiques in vitro et in vivo avant et après passage chez le réservoir et le vecteur. 3) Étudier les marqueurs moléculaires cibles de la chimiorésistance de Leishmania, et analyser leur variation sous la pression exercée par le changement d’hôtes. Les résultats de cette étude ont révélé que le rongeur M. shawi joue un rôle potentiellement mineur dans la transmission de parasites chimiorésistants, contrairement au vecteur compétent Ph. papatasi, qui véhicule des formes métacycliques infectantes chimiorésistantes. Par ailleurs, la sélection expérimentale in vitro, de parasites SbIII-résistants sans maintien de la résistance in vivo réduit le fitness et la virulence des parasites aussi bien chez le rongeur et le vecteur, qu’en culture in vitro. Enfin, sans maintien d’une pression médicamenteuse chez l’animal, il ne se produit aucune variation dans l’amplification des gènes des transporteurs membranaires, alors que la voie de détoxification semble être essentielle à la survie et à la persistance du parasite SbIII-R dans l’organisme de l’hôte. Ceci ouvre un grand champ de perspectives pour la compréhension de la chimiorésistance comme : i) L’étude de la réponse immunitaire associée à la chimiorésistance au SbIII chez le réservoir M. shawi, ii) L’analyse des gènes impliqués dans le développement des parasites chimiorésistants dans un cycle complet de L. major, iii) Le séquençage du génome entier de Leishmania WT et SbIII-R isolés après passage chez l’animal réseroir et après passage chez l'insecte vecteur.

Cutaneous leishmaniasis (CL), a neglected disease prevalent in 88 countries, is commonly caused by Leishmania (L.) major in the Old World, posing significant public health concerns. Isolation from sand flies or infected mammals and in vitro cultivation of Leishmania parasites are critical for epidemiological studies, but these cultures are often compromised by bacterial and fungal contamination, especially when outsourced from vector digestive tracts. While the Leishmania parasite's natural resistance to antibiotics simplifies bacterial control, most antifungals also inhibit the parasites growth, complicating efforts to manage fungal contamination. This study aimed to identify antifungal agents that could protect Leishmania cultures from yeast contamination with minimal impact on parasite growth. Five antifungal drugs: griseofulvin (GRF), caspofungin diacetate (CSF), 5-fluorocytosine (5-FC), po(ly)vidone-iodine (PVI), and undecylenic acid (UCA) were assessed in vitro for their effects on promastigote forms of L. major. The IC₅₀ values indicated strong antileishmanial activity for 5-FC, GRF, UCA, and PVI (<13 µM), whereas CSF exhibited higher IC₅₀ value (17 µM), suggesting relatively lower toxicity to the parasites. Under continuous CSF exposure, L. major promastigotes demonstrated substantial survival, with only a modest reduction in maximum parasite growth curves (peak of 2.65 × 10⁶ parasites/mL) compared to untreated controls (4.31 × 10⁶ parasites/mL maximum growth). These findings suggest that caspofungin diacetate could be used in the field to decontaminate Leishmania cultures from yeasts without significant parasite loss, facilitating L. major isolation and epidemiological investigations in endemic regions.