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To evaluate the efficacy of a synthetic sex-aggregation pheromone of the sand fly vector Lu. longipalpis, co-located with residual insecticide, to reduce the infection incidence of Leishmania infantum in the canine reservoir. A stratified cluster randomised trial was designed to detect a 50% reduction in canine incident infection after 24 months in 42 recruited clusters, randomly assigned to one of three intervention arms (14 cluster each): synthetic pheromone + insecticide, insecticide-impregnated dog collars, or placebo control. Infection incidence was measured by seroconversion to anti-Leishmania serum antibody, Leishmania parasite detection and canine tissue parasite loads. Changes in relative Lu. longipalpis abundance within households were measured by setting three CDC light traps per household. A total 1,454 seronegative dogs were followed-up for a median 15.2 (95% C.I.s: 14.6, 16.2) months per cluster. The pheromone + insecticide intervention provided 13% (95% C.I. 0%, 44.0%) protection against anti-Leishmania antibody seroconversion, 52% (95% C.I. 6.2%, 74·9%) against parasite infection, reduced tissue parasite loads by 53% (95% C.I. 5.4%, 76.7%), and reduced household female sand fly abundance by 49% (95% C.I. 8.2%, 71.3%). Variation in the efficacy against seroconversion varied between trial strata. Equivalent protection attributed to the impregnated-collars were 36% (95% C.I. 14.4%, 51.8%), 23% (95% C.I. 0%, 57·5%), 48% (95% C.I. 0%, 73.4%) and 43% (95% C.I. 0%, 67.9%), respectively. Comparison of the two interventions showed no statistically consistent differences in their efficacies; however, the errors were broad for all outcomes. Reductions in sand fly numbers were predominant where insecticide was located (chicken and dog sleeping sites), with no evidence of insecticide-induced repellence onto humans or dogs. The synthetic pheromone co-located with insecticide provides protection particularly against canine L. infantum parasite transmission and sand fly vector abundance. The effect estimates are not dissimilar to those of the insecticide-impregnated collars, which are documented to reduce canine infection incidence, human infection and clinical VL disease incidence, in different global regions. The trialled novel lure-and-kill approach is a low-cost potential vector control tool against ZVL in the Americas.

Background Dogs are the reservoir host of Leishmania infantum , the agent of zoonotic visceral leishmaniasis (VL), which is transmitted by the bite of phlebotomine sand flies. The sand fly Phlebotomus perniciosus is the main vector of zoonotic VL in the western Mediterranean region. Fluralaner has been shown to effectively kill this vector. The aim of this study was to evaluate the insecticidal efficacy of oral fluralaner in dogs bitten by P . perniciosus . Methods Two parallel-group, negative-controlled, randomized, masked laboratory trials with equivalent designs were performed in two different locations using two different pathogen-free laboratory-bred P. perniciosus strains for the challenge. In each trial, 12 purpose-bred beagles, initially ranked on natural attractiveness to sand flies, were randomly allocated to two groups (6 animals/group). Dogs in one group received fluralaner orally at the approved dose on day 0, and dogs in the control group were not treated. Each dog was subsequently exposed to an average of 70 unfed live sand fly females on days 1, 28, 56 and 84. Viability of blood-fed females was then evaluated for up to 96 h after exposure, and insecticidal efficacy was measured as the survival rate of flies fed on the fluralaner-treated dogs versus that of dogs in the control group. Significance was calculated for the proportion of live fed sand fly counts from treated versus control group dogs. Results Comparison of the survival proportions between treated and control groups showed that fluralaner insecticidal efficacy was highly significant in both trials ( P  < 0.001 or P  < 0.01 in different assessments) through to day 56. In the first trial, efficacy reached 100% on days 1 and 28, and 99.1% on day 56; in the second trial, the insecticidal efficacy was 98.5, 100 and 85.9%, respectively on the same days. On day 84, efficacy was in the range of 53–57% ( P  < 0.05) in the first trial and 0% in the second trial. Conclusion A single oral fluralaner administration to dogs under laboratory conditions results in strong and reproducible insecticidal efficacy against P. perniciosus for at least 8 weeks. Graphical Abstract

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi. Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the β5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the β4 and β5 proteasome subunits. This induced pocket exploits β4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Deltamethrin-impregnated dog collars reduce sandfly bite rates on dogs, and are effective in killing sandflies that attempt to feed. Because domestic dogs are the principal reservoir hosts of zoonotic visceral leishmaniasis, we tested whether community-wide application of dog collars could protect children against infection with Leishmania infantum, the parasite that causes the disease. 18 villages were paired, matched by preintervention child prevalence of L infantum infection. Within pairs, villages were randomly assigned to either control or intervention. All domestic dogs in intervention villages were provided with collars for the transmission season. The main outcome measure was incidence of L infantum infection after 1 year measured by seroconversion. Secondary outcomes were leishmanin skin test (LST) conversion and seroconversion in dogs. The seroconversion rate in children was 1.49% (17/1141) in the intervention villages and 2.41% (26/1078) in control villages (odds ratio 0.57, 95% CI 0.36-0.90, p=0.017). LST conversion was also lowered, but not significantly (odds ratio 0.66, 0.41-1.08, p=0.096). The seroconversion rate in dogs in intervention villages was also significantly reduced (0.46, 0.30-0.70, p=0.0003). Community-wide application of deltamethrin-impregnated dog collars not only protects domestic dogs from L infantum infections, but might also reduce the risk of L infantum infection in children. These dog collars could have a role in control of visceral leishmaniasis and replace controversial dog culling programmes in some countries. However, the effectiveness of dog collars will depend on the importance of wild versus domestic canids as reservoir hosts of L infantum.

Leishmaniasis is a Neglected Tropical Disease caused by protozoa of the genus. No human vaccine is available, and current treatments are limited by toxicity and drug resistance. Thus, the identification of new active molecules remains an urgent priority. However, drug discovery is severely hampered by the lack of fast, robust, and high-throughput screening assays, particularly for the clinically relevant intracellular amastigote stage. Here, a transgenic line stably co-expressing the bioluminescent marker PpyRE9H fused to the fluorescent reporter tdTomato was generated and used to develop a novel dual-reporter microplate-based platform to identify compounds active against promastigotes or intracellular parasite stages. The engineered strain exhibited comparable growth and infectivity to the wild-type strain, validating its suitability for drug screening assays. The activity of reference drugs was evaluated, and results were compared to those obtained using standard methods, such as MTT assay for promastigotes and Giemsa staining with microscopic quantification for amastigotes. Comparable IC values were obtained using fluorescent/bioluminescent assays and conventional methods. These proposed assays provide a sensitive and reproducible alternative to conventional methods. The microplate format enables higher throughput screening, while the dual bioluminescence and fluorescence readouts enable cross-validation of results and reduce the risk of compound-specific signal interference.

Dual-specificity protein phosphatases (DUSPs) are key modulators of mitogen-activated protein kinase (MAPK) signaling pathways that regulate cellular proliferation, differentiation, and stress adaptation. Although extensively characterized in higher eukaryotes, the functional roles of DUSPs in Leishmania remain poorly understood. In this study, we investigated the function of an atypical dual-specificity phosphatase/kinatase (LINF_340027100) in Leishmania infantum using CRISPR/Cas9-mediated genome editing. Repeated attempts to generate DUSP -null mutants were unsuccessful, and episomal complementation prior to chromosomal disruption did not permit deletion of the endogenous locus, suggesting that DUSP is essential for promastigote viability. To investigate DUSP function, we generated parasite lines with altered gene expression, including a heterozygous knockout ( Li DUSP +/− ) and an episomal overexpressor ( Li WT + pIR1_SAT-DUSP). Modulation of DUSP expression resulted in pronounced alterations in parasite morphology, cell-cycle progression, differentiation, and intracellular proliferation in macrophages. The Li DUSP +/− line exhibited a twofold reduction in transcript abundance and maintained normal growth kinetics, but displayed elongated morphology, increased flagellar length, and a reduced G0/G1 population with concomitant S-phase accumulation. In addition, Li DUSP +/− parasites showed decreased tolerance to hydrogen peroxide, being 1.5- and 5.7-fold more sensitive in promastigote and intracellular amastigote forms, respectively. Conversely, the DUSP -overexpressing line exhibited a 2.3-fold increase in DUSP transcript levels, accompanied by modest impairment of promastigote proliferation, rounded morphology, altered cell-cycle distribution, and increased resistance to trivalent antimony, with 2.6- and 3.1-fold higher resistance observed in promastigote and intracellular amastigote forms, respectively. Both mutant lines showed reduced intracellular proliferation at 72 h post-infection. Transcript level analysis revealed altered expression of MAPK1 , MAPK3 , and MAPK10 in both promastigote and axenic amastigote-like forms, supporting a functional link between DUSP activity and MAPK signaling homeostasis. Together, these findings identify DUSP as a key regulator of parasite biology that integrates MAPK signaling with oxidative stress response, differentiation, and antimony susceptibility in L. infantum . This study provides the first functional characterization of this atypical DUSP/kinatase in Leishmania and highlights its potential as a target for therapeutic intervention.

During the last decade miltefosine (MIL) has been used as first-line treatment for visceral leishmaniasis in endemic areas with antimonial resistance, but a decline in clinical effectiveness is now being reported. While only two MIL-resistant Leishmania infantum strains from HIV co-infected patients have been documented, phenotypic MIL-resistance for L. donovani has not yet been identified in the laboratory. Hence, a better understanding of the factors contributing to increased MIL-treatment failure is necessary. Given the paucity of defined MIL-resistant L. donovani clinical isolates, this study used an experimental amastigote-selected MIL-resistant L. infantum isolate (LEM3323). In-depth exploration of the MIL-resistant phenotype was performed by coupling genomic with phenotypic data to gain insight into gene function and the mutant phenotype. A naturally MIL-resistant L. infantum clinical isolate (LEM5159) was included to compare both datasets. Phenotypically, resistance was evaluated by determining intracellular amastigote susceptibility in vitro and actual MIL-uptake. Genomic analysis provided supportive evidence that the resistance selection model on intracellular amastigotes can be a good proxy for the in vivo field situation since both resistant strains showed mutations in the same inward transporter system responsible for the acquired MIL-resistant phenotype. In line with previous literature findings in promastigotes, our data confirm a defective import machinery through inactivation of the LiMT/LiRos3 protein complex as the main mechanism for MIL-resistance also in intracellular amastigotes. Whole genome sequencing analysis of LEM3323 revealed a 2 base pair deletion in the LiMT gene that led to the formation an early stop codon and a truncation of the LiMT protein. Interestingly, LEM5159 revealed mutations in both the LiMT and LiRos3 genes, resulting in an aberrant expression of the LiMT protein. To verify that these mutations were indeed accountable for the acquired resistance, transfection experiments were performed to re-establish MIL-susceptibility. In LEM3323, susceptibility was restored upon expression of a LiMT wild-type gene, whereas the MIL-susceptibility of LEM5159 could be reversed after expression of the LiRos3 wild-type gene. The aberrant expression profile of the LiMT protein could be restored upon rescue of the LiRos3 gene both in the LEM5159 clinical isolate and a ΔLiRos3 strain, showing that expression of LdMT is dependent on LdRos3 expression. The present findings clearly corroborate the pivotal role of the LiMT/LiRos3 complex in resistance towards MIL.

Background Drug-resistant strains of Leishmania infantum challenge the effectiveness of treatments for clinical leishmaniosis and may lead to more frequent relapses. Copy number variation (CNV) at specific genetic loci is associated with drug resistance and virulence, but information about its prevalence in endemic regions is limited. This study examines the drug resistance and virulence status of Leishmania strains in human and canine isolates from the Mediterranean region. Methods Forty-eight Leishmania infantum isolates were whole-genome sequenced with nanopore long reads, followed by de novo assembly. We analyzed chromosomal aneuploidies and gene copy number variation in loci linked to drug resistance and virulence in Leishmania , alongside the genomic structure and rearrangements responsible for these variations. Results Complete genomes were de novo assembled for 35 L. infantum isolates (22 from dogs and 13 from humans), revealing significant chromosomal variability. We assessed copy number variation for 22 potential biomarkers: 15 genes related to drug resistance to first-line drugs (METK for allopurinol; LdSMT for amphotericin B; AQP1 and H-locus for antimonials; LdMT, LdRos3, and MSL for miltefosine; and PPM for paramomycin) and 7 genes related to virulence (lipophosphoglycan and proteophosphoglycan biosynthesis, and the Lack protein). Drug-resistance biomarkers were identified in 80% of the isolates. Canine strains primarily showed resistance to allopurinol and antimonials, while human isolates exhibited a broader resistance spectrum, especially to antimonials and paromomycin. The co-occurrence of resistance biomarkers was common, especially for allopurinol and antimonial resistance. Distinct mechanisms underlie the observed copy number variations. Virulence-associated genes were less variable among isolates. Conclusions The prevalence of drug-resistance biomarkers in Leishmania infantum strains from the Mediterranean region, as revealed by this study, underscores the critical need for routine resistance surveillance in managing clinical leishmaniosis. These findings not only inform current clinical practice but also pave the way for more effective management strategies in the future. Graphical Abstract

Leishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum , unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania . This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.

This article reviews essential topics of canine visceral leishmaniasis (CVL) due to Leishmania infantum infection. It focuses on the current serological and molecular diagnostic methods used in epidemiological research and veterinary clinics to diagnose CVL and includes new point-of-care (POC) tests under development. The efficacy of different treatment regimens on the clinical improvement and infectiousness of dogs is also addressed. In the last section, the review provides a critical appraisal of the effectiveness of different control measures that have been implemented to curb disease transmission.