Antileishmanial natural products as potential inhibitors of the Leishmania pteridine reductase: insights from molecular docking and molecular dynamics simulations.

Although many natural product-derived compounds possess anti-leishmanial activities in vitro and in vivo, their molecular targets in the Leishmania parasite remain elusive. This is a major challenge in optimizing these compounds into leads. The Leishmania pteridine reductase (PTR1) is peculiar for folate and pterin metabolism and has been validated as a drug target. In this study, 17 compounds with anti-leishmanial activities were screened against Leishmania major PTR1 (LmPTR1) using molecular docking and molecular dynamics (MD) simulations. All ligands were bound in the active site pocket of LmPTR1 with binding affinities ranging from -11.2 to -5.2 kcal/mol. Agnuside, betulin, betulinic acid, gerberinol, ismailin, oleanolic acid, pristimerin, and ursolic acid demonstrated binding affinities similar to a known inhibitor, methyl 1-(4-{[2,4-diaminopteridin-6-yl) methyl] amino} benzoyl) piperidine-4-carboxylate (DVP). MD simulations revealed that betulin, betulinic acid, ismailin, oleanolic acid, pristimerin, and ursolic acid formed stable complexes with LmPTR1. The binding free energies of the complexes were very good (-87 to -148 kJ/mol), and much higher than the complex of the standard DVP inhibitor and LmPTR1 (-27 kJ/mol). Betulin, betulinic acid, ismailin, oleanolic acid, pristimerin, and ursolic acid likely exert their antileishmanial action by inhibiting PTR1 and could thus be used as a basis for the development of potential antileishmanial chemotherapeutic agents. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00247-8.

Serosurvey of canine leishmaniasis in five departments near an identified human clinical case in Marseille (France).

Leishmania infantum is a protozoan parasite of the family Trypanosomatidae, transmitted by the bite of phlebotomine sand flies (Diptera: Psychodidae). It is responsible for human and canine leishmaniasis in countries bordering the Mediterranean basin. Here we describe a clinical case of human cutaneous leishmaniasis in a 76-year-old female patient living in Marseille. Upon interrogation, the patient had no history of recent travel or contact with animals. The study involved clinical, serological, and molecular investigation of the occurrence of Leishmania infection in 718 dogs from five departments within a 130 km radius perimeter around Marseille. Five dogs showed signs of moderate leishmaniasis. Additionally, the serological survey of dogs revealed a global seroprevalence of 5.1%, with a significantly different prevalence in the Vaucluse department. Molecular analysis and phylogenetic studies highlighted the close relatedness of Leishmania strains between human and canine hosts with 99.6% of identity, indicating local transmission. The findings underscore the importance of serological surveillance in dogs and human. In a One Health approach, it is necessary to continue investigating Leishmania infection in all potential reservoirs, especially for zoonotic L. infantum in wildlife (red foxes, leporids, rodents, etc.) but also in dogs, cats, and equids.

CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

Contribution of imaging in the diagnosis of three neglected diseases in the Southern Cone: Leishmaniasis, Dengue, and Chikungunya.

Neglected diseases such as leishmaniasis, dengue, and chikungunya pose significant challenges to public health due to their high prevalence and wide geographic distribution in the Southern Cone region. These diseases are transmitted through insect bites, which serve as natural reservoirs. While their imaging findings are not always conclusive, they can play a crucial role in the diagnosis and monitoring. This review provides a concise overview of the clinical manifestations, epidemiological context, and imaging findings associated with these diseases. The primary purpose of this article is to share our experience and offer valuable insights into the use of imaging for the diagnosis and monitoring of patients suspected to have these diseases.

Oral Leishmaniasis in HIV-Positive and HIV-Negative Patients: A Comparative Analysis with Two New Case Reports.

PURPOSE: This case report examines the clinical presentation, diagnosis, treatment, and outcomes of mucocutaneous leishmaniasis with primary oral involvement in HIV-positive and HIV-negative patients diagnosed in Brazil. METHODS: We discuss the clinical manifestations, diagnostic methods, and therapeutic strategies, highlighting the clinical and histopathologic diagnostic features and distinct progression patterns based on HIV status. Our findings are compared with patterns observed in other countries, emphasizing the differences between the Americas and Europe, Asia, and Africa. RESULTS: In the Americas, particularly in Brazil, mucocutaneous leishmaniasis often presents with localized oral lesions, even in the presence of systemic immunosuppression, whereas in the Europe, Asia, and Africa, oral involvement is typically associated with visceral leishmaniasis in immunocompromised patients. These differences were due to variations in the parasite species involved. CONCLUSION: This comparison underscores the importance of regional and immunological factors in diagnosing and managing this neglected infectious disease.

Microemulsions strongly promoted the activity of α-bisabolol against different Leishmania species and its skin permeation.

This study aimed to develop microemulsions (MEs) containing α-bisabolol for the topical treatment of cutaneous leishmaniasis (CL). Initially, pseudoternary phase diagrams were developed using α-bisabolol as the oil phase, Eumulgin® CO 40 as the surfactant, Polymol® HE as the co-surfactant, and distilled water as the aqueous phase. Two transparent liquid systems (TLS) containing 5% of α-bisabolol were selected and characterized (F5E25 and F5EP25). Next, skin permeation and retention assays were performed using Franz cells. The interaction of the formulation with the stratum corneum (SC) was evaluated using the FTIR technique. The cytotoxicity was evaluated in murine peritoneal macrophages. Finally, the antileishmanial activity of microemulsions was determined in promastigotes and amastigotes of L. amazonensis (strain MHOM/BR/77/LTB 0016). As a result, the selected formulations showed isotropy, nanometric size (below 25 nm), Newtonian behavior and pH ranging from 6.5 to 6.9. The MEs achieved a 2.5-fold increase in the flux and skin-permeated amount of α-bisabolol. ATR-FTIR results showed that microemulsions promoted fluidization and extraction of lipids and proteins of the stratum corneum, increasing the diffusion coefficient and partition coefficient of the drug in the skin. Additionally, F5E25 and F5EP25 showed higher activity against promastigotes (IC50 13.27 and 18.29, respectively) compared to unencapsulated α-bisabolol (IC50 53.8). Furthermore, F5E25 and F5EP25 also showed antileishmanial activity against intracellular amastigotes of L. amazonensis, with IC50 50 times lower than free α-bisabolol and high selectivity index (up to 15). Therefore, the systems obtained are favorable to topical administration, with significant antileishmanial activity against L. amazonensis promastigotes and amastigotes, being a promising system for future in vivo trials.

Nicotinamide mitigates visceral leishmaniasis by regulating inflammatory response and enhancing lipid metabolism.

BACKGROUND: Currently, treatment regimens for visceral leishmaniasis (VL) are limited because of the presence of numerous adverse effects. Nicotinamide, a readily available and cost-effective vitamin, has been widely acknowledged for its safety profile. Several studies have demonstrated the anti-leishmanial effects of nicotinamide in vitro. However, the potential role of nicotinamide in Leishmania infection in vivo remains elusive. METHODS: In this study, we assessed the efficacy of nicotinamide as a therapeutic intervention for VL caused by Leishmania infantum in an experimental mouse model and investigated its underlying molecular mechanisms. The potential molecular mechanism was explored through cytokine analysis, examination of spleen lymphocyte subsets, liver RNA-seq analysis, and pathway validation. RESULTS: Compared to the infection group, the group treated with nicotinamide demonstrated significant amelioration of hepatosplenomegaly and recovery from liver pathological damage. The NAM group exhibited parasite reduction rates of 79.7% in the liver and 86.7% in the spleen, respectively. Nicotinamide treatment significantly reduced the activation of excessive immune response in infected mice, thereby mitigating hepatosplenomegaly and injury. Furthermore, nicotinamide treatment enhanced fatty acid ß-oxidation by upregulating key enzymes to maintain lipid homeostasis. CONCLUSIONS: Our findings provide initial evidence supporting the safety and therapeutic efficacy of nicotinamide in the treatment of Leishmania infection in BALB/c mice, suggesting its potential as a viable drug for VL.

Targeting Leishmania Promastigotes and Amastigotes Forms through Amino Acids and Peptides: A Promising Therapeutic Strategy.

Millions of people worldwide are affected by leishmaniasis, caused by the Leishmania parasite. Effective treatment is challenging due to the biological complexity of the parasite, drug toxicity, and increasing resistance to conventional drugs. To combat this disease, the development of specific strategies to target and selectively eliminate the parasite is crucial. This Review highlights the importance of amino acids in the developmental stages of Leishmania as a factor determining whether the infection progresses or is suppressed. It also explores the use of peptides as alternatives in parasite control and the development of novel targeted treatments. While these strategies show promise for more effective and targeted treatment, further studies to address the remaining challenges are imperative.

Thiourea-functionalized aminoglutethimide derivatives as anti-leishmanial agents.

Aim: We aim to develop new anti-leishmanial agents against Leishmania major and Leishmania tropica. Materials & methods: A total of 23 thiourea derivatives of (±)-aminoglutethimide were synthesized and evaluated for in vitro activity against promastigotes of L. major and L. tropica. Results & conclusion: The N-benzoyl analogue 7p was found potent (IC50 = 12.7 µM) against L. major and non toxic to normal cells. The docking studies, indicates that these inhibitors may target folate and glycolytic pathways of the parasite. The N-hexyl compound 7v was found strongly active against both species, and lacked cytotoxicity against normal cells, whereas compound 7r, with a 3,5-bis-(tri-fluoro-methyl)phenyl unit, was active against Leishmania, but was cytotoxic in nature. Compound 7v was thus identified as a hit for further studies.

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The impact of cholesteryl ester transfer protein on the progression of cutaneous leishmaniasis.

Introduction: Pathogenesis of cutaneous leishmaniases involves parasite growth, persistent inflammation, and likely participation of lipoproteins (LP). The cholesteryl ester transfer protein (CETP), involved in LP remodeling, has been shown to participate in the inflammatory response and the evolution of infectious conditions. Methods: We evaluated the impact of the presence of CETP on infection by Leishmania (L.) amazonensis in an experimental model of cutaneous leishmaniasis using C57BL6/J mice transgenic for human CETP (CETP), having as control their littermates that do not express the protein, wild-type (WT) mice. The progression of the lesion after infection in the footpad was monitored for 12 weeks. Two groups of animals were formed to collect the plantar pad in the 4th and 12th week post-infection. Results: The lesion increased from the 3rd week onwards, in both groups, with a gradual decrease from the 10th week onwards in the CETP group compared to the WT group, showing a reduction in parasitism and an improvement in the healing process, a reduction in CD68+ cells, and an increase in CD163+ and CD206, characterizing a population of M2 macrophages. A reduction in ARG1+ cells and an increase in INOS+ cells were observed. During infection, the LP profile showed an increase in triglycerides in the VLDL fraction in the CETP group at 12 weeks. Gene expression revealed a decrease in the CD36 receptor in the CETP group at 12 weeks, correlating with healing and parasite reduction. In vitro, macrophages derived from bone marrow cells from CETP mice showed lower parasite load at 48 h and, a reduction in arginase activity at 4 h accompanied by increased NO production at 4 and 24 h compared to WT macrophages, corroborating the in vivo findings. Discussion: The data indicate that the presence of CETP plays an important role in resolving Leishmania (L.) amazonensis infection, reducing parasitism, and modulating the inflammatory response in controlling infection and tissue repair.

rPagSP02+rPagSP06 recombinant salivary antigen is a reliable biomarker for evaluating exposure to Phlebotomus argentipes in Sri Lanka.

Phlebotomus argentipes is the established vector of leishmaniasis in the Indian sub-continent. Antibodies to sand fly salivary antigens are biomarkers for vector-host exposure in leishmaniasis-endemic regions. Ph. argentipes transmits Leishmania donovani in Sri Lanka, primarily causing cutaneous leishmaniasis (CL). Our study compared the performance of salivary gland homogenate (SGH) from a lab-reared local strain of Ph. argentipes females to a composite recombinant salivary biomarker (rPagSP02 + rPagSP06) in a CL-endemic population. Sera from 546 healthy individuals, 30 CL patients, and 15 non-endemic individuals were collected. Western blot analysis of Ph. argentipes SGH identified immunogenic bands between 15 kDa and 67 kDa, with bands of predicted molecular weight õf 15 kDa (SP02) and ~28-30 kDa (SP06) as the major antibody targets. Indirect ELISAs using SGH or rPagSP02 + rPagSP06 antigens showed high sensitivity (96.7%) and specificity (100%), detecting comparable seropositivity in endemic populations. rPagSP02 + rPagSP06 exhibited enhanced discriminatory ability, supported by a strong positive correlation (r = 0.869) with SGH. Our findings indicate that the composite rPagSP02 + rPagSP06 salivary biomarker effectively identifies Ph. argentipes exposure in individuals living in Sri Lanka, showing promising potential for use in surveillance. These findings should be further validated to confirm the epidemiological applications in leishmaniasis-endemic regions.

Epidemiological Characteristics and Spatiotemporal Patterns of Visceral Leishmaniasis in Xinjiang, China, during 2004-2021.

The spread of visceral leishmaniasis (VL), a serious global zoonotic parasitic disease, is mostly under control; however, several cases have been reported in recent decades in Xinjiang, China. This study aimed to analyze the epidemiological status and spatiotemporal clustering characteristics of VL in Xinjiang, China, between 2004 and 2021 to provide a basis for the development and implementation of surveillance and response measures. Data on VL incidence during 2004-2021 were collected from the National Diseases Reporting Information System of China. Global spatial autocorrelation analysis, identification of local indicators of spatial association, and spatial-temporal clustering analysis were conducted to identify the distribution and high-risk areas. A total of 2034 VL cases were reported, with a mean annual incidence of 0.50 per 100,000. There was a general decreasing trend in the incidence of VL during our study period. The majority of the cases were reported from October to February of the following year, and fewer cases were reported from April to July. Spatial autocorrelation analysis revealed that the incidence of VL was spatially clustered within a few counties. Significant differences were observed during the study period (Moran's I = 0.74, Z = 4.900, p < 0.05). The male-to-female ratio was 1.37:1, and most patients were in the age group 0-3 years. Cases were primarily distributed in seven regions and two autonomous prefectures, and Kashgar reported the highest number of cases (1688, 82.98%). Spatial analysis revealed that the aggregation of VL was predominantly observed in southwest Xinjiang. This was in alignment with the high-risk areas identified by spatiotemporal clustering analysis. The H-H clustering region was primarily observed in Gashi, Atushi, Shufu, Injisha, Kashgar, Yepuhu, and Bachu. These findings indicate that integrated control measures must be taken in different endemic areas to strengthen the VL control program in Xinjiang, China.

Prevalence of Leishmaniasis among Blood Donors: A Systematic Review and Meta-Analysis.

Our study seeks to provide a comprehensive assessment of leishmaniasis prevalence among blood donors, employing rigorous methodologies to inform public health initiatives and transfusion safety measures. A thorough literature search was conducted using electronic databases (Medline, Scopus, Web of Science, and Google Scholar) to identify the relevant studies reporting the prevalence of leishmaniasis among blood donors, gathering a wide range of studies encompassing different geographic locations and time periods. The pooled prevalence with a 95% confidence interval (CI) was estimated, and quality assessment, outlier analysis, and influential analysis were performed to ensure the robustness and validity of the findings. Our search and subsequent analyses led to the inclusion of thirty-five studies in our review. Using molecular diagnostic methods, the prevalence was estimated at 2.3% (95% CI 1-3.9%), while serological diagnostic methods indicated a higher prevalence rate of 4.5% (95% CI 2.8-6.7%). Notably, we observed significant heterogeneity among the included studies for each analysis. The observed heterogeneity highlights the need for future research to delve into the factors influencing leishmaniasis prevalence, with prospective and retrospective studies addressing the limitations identified in this review.

Epidemiology and Genetic Characterization of Leishmania RNA Virus in Leishmania (Viannia) spp. Isolates from Cutaneous Leishmaniasis Endemic Areas in Panama.

Leishmania (Viannia) spp. can harbor a double-stranded RNA virus known as Leishmania RNA virus 1 (LRV-1), whose presence has been reported in nine countries across the Americas and seven Leishmania species. Here, we studied 100 Leishmania (Viannia) isolates from patients with cutaneous leishmaniasis collected from different endemic areas in Panama from 2016 to 2022. We identified L. (V.) panamensis, L. (V.) guyanensis, L. (V.) braziliensis/guyanensis hybrid, and L. (V.) panamensis sp.1. (genetic variant). LRV-1 was detected by RT-PCR in 9% of L. (Viannia) isolates (eight cases in L. (V.) panamensis, and one in L. (V.) guyanensis). Phylogenetic analysis based on sequencing data classified all LRV-1 isolates within genotype A, suggesting that LRV phylogenetic proximity is closely aligned with geographical distribution or to the phylogenetic proximity of the Leishmania host in the case of the L. (V.) panamensis and L. (V.) guyanensis in Panama.

Serological Screening and Risk Factors Associated with Leishmania infantum Positivity in Newly Diagnosed HIV Patients in Greece.

A serological screening was conducted to detect IgG antibodies against Leishmania infantum (L. infantum) in newly diagnosed human immunodeficiency virus (HIV) patients in Greece. The study also examined potential risk factors and the agreement of commercially available serological methods. IgG antibodies against L. infantum were detected using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence antibody test (IFAT), and Western blot (WB). Out of 155 samples, 14 (9.0%) tested positive for IgG antibodies against L. infantum using at least two methods. Statistical analysis showed substantial agreement between WB and IFAT methods (Cohen's kappa = 0.75) but moderate overall agreement among the three methods (Fleiss' kappa = 0.42). Additionally, HIV+ intravenous drug users faced 3.55 times (p = 0.025) higher risk of testing positive for L. infantum IgG, positing that anthroponotic transmission between these patients is a plausible hypothesis based on existing literature. Non-invasive and cost-effective techniques are preferred to detect asymptomatic infections, and leishmaniasis screening should be conducted immediately after HIV diagnosis in endemic regions to enable prophylactic treatment for leishmaniasis in addition to antiretroviral therapy. To maximize sensitivity, performing at least two different serological methods for each patient is recommended.

A Second Wind for Inorganic APIs: Leishmanicidal and Antileukemic Activity of Hydrated Bismuth Oxide Nanoparticles.

American cutaneous leishmaniasis is a disease caused by protozoa of the genus Leishmania. Currently, meglumine antimoniate is the first-choice treatment for the disease. The limited efficacy and high toxicity of the drug results in the necessity to search for new active principles. Nanotechnology is gaining importance in the field, since it can provide better efficacy and lower toxicity of the drugs. The present study aimed to synthesize, characterize, and evaluate the in vitro leishmanicidal and antileukemic activity of bismuth nanoparticles (BiNPs). Promastigotes and amastigotes of L. (V.) guyanensis and L. (L.) amazonensis were exposed to BiNPs. The efficacy of the nanoparticles was determined by measurement of the parasite viability and the percentage of infected cells, while the cytotoxicity was characterized by the colorimetry. BiNPs did not induce cytotoxicity in murine peritoneal macrophages and showed better efficacy in inhibiting promastigotes (IC50 < 0.46 nM) and amastigotes of L. (L.) amazonensis. This is the first report on the leishmanicidal activity of Bi-based materials against L. (V.) guayanensis. BiNPs demonstrated significant cytotoxic activity against K562 and HL60 cells at all evaluated concentrations. While the nanoparticles also showed some cytotoxicity towards non-cancerous Vero cells, the effect was much lower compared to that on cancer cells. Treatment with BiNPs also had a significant effect on inhibiting and reducing colony formation in HL60 cells. These results indicate that bismuth nanoparticles have the potential for an inhibitory effect on the clonal expansion of cancer cells.

Synthesis of Nitrostyrylthiazolidine-2,4-dione Derivatives Displaying Antileishmanial Potential.

A series of 61 thiazolidine-2,4-diones bearing a styryl group at position 5 was synthesized in 2-5 steps and their structure was proved by elemental and spectral analyses. The compounds obtained were evaluated in vitro against the promastigote stage of the kinetoplastid parasite Leishmania infantum and the human HepG2 cell line, to determine selectivity indices and to compare their activities with those of antileishmanial reference drugs. The study of structure-activity relationships indicated the potential of some derivatives bearing a nitro group on the phenyl ring, especially when located at the meta position. Thus, among the tested series, compound 14c appeared as a hit compound with good antileishmanial activity (EC50 = 7 µM) and low cytotoxicity against both the hepatic HepG2 and macrophage THP-1 human cell lines (CC50 = 101 and 121 µM, respectively), leading to good selectivity indices (respectively, 14 and 17), in comparison with the reference antileishmanial drug compound miltefosine (EC50 = 3.3 µM, CC50 = 85 and 30 µM, SI = 26 and 9). Regarding its mechanism of action, among several possibilities, it was demonstrated that compound 14c is a prodrug bioactivated, predominantly by L. donovani nitroreductase 1, likely leading to the formation of cytotoxic metabolites that form covalent adducts in the parasite. Finally, compound 14c is lipophilic (measured CHI LogD7.7 = 2.85) but remains soluble in water (measured PBS solubility at pH7.4 = 16 µM), highlighting the antileishmanial potential of the nitrostyrylthiazolidine-2,4-dione scaffold.

Deciphering the intricate dynamics of inflammasome regulation in visceral and post-kala-azar dermal leishmaniasis: A meta-analysis of consistencies.

Post Kala-azar dermal leishmaniasis (PKDL) arises as a significant dermal sequel following Visceral leishmaniasis (VL) caused by protozoan parasite Leishmania donovani (LD). PKDL acts as a significant constrain for VL elimination serving as a crucial reservoir for LD. PKDL patients exhibit depigmented macular and papular lesions on their skin, which results in social discrimination due to loss of natural skin color. Inflammatory reactions, prevalent in both VL and PKDL, potentially lead to tissue damage in areas harboring the parasite. Disruption of the immune-inflammasomal network not only facilitates LD persistence but also leads to the skin hypopigmentation seen in PKDL, impacting social well-being. Activation of inflammasomal markers like STAT1, NLRP1, NLRP3, AIM2, CASP11, and NLRP12 have been identified as a common host-defense mechanism across various Leishmania infections. Conversely, Leishmania modulates inflammasome activation to sustain its presence within the host. Nevertheless, in specific instances of Leishmania infection, inflammasome activation can worsen disease pathology by promoting parasite proliferation and persistence. This study encompasses recent transcriptomic analyses conducted between 2016 and 2023 on human and murine subjects afflicted with VL/PKDL, elucidating significant alterations in inflammasomal markers in both conditions. It offers a comprehensive understanding how these markers contribute in disease progression, drawing upon available literature for logical analysis. Furthermore, our analysis identifies validated miRNA network that could potentially disrupt this crucial immune-inflammasomal network, thereby offering a plausible explanation on how secreted LD-factors could enable membrane-bound LD, isolated from the host cytoplasm, to modulate cytoplasmic inflammasomal markers. Insights from this study could guide the development of host-directed therapeutics to impede transmission and address hypopigmentation, thereby mitigating the social stigma associated with PKDL.