Analysis of clinical cure outcome, macrophages number, cytokines levels and expression of annexin-A1 in the cutaneous infection in patients with Leishmania braziliensis.

BACKGROUND: Leishmania braziliensis, a protozoan prevalent in Brazil, is the known causative agent of cutaneous leishmaniasis (CL). The activation of M1 macrophages is a pivotal factor in the host's ability to eliminate the parasite, whereas M2 macrophages may facilitate parasite proliferation. This study analyzed the clinical outcomes of CL and the patients' immunological profiles, focusing on the prevalence of M1 and M2 macrophages, cytokine production, and annexin-A1 (ANXA1) expression in the lesion. METHODS: Data were obtained by polymerase chain reaction (PCR) and histopathological, immunofluorescence, and cytokine analyses. RESULTS: Patients with exudative and cellular reaction-type (ECR)-type lesions that healed within 90 days showed a significant increase in M1. Conversely, patients with ECR and exudative and granulomatous reaction (EGR)types, who healed within 180 days, showed an elevated number of M2. Cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α were higher in ECR lesions that resolved within 90 days (P<0.05). In contrast, IL-9 and IL-10 levels significantly increased in both ECR and EGR lesions that healed after 180 days (P<0.001). The production of IL-21, IL-23 and TGF-ß was increased in patients with ECR or EGR lesions that healed after 180 days (P<0.05). The expression of ANXA1 was higher in M2 within ECR-type lesions in patients who healed after 180 days (P<0.05). CONCLUSIONS: These findings suggest that the infectious microenvironment induced by L. braziliensis affects the differentiation of M1 and M2 macrophages, cytokine release, and ANXA1 expression, thereby influencing the healing capacity of patients. Therefore, histopathological and immunological investigations may improve the selection of CL therapy.

Assessing proteases and enzymes of the trypanothione system in subpopulations of Leishmania (Viannia) braziliensis Thor strain during macrophage infection.

BACKGROUND: Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES: Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS: Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS: Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes' expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS: The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells.

A Case of Cutaneous Leishmaniasis with Mucosal Involvement in the Northern United States.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-borne parasitic infection endemic to many sub-tropical regions worldwide. In the Americas, Leishmania braziliensis is responsible for most reported CL cases. Variable symptom presentation and susceptibility to secondary infection make diagnosing CL a difficult proposition for physicians who may not encounter cases frequently. CASE REPORT: We present the case of a 50-year-old man with multiple progressive lesions, diagnosed initially as a bacterial infection, who presented to a North American emergency department after several unsuccessful trials of antibiotic therapy. Eventually, polymerase chain reaction testing of a wound biopsy sample confirmed the presence of L. braziliensis. After a complicated course, the patient's infection resolved after tailored antiparasitic therapy. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: This case highlights the need to include travel history in the evaluation of atypical dermatologic infections.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

Piperine, quercetin, and curcumin identified as promising natural products for topical treatment of cutaneous leishmaniasis.

Leishmania braziliensis (L. braziliensis) causes cutaneous leishmaniasis (CL) in the New World. The costs and the side effects of current treatments render imperative the development of new therapies that are affordable and easy to administer. Topical treatment would be the ideal option for the treatment of CL. This underscores the urgent need for affordable and effective treatments, with natural compounds being explored as potential solutions. The alkaloid piperine (PIP), the polyphenol curcumin (CUR), and the flavonoid quercetin (QUE), known for their diverse biological properties, are promising candidates to address these parasitic diseases. Initially, the in vitro cytotoxicity activity of the compounds was evaluated using U-937 cells, followed by the assessment of the leishmanicidal activity of these compounds against amastigotes of L. braziliensis. Subsequently, a golden hamster model with stationary-phase L. braziliensis promastigote infections was employed. Once the ulcer appeared, hamsters were treated with QUE, PIP, or CUR formulations and compared to the control group treated with meglumine antimoniate administered intralesionally. We observed that the three organic compounds showed high in vitro leishmanicidal activity with effective concentrations of less than 50 mM, with PIP having the highest activity at a concentration of 8 mM. None of the compounds showed cytotoxic activity for U937 macrophages with values between 500 and 700 mM. In vivo, topical treatment with QUE daily for 15 days produced cured in 100% of hamsters while the effectiveness of CUR and PIP was 83% and 67%, respectively. No failures were observed with QUE. Collectively, our data suggest that topical formulations mainly for QUE but also for CUR and PIP could be a promising topical treatment for CL. Not only the ease of obtaining or synthesizing the organic compounds evaluated in this work but also their commercial availability eliminates one of the most important barriers or bottlenecks in drug development, thus facilitating the roadmap for the development of a topical drug for the management of CL caused by L. braziliensis.

Leishmania braziliensis enhances monocyte responses to promote anti-tumor activity.

Innate immune cells can undergo long-term functional reprogramming after certain infections, a process called trained immunity (TI). Here, we focus on antigens of Leishmania braziliensis, which induced anti-tumor effects via trained immunity in human monocytes. We reveal that monocytes exposed to promastigote antigens of L. braziliensis develop an enhanced response to subsequent exposure to Toll-like receptor (TLR)2 or TLR4 ligands. Mechanistically, the induction of TI in monocytes by L. braziliensis is mediated by multiple pattern recognition receptors, changes in metabolism, and increased deposition of H3K4me3 at the promoter regions of immune genes. The administration of L. braziliensis exerts potent anti-tumor capabilities by delaying tumor growth and prolonging survival of mice with non-Hodgkin lymphoma. Our work reveals mechanisms of TI induced by L. braziliensis in vitro and identifies its potential for cancer immunotherapy.

Photodynamic therapy of cationic and anionic BSA-curcumin nanoparticles on amastigotes of Leishmania braziliensis and Leishmania major and Leishmania amazonensis.

Cutaneous leishmaniasis is a neglected disease prevalent in tropical countries, and conventional treatment can cause several serious side effects. Photodynamic therapy (PDT) can be considered a promising treatment alternative, as it is non-invasive therapy that has no side effects and uses accessible and low-cost substances, such as curcumin. This study evaluated the PDT response with cationic and anionic BSA nanoparticles encapsulated with curcumin in macrophages infected with L. braziliensis, L. major, and L. amazonensis. The nanoparticle system was characterized using a steady-state technique, scanning electron microscopy (SEM) study, and its biological activity was evaluated using macrophage cell lines infected with different Leishmania species. All spectroscopy measurements demonstrated that BSA curcumin (BSACur) has good photophysical properties, and confocal microscopy shows that macrophages and protozoa internalized the nanoparticles. The viability test demonstrated that at low concentrations, such as 0.1, 0.7, and 1.0 µmol. L-1, there was a decrease in cell viability after PDT application. Furthermore, a decrease in the number of parasites recovered was observed in the PDT groups. The results allowed us to conclude that curcumin loaded into BSA nanoparticles may have potential application in drug delivery systems for PDT protocols, demonstrating reduced cell viability at lower concentrations than free curcumin.

Short communication: The miR-155a-5p is correlated with increased ROS and impaired apoptosis in macrophages infected by Leishmania braziliensis.

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, is a disease characterized by well-limited ulcerated lesions with raised borders in exposed parts of the body. miRNAs are recognized for their role in the complex and plastic interaction between host and pathogens, either as part of the host's strategy to neutralize infection or as a molecular mechanism employed by the pathogen to modulate host inflammatory pathways to remain undetected. The mir155 targets a broad range of inflammatory mediators, following toll-like receptors (TLRs) signaling. In this work, we evaluated the effects of the expression of miR155a-5p in human macrophages infected with L. braziliensis. Our results show that miR155a-5p is inversely correlated with early apoptosis and conversely, seems to influence an increment in the oxidative burst in these cells. Altogether, we spotted a functional role of the miR155a-5p in CL pathogenesis, raising the hypothesis that an increased miR-155 expression by TLR ligands influences cellular mechanisms settled to promote both killing and control of parasite density after infection.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Leishmania braziliensis exosomes activate human macrophages to produce proinflammatory mediators.

Exosomes, organelles measuring 30-200nm, are secreted by various cell types. Leishmania exosomes consist of many proteins, including heat shock proteins, annexins, Glycoprotein 63, proteins exerting signaling activity and those containing mRNA and miRNA. Studies have demonstrated that Leishmania donovani exosomes downregulate IFN-γ and inhibit the expression of microbicidal molecules, such as TNF and nitric oxide, thus creating a microenvironment favoring parasite proliferation. Despite lacking immunological memory, data in the literature suggest that, following initial stimulation, mononuclear phagocytes may become "trained" to respond more effectively to subsequent stimuli. Here we characterized the effects of macrophage sensitization using L. braziliensis exosomes prior to infection by the same pathogen. Human macrophages were stimulated with L. braziliensis exosomes and then infected with L. braziliensis. Higher levels of IL-1ß and IL-6 were detected in cultures sensitized prior to infection compared to unstimulated infected cells. Moreover, stimulation with L. braziliensis exosomes induced macrophage production of IL-1ß, IL-6, IL-10 and TNF. Inhibition of exosome secretion by L. braziliensis prior to macrophage infection reduced cytokine production and produced lower infection rates than untreated infected cells. Exosome stimulation also induced the consumption/regulation of NLRP3 inflammasome components in macrophages, while the blockade of NLRP3 resulted in lower levels of IL-6 and IL-1ß. Our results suggest that L. braziliensis exosomes stimulate macrophages, leading to an exacerbated inflammatory state that may be NLRP3-dependent.

Vaterite microparticle-loaded methylene blue for photodynamic activity in macrophages infected with Leishmania braziliensis.

Calcium carbonate (CaCO3) exhibits a variety of crystalline phases, including the anhydrous crystalline polymorphs calcite, aragonite, and vaterite. Developing porous calcium carbonate microparticles in the vaterite phase for the encapsulation of methylene blue (MB) as a photosensitizer (PS) for use in photodynamic therapy (PDT) was the goal of this investigation. Using an adsorption approach, the PS was integrated into the CaCO3 microparticles. The vaterite microparticles were characterized by scanning electron microscopy (SEM) and steady-state techniques. The trypan blue exclusion method was used to measure the biological activity of macrophages infected with Leishmania braziliensis in vitro. The vaterite microparticles produced are highly porous, non-aggregated, and uniform in size. After encapsulation, the MB-loaded microparticles kept their photophysical characteristics. The carriers that were captured allowed for dye localization inside the cells. The results obtained in this study indicated that the MB-loaded vaterite microparticles show promising photodynamic activity in macrophages infected with Leishmania braziliensis.

Low doses of 3-phenyl-lawsone or meglumine antimoniate delivery by tattooing route are successful in reducing parasite load in cutaneous lesions of Leishmania (Viannia) braziliensis-infected hamsters.

Current therapeutic ways adopted for the treatment of leishmaniasis are toxic and expensive including parasite resistance is a growing problem. Given this scenario, it is urgent to explore treatment alternatives for leishmaniasis. The aim of this study was to evaluate the effect of 3-phenyl-lawsone (3-PL) naphthoquinone on Leishmania (Viannia) braziliensis infection, both in vitro and in vivo, using two local routes of administration: subcutaneous (higher dose) and tattoo (lower dose). In vitro 3-PL showed low toxicity for macrophages (CC50 >3200 µM/48h) and activity against intracellular amastigotes (IC50 = 193 ± 19 µM/48h) and promastigotes (IC50 = 116 ± 26 µM/72h), in which induced increased ROS generation. Additionally, 3-PL up-regulated the production of cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6) and IL-10 in infected macrophages. However, the anti-amastigote action was independent of nitric oxide production. Treatment of hamsters infected with L. (V.) braziliensis from one week after infection with 3-PL by subcutaneous (25 µg/Kg) or tattooing (2.5 µg/Kg) route, during 3 weeks (3 times/week) or 2 weeks (2 times/week) significantly decreased the parasite load (p<0.001) in the lesion. The reduction of parasite load by 3-PL treatment was comparable to reference drug meglumine antimoniate administered by the same routes (subcutaneous 1mg/Kg and tattoo 0.1mg/Kg). In addition, treatment started from five weeks after infection with 3-PL per tattoo also decreased the parasite load. These results show the anti-leishmanial effect of 3-PL against L. (V.) braziliensis and its efficacy by subcutaneous (higher dose) and tattoo (lower dose) routes. In addition, this study shows that drug delivery by tattooing the lesion allows the use of lower doses than the conventional subcutaneous route, which may support the development of a new therapeutic strategy that can be adopted for leishmaniasis.

Mucosal Leishmaniasis of the lip: Report of an Exuberant case in a Young man.

BACKGROUND: Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania. Mucosal leishmaniasis has been described as secondary to the cutaneous form; however, isolated mucosal involvement can also occur. Specifically, mucosal leishmaniasis of the lip is poorly described and its diagnosis challenges clinicians. METHODS: We herein report a case of mucosal leishmaniasis affecting the lower lip without cutaneous involvement in a 20-year-old Venezuelan man. The patient had no relevant past medical history. Clinically, a mass-like lesion with ulcerations and crusts was observed. RESULTS: Microscopically, the lesion was composed of granulomatous inflammation along with macrophages containing intracytoplasmic inclusions similar to round-shaped Leishmania. The species Leishmania (Viannia) braziliensis was confirmed. Treatment with meglumine antimonate was effective. The lesion healed satisfactorily, and no side effects or recurrences were observed. CONCLUSION: Clinicians should be aware of isolated forms of mucosal leishmaniasis of the lip, even in cases where the cutaneous lesion is undetected or clinically manifests as self-limiting. Knowing the endemic areas in the scenario of the dynamics of the ecoepidemiology of leishmaniasis is also essential for surveillance and counselling of the population.

Desenvolvimento e avaliação de uma proteína quimérica recombinante para o diagnóstico e prognóstico das leishmanioses tegumentar e visceral / Development and evaluation of a recombinant chimeric protein for the diagnosis and prognosis of tegumentary and visceral leishmaniasis

As leishmanioses são doenças tropicais negligenciadas com alta endemicidade e que afetam milhares de pessoas no mundo. Sua infecção é causada por parasitos protozoários do gênero Leishmania. A diversidade biológica entre as espécies é quem permite determinar as manifestações clínicas, sendo elas na forma de leishmaniose visceral (LV) ou leishmaniose tegumentar (LT). Dentre estas manifestações, a LV é considerada a mais grave, devido sua alta letalidade e grande emergência em indivíduos com a infecção provocada pelo vírus da imunodeficiência humana (HIV). Atualmente, as medidas de controle e prevenção adotadas pela Organização Mundial da Saúde (OMS), baseiam-se em uma combinação de estratégias de intervenção contra a infecção, uma vez que o diagnóstico eficaz e precoce é indispensável para que se possa intervir com o tratamento adequado, diminuindo índices de mortalidade e a evolução de complicações clínicas. Entretanto, os testes sorológicos utilizados apresentam sensibilidade e especificidade prejudicadas em pacientes com leishmanioses e/ou coinfectados LV/HIV, devido a baixos ní-veis de anticorpos antileishmanial ou pela presença de doenças que causem reação cruzada, levando a resultados falso-positivos. A sensibilidade torna-se também variável em pacientes tratados, uma vez que a sorologia pode manter-se positiva por meses ou anos após o fim do tratamento e cura da doença. Buscando resolver tal problemática, a identificação de novos antígenos, por meio de análises de bioinformática associadas à imunoproteômica, tem permitido a detecção de novas proteínas com potencial aplicação diagnóstica. Em estudos anteriores, as proteínas hipotéticas LiHyT, LiHyD, LiHyV e LiHyP foram encontradas em espécies de Leishmania spp, e avaliadas em suas versões recombinantes por meio de ensaios de ELISA, obtendo resultados satisfatórios para a detecção da LV humana e canina. Com base nessas informações, o presente trabalho teve como objetivo desenvolver uma proteína quimera recombinante base-ada na predição de epítopos lineares específicos de células B das quatro proteínas antigênicas de L. infantum citadas e avaliar o potencial diagnóstico, assim como dos peptídeos individuais que a constituíram, frente à leishmaniose humana, bem como com a coinfecção com HIV, além de testar os antígenos como marcadores prognóstico após o tratamento da LV e LT. As sequências de aminoácidos das proteínas foram avaliadas e oito epítopos de células B foram preditos e utilizados na construção de uma nova proteína quimérica. A proteína foi expressa, purificada e avaliada como antígeno recombinante em ELISA para o diagnóstico de LV, LT, coinfecção LV/HIV e prognóstico em amostras de pacientes tratados de LV e LT. Os epítopos de células B usados na construção da quimera foram sintetizados e também testados em ELISA frente às mesmas amostras, assim como um extrato antigênico solúvel de Leishmania braziliensis (SLA). Os resultados mostraram que a proteína quimera apresentou sensibilidade e especificidade de 100% para diagnosticar a LV, LT e LV/HIV, enquanto os peptídeos sintéticos apresentaram sensibilidade variando entre eles de 9,1% a 90,9% para amostras de LT e 76,8% a 99,2% para amostras de LV e LV/HIV, já os valores de especificidade atingiram 98,3% a 99,1% para LT e 67,1% a 95,7% para LV e LV/HIV. O SLA apresentou sensibilidade e especificidade de 18,2% e 98,3% para LT, e 56,8% a 69,5% para amostras de LV e LV/HIV, respectivamente. Uma avaliação prognóstica preliminar mostrou ainda que os anticorpos anti-quimera diminuíram em níveis significativos, quando comparada a reatividade sorológica antes e seis meses após o tratamento, sugerindo um possível papel prognóstico da quimera para as leishmanioses. O presente estudo, mostrou-se eficaz na construção e avaliação de novos candidatos, que demonstram ter um bom desempenho na detecção diagnóstica e prognóstica para as leishmanioses e dos casos de coinfecção LV/HIV.

Leishmaniasis are neglected tropical diseases with high endemicity that affect thousands of people in the world. Infection is caused by protozoan parasites of the genus Leishmania. The biological diversity between species is what allows determining the clinical manifestations, either in the form of visceral leishmaniasis (VL) or tegumentary leishmaniasis (TL). Among these clinical manifestations, VL is considered the most serious, due to its high lethality and great emergence in individuals with infection caused by the human immunodeficiency virus (HIV). Currently, the control and prevention measures adopted by the World Health Organiza-tion (WHO) are based on a combination of intervention strategies against the infection, since an effective and early diagnosis is essential to intervene with the appropriate treatment, decrea-sing mortality rates and evolution of clinical complications. However, the serological tests used show impaired sensitivity and specificity in patients with leishmaniasis and/or coinfected with VL/HIV, due to low levels of anti-leishmanial antibodies or the presence of diseases that cause cross-reaction, leading to false-positive results. The sensitivity also becomes variable in treated patients, since the serology can remain positive for months or years after the end of the trea-tment and cure of the disease. Seeking to solve this problem, the identification of new antigens, through bioinformatic analysis associated with immunoproteomic, has allowed the detection of new proteins with potential diagnostic application. In previous studies, the hypothetical proteins LiHyT, LiHyD, LiHyV and LiHyP were found in species of Leishmania spp, and evaluated in their recombinant versions through ELISA assays, and satisfactory results were obtained for the detection of human and canine VL. Based on this information, the present work aimed to develop a recombinant chimera protein through on the prediction of specific linear epitopes of B cells derived from these four antigenic proteins of L. infantum and to evaluate its diagnostic potential, as well as the individual peptides that constitute it, against human leishmaniasis, as well as co-infection with HIV, in addition to testing them as possible prognostic markers of patients after VL and TL treatment. The amino acid sequences of the proteins were evaluated and eight B cell epitopes were predicted and used in the construction of a new chimeric protein. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA for the diagnosis of VL, TL, VL/HIV co-infection and prognosis in samples from patients treated for VL and TL. The B cell epitopes used in the construction of the chimera were synthesized and also tested in ELISA against the same samples, as well as a soluble Leishmania braziliensis antigenic extract (SLA). The results showed that the chimera protein apresented sensitivity and specificity of 100% for diagnosing VL, TL and VL/HIV, while the synthetic peptides showed sensitivity ranging from 9.1% to 90.9% for TL samples and 76.8 % to 99.2% for VL and VL/HIV samples, while the specificity values reached from 98.3% to 99.1% for TL and 67.1% to 95.7% for VL and VL/HIV. The SLA showed sensitivity and specificity of 18.2% and 98.3% for TL, and 56.8% to 69.5% for VL and VL/HIV samples, respectively. A preliminary prog-nostic evaluation also showed that anti-chimera antibodies significantly decreased when com-pared to serological reactivity before and six months after treatment, suggesting a possible prognostic role of the antigen for leishmaniasis. The present study proved to be effective in the construction and evaluation of new candidates, who demonstrate good performance in diagnos-tic and prognostic detection for leishmaniasis and VL/HIV co-infection.

Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil.

This study was conducted to characterize the transmission cycle of the tegumentary leishmaniasis (TL) in an old colonization area at Pernambuco State, Brazil. The aims were to identify autochthonous cases, sandflies fauna, domestic animals as possible reservoir hosts and the Leishmania species involved in this endemic area. A total of 168 suspected human cases of TL and 272 domestic animals (canine, feline, equine, goat, and sheep) were included. The sandflies were captured and identified by species. Patients were predominantly male and the average age was 37+18.1 years old. Of 85 patients who had skin lesions, 25.6% of them had direct positive smears for TL and 34 isolates were identified as Leishmania (Viannia) braziliensis. The confirmation for TL diagnosed by molecular detection (PCR) was almost three times more sensitive than the direct test [p < 0.001; PR = 2.72] associated with clinical examination. The Kappa test on PCR between two different specimens, biopsy, and skin lesion swab was 60.8% (p < 0.001). More than 200 specimens of sandflies (80 males and 159 females) were captured and identified as Lutzomyia whitmani (99.6%) and Lu. evandroi (0.4%). The detection of L. (V.) braziliensis by Real-Time PCR in the blood of a captured fed female was positive in 59.3% of Lu. whitmani. Of the 272 domestic animals included, 61.76% were male (n = 168). Thirty-six animals (13.2%) had lesions compatible with TL (34 dogs, 1 cat and 1 sheep) and 3 of them, all dogs, had lesions on the snout, showing destruction of cartilage and mucosa. The study suggests the participation of domestic animals as possible reservoirs. However, further studies are necessary to better understand the transmission cycle and take recommended measures in order to control the disease.

In and out: Leishmania metastasis by hijacking lymphatic system and migrating immune cells.

The lymphatic system plays a crucial role in mounting immune response against intracellular pathogens, and recent studies have documented its role in facilitating tumor dissemination linked largely with cancer cells. However, in mucocutaneous leishmaniasis (MCL) caused by Leishmania Viannia subgenus showing infectious metastasis and resulting in severe distant secondary lesions, the route of escape of these parasites to secondary sites has not yet been investigated in detail. Our results demonstrated that when infection was associated with inflammation and additionally exacerbated by the presence of dsRNA viral endosymbiont (LRV1), lymphatic vessels could serve as efficient routes for infected cells to egress from the primary site and colonize distant organs. We challenged this hypothesis by using the intracellular Leishmania protozoan parasites Leishmania guyanensis (Lgy) associated with or without a dsRNA viral endosymbiont, exacerbating the infection and responsible for a strong inflammatory response, and favoring metastasis of the infection. We analyzed possible cargo cells and the routes of dissemination through flow cytometry, histological analysis, and in vivo imaging in our metastatic model to show that parasites disseminated not only intracellularly but also as free extracellular parasites using migrating immune cells, lymph nodes (LNs), and lymph vessels, and followed intricate connections of draining and non-draining lymph node to finally end up in the blood and in distant skin, causing new lesions.

Murine macrophages do not support the proliferation of Leishmania (Viannia) braziliensis amastigotes even in absence of nitric oxide and presence of high arginase activity.

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Clinical-therapeutic follow-up of patients with American cutaneous leishmaniasis caused by different Leishmania spp. in Brazil.

American cutaneous leishmaniasis (ACL) may present different clinical manifestations, immune and therapeutic responses, depending on the Leishmania species, as well as inoculum size and factors inherent to the affected individual. Thus, the aim of this study was to carry out clinical-therapeutic follow-up of Brazilian patients with ACL caused by different Leishmania species. Between 2015 and 2018, patients with ACL from Amazonas and Pernambuco states (Brazil) were submitted to blood collection before and after treatment. The qPCR technique was used to quantify the parasite load. To identify the Leishmania species, one of the following techniques was employed: a conventional PCR performed from biopsy or blood DNA, followed by sequencing; or Multilocus Enzyme Electrophoresis from Leishmania isolated from biopsy/aspirated lesion. A total of 10.8% (23/213) of the patients included in positive cases were followed-up. All 23 patients were clinically and epidemiologically compatible with ACL and were also positive in parasitological tests (86.96%), molecular tests (73.91%) or both (60.87%). Seventeen samples collected before treatment and 11 collected after treatment were positive in the qPCR assay, with a mean parasite load (MPL) of 38.33 fg/µL and 11.81 fg/µL, respectively. Eight samples were positive in both collections. Thirteen patients (56.52%) were clinically cured (wound healing). Ten patients (43.47%) were not clinically cured at the time of return with the attending physician. Identification of Leishmania species was carried out in samples from nine patients, and six were identified as L. (Viannia) braziliensis, 2 as L (Viannia) guyanensis and 1 as L (Leishmania) amazonensis. One patient infected with L. guyanensis and other with L. braziliensis were not clinically cured and increased the mean parasite load after treatment. The data obtained from the followed-up patients and the relationship between clinical evolution and the infecting species demonstrate the need to understand its etiology to define the effective therapeutic protocol.

Impact of collaborative actions of Leishmania (Viannia) braziliensis subpopulations on the infection profile.

This study focuses on the role of the population structure of Leishmania spp. on the adaptive capacity of the parasite. Herein, we investigate the contribution of subpopulations of the L. (V.) braziliensis Thor strain (Thor03, Thor10 and Thor22) in the profile of murine macrophages infection. Infection assays were performed with binary combinations of these subpopulations at stationary phases. The initial interaction time showed major effects on the combination assays, as demonstrated by the significant increase in the infection rate at 5 h. Based on the endocytic index (EI), Thor10 (EI = 563.6) and Thor03 (EI = 497) showed a higher infection load compared to Thor22 (EI = 227.3). However, the EI decreased in Thor03 after 48 h (EI = 447) and 72 h (EI = 388.3) of infection, and showed changes in the infection level in all Thor10/Thor22 combinations. Assays with CellTrace CFSE-labelled Thor22 promastigotes indicated an increase (~1.5 fold) in infection by this subpopulation in the presence of Thor10 when compared to the infection profile of Thor03/Thor22 combinations in the same proportions. In addition, the potential of these subpopulations, alone or in binary combinations, to modulate the expression of cytokines and nitric oxide (NO) in vitro was investigated. Lower NO and tumour necrosis factor-α production levels were observed for all Thor10/Thor22 combinations at 24 h compared to these subpopulations alone. In contrast, Thor03/Thor22 combination assays increased IL-10 production at this time. Collectively, these results provide in vitro evidence on the potential of L. (V.) braziliensis population structure to play a relevant role in a host infection by this parasite.