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With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings. [ABSTRACT FROM AUTHOR]

• Two thousand (n = 2000) consecutive healthy voluntary blood donors were tested for 18 s rRNA by Real time Polymerase chain reaction. • One thousand (n = 1000) subject's permanent resident of Azad Kashmir along with a thousand (n = 1000) healthy voluntary blood donors from rest of Pakistan were included. • No donor was diagnosed as an asymptomatic carrier. • Screening of blood donors for Leismania Donovan's is not recommended even in endemic areas such as Pakistan and Azad Kashmir. • Future research on healthy donors with follow-up is necessary in order to investigate persistence of Leismania in asymptomatic blood donors for longer than a year following exposure. To check incidence of Asymptomatic Leishmania donovani reporting to Armed Forces Institute of Transfusion Rawalpindi. Two thousand (n = 2000) consecutive healthy voluntary blood donors were tested for 18 s rRNA by Real time Polymerase chain reaction. One thousand (n = 1000) subject's permanent resident of Azad Kashmir along with a thousand (n = 1000) healthy voluntary blood donors from rest of Pakistan were included. The study was carried out over a period of three months Jun – Aug 2020. Total of 2000 blood donors were enrolled in the study, all were males with age ranging from 16 to 60 years. Stratification based on residence, 1000 (50 %) resided in the Azad kashmir, 349 (17.45 %) were from Islamabad and Rawalpindi, 541 (27.05 %) from Punjab mainly residing in Lahore and Multan, 110 (5.5 %) were from other cities of Pakistan. Grouping on the basis of age, 55.25 % (n = 1105) of the donors were 16–25 years old, 19.45 % (n = 389) were in age range of 26–40 years old, 15.55 % (n = 311) were 41–50 years old and 9.75 % (n = 195) 51–60 years old. No donor was diagnosed as an asymptomatic carrier. Screening of blood donors for Leishmania donovani is not recommended. [ABSTRACT FROM AUTHOR]

Cutaneous leishmaniasis caused by Leishmania major or L. tropica and visceral leishmaniasis caused by L. infantum have been reported in Israel. We collected Phlebotomus spp. sand flies in the Negev desert of southern Israel to identify circulating Leishmania spp. Of 22,636 trapped sand flies, 80% were P. alexandri. We sequenced Leishmania-specific internal transcribed spacer 1 fragments and K26 genes. Of 5,019 Phlebotomus female sand flies, 2.5% were Leishmania DNA–positive; 92% of infections were L. donovani. Phylogenetic analyses showed separate clustering of L. donovani and L. infantum. P. alexandri flies positive for L. donovani harbored blood meals from European hares. Leishmania DNA isolated from a patient with cutaneous leishmaniasis who lived in the survey area was identical to L. donovani from P. alexandri flies. We report circulation of L. donovani, a cause of visceral leishmaniasis, in southern Israel. Prompt diagnosis and Leishmania spp. identification are critical to prevent leishmaniasis progression. [ABSTRACT FROM AUTHOR]

ABSTRACT Polyclonal B cell activation and the resulting hypergammaglobulinemia are a detrimental consequence of visceral leishmaniasis (VL); however, the mechanisms underlying this excessive production of nonprotective antibodies are still poorly understood. Here, we show that a causative agent of VL, Leishmania donovani, induces CD21-dependent formation of tunneling nanotubule (TNT)-like protrusions in B cells. These intercellular connections are used by the parasite to disseminate among cells and propagate B cell activation, and close contact both among the cells and between B cells and parasites is required to achieve this activation. Direct contact between cells and parasites is also observed in vivo, as L. donovani can be detected in the splenic B cell area as early as 14 days postinfection. Interestingly, Leishmania parasites can also glide from macrophages to B cells via TNT-like protrusions. Taken together, our results suggest that, during in vivo infection, B cells may acquire L. donovani from macrophages via TNT-like protrusions, and these connections are subsequently exploited by the parasite to disseminate among B cells, thus propagating B cell activation and ultimately leading to polyclonal B cell activation. IMPORTANCE Leishmania donovani is a causative agent of visceral leishmaniasis, a potentially lethal disease characterized by strong B cell activation and the subsequent excessive production of nonprotective antibodies, which are known to worsen the disease. How Leishmania activates B cells is still unknown, particularly because this parasite mostly resides inside macrophages and would not have access to B cells during infection. In this study, we describe for the first time how the protozoan parasite Leishmania donovani induces and exploits the formation of protrusions that connect B lymphocytes with each other or with macrophages and glides on these structures from one cell to another. In this way, B cells can acquire Leishmania from macrophages and become activated upon contact with the parasites. This activation will then lead to antibody production. These findings provide an explanation for how the parasite may propagate B cell activation during infection.

Visceral leishmaniasis (VL) is an infectious disease caused by parasitic protozoa of the genus Leishmania and manifests clinical symptoms such as splenomegaly, hepatomegaly, anemia, and fever. It has previously been shown that B-cell-activating factor (BAFF) is involved in splenomegaly during VL. Although BAFF is known to be expressed by a variety of cells, the mechanism of elevated BAFF expression in VL is not clear. In this study, we aimed to identify BAFF-producing cells in the spleens of mice infected with Leishmania donovani. Splenocytes of L. donovani-infected mice showed elevated BAFF expression compared to that of naive mice. In the infected spleen, the number of both CD11b+ and F4/80+ cells increased, and the major BAFF-producing cells were CD11b+ cells, which did not serve as host cells of Leishmania. Immunohistochemical/immunofluorescent staining of spleens of infected mice revealed that the increased CD11b+ cells were primarily MRP14+ mononuclear cells. Together, these results suggest the increased BAFF expression in the spleen of L. donovani-infected mice involves a recruitment of inflammatory macrophages distinct from host macrophages for the parasites.

A study was carried out to compare the infection rates of Leishmania donovani in Phlebotomus orientalis sandflies at different microhabitats of a VL endemic village in Gedarif state, Sudan. DNA extracts of 1078 P. orientalis sand fly females sampled by CDC light traps from indoor, outdoor, peri-domestic, and sylvatic sites, in three transmission seasons, March–June 2016–18, in Helat-Belo village, were subjected to independent PCR amplifications targeting Leishmania kDNA and the cpb gene followed by ITS1 region sequencing. Leishmania kDNA was detected in 1.4% of the 1078 P. orientalis females captured in the area. Two of these specimens showed a characteristic 741 bp band of L. donovani after cpb gene amplification. The DNA sequence of the ITS1 region of the parasites matched the ITS1 L. donovani genotype F. There were no signficant differences between rates of infection of L. donovani in P. orientalis captured at different sites. Blood meals found in infected flies origninated from human (5 specimens), cattle (4 specimens) and donkey (2 specimens). The finding of fresh cow and donkey blood in the infected flies suggests the possible role of these animals in the zoopotentiation and/or zooprophylaxis against VL. The study provides important information for VL transmission models and control programs in East Africa.

Leishmaniasis, a neglected tropical disease, is on the decline in South Asia. However, cases of cutaneous leishmaniasis have risen in Sri Lanka since 2001, and the lack of in-depth research on its epidemiologic characteristics hampers control efforts. We analyzed data collected from patients with cutaneous leishmaniasis in Sri Lanka during 2001-2018 to study temporal and geographic trends and identify and monitor disease hotspots. We noted a progression in case rates, including a sharp rise in 2018, showing temporal expansion of disease-prevalent areas and 2 persistent hotspots. The northern hotspot shifted and shrank over time, but the southern hotspot progressively expanded and remained spatially static. In addition, we noted regional incidence differences for age and sex. We provide evidence of temporally progressive and spatially expanding incidence of leishmaniasis in Sri Lanka with distinct geographic patterns and disease hotspots, signaling an urgent need for effective disease control interventions.

To know the effect of the Capparies silver nanoparticles (AgNPs) synthesis from the leaves of the Capparis spinosa plant on the liver enzymes GOT, GPT and the histology of the liver in infected mice with visceral leishmaniasis (VL), they were dosed with 0.1 mL day-1 and pentostam drug (0.01 mL day-1) for 21 days. The results showed that the silver nanoparticles of the Capparies spinosa, led to a significant decrease in liver enzymes, i.e., GOT and GPT after three weeks, compared to the positive control group. Also, it was noted that the liver tissue was less affected with a slight expansion of the hepatic sinusoids and a decrease in lymphocyte infiltration.

Abstract Background Visceral Leishmaniasis (VL) is a fatal vector-borne parasitic disorder occurring mainly in tropical and subtropical regions. VL falls under the category of neglected tropical diseases with growing drug resistance and lacking a licensed vaccine. Conventional vaccine synthesis techniques are often very laborious and challenging. With the advancement of bioinformatics and its application in immunology, it is now more convenient to design multi-epitope vaccines comprising predicted immuno-dominant epitopes of multiple antigenic proteins. We have chosen four antigenic proteins of Leishmania donovani and identified their T-cell and B-cell epitopes, utilizing those for in-silico chimeric vaccine designing. The various physicochemical characteristics of the vaccine have been explored and the tertiary structure of the chimeric construct is predicted to perform docking studies and molecular dynamics simulations. Results The vaccine construct is generated by joining the epitopes with specific linkers. The predicted tertiary structure of the vaccine has been found to be valid and docking studies reveal the construct shows a high affinity towards the TLR-4 receptor. Population coverage analysis shows the vaccine can be effective on the majority of the world population. In-silico immune simulation studies confirms the vaccine to raise a pro-inflammatory response with the proliferation of activated T and B cells. In-silico codon optimization and cloning of the vaccine nucleic acid sequence have also been achieved in the pET28a vector. Conclusion The above bioinformatics data support that the construct may act as a potential vaccine. Further wet lab synthesis of the vaccine and in vivo works has to be undertaken in animal model to confirm vaccine potency.

Drug repurposing has emerged as an effective strategy against infectious diseases such as visceral leishmaniasis. Here, we evaluated four FDA‐approved drugs–valrubicin, ciclesonide, deflazacort, and telithromycin—for their anti‐leishmanial activity on Leishmania donovani parasites, especially their ability to target the enzyme glutathione synthetase (LdGS), which enables parasite survival under oxidative stress in host macrophages. Valrubicin and ciclesonide exhibited superior inhibitory effects compared to deflazacort and telithromycin, inhibiting the promastigotes at very low concentrations, with IC50 values of 1.09 ± 0.09 μm and 2.09 ± 0.09 μm, respectively. Subsequent testing on amastigotes revealed the IC50 values of 1.74 ± 0.05 μm and 3.32 ± 0.21 μm for valrubicin and ciclesonide, respectively. Molecular and cellular level analysis further elucidated the mechanisms underlying the anti‐leishmanial activity of valrubicin and ciclesonide. [ABSTRACT FROM AUTHOR]

Here we report a new polyhydroxylated triterpene, 2β,6β,21α-trihydroxyfriedelan-3-one (4) isolated from the root and stem bark of Dichapetalum albidum A. Chev (Dichapetalaceae), along with six known triterpenoids (1–3, 5, 6, 8), sitosterol-3β-O-D-glucopyranoside (9), a dipeptide (7), and a tyramine derivative of coumaric acid (10). Friedelan-3-one (2) showed an antimicrobial activity (IC50) of 11.40 μg/mL against Bacillus cereus, while friedelan-3α-ol (1) gave an IC50 of 13.07 μg/mL against Staphylococcus aureus with ampicillin reference standard of 19.52 μg/mL and 0.30 μg/mL respectively. 3β-Acetyl tormentic acid (5) showed an IC50 of 12.50 μg/mL against Trypanosoma brucei brucei and sitosterol-3β-O-d-glucopyranoside (9) showed an IC50 of 5.06 μg/mL against Leishmania donovani with respective reference standards of IC50 5.02 μg/mL for suramin and IC50 0.27 μg/mL for amphotericin B. Molecular docking of the isolated compounds on the enzyme glucose-6-phosphate dehydrogenase (G6PDH) suggested 3β-acetyl tormentic acid (5) and sitosterol-3β-O-D-glucopyranoside (9) as plausible inhibitors of the enzyme in accordance with the experimental biological results observed.

ABSTRACT To initiate an antileishmanial adaptive immune response, dendritic cells (DCs) must carry Leishmania antigens from peripheral tissues to local draining lymph nodes. However, the migratory capacity of DCs is largely compromised during Leishmania donovani infection. The molecular mechanism underlying this defective DC migration is not yet fully understood. Here, we demonstrate that L. donovani infection impaired the lymph node homing ability of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression. L. donovani exerted this inhibitory effect by inducing transforming growth factor-β (TGF-β) secretion from DCs. Indeed, TGF-β produced in this manner inhibited nuclear factor-κB (NF-κB)-mediated CLEC-2 expression on DCs by activating c-Src. Notably, suppression of c-Src expression significantly improved the arrival of DCs in draining lymph nodes by preventing L. donovani-induced CLEC-2 downregulation on DCs. These findings reveal a unique mechanism by which L. donovani inhibits DC migration to lymph nodes and suggest a key role for TGF-β, c-Src, and CLEC-2 in regulating this process. IMPORTANCE Dendritic cells (DCs) play a key role in initiating T cell-mediated protective immunity against visceral leishmaniasis (VL), the second most lethal parasitic disease in the world. However, the T cell-inducing ability of DCs critically depends on the extent of DC migration to regional lymph nodes. Notably, the migration of DCs is reported to be impaired during VL. The cause of this impaired DC migration, however, remains ill-defined. Here, we provide the first evidence that L. donovani, the causative agent of VL, attenuates the lymph node homing capacity of DCs by decreasing C-type lectin receptor 2 (CLEC-2) expression on DCs. Additionally, we have demonstrated how L. donovani mediates this inhibitory effect. Overall, our work has revealed a unique mechanism underlying L. donovani-induced impairment of DC migration and suggests a potential strategy to improve antileishmanial T cell activity by increasing DC arrival in lymph nodes.

BackgroundVisceral leishmaniasis (VL) is a neglected vector-borne tropical disease caused by Leishmania donovani (L. donovani) and Leishmania infantum (L. infantum). Due to the very small dimensions of the protozoa impounded within blood cells and reticuloendothelial structure, diagnosing VL remains challenging.Case presentationHerein, we reported a case of VL in a 17-month-old boy with acute lymphoblastic leukemia (ALL). The patient was admitted to West China Second University Hospital, Sichuan University, due to repeated fever after chemotherapy. After admission, chemotherapy-related bone marrow suppression and infection were suspected based on clinical symptoms and laboratory test results. However, there was no growth in the conventional peripheral blood culture, and the patient was unresponsive to routine antibiotics. Metagenomics next-generation sequencing (mNGS) of peripheral blood identified 196123 L. donovani reads, followed by Leishmania spp amastigotes using cytomorphology examination of the bone marrow specimen. The patient was given pentavalent antimonials as parasite-resistant therapy for 10 days. After the initial treatment, 356 L. donovani reads were still found in peripheral blood by mNGS. Subsequently, the anti-leishmanial drug amphotericin B was administrated as rescue therapy, and the patient was discharged after a clinical cure.ConclusionOur results indicated that leishmaniasis still exists in China. Unbiased mNGS provided a clinically actionable diagnosis of a specific infectious disease from an uncommon pathogen that eluded conventional testing.

Background: Ethiopia is one of the top 10 countries in the world where 90% visceral leishmaniasis cases are reported. Metema-Humera lowlands are the most important foci in Ethiopia. Blood transfusion in visceral leishmaniasis endemic foci in Ethiopia does not consider screening of visceral leishmaniasis during blood donation. The aim of this study is therefore, was to assess asymptomatic Leishmania donovani infection and associated factors among blood donors attending at Metema district Blood Bank, Northwest Ethiopia. Methods: A Health facility based cross-sectional study was conducted at Metema Blood Bank from February to March 2020. A total of 205 blood donors were eligible and participated in this study. Structured questionnaire were used to collect data on socio-demographic characteristics and perceived risk factors associated with asymptomatic visceral leishmaniasis among blood donors. Blood donors were screened using both rK39 and direct agglutination tests based on the manufactures' instructions. Data were analyzed using SPSS version 20.0. Chi-square test was used to assess associations of Leishmania donovani infection with predisposing factors. Associations were considered statstically significant on P-value < 0.05 at 95% confidence level. Results: Of the total 205 participants, 32(15.6%) were positive for asymptomatic Leishmania donovani infection at least by one of the diagnostic tests used. Eight (3.9%) and 30(14.6%) of the participants' were positive by the rK39 and direct agglutination tests, respectively. Six (2.9%) donors were tested positive by both diagnostic tests. Family history of visceral leishmaniasis (X²=11.334, P = 0.003) and having neighbors with history of visceral leishmaniasis (X²=5.923, P = 0.015) were significantly associated with asymptomatic Leishmania donovani infection among blood donors. Conclusions: The prevalence of asymptomatic Leishmania donovani infection was 15.6%. Asymptomatic visceral leishmaniasis was significantly associated with donors' family and neighbors' history of infection. Therefore, laboratory screening of blood donors for visceral leishmaniasis in endemic areas will be mandatory. Moreover, this study will give base line information for future study in the country. [ABSTRACT FROM AUTHOR]

Background: The potential reservoirs of visceral leishmaniasis (VL) in South Asia include asymptomatic and relapsed cases of VL, along with patients with post kala-azar dermal leishmaniasis (PKDL). Accordingly, accurate estimation of their parasite load is pivotal for ensuring disease elimination, presently targeted for 2023. Serological tests cannot accurately detect relapses and/or monitor treatment effectiveness, and therefore, parasite antigen/nucleic acid based detection assays remain the only viable option. An excellent option is the quantitative polymerase chain reaction (qPCR) but the high cost, technical expertise and time involved precludes its wider acceptability. Accordingly, the recombinase polymerase amplification (RPA) assay operated in a mobile suitcase laboratory has emerged not simply as a diagnostic tool for leishmaniasis but also to monitor the disease burden. Methodology/Principal findings: Using total genomic DNA isolated from peripheral blood of confirmed VL cases (n = 40) and lesional biopsies of PKDL cases (n = 64), the kinetoplast-DNA based qPCR and RPA assay was performed and parasite load expressed as Cycle threshold (Ct) and Time threshold (Tt) respectively. Using qPCR as the gold standard, the diagnostic specificity and sensitivity of RPA in naïve cases of VL and PKDL was reiterated. To assess the prognostic potential of the RPA, samples were analyzed immediately at the end of treatment or ≥6 months following completion of treatment. In cases of VL, the RPA assay in terms of cure and detection of a relapse case showed 100% concordance with qPCR. In PKDL following completion of treatment, the overall detection concordance between RPA and qPCR was 92.7% (38/41). At the end of treatment for PKDL, 7 cases remained qPCR positive, whereas RPA was positive in only 4/7 cases, perhaps attributable to their low parasite load. Conclusions/Significance: This study endorsed the potential of RPA to evolve as a field applicable, molecular tool for monitoring parasite load, possibly at a point of care level and is worthy of consideration in resource limited settings. Author summary: Visceral leishmaniasis (VL) or kala-azar caused by the digenetic parasite Leishmania donovani is a fatal form of leishmaniasis, unless treated. In South Asia, approximately 10–20% of apparently cured cases of VL develop a dermal sequela termed post kala-azar dermal leishmaniasis (PKDL). It presents with macular or papulonodular lesions that harbour Leishmania parasites. Importantly, these cases of PKDL in addition to asymptomatic and relapsed VL cases serve as mobile disease reservoirs and sustain disease transmission. In macular PKDL, that presently comprise over 50% of cases, the diagnosis is particularly challenging as parasites are often not detectable by microscopy. Accordingly, development of strategies for molecular diagnosis is one of the major foci of the ongoing leishmaniasis elimination programme targeted for 2023, that aims to decrease the burden to <1/10,000 cases of VL at the block/zilla level. Serological tests have limited applicability when it is necessary to distinguish present from past infection of VL, and in cases of PKDL when the positivity maybe due to a past history of VL. An excellent option is the quantitative polymerase chain reaction (qPCR) but limitations for its wider field applicability include the high cost, technical expertise and time factor. Accordingly, an urgent need exists for availability of field applicable molecular tests. The RPA tested in this study could well be the desired game-changer as it provided accurate diagnosis of VL and PKDL, can be performed in a primary health centre, and demonstrated the potential to be a monitoring tool for parasite load after treatment, that eventually can accelerate achieving the end-goal of elimination of leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: There are three main forms of leishmaniases: visceral (the most serious form because it is almost always fatal without treatment), cutaneous (the most common, usually causing skin ulcers), and mucocutaneous (affecting mouth, nose, and throat). Leishmaniasis is caused by protozoan parasites, which are transmitted by the bite of infected female phlebotomine sandflies. The disease affects some of the world's poorest people and is associated with malnutrition, population displacement, poor housing, a weak immune system, and lack of financial resources. An estimated 700,000 to 1 million new cases occur annually. Only a small fraction of those infected by parasites causing leishmaniasis will eventually develop the disease. We report a case of exclusive lymph node involvement in leishmaniasis, presenting as localized lymphadenopathies. The diagnosis of lymphatic leishmaniasis was confirmed by the presence of Leishmania donovani bodies in fine needle aspiration cytology, and positive anti-rK39 antibodies. The bone marrow aspiration was negative for Leishmania donovani bodies. Abdominal ultrasound was done and there was no organomegaly. Furthermore, localized lymphadenopathies may provide a diagnostic challenge by clinically mimicking a lymphoma or other causes of lymphadenopathy. Due to its rarity and its tendency to pose a clinical diagnostic challenge, we decided to report a case of lymphatic leishmaniasis. Case presentation: A 12-year-old Amara male patient presented to the University of Gondar comprehensive specialized hospital, Northwestern Ethiopia, with six discrete right lateral cervical lymphadenopathies, the largest measuring 3 × 2 cm2, with no cutaneous lesion. Fine needle aspiration cytology confirmed the diagnosis of leishmaniasis in lymph node, and he was put on sodium stibogluconate (20 mg/kg body weight/day) and paromomycin (15 mg/kg body weight/day) injections, which are given intramuscularly for 17 days. Having completed his medication at the University of Gondar comprehensive specialized hospital, he had a smooth course and was discharged with appointment scheduled for follow-up after 3 months. Conclusion: In the clinical evaluation of a patient with isolated lymphadenopathies, leishmaniasis must be considered as a differential diagnosis in immunocompetent subjects in endemic areas for early diagnostic workup and management. [ABSTRACT FROM AUTHOR]

Recently, autophagy has been implicated as a host defense mechanism against intracellular pathogens. On the other hand, certain intracellular pathogens such as Leishmania can manipulate the host's autophagy to promote their survival. Our recent findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of regulation by the mammalian target of rapamycin complex 1. This suggests the fine-tuning of autophagy to optimally promote parasite survival, possibly by the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Selected proteomic results were validated by Western blotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes, while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level. A comprehensive analysis of the Leishmania-induced autophagosome proteome will be instrumental in the advancement of understanding leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a neglected tropical disease that is globally distributed and has the potential to cause very serious illness. Prior literature highlights the emergence and spread of VL is influenced by multiple factors, such as socioeconomic status, sanitation levels or animal and human reservoirs. The study aimed to retrospectively investigate the presence and infectiousness of VL in Rio Grande do Norte (RN), Brazil between 2007 and 2020. We applied a hierarchical Bayesian approach to estimate municipality-specific relative risk of VL across space and time. The results show evidence that lower socioeconomic status is connected to higher municipality-specific VL risk. Overall, estimates reveal spatially heterogeneous VL risks in RN, with a high probability that VL risk for municipalities within the West Potiguar mesoregion are more than double the expected VL risk. Additionally, given the data available, results indicate there is a high probability of increasing VL risk in the municipalities of Natal, Patu and Pau dos Ferros. These findings demonstrate opportunities for municipality-specific public health policy interventions and warrant future research on identifying epidemiological drivers in at-risk regions. Author summary: As a neglected tropical disease (NTD), Leishmaniasis contributes to poor health outcomes, suffering, and death of vulnerable populations. Leishmania infantum or Leishmania donovani is transmitted from animals to humans through the sandfly vector and can be fatal in 5% of the cases even with treatment. Visceral leishmaniasis is a severe form of Leishmaniasis. The purpose of this study was to estimate regions of high Visceral leishmaniasis risk in Rio Grande do Norte, Brazil between 2007 and 2020 to help inform public health initiatives and potential health policy interventions for this disease. Visceral leishmaniasis is a reportable disease and therefore VL cases are documented to the Brazilian Ministry of Health database through passive surveillance. Passive surveillance data can suffer from missing information; individuals may be asymptomatic, or may not seek medical care allowing for diagnosis. When modeling disease risk from passive surveillance data, assuming there is an absence of disease in regions without documented cases can underestimate disease risk. In this paper we estimate disease risk and identify areas with high-risk and increasing infection trends over time while considering data characteristics such as missing information and high correlation of risk factors. [ABSTRACT FROM AUTHOR]

The shedding of cell surface receptors may bring synergistic outcomes through the loss of receptor-mediated cell signaling and competitive binding of the shed soluble receptor to its ligand. Thus, soluble receptors have both biological importance and diagnostic importance as biomarkers in immunological disorders. Signal regulatory protein α (SIRPα), one of the receptors responsible for the 'don't-eat-me' signal, is expressed by myeloid cells where its expression and function are in part regulated by proteolytic cleavage. However, reports on soluble SIRPα as a biomarker are limited. We previously reported that mice with experimental visceral leishmaniasis (VL) manifest anemia and enhanced hemophagocytosis in the spleen accompanied with decreased SIRPα expression. Here, we report increased serum levels of soluble SIRPα in mice infected with Leishmania donovani, a causative agent of VL. Increased soluble SIRPα was also detected in a culture supernatant of macrophages infected with L. donovani in vitro, suggesting the parasite infection promotes ectodomain shedding of SIRPα on macrophages. The release of soluble SIRPα was partially inhibited by an ADAM proteinase inhibitor in both LPS stimulation and L. donovani infection, suggesting a shared mechanism for cleavage of SIRPα in both cases. In addition to the ectodomain shedding of SIRPα, both LPS stimulation and L. donovani infection induced the loss of the cytoplasmic region of SIRPα. Although the effects of these proteolytic processes or changes in SIRPα still remain unclear, these proteolytic regulations on SIRPα during L. donovani infection may explain hemophagocytosis and anemia induced by infection, and serum soluble SIRPα may serve as a biomarker for hemophagocytosis and anemia in VL and the other inflammatory disorders. [ABSTRACT FROM AUTHOR]

The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-β). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Antrocaryon klaineanum is traditionally used for the treatment of back pain, malaria, female sterility, chlamydiae infections, liver diseases, wounds, and hemorrhoid. This work aimed at investigating the bioactive compounds with antileishmanial and antiplasmodial activities from A. klaineanum. An unreported glucocerebroside antroklaicerebroside (1) together with five known compounds (2–6) were isolated from the root barks of Antrocaryon klaineanum using chromatographic techniques. The NMR, MS, and IR spectroscopic data in association with previous literature were used for the characterization of all the isolated compounds. Compounds 1–4 are reported for the first time from A. klaineanum. The methanol crude extract (AK-MeOH), the n-hexane fraction (AK-Hex), the dichloromethane fraction (AK-DCM), the ethyl acetate fraction (AK-EtOAc), and compounds 1–6 were all evaluated for their antiparasitic effects against Plasmodium falciparum strains susceptible to chloroquine (3D7), resistant to chloroquine (Dd2), and promastigotes of Leishmania donovani (MHOM/SD/62/1S). The AK-Hex, AK-EtOAc, AK-MeOH, and compound 2 were strongly active against Dd2 strain with IC50 ranging from 2.78 ± 0.06 to 9.30 ± 0.29 µg/mL. Particularly, AK-MeOH was the most active—more than the reference drugs used—with an IC50 of 2.78 ± 0.06 µg/mL. The AK-EtOAc as well as all the tested compounds showed strong antileishmanial activities with IC50 ranging from 4.80 ± 0.13 to 9.14 ± 0.96 µg/mL. [ABSTRACT FROM AUTHOR]

The chemical investigation of the n-hexane fraction from the methanol extract of the stem bark of Symphonia globulifera Linn f., which displayed good in vitro activity against Leishmania donovani NR-48822 promastigotes (IC50 43.11 µg/mL), led to the isolation of three previously unreported polyprenylated benzophenones, guttiferone U (1), V (2)/W (3), and a new tocotrienol derivative named globuliferanol (4), along with 11 known compounds (5–15). Their structures were elucidated based on their NMR and MS data. Some isolated compounds were assessed for both their antileishmanial and cytotoxic activities against L. donovani and Vero cells, respectively. Guttiferone K (5) exhibited the best potency (IC50 3.30 μg/mL), but with low selectivity to Vero cells. The n-hexane fraction and some compounds were also assessed in vitro for their antibacterial activity against seven bacterial strains. All the samples exhibited moderate to potent antibacterial activity (MICs ≤ 15.6 µg/mL) against at least one of the tested strains. [ABSTRACT FROM AUTHOR]

The recent outlook of leishmaniasis as a global public health concern coupled with the reportage of resistance and lack of efficacy of most antileishmanial drugs calls for a concerted effort to find new leads. The study combined In silico and in vitro approaches to identify novel potential synthetic small-molecule inhibitors targeting the Leishmania donovani sterol methyltransferase (LdSMT). The LdSMT enzyme in the ergosterol biosynthetic pathway is required for the parasite's membrane fluidity, distribution of membrane proteins, and control of the cell cycle. The lack of LdSMT homologue in the human host and its conserved nature among all Leishmania parasites makes it a viable target for future antileishmanial drugs. Initially, six known inhibitors of LdSMT with IC50 < 10 μM were used to generate a pharmacophore model with a score of 0.9144 using LigandScout. The validated model was used to screen a synthetic library of 95,630 compounds obtained from InterBioScreen limited. Twenty compounds with pharmacophore fit scores above 50 were docked against the modelled three-dimensional structure of LdSMT using AutoDock Vina. Consequently, nine compounds with binding energies ranging from −7.5 to −8.7 kcal/mol were identified as potential hit molecules. Three compounds comprising STOCK6S-06707, STOCK6S-84928, and STOCK6S-65920 with respective binding energies of −8.7, −8.2, and −8.0 kcal/mol, lower than 22,26-azasterol (−7.6 kcal/mol), a known LdSMT inhibitor, were selected as plausible lead molecules. Molecular dynamics simulation studies and molecular mechanics Poisson–Boltzmann surface area calculations showed that the residues Asp25 and Trp208 were critical for ligand binding. The compounds were also predicted to have antileishmanial activity with reasonable pharmacological and toxicity profiles. When the antileishmanial activity of the three hits was evaluated in vitro against the promastigotes of L. donovani, mean half-maximal inhibitory concentrations (IC50) of 21.9 ± 1.5 μM (STOCK6S-06707), 23.5 ± 1.1 μM (STOCK6S-84928), and 118.3 ± 5.8 μM (STOCK6S-65920) were obtained. Furthermore, STOCK6S-84928 and STOCK6S-65920 inhibited the growth of Trypanosoma brucei, with IC50 of 14.3 ± 2.0 μM and 18.1 ± 1.4 μM, respectively. The identified compounds could be optimised to develop potent antileishmanial therapeutic agents. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a neglected disease with a broad spectrum of clinical manifestations and involvement of visceral organs. Organ‐specific immune response against the Leishmania donovani (Ld) complex is not yet understood due to the unavailability of an appropriate experimental model. In reference to our recent work on comparing the hamster model with VL patients, it is now possible to understand immune profiling in different visceral organs. This may offer an answer to varying parasite loads in different visceral organs in the same host. Herein, we analysed a panel of immune markers (Th‐2/Th‐1) in visceral organs of Ld‐infected hamsters and quantified parasitic load in the same tissues using qPCR assay. In spleen, liver, bone marrow and lymph node (mesenteric) from Ld‐infected hamsters, the parasite burden was quantified along with mRNA expression of a panel of Th‐2 and Th‐1 type immune markers, namely IL‐10, IL‐4, Arginase‐I, GATA‐3, SOCS‐3, IL‐12, IFN‐γ, iNOS, T‐bet and SOCS‐5. A clear dichotomy was absent between Th‐2 and Th‐1 type immune markers and the major players of this immune response were IFN‐γ, IL‐10, T‐bet, GATA‐3, SOCS‐5 and SOCS‐3. [ABSTRACT FROM AUTHOR]

The amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) occurs only in isoforms of eukaryotic translation factor 5A (eIF5A) and has a role in initiating protein translation. Hypusinated eIF5A promotes translation and modulates mitochondrial function and oxygen consumption rates. The hypusination of eIF5A involves two enzymes, deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH is the second enzyme that completes the synthesis of hypusine and the maturation of eIF5A. Our current study aims to identify inhibitors against DOHH from Leishmania donovani (Ld DOHH), an intracellular protozoan parasite causing Leishmaniasis in humans. The Ld DOHH protein was produced heterologously in Escherichia coli BL21(DE3) cells and characterized biochemically. The three-dimensional structure was predicted, and the compounds folic acid, scutellarin and homoarbutin were selected as top hits in virtual screening. These compounds were observed to bind in the active site of Ld DOHH stabilizing the structure by making hydrogen bonds in the active site, as observed by the docking and molecular dynamics simulation studies. These results pave the path for further investigation of these molecules for their anti-leishmanial activities. [ABSTRACT FROM AUTHOR]

As part of our ongoing scaffold-hopping work on an antikinetoplastid 3-nitroimidazo[1,2-a]pyridine scaffold, we explored 3-nitroimidazo[1,2-b]pyridazine as a potential new antikinetoplastid series. Using conditions previously described by our lab, we obtained 6-chloro-3-nitro-2-[(phenylsulfonyl)methyl]imidazo[1,2-b]pyridazine with 54% yield. In vitro activity of this compound was evaluated against both the promastigote form of Leishmania donovani, the axenic amastigote form of Leishmania infantum and the trypomastigote blood stream form of Trypanosomabrucei brucei, and its influence on cell viability was assessed on the HepG2 cell line. However, despite good activity against the trypomastigote blood stream form of T. b. brucei (EC50 = 0.38 µM), it showed poor solubility in both HepG2 (CC50 > 7.8 µM) and L. infantum axenic amastigotes (EC50 > 1.6 µM) culture media, associated with a loss of activity against the promastigote form of L. infantum (EC50 > 15.6 µM). [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is among the most neglected tropical diseases in the world. Drug cell permeability is essential for killing the intracellular residing parasites responsible for VL, making cell‐permeating peptides a logical choice to address VL. Unfortunately, the limited biological stability of peptides restricts their usage. Sequence‐specific oligo‐N‐substituted glycines ('peptoids') are a class of peptide mimics that offers an excellent alternative to peptides in terms of ease of synthesis and good biostability. We tested peptoids against the parasite Leishmania donovani in both forms, that is, intracellular amastigotes and promastigotes. N‐alkyl hydrophobic chain addition (lipidation) and bromination of oligopeptoids yielded compounds with good antileishmanial activity against both forms, showing the promise of these antiparasitic peptoids as potential drug candidates to treat VL. [ABSTRACT FROM AUTHOR]

Purpose: Leishmaniasis is a parasitic disease transmitted by the bite of the phlebotomine female sand fly. Currently, no reported effective vaccines are available for the treatment of leishmaniasis; consequently, restricting this disease completely depends on controlling its transmission. Mitogen-activated protein (MAP) kinases have been reported to be involved in the regulation of the flagellum length and hence play an important role in disease transmission, especially the MAPK3 protein. Therefore, the current work focused on identifying approved drugs that can inhibit the MAPK3 protein. Methods: First, the recombinant plasmid (pET28b(+) MAPK3) was cloned into E. coli strain BL21 using the heatshock method. Afterward, E. coli was induced using IPTG, and cells were harvested for protein purification in the next step. After that, the MAPK3 protein was purified using Ni–NTA column. Then, the inhibition kinase activity of the purified MAPK3 protein was performed using an ADP-Glo™ Kinase Assay kit. Furthermore, the cytotoxicity of Leishmania cells were detected by alamarBlue™ Cell Viability Reagent. Finally, the binding affinity within the binding site of MAPK3 protein was performed by computational methods. Results: Purification of the MAPK3 protein was done using an Ni–NTA column and a protein band was identified at the expected 44 kDa molecular weight. Afterward, the ability of commercial drugs (afatinib and lapatinib) to inhibit the purified MAPK3 kinase activity was performed using an ADP-Glo™ Kinase Assay kit. The half-maximal inhibitory concentrations (IC50) of two drugs inhibited the MAPK3 protein within the same range of IC50 values (3.27 and 2.22 µM for afatinib and lapatinib, respectively). Furthermore, the cytotoxicity assay of compounds toward the extracellular promastigote and intracellular amastigote stages was investigated using alamarBlue™ Cell Viability Reagent. The results showed that both drugs were more efficient against extracellular promastigotes and intracellular amastigotes of both Leishmania donovani and Leishmania martiniquensis. Finally, the molecular dynamics simulation (MD) was performed to study the intermolecular interactions of both drugs with MAPK3 protein. From 100 ns molecular dynamics simulation, the structural stability of both drugs in a complex with MAPK3 was quite stable. Conclusion: This work was suggesting that afatinib and lapatinib act as MAPK3 inhibitors and might be developed for leishmaniasis treatment. [ABSTRACT FROM AUTHOR]

Background: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are pivotal for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. Methodology: We conducted a retrospective study on stored microscopically negative spleen and bone marrow smears from suspected VL patients collected at the Leishmaniasis Research and Treatment Center (LRTC) in Gondar, northern Ethiopia between June 2019 and November 2020. Sociodemographic, clinical and treatment data were collected and samples were tested by real-time PCR targeting kinetoplast DNA. Principle findings: Among the 191 eligible samples (135 spleen and 56 bone marrow) with a microscopically negative and valid PCR result, 119 (62.3%) were positive by PCR, although Ct values for some were high (median 33.0). Approximately three quarters of these undiagnosed primary VL (77.3%) and relapse (69.6%) patients did not receive antileishmanial treatment. Of the 56 microscopically negative bone marrow samples, 46 (82.1%) were PCR positive, which is considerably higher compared to the microscopically negative spleen samples, for which 73 out of 135 (54.1%) were PCR positive. The odds of being PCR positive were significantly higher for bone marrow aspirates and higher when white blood cell values were lower and splenomegaly (in cm) was more pronounced. Conclusions: This study demonstrates that a lot of suspected VL patients remain undiagnosed and untreated. This indicates the urgent need for better diagnostics for VL in the East-African region. The outcomes of PCR positive should be closely monitored and treatment should be provided if the patient deteriorates. In resource limited settings, implementation of PCR on bone marrow aspirate smears of patients with low WBC values and splenomegaly could lead to considerable improvements in patient management. Author summary: As untreated visceral leishmaniasis (VL) is fatal, reliable diagnostics are important for accurate treatment allocation. The current diagnostic algorithm for VL in Ethiopia, which is based on the rK39 rapid diagnostic test and microscopy of tissue smears, lacks sensitivity. This probably leads to missed cases and patients not receiving treatment. To prove this, we conducted a study on stored microscopically negative spleen and bone marrow aspirate smears from suspected VL patients in Gondar, Ethiopia. Clinical and treatment data were collected and samples were tested for Leishmania by PCR. We found that about 60% of these microscopically negative samples were PCR positive. This PCR positivity rate was considerably higher in patients with a microscopically negative bone marrow compared to splenic aspirate. Importantly, more than three quarters of the patients with a PCR positive sample was not treated for VL. Overall, our study demonstrates the gap in the diagnostic algorithm for VL in northern Ethiopia, especially when bone marrow samples are used. In resource limited settings, we advise to challenge the current diagnostic algorithm and implement molecular tools to accurately diagnose patients. This could lead to considerable improvements in patient management in Ethiopia and beyond. [ABSTRACT FROM AUTHOR]

Background: In Nepal, the burden of post kala-azar dermal leishmaniasis (PKDL) is not known since there is no active case detection of PKDL by the national programme. PKDL patients could pose a challenge to sustain visceral leishmaniasis (VL) elimination. The objective of this study was to determine the prevalence of PKDL and assess PKDL patients' knowledge on VL and PKDL, and stigma associated with PKDL. Methodology/Principal findings: Household surveys were conducted in 98 VL endemic villages of five districts that reported the highest number of VL cases within 2018–2021. A total of 6,821 households with 40373 individuals were screened for PKDL. Cases with skin lesions were referred to hospitals and examined by dermatologists. Suspected PKDL cases were tested with rK39 and smear microscopy from skin lesions. An integrated diagnostic approach was implemented in two hospitals with a focus on management of leprosy cases where cases with non-leprosy skin lesions were tested for PKDL with rK39. Confirmed PKDL patients were interviewed to assess knowledge and stigma associated with PKDL, using explanatory model interview catalogue (EMIC) with maximum score of 36. Among 147 cases with skin lesions in the survey, 9 (6.12%) were confirmed as PKDL by dermatologists at the hospital. The prevalence of PKDL was 2.23 per 10,000 population. Among these 9 PKDL cases, 5 had a past history of VL and 4 did not. PKDL cases without a past history of VL were detected among the "new foci", Surkhet but none in Palpa. None of the cases negative for leprosy were positive for PKDL. There was very limited knowledge of PKDL and VL among PKDL cases. PKDL patients suffered to some degree from social and psychological stigma (mean ± s.d. score = 17.89 ± 12.84). Conclusions/Significance: Strengthening the programme in PKDL case detection and management would probably contribute to sustenance of VL elimination. Awareness raising activities to promote knowledge and reduce social stigma should be conducted in VL endemic areas. Author summary: Post kala-azar dermal leishmaniasis (PKDL) is a sequel of visceral leishmaniasis (VL) and occurs in VL endemic areas. PKDL patients can be a reservoir for Leishmania donovani parasites and thus sustaining the occurrence of VL. Since Bangladesh, India and Nepal have targeted the elimination of VL, it is important to eliminate PKDL as well. However, in Nepal there is no active case detection of PKDL and the real burden of PKDL is not known. This community based survey encountered a PKDL prevalence of 2.23 per 10,000 population. PKDL patients had limited knowledge of VL and PKDL. They suffered to some degree from social and psychological stigma. Therefore, PKDL case detection and management should be strengthened in the national programme in the context of the VL elimination initiative. [ABSTRACT FROM AUTHOR]

Objectives Previously, a series of side chain-modified quinolinyl β-enaminones was identified to possess significant activity against chloroquine-sensitive or -resistant Plasmodium falciparum and Brugia malayi microfilariae. The present study evaluates in vitro and in vivo activity of the series against Leishmania donovani and reports their mode of action. Methods The in vitro activity of 15 quinolinyl β-enaminone derivatives against Leishmania promastigotes and amastigotes was assessed by luciferase assay. The reduction of organ parasite burden was assessed by Giemsa staining in L. donovani -infected BALB/c mice and hamsters. Intracellular Ca2+ and ATP level in active derivative (3D)-treated promastigotes were determined by fluorescence and luminescence assays. Flow cytometry was performed to determine loss of mitochondrial membrane potential (MMP) using JC-1 dye, reactive oxygen species (ROS) generation using 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dye, phosphatidylserine externalization by Annexin V-FITC staining and cell-cycle arrest by propidium iodide (PI) staining. Results Compounds 3A, 3B and 3D showed significant in vitro efficacy against L. donovani with IC50 < 6 µM and mild cytotoxicity (∼75% viability) at 25 µM on J774 macrophages. 3A and 3D at 50 mg/kg and 100 mg/kg reduced parasite burden (>84%) in infected mice and hamsters, respectively, whereas 3D-treated animals demonstrated maximum parasite burden reduction without organ toxicity. Mode-of-action analysis revealed that 3D induced apoptosis by inhibiting mitochondrial complex II, reducing MMP and ATP levels, increasing ROS and Ca2+ levels, ultimately triggering phosphatidylserine externalization and sub-G0/G1 cell-cycle arrest in promastigotes. Conclusions Compound 3D-mediated inhibition of L. donovani mitochondrial complex induces apoptosis, making it a promising therapeutic candidate for visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), the second-most-serious parasitic illness after malaria, is currently endemic in more than 88 countries. Need for new antileishmanial compounds is currently being taken into consideration by researchers due to resistance and lack of effective vaccinations. This research was conducted to find out more about the effect of artemisinin (ART). ART was examined in vitro promastigotes stages and ex vivo amastigotes stages of the Iraqi strain of Leishmania donovani in U937 cell line after 24, 48 and 27 hours using MTT assay. In addition, the level of macrophage nitric oxide (NO) was measured using Griess assay in U937 cell line. The results of promastigotes viability percentage were less than 50 %, where the IC50 was 43.8, 37.9 and 36.6 µM respectively. Similar cytotoxic effect of ART was observed against amastigote forms, where the cell viability was 36.11, 40.2 and 37.4 µM respectively, after the three times of follow up. Furthermore, by using Griess assay the level of nitrite in infected macrophages was measured. The results after 48hr were 6.58 in high concentration and 1.22 in lower concentration. However, considering the absence of a significant difference, the observed concentrations are still higher than the control when compared to Griess assay. These findings demonstrated that ART has a cytotoxic effect on both forms of L. donovani. It also increased iNOS production in infected macrophages which can define artemisinin therapeutic efficacy against L. donovani infection. [ABSTRACT FROM AUTHOR]

Parasite of genus Leishmania viz. L. donovani and L. infantum cause visceral leishmaniasis (VL) or Kala-azar, systemic disease with significant enlargement of the liver and spleen, weight loss, anemia, fever and immunosuppression. The silent expansion of vectors, reservoir hosts and resistant strains is also of great concern in VL control. Considering all these issues, the present study focused on in vitro and in vivo antileishmanial screening of ellagic acid (EA) against L. donovani. The in vitro study was performed against the protozoan parasite L. donovani and a 50% inhibitory concentration was calculated. The DNA arrest in the sub-G0/G1 phase of the cell cycle was studied. In vivo studies included the assessment of parasite burden and immunomodulation in response to treatment of ellagic acid in BALB/c mice. The levels of Th1 and Th2 cytokines and isotype antibodies were assessed in different groups of mice. EA showed in vitro parasiticidal activity with IC50 18.55 µg/mL and thwarted cell-cycle progression at the sub-G0/G1 phase. Administration of ellagic acid to the BALB/c mice reported diminution of splenic and hepatic parasite burden coupled with an expansion of CD4+ and CD8+ T lymphocytes. EA further potentiated a protective immune response with augmentation of Th1 type immune response evidenced by elevation of serum IgG2a levels and DTH response. EA was reported to be safe and non-toxic to the THP-1 cell line as well as to the liver and kidneys of mice. These findings endorse the therapeutic potential of EA with significant immunomodulation and can serve as a promising agent against this debilitating parasitic disease. [ABSTRACT FROM AUTHOR]

Arachis hypogea L. protein fraction-2 (AHP-F2) from the Peanut shell was extracted and characterized and its potent immunomodulatory and anti-leishmanial role was determined in this present study. AHP-F2 was found to be a glycoprotein as the presence of carbohydrates were confirmed by the analysis of high-performance liquid chromatography (HPLC) yielded glucose, galactose, mannose, and xylose. AHP-F2 molecular mass was found to be ∼28 kDa as indicated in MALDI-TOF and peptide mass fingerprinting analysis followed by Mascot search. The peptide matches revealed the similarity of the mannose/glucose binding lectin with 71.07% in the BLAST analysis. After that, the 3D structure of the AHP-F2 model was designed and validated by the Ramachandran plot. The immunomodulatory role of AHP-F2 was established in murine peritoneal macrophages as induction of nitric oxide (NO), and stimulation of proinflammatory cytokines (IL-12 and IFN-γ) in a dose-dependent manner was observed. Interestingly, it was also found that AHP-F2 has interacted with the innate immune receptor, toll-like receptors (TLRs) as established in molecular docking as well as mRNA expression. The anti-leishmanial potential of AHP-F2 was revealed with a prominent inhibition of amastigote growth within the murine macrophages with prompt induction of nitrite release. Altogether, the isolated AHP-F2 from Arachis hypogea L. has strong immunomodulatory and anti-leishmanial potential which may disclose a new path to treat leishmaniasis. [ABSTRACT FROM AUTHOR]

Protozoal infections caused by species belonging to Leishmania donovani complex are responsible for the most severe form of leishmaniasis, especially in Sudan and other developing countries. Drugs commonly used for the treatment of the disease show varying levels of effectiveness and also have associated side effects. Thus, the present work highlights the synthesis of some chalcones to be used as potential anti-leishmanial agents. The activity of the synthesized chalcones has been evaluated against L. donovani. The ADMET profile of the synthesized compounds were tested using various integrated web-based tools. Moreover, in order to investigate the molecular mechanism of action, the chalcone compounds were docked into L. donovani trypanothione reductase (TR) using Autodock 4.0 and molecular dynamics were studies. Eight compounds showed the highest activity against the morphological forms. Among these compounds, chalcones 15 has shown the highest inhibitory effect with IC50 value of 1.1 µM. In addition, pharmacokinetic and toxicological investigations revealed its good oral bioavailability and low toxicity. Furthermore, chalcone 15 was found to interact with high affinity (−13.7 kcal/mol) with TR, an essential enzyme for the leishmanial parasite. Thus, this promising activity against L. donovani supports the use of chalcone 15 as a potential new therapy for visceral leishmaniasis.

Host-tissue preference is a critical aspect of parasitic infections and is directly correlated with species diversity. Even the same species, Leishmania donovani , infects the host's bone marrow, spleen, and liver differentially. The tissue-specific persistence of Leishmania results from host-pathogen immune conflicts and arguments. The protective pro-host or destructive pro-parasitic role of TLRs during L. donovani infection has been well established, but what entirely missing is the influence of TLRs on tissue-specific parasite persistence. We observed that the parasites induced differential expression of TLR1/2 in the bone marrow but not in the spleen. Interestingly, the rate of Leishmania infection was found to be positively correlated with TLR1/2-mediated upregulation of myelopoietic cytokines, M-CSF, GM-CSF, IL-6, and IL-3, leading to the expansion of Ly6ChiCCR2+ monocytes, however, negatively correlated with the expression of the disease hallmark cytokines, TNF-α, TGF-β, and IL-10, along the course of infection in the bone marrow. Leishmania induced the activation of bone marrow-specific TLR1/2 to promote Ly6ChiCCR2+ monocytes for its safe shelter vis-à-vis infection establishment. Consequently, the established infection initiated the release of TNF-α, TGF-β, and IL-10 in the bone marrow. Post-infection time-kinetic study affirmed that TGF-β had a significant negative influence on the expression of TLR1/2 heterodimer in the bone marrow niche. To the best of our knowledge, this is the first report to show that the inverse correlation of TLR1/2 - TGF-β can be instrumental in tissue-specific parasite persistence during Leishmania infection. Leishmania -specific induction of myelopoiesis leading to the proliferation of Ly6ChiCCR2+ monocytes, the preferred host cells for the parasites, is guided by TLR1/2 in bone marrow. The production of TGF-β starts increasing after the highest parasite establishment on day 42pi and peaks at day 56pi leading to a suppressed expression of TLR1/2 and expectedly lower parasitic incidence. An inverse correlation of TLR1/2 with TGF-β ultimately favors the host as it disrupts the exploitation of TLR1/2 by L. donovani to safely reside in the bone marrow. Pro-host mechanism (▪); Pro-parasitic mechanism (▪). [Display omitted] • Leishmania donovani displays differential tissue-infectivity in the same host. • TLR1/2 positively correlates with the parasite persistence in bone marrow. • Leishmania -specific TLR1/2 induces myelopoiesis in bone marrow. • TGF-β disrupts the exploitation of bone marrow TLR1/2 by the parasites in the chronic phase. • The inverse correlation between Leishmania -induced TLR1/2 and TGF-β favors the host. [ABSTRACT FROM AUTHOR]

The majority of anti-leishmanial drugs used for treating trypanosomatid parasites help to reduce human morbidity and mortality. However, parasites have developed drug resistance, which has made it challenging to treat leishmaniasis. Therefore, new drugs and drug targets need to be identified. Protein synthesis is a crucial anabolic mechanism necessary for parasite survival. Histidyl-tRNA synthetase (HisRS) is an essential enzyme that is required for histidine incorporation into proteins. Recent studies on HisRS have shown differences between trypanosomatid HisRS and human HisRS, which could lead to the development of trypanosomatid HisRS structure-based inhibitors. This study aims to determine the role of L. donovani HisRS (LdHisRS) in parasite growth and virulence in vitro using RNAi. The silencing effect of LdHisRS expression was determined using qPCR. The results showed that after 24 and 48 h of incubation with 90 ng siRNAs, LdHisRS mRNA expression levels were significantly reduced by ∼3.14-fold and ∼3.90-fold, respectively. SiRNA-treated parasites also exhibited ∼46.6 % delayed growth and ∼47 % reduced virulence. Additionally, homology modeling, virtual screening, and molecular docking studies were performed with potential inhibitors that have significant suppressive activity in bacteria, fungi, and viruses. Halofuginone was found to have the best binding affinity of −9.09 kcal/mol as a potent inhibitor against LdHisRS. The molecular dynamics (MD) results showed that halofuginone could interact with the various active site segments, potentially blocking substrate access. The data on gene silencing through siRNA suggests that LdHisRS is essential for the parasite's growth and survival. The computational findings could lead to the development of a potent ligand (halofuginone) as a future anti-leishmanial drug, paving the way for an effective therapeutic treatment. • LdHisRS silencing has reduced the growth and virulence of the promastigotes stage of the parasite. • LdHisRS is localized in the cytosol of the parasite and is crucial for the intracellular proliferation of amastigotes. • Low expression of LdHisRS in siRNA-treated promastigotes as compared to control promastigotes. • LdHisRS has high homology with the HisRS gene of trypanosomatid parasites while having less similarity with humans. • Molecular docking and simulation results revealed that the halofuginone compound exhibited the strongest binding interaction. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is caused by Leishmania donovani which affects the poorer sections of society, and despite the global spread, effective treatment is unavailable. The current study investigates the potential of leishmanial histidyl-tRNA synthetase (Ld HisRS) as a drug target. Ld HisRS delineated more closeness to other protozoan parasites than its mammalian counterparts and contained relevant differences in the active site residues. The important ATP-binding residues were mutated to alanine and all the proteins, including human HisRS , were purified to homogeneity. Ld HisRS exhibited a dimeric state in solution and showed maximal amino acid activation activity in physiological conditions. It also demonstrated a greater affinity for substrate over cofactor, while magnesium and potassium enhanced its activity better than other tested metal ions. Comp-7m, a benzothiazolo-coumarin derivative, proved to be specific inhibitor of Ld HisRS with competitive mode of inhibition for ATP whereas it displayed lower binding affinity towards mutants. Ld HisRS majorly contained α-helices and most of the aromatic residues were present in its hydrophobic core. Additionally, Comp-7m superimposed on ATP adenine ring during docking analysis and Ld HisRS-ligand complexes had comparable stability as well as rigidity in molecular dynamics simulation. We thus provide structural and functional insights of Ld HisRS which can be useful for devising antileishmanials. • Ld HisRS is evolutionarily distant from Hs HisRS with differences in active site. • Ld HisRS is a dimer in solution and depicts more affinity for substrate than cofactor. • Mutation of key ATP-binding residues reduces catalytic efficiency of the enzyme. • Comp-7m is a potent and specific inhibitor of Ld HisRS that binds at ATP pocket. • Comp-7m exhibits lower binding energy towards Ld HisRS in comparison to ATP. [ABSTRACT FROM AUTHOR]

Antigenic inefficacy to induce robust immune responses and durable memory are major causes of constantly failing prophylactic approaches in leishmaniasis. Here, we determine the potential of a standardized whole-killed Leishmania vaccine (Leishvacc) adjuvanted with anti-CD200 and anti-CD300a antibodies, either alone or with monophosphoryl lipid A (MPL-SE) emulsified in squalene oil, in restoring the compromised antigen presenting abilities of dendritic cells (DCs), effector properties of CD4+T cells and providing protection against Leishmania donovani parasites. In animals vaccinated with antibodies adjuvanted vaccines, either alone or with MPL-SE, the antigen presenting abilities of CD11c+ DCs against Leishmania antigens, measured in terms of CD80, CD86, MHC-I, and MHC-II surface receptors and intracellular IL-12 were found enhanced than non-adjuvanted vaccine. We observed more proliferative and pro-inflammatory cytokines i.e. IL-2, IFN-γ, IL-23, and IL-12 producing CD4+T cells in antibodies/MPL-SE adjuvanted vaccinated animals further suggesting that this approach helps antigen activated CD4+T cells to acquire pro-inflammatory cytokines producing abilities. In antibodies, either alone or with MPL-SE, vaccinated animals, the number of CD4+ central memory T cells and their longevity were found significantly enhanced that further evidenced the impact of this vaccination approach in inducing long term protective immunity. The animals, receiving antibodies adjuvanted vaccines, either alone or with MPL-SE, exhibited excellent protection against virulent parasites by restricting their growth, which correlated with the significantly reduced parasitemia, splenomegaly, and hepatomegaly, along with fewer numbers of liver granulomas. Our findings provide an insight to a new immunoprophylactic approach against visceral leishmaniasis, which not only satisfies the safety criteria, but also provides a robust immunogenic response with remarkable potential for parasites control. However, further in-depth investigations are needed to ascertain its ability in inducing long-lasting immunity. • Anti-CD200 & Cd300a antibodies adjuvantation enhances Leishmania vaccine efficacy. • MPL-SE foster the efficacy of antibodies adjuvanted Leishvacc. • MPL-SE and antibodies adjuvantation enhances dendritic and CD4+T cells functions. • Antibodies adjuvanted Leishvacc, with or without MPL-SE meets the safety criteria. [ABSTRACT FROM AUTHOR]

• High-throughput screening has revolutionized drug discovery by rapidly sifting through vast libraries of chemical compounds to identify potential therapeutic leads. • Leishmaniasis belongs to a class of vector-borne diseases caused by protozoan parasites of the genus Leishmania. • The present study employs High-throughput screening strategy to identify potent hits against Leishmania donovani sterol C-24 methyltransferase from Chembridge library. • Three potent hits (IIIM-CB-14, IIIM-CB-29 , and IIIM-CB-45) were identified with 50 % inhibitory concentration (IC 50) ≤10 µg/mL. Leishmaniasis is a neglected tropical disease and is one of the major causes of mortality in poverty-stricken areas. A limited chemotherapeutics arsenal is available to tackle this deadly infection. Thus, identifying novel potent scaffolds using innovative strategies is the need of the hour. High-throughput screening (HTS) is a critical technique that can accelerate the process of drug discovery by evaluating millions of drug-like molecules using various automation tools and biological assays. In the present study, we have employed the HTS strategy to identify potent hits against Leishmania donovani sterol C-24 methyltransferase (LdSMT) from the in-house ChemBridge library. Firstly, a robust dataset was prepared with previously reported sterol C-24 methyltransferase inhibitors, belonging to diverse structural classes. Then, ligand-based virtual screening using similarity search was performed to screen the ChemBridge library having ∼20,000 molecules. This computational approach yielded 81 candidate compounds, which were selected for further molecular docking and biological evaluation. Anti-leishmanial assays revealed that out of 81 molecules, seven showed potential parasitic killing. Three molecules namely IIIM-CB-14, IIIM-CB-29 , and IIIM-CB-45 were the most potent ones with 50 % inhibitory concentration (IC 50) of 5.76, 8.08, and 10.64 µg/mL, respectively. SEM analyses suggest that these potent hits cause considerable morphological alterations. ADME studies of the potent hit molecules indicate that all the hits have considerable drug-likeness properties. Further, molecular dynamics studies were also performed to check the stable confirmation of LdSMT protein with the top two hits (IIIM-CB-14 and IIIM-CB-45). Thus, the present study harnesses computational and experimental approaches to unravel potent anti-leishmanial scaffolds. [ABSTRACT FROM AUTHOR]

• New markers for Leishmania species identification were tested. • Specific primers were designed and standardized for use in multiplex PCR assay. • The primers developed were highly specific. • The multiplex PCR assay can differentiate five Leishmania species and one species complex. Leishmaniasis is a diverse group of clinical diseases caused by protozoan parasites of the Leishmania genus. Species-specific identification of Leishmania spp. is challenging due to the high number of different pathogenic species that sometimes co-circulate in the same foci, hampering efforts to effectively control the disease. Multiplex PCR is an attractive alternative for rapid differentiation of Leishmania species with high sensitivity and specificity. We aimed to generate a panel of primers optimized for a multiplex PCR assay capable of identifying different Leishmania species in a single reaction. Species-specific primers were designed based on genomic data using the TipMT too L. Potential non-specific amplifications of other trypanosomatids as well as human, dog, and sandfly hosts were first evaluated in silico using the Primer-Blast too L. Species-specific primers for Leishmania amazonensis, Leishmania braziliensis, Leishmania donovani, Leishmania infantum, Leishmania mexicana and for the Leishmania guyanensis complex were tested in vitro. The primers have a limit of detection ranging from 1 to 0.01 ng of parasite gDNA using the same annealing temperature of 66 °C. The primers were specific for their targets when tested against 13 species of Leishmania , six trypanosomatids, and Babesia sp., and to detect the target species in a prepared pool with gDNA of six pathogenic Leishmania species. The designed primers were optimized for multiplex PCR, enabling species-specific identification of all five Leishmania species and one species complex. This new primer set could allow for efficient, fast, and reliable identification of Leishmania parasites. [Display omitted] [ABSTRACT FROM AUTHOR]

• Antibodies against r Ld APx in VL patients' sera confirm its antigenicity. • In-silico-based peptides were predicted against MHC-I and MHC-II for further study. • In vitro immunogenicity of Ld APx-derived peptides was tested on human PBMCs. • These peptides induced PBMC proliferation and significant IFN-ϒ production. • Enhanced Th1-type immune responses in treated VL suggest its vaccine potential. The control and eradication of any infectious disease is only possible with a potential vaccine, which has not been accomplished for human visceral leishmaniasis (VL). The lack of vaccines may increase the risk of VL outbreaks periodically in endemic zones. Identifying a reliable vaccine candidate for Leishmania is a major challenge. Here, we considered Leishmania donovani ascorbate peroxidase (Ld APx) for its in vitro evaluation with the hope of future vaccine candidates for VL. Ld APx was selected based on its unique presence in Leishmania and virulence in VL pathogenesis. Initially, we found antibodies against recombinant Ld APx (r Ld APx) in the serum of VL patients. Therefore, using bioinformatics, we predicted and selected ten (MHC class I and II) peptides. These peptides, evaluated in vitro with PBMCs from healthy, active VL, and treated VL individuals induced PBMC proliferation, IFN-γ secretion, and Nitric Oxide (NO) production, indicating host-protective immune responses. Among them, three peptides (PEP6, PEP8, and PEP9) consistently elicited a Th1-type immune response in PBMCs. Treated VL individuals showed a stronger Th1 response compared to active VL patients and healthy subjects, highlighting these peptides' potential as vaccine candidates. Further studies are on the way toward evaluating the Ld APx-derived peptides or sub-unit vaccine in animal models against the L. donovani challenge. [ABSTRACT FROM AUTHOR]

• Visceral and cutaneous leishmaniasis is an emerging infection in the Western Ghats. • Both manifestations were proven to be caused by Leishmania donovani. • This dual tropism of L. donovani has necessitated the genetic analysis of parasite. • k-DNA sequence analysis revealed variation among L. donovani from various countries. The visceral and atypical cutaneous leishmaniasis (VL and CL) caused by Leishmania donovani is an emerging infectious disease in the Western Ghats, Kerala, India. In this study, L. donovani specific kinetoplast minicircle DNA (k-DNA) sequence analysis was conducted to ascertain the genetic variability among the L. donovani isolates from the Western Ghats. Out of 23 CL and 5 VL suspected patient samples, 18 CL and 3 VL tested positive for k-DNA diagnostic PCR. Subsequently, 17 CL and 3 VL samples were found positive for L. donovani specific k-DNA PCR. Although the genetic diversity among the VL and CL isolates was low, there was clear variation from the parasites reported from other countries. The parasites characterized from the current study were more related to those reported from East Africa and India. [ABSTRACT FROM AUTHOR]

Citrate synthase is a crucial enzyme in the TCA cycle and represents a potential therapeutic target. However, knowledge about this enzyme in Leishmania parasites remains limited. In this study, we have successfully cloned, expressed, and purified citrate synthase from Leishmania donovani (LdCS) using a bacterial system, and characterized it through various biophysical and biochemical methods. Circular dichroism analysis at physiological pH indicates that LdCS is properly folded. Further investigation into its tertiary structure using a quencher reveals that most tryptophan residues are located within the protein's hydrophobic core. Biochemical assays show that the recombinant enzyme is catalytically active, with optimal activity at pH 7.0. Kinetic studies provided parameters such as Km and Vmax. Enzyme inhibition assays revealed that LdCS activity is competitively inhibited by FDA-approved compounds—Abemaciclib, Bazedoxifene, Vorapaxar, and Imatinib—with Ki values ranging from 2 to 3 μM, demonstrating significant binding affinity. This research paves the way for exploring LdCS as a potential drug target for treating leishmaniasis. [ABSTRACT FROM AUTHOR]

M1 aminopeptidase is a metallopeptidase that plays a vital role in protein catabolism and has been identified as a validated drug target in various parasites; however, our understanding of this enzyme is restricted for leishmanial parasite. The present investigation involved the purification of Leishmania donovani M1 aminopeptidase (Ld M1AP) to homogeneity by affinity chromatography. Purified Ld M1AP was observed to be enzymatically active and displayed maximal activity in the presence of cobalt ions, whereas secondary structure analysis confirmed the dominance of α-helices. Intrinsic fluorescence and quenching studies of Ld M1AP has revealed that tryptophan residues were predominantly concealed within the hydrophobic areas. The synthesized 8-hydroxy-2-quinoline carbaldehyde derivatives were screened, wherein HQ2 and HQ12 were found as potent inhibitors for Ld M1AP that compete with the substrate and exhibit pharmacokinetic properties as well as no toxicity for macrophages. Moreover, structural insights of protein and ligand complexes demonstrated that lead compounds mostly interact via hydrophobic contacts into the substrate binding pocket of Ld M1AP. Furthermore, lead compounds exhibited a greater affinity for Ld M1AP compared to the substrate during in vitro and in silico studies. This report establishes the possibility of quinoline derivatives to target the Ld M1AP activity and provide a platform to design the specific antileishmanial drugs. • Ld M1AP is a metal dependent enzyme possessing more alpha-helices as secondary structure. • The activity of Ld M1AP is potently inhibited by two quinoline-carbaldehyde derivatives (HQ2 and HQ12). • HQ2 and HQ12 inhibit Ld MetAP1 competitively and demonstrate higher binding along with druglikeness. • The stable complexes between Ld MetAP1 and lead molecules provide structural insights of inhibition. [ABSTRACT FROM AUTHOR]

Cholesterol reduction by intracellular protozoan parasite Leishmania donovani (L. donovani) , causative agent of leishmaniasis, impairs antigen presentation, pro-inflammatory cytokine secretion and host-protective membrane-receptor signaling in macrophages. Here, we studied the miRNA mediated regulation of cholesterol biosynthetic genes to understand the possible mechanism of L. donovani- induced cholesterol reduction and therapeutic importance of miRNAs in leishmaniasis. System-scale genome-wide microtranscriptome screening was performed to identify the miRNAs involved in the regulation of expression of key cholesterol biosynthesis regulatory genes through miRanda3.0. 11 miRNAs out of 2823, showing complementarity with cholesterol biosynthetic genes were finally selected for expression analysis. These selected miRNAs were differentially regulated in THP-1 derived macrophages and in primary human macrophages by L. donovani. Correlation of expression and target validation through luciferase assay suggested two key miRNAs, hsa-miR-1303 and hsa-miR-874-3p regulating the key genes hmgcr and hmgcs1 respectively. Inhibition of hsa-mir-1303 and hsa-miR-874-3p augmented the expression of targets and reduced the parasitemia in macrophages. This study will also provide the platform for the development of miRNA-based therapy against leishmaniasis. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a neglected tropical disease that can be lethal if not treated. The available medicines have severe side effects, such as toxicity and drug resistance. Various investigations are looking into new anti-leishmanial compounds from natural products that have little impact on host cells. Lupeol, a triterpenoid present in the flora of many edible plants, has been shown to have antimicrobial properties. The present study investigated the immunomodulatory effects of lupeol on U937 macrophages infected with Leishmania donovani , focusing on the expression of key cytokines and enzymes involved in the immune response. U937 macrophages were infected with Leishmania donovani amastigotes and treated with varying concentrations of lupeol throughout three days. The expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured using real-time polymerase chain reaction (RT-PCR). A positive simulation of gene expression was estimated using ΔΔCT to assess relative expression. The results demonstrated that lupeol significantly upregulated iNOS and TNF-α expression, especially at higher concentrations, indicating enhanced pro-inflammatory and anti-leishmanial activity. Interestingly, IL-10 expression also increased, suggesting a complex immunomodulatory role of lupeol that involves both pro-inflammatory and anti-inflammatory pathways. Pearson correlation analysis revealed a strong association between iNOS and TNF-α (0.97692), as well as a moderate correlation between iNOS and IL-10 (0.51603). These findings suggest that lupeol may promote a balanced immune response, enhancing the body's ability to combat L. donovani while potentially mitigating excessive inflammation. Lupeol can potentially serve as a novel therapeutic agent against visceral leishmaniasis. [ABSTRACT FROM AUTHOR]

Sri Lanka reports a large epidemic of cutaneous leishmaniasis (CL) caused by an atypical L. donovani while regional leishmaniasis elimination drive aims at achieving its targets in 2020. Visceralization, mucotrophism, and CL associated poor treatment response were recently reported. Long-term clinico-epidemiological trends (2001-2013) in this focus were examined for the first time. Both constant and changing features were observed. Sociodemographic patient characteristics that differ significantly from those of country profile, microchanges within CL profile, spatial expansion, constant biannual seasonal variation, and nondependency of clinical profile on age or gender were evident. Classical CL remains the main clinical entity without clinical evidence for subsequent visceralization indicating presence of parasite strain variation. These observations make a scientific platform for disease control preferably timed based on seasonal variation and highlights the importance of periodic and continued surveillance of clinic-epidemiological and other characteristics.

Abstract Background We report a clinically challenging and unusual case of L. donovani oral mucosal leishmaniasis. Case presentation Israeli resident with a former travel to central and North Africa, with no documented or prior cutaneous lesions presented with oral lesions of the maxillary gingiva and the upper lip. A delay in diagnosis and treatment have led to progression of the maxillary gingival lesions towards the hard palatal and the soft palate that could have potentially compromised the upper airway. Conclusions This case highlights the importance of early diagnosis of leishmaniasis in patients with oral lesions and the laboratory workup necessary to appropriately characterize and treat the disease.