Role of antigen-presenting cells in non-ulcerated skin lesions caused by Leishmania (Leishmania) infantum chagasi.

In Central America, infection by Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis (NUCL). This work aimed to evaluate the participation of subpopulations of antigen-presenting cells in skin lesions of patients affected by NUCL through double-staining immunohistochemistry using cellular and intracellular markers. Twenty-three skin biopsies from patients affected by NUCL were used. Histological sections stained by HE were used for histopathological study. Immunohistochemical studies were performed using primary antibodies against Langerhans cells, dermal dendritic cells, T lymphocytes, and the cytokines IL-12, IFN-γ, TNF-α, iNOS, and IL-10. The histopathological lesions were characterized by an inflammatory infiltrate, predominantly lymphohistiocytic, of variable intensity, with a diffuse arrangement associated with epithelioid granulomas and discreet parasitism. Double-staining immunohistochemistry showed higher participation of dendritic cells producing the proinflammatory cytokine IL-12 in relation to the other evaluated cytokines. Activation of the cellular immune response was marked by a higher density of CD8 Tc1-lymphocytes followed by CD4 Th1-lymphocytes producing mainly IFN-γ. The data obtained in the present study suggest that antigen-presenting cells play an important role in the in situ immune response through the production of proinflammatory cytokines, directing the cellular immune response preferentially to the Th1 and Tc1 types in NUCL caused by L. (L.) infantum chagasi.

Leishmania amazonensis from distinct clinical forms/hosts has polymorphisms in Lipophosphoglycans, displays variations in immunomodulatory properties and, susceptibility to antileishmanial drugs.

Lipophosphoglycan (LPG), the major Leishmania glycoconjugate, induces pro-inflammatory/immunosuppressive innate immune responses. Here, we evaluated functional/biochemical LPG properties from six Leishmania amazonensis strains from different hosts/clinical forms. LPGs from three strains (GV02, BA276, and LV79) had higher pro-inflammatory profiles for most of the mediators, including tumor necrosis factor alpha and interleukin 6. For this reason, glycoconjugates from all strains were biochemically characterized and had polymorphisms in their repeat units. They consisted of three types: type I, repeat units devoid of side chains; type II, containing galactosylated side chains; and type III, containing glucosylated side chains. No relationship was observed between LPG type and the pro-inflammatory properties. Finally, to evaluate the susceptibility against antileishmanial agents, two strains with high (GV02, BA276) and one with low (BA336) pro-inflammatory activity were selected for chemotherapeutic tests in THP-1 cells. All analyzed strains were susceptible to amphotericin B (AmB) but displayed various responses against miltefosine (MIL) and glucantime (GLU). The GV02 strain (canine visceral leishmaniasis) had the highest IC50 for MIL (3.34 µM), whereas diffuse leishmaniasis strains (BA276 and BA336) had a higher IC50 for GLU (6.87-12.19 mM). The highest IC50 against MIL shown by the GV02 strain has an impact on clinical management. Miltefosine is the only drug approved for dog treatment in Brazil. Further studies into drug susceptibility of L. amazonensis strains are warranted, especially in areas where dog infection by this species overlaps with those caused by Leishmania infantum.

Histological and neuronal changes in the duodenum of hamsters infected with Leishmania (Leishmania) infantum.

Visceral leishmaniasis is a neglected tropical disease caused by parasites belonging to the Leishmania genus that infect macrophages in different tissues such as the spleen, liver, lymph nodes, bone marrow, and intestine. Therefore, this study aimed to investigate the integrity of the intestinal tract and the nitrergic (NADPH-dp) and metabolically active (NADH-dp) myenteric neurons of the duodenum of golden hamsters infected with L. (L.) infantum. Therefore, thirty golden hamsters were divided into six groups (n = 5); three of them were infected with 2 × 107 promastigote forms of L. (L.) infantum by intraperitoneal route (Infected Group - IG) and three were inoculated with saline solution (control group - CG). After 30, 60 and 90 days post-infection (DPI) infected animals were euthanized and the liver, spleen and duodenum were collected to analyze tissue parasitism. The duodenum was processed using usual histological techniques to analyze the main changes that occurred during infection and histochemical techniques to phenotype myenteric neurons. Amastigote forms were observed in the spleen, liver, and duodenum during all experimental periods, and tissue parasitism in these organs increased significantly over time. At 30 DPI, reduction in muscle tunic, increase in the total intestinal wall and the number of goblet cells PAS+ was observed. At 60 DPI, an increase in intestinal crypts and intraepithelial lymphocytes was observed, and a reduction in intestinal villi was observed at 90 DPI, along with an increase in crypt size. Regarding neurons, an increase in the density of the NADPH-dp population was observed at 30 DPI, but at 60 and 90 DPI a significant reduction of this population was observed. In general, infection progression was observed to cause significant morphofunctional changes in the duodenum of infected hamsters.

Mechanistic Insights into the Leishmanicidal and Bactericidal Activities of Batroxicidin, a Cathelicidin-Related Peptide from a South American Viper (Bothrops atrox).

Snake venoms are important sources of bioactive molecules, including those with antiparasitic activity. Cathelicidins form a class of such molecules, which are produced by a variety of organisms. Batroxicidin (BatxC) is a cathelicidin found in the venom of the common lancehead (Bothrops atrox). In the present work, BatxC and two synthetic analogues, BatxC(C-2.15Phe) and BatxC(C-2.14Phe)des-Phe1, were assessed for their microbicidal activity. All three peptides showed a broad-spectrum activity on Gram-positive and -negative bacteria, as well as promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. Circular dichroism (CD) and nuclear magnetic resonance (NMR) data indicated that the three peptides changed their structure upon interaction with membranes. Biomimetic membrane model studies demonstrated that the peptides exert a permeabilization effect in prokaryotic membranes, leading to cell morphology distortion, which was confirmed by atomic force microscopy (AFM). The molecules considered in this work exhibited bactericidal and leishmanicidal activity at low concentrations, with the AFM data suggesting membrane pore formation as their mechanism of action. These peptides stand as valuable prototype drugs to be further investigated and eventually used to treat bacterial and protozoal infections.

Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil.

Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.

Lipophosphoglycans from dermotropic Leishmania infantum are more pro-inflammatory than those from viscerotropic strains.

Although Leishmania infantum is well-known as the aethiological agent of visceral leishmaniasis (VL), in some Central American countries it may cause atypical non-ulcerated cutaneous leishmaniasis (NUCL). However, the mechanisms favoring its establishment in the skin are still unknown. Lipophosphoglycan (LPG) is the major Leishmania multivirulence factor involved in parasite-host interaction. In the case of viscerotropic L. infantum, it causes an immunosuppression during the interaction with macrophages. Here, we investigated the biochemical and functional roles of LPGs from four dermotropic L. infantum strains from Honduras during in vitro interaction with murine macrophages. LPGs were extracted, purified and their repeat units analysed. They did not have side chains consisting of Gal(ß1,4)Man(α1)-PO4 common to all LPGs. Peritoneal macrophages from BALB/c and C57BL/6 were exposed to LPG for nitric oxide (NO) and cytokine (TNF-α and, IL-6) production. LPGs from dermotropic strains from Honduras triggered higher NO and cytokine levels compared to those from viscerotropic strains. In conclusion, LPGs from dermotropic strains are devoid of side-chains and exhibit high pro-inflammatory activity.

Antileishmanial activity and ultrastructural changes of sesquiterpene lactones isolated from Calea pinnatifida (Asteraceae).

Bioactivity-guided fractionation of antileishmanial active CH2Cl2 phase of MeOH extract from leaves of Calea pinnatifida led to isolation of two sesquiterpene lactones calein C (1) and calealactone C (2), which structures were stablished on the basis of spectroscopic analysis. Compounds 1 and 2 displayed potent activity against Leishmania amazonensis promastigotes with EC50 of 1.7 and 4.6 µg mL-1, respectively. Compound 2 presented low cytotoxicity for J774 macrophages and displayed activity against amastigote forms of L. amazonensis similar to miltefosine with CC50 values of 31.73 and 27.18 µg mL-1, respectively. Additionally, compounds 1 and 2 caused ultrastructural changes in promastigotes leading to a loss of their classical structural morphology, as evidenced by electron microscopy. Also compound 2 decreased the mitochondria membrane potential. To the best of our knowledge, this is the first occurrence of 1 and 2 in C. pinnatifida. The results obtained highlighted the importance of studying sesquiterpene lactones isolated from Calea pinnatifida in terms of antileishmanial activity, in order to understand the mechanism of action of the isolated compounds in promastigotes forms of L. amazonensis.

Antileishmanial activity of meroditerpenoids from the macroalgae Cystoseira baccata.

The development of novel drugs for the treatment of leishmaniases continues to be crucial to overcome the severe impacts of these diseases on human and animal health. Several bioactivities have been described in extracts from macroalgae belonging to the Cystoseira genus. However, none of the studies has reported the chemical compounds responsible for the antileishmanial activity observed upon incubation of the parasite with the aforementioned extracts. Thus, this work aimed to isolate and characterize the molecules present in a hexane extract of Cystoseira baccata that was found to be bioactive against Leishmania infantum in a previous screening effort. A bioactivity-guided fractionation of the C. baccata extract was carried out and the inhibitory potential of the isolated compounds was evaluated via the MTT assay against promastigotes and murine macrophages as well as direct counting against intracellular amastigotes. Moreover, the promastigote ultrastructure, DNA fragmentation and changes in the mitochondrial potential were assessed to unravel their mechanism of action. In this process, two antileishmanial meroditerpenoids, (3R)- and (3S)-tetraprenyltoluquinol (1a/1b) and (3R)- and (3S)-tetraprenyltoluquinone (2a/2b), were isolated. Compounds 1 and 2 inhibited the growth of the L. infantum promastigotes (IC50 = 44.9 ± 4.3 and 94.4 ± 10.1 µM, respectively), inducing cytoplasmic vacuolization and the presence of coiled multilamellar structures in mitochondria as well as an intense disruption of the mitochondrial membrane potential. Compound 1 decreased the intracellular infection index (IC50 = 25.0 ± 4.1 µM), while compound 2 eliminated 50% of the intracellular amastigotes at a concentration > 88.0 µM. This work identified compound 2 as a novel metabolite and compound 1 as a biochemical isolated from Cystoseira algae displaying antileishmanial activity. Compound 1 can thus be an interesting scaffold for the development of novel chemotherapeutic molecules for canine and human visceral leishmaniases studies. This work reinforces the evidence of the marine environment as source of novel molecules.

Antileishmanial Activity and Immunomodulatory Effects of Tricin Isolated from Leaves of Casearia arborea (Salicaceae).

Bioactivity-guided fractionation of antileishmanial active extract from leaves of Casearia arborea led to isolation of three metabolites: tricin (1), 1',6'-di-O-ß-d-vanilloyl glucopyranoside (2) and vanillic acid (3). Compound 1 demonstrated the highest activity against the intracellular amastigotes of Leishmania infantum, with an IC50 value of 56 µm. Tricin (1) demonstrated selectivity in mammalian cells (SI > 7) and elicited immunomodulatory effect on host cells. The present work suggests that tricin modulated the respiratory burst of macrophages to a leishmanicidal state, contributing to the parasite elimination. Therefore, the natural compound tricin could be further explored in drug design studies for leishmaniasis treatment.

Genome-Wide Association Study of Cell-Mediated Response in Dogs Naturally Infected by Leishmania infantum.

A genome-wide association study (GWAS) could unravel the complexity of the cell-mediated immunity (CMI) to canine leishmaniasis (CanL). Therefore, we scanned 110,165 single-nucleotide polymorphisms (SNPs), aiming to identify chromosomal regions associated with the leishmanin skin test (LST), lymphocyte proliferation assay (LPA), and cytokine responses to further understand the role played by CMI in the outcome of natural Leishmania infantum infection in 189 dogs. Based on LST and LPA, four CMI profiles were identified (LST-/LPA-, LST+/LPA-, LST-/LPA+, and LST+/LPA+), which were not associated with subclinically infected or diseased dogs. LST+/LPA+ dogs showed increased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) levels and mild parasitism in the lymph nodes, whereas LST-/LPA+ dogs, in spite of increased IFN-γ, also showed increased interleukin-10 (IL-10) and transforming growth factor ß (TGF-ß) levels and the highest parasite load in lymph nodes. Low T cell proliferation under low parasite load suggested that L. infantum was not able to induce effective CMI in the early stage of infection. Altogether, genetic markers explained 87%, 16%, 15%, 11%, 0%, and 0% of phenotypic variance in TNF-α, TGF-ß, LST, IL-10, IFN-γ, and LPA, respectively. GWAS showed that regions associated with TNF-α include the following genes: IL12RB1, JAK3, CCRL2, CCR2, CCR3, and CXCR6, involved in cytokine and chemokine signaling; regions associated with LST, including COMMD5 and SHARPIN, involved in regulation of NF-κB signaling; and regions associated with IL-10, including LTBP1 and RASGRP3, involved in T regulatory lymphocytes differentiation. These findings pinpoint chromosomic regions related to the cell-mediated response that potentially affect the clinical complexity and the parasite replication in canine L. infantum infection.

Cutaneous Leishmaniasis in dogs: is high seroprevalence indicative of a reservoir role?

American cutaneous leishmaniasis (ACL) is a complex disease with a rich diversity of animal host species. This diversity imposes a challenge, since understanding ACL transmission requires the adequate identification of reservoir hosts, those species able to be a source of additional infections. In this study we present results from an ACL cross-sectional serological survey of 51 dogs (Canis familiaris), where we used diagnostic tests that measure dog's exposure to Leishmania spp. parasites. We did our research in Panamá, at a village that has undergone significant ecosystem level transformations. We found an ACL seroprevalence of 47% among dogs, and their exposure was positively associated with dog age and abundance of sand fly vectors in the houses of dog owners. Using mathematical models, which were fitted to data on the proportion of positive tests as function of dog age, we estimated a basic reproductive number (R 0 ± s.e.) of 1·22 ± 0·09 that indicates the disease is endemically established in the dogs. Nevertheless, this information by itself is insufficient to incriminate dogs as ACL reservoirs, given the inability to find parasites (or their DNA) in seropositive dogs and previously reported failures to experimentally infect vectors feeding on dogs with ACL parasites.

Salivary gland homogenates from wild-caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice.

During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu. flaviscutellata and Lu. (P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used.

Prevalence and incidence of canine visceral leishmaniasis and its clinical-immunological features in an endemic area of the Brazilian Amazon.

BACKGROUND: A cohort study for 2 years period analysed the prevalence, incidence and clinical-immunological features of canine Leishmania (L.) chagasi-infection in 316 mongrel dogs in a visceral leishmaniasis-endemic area in Pará State, Brazil. OBJECTIVE/METHODS: Diagnosis of infection was performed by the indirect fluorescent antibody test (IFAT-IgG), the leishmanin skin test (LST) and a parasite search (from the popliteal lymph node aspiration) at the beginning of the study and at 6, 12 and 24 months intervals. RESULTS: IFAT/LST revealed three immune profiles of infection: (I) IFAT(+) /LST(-) (81), (II) IFAT(-) /LST(+) (17) and (III) IFAT(+) /LST(+) (13). Prevalence of profiles I, II and III were 25.6, 5.4 and 4.1%, and an overall prevalence 35.1%. Incidence of profiles I, II and III were 5.4, 0.3 and 0.0%, and an overall incidence 5.7% dogs per month. Incidence at the age ranges <1 year, ≥1 year, <7 years and ≥7 years evidenced a highest rate in the age range <1 year (6.6% dogs per month). Parasitological diagnosis was positive in 19% dogs at the prevalence (85.7% profile I), and in 11% at the incidence (100% profile I). The clinical picture of 179 infected dogs showed 145 (81%) of profile I (82% subclinical); 21 (11.7%) of profile II (100% subclinical); and 13 (7.3%) of profile III (84.6% subclinical). Conversion from subclinical to sick dogs was higher (p < 0.05) in profile I (40.2%) than in profiles II (5.8%) and III (9%). Immunological conversion showed that only 3.2% of profile I dogs (prevalence) converted to LST(+) (two at the end of the first 6 months and 1 after 24 months), while 82.3% of profile II dogs converted to IFAT(+) (11 in the first 6 months, whereas three after 12 months). A 100% death rate was observed in dogs from profile I alone. CONCLUSION: These results reinforce the need of adopting preventive strategies against CVL as early as in the first semester of the dog's life.

New Immunological Markers in Chromoblastomycosis-The Importance of PD-1 and PD-L1 Molecules in Human Infection.

The pathogenesis of chromoblastomycosis (CBM) is associated with Th2 and/or T regulatory immune responses, while resistance is associated with a Th1 response. However, even in the presence of IFN-γ, fungi persist in the lesions, and the reason for this persistence is unknown. To clarify the factors associated with pathogenesis, this study aimed to determine the polarization of the cellular immune response and the densities of cells that express markers of exhaustion in the skin of CBM patients. In the skin of patients with CBM, a moderate inflammatory infiltrate was observed, characterized primarily by the occurrence of histiocytes. Analysis of fungal density allowed us to divide patients into groups that exhibited low and high fungal densities; however, the intensity of the inflammatory response was not related to mycotic loads. Furthermore, patients with CBM exhibited a significant increase in the number of CD4+ and CD8+ cells associated with a high density of IL-10-, IL-17-, and IFN-γ-producing cells, indicating the presence of a chronic and mixed cellular immune response, which was also independent of fungal load. A significant increase in the number of PD-1+ and PD-L1+ cells was observed, which may be associated with the maintenance of the fungus in the skin and the progression of the disease.

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters.

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.

Gene Signatures of Symptomatic and Asymptomatic Clinical-Immunological Profiles of Human Infection by Leishmania (L.) chagasi in Amazonian Brazil.

Individuals infected with Leishmania (L.) chagasi may present different asymptomatic and symptomatic stages of infection, which vary in the clinical-immunological profiles that can be classified as asymptomatic infection (AI), subclinical resistant infection (SRI), indeterminate initial infection (III), subclinical oligosymptomatic infection (SOI), and symptomatic infection (SI) (=American visceral leishmaniasis, AVL). However, little is known about the molecular differences between individuals having each profile. Here, we performed whole-blood transcriptomic analyses of 56 infected individuals from Pará State (Brazilian Amazon), covering all five profiles. We then identified the gene signatures of each profile by comparing their transcriptome with those of 11 healthy individuals from the same area. Symptomatic individuals with SI (=AVL) and SOI profiles showed higher transcriptome perturbation when compared to those asymptomatic III, AI and SRI profiles, suggesting that disease severity may be associated with greater transcriptomic changes. Although the expression of many genes was altered on each profile, very few genes were shared among the profiles. This indicated that each profile has a unique gene signature. The innate immune system pathway was strongly activated only in asymptomatic AI and SRI profiles, suggesting the control of infection. In turn, pathways such as MHC Class II antigen presentation and NF-kB activation in B cells seemed to be specifically induced in symptomatic SI (=AVL) and SOI profiles. Moreover, cellular response to starvation was down-regulated in those symptomatic profiles. Overall, this study revealed five distinct transcriptional patterns associated to the clinical-immunological (symptomatic and asymptomatic) profiles of human L. (L.) chagasi-infection in the Brazilian Amazon.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs.

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH((-)) (107 dogs), (2) IFAT((-))/DTH((+)) (18 dogs), and (3) IFAT((+))/DTH((+)) (13 dogs), providing an overall prevalence of infection of 43% (138/320). Thus, the specific prevalence of IFAT( (+) )/DTH( (-) ) 33.4% (107/320) was higher than those of IFAT( (-) )/DTH( (+) ) 5.6% (18/320) and IFAT( (+) )/DTH( (+) ) 4.0% (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT( (+) )/DTH( (-) ) rate of 77.5% (107/138) was also higher than those of 13.0% (18/138) of IFAT( (-) )/DTH( (+) ) and 9.5% (13/138) of IFAT( (+) )/DTH( (+) ) rates. The frequency of asymptomatic dogs (76%-105) was higher than those of symptomatic (16.6%-23) and oligosymptomatic ones (7.4%-10). A total of 16 (11.6%) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT( (+) ) /DTH( (-) ) profile: 41% (9/22) from symptomatic, 33.3% (3/9) from oligosymptomatic, and 5.2% (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76%-105), the majority (72.4%-76) was characterized by the IFAT( (+) ) /DTH( (-) ) profile with a doubtful immunogenetic resistance against infection.

Absolute Configuration and Antileishmanial Activity of (-)-Cyclocolorenone Isolated from Duguetia lanceolata (Annonaceae).

BACKGROUND: The fractionation of the n-hexane phase of the EtOH extract from the leaves of Duguetia lanceolata (Annonaceae) led to the identification of the sesquiterpene (-)-cyclocolorenone. OBJECTIVES: Chemical characterization, including determination of the absolute stereochemistry, and in vitro evaluation of antileishmanial activity of the sesquiterpene (-)-cyclocolorenone, isolated from D. lanceolata, were carried out. METHODS: (-)-Cyclocolorenone was isolated from D. lanceolata leaves using different chromatographic steps and its structure was defined by analysis of NMR and ESI-HRMS data. Additionally, the absolute configuration of (-)-cyclocolorenone was ambiguously assigned by means of vibrational circular dichroism (VCD). Antileishmanial activity of (-)-cyclocolorenone was evaluated on promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The integrity of the cell membrane of L. (L.) amazonensis was analyzed using the SYTOX green probe. RESULTS: (-)-(1R,6S,7R,10R)-Cyclocolorenone displayed activity against promastigotes and amastigotes forms of L. (L.) amazonensis with IC50 of 4.54 and 28.44 µM, respectively. Furthermore, this compound was non-toxic in J774 macrophage cells (CC50 > 458.71 µM) with a selectivity index > 100 (promastigotes) and > 32.2 (amastigotes). Additionally, (-)-cyclocolorenone was observed to target the parasite cell membrane. CONCLUSION: Obtained data suggested that (-)-cyclocolorenone, in which absolute configuration was determined, can be considered as a scaffold for the development of new drugs for the treatment of leishmaniasis.

Visceral Leishmaniasis Urbanization in the Brazilian Amazon Is Supported by Significantly Higher Infection Transmission Rates Than in Rural Area.

This was an open cohort prospective study (2016−2018) that analyzed the prevalence and incidence rates of human Leishmania (L.) infantum chagasi-infection and the evolution of their clinical-immunological profiles in distinct urban and rural scenarios of American visceral leishmaniasis (AVL) in Pará State, in the Brazilian Amazon. These infection profiles were based on species-specific DTH/IFAT-IgG assays and clinical evaluation of infected individuals, comprising five profiles: three asymptomatic, Asymptomatic Infection [AI], Subclinical Resistant Infection [SRI], and Indeterminate Initial Infection [III]; and two symptomatic, Subclinical Oligosymptomatic Infection [SOI] and Symptomatic Infection [SI = AVL]. The two distinct scenarios (900 km away) were the urban area of Conceição do Araguaia municipality and the rural area of Bujaru municipality in the southeast and northeast of Pará State. Human populations were chosen based on a simple convenience sampling design (5−10% in each setting), with 1723 individuals (5.3%) of the population (32,464) in the urban area and 1568 individuals (8.9%) of the population (17,596) in the rural one. A serological survey (IFAT-IgG) of canine infection was also performed in both scenarios: 195 dogs in the urban area and 381 in the rural one. Prevalence and incidence rates of human infection were higher in the urban area (20.3% and 13.6/100 person-years [py]) than in the rural setting (14.1% and 6.8/100-py). The AI profile was the most prevalent and incident in both urban (13.4% and 8.1/100-py) and rural (8.3% and 4.2/100-py) scenarios, but with higher rates in the former. An III profile case evolved to SOI profile after four weeks of incubation and another to SI (=AVL) after six. The prevalence of canine infection in an urban setting (39.2%) was also higher (p < 0.05) than that (32%) in the rural zone. AVL urbanization in Pará State, in the Brazilian Amazon, has led to infection rates significantly higher than those in rural sites, requiring more intense control measures.

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs.

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.