Treatment using vanillin-derived synthetic molecules incorporated into polymeric micelles is effective against infection caused by Leishmania amazonensis species.

Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.

Re-emergence of human leishmaniasis in northern Italy, 2004 to 2022: a retrospective analysis.

BackgroundHuman leishmaniasis is a protozoan disease transmitted by sand flies and endemic in the Mediterranean region. In Italy, leishmaniasis is present in the south and the western coastal regions, with an epidemic peak detected in northern Italy in the early 1970s.AimTo examine temporal trends, and demographic, clinical, geographical and environmental features of human leishmaniasis cases recorded by the local health unit (LHU) of Bologna, northern Italy.MethodsIn this retrospective observational study, we analysed human leishmaniasis cases recorded from 2004 to 2022 within the Bologna LHU. We also conducted serological investigations for canine leishmaniasis in owned dogs living near the place of infection of human cases.ResultsIn total, 173 cases of human leishmaniasis were detected, and 154 cases were considered autochthonous. An increase of human cases was observed since 2004, with incidence peaks above 2 cases/100,000 inhabitants in 2013, 2018 and 2022; epidemic peaks were preceded by dry summers. Most cases lived in the plain and hilly areas less than 400 m above sea level and many resided in isolated housing, in city outskirts, and/or near uncultivated areas, watercourses and railway sections. The incidence of canine leishmaniasis did not increase in the study period.ConclusionAn epidemic of human leishmaniasis with fluctuating annual numbers of cases, probably related to environmental and climatic factors, was identified in the Bologna LHU. Understanding the risk factors and the environmental characteristics related to places of infection is crucial to evaluate the public health implications of leishmaniasis.

Exploring drug repositioning for leishmaniasis treatment: Ivermectin plus polymeric micelles induce immunological response and protection against tegumentary leishmaniasis.

Leishmania amazonensis can cause a wide spectrum of the clinical manifestations of leishmaniasis in humans. The development of new therapeutics is a long and expensive task; in this context, drug repositioning could be considered a strategy to identify new biological actions of known products. In the present study, ivermectin (IVE) was tested against distinct Leishmania species able to cause disease in humans. In vitro experiments showed that IVE was effective to reduce the infection degree and parasite load in Leishmania donovani- and L. amazonensis-infected macrophages that were treated with it. In addition, using the culture supernatant of treated macrophages, higher production of IFN-γ and IL-12 and lower levels of IL-4 and IL-10 were found. Then, IVE was used in a pure form or incorporated into Poloxamer 407-based polymeric micelles (IVE/M) for the treatment of L. amazonensis-infected BALB/c mice. Animals (n = 16 per group) were infected and later received saline, empty micelles, amphotericin B (AmpB), IVE, or IVE/M. They were euthanized at one (n = 8 per group) and 30 (n = 8 per group) days after treatment and, in both endpoints, immunological, parasitological, and biochemical evaluations were performed. Results showed that both IVE and IVE/M induced higher levels of IFN-γ, IL-12, GM-CSF, nitrite, and IgG2a antibodies, as well as higher IFN-γ expression evaluated by RT-qPCR in spleen cell cultures. Such animals showed low organic toxicity, as well as significant reductions in the lesion's average diameter and parasite load in their infected tissue, spleen, liver, and draining lymph node. The efficacy was maintained 30 days post-therapy, while control mice developed a polarized Th2-type response and high parasite load. In this context, IVE could be considered as a new candidate to be applied in future studies for the treatment against distinct Leishmania species.

In vitro miltefosine and amphotericin B susceptibility of strains and clinical isolates of Leishmania species endemic in Brazil that cause tegumentary leishmaniasis.

Tegumentary leishmaniasis encompasses a spectrum of clinical manifestations caused by the parasitic protozoa of the genus Leishmania. In Brazil, there are at least seven Leishmania species that are endemic and responsible for this set of clinical manifestations of the disease. Current treatment is limited to a restricted number of drugs that in general have several drawbacks including parenteral use, toxicity, and severe side effects. Amphotericin B is considered a second-line drug for tegumentary leishmaniasis in Brazil, while miltefosine was recently approved for clinical use in the treatment of this disease. In this study, we investigated the in vitro susceptibility of Leishmania strains representative of the species endemic to Brazil, as well as a panel of thirteen clinical isolates of tegumentary leishmaniasis, to both amphotericin B and miltefosine. A moderate variation in the susceptibility to both drugs was found, where the EC50 values varied from 11.43 to 52.67 µM for miltefosine and from 12.89 to 62.36 nM for amphotericin B in promastigotes, while for the intracellular amastigotes, values ranged from 1.08 to 9.60 µM and from 1.69 to 22.71 nM for miltefosine and amphotericin B respectively. Furthermore, the clinical isolates and strains of the subgenus Viannia were evaluated for the presence of Leishmania RNA virus 1 (LRV1), as this is an important factor associated with disease severity and treatment outcome. These findings provide a preclinical dataset of the activity of these drugs against the causative species of tegumentary leishmaniasis in Brazil.

Immunization with recombinant LiHyp1 protein plus adjuvant is protective against tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is the main clinical manifestation of leishmaniasis, and it can cause the infected hosts to self-healing cutaneous lesions until mutilating scars in mucosal membranes, particularly in the nose and throat. The treatment against disease presents problems, and the diagnosis is hampered by variable sensitivity and/or specificity of the tests. In this context, the development of prophylactic vaccines could be considered as a strategy to control the disease. Previously, we showed that the recombinant LiHyp1 protein plus adjuvant protected mice from infection with Leishmania infantum, which causes visceral leishmaniasis. In the present study, we tested whether rLiHyp1 could induce protection against infection with L. amazonensis, a parasite species able to cause TL. We immunized BALB/c mice with rLiHyp1 plus saponin (rLiHyp1/S) or incorporated in micelles (rLiHyp1/M) as adjuvants and performed parasitological and immunological evaluations before and after infection. Results showed that after in vitro stimulation from spleen cell cultures using rLiHyp1 or a Leishmania antigenic extract (SLA), rLiHyp1/S and rLiHyp1/M groups developed a Th1-type immune response, which was characterized by high levels of IFN-γ, IL-2, TNF-α and IL-12 cytokines, nitrite, and IgG2a isotype antibodies when compared to values found in the control (saline, saponin, micelles alone) groups, which showed higher levels of anti-SLA IL-4, IL-10, and IgG1 antibodies before and after challenge. In addition, mice receiving rLiHyp1/S or rLiHyp1/M presented significant reductions in the lesion average diameter and parasite load in the infected tissue and internal organs. Blood samples were collected from healthy subjects and TL patients to obtain PBMC cultures, which were in vitro stimulated with rLiHyp1 or SLA, and results showed higher lymphoproliferation and IFN-γ production after stimulus using rLiHyp1, as compared to values found using SLA. These results suggest that rLiHyp1 plus adjuvant was protective against experimental TL and could also be considered for future studies as a vaccine candidate against human disease.

Antileishmanial Screening, Cytotoxicity, and Chemical Composition of Essential Oils: A Special Focus on Piper callosum Essential Oil.

Leishmania amazonensis is the etiological agent of tegumentary leishmaniasis, a disease characterized by the emergence of cutaneous and mucocutaneous ulcerated lesions that can evolve into severe destruction of skin tissue. Treatment of the disease is often accompanied by high toxicity and variable efficacy. Essential oils stand out for having diverse pharmacological properties. Here, we screened a panel of fourteen essential oils for their anti-L. amazonensis activity, cytotoxicity, and chemical profile. Lippia sidoides (LSEO) and Piper callosum (PCEO) oils displayed the best anti-promastigote and anti-amastigote activities with IC50 of 31 and 21 µg/ml, respectively. PCEO was the safest oil with a desirable selectivity index >10. In addition, PCEO showed no cytotoxicity against the VERO line and erythrocytes. PCEO-treated amastigotes displayed mitochondrial membrane depolarization and high levels of intracellular ROS. Safrole (54.72 %) was the main component of PCEO. The results described here highlight the use of essential oils to combat tegumentary leishmaniasis.

Sensitive and specific serodiagnosis of tegumentary leishmaniasis using a new chimeric protein based on specific B-cell epitopes of Leishmania antigenic proteins.

Serological tests used for the diagnosis of tegumentary leishmaniasis (TL) presents problems, mainly related to their variable sensitivity and/or specificity, which can be caused by low levels of antileishmanial antibodies or by presence of cross-reactive diseases, respectively. In this context, the search for new antigenic candidates presenting higher sensitivity and specificity is urgently required. In the present study, the amino acid sequences of the LiHyT, LiHyD, LiHyV, and LiHyP proteins, which were previously showed to be antigenic in the visceral leishmaniasis (VL), were evaluated and eight B-cell epitopes were predicted and used for construction of gene codifying a chimeric protein called ChimLeish. The protein was expressed, purified and evaluated as a recombinant antigen in ELISA (Enzyme-Linked Immunosorbent Assay) for the diagnosis of TL. The own B cell epitopes used to construct the chimera were synthetized and also evaluated as antigens, as well as a soluble Leishmania braziliensis antigenic extract (SLA). Results showed that ChimLeish presented 100% sensitivity and specificity to diagnose TL, while synthetic peptides showed sensitivity varying from 9.1% to 90.9%, while specificity reached from 98.3% to 99.1%. SLA showed sensitivity and specificity of 18.2% and 98.3%, respectively. A preliminary prognostic evaluation showed that anti-ChimLeish IgG antibodies declined in significant levels, when serological reactivity was compared before and six months after treatment, suggesting also a possible prognostic role of this antigen for TL.

Different inoculum of Leishmania braziliensis concentrations influence immunopathogenesis and clinical evolution in the ear dermis hamster model of cutaneous leishmaniasis.

The golden hamster (Mesocricetus auratus) is commonly used as a promising model for Leishmania braziliensis infection developing skin-ulcerated lesions. However, different protocols using high concentration of parasites inoculated in the footpad result in severe clinical disease. Here, we further investigate the outcome of the site of infection and concentration of L. braziliensis parasites inoculated on the immunopathogenesis and clinical evolution. Initially, hamsters were infected in the ear dermis or hind footpad with a concentration of 1 × 105 parasites. Animals infected in the ear dermis developed a disease, with an increased parasite load that more closely resembled human cutaneous leishmaniasis lesions comparing to the group infected in the footpad. Next, we evaluated if different parasite concentrations (104 , 105 and 106 ) inoculated in the ear dermis would impact the course and clinical aspects of infection. Hamsters infected with 104 and 105 parasites developed mild lesions compared to the group infected with 106 that presented severe and persistent lesions. The parasite load varied between the different parasite concentrations. The inflammatory response was more intense when infection was initiated with 106 parasites accompanied by an increased initial expression of IL-4, IL-10 and arginase in the lymph node followed by expression of both pro-and anti-inflammatory cytokines comparing to groups infected with 104 and 105 parasites. In conclusion, the number of parasites inoculated, and the initial site of infection could influence the inflammatory response, and clinical presentation. Our results suggest that the ear dermis infection model induces a chronic disease that relates to immunopathological aspects of CL natural infection.

Development of an immunogen containing CD4+/CD8+ T-cell epitopes for the prophylaxis of tegumentary leishmaniasis.

Tegumentary leishmaniasis (TL) is a disease of high severity and incidence in Brazil, and Leishmania braziliensis is its main etiological agent. The inefficiency of control measures, such as high toxicity and costs of current treatments and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present work developed a gene encoding multiple T-cell (CD4+/CD8+) epitope, derived from conserved proteins found in Leishmania species and associated with TL, to generate a chimeric protein (rMEP/TL) and compose a vaccine formulation. For this, six T-cell epitopes were selected by immunoinformatics approaches from proteins present in the amastigote stage and associated with host-parasite interactions. The following formulations were then tested in an L. braziliensis murine infection model: rMEP/TL in saline or associated with MPLA-PHAD®. Our data revealed that, after immunization (three doses; 14-day intervals) and subsequent challenging, rMEP/TL and rMEP/TL + MPLA-vaccinated mice showed an increased production of key immunological biomarkers of protection, such as IgG2a, IgG2a/IgG1, NO, CD4+, and CD8+ T-cells with IFN-γ and TNF-α production, associated with a reduction in CD4+IL-10+ and CD8+IL-10+ T-cells. Vaccines also induced the development of central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory of CD4+ and CD8+ T-cells. These findings, associated with the observation of lower rates of parasite burdens in the vaccinated groups, when compared to the control groups, suggest that immunization with rMEP/TL and, preferably, associated with an adjuvant, may be considered an effective tool to prevent TL. KEY POINTS: • Rational design approaches for vaccine development. • Central and effector memory of CD4+ and CD8+ T-cells. • Vaccine comprised of rMEP/TL plus MPLA as an effective tool to prevent TL.

[Tegumentary leishmaniasis and sandflies in Colonia Santa Rosa locality in northern Argentina]. / Leishmaniasis tegumentaria y flebótomos en la localidad de Colonia Santa Rosa del norte de Argentina.

Tegumentary leishmaniasis (TL) is caused by parasites of the genus Leishmania and transmitted by the sandfly species, insects belonging to the order Diptera, family Psychodidae. Historically, the most endemic area of TL in Argentina has been the northern region. The aim of this work was to analyze the presence and temporal variation of TL cases reported between 1985 and 2019 in Colonia Santa Rosa locality, northern Argentina. Furthermore, its clinical forms were characterized and sandflies were captured. Patients were diagnosed by smear and the Montenegro skin test. For sampling, CDC light traps were placed at 14 sites from 7pm to 7am. The correlation between vegetation cover and sandfly abundance was also studied. One hundred and twenty TL cases were diagnosed and the overall prevalence was 0.75% (≈16 000 inhabitants). Patients presented simple and multiple cutaneous leishmaniasis (88.79%) and the mucocutaneous form (10.83%). Skin lesions were more frequent on the lower extremities (46.73%). Of the total number of sandflies, Nyssomyia neivai (95%) was the predominant species followed by Migonemyia migonei (1.9%), cortelezzii complex (1.3%) and Evandromyia sallesi (0.09%). The persistent occurrence of cases and the presence of sandflies in the locality suggest the existence of endemic transmission in the area. This highlights the need to design prevention and control measures for TL in northern Argentina.

Chemokines in Leishmaniasis: Map of cell movements highlights the landscape of infection and pathogenesis.

Pathogen interactions with the host immune response components are critical for establishing protective immunity and pathological responses against Leishmania parasites. A predominant proinflammatory profile associated with enhanced phagocytosis trigger a cell-mediated immune response that is relevant to infection control. On the other hand, an anti-inflammatory phenotype, correlated with a predominant modulated/regulatory response, favors intracellular proliferation of Leishmania parasites and disease progression. In this context, chemokines play an important role in determining cellular composition at inflammatory sites. Leishmania infection induces the expression of various chemokines and chemokine receptors in the mammalian host, which can subvert the host immune responses. Indeed, the balance and dynamic changes in cytokines and chemokines may control or predict the disease outcome. In this review, we address our current knowledge regarding the chemokines and chemokines receptors' role in the immunopathogenesis of Tegumentary and Visceral Leishmaniasis.

Development of a chimeric protein based on a proteomic approach for the serological diagnosis of human tegumentary leishmaniasis.

Leishmania braziliensis is responsible for most cases of human tegumentary leishmaniasis (HTL) and has caused a wide range of clinical manifestations, including cutaneous (CL) and mucosal leishmaniasis (ML). The diagnosis is based on criteria that consider epidemiological data, clinical findings, and laboratory tests and is hard to establish. For laboratory tests, none of the assays available can be considered gold standards for disease detection. In addition, the Montenegro skin test, essential to supporting infectologists in the clinical management of the disease, is no longer available in Brazil. Thus, the aim of this study was to develop new targets to be used in diagnostic tests for HTL. In the first step, we carried out two-dimensional gel electrophoresis, followed by mass spectrometry, combined with heat map analysis and immunoproteomics approach, and disclosed eight proteins expressed in the amastigote stage specifically recognized by serum from CL and ML patients. A chimeric protein was designed based on the combination of thirteen linear B-cell epitopes, identified by immunoinformatics analysis, from L. braziliensis proteins. Our results showed that the strategy used in this work was successful in developing an antigen to be used in immunological assays (100.0% sensitivity and specificity) in the detection of HTL cases and in comparison with results obtained from an ELISA using soluble L. braziliensis antigen (SLb-Antigen) and immunofluorescence assay (Bio-Manguinhos/FIOCRUZ). The present technology opens the door for its use in field exams by means of an immunochromatographic test, which will be even more helpful in regions without laboratory structures.Key points• Rational strategy to develop antigens.• Integration between immunoproteomic and immunoinformatics analysis.• Chimeric protein shows high performance in HTL diagnosis.

Accuracy of diagnostic tests for American tegumentary leishmaniasis: a systematic literature review with meta-analyses.

OBJECTIVES: To analyse the accuracy of American tegumentary leishmaniasis (ATL) diagnostic methods and evaluate the quality of the existing publications by means of a systematic review. METHODS: Diagnostic tests evaluated in at least two studies with common reference standards were included in the sensitivity and/or specificity meta-analyses. Quality and susceptibility to bias were analysed using the QUADAS-2 and STARD tools. RESULTS: The title and abstract of 3387 publications were evaluated after deduplication resulting from database searches. Thirty-eight studies were included in the review, and 26 of them had results inserted in meta-analyses. The diagnostic methods with the highest pooled sensitivity values were ELISA, polymerase chain reaction (PCR), indirect immunofluorescence reaction and Montenegro's intradermal reaction. Cytometry was assessed in only two studies and presented 100% sensitivity in both. Smear slide microscopy and histopathology showed low pooled values of sensitivity. For specificity, the highest pooled values were identified for PCR. High values were also identified for ELISA, except for studies in which the reference standard for defining negative participants included individuals with Chagas' disease or paracoccidioidomycosis, which also occurred for cytometry. IFR had lower specificities than ELISA. There was a predominance of case-control designs of phase 1 or 2 and only four studies were strongly recommended as evidence generators. Several reference standards were adopted, and different methods were assessed in a small number of studies. CONCLUSION: PCR showed the highest accuracy for the diagnosis of ATL, and its use should be encouraged in clinical practice. ELISA is recommended for the screening of suspected individuals, but the possibility of cross-reactions should be considered. New validation studies for the tests evaluated in few publications and studies of phase 3 with appropriate methods are needed.

OBJECTIFS: Analyser l'exactitude des méthodes de diagnostic de la leishmaniose tégumentaire américaine (LTA) et évaluer la qualité des publications existantes au moyen d'une analyse systématique. MÉTHODES: Les tests diagnostiques évalués dans au moins deux études avec des étalons de référence communs ont été inclus dans les méta-analyses de sensibilité et/ou de spécificité. La qualité et la sensibilité au biais ont été analysées à l'aide des outils QUADAS-2 et STARD. RÉSULTATS: Le titre et le résumé de 3387 publications ont été évalués après déduplication résultant de recherches dans la base de données. 38 études ont été incluses dans la revue et 26 d'entre elles ont eu des résultats inclus dans des méta-analyses. Les méthodes de diagnostic avec les valeurs de sensibilité poolées les plus élevées étaient ELISA, la réaction en chaîne par polymérase (PCR), la réaction d'immunofluorescence indirecte et la réaction intradermique du Monténégro. La cytométrie a été évaluée dans seulement deux études et présentait une sensibilité de 100% dans les deux. La microscopie et l'histopathologie sur lame de frottis ont montré de faibles valeurs poolées de sensibilité. Pour la spécificité, les valeurs poolées les plus élevées ont été identifiées pour la PCR. Des valeurs élevées ont également été identifiées pour l'ELISA, à l'exception des études dans lesquelles la norme de référence pour définir les participants négatifs incluait des individus atteints de la maladie de Chagas ou de paracoccidioïdomycose, qui s'est également produite pour la cytométrie. L'IFR avait des spécificités plus faibles que l'ELISA. Il y avait une prédominance des essais cas-témoins de phases 1 ou 2 et seules quatre études étaient fortement recommandées comme génératrices de preuves. Plusieurs normes de référence ont été adoptées et différentes méthodes ont été évaluées dans un petit nombre d'études. CONCLUSION: la PCR a montré la plus grande exactitude pour le diagnostic de l'ATL et son utilisation doit être encouragée dans la pratique clinique. L'ELISA est recommandé pour le dépistage des personnes suspectées, mais la possibilité de réactions croisées doit être considérée. De nouvelles études de validation des tests évalués dans quelques publications et des études de phase 3 avec des méthodes appropriées sont nécessaires.

High-through identification of T cell-specific phage-exposed mimotopes using PBMCs from tegumentary leishmaniasis patients and their use as vaccine candidates against Leishmania amazonensis infection.

In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection.

Influence of Leishmania RNA Virus 1 on Proinflammatory Biomarker Expression in a Human Macrophage Model of American Tegumentary Leishmaniasis.

Backgound: Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Methods: Expression of proinflammatory biomarkers, including interleukin (IL) 1ß, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. Results: LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1ß (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Conclusions: Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.

Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL.

A recombinant fusion protein displaying murine and human MHC class I- and II-specific epitopes protects against Leishmania amazonensis infection.

Tegumentary leishmaniasis (TL) constitutes a major public health problem with significant morbidity worldwide. Synthetic peptide-based vaccines are attractive candidates to protect against leishmaniasis, since T cell-specific epitopes can be delivery to antigen-presenting cells, leading to the generation of a Th1 cell-mediated immunity. In this context, the present study aims to evaluate the immunogenicity and protective efficacy of a vaccine composed of major histocompatibility complex class I and II-restricted epitopes derived from four Leishmania infantum proteins to protect mice against Leishmania amazonensis infection. This recombinant fusion protein was administered in BALB/c mice alone or with saponin. As controls, animals received saline or saponin. In the results, the administration of the recombinant protein plus saponin induced a specific IFN-γ, IL-12 and GM-CSF production, as well as high IgG2a isotype antibody levels, which protected mice against a challenge using L. amazonensis promastigotes. Lower parasite burden was found in the infected footpads, liver, spleen and draining lymph node of vaccinated mice, when compared to those from the control groups. In addition, protection was associated with a lower IL-4 and IL-10 response, which was accompanied by the antileishmanial nitrite production by spleen cells of the animals. Interestingly, the recombinant protein administered alone induced a partial protection against challenge. In conclusion, this study shows a new vaccine candidate based on T cell-specific epitopes that was able to induce protection against L. amazonensis infection.

Clinical and laboratory profiles of patients with early spontaneous healing in cutaneous localized leishmaniasis: a historical cohort study.

BACKGROUND: Skin ulcers in American cutaneous leishmaniasis (ACL) may heal spontaneously after months/years. However, few cases may present quick heal even during diagnosis procedure (early spontaneous healing- ESH). The main objective of this study was to compare ESH patients with cases requiring specific treatment [non-ESH (NESH)]. METHODS: A historical cohort study of ACL patients (n = 445) were divided into 2 groups: ESH - spontaneously healed patients (n = 13; 2.90%), and NESH- treated patients (n = 432; 97.10%). We compared clinical and laboratorial findings at diagnosis, including the lesion healing process. RESULTS: ESH patients had a higher percentage of single lesions (p = 0.027), epithelialized lesion on initial examination (p = 0.001), lesions located in the dorsal trunk (p = 0.017), besides earlier healing (p < 0.001). NESH presents higher frequency of ulcerated lesions (p = 0.002), amastigotes identified in histopathology exams (p = 0.005), positive cultures (p = 0.001), and higher positivity in ≥3 parasitological exams (p = 0.030). All ESH cases were positive in only a single exam, especially in PCR. CONCLUSIONS: ESH group apparently presented a lower parasitic load evidenced by the difficulty of parasitological confirmation and its positivity only by PCR method. The absence or deficiency of specific treatment is commonly identified as predisposing factors for recurrence and metastasis in ACL. However, due to the drugs toxicity, the treatment of cases which progress to early spontaneous healing is controversial. ESH patients were followed for up to 5 years after cure, with no evidence of recrudescence, therefore suggesting that not treating these patients is justifiable, but periodic dermatological and otorhinolaryngological examinations are advisable to detect a possible relapse.

Evaluation of a hypothetical protein for serodiagnosis and as a potential marker for post-treatment serological evaluation of tegumentary leishmaniasis patients.

The serodiagnosis for tegumentary leishmaniasis (TL) presents problems related to the sensitivity and/or specificity of the tests. In the present study, an enzyme-linked immunosorbent assay (ELISA) technique was used to evaluate the performance from a Leishmania braziliensis hypothetical protein, LbHyM, in an attempt to compare its serological reactivity with a soluble Leishmania antigenic preparation (SLA) for the serodiagnosis of cutaneous (CL) and mucosal (ML) leishmaniasis. LbHyM was predicted to be a kinesin-like protein by bioinformatics tools. Serum samples were collected from both CL and ML patients, as well as from those with Chagas disease and from healthy subjects living in endemic or non-endemic areas of TL. Also, sera were collected from patients before and after the treatments, seeking to evaluate their serological follow-up in relation to the anti-protein and anti-parasite antibody levels. When an ELISA-rLbHyM assay was performed, it proved to be significantly more sensitive than ELISA-L. braziliensis SLA in detecting both CL and ML patients. Also, when using sera from Chagas disease patients, the ELISA-rLbHyM proved to be more specific than ELISA-SLA. The anti-protein and anti-parasite antibody levels were also evaluated 6 months after the treatments, and treated patients showed significantly lower levels of specific-rLbHyM antibodies, when compared to the anti-parasite antibody levels. In conclusion, the ELISA-rLbHyM assay can be considered a confirmatory serological technique for the serodiagnosis of L. braziliensis infection and can also be used in the serological follow-up of treated patients, aiming to correlate the low anti-protein antibody levels with the improvement of the healthy state of the patients.

Differential Expression of the Eicosanoid Pathway in Patients With Localized or Mucosal Cutaneous Leishmaniasis.

Unfettered inflammation is thought to play critical role in the development of different clinical forms of tegumentary leishmaniasis. Eicosanoids are potent mediators of inflammation and tightly associated with modulation of immune responses. In this cross-sectional exploratory study, we addressed whether targets from the eicosanoid biosynthetic pathway, assessed by multiplexed expression assays in lesion biopsy and plasma specimens, could highlight a distinct biosignature in patients with mucocutaneous leishmaniasis (MCL) or localized cutaneous leishmaniasis (LCL). Differences in immunopathogenesis between MCL and LCL may result from an imbalance between prostaglandins and leukotrienes, which may serve as targets for future host-directed therapies.