Subject(s)
Leishmania mexicana , Pseudomonas Infections , Pseudomonas aeruginosa , Superinfection , Humans , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/genetics , Superinfection/microbiology , Pseudomonas Infections/microbiology , Pseudomonas Infections/diagnosis , Leishmania mexicana/genetics , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/microbiology , Male , Communicable Diseases, Imported/microbiology , Communicable Diseases, Imported/parasitology , Communicable Diseases, Imported/diagnosis , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/pharmacologyABSTRACT
In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.
Subject(s)
Amphotericin B , Antiprotozoal Agents , Drug Resistance , Amphotericin B/pharmacology , Amphotericin B/therapeutic use , Animals , Brazil , Middle Aged , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/therapeutic use , Humans , Male , Mice , Leishmania/drug effects , Leishmania/isolation & purification , Leishmania/classification , Leishmania mexicana/drug effects , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/parasitology , HIV Infections/complications , HIV Infections/drug therapy , Parasitic Sensitivity Tests , Mice, Inbred BALB C , Leishmaniasis, Diffuse Cutaneous/parasitology , Leishmaniasis, Diffuse Cutaneous/drug therapyABSTRACT
Background: Leishmaniases are a group of vector-borne zoonotic diseases of public health relevance within the tropical and subtropical regions of the world. The state of Yucatan is a vulnerable and receptive area to localized cutaneous leishmaniasis (LCL) due to its proximity to the high-transmission endemic states of Campeche and Quintana Roo. Autochthonous cases of LCL caused by Leishmania (Leishmania) mexicana have been documented in the state, showing a geographical expansion of the disease. Materials and Methods: Using CO2-supplemented Centers for Disease Control and Prevention light traps and Shannon traps, we captured anthropophilic sandflies in the surroundings of a locality with recent records of autochthonous cases of LCL. Sandflies carrying Leishmania DNA were evidenced using PCR. Results: A total of 140 Phlebotominae (Diptera: Psychodidae) females of four species were captured: Lutzomyia (Tricholateralis) cruciata (Coquillett), Psathyromyia (Psathyromyia) shannoni (Dyar), Lutzomyia (Lutzomyia) longipalpis (Lutz and Neiva), and Dampfomyia (Coromyia) deleoni (Fairchild and Hertig). Molecular results showed that 6.1% (95% confidence interval [CI] = 2.3-12.9%) of Lu. cruciata and 43.8% (95% CI = 19.8-70.1%) of Pa. shannoni showed evidence of carrying L. (L.) mexicana DNA. Conclusion: We provide evidence of anthropophilic sandflies carrying L. mexicana DNA in a municipality with recorded autochthonous cases of LCL caused by this parasite species in the state of Yucatan, suggesting the emergence of new focus of LCL in Mexico.
Subject(s)
Leishmania mexicana , Psychodidae , Animals , Leishmania mexicana/classification , Leishmania mexicana/genetics , Leishmania mexicana/isolation & purification , Mexico , Psychodidae/parasitologyABSTRACT
Leishmaniasis is prevalent in Southern Europe, the Middle East, India, Africa, and Central and South America. Cutaneous leishmaniasis may spontaneously heal over time without treatment; however, risk of visceral dissemination and the impact of cosmetic defect are important concerns. We report a Case of cutaneous leishmaniasis in a patient who ever traveled to Mexico before the onset of a deteriorating wound around the swollen left eyebrow. A diagnosis of infection with Leishmania mexicana was made based on histopathological examination and molecular identification. Systemic treatment with liposomal amphotericin B and ketoconazole were administered with gradual healing of the lesion. Also, this traveler case implicates that the spread of endemic parasitic diseases may be a concealed risk on the public health for Taiwan underlying globalization.
Subject(s)
Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/drug therapy , Travel-Related Illness , Adult , Amphotericin B/therapeutic use , DNA, Protozoan/genetics , Humans , Ketoconazole/therapeutic use , Leishmania mexicana/genetics , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/pathology , Male , Treatment OutcomeABSTRACT
Trypanosoma cruzi and Leishmania mexicana are parasites of humans and other mammals, causing American Trypanosomiasis and Cutaneous Leishmaniasis, respectively. Domestic dogs are considered key hosts for these parasites in the domicile and peridomicile cycles of transmission, due to their abundance and contact with human population. In Mexico, there are few studies that involve the study of infection with these parasites in dogs, and have only been carried out mainly in the endemic areas for these diseases. In the state of Querétaro (Mexico), infections with both parasites have been reported for dogs only from rural areas, with no records for the metropolitan zone. We analyzed the seropositivity to T. cruzi and L. mexicana in dogs from localities within of the metropolitan zone of Querétaro City in order to determine if these animals are exposed to these parasites and thus, could be an important part of the transmission cycle of these trypanosomatids in a densely populated urban region within the state of Querétaro, Mexico. Serum samples were collected from 303 dogs housed in the Animal Control centers of the municipalities of Querétaro and El Marques, analyzed by indirect ELISA and Western Blot using as an antigen the Iron Superoxide Dismutase (FeSODe) of the parasites. From the total serum samples, we detected 10.2% of seropositivity for T. cruzi and 2.9% for L. mexicana. Our results represent the first evidence of infection with T. cruzi in domestic dogs from the Metropolitan Zone of Querétaro, and the first record for L. mexicana in Central Mexico. Ongoing investigations seek to confirm the circulation of these parasites in the area to evaluate the risk associated to the human population.
Subject(s)
Chagas Disease/veterinary , Dog Diseases/epidemiology , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/veterinary , Trypanosoma cruzi/isolation & purification , Animals , Blotting, Western/veterinary , Chagas Disease/epidemiology , Chagas Disease/parasitology , Dog Diseases/parasitology , Dogs , Enzyme-Linked Immunosorbent Assay/veterinary , Leishmaniasis, Cutaneous/epidemiology , Leishmaniasis, Cutaneous/parasitology , Mexico/epidemiology , Prevalence , Seroepidemiologic StudiesABSTRACT
Leishmania mexicana can produce chronic infections leading to exhausted T cell phenotypes, mediated by PD-1/PD-L1. Little is known on mechanisms that induce these inhibitory molecules in chronic leishmaniasis. We analyzed factors that contribute to exhausted phenotypes in chronic L. mexicana infections of mice. Our results show that draining lymph node cells express enhanced levels of PD-1/PD-L1. T lymphocytes producing low cytokine levels were also found. L. mexicana infection of dendritic cells (DCs) produced elevated amounts of TNF and showed up-regulation of PD-L1 expression. We provide evidence that T cells of chronic L. mexicana infections in mice are functionally exhausted due to chronic TNF production, which leads to PD-L1 up-regulation in DCs. We conclude that TNF has a fundamental role in promoting T cell exhaustion during chronic L. mexicana infections, which contributes to the inability of T cells to proliferate and produce pro-inflammatory cytokines, thus favoring disease progression.
Subject(s)
B7-H1 Antigen/immunology , Leishmaniasis, Cutaneous/immunology , Programmed Cell Death 1 Receptor/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/metabolism , Animals , B7-H1 Antigen/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cytokines/genetics , Cytokines/immunology , Dendritic Cells/immunology , Disease Models, Animal , Female , Leishmania mexicana/immunology , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/metabolism , Mice , Mice, Inbred BALB C , Programmed Cell Death 1 Receptor/genetics , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/physiology , Up-RegulationABSTRACT
As the causative agent of hard-to-treat diffuse cutaneous leishmaniasis, Leishmania (L.) amazonensis persists in the host organism sheltered within large Parasitophorous Vacuoles (PVs) formed mainly in macrophages. In the present study, I present a simple and efficient method for L. amazonensis PV isolation. Isolated PVs are intact as demonstrated by the conservation of lysosomal probes loaded into PVs before the procedure. The method is useful for studies aiming at a complete and accurate molecular profile of these structures, to better understand the biogenesis of this pathogen-containing vacuole and its implication in parasite persistence and in leishmaniasis pathogenesis.
Subject(s)
Leishmania mexicana/isolation & purification , Leishmaniasis, Diffuse Cutaneous/parasitology , Macrophages/parasitology , Animals , Humans , Leishmania mexicana/growth & development , Lysosomal-Associated Membrane Protein 1/immunology , Lysosomal-Associated Membrane Protein 2/immunology , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Species Specificity , Vacuoles/parasitologyABSTRACT
The inadequacy of available treatments for leishmaniasis has presented up to 40% therapeutic failure. This fact suggests an urgency in the discovery of new drugs or alternative approaches for treating this disease. The objective of this study was to evaluate the antileishmanial activity of combined therapy between crotamine (CTA) from Crotalus durissus terrificus and the pentavalent antimonial Glucantime® (GLU). The assays were in vitro performed measuring the inhibition of Leishmania amazonensis amastigotes, followed by the evaluation of cellular production of cytokines and nitrites. After that, analytical methods were performed in order to characterize the molecules involved in the study by Mass Spectrometry, molecular affinity through an in silico assay and Surface Plasmon Resonance. In vivo experiments with BALB/c mice were performed by analyzing parasitemia, lesion size and immunological mediators. In the in vitro experiments, the pharmacological association improved the inhibition of the amastigotes, modulated the production of cytokines and nitric oxide. The therapy improved the effectiveness of the GLU, demonstrating a decreased parasitemia in the infected tissues. Altogether, the results suggest that the combined approach with CTA and GLU may be a promising alternative for the treatment of cutaneous leishmaniasis.
Subject(s)
Antiprotozoal Agents/therapeutic use , Crotalid Venoms/therapeutic use , Crotalus , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/drug therapy , Meglumine Antimoniate/therapeutic use , Animals , Antiprotozoal Agents/pharmacology , Crotalid Venoms/pharmacology , Drug Combinations , Interleukin-12/blood , Interleukin-12/metabolism , Leishmania mexicana/isolation & purification , Lymph Nodes/parasitology , Macrophages, Peritoneal , Mass Spectrometry , Meglumine Antimoniate/pharmacology , Mice , Mice, Inbred BALB C , Molecular Docking Simulation , Nitric Oxide/metabolism , Nitrites/analysis , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In the United States, phlebotomine sand flies carrying Leishmania (Leishmania) mexicana are endemic along the southern border. However, relatively little is known about the enzootic and zoonotic transmission of L. (L.) mexicana within the United States, and autochthonous cases of the consequent disease are rarely reported. We investigated an atypical case of cutaneous leishmaniasis (CL) caused by L. (L.) mexicana in a patient from central Texas which did not respond to a typical antileishmanial chemotherapy. We also investigated sand fly vectors around the patient's residence. PCR followed by DNA sequencing was used for determination of Leishmania spp., sand fly species, and host blood meal source. The L. (L.) mexicana genotype from the patient was identical to one found in a positive sand fly. Moreover, this genotype presented the same single-nucleotide polymorphisms as other historical CL cases acquired in Texas over the last 10 years, but distinct from those originating in Mexico and Central America. Three sand fly species were identified among the samples analyzed (n = 194), the majority of which were Lutzomyia (Dampfomyia) anthophora (n = 190), of which four specimens tested positive for Leishmania and two blood-fed specimens showed the presence of a human blood meal. This study highlights the complexity of clinical management of CL in a setting where the disease is infrequently encountered. The detection of human blood in Lu. (D.) anthophora is the first documentation of anthropophagy in this species. This is the first report of wild-caught, naturally infected sand flies found in association with an autochthonous case of human leishmaniasis and the specific strain of Leishmania (Leishmania) mexicana in the United States.
Subject(s)
Insect Vectors/parasitology , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Phlebotomus/parasitology , Aged , Animals , Humans , Leishmania mexicana/genetics , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/pathology , Male , Polymerase Chain Reaction , Sequence Analysis, DNA , TexasABSTRACT
l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with Leishmania mexicana can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an in vivo model the capacity of two L. mexicana isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with L. mexicana isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that L. mexicana isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.
Subject(s)
Arginase/metabolism , Arginine/metabolism , Host-Pathogen Interactions , Leishmania mexicana/physiology , Leishmaniasis, Diffuse Cutaneous/metabolism , Leishmaniasis, Diffuse Cutaneous/parasitology , Animals , Disease Models, Animal , Disease Susceptibility , Humans , Leishmania mexicana/isolation & purification , Macrophages/immunology , Macrophages/metabolism , Macrophages/parasitology , Mice , Nitric Oxide Synthase Type II/metabolism , Time FactorsABSTRACT
This protocol describes the use of heavy water (2H2O) labeling to determine the growth rate and metabolic state of Leishmania parasites in culture and in infected animals. In vitro labeling studies are undertaken by cultivating defined parasite developmental stages in standard medium supplemented with 5% 2H2O, resulting in the incorporation of deuterium (2H) into a range of metabolic precursors used in macromolecule (DNA, RNA, protein, lipid, and glycan) synthesis. The rate of turnover of different parasite macromolecules can subsequently be determined by analysis of deuterium enrichment in the different constituents of these macromolecules by gas chromatography-mass spectrometry (GC-MS). To measure the growth rate and physiological state of parasite stages in lesion tissue, infected mice were provided with 9% 2H2O in their drinking water for various periods of time and 2H-enrichment in the macromolecular constituents of isolated lesion-derived parasite stages determined by GC-MS. This protocol provides quantitative information on key cellular processes, such as replication (DNA turnover), transcription (RNA turnover), translation (protein turnover), membrane biogenesis (lipid turnover), and central carbon metabolism (glycan turnover) that define the growth state and phenome of different parasite stages in vitro and in vivo. This approach can be used to assess the impact of host immune responses on parasite growth and physiology (using different Leishmania strains/species, mouse lines), characterize different parasite populations during chronic and acute infections, and assess parasite responses to drug treatments. It is also broadly applicable to other microbial pathogens.
Subject(s)
Deuterium Oxide/chemistry , Isotope Labeling/methods , Leishmania mexicana/metabolism , Leishmaniasis, Cutaneous/diagnosis , Animals , DNA, Protozoan/analysis , DNA, Protozoan/chemistry , DNA, Protozoan/metabolism , Disease Models, Animal , Female , Gas Chromatography-Mass Spectrometry/methods , Humans , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/pathology , Life Cycle Stages/physiology , Metabolomics/methods , Mice , Polysaccharides/analysis , Polysaccharides/chemistry , Polysaccharides/metabolism , Protozoan Proteins/analysis , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , RNA, Protozoan/analysis , RNA, Protozoan/chemistry , RNA, Protozoan/metabolism , Skin/parasitologyABSTRACT
In the present study, we reported the natural infection by Leishmania sp. in a domestic cat, in which the amastigote forms of the parasite were observed within a lesion on its ear-tip. Fragment of the lesion was obtained and cultured in NNN medium, and PCR-RFLP analysis of the isolated sample was performed, which revealed that the profile was compatible with Leishmania (L.) amazonensis. This is the first proven case of a cat infected by L. (L.) amazonensis reported in Belém city, Pará state, northern Brazil.
Subject(s)
Allopurinol/therapeutic use , Azithromycin/therapeutic use , Cat Diseases/diagnosis , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/veterinary , Skin Diseases/veterinary , Trypanocidal Agents/therapeutic use , Animals , Brazil , Cat Diseases/drug therapy , Cat Diseases/parasitology , Cats , Diagnosis, Differential , Female , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/parasitology , Skin Diseases/diagnosis , Skin Diseases/drug therapy , Skin Diseases/parasitologyABSTRACT
To elucidate the transmission mode of Andean cutaneous leishmaniasis (Andean-CL), natural Leishmania infection and biting activity of sand flies were tested in a selected sylvatic focus of the endemic area of the Ecuadorian Andes. Monthly sand fly collections and dissections were conducted during 12 months from July 2018 to June 2019. The Leishmania positive specimens/slides with innumerable amounts of actively mobile flagellates made us easy to detect positive sand flies. The promastigotes observed located in the anterior and posterior midgut, without the hindgut localization. The parasite isolated was identified as L. (L.) mexicana by cytochrome b gene analysis. No other Leishmania or flagellate species parasitic in sand flies was observed in the area. Only Lu. ayacuchensis was caught throughout. Monthly microscopic examination of Lu. ayacuchensis revealed 0.75-8.33% of natural L. (L.) mexicana infection rates. Higher Leishmania infection months were present at the end of the wet season of the Andes, while higher sand fly numbers occurred during the dry season. Diurnal biting (blood meal seeking) activity of sand flies started around 17:30 before sunset, increased between 18:00 and 19:30, and thereafter decreased drastically probably because of low temperature (15-18⯰C) in the area. The results provide information important for the planning of vector control strategy and management of the disease in the Andean-CL endemic area of Ecuador.
Subject(s)
Insect Bites and Stings , Insect Vectors/parasitology , Leishmaniasis, Cutaneous/transmission , Psychodidae/parasitology , Animals , Ecuador/epidemiology , Humans , Leishmania mexicana/genetics , Leishmania mexicana/isolation & purificationABSTRACT
Macrophages can reprogram their metabolism in response to the surrounding stimuli, which affects their capacity to kill intracellular pathogens. We have investigated the metabolic and immune status of human macrophages after infection with the intracellular trypanosomatid parasites Leishmania donovani, L. amazonensis and T. cruzi and their capacity to respond to a classical polarizing stimulus (LPS and IFN-γ). We found that macrophages infected with Leishmania preferentially upregulate oxidative phosphorylation, which could be contributed by both host cell and parasite, while T. cruzi infection did not significantly increase glycolysis or oxidative phosphorylation. Leishmania and T. cruzi infect macrophages without triggering a strong inflammatory cytokine response, but infection does not prevent a potent response to LPS and IFN-γ. Infection appears to prime macrophages, since the cytokine response to activation with LPS and IFN-γ is more intense in infected macrophages compared to uninfected ones. Metabolic polarization in macrophages can influence infection and immune evasion of these parasites since preventing macrophage cytokine responses would help parasites to establish a persistent infection. However, macrophages remain responsive to classical inflammatory stimuli and could still trigger inflammatory cytokine secretion by macrophages.
Subject(s)
Chagas Disease/immunology , Cytokines/metabolism , Leishmaniasis/immunology , Macrophage Activation , Macrophages/immunology , 3T3 Cells , Animals , Cells, Cultured , Chagas Disease/blood , Chagas Disease/parasitology , Cytokines/immunology , Healthy Volunteers , Humans , Leishmania donovani/immunology , Leishmania donovani/isolation & purification , Leishmania mexicana/immunology , Leishmania mexicana/isolation & purification , Leishmaniasis/blood , Leishmaniasis/parasitology , Macrophages/metabolism , Metabolome/immunology , Mice , Oxidative Phosphorylation , Primary Cell Culture , Trypanosoma cruzi/immunology , Trypanosoma cruzi/isolation & purification , Up-RegulationABSTRACT
BACKGROUND: American cutaneous leishmaniasis (ACL) is endemic in French Guiana. Its epidemiology is evolving, notably because of immigration, anthropization of natural areas, and new microbiological methods. Our first objective was to update epidemiological data. Our second objective was to look for risk factors of ACL. METHODS: This multicentric study was conducted from October 2017 to June 2018 in French Guiana. Patients with suspicion of mucocutaneous leishmaniasis were included in case of positive smear, culture, or PCR-RFLP on skin biopsy. RESULTS: One hundred and twenty-three patients met the inclusion criteria. Among those patients, 59.3% were Brazilian, mostly gold miners. Most of them (58%) were between 16 and 40 years old, and 69% were male. A large proportion of patients lived in traditional wooden houses (51%). Patients living in coastal towns were usually infected during trips to the primary forest (60%) and had a shorter time to diagnosis than workers of the hinterland. Among environmental risk factors, the presence of a water spring (40%) and dogs around houses (40%) were frequently reported. Leishmania guyanensis represented 80% of cases, followed by Leishmania braziliensis (6%), Leishmania naiffi (2%), and Leishmania amazonensis (1%). CONCLUSIONS: Gold mining and trips to the primary forest represent high-risk situations for ACL in French Guiana, where the population of infected patients is dominated by Brazilian immigrants. Possible environmental risk factors such as the presence of dogs, water sources, and traditional wooden houses require further investigation.
Subject(s)
Endemic Diseases , Leishmaniasis, Cutaneous/epidemiology , Skin/parasitology , Adolescent , Adult , Aged , Biopsy , Environmental Exposure/adverse effects , Female , Forests , French Guiana/epidemiology , Gold , Humans , Leishmania braziliensis/isolation & purification , Leishmania guyanensis/isolation & purification , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/parasitology , Male , Middle Aged , Mining/statistics & numerical data , Risk Factors , Young AdultABSTRACT
Leishmaniasis is a disease that affects millions of people and it is an important public health problem. The drugs currently used for the treatment of leishmaniasis present undesirable side effects and low efficacy. In this study, we evaluated the in vitro activity of Melampodium divaricatum (MD-EO) and Casearia sylvestris (CS-EO) essential oils (EO) against promastigote and amastigote forms of Leishmania amazonensis. Sesquiterpenes E-caryophyllene (56.0%), germacrene D (12.7%) and bicyclogermacrene (9.2%) were identified as the main components of MD-EO, whereas E-caryophyllene (22.2%), germacrene D (19.6%) and bicyclogermacrene (12.2%) were the main constituents of CS-EO. CS-EO and E-caryophyllene were active against promastigote forms of L. amazonensis (IC50 24.2, 29.8 and 49.9 µg/mL, respectively). However, MD-EO, CS-EO and E-caryophyllene were more active against amastigote forms, with IC50 values of 10.7, 14.0, and 10.7 µg/mL, respectively. E-caryophyllene presented lower cytotoxicity against macrophages J774-A1 (CC50 of 62.1 µg/mL) than the EO. The EOs and E-caryophyllene should be further studied for the development of new antileishmanial drugs.
Subject(s)
Antiprotozoal Agents/pharmacology , Asteraceae/chemistry , Casearia/chemistry , Leishmania mexicana/drug effects , Oils, Volatile/pharmacology , Antiprotozoal Agents/isolation & purification , Humans , Inhibitory Concentration 50 , Leishmania mexicana/isolation & purification , Parasitic Sensitivity TestsABSTRACT
In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50â¯ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.
Subject(s)
Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/diagnosis , Skin/parasitology , Animals , Base Sequence , Colorimetry , Cricetinae , DNA, Kinetoplast/chemistry , DNA, Kinetoplast/genetics , DNA, Protozoan/isolation & purification , Electrophoresis, Polyacrylamide Gel , Female , Leishmania mexicana/genetics , Leishmaniasis, Cutaneous/parasitology , Male , Mesocricetus , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Silver StainingABSTRACT
For years, mammals of the order Pilosa have been considered Leishmania reservoirs. But while most studies have focused on sloth species, anteaters have been overlooked, and in many Leishmania endemic countries like Mexico, no studies have been carried out. The aims of this work were to identify the presence of Leishmania spp. in tissue samples from road-killed northern tamanduas (Tamandua mexicana), using PCR amplification and sequencing of ITS1 DNA, and to discuss the role of Pilosa mammals as reservoirs of Leishmania based on available scientific records. This is the first study that identifies Leishmania in T. mexicana, from 1 of 16 individuals analyzed, so the estimated prevalence (CI 95%) of infection was 6.3% (0.3-27.2). Amplified sequence exhibited a 98.9% (727/735) similarity with L. mexicana, and phylogenetic analysis grouped the species in the L. mexicana-amazonensis cluster. The literature review revealed 241 cases of Leishmania spp. infection among 1219 Pilosa mammals evaluated, with prevalence between studies ranging from 3.5% in the brown-throated three-toed sloth (Bradypus variegatus) to 78% in the Hoffman's two-toed sloth (Choloepus hoffmanni). Current scientific information indicates that C. hoffmanni sloths are reservoirs of Leishmania, and further studies are needed in order to clarify if other Pilosa species play a role in Leishmania transmission.
Subject(s)
Disease Reservoirs/parasitology , Leishmania mexicana/isolation & purification , Leishmaniasis/epidemiology , Leishmaniasis/veterinary , Sloths/parasitology , Xenarthra/parasitology , Animals , DNA, Protozoan/genetics , Leishmania mexicana/genetics , Mexico/epidemiology , PhylogenyABSTRACT
Leishmaniasis is a neglected tropical disease caused by different species of protozoan parasites of the genus Leishmania. Dogs have been proven as primary hosts of the parasite. Cases of cutaneous leishmaniasis in humans caused by Leishmania mexicana have been reported in Sinaloa; however, the vectors and hosts involved in the epidemiology of the parasite in northwestern Mexico are still unknown. Given the public health implications of this parasite's domestic hosts regarding the permanence and transmission of the disease to humans, the objective of the present study was to detect and determine the species of Leishmania that caused the first three cases of autochthonous canine leishmaniasis in the state of Sinaloa, Mexico. Three domestic dogs showing symptoms similar to canine leishmaniasis were identified, including chronic eye inflammation, corneal opacity, ocular exudate, emaciation and hyporexia. DNA was extracted from venous blood of the infected animals using a commercial kit. The internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by specific primers for Leishmania from the extracted DNA, and the PCR products were digested with the restriction enzyme HaeIII. In addition, PCR products were subjected to automated sequencing. Molecular analysis showed that the infecting species was L. mexicana. This is the first report of autochthonous canine leishmaniasis caused by L. mexicana in Sinaloa, Mexico. Further studies are required to identify the species that serve as vectors and other wild and domestic hosts of the parasite, as well as to determine if there are more species of Leishmania circulating in Sinaloa.
Subject(s)
Dog Diseases/parasitology , Leishmania mexicana/isolation & purification , Leishmaniasis, Cutaneous/veterinary , Animals , Dogs , Leishmania mexicana/genetics , Leishmaniasis, Cutaneous/parasitology , Polymerase Chain ReactionABSTRACT
BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.