CD4+ and CD8+ T-cell multi-epitope chimeric protein associated with an MPLA adjuvant induce protective efficacy and long-term immunological memory for the immunoprophylaxis of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is a disease of high severity and incidence in Brazil, in addition to being a worldwide concern in public health. Leishmania amazonensis is one of the etiological agents of ATL, and the inefficiency of control measures, associated with the high toxicity of the treatment and the lack of effective immunoprophylactic strategies, makes the development of vaccines indispensable and imminent. In this light, the present study proposes to elaborate a chimeric protein (rChiP), based on the fusion of multiple epitopes of CD4+/CD8+ T cells, identified in the immunoproteome of the parasites L. amazonensis and L. braziliensis. The designed chimeric protein was tested in the L. amazonensis murine model of infection using the following formulations: 25 µg of the rChiP in saline (rChiP group) and 25 µg of the rChiP plus 25 µg of MPLA-PHAD® (rChiP+MPLA group). After completing immunization, CD4+ and CD8+ T cells, stimulated with SLa-Antigen or rChiP, showed an increased production of nitric oxide and intracytoplasmic pro-inflammatory cytokines, in addition to the generation of central and effector memory T cells. rChiP and rChiP+MPLA formulations were able to promote an effective protection against L. amazonensis infection determined by a reduction in the development of skin lesions and lower parasitic burden. Reduction in the development of skin lesions and lower parasitic burden in the vaccinated groups were associated with an increase of nitrite, CD4+/CD8+IFN-γ+TNF-α+ and CD4+/CD8+CD44highCD62Lhigh/low T cells, IgGTotal, IgG2a, and lower rates of IgG1 and CD4+/CD8+IL-10+. This data suggests that proposed formulations could be considered potential tools to prevent ATL.

Oral Leishmaniasis in HIV-Positive and HIV-Negative Patients: A Comparative Analysis with Two New Case Reports.

PURPOSE: This case report examines the clinical presentation, diagnosis, treatment, and outcomes of mucocutaneous leishmaniasis with primary oral involvement in HIV-positive and HIV-negative patients diagnosed in Brazil. METHODS: We discuss the clinical manifestations, diagnostic methods, and therapeutic strategies, highlighting the clinical and histopathologic diagnostic features and distinct progression patterns based on HIV status. Our findings are compared with patterns observed in other countries, emphasizing the differences between the Americas and Europe, Asia, and Africa. RESULTS: In the Americas, particularly in Brazil, mucocutaneous leishmaniasis often presents with localized oral lesions, even in the presence of systemic immunosuppression, whereas in the Europe, Asia, and Africa, oral involvement is typically associated with visceral leishmaniasis in immunocompromised patients. These differences were due to variations in the parasite species involved. CONCLUSION: This comparison underscores the importance of regional and immunological factors in diagnosing and managing this neglected infectious disease.

Non-invasive urine-based ELISA using a recombinant Leishmania protein to diagnose tegumentary leishmaniasis.

The diagnosis of tegumentary leishmaniasis (TL) is hampered by variable sensitivity and/or specificity of the tests. Serological assays are suitable to diagnose visceral leishmaniasis (VL); however, they present low performance for the detection of TL cases. Additionally, blood collection to obtain patient serum represents a challenge, as it is an invasive and uncomfortable procedure, requiring laboratorial infrastructure and trained professionals. In this context, the present study proposed to evaluate patient urine to detect TL, given that this analyte has proven to be effective in ELISA experiments for the detection of VL cases. For this, a Leishmania protein called LiHyV, two specific B-cell epitopes derived from protein amino acid sequence, and a Leishmania antigenic extract (SLA) were used as antigens. A total of 215 paired urine and serum samples were evaluated, and results showed that, when serum was employed as an analyte, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 85 %, 29 %, 58 %, and 31 %, respectively, and a specificity of 97.5 %, 98 %, 100 %, and 97.5 %, respectively, in the diagnosis of TL. When urine was used, rLiHyV, Peptide1, Peptide2, and SLA presented a sensitivity of 95 %, 74 %, 67 %, and 52 %, respectively, and a specificity of 100 %, 99 %, 98 %, and 86 %, respectively. In conclusion, preliminary data suggest that urine could be considered as an alternative biological sample for the detection of TL cases.

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.

A Review on the use of Synthetic and Recombinant Antigens for the Immunodiagnosis of Tegumentary Leishmaniasis.

Improving the diagnostic technology used to detect tegumentary leishmaniasis (TL) is essential in view of it being a widespread, often neglected tropical disease, with cases reported from the Southern United States to Northern Argentina. Recombinant proteins, recombinant multiepitope proteins, and synthetic peptides have been extensively researched and used in disease diagnosis. One of the benefits of applying these antigens is a measurable increase in sensitivity and specificity, which improves test accuracy. The present review aims to describe the use of these antigens and their diagnostic effectiveness. With that in mind, a bibliographic survey was conducted on the PudMed platform using the search terms "tegumentary leishmaniasis" AND "diagno", revealing that recombinant proteins have been described and evaluated for their value in TL diagnosis since the 1990s. However, there was a spike in the number of publications using all of the antigens between 2013 and 2022, confirming an expansion in research efforts to improve diagnosis. Moreover, all of the studies involving different antigens had promising results, including improved sensitivity and specificity. These data recognize the importance of doing research with new technologies focused on developing quick, more effective diagnostic kits as early diagnosis facilitates treatment.

The need for culture in tegumentary leishmaniasis diagnosis in Bolivia: A comparative evaluation of four parasitological techniques using two sampling methods.

Leishmaniases are zoonotic diseases caused by protozoa of the genus Leishmania. In Bolivia, leishmaniasis occurs mainly in the cutaneous form (CL) followed by the mucosal or mucocutaneous form (ML or MCL), grouped as tegumentary leishmaniosis (TL), while cases of visceral leishmaniasis (VL) are rare. The cases of TL are routinely diagnosed by parasitological methods: Direct Parasitological Exam (DPE) and axenic culture, the latter being performed only by specialized laboratories. The aim of the present study was to optimize the parasitological diagnosis of TL in Bolivia, using two sampling methods. Samples from 117 patients with suspected TL, obtained by aspiration (n = 121) and scraping (n = 121) of the edge of the lesion were tested by: direct parasitological exam, culture in TSTB medium, and miniculture and microculture in Schneider's medium. A positive laboratory result by any of the four techniques evaluated using either of the two sampling methods was considered the gold standard. Of the 117 suspected patients included, TL was confirmed in 96 (82 %), corresponding 79 of the confirmed cases (82.3 %) to CL and 16 (16.7 %) to ML. Parasitological techniques specificity was 100 % and their analytical sensitivity was greater with scraping samples in TSTB culture (98 %). Scraping samples in TSTB and miniculture correlated well with the reference (Cohen's kappa coefficient=0.88) and showed good reliability (Cronbach's alpha coefficient ≥0.91). Microculture provided positive results earlier than the other culture methods (mean day 4.5). By day 14, 98 % of positive cultures had been detected. Scraping sampling and miniculture were associated with higher culture contamination (6 % and 17 %, respectively). Bacterial contamination predominated, regardless of the sampling and culture method, while filamentous fungi and mixed contamination were more frequently observed in cultures from scraping samples. In conclusion: (i) scraping samples proved more suitable for the diagnosis of TL as they increased analytical sensitivity, are less traumatic for the patient and are safer for laboratory personnel than aspirates; (ii) culture, mainly in TSBT medium, should be used for the diagnosis of TL due to its high sensitivity (doubling the number of cases diagnosed by DPE) and its low cost compared to other culture media.

Chlorella vulgaris extract and Imiquimod as new therapeutic targets for leishmaniasis: An immunological approach.

The therapeutic regimen for the treatment of American Tegumentary Leishmaniasis (ATL) is targeted at the death of the parasite; therefore, it is essential to develop a treatment that can act on the parasite, combined with the modulation of the inflammatory profile. Thus, the aim of this study was to make an in vitro evaluation of the therapeutic potential of Chlorella vulgaris extract (CV) and Imiquimod for ATL. Selectivity indices (SI) were determined by inhibitory concentration assays (IC50) in L. braziliensis cells and cytotoxic concentrations (CC50) were measured in human cells using the MTT method, based on the CV microalgae extract (IC50 concentrations of 15.63 to 500 µg/mL; CC50 concentrations of 62.5-1000 µg/mL) in comparison with the reference drugs and Imiquimod. The immune response was evaluated in healthy human cells by gene expression (RT-qPCR) and cytokine production (Flow Cytometry). The CV extract (SI = 6.89) indicated promising results by showing higher SI than meglumine antimoniate (SI = 3.44) (reference drug). In all analyses, CV presented a protective profile by stimulating the production of Th1 profile cytokines to a larger extent than the reference drugs. Imiquimod showed a high expression for Tbx21, GATA3, RORc and Foxp3 genes, with increased production only of the TNF cytokine. Therefore, the data highlight the natural extract and Imiquimod as strong therapeutic or adjuvant candidates against ATL, owing to modulation of immune response profiles, low toxicity in human cells and toxic action on the parasite.

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.

Isolation and characterisation of Leishmania (Leishmania) infantum from cutaneous leishmaniasis patients in northeast Brazil

BACKGROUND In Brazil, Leishmania (Leishmania) infantum is a widely distributed protozoan parasite. The human leishmaniasis caused by this species is often associated with visceral form. Tegumentary leishmaniasis (TL) cases due to L. (L.) infantum in the country are considered rare but may be underestimated. Although probably uncommon, these cases represent a new challenge to the prevention and control of leishmaniasis. OBJECTIVES Here, we describe two distinct cases of TL with atypical clinical presentations caused by L. (L.) infantum. METHODS AND FINDINGS Parasites were isolated from cutaneous lesions of the two patients and typed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer. The dermotropic L. (L.) infantum isolates were compared in terms of growth culture patterns, metacyclogenesis and in vitro infectivity in macrophages. MAIN CONCLUSIONS This study addresses the emergence of L. (L.) infantum as a causative agent of cutaneous disease in a visceral leishmaniasis hotspot located in northeast Brazil. The data presented provides novel information about the presence of dermotropic L. (L.) infantum in the country and demonstrates the infectivity potential of theses isolates.

In Vitro Leishmanicidal Activity of Copaiba Oil and Kojic Acid Combination on the Protozoan Leishmania (Leishmania) amazonensis and Host Cell.

(1) Background: Leishmaniasis refers to a group of anthropozoonotic diseases caused by Leishmania. The major chemotherapeutic agent used for its treatment is Glucantime®®, but the search continues for new compounds that are economically viable and act on the protozoan without causing damage to the host cell. As an alternative approach, this study used a combination of copaiba oil (CO) and kojic acid (KA) to determine their in vitro action on host cells, on the Leishmania (Leishmania) amazonensis protozoan and its interaction with macrophages. (2) Methods: In vitro culture, analysis of cytokine release and microscopy assays were performed. Statistical analysis was performed with ANOVA (GraphPad Prism). (3) Results: The combination did not induce cytotoxic effects on macrophages after treatment but promoted morphological changes in the protozoan, such as nuclear alterations (apoptotic characteristics), alterations in the cellular body and an increase in the number of electrodense structures and acidocalcisomes, observed mainly at the concentrations of CO20KA50 and CO30KA50 µg/mL. We observed reductions in the intracellular amastigote number and in the production of proinflammatory cytokines, such as IL-6 and TNF-α, after treatment with CO30KA at 50 µg/mL. (4) Conclusions: We report here, for the first time, that the combination of CO and KA may be a promising approach against Leishmania (Leishmania) amazonensis.

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.

A byproduct from the Valles Calchaquíes vineyards (Argentina) rich in phenolic compounds: a tool against endemic Leishmania dissemination.

Vineyard-derived pomace is a byproduct of the wine industry that can have a negative impact on the environment if it is only disposed of or used as a fertilizer. Owing to its polyphenol content, grape pomace is an alternative to biocontrol undesirable microorganisms. In the present study, we characterized the phenolic composition of red and white grape pomace from Valles Calchaquíes, Argentina, and explored its activity against Leishmania (Leishmania) amazonensis, an etiological agent of American tegumentary leishmaniasis, a neglected endemic disease in northern Argentina. Red and white pomace extracts similarly reduced Leishmania viability after a 48-h treatment, with the fractions containing a higher proportion of phenolic compounds being more active. Both extracts stimulated ATPase activity on the parasite plasma membranes, with white grape pomace having a stronger effect than red grape pomace. In addition, the extracts displayed fairly good anticholinesterase activity, which may have contributed to their anti-Leishmania activity. These results reinforce the potential applicability of grape pomace as an antimicrobial agent for the development of biopesticides.

Synthesis of 1,2,3-Triazole-Containing Methoxylated Cinnamides and Their Antileishmanial Activity against the Leishmania braziliensis Species.

Leishmaniasis is a group of infectious diseases caused by protozoan parasites that belong to the genus Leishmania. Currently, there is no human vaccine, and the available treatments are associated with toxicity, high cost, and the emergence of resistant strains. These factors highlight the need to identify new antileishmanial candidates. In this study, we synthesized twenty-four methoxylated cinnamides containing 1,2,3-triazole fragments and evaluated their antileishmanial activity against the Leishmania braziliensis species, which is the main etiological agent responsible for American Tegumentary Leishmaniasis (ATL). The cinnamides were synthetically prepared using nucleophilic acyl substitution and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The compounds were characterized using infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. We performed preliminary studies to evaluate the biological activity of these compounds against L. braziliensis promastigotes and axenic amastigotes. Compound 28, N-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazole-4-yl) methyl)-3,4-dimethoxy cinnamide, demonstrated relevant antileishmanial activity with low toxicity in murine cells. The selectivity index values for this compound were superior compared with data obtained using amphotericin B. Furthermore, this cinnamide derivative reduced the infection percentage and number of recovered amastigotes in L. braziliensis-infected macrophages. It also induced an increase in reactive oxygen species production, depolarization of the mitochondrial potential, and disruption of the parasite membrane. Taken together, these findings suggest that this synthetic compound holds potential as an antileishmanial candidate and should be considered for future studies in the treatment of ATL.

Dogs Harbor Leishmania braziliensis and Participate in the Transmission Cycle of Human Tegumentary Leishmaniasis.

Dogs play an important role in transmission of Leishmania infantum, but epidemiologic and clinical studies of canine tegumentary leishmaniasis (CTL) are scarce. In an endemic area of human American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis, we determine the prevalence and incidence of both CTL and subclinical (SC) L. braziliensis infection in dogs and evaluated if the presence of dogs with CTL or SC L. braziliensis infection is associated with the occurrence of human ATL. SC infection in healthy animals and CTL in animals with ulcers were determined by PCR on biopsied healthy skin or on ulcers or by detecting antibodies against soluble leishmania antigen. We compared the occurrence of human ATL in homes with dogs with CTL or SC infection with control homes without dogs or with dogs without CTL or SC infection. The prevalence of SC infection was 35% and of CTL 31%. The incidence of SC infection in dogs was 4.6% and of CTL 9.3%. The frequency of ATL in humans was 50% in homes with infected dogs and 13% in homes without L. braziliensis infection in dogs. CTL and SC infection is highly prevalent, and dogs may participate in the transmission chain of L. braziliensis.

Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil.

Human tegumentary leishmaniasis (HTL) is a serious tropical disease caused by Leishmania amazonensis. Developing new leishmanicidal agents can help overcome current treatment challenges, such as drug resistance and toxicity. Essential oils are a source of lipophilic substances with diverse therapeutic properties. This study aimed to determine the anti-L. amazonensis activity, cytotoxicity, and chemical profile of Allium sativum essential oil (ASEO). The effect of ASEO on parasite and mammalian cells viability was evaluated using resazurin and MTT assays, respectively. The oil's effect against intracellular amastigotes was also determined. Transmission electron microscopy was used to assess the ultrastructural changes induced by ASEO. In addition, the chemical constituents of ASEO were identified by gas chromatography-mass spectrometry (GC-MS). The cytotoxic potential was evaluated in vitro and in silico. The oil displayed IC50 of 1.76, 3.46, and 3.77 µg/mL against promastigotes, axenic, and intracellular amastigotes, respectively. Photomicrographs of treated parasites showed plasma membrane disruption, increased lipid bodies, and autophagic-like structures. ASEO chemical profiling revealed 1,2,4,6-tetrathiepane (24.84%) and diallyl disulfide (16.75%) as major components. Computational pharmacokinetics and toxicological analysis of ASEO's major components demonstrated good oral bioavailability and better toxicological endpoints than the reference drugs. Altogether, the results suggest that ASEO could be an alternative drug candidate against HTL.

Population dynamics of Nyssomyia whitmani (Diptera: Psychodidae) in domestic and peridomestic environments in Northeast Argentina, a tegumentary leishmaniasis outbreak area.

In the present study, the temporal dynamics of the main vector of Leishmania braziliensis, Nyssomyia whitmani, was measured by monthly captures of phlebotominae sandflies during 5 consecutive years (from 2011 to 2016) in the Paranaense region of South America. The captures were performed in environments where the human-vector contact risk is high: domiciliary and peridomiciliary environments in a rural area endemic of tegumentary leishmaniasis. Nyssomyia whitmani was recorded as the dominant species of the phlebotominae ensemble in all domiciliary and peridomiciliary environments (House, Chicken Shed, Pigsty, and Forest Edge). Using generalized additive models, intra- and interannual fluctuations were observed, modulated by meteorological variables such as the minimum temperature and the accumulated precipitation 1 wk prior to capture. The installation of a pigsty by the farmer during the study period allowed us to observe and describe the so-called "pigsty effect" where the Ny. whitmani population was spatially redistributed, turning the pigsty as the environment that obtained the highest phlebotominae record counts, thus maintaining the farm overall abundance, supporting the idea that the environmental management of the peridomicile could have an impact on the reduction of epidemiological risk by altering the spatial distribution of the phlebotominae ensemble in the environments.

Insights into the Genetic Diversity of Leishmania (Viannia) panamensis in Panama, Inferred via Multilocus Sequence Typing (MLST).

Leishmaniasis is a disease caused by parasites of the genus Leishmania and transmitted by sand fly vectors. Tegumentary leishmaniasis is the most prevalent clinical outcome in Latin America, afflicting people from 18 countries. In Panama, the annual incidence rate of leishmaniasis is as high as 3000 cases, representing a major public health problem. In endemic regions, L. panamensis is responsible for almost eighty percent of human cases that present different clinical outcomes. These differences in disease outcomes could be the result of the local interplay between L. panamensis variants and human hosts with different genetic backgrounds. The genetic diversity of L. panamensis in Panama has only been partially explored, and the variability reported for this species is based on few studies restricted to small populations and/or with poor resolutive markers at low taxonomic levels. Accordingly, in this study, we explored the genetic diversity of sixty-nine L. panamensis isolates from different endemic regions of Panama, using an MLST approach based on four housekeeping genes (Aconitase, ALAT, GPI and HSP70). Two to seven haplotypes per locus were identified, and regional differences in the genetic diversity of L. panamensis were observed. A genotype analysis evidenced the circulation of thirteen L. panamensis genotypes, a fact that might have important implications for the local control of the disease.

Immunotherapy Using Immunogenic Mimotopes Selected by Phage Display plus Amphotericin B Inducing a Therapeutic Response in Mice Infected with Leishmania amazonensis.

Leishmania amazonensis can cause cutaneous and visceral clinical manifestations of leishmaniasis in infected hosts. Once the treatment against disease is toxic, presents high cost, and/or there is the emergence of parasite-resistant strains, alternative means through which to control the disease must be developed. In this context, immunotherapeutics combining known drugs with immunogens could be applied to control infections and allow hosts to recover from the disease. In this study, immunotherapeutics protocols associating mimotopes selected by phage display and amphotericin B (AmpB) were evaluated in L. amazonensis-infected mice. Immunogens, A4 and A8 phages, were administered alone or associated with AmpB. Other animals received saline, AmpB, a wild-type phage (WTP), or WTP/AmpB as controls. Evaluations performed one and thirty days after the application of immunotherapeutics showed that the A4/AmpB and A8/AmpB combinations induced the most polarized Th1-type immune responses, which reflected in significant reductions in the lesion's average diameter and in the parasite load in the infected tissue and distinct organs of the animals. In addition, the combination also reduced the drug toxicity, as compared to values found using it alone. In this context, preliminary data presented here suggest the potential to associate A4 and A8 phages with AmpB to be applied in future studies for treatment against 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.