Leishmaniasis In South America Research Articles

In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses.

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil.

The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07μM). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.

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

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

Validation of Swab Sampling and SYBR Green-Based Real-Time PCR for the Diagnosis of Cutaneous Leishmaniasis in French Guiana.

Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of Leishmania species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold (CT ) was 24.4 (minimum CT , 17; maximum CT , 36) and was <35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae), an important vector of Leishmaniosis in South America, described by scanning electron microscopy.

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the etiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of the egg, all instar larvae, and the pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy have been highlighted. Different sensillary subtypes—trichoidea, basiconica, coelonica and campanoformia—were observed in the larval stages. The results presented herein contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important vector species.

Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation decrease and sterol biosynthesis of Leishmania braziliensis

Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF3•OEt2-catalyzed imino Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole derivatives were selected and evaluated at different stages of the life cycle of Leishmania braziliensis parasites, responsible for cutaneous leishmaniasis in South America. Among them, the 8′-ethyl-4‘-(4-hydroxy-3-methoxyphenyl)-3′-methyl-3′,4′-dihydro-1′H-spiro[indoline-3,2′-quinolin]-2-one called JS87 was able to inhibit the growth of promastigotes without affecting the mammalian cells viability, and to decrease the number of intracellular amastigotes of L. braziliensis. This spiro compound was found to act through the alteration of parasite internal regulation by disrupting the regulatory volume decrease (RVD), and to affect the sterol biosynthetic pathway at level of squalene epoxidase (SE) enzyme. These results revealed that the spiro annulation between quinoline and oxindole scaffolds enhances the anti-leishmanial activity, and could assist in the development of potent quinoline-oxindole hybrids against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in South America.

Establishment of a method for Lutzomyia longipalpis sand fly egg microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.)longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a 'marker' for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

Establishment of a method for Lutzomyia longipalpis sand fly embryo microinjection: The first step towards potential novel control strategies for leishmaniasis

Leishmaniasis is a vector-borne parasitic disease transmitted by sand flies that affects 1.3 million people across 98 countries, with limited control strategies due to the lack of an available vaccine and the emergence of insecticide resistance. Novel control strategies that are being explored for mosquito-borne diseases, such as Wolbachia bacterial inhibition of pathogens and genetically modified insects (e.g. using CRISPR-Cas9 editing), rely on the ability to consistently inject embryos of the target species. Here we present a novel method to obtain and inject preblastoderm sand fly embryos of the genus Lutzomyia (Lu.) longipalpis, the principle vector of zoonotic visceral leishmaniasis in South America. The procedures required to obtain sufficiently young Lu. longipalpis colony embryos are described alongside a microinjection technique that permits rapid injection and minimal handling of small sand fly embryos post-injection. Using a strain of Wolbachia as a ‘marker’ for successful injection, our protocol produced early generation Wolbachia transinfected Lu. longipalpis lines, demonstrating its potential as the first step for use in novel applied strategies for sand fly control.

Species-specific ecological niche modelling predicts different range contractions for Lutzomyia intermedia and a related vector of Leishmania braziliensis following climate change in South America

BackgroundBefore 1996 the phlebotomine sand fly Lutzomyia neivai was usually treated as a synonym of the morphologically similar Lutzomyia intermedia, which has long been considered a vector of Leishmania braziliensis, the causative agent of much cutaneous leishmaniasis in South America. This report investigates the likely range changes of both sand fly species in response to a stabilisation climate change scenario (RCP4.5) and a high greenhouse gas emissions one (RCP8.5).MethodsEcological niche modelling was used to identify areas of South America with climates currently suitable for each species, and then the future distributions of these climates were predicted based on climate change scenarios. Compared with the previous ecological niche model of L. intermedia (sensu lato) produced using the GARP algorithm in 2003, the current investigation modelled the two species separately, making use of verified presence records and additional records after 2001. Also, the new ensemble approach employed ecological niche modelling algorithms (including Maximum Entropy, Random Forests and Support Vector Machines) that have been widely adopted since 2003 and perform better than GARP, as well as using a more recent climate change model (HadGEM2) considered to have better performance at higher resolution than the earlier one (HadCM2).ResultsLutzomyia intermedia was shown to be the more tropical of the two species, with its climatic niche defined by higher annual mean temperatures and lower temperature seasonality, in contrast to the more subtropical L. neivai. These different latitudinal ranges explain the two species' predicted responses to climate change by 2050, with L. intermedia mostly contracting its range (except perhaps in northeast Brazil) and L. neivai mostly shifting its range southwards in Brazil and Argentina. This contradicts the findings of the 2003 report, which predicted more range expansion. The different findings can be explained by the improved data sets and modelling methods.ConclusionsOur findings indicate that climate change will not always lead to range expansion of disease vectors such as sand flies. Ecological niche models should be species specific, carefully selected and combined in an ensemble approach.

What pre-Columbian mummies could teach us about South American leishmaniases?

A recent report on the taxonomic profile of the human gut microbiome in pre-Columbian mummies (Santiago-Rodriguez etal. 2016) gives for the first time evidence of the presence of Leishmania DNA (sequences similar to Leishmania donovani according to the authors) that can be reminiscent of visceral leishmaniasis during the pre-Columbian era. It is commonly assumed that Leishmania infantum, the etiological agent of American visceral leishmaniasis (AVL) was introduced into the New World by the Iberian conquest. This finding is really surprising and must be put into perspective with what is known from an AVL epidemiological and historical point of view. Beside L. infantum, there are other species that are occasionally reported to cause AVL in the New World. Among these, L. colombiensis is present in the region of pre-Columbian mummies studied. Other explanations for these findings include a more ancient introduction of a visceral species of Leishmania from the Old World or the existence of a yet unidentified endemic species causing visceral leishmaniasis in South America. Unfortunately, very few molecular data are known about this very long pre-Columbian period concerning the circulating species of Leishmania and their diversity in America.

A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome.

BackgroundLeishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine.Methodology/Principal FindingsA L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine.Conclusions/SignificanceHere we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.

South American collaboration in scientific publications on leishmaniasis: bibliometric analysis in SCOPUS (2000-2011).

Objectives: Evaluate the production and the research collaborative network on Leishmaniasis in South America. Methods: A bibliometric research was carried out using SCOPUS database. The analysis unit was original research articles published from 2000 to 2011, that dealt with leishmaniasis and that included at least one South American author. The following items were obtained for each article: journal name, language, year of publication, number of authors, institutions, countries, and others variables. Results: 3,174 articles were published, 2,272 of them were original articles. 1,160 different institutional signatures, 58 different countries and 398 scientific journals were identified. Brazil was the country with more articles (60.7%) and Oswaldo Cruz Foundation (FIOCRUZ) had 18% of Brazilian production, which is the South American nucleus of the major scientific network in Leishmaniasis. Conclusions: South American scientific production on Leishmaniasis published in journals indexed in SCOPUS is focused on Brazilian research activity. It is necessary to strengthen the collaboration networks. The first step is to identify the institutions with higher production, in order to perform collaborative research according to the priorities of each country.

Leishmaniasp. Amastigotes Identification in Canine Transmissible Venereal Tumor

Leishmaniasis is a vector-borne disease withLeishmania chagasibeing the etiological agent of canine visceral leishmaniasis in South America. Canine venereal tumor is a transplantable round cell tumor of histiocytic origin which is mostly observed in sexually active male and female intact dogs. It has been shown thatLeishmaniaamastigotes have higher tropism for the canine male genital tract tissues and venereal leishmaniasis transmission has been documented in dogs but, to date, a canine venereal tumor-dependent transmission route has not been fully demonstrated. In this report, a 10-year-old, mixed breed, intact female dog presented a vaginal venereal transmissible tumor but no other clinical abnormalities otherwise. Unexpectedly, tumor tissue imprint smears examination revealedLeishmaniasp. amastigotes within infiltrating macrophages. In addition to the cytological direct identification, the protozoan was confirmed within the neoplastic tissue by means of immunohistochemistry and polymerase chain reaction. This report illustrates an asymptomaticLeishmaniasp. infection that may have started on or from the canine venereal tumor tissue, the latter option further supporting previous evidence of such an alternative vector-independent route of transmission for canine visceral leishmaniasis in areas where these diseases coexist.

Dynamic of the cellular immune response at the dermal site of Leishmania (L.) amazonensis and Leishmania (V.) braziliensis infection in Sapajus apella primate.

The purpose of this study was to characterize the immunopathological response in the skin of S. apella infected with Leishmania (L.) amazonensis and L. (V.) braziliensis parasites, the main causative agents of localized cutaneous leishmaniasis in South America. In infected animals, amastigote forms of L. (L.) amazonensis could be detected till 120 days postinfection (PI), while, in L. (V.) braziliensis infection, parasites could be detected until 180 days PI in the skin sections. CD20+ cells were detected throughout the experimental time in both groups as well as in CD3+ cells, which appeared to be activated because high densities of inducible nitric oxide synthase (iNOS+) cells were detected at 60 and 90 days PI in both studied groups. After 60 and 120 days PI, decrease in iNOS+ cells was observed in L. (L.) amazonensis and L. (V.) braziliensis, respectively, which was associated with parasite clearance. Increase in lysozyme+ cells was observed during the experimental infections, which also can be associated with parasite killing.

Leishmania amazonensis: Characterization of an ecto-pyrophosphatase activity

Several ecto-enzymatic activities have been described in the plasma membrane of the protozoan Leishmania amazonensis, which is the major etiological agent of diffuse cutaneous leishmaniasis in South America. These enzymes, including ecto-phosphatases, contribute to the survival of the parasite by participating in phosphate metabolism. This work identifies and characterizes the extracellular hydrolysis of inorganic pyrophosphate related to an ecto-pyrophosphatase activity of the promastigote form of L. amazonensis. This ecto-pyrophosphatase activity is insensitive to MnCl2 but is strongly stimulated by MgCl2. This stimulation was not observed during the hydrolysis of p-nitrophenyl phosphate (p-NPP) or β-glycerophosphate, two substrates for different ecto-phosphatases present in the L. amazonensis plasma membrane. Furthermore, extracellular PPi hydrolysis is more efficient at alkaline pHs, while p-NPP hydrolysis occurs mainly at acidic pHs. These results led us to conclude that extracellular PPi is hydrolyzed not by non-specific ecto-phosphatases but rather by a genuine ecto-pyrophosphatase. In the presence of 5mM MgCl2, the ecto-pyrophosphatase activity from L. amazonensis is sensitive to micromolar concentrations of NaF and millimolar concentrations of CaCl2. Moreover, this activity is significantly higher during the first days of L. amazonensis culture, which suggests a possible role for this enzyme in parasite growth.

Clinical complexity of Leishmania (Viannia) braziliensis infections amongst travelers

The protozoan parasite Leishmania (Viannia) braziliensis is one of the main causes of cutaneous and mucocutaneous leishmaniasis in South America. Here, we describe three cases of L.(V.)braziliensis infection which were acquired during travelling in Bolivia, Peru or Paraguay and illustrate the phenotypic heterogeneity and therapeutic complexity of the disease. Two patients presented with unusual clinical manifestations, i.e. with prominent regional lymphadenopathy ("bubonic leishmaniasis") and with simultaneously emerged skin and mucosal lesions, respectively. Both patients insufficiently responded to oral treatment with miltefosine; resolution of the lesions was only achieved after a course of intravenous liposomal amphotericin B.

The Association of Leishmania RNA Viruses with metastatic or mucocutaneous Leishmaniasis in South America

Leishmaniasis is a spectral disease caused by the Trypanosomatid protozoan parasite Leishmania. One manifestation is a disfiguring mucocutaneous form (MCL) involving dissemination to nasopharyngeal areas and tissue destruction, caused by species from the South American Leishmania subgenus Viannia. We reported previously that metastatic L. guyanensis bear high levels of a novel dsRNA totivirus (LRV1), while non‐metastatic lines have low levels or lack LRV1. LRV1‐infected Leishmania were associated with a hyper‐inflammatory response dependent on the host Toll‐like receptor 3 (TLR3) signaling. Thus, LRV in metastatic parasites subverts the host immune response and promotes dissemination. This is the first parasite factor implicated causally in phenotypes relevant to metastatic mucocutaneous disease, with LRV acting as a ‘parasite within a parasite’. The closely related species L. braziliensis is responsible for the great majority of human MCL. Thus we are surveying L. braziliensis strains to establish the distribution and potential for clinical association(s) of LRVs in parasites taken from humans. Several novel LRVs were identified; these are currently being characterized by virus and genome sequencing, classical virological approaches, and infectivity tests of the LRV‐bearing L. braziliensis in animal models.

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L. chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L. chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly ( p < 0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N G-monomethyl- l-arginine (LNMMA) caused a significant reduction of NO production and DNA damage ( p < 0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells.

Guns, germs and dogs: On the origin of Leishmania chagasi

The evolutionary history of Leishmania chagasi, the aetiological agent of visceral leishmaniasis in South America has been widely debated. This study addresses the problem of the origin of L. chagasi, its timing and demography with fast evolving genetic markers, a suite of Bayesian clustering algorithms and coalescent modelling. Here, using 14 microsatellite markers, 450 strains from the Leishmania donovani complex, we show that the vast majority of the Central and South American L. chagasi were nested within the Portuguese Leishmania infantum clade. Moreover, L. chagasi allelic richness was half that of their Old World counterparts. The bottleneck signature was estimated to be about 500 years old and the settlement of L. chagasi in the New World, probably via infected dogs, was accompanied by a thousand-fold population decrease. Visceral leishmaniasis, lethal if untreated, is therefore one more disease that the Conquistadores brought to the New World.

Vaccination with the Leishmania infantum ribosomal proteins induces protection in BALB/c mice against Leishmania chagasi and Leishmania amazonensis challenge

Leishmania chagasi and Leishmania amazonensis are the etiologic agents of different clinical forms of human leishmaniasis in South America. In an attempt to select candidate antigens for a vaccine protecting against different Leishmania species, the efficacy of vaccination using Leishmania ribosomal proteins and saponin as adjuvant was examined in BALB/c mice against challenge infection with both parasite species. Mice vaccinated with parasite ribosomal proteins purified from Leishmania infantum plus saponin showed a specific production of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with L. infantum ribosomal proteins. Vaccinated mice showed a reduction in the liver and spleen parasite burdens after L. chagasi infection. After L. amazonensis challenge, vaccinated mice showed a decrease of the dermal pathology and a reduction in the parasite loads in the footpad and spleen. In both models, protection was correlated to an IL-12-dependent production of IFN-γ by CD4+ and CD8+ T cells that activate macrophages for the synthesis of NO. In the protected mice a decrease in the parasite-mediated IL-4 and IL-10 responses was also observed. In mice challenged with L. amazonensis, lower levels of anti-parasite-specific antibodies were detected. Thus, Leishmania ribosomal proteins plus saponin fits the requirements to compose a pan-Leishmania vaccine.