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Leishmaniasis causes high mortality and morbidity in tropical and subtropical regions of Africa, Asia, the Americas and southern Europe, and is characterized by diverse clinical manifestations. As a neglected tropical disease, limited resources are allocated for antileishmanial drug discovery. The Leishmania parasite is deficient in de novo purine synthesis, and therefore acquires purines from the host and processes these using a purine salvage pathway. By making use of purine transport systems and interfering with this salvage pathway, purine (nucleoside) analogues might exert a selective detrimental impact on its growth and survival. In vitro screening of an in-house purine nucleoside library and analogue synthesis afforded the 6-methyl-7-(2-pyridyl)-7-deazapurine ribonucleoside analogue 18 as a promising hit. Optimization of the 7-substituent afforded 31 and 32 which displayed potent activity against wild-type and resistant L. infantum, intracellular amastigote and extracellular promastigote forms, and favorable selectivity versus primary mouse macrophages (Mφ) and MRC-5 cells. Encouraged by the favorable in vitro metabolic stability of 32, an in vivo study was performed using an early curative L. infantum hamster model. When orally administrated at 50 mg/kg once daily (s.i.d) for 10 days, 32 was devoid of side effects, however, it only poorly reduced amastigote burdens in the major target organs., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Chagas disease and leishmaniasis are two poverty-related neglected tropical diseases that cause high mortality and morbidity. Current treatments suffer from severe limitations and novel, safer and more effective drugs are urgently needed. Both Trypanosoma cruzi and Leishmania are auxotrophic for purines and absolutely depend on uptake and assimilation of host purines. This led us to successfully explore purine nucleoside analogues as chemotherapeutic agents against these and other kinetoplastid infections. This study extensively explored the modification of the 6-amino group of tubercidin, a natural product with trypanocidal activity but unacceptable toxicity for clinical use. We found that mono-substitution of the amine with short alkyls elicits potent and selective antitrypanosomal and antileishmanial activity. The methyl analogue 15 displayed the best in vitro activity against both T. cruzi and L. infantum and high selectivity versus host cells. Oral administration for five consecutive days in an acute Chagas disease mouse model resulted in significantly reduced peak parasitemia levels (75, 89 and 96% with 12.5, 25 and 50 mg/kg/day, respectively). as well as increased animal survival rates with the lower doses (83 and 67% for 12.5 and 25 mg/kg/day, respectively)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Background: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed., Methodology: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection., Principal Findings: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed., Conclusions/significance: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts., Methods: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve., Results: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10 -3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents., Conclusions: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Background: Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia., Methods: Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active., Principal Findings: A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight., Conclusion: In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control., Competing Interests: The authors have declared that no competing interests exist.

In vitro growth (inhibition) assays have a dual application, either supporting the discovery of novel drugs or as a monitoring tool of drug resistance in patient isolates. From an experimental design point of view, both are quite different with regard to the infecting Leishmania species and strain, the wide variety of permissive host cells (primary cells versus cell lines), drug exposure times, detection methods and endpoint criteria. Recognizing the need for enhanced assay standardization to decrease interlaboratory variation and improve proper interpretation of results, a detailed description is given of the basic fundamental procedures and requirements for routine in vitro growth of Leishmania spp. with specific focus on the intracellular amastigote susceptibility assay. Although the described experimental procedures focus on visceral Leishmania species, the same assay principles may apply for the cutaneous species as well.

Leishmaniasis is a neglected tropical disease that affects about 350 million individuals worldwide. The protozoan parasite has a relatively simple life cycle with two principal stages: the flagellated mobile promastigote living in the gut of the sandfly vector and the intracellular amastigote within phagolysosomal vesicles of the vertebrate host macrophage. This review presents a state-of-the-art overview of the redox biology at the parasite-macrophage interface. Although Leishmania species are susceptible in vitro to exogenous superoxide radical, hydrogen peroxide, nitric oxide, and peroxynitrite, they manage to survive the endogenous oxidative burst during phagocytosis and the subsequent elevated nitric oxide production in the macrophage. The parasite adopts various defense mechanisms to cope with oxidative stress: the lipophosphoglycan membrane decreases superoxide radical production by inhibiting NADPH oxidase assembly and the parasite also protects itself by expressing antioxidant enzymes and proteins. Some of these enzymes could be considered potential drug targets because they are not expressed in mammals. In respect to antileishmanial therapy, the effects of current drugs on parasite-macrophage redox biology and its involvement in the development of drug resistance and treatment failure are presented., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani, L. guyanensis, and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering log-phase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25 degrees C to pH 5.4 for 24 h in Schneider's medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (Sb(V); sodium stibogluconate), trivalent antimony (Sb(III); potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC(50)] < 10 microM) and PX-6518 (IC(50) < 2 microg/ml) but showed distinct susceptibility to Sb(V) and/or Sb(III), depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous Sb(V)-Sb(III) resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of Sb(V)-Sb(III) susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.

Introduction: Saponins are natural products that are well known for a wide range of biological activities. For saponins of Maesa balansae, selective antileishmanial activity has been described., Objective: In view of their pharmacological interest, several Maesa species from the National Botanical Garden of Meise (Belgium) and wild-grown plants from Vietnam were screened for their antileishmanial potential and saponin content., Methodology: Different parts of the plants (mainly leaves and twigs) were collected, dried and extracted. Plant extracts were evaluated by liquid chromatography/mass spectrometry (LC-MS) using electrospray ionisation in the negative ion mode and their saponin content was compared with those of Maesa balansae (maesabalides) and Maesa lanceolata (maesasaponins)., Results: Several Maesa species (M. ambigua, M. argentea, M. brevipaniculata, M. japonica and M. perlarius) showed potent antileishmanial activity (<0.1 microg/mL) and indeed contained known maesasaponins and maesabalides. However the leaves of M. argentea also revealed two new compounds. Two saponins with [M - H]- ions at m/z 1465 and 1477 were characterised. Their mass spectrometric fragmentation pattern revealed a structure that was the same or closely related to maesasaponin V.3 and VI.2, respectively, but had a glycan part with one additional hexose residue., Conclusion: Several known as well as new saponins from Maesa species active against leishmaniasis were characterised using LC-MS., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2–3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits. Author summary: Quiescence and post-treatment relapse are crucial aspects of treatment failure across the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence in both human and mouse bone marrow stem cells, but not in macrophages. Besides a reduced size, quiescent amastigotes show signs of a rapid evolutionary adaptation with notable genetic alterations. Transitioning through a quiescent state allows escape from treatment, efficient transmission by sand flies and the acquisition of an increased cellular infectivity. Transcriptional profiling of quiescent and non-quiescent parasites isolated from the stem cell niche confirmed a generalized transcriptional downregulation as a hallmark of quiescence. [ABSTRACT FROM AUTHOR]

Type I interferons (IFNs) induced by an endogenous Leishmania RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous Leishmania parasites, however, the impact of type I IFNs in visceral Leishmania infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with L. infantum and L. donovani and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent L. infantum promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral Leishmania promastigotes and that targeting of Sn may have some protective effects in early infection. [ABSTRACT FROM AUTHOR]

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity. Author summary: Leishmaniasis is a neglected tropical disease caused by parasites belonging to the genus of Leishmania and transmitted by the bite of infected female sand flies. Concerns about the effective control of the disease are rising in view of the increasing number of treatment failures that may be related to drug resistance. Monitoring of drug susceptibility in the field should become an essential asset, however, there is still insufficient harmonization in the laboratory assays. This study focused on the standard intracellular amastigote susceptibility assay and compared protocol variables, such as type of macrophage host cell (primary versus cell lines), multiplicity of infection and duration of drug exposure. Primary cells perform best with little difference between cells derived from Swiss mice or BALB/c mice. From a practical point of view, mouse peritoneal exudate cells can be recommended. If mice would not be available, THP-1 cells are the best alternative. For field strains, metacyclic promastigotes should be used at a multiplicity of infection of 10–15 parasites per cell with drug exposure starting at 24h post-infection and continued for 120h. Unfortunately, susceptibility testing of clinical isolates will remain problematic because of the reliance on promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity. Opting for cell-based assays may be complicated by the fact that dedicated laboratory infrastructure may sometimes be lacking in disease-endemic countries. [ABSTRACT FROM AUTHOR]

Background: Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. Principal findings: Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. Conclusions: These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance. [ABSTRACT FROM AUTHOR]

Objectives: This study evaluated the pharmacokinetic properties of oleylphosphocholine (OlPC) in hamsters following a single oral dose. Its prophylactic activity was tested to establish exposure-activity relationships, while a 5 + 5 day oral regimen at 20 mg/kg with long post-treatment follow-up was performed to assess its curative potential.Methods: Single oral doses of 20, 50 and 100 mg/kg were administered for pharmacokinetic analysis while a 100 mg/kg single oral dose was given on day 7, 4 or 1, or 4 h prior to infection in the prophylactic efficacy study. The animals were infected on day 0 with Leishmania infantum and the resulting parasite burdens were measured in target organs on day 21. In the curative model, treatment started on day 21 post-infection at 20 mg/kg for 5 + 5 days and amastigote burdens were determined in target organs either on day 42 [10 days after the end of treatment (dpt)] or day 72 (40 dpt).Results: OlPC showed elimination t1/2 of ∼50 h and dose-proportional exposure. The prophylactic action of OlPC was in agreement with model-simulated drug exposures, showing dose-dependent residual activity. Interestingly, the 100 mg/kg single dose administered 4 days before infection (day -4) still reduced the overall parasite burden by ∼50%. In the curative model, >99% clearance of infection was observed at 10 dpt in all OlPC-treated animals and remained so at 40 dpt.Conclusions: This study reveals that total plasma exposure (AUCt-∞) correlates well with the prophylactic and curative efficacy of OlPC in the L. infantum hamster model. [ABSTRACT FROM AUTHOR]

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

Objectives: Increasing numbers of miltefosine treatment failures in visceral leishmaniasis therapy and reports of miltefosine resistance in the Indian subcontinent resulted in the recommendation to use liposomal amphotericin B as first-line therapy. Cross-resistance between miltefosine and amphotericin B has recently been documented, suggesting a role of mutations in the miltefosine transporter, a complex encoded by the MT and ROS3 genes. This study aimed to further explore the putative role of MT/ROS3 defects in the molecular basis of amphotericin B cross-resistance.Methods: The susceptibility profiles of different miltefosine-resistant Leishmania infantum strains with well-characterized mutations in the transporter complex and the corresponding episomally restored susceptible parasite lines were determined using both the routine extracellular promastigote assay and the intracellular amastigote assay.Results: In vitro amastigote and promastigote susceptibility testing of the two miltefosine-resistant and the episomally reconstituted L. infantum lines revealed full susceptibility to amphotericin B, despite the variable miltefosine susceptibility profile.Conclusions: Mutations present in either the MT and/or ROS3 gene are not sufficient to elicit higher tolerance to amphotericin B. Additional synergistic adaptations may be responsible for the miltefosine/amphotericin B cross-resistance described earlier. [ABSTRACT FROM AUTHOR]

The search for new therapeutic agents from natural sources has been a constant for the treatment of diseases such as leishmaniasis. Herein, in vitro and in vivo pharmacological activities of pure major phenolic constituents (caffeic acid, chlorogenic acid, ferulic acid, quercetin, and rosmarinic acid) from Pluchea carolinensis against Leishmania amazonensis are presented . Pure compounds showed inhibitory activity against promastigotes (IC = 0.2-0.9 μg/mL) and intracellular amastigotes (IC = 1.3-2.9 μg/mL). Four of them were selected after testing against macrophages of BALB/c mice: caffeic acid, ferulic acid, quercetin, and rosmarinic acid, with selective indices of 11, 17, 10, and 20, respectively. Ferulic acid, rosmarinic acid, and caffeic acid controlled lesion size development and parasite burden in footpads from BALB/c experimentally infected mice, after five injections of compounds by intralesional route at 30 mg/kg every 4 days. Pure compounds from P. carolinensis demonstrated antileishmanial properties. [ABSTRACT FROM AUTHOR]

Chromatographic separation of the n-hexane extract of the aerial part of Plectranthus barbatus led to the isolation of five abietane-type diterpenes: dehydroabietane (1); 5,6-didehydro-7-hydroxy-taxodone (2); taxodione (3); 20-deoxocarnosol (4) and 6a,11,12,-trihydroxy-7β,20-epoxy-8,11,13-abietatriene (5). The structures were determined using spectroscopic methods including one- and two-dimensional NMR methods. Compounds (1)-(3) and (5) are isolated here for the first time from the genus Plectranthus. The isolated abietane-type diterpenes tested in vitro for their antiprotozoal activity against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxicity was determined against fibroblast cell line MRC-5. Compound (2) 5,6-didehydro-7-hydroxy-taxodone showed remarkable activity with acceptable selectivity against P. falciparum (IC50 9.2 µM, SI 10.4) and T. brucei (IC50 1.9 µM, SI 50.5). Compounds (3)-(5) exhibited non-specific antiprotozoal activity due to high cytotoxicity. Compound (1) dehydroabietane showed no antiprotozoal potential. [ABSTRACT FROM AUTHOR]

Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases ( n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly ( r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation ( r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions ( n = 7, r = 0.78, p = 0.046) and MIL-induced parasites ( r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients. [ABSTRACT FROM AUTHOR]

Background: Worldwide particularly in developing countries, a large proportion of the population is at risk for tropical parasitic diseases. Several medicinal plants are still used traditionally against protozoal infections in Yemen and Saudi Arabia. Thus the present study investigated the in vitro antiprotozoal activity of twenty-five plants collected from the Arabian Peninsula. Methods: Plant materials were extracted with methanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi and free trypomastigotes of T. brucei. Cytotoxic activity was determined against MRC-5 cells to assess selectivity. The criterion for activity was an IC50<10 µg/ml (<5 µg/ml for T. brucei) and selectivity index of >4. Results: Antiplasmodial activity was found in the extracts of Chrozophora oblongifolia, Ficus ingens, Lavandula dentata and Plectranthus barbatus. Amastigotes of T. cruzi were affected by Grewia erythraea, L. dentata, Tagetes minuta and Vernonia leopoldii. Activity against T. brucei was obtained in G. erythraea, L. dentata, P. barbatus and T. minuta. No relevant activity was found against L. infantum. High levels of cytotoxicity (MRC-5 IC50<10 µg/ml) and hence non-specific activities were noted in Cupressus sempervirens, Kanahia laniflora and Kniphofia sumarae. Conclusion: The results endorse that medicinal plants can be promising sources of natural products with antiprotozoal activity potential. The results support to some extent the traditional uses of some plants for the treatment of parasitic protozoal diseases. [ABSTRACT FROM AUTHOR]

Background: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study. Methodology/Principal Findings: We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered. Conclusion/Significance: Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them. Author Summary: Leishmania is a group of parasites (Protozoa, Trypanosomatidae) responsible for a wide spectrum of clinical forms. Among the factors explaining this phenotypic polymorphism, parasite features are important contributors. One approach to identify them consists in characterizing the gene expression profiles throughout the life cycle. In a recent study, the transcriptome of 3 Leishmania species was compared and this revealed species-specific differences, albeit in a low number. A key issue, however, is to ensure that the observed differences are indeed species-specific and not specific of the strains selected for representing the species. In order to illustrate the relevance of this concern, we analyzed here the gene expression profiles of 5 clinical isolates of L. braziliensis at seven time points of the life cycle. Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics: one Leishmania strain is not necessarily representative of a given species. [ABSTRACT FROM AUTHOR]

Objectives The oleanane triterpene saponin PX-6518, with known potent in vitro and in vivo activity against Leishmania donovani, was investigated for its spectrum against the cutaneous species Leishmania mexicana, Leishmania panamensis and Leishmania major. Methods In vitro activity was based on the reduction of amastigotes in primary peritoneal mouse macrophages. BALB/c mice were injected with 2 × 106 amastigotes in the base of the tail (L. panamensis and L. major) or the foot (L. mexicana) and subcutaneously treated with PX-6518 [1–10 mg/kg body weight (BW)] or Pentostam® (250 mg/kg BW SbV eq). Evolution of skin lesions was monitored in a prophylactic dose-finding study, and early curative [6 weeks post-infection (pi)] and late curative (>8–10 weeks pi) studies. Results While moderate susceptibility to PX-6518 was obtained in vitro (IC50: 1–4 µg/mL), excellent in vivo activity was demonstrated. In the prophylactic study (six administrations on alternate days, starting at 1day pi), PX-6518 was 100% effective at 1 mg/kg BW against L. mexicana and L. panamensis, whereas L. major lesions could be prevented at 2 mg/kg BW. In the early curative (1 mg/kg BW once a week for 4 weeks) and late curative (1 mg/kg BW twice a week for 4 weeks) studies, PX-6518 completely healed L. mexicana and L. panamensis lesions, whereas L. major lesions were reduced by ∼50%. Conclusions This study demonstrates that PX-6518 possesses potent and broad-spectrum prophylactic and curative efficacy against cutaneous leishmaniasis in the BALB/c mouse model. L. major was the least susceptible species tested and parasitological cure could not be obtained. [ABSTRACT FROM PUBLISHER]

Background: Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these 'hostile' environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent. Methodology/Principal Findings: We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain. Conclusions/Significance: The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis. [ABSTRACT FROM AUTHOR]

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms. [ABSTRACT FROM AUTHOR]

The kinetoplastid parasites Trypanosoma brucei , Trypanosoma cruzi and Leishmania spp. are the causative agents of neglected tropical diseases with a serious burden in several parts of the world. These parasites are incapable of synthesizing purines de novo , and therefore rely on ingenious purine salvage pathways to acquire and process purines from their host. Purine nucleoside analogs that may interfere with these pathways therefore constitute a privileged source of new antikinetoplastid agents. In this study, we synthetized a collection of C -nucleosides employing five different heterocyclic nucleobase surrogates. C -nucleosides are chemically and enzymatically stable and allow for extensive structural modification. Inspired by earlier 7-deazaadenosine nucleosides and known antileishmanial C -nucleosides, we introduced different modifications tailored towards antikinetoplastid activity. Both adenosine and inosine analogs were synthesized with the aim of discovering new antikinetoplastid hits and expanding knowledge of structure-activity relationships. Several promising hits with potent activity against Trypanosoma brucei , Trypanosoma cruzi and Leishmania infantum were discovered, and the nature of the nucleobase surrogate was found to have a profound influence on the selectivity profile of the compounds. Image 1 • A collection of purine-like C -nucleosides comprising five different heterocyclic nucleobase surrogates was synthesized. • Modifications tailored towards antikinetoplastid activity were introduced. • Promising hits for Trypanosoma brucei , Trypanosoma cruzi and Leishmania infantum were discovered. • The nature of the heterocyclic nucleobase surrogate had a profound influence on the selectivity profile of the compounds. [ABSTRACT FROM AUTHOR]

The article discusses a study on the drug susceptibility of HIV-positive and HIV-negative patients to Leishmania infantum isolates. For the study, researchers examined the in vitro drug susceptibility phenotype of Brazilian Leishmania infantum isolates from the patients diagnosed with clinical visceral leishmaniasis (VL) before starting on antileishmanial treatment. Based on the findings, they concluded that there is a need for epidemiological monitoring of Leishmania infantum antimony.

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes. Drug resistance represents a major threat for the control of visceral leishmaniasis (VL). Monitoring the spread of resistance is essential to adjust control interventions and safeguard current and future drugs. Sand fly infection studies represent a useful laboratory tool to assess the transmission potential of resistant phenotypes. The resistance-conferring genetic alterations determine transmissibility by the vector. The possibility of recombination between different Leishmania strains and species in the vector may result in the formation of transmissible hybrids. [ABSTRACT FROM AUTHOR]

Sterol 14α-demethylase (CYP51) is a key enzyme involved in the survival and virulence of many parasite protozoa, such as Trypanosoma and Leishmania species, thus representing a valuable drug target for the treatment of Kinetoplastid diseases. A set of azole-based compounds selected from an in-house compound library was in vitro screened against different human protozoan parasites. Several compounds showed selective activity against Trypanosoma cruzi , with compound 7 being the most active (IC 50 = 40 nM). Given the structural similarity between the compounds here reported and known CYP51 inhibitors, a molecular docking study was performed to assess their binding with protozoal target and to rationalize the biological activity data. [ABSTRACT FROM AUTHOR]

Highlights: [•] Chenopodium ambrosioides oil showed potential antileishmanial activity. [•] Chenopodium oil caused better selectivity compared with its major components. [•] Products cause a breakdown of mitochondrial membrane potential and redox indexes. [•] Oil showed effect against Plasmodium falciparum and Trypanosoma brucei. [ABSTRACT FROM AUTHOR]

The antiplasmodial, antileishmanial, and antitrypanosomal activity of twenty-five medicinal plants distributed in Saudi Arabia and Yemen was evaluated. The plants were extracted withmethanol and screened in vitro against erythrocytic schizonts of Plasmodium falciparum, intracellular amastigotes of Leishmania infantum and Trypanosoma cruzi, and free trypomastigotes of T. brucei. To assess selectivity, cytotoxicity was determined on MRC-5 cells. Criteria for activity were an IC50 < 10 μg/mL and high selectivity (SI). Seven plants showed interesting antiprotozoal activity in one or more models. Extracts of Caralluma penicillata and Acalypha ciliata showed fairly good activity against P. falciparum with IC50 of 6.7 and 10.8 μg/mL and adequate selectivity (SI > 9.6 and > 5.9). Interesting activity against L. infantum was obtained with Verbascum bottae (IC50 of 3.2 μg/mL, SI 10.2) and Solanum glabratum (IC50 8.1 μg/mL, SI 3.4).The extracts of C. penicillata, Leucas virgata, Loranthus regularis, and V. bottae exhibited moderate activity against T. brucei (IC50 8.5, 8.1, 8.3, and 2.3 μg/mL; SI > 7.6, 7.7, 4.3, and > 14.1).These results partly support the traditional use of some of the selected medicinal plants and warrant further investigations into the putative active constituents. [ABSTRACT FROM AUTHOR]

Abstract: In order to understand the epidemiological dynamics of antimonial (SbV) resistance in zoonotic tegumentary leishmaniasis and its link with treatment outcome, we analyzed the population structure of 24 Peruvian Leishmania braziliensis clinical isolates with known in vitro antimony susceptibility and clinical phenotype by multilocus microsatellite typing (14 microsatellite loci). The genetic variability in the Peruvian isolates was high and the multilocus genotypes were strongly differentiated from each other. No correlation was found between the genotypes and in vitro drug susceptibility or clinical treatment outcome. The finding of a polyphyletic pattern among the SbV-resistant L. braziliensis might be explained by (i) independent events of drug resistance emergence, (ii) sexual recombination and/or (iii) other phenomena mimicking recombination signals. Interestingly, the polyphyletic pattern observed here is very similar to the one we observed in the anthroponotic Leishmania donovani (), hereby questioning the role of transmission and/or chemotherapeutic drug pressure in the observed population structure. [Copyright &y& Elsevier]