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

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

FVB/NJ strain as a mouse model for cutaneous leishmaniasis by Leishmania (L.) amazonensis

BACKGROUND Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.

Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein­protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.

Assessing nystatin cream treatment efficacy against Leishmania (L.) amazonensis infection in BALB/c model.

The current scenario for cutaneous leishmaniasis treatment includes the use of first and second-choice drugs, both therapeutic strategies presenting several adverse effects and being related to an increment of treatment-refractory parasite strains. These facts encourage the search for new treatment approaches, including repositioning drugs, such as nystatin. Although in vitro assays show that this polyene macrolide compound has leishmanicidal activity, no in vivo evidence for a similar activity has been shown so far for the commercial nystatin cream formulation. This work assessed the effects of nystatin cream (25,000 IU/g) administered on mice in an amount to completely cover the paw surface of BALB/c mice infected with Leishmania (L.) amazonensis once a day, until a total of up to 20 doses. The data presented herein points to unequivocal evidence that treatment with this formulation causes a statistically significant reduction of swelling/edema in mice paws when compared to animal groups not submitted to this treatment regimen after the fourth week of infection: lesion sizes at the sixth (p = 0.0159), seventh (p = 0.0079) and eighth (p = 0.0079) week. Furthermore, swelling/edema reduction relates to a decrease in parasite load in the footpad (∼48%) and in draining lymph nodes (∼68%) at eight weeks post-infection. This is the first report of the effectiveness of nystatin cream used as a topical treatment in BALB/c model for cutaneous leishmaniasis.

Unsaturated lipids modulating the interaction of the antileishmanial isolinderanolide E with models of cellular membranes.

The present work evaluated the antiprotozoal activity of isolinderanolide E, isolated from the Brazilian plant Nectandra oppositifolia, against promastigote forms of Leishmania (Leishmania) amazonensis. The compound exhibited an EC50 value of 20.3 µM, similar to the positive control miltefosine (IC50 of 19.4 µM), and reduced toxicity to macrophages (CC50 > 200 µM). Based on these results, Langmuir monolayers of two unsaturated lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were employed as a model of mammalian and parasite membranes, respectively, to study the interaction of isolinderanolide E at a molecular level. The films were characterized with tensiometry (surface pressure-area isotherms and surface pressure-time curves), infrared spectroscopy, and Brewster angle microscopy (BAM). This compound changed the profile of the isotherms leading to fluid DOPC and DOPE monolayers, which were not able to attain rigid states even with compression. Infrared spectroscopy showed that the bioactive compound decreases the trans/gauche ratio conformers related to the molecular conformational disorder. BAM showed the formation of specific aggregates upon drug incorporation. In conclusion, isolinderanolide E changes the thermodynamic, mechanical, structural, and morphological characteristics of the monolayer of these unsaturated lipids, which may be essential to understand the action at the molecular level bioactives in biointerfaces.

Innate Resistance to Leishmania amazonensis Infection in Rat Is Dependent on NOS2.

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2 -/- rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2 -/- rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

Galectin-3 plays a protective role in Leishmania (Leishmania) amazonensis infection.

Leishmania (L.) amazonensis is one of the species responsible for the development of cutaneous leishmaniasis in South America. After entering the vertebrate host, L. (L.) amazonensis invades mainly neutrophils, macrophages and dendritic cells. Studies have shown that gal-3 acts as a pattern recognition receptor. However, the role of this protein in the context of L. (L.) amazonensis infection remains unclear. Here, we investigated the impact of gal-3 expression on experimental infection by L. (L.) amazonensis. Our data showed that gal-3 plays a role in controlling parasite invasion, replication and the formation of endocytic vesicles. Moreover, mice with gal-3 deficiency showed an exacerbated inflammatory response. Taken together, our data shed light to a critical role of gal-3 in the host response to infection by L. (L.) amazonensis.

Constitutive nitric oxide synthase-like enzyme in two species involved in cutaneous and mucocutaneous leishmaniasis.

Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/- 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/- 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.

Effects of extracellular vesicles released by peritoneal B-1 cells on experimental Leishmania (Leishmania) amazonensis infection.

B-1 cells are a B-lymphocyte subtype whose roles in immunity are not completely defined. These cells can produce cytokines (mainly IL-10) and natural and specific antibodies. Currently, extracellular vesicles (EVs) released by immune cells have emerged as new important entities in cell-cell communication. Immune cells release EVs that can activate and/or modulate other immune cells. Here, we characterized the EVs released by peritoneal B-1 cells infected or not with Leishmania (Leishmania) amazonensis. This Leishmania species causes cutaneous leishmaniasis and can infect macrophages and B-1 cells. Our results showed that peritoneal B-1 cells spontaneously release EVs, but the parasite stimulated an increase in EVs production by peritoneal B-1 cells. The treatment of BALB/c and C57BL/6 bone marrow-derived macrophages (BMDM) with EVs from infected peritoneal B-1 cells led to differential expression of iNOS, IL-6, IL-10, and TNF-α. Additionally, BALB/c mice previous treated with EVs released by peritoneal B-1 cells showed a significant lower lesion size and parasite burden. Thus, this study demonstrated that peritoneal B-1 cells could release EVs that can alter the functions of macrophages in vitro and in vivo these EVs altered the course of L. amazonensis infection. These findings represent the first evidence that EVs from peritoneal B-1 cells can act as a new mechanism of cellular communication between macrophages and B-1 cells, contributing to immunity against experimental leishmaniasis.

Efficacy of Topical Treatment with (-)-Epigallocatechin Gallate, A Green Tea Catechin, in Mice with Cutaneous Leishmaniasis.

The treatment of leishmaniasis includes pentavalent antimony drugs but, because of the side effects, toxicity and cases of treatment failure or resistance, the search of new antileishmanial compounds are necessary. The aims of this study were to evaluate and compare the in vitro antileishmanial activity of four green tea catechins, and to assess the efficacy of topical (-)-epigallocatechin gallate in a cutaneous leishmaniasis model. The antileishmanial activity of green tea catechins was evaluated against intracellular amastigotes, and cytotoxicity was performed with human monocytic cell line. BALB/c mice were infected in the ear dermis with Leishmania (Leishmania) amazonensis and treated with topical 15% (-)-epigallocatechin gallate, intraperitoneal Glucantime, and control group. The efficacy of treatments was evaluated by quantifying the parasite burden and by measuring the lesions size. (-)-Epigallocatechin gallate and (-)-epigallocatechin were the most active compounds with IC50 values <59.6 µg/mL and with a selectivity index >1. Topical treatment with (-)-epigallocatechin gallate decreased significantly both lesion size and parasite burden (80.4% inhibition) compared to control group (p < 0.05), and moreover (-)-epigallocatechin gallate showed a similar efficacy to Glucantime (85.1% inhibition), the reference drug for leishmaniasis treatment.

Nitric oxide-loaded chitosan nanoparticles as an innovative antileishmanial platform.

Leishmaniasis is a neglected tropical disease that demands for new therapeutic strategies due to adverse side effects and resistance development promoted by current drugs. Nitric oxide (NO)-donors show potential to kill Leishmania spp. but their use is limited because of their instability. In this work, we synthesize, characterize, and encapsulate S-nitroso-mercaptosuccinic acid into chitosan nanoparticles (NONPs) and investigate their activity on promastigotes and intracellular amastigotes of Leishmania (Leishmania) amazonensis. Cytotoxicity on macrophages was also evaluated. We verified that NONPs reduced both forms of the parasite in a single treatment. We also noticed reduction of parasitophorous vacuoles as an evidence of inhibition of parasite growth and resolution of infection. No substantial cytotoxicity was detected on macrophages. NONPs were able to provide a sustained parasite killing for both L. (L.) amazonensis infective stages with no toxicity on macrophages, representing a promising nanoplatform for cutaneous leishmaniasis.

Nitro-Heterocyclic compounds induce apoptosis-like effects in Leishmania (L). amazonensis promastigotes.

BACKGROUND: Three drugs - pentavalent antimonials, amphotericin B and pentamidine - are currently used for leishmaniasis treatment. They are administered for long periods, only parenterally, and have high cardiac, renal and hepatic toxicities. Therefore, the investigation of new compounds is required. Nitro-heterocyclic derivatives have been used as possible drug candidates to treat diseases caused by trypanosomatids. METHODS: Leishmania (L.) amazonensis promastigotes (MHO/BR/73/M2269), maintained in the Laboratório de Soroepidemiologia - Instituto de Medicina Tropical- USP, were exposed to five nitroheterocyclic derivatives, with differences at phenyl-ring position 4: BSF-C4H9, BSF-H, BSF-NO2, BSF-CH3 and BSF-Cl, for 48 hours. After analyzing viability (MTT assay), we evaluated cellular-morphology activity of compounds by transmission electron microscopy (TEM) and measurement of apoptosis (phosphatidylserine expression) by flow cytometry. RESULTS: EC50 of amphotericin B and BSF-CH3 were 0.50 (M and 0.39 (M respective. Other nitro-heterocyclic compounds presented EC50 higher than amphotericin B. All compounds showed greater AV- and PI-positive expression than amphotericin B at 100 (M, except BSF-NO2. TEM showed complete nuclear disfigurement with 100 (M of BSF-NO2, 25 and 6.25 (M of BSF-H, and 6.25 (M BSF-Cl; presence of vesicles within the flagellar pocket with 25 (M BSF-H; alteration of the kinetoplast with 25 (M BSF-C4H9, 25 (M of BSF-H, 6.25 (M BSF-CH3 and 6.25 (M of BSF-Cl. CONCLUSIONS: Nitro-heterocyclic compounds have shown activity against promastigotes of L. amazonensis, at lower concentrations. However, improvement of compound scaffolds are needed to assist the elucidation of the mechanism of action and to achieve greater activity.

What makes mucosal and anergic diffuse cutaneous leishmaniases so clinically and immunopathogically different? A review in Brazil.

American cutaneous leishmaniasis (ACL) is a parasitic protozoan disease caused by different Leishmania species widely distributed throughout Latin America. Fifteen Leishmania species belonging to the subgenera Viannia, Leishmania and Mundinia are known to cause ACL. Seven of these species are found in Brazil, of which Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis have the highest potential to cause mucosal (ML) and anergic diffuse cutaneous leishmaniasis (DCL), respectively, the most severe forms of ACL. The clinical and immunopathological differences between these two clinical forms are reviewed here, taking into account their different physiopathogenic mechanisms of dissemination from cutaneous lesions to mucosal tissues in the case of ML and to almost all body surfaces in the case of anergic DCL. We also discuss some immunopathogenic mechanisms of species-specific Leishmania antigens (from the subgenera Viannia and Leishmania) that are most likely associated with the clinical and immunopathological differences between ML and anergic DCL. Those discussions emphasize the pivotal importance of some surface antigens of L. (V.) braziliensis and L. (L.) amazonensis, such as lipophosphoglycan, phosphatidylserine and CD200 (an immunoregulatory molecule that inhibits macrophage activation), that have been shown to exert strong influences on the clinical and immunopathological differences between ML and anergic DCL.

Tocopherol biosynthesis in Leishmania (L.) amazonensis promastigotes.

Leishmaniasis is a neglected disease caused by a trypanosomatid protozoan of the genus Leishmania. Most drugs used to treat leishmaniasis are highly toxic, and the emergence of drug-resistant strains has been observed. Therefore, new therapeutic targets against leishmaniasis are required. Several isoprenoid compounds, including dolichols or ubiquinones, have been shown to be important for cell viability and proliferation in various trypanosomatid species. Here, we detected the biosynthesis of tocopherol in Leishmania (L.) amazonensis promastigotes in vitro through metabolic labelling with [1-(n)-3H]-phytol. Subsequently, we confirmed the presence of vitamin E in the parasite by gas chromatography-mass spectrometry. Treatment with usnic acid or nitisinone, inhibitors of precursors of vitamin E synthesis, inhibited growth of the parasite in a concentration-dependent manner. This study provides the first evidence of tocopherol biosynthesis in a trypanosomatid and suggests that inhibitors of the enzyme 4-hydroxyphenylpyruvate dioxygenase may be suitable for use as antileishmanial compounds. Database: The amino acid sequence of a conserved hypothetical protein [Leishmania mexicana MHOM/GT/2001/U1103] has been deposited in GenBank (CBZ28005.1).

Insights into the tracking of the cysteine proteinase B COOH-terminal polypeptide of Leishmania (Leishmania) amazonensis by surface plasmon resonance.

Leishmania (Leishmania) amazonensis has adaptive mechanisms to the host environment that are guided by its proteinases, including cysteine proteinase B (CPB), and primarily its COOH-terminal region (Cyspep). This work aimed to track the fate of Cyspep by surface plasmon resonance (SPR) of promastigotes and amastigotes to gain a greater understanding of the adaptation of this parasite in both hosts. This strategy consisted of antibody immobilization on a COOH1 surface, followed by interaction with parasite proteins and epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64). Pro-CPB and Cyspep were detected using specific polyclonal antibodies against a recombinant Cyspep in both parasite forms. The parasitic supernatants from amastigotes and promastigotes exhibited higher anti-Cyspep recognition compared with that in the subcellular fractions. As the supernatant of the promastigote cultures exhibited resonance unit values indicative of an effective with to E-64, this result was assumed to be Pro-CPB detection. Finally, after using three sequential SPR assay steps, we propose that amastigotes and promastigotes release Cyspep into the extracellular environment, but only promastigotes release this polypeptide as Pro-CPB.

Nitro-Heterocyclic compounds induce apoptosis-like effects in Leishmania (L). amazonensis promastigotes

Abstract Background: Three drugs - pentavalent antimonials, amphotericin B and pentamidine - are currently used for leishmaniasis treatment. They are administered for long periods, only parenterally, and have high cardiac, renal and hepatic toxicities. Therefore, the investigation of new compounds is required. Nitro-heterocyclic derivatives have been used as possible drug candidates to treat diseases caused by trypanosomatids. Methods: Leishmania (L.) amazonensis promastigotes (MHO/BR/73/M2269), maintained in the Laboratório de Soroepidemiologia - Instituto de Medicina Tropical- USP, were exposed to five nitroheterocyclic derivatives, with differences at phenyl-ring position 4: BSF-C4H9, BSF-H, BSF-NO2, BSF-CH3 and BSF-Cl, for 48 hours. After analyzing viability (MTT assay), we evaluated cellular-morphology activity of compounds by transmission electron microscopy (TEM) and measurement of apoptosis (phosphatidylserine expression) by flow cytometry. Results: EC50 of amphotericin B and BSF-CH3 were 0.50 (M and 0.39 (M respective. Other nitro-heterocyclic compounds presented EC50 higher than amphotericin B. All compounds showed greater AV- and PI-positive expression than amphotericin B at 100 (M, except BSF-NO2. TEM showed complete nuclear disfigurement with 100 (M of BSF-NO2, 25 and 6.25 (M of BSF-H, and 6.25 (M BSF-Cl; presence of vesicles within the flagellar pocket with 25 (M BSF-H; alteration of the kinetoplast with 25 (M BSF-C4H9, 25 (M of BSF-H, 6.25 (M BSF-CH3 and 6.25 (M of BSF-Cl. Conclusions: Nitro-heterocyclic compounds have shown activity against promastigotes of L. amazonensis, at lower concentrations. However, improvement of compound scaffolds are needed to assist the elucidation of the mechanism of action and to achieve greater activity.

PD-L1 May Mediate T-Cell Exhaustion in a Case of Early Diffuse Leishmaniasis Caused by Leishmania (L.) amazonensis.

Introduction: Diffuse cutaneous leishmaniasis (DCL) is a rare disease form associated with Leishmania (L.) amazonensis in South America. It represents the "anergic" pole of American Tegumentary Leishmaniasis, and the explanation for its resistance to treatment remains elusive. We aimed to study some possible immunological mechanisms involved in the poor DCL treatment response by evaluating some cell surface molecules obtained from a patient with DCL by flow cytometry. Case presentation: A 65-year-old DCL patient who initially failed to respond to the standard treatment for the disease showed vacuolated macrophages filled with amastigotes in lesion biopsy, and L. (L.) amazonensis was identified through ITS1PCR amplification. The Leishmania skin test and indirect immunofluorescence analysis revealed negative results. Peripheral blood from the patient was collected after a few months of treatment, when the patient presented with no lesion. Peripheral blood mononuclear cells were analyzed ex vivo and in vitro after 48 h of stimulation with soluble L. (L.) amazonensis antigen (SLA). Cell death, surface molecules, and intracellular molecules, such as IFN-γ and granzyme B, were analyzed in the cells using flow cytometry. Analysis of the surface markers showed an increased expression of the inhibitory molecule programmed death ligand 1 (PD-L1) in the monocytes restimulated with SLA (approximately 65%), whereas the negative controls were 35% positive for PD-L1. Conversely, compared with the negative controls, we observed a decrease in CD4+IFN-γ+ T cells (8.32 versus 1.7%) and CD8+IFN-γ+ T cells (14% versus 1%). We also observed a relevant decrease in the granzyme B levels in the CD8+ T cells, from 31% in the negative controls to 5% after SLA restimulation. Conclusion: The dysfunctional activation of PD-L1 inhibitory pathway after Leishmania antigen stimulation and reduced levels of IFN-gamma and granzyme B-producing cells could be closely related to unresponssiveness to standard drug treatment of DCL patient.

The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis.

BACKGROUND: Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. METHODS: DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. RESULTS: The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. CONCLUSIONS: A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

Distinct courses of infection with Leishmania (L.) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice.

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

Clinical and epidemiological features of leishmaniasis in northwestern-Argentina through a retrospective analysis of recent cases.

Leishmaniasis is a parasitic disease caused by hemoflagellates of the genus Leishmania and is transmitted to humans by the bite of infected phlebotomine sandflies. Depending on the Leishmania species, the disease has different clinical forms including cutaneous, mucocutaneous, and visceral manifestations. Previous studies performed in endemic zones of northwestern-Argentina, during epidemic outbreaks, have been important for detecting patients suffering from the acute phase of the disease, but have not given a complete representation of the clinical and epidemiological features in the region. Furthermore, due to the resurgence of leishmaniasis worldwide and in particular the large increase of international tourism to the region, it seems pertinent to update the current epidemiological and clinical profile of leishmaniasis in northwestern-Argentina. Here we present a retrospective analysis of 95 Leishmania positive cases, presenting between 2000 and 2014. Patients were derived from hospitals and diagnosed in our lab at the University of Salta, located in a non-endemic area in Salta, Argentina. We detected numerous extensive mucocutaneous cases (34/95, 35.8%) distinct from mucosal affected patients, some instances originating in locations with no previously reported human cases. Additionally patients suffering from concomitant diseases, besides leishmaniasis, were assessed. These included Chagas disease, syphilis, deep mycoses, tuberculosis, toxoplasmosis and intestinal parasitosis. This study updates the clinical and epidemiological features of leishmaniasis in northwestern-Argentina, and discusses the implications and management strategy for patients who acquire the disease in this region.