Combination therapy with amphotericin B and Ultradiluted Leishmania donovani antigen skews cytokine balance towards protective immunity in visceral leishmaniasis.

Differentiation between leishmaniasis and histoplasmosis remains a significant diagnostic conundrum, especially in immunosuppressed patients, due to overlapping clinical presentations and histopathological features. We report a case of a 53-year-old Thai man with advanced HIV disease from Southern Thailand who presented in 2020 with a chronic palate ulcer and hard palate perforation, initially diagnosed as histoplasmosis and treated with itraconazole for a year. Three years later (2023), the patient developed systemic symptoms, and biopsies confirmed visceral leishmaniasis. Given the increasing number of autochthonous leishmaniasis cases, the patient's original diagnosis from 2020 was reexamined. Retrospective analysis of the original palate biopsy revealed amastigotes with kinetoplasts, whereas PAS and GMS staining were negative for fungi, excluding histoplasmosis. Molecular diagnostics, which combined PCR for Leishmania spp. and Histoplasma capsulatum with Sanger sequencing, confirmed Leishmania martiniquensis and ruled out Histoplasma capsulatum, leading to a corrected diagnosis of mucocutaneous leishmaniasis. This report documents the second case of mucocutaneous leishmaniasis caused by L. martiniquensis in Thailand, with primary mucocutaneous presentation that progressed to visceral involvement. This highlights diagnostic challenges due to morphological similarities between Leishmania amastigotes and Histoplasma capsulatum yeasts, emphasizing the need for integrative histopathological (including specific staining techniques) and molecular diagnostics for accurate diagnosis and effective treatment.

The Editor and Publisher of the Journal of the Nigerian Society of Physical Sciences hereby issue this formal retraction notice for the article titled “Fractional-order modeling of visceral leishmaniasis disease transmission dynamics: strategies in eastern Sudan” by Ghada A. Ahmed, published in Journal of the Nigerian Society of Physical Sciences, Volume 5, 2023, Article ID 1693, DOI: https://doi.org/10.46481/jnsps.2023.1693. The article has been retracted due to errors in the calculations reported in the manuscript. Since the calculations form the basis of the fractional-order mathematical modelling, disease transmission analysis, and resulting conclusions, the identified errors affect the reliability of the findings presented in the article. The retraction decision had previously been reflected on the article page; however, a separate formal retraction notice was not published at that time. This notice is therefore being published now to correct that omission, improve transparency, and ensure that the retraction is properly visible to readers, indexing services, and metadata platforms. The original article should be regarded as retracted and should not be cited as reliable scientific evidence except in discussions relating to the retraction itself. Retracted article: Ghada A. Ahmed, “Fractional-order modeling of visceral leishmaniasis disease transmission dynamics: strategies in eastern Sudan,” Journal of the Nigerian Society of Physical Sciences 5 (2023) 1693. https://doi.org/10.46481/jnsps.2023.1693 Retraction decision date: 06-12-2023Retraction notice publication date: 26-04-2026

Colonic involvement in visceral leishmaniasis (VL) is exceptionally rare. We report a case of a 39-year-old man who presented with anorexia, weight loss, and diarrhea. Imaging revealed hepatosplenomegaly and extensive retroperitoneal lymphadenopathy, while laboratory tests showed pancytopenia and renal dysfunction. He was newly diagnosed with HIV (CD4⁺ count: 3 cells/μL), and VL was confirmed by identification of amastigotes and positive PCR in bone marrow aspirate. During hospitalization, he developed severe hematochezia. Colonoscopy revealed a pseudo-tumoral, ulcerated, and stricturing lesion in the ascending colon; histology confirmed colonic leishmaniasis. The patient progressed to hemorrhagic shock, requiring urgent right hemicolectomy. This case underscores an uncommon and fulminant presentation of VL in the setting of profound immunosuppression, highlighting the importance of considering parasitic infections in the differential diagnosis of gastrointestinal bleeding in HIV-positive individuals.

Global control strategies for vector-borne diseases (VBDs) remain largely anchored in the detection and management of symptomatic cases, despite substantial evidence that a considerable proportion of infections-often exceeding 60% in several major arboviral diseases-are asymptomatic or minimally symptomatic. These asymptomatic human reservoirs can sustain low-level pathogen circulation during interepidemic periods and may amplify transmission once ecological, immunological, or entomological thresholds are crossed. Because vector-dependent transmission is shaped by environmental variability, vector density, and host immunity dynamics, reliance on symptom-based surveillance alone risks delayed outbreak detection and missed opportunities to prevent severe health outcomes. Evidence from dengue, Zika, West Nile virus, Japanese encephalitis, chikungunya, and Chandipura virus, as well as parasitic diseases such as malaria and visceral leishmaniasis, illustrates how silent human infections complicate surveillance and undermine outbreak anticipation. We propose the establishment of Enhanced Vector-Borne Disease Vigilance Teams (EVBD-VTs) that would serve as mobile, multidisciplinary units integrated with laboratory networks and sentinel systems to strengthen early detection of asymptomatic infections with epidemic potential. Anchored within a One Health framework and supported by laboratory infrastructure, climate-informed risk assessment, and targeted active case detection, this model seeks to shift VBD control from reactive containment to anticipatory preparedness in an era of ecological change.

Visceral leishmaniasis elimination in Ethiopia: insights into epidemiology, transmission, treatment access, and outcome - a systematic review and meta-analysis.

Visceral leishmaniasis caused by Leishmania infantum remains a lethal disease with few therapeutic options, necessitating innovative computational methods and approaches to accelerate drug discovery. Here, we present a graph neural network (GNN) framework incorporating well-established multiscale mechanisms to improve the identification of novel antileishmanial compounds. Across two classificatory antileishmanial data sets, our GNNs demonstrated significant improvements in predictive performance, with area under the receiver operating characteristic curve (AUC) increases of 2.2-29.2% on the imbalanced data set (activity cutoff: 1 μM) and 3.4-22.5% on the balanced data set (activity cutoff: 10 μM) compared to default GNNs. Subsequently, the framework was applied to screen a library of approximately 1.3 million compounds, pinpointing LC-61 as a potent antileishmanial agent with nanomolar activity against intracellular L. infantum (IC50 = 0.076 μM) and minimal cytotoxicity to macrophages (THP-1 CC50 = 157 μM). A comprehensive in vitro ADME profiling revealed that LC-61 combines high solubility at both acidic and physiological pH (>28 μg/mL), balanced lipophilicity (eLogD = 4.07), and favorable passive permeability (PAMPA = 4.86 × 10-6 cm/s), while exhibiting lower microsomal stability. Overall, our GNN framework effectively accelerated the discovery of LC-61, a novel and biologically validated hit suitable for hit-to-lead optimization.

Robust T helper 1 (Th1) cell responses, which activate macrophages to kill intracellular parasites, are required to control Leishmania infection. Yet, visceral leishmaniasis (VL) patients do not control the infection despite the expansion of CD4+ T cells and increased IFN-γ expression in the spleen. Chemokines and/or chemokine receptors are involved in cellular migration and are critical in the inflammatory response. In a recent study that defined a transcriptional signature for CD4+ T cells from active VL patients, we found several differentially expressed chemokine receptor genes in CD4+ T cells compared to healthy endemic controls (EC). Since CD4+ T cells play crucial roles in parasite clearance, a better understanding of the role of altered chemokine receptor expression on CD4+ T cells and their different subsets during VL could inform future treatment strategies. In this study, we examined the gene expression and surface protein expression of differentially expressed chemokine receptors found in human VL subjects, relative to endemic controls (EC) by real-time qPCR and multicolor flow cytometry, respectively. We measured chemokine levels in plasma by enzyme-linked immunosorbent assay (ELISA) and performed transwell migration assays and flow cytometry to measure the migratory potential of CD4+ T cell subsets. We found elevated mRNA and surface protein expression of CCR5, while reduced CCR4 and CCR6 expression in CD4+ T cells from VL patients compared to EC. The frequency of CCR5 expressing Th1 cells was increased in peripheral blood, indicating the expansion of CCR5+ Th1 cells that may be responsible for Th1 cells trafficking towards infected tissues. Lower CCR4 expression was found on regulatory T (Treg) cells and central memory T (Tcm) cells, possibly explaining the reduced frequencies of these cells in peripheral blood during VL. The frequency of CCR6 expressing CD4+ T cells was also found to be lower in VL patients. Our results show that VL patients possess unique chemokine receptor expression patterns on the cell surface of CD4+ T cells, compared to EC. We also found increased levels of CCL3, CCL5 and CCL20 in VL plasma compared to EC. However, no changes were observed for CCL17 levels in VL plasma compared to EC, but their levels increased following treatment. We also observed reduced migration of VL CD4+ T cells, relative to other lymphocytes and mononuclear cells, irrespective of exogenous CCL5 presence. However, the frequency of CD4+ T cells migrating in response to CCL5 in EC individuals was similar. Additionally, we noted the frequency of CCR5+ Th1 cells was increased in VL patients compared to EC. However, no enhancement was seen in the migratory capacity of CCR5+ CD4+ T cells in the presence of recombinant CCL5. This suggests that the CCR5 receptor signalling pathway is less responsive towards exogenous CCL5, potentially due to high levels of CCL3 and CCL5 present in VL plasma, which may saturate surface CCR5. The upregulation of CCR5 expression and downregulation of CCR4 and CCR6 by CD4+ T cells distinguished VL patients from healthy individuals. These findings provide new insights into VL pathogenesis and could direct the development of new and improved disease diagnostics and therapeutics for treatment of VL.

Visceral Leishmaniasis (VL), a severe systemic neglected tropical disease (NTD) caused by Leishmania donovani complex protozoa, poses a significant public health threat, particularly in East Africa, where it is fatal if untreated. Somalia is known to be endemic, but the true burden and programmatic challenges are poorly documented due to long-standing conflict, population displacement, and a fragmented, resource-constrained health system. We report the case of a 40-year-old male farmer from Borama, Awdal region (Somaliland), presenting with a 20-day history of intermittent high-grade fever, chills, progressive weakness, anorexia, and vomiting. Physical examination revealed marked hepatosplenomegaly and pallor. Laboratory investigations showed moderate anemia (Hemoglobin 9.4 g/dL), thrombocytopenia (114,000/μL), and a highly elevated ESR (50 mm/h). Mild hyperbilirubinemia was noted, likely secondary to hemolysis. Tests for malaria, tuberculosis, and enteric fever were negative. Definitive parasitological confirmation was not feasible due to local resource limitations. A rapid diagnostic test (RDT) for VL based on rK39 antigen-Kalazar Detect (Human) Rapid Test-was positive. Ultrasound confirmed significant hepato-splenomegaly. Based on the clinical presentation, positive rK39 RDT, and exclusion of other etiologies, a diagnosis of Visceral Leishmaniasis was established. The patient was treated according to WHO regional guidelines with Sodium Stibogluconate (SSG, 20 mg/kg/day) and Paromomycin (PM, 15 mg/kg/day) for 17 days. The patient tolerated treatment well; the fever resolved within five days. At one-week post-therapy follow-up, he showed significant clinical improvement. This case highlights the classic presentation of VL in a fragile setting and underscores the critical role of RDTs where parasitological confirmation is unavailable. It demonstrates the feasibility of the WHO-recommended SSG+PM combination therapy despite systemic challenges.

Identification, selection and testing of new antigens for tegumentary Leishmaniasis serological diagnosis.

Leishmaniasis is a neglected tropical disease caused by infection with the protozoan parasite Leishmania and it is a significant global health problem. The disease has a wide clinical spectrum, from tegumentary leishmaniasis (TL) that encompasses cutaneous (CL), mucosal (ML) and cutaneous-diffuse (CDL) forms, to the potentially fatal systemic visceral leishmaniasis (VL). Neurological manifestations are not generally considered as classical clinical signs or symptoms of leishmaniasis, but in this review, we present evidence that this is a false assumption. We examine brain involvement in human and canine leishmaniasis and the contribution of animal models for studying cerebral Leishmania infection. Clinical descriptions of brain involvement are presented, and evidence of Leishmania invasion of the central nervous system. Notably, evidence for brain involvement comes from considerable studies in the dog and covers aspects of disruption of the blood-brain and blood-cerebrospinal fluid barriers and the nature of the inflammatory response.

Leishmaniasis elimination and control remain challenging due to the lack of successful treatment and possible prevention. A suitable anti-leishmanial medication that is acceptable in terms of price and safety is currently being researched. In light of this, the current study examined the effectiveness of imidazoquinoline based selective Toll-like receptor (TLR)-7 agonists as anti-leishmanial agents. A library of TLR7 agonists was evaluated for anti-leishmanial efficacy against experimental visceral leishmaniasis (VL). Among all the agonists, 2-butyl-1-(3,4-dimethoxybenzyl)-1H-imidazo[4,5-c]quinolin-4-amine (7) and its niosomal formulation 18 exhibited better activity than Miltefosine. These were then further studied against Leishmania donovani for their ability to arrest the parasites at sub G0/G1 phase of cell cycle. The selected TLR7 agonists depicted their ability to arrest higher percentage of parasites in comparison to the standard antileishmanial drugs. Similar observations were seen in case of reactive oxygen species (ROS) generation where parasites treated with the lead TLR7 agonist produced higher ROS than the untreated parasites. These results suggest that TLR7 agonist 7 and its niosomal formulation 18 could be the promising hits for development of a novel drug treating VL. However additional in-vivo studies could aid in their future advancement.

ABSTRACTZoonotic visceral leishmaniasis is caused by Leishmania (Leishmania) infantum. Dogs are considered the most critical urban reservoirs of L. (L.) infantum due to their high infection rate and direct transmission to humans. The parasite has developed mechanisms to evade the host's defence system by inhibiting macrophage activation, thereby allowing it to replicate and survive. Pathogens such as viruses and bacteria can modify the host's epigenome, thereby facilitating their survival. Cellular reprogramming leads to epigenetic alterations that modulate chromatin, modify histones and DNA methylation, disrupt normal progression and compromise the continuity of cell differentiation. Histone deacetylases (HDACs) remove lysine acetyl groups from histones, resulting in chromatin alteration and gene silencing. Histone acetyltransferases regulate their function. In this study, we analysed the involvement of HDAC‐1 in the defen mechanisms of DH82 macrophages infected with L. infantum. We observed that L. infantum infection increases HDAC1 levels and expression. Silencing HDAC1 with siRNA and the pharmacological inhibitor NaB decreased parasite load and increased iNOS expression, associated with increased histone acetylation at the iNOS promoter. The pharmacological inhibitor NaB also decreased IL‐6, IL‐10 and TNF‐α levels in the culture supernatant of DH82 macrophages infected with L. infantum. Together, these findings indicate that L. infantum‐induced HDAC1 upregulation modulates the expression of key innate immune response genes, contributing to the establishment of infection in canine macrophages.

Visceral leishmaniasis, caused by Leishmania donovani and transmitted by Phlebotomus argentipes, remains a major public health challenge in the Indian subcontinent. Sand fly populations are controlled by using different insecticides, particularly dichlorodiphenyltrichloroethane (DDT) and pyrethroids such as alpha-cypermethrin. Prolonged and irregular use of these insecticides raises concern about the development of resistance in sand fly populations. This study aimed to compare metabolic detoxification and knockdown resistance (kdr) mutations in Vgsc and ace-1 between nine sprayed (indoor residual spray, IRS) villages and one unsprayed (non-IRS) village in Muzaffarpur, Bihar, India. A total of 10 Ph.argentipes from each village were used for metabolic detoxification and 10-25 Ph.argentipes were used for target site insensitivity mutations. Homogenized Ph.argentipes aliquots were used for different enzymes activity and DNA was used for the sequence analysis of Vgsc and ace-1 genes to access the presence of kdr mutations. Biochemical assays revealed that levels of detoxification enzymes, including glutathione S-transferase (GST), esterase (PNPA), and cytochrome P450 monooxygenase, were elevated in IRS villages, suggesting localized metabolic resistance. Molecular screening of the voltage-gated sodium channel (Vgsc) gene revealed high frequencies of knockdown resistance (kdr) mutations at codon 1014, with serine (L1014S) (48.5%) being the most prevalent, followed by wild type leucine (L1014) (39.5%). No mutations were detected at codon 119 of the ace-1 gene, indicating the sensitivity to organophosphates in the sand fly population. The results suggested that continuous and repeated exposure to the synthetic pyrethroid may exert selective pressure, leading to early signs of resistance in Ph.argentipes, mediated through metabolic detoxification mechanisms and mutation in the kdr gene. These findings underscore the importance of ongoing resistance monitoring and the implementation of rotational insecticide strategies to support sustained efforts toward the elimination of visceral leishmaniasis.

Development of highly sensitive and specific diagnostic tests for cutaneous leishmaniasis (CL) remains a significant challenge. Localized immune response in dermal leishmaniasis results in low systemic antibody production. Consequently, serologic diagnostic platform should focus on detecting Leishmania antigens directly from the skin lesions. Designing multiepitope-based proteins as an alternative to crude antigens can enhance sensitivity and specificity, lower production costs, and facilitate development of more field-amenable diagnostic tools. A chimeric multiepitope protein construct (THAG) was designed from four Leishmania spp. genes (txnpx, gp63, hsp70, and amastin). Bioinformatic tools were employed to ensure that the construct was optimized for expression in Escherichia coli and had favorable physicochemical properties. The gene was synthesized, subcloned into the pET28a(+) vector, and expressed in E. coli BL21 (DE3). Protein purification was carried out using Ni-NTA affinity chromatography under hybrid conditions. The purified protein was used to generate polyclonal antibodies in rabbits, and the immunoreactivity of antiserum was assessed against Leishmania antigens from lesions of 54 volunteers with suspected active skin lesions mimic CL through an in-house ELISA. The THAG multiepitope recombinant protein of Leishmania has successfully designed, expressed, purified and confirmed in our study. The results demonstrated sensitivity, specificity and accuracy of the ELISA test in detection of Leishmania antigens in CL lesions to be 90.91% (75.67%-98.08%), 95.24% (76.18%-99.88%), and 92.59% (82.11%-97.94%), respectively. This multiepitope protein has the potential to be further evaluated as a diagnostic tool for CL patients. While we have evaluated this multiepitope protein in an ELISA format, it might be applied to other test formats especially in immunochromatographic assays for the rapid detection ofLeishmania antigens in skin lesions. Furthermore, it has the potential to be used as a tool to detect anti-Leishmania antibodies in serum samples of canine and human visceral leishmaniasis (VL) patients.

Phlebotomine sand flies (Diptera: Psychodidae) are the principal vectors of Leishmania spp., the causative agents of leishmaniasis. Since 2008, the Ministry of Health and the Ministry of Environmental Protection of Israel have conducted nationwide, periodic sand fly surveys. Initially, these surveys focused on localities with known endemic transmission of cutaneous leishmaniasis, but in recent years, entomological trapping has expanded to areas where prior information on sand flies was unavailable. Here we report the first confirmed occurrence of Phlebotomus (Larroussius) orientalis (Parrot, 1936) in Israel and place the Israeli material in a comparative phylogeographic context. Entomological surveys by CO2 trapping were conducted in the Negev Desert, southern Israel, between 2020 and 2024. Morphological sand fly identification was confirmed by sequencing fragments of the mitochondrial COI and Cytb/NADH1 genes. For a newly reported species, we inferred intraspecific phylogenetic relationships and divergence times between major clades. A subset of females was additionally screened for Leishmania DNA and vertebrate blood-meal sources by real-time polymerase chain reaction (PCR) coupled with high-resolution melt analysis. Targeted surveys and routine surveillance in the Negev region between 2020 and 2024 yielded 269 Phlebotomus orientalis (96 males, 173 females) among other species of local sand fly fauna from multiple wadi systems in the central Negev. These detections constitute the first confirmed records of Ph. orientalis in Israel. Species identification was confirmed through both morphological examination and molecular analyses of partial COI and Cytb/NADH1 genes. Phylogenetic analysis indicated that the Israeli Ph. orientalis specimens constitute a distinct lineage that diverged from East African conspecifics during the Early to Middle Pleistocene. Blood-meal analysis of engorged Ph. orientalis females identified the European hare as a vertebrate host, and none of the tested Ph. orientalis specimens were positive for Leishmania DNA. Phlebotomus orientalis is a confirmed vector of L. donovani, the main agent of visceral leishmaniasis in East Africa. Its detection as a distinct and apparently long-established lineage in the Negev, in a region where parasites of the L. donovani complex are already involved in cutaneous leishmaniasis transmission, highlights the need to clarify the distribution, ecology, and host preferences of Ph. orientalis in Israel. Further studies are required to characterize its spatial and seasonal occurrence, evaluate its vector competence for L. donovani and L. infantum, and assess its potential contribution to current and future leishmaniasis transmission risks.

MicroRNAs (miRNAs) regulate gene expression and play a crucial role in numerous diseases, including infections. Leishmaniasis is a neglected infectious disease occurring in different forms (i.e., cutaneous, mucocutaneous, and visceral) caused by a protozoan belonging to the Leishmania genus. The parasite infects mainly the macrophages, establishing a niche permissive for its proliferation. Leishmania parasites are known to modulate host gene expression, including miRNAs; however, the specific role of these miRNAs in driving host transcriptomic changes remains to be fully characterized. The aim of this work was to study miRNA expression profile in human macrophage-like cells infected by L. infantum, the causative agent of visceral and cutaneous leishmaniasis in the Mediterranean region. Moreover, we attempted to identify putative miRNA-mRNA interactions based on the mRNA expression changes previously described. To this end, small RNA-seq was performed in U937 cells infected with L. infantum after 24 h and 48 h, and differentially expressed miRNA were identified and validated through qPCR. For identifying miRNA-mRNA interactions, the upregulated and downregulated miRNAs at 24 h (n = 24, 10) and 48 h (n = 25, 12) post-infection were analyzed against downregulated and upregulated mRNA, respectively, through the mirDIP (microRNA Data Integration Portal) database. A large fraction of dysregulated protein-coding transcripts was predicted to be affected by dysregulated miRNAs identified in the same samples. We focused in particular on protein-coding genes involved in previously identified dysregulated pathways characterizing L. infantum infection (i.e., cholesterol and lipid metabolism, VEGF-VEGFR2 and NF2EL2-related pathways) and transcription factors (TFs). Notably, the fraction of protein coding transcripts predicted to be targeted by dysregulated miRNAs was particularly high in TFs, indicating that changes in a small set of miRNAs may have great impact on macrophage expression profile and phenotype.

Visceral leishmaniasis (VL), a potentially fatal disease caused by Leishmania donovani and transmitted by Phlebotomus argentipes sand flies, remains a public health issue in India, as evidenced by 385 cases reported in 2024. Despite low incidence, sporadic fluctuations necessitate vigilant vector control. Indoor residual spraying (IRS) is the principal strategy for controlling Ph. argentipes. After resistance to dichloro-diphenyl-trichloroethane (DDT) became widespread, alpha-cypermethrin IRS replaced it in 2016 for VL control in India. Although prior studies have assessed sand fly susceptibility to insecticides at WHO-recommended discriminatory concentrations, the present study is the first to simultaneously evaluate the residual efficacy of alpha-cypermethrin 5% wettable powder on common household substrates across three VL-endemic Indian states. From 2023 to 2024, three Indian Council of Medical Research institutes in Bihar, Jharkhand, and Uttar Pradesh assessed the susceptibility of wild Ph. argentipes to DDT (4.0%), malathion (5.0%), alpha-cypermethrin (0.05%, 0.1%), and deltamethrin (0.05%) using WHO tube tests. Residual efficacy of alpha-cypermethrin (25 mg/m2) was tested on cement concrete, limewashed cement, mud, and wood. Ph. argentipes were resistant to DDT but susceptible to malathion, deltamethrin, and alpha-cypermethrin. Residual efficacy of alpha-cypermethrin dropped below 80% 1-month post spray on cement and limewashed cement, and after 3 months on mud. Mortality remained 100% for 4 months on wood. These results support the use of one to two rounds of alpha-cypermethrin IRS to interrupt VL transmission in Indian villages, given wall substrate types and seasonal transmission patterns.

The aim for the present study was to analyze clinical features, diagnostic approaches, and therapeutic strategies for visceral leishmaniasis (VL)-associated hemophagocytic lymphohistiocytosis (HLH) in pediatric patients. The clinical characteristics and test results of the children were summarized. Among the four patients, three resided in VL-endemic regions, and one had traveled to a VL-endemic region. All patients presented with recurrent fever (>38.5°C), hepatosplenomegaly, and decreased hemoglobin (HGB) levels ([78.75 ± 8.50] g/L) and platelet (PLT) counts ([59.50 ± 17.48] × 109/L). Before a definitive diagnosis could be made, patients exhibited progressive declines in white blood cell counts, HGB levels, and PLT counts, along with elevated triglyceride, serum cytokine (interleukin [IL]-6, IL-10, IL-2R, and tumor necrosis factor α) levels. Bone marrow aspirate smears revealed hemophagocytosis and Leishmania donovani (LD) bodies in all cases: two were diagnosed via direct identification of LD bodies, one was diagnosed through re-examination of bone marrow smears after confirming a travel history, and one was diagnosed via re-examination prompted by metagenomic next-generation sequencing, which revealed leishmaniasis. All the patients were initially diagnosed with HLH and received HLH-directed immunochemotherapy before VL diagnosis, with suboptimal response. After confirmation of VL, sodium stibogluconate therapy was initiated, resulting in a partial response in all cases. Etiological investigation is critical for HLH diagnosis. For VL-associated HLH, sodium stibogluconate as targeted therapy rapidly controls HLH, facilitates immunosuppression withdrawal, and significantly improves patient outcomes. White blood cell count, HGB level, PLT count, and lactate dehydrogenase level may serve as critical prognostic biomarkers for VL-associated HLH.

Visceral leishmaniasis (VL) is a fatal parasitic disease for which safe and effective immunotherapeutic strategies remain limited. Modulation of innate immune signaling using small-molecule Toll-like receptor (TLR) agonists represents a promising approach to enhance host-directed antiparasitic responses. In this study, we investigated the immunomodulatory efficacy of a synthetic TLR7/8 agonist, HYBRID2, formulated with formalin-killed Leishmania donovani antigen (FK), in a BALB/c mouse model of VL. Immunized mice challenged with L. donovani exhibited a marked reduction in splenic parasite burden following FK + HYBRID2 administration. Mechanistic evaluation of splenocytes revealed enhanced redox activity, with significantly elevated reactive oxygen species and nitric oxide production, accompanied by up-regulation of inducible nitric oxide synthase and NF-κB expression. Cytokine analysis demonstrated a pronounced Th1-biased immune response, characterized by increased INF-γ and TNF-α levels. Comparative assessment showed that FK + HYBRID2 elicited superior immunostimulatory and antileishmanial effects relative to FKAlone or FK combined with Resiquimod. Collectively, these findings establish HYBRID2 as a potent innate immune modulator enhancing redox responses and NF-κB activation alongside Th1 polarization, highlighting its potential utility as an immunomodulatory adjuvant platform against intracellular parasitic infections.