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1.
Rev. Soc. Bras. Med. Trop ; 54: e00472020, 2021. tab, graf
Article in English | LILACS, ColecionaSUS, SES-SP | ID: biblio-1143886

ABSTRACT

Abstract INTRODUCTION: The objective of this study was to evaluate the performance of filter paper (FP) for lesion scraping collection in a polymerase chain reaction (PCR) for cutaneous leishmaniasis (CL) diagnosis. METHODS: Lesion scrapings from 48 patients were collected and analyzed for PCR. RESULTS: PCR with FP detected up to three Leishmania braziliensis promastigotes. Considering the direct search by microscopy or PCR of samples collected in STE buffer as standards, the sensitivity of PCR with FP was 100%. CONCLUSIONS: FP can be useful for CL diagnosis in remote regions, allowing high sensitivity in the detection of the parasite by PCR.


Subject(s)
Humans , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/diagnosis , Polymerase Chain Reaction , DNA, Protozoan/genetics , Sensitivity and Specificity , Microscopy
2.
Rev. Soc. Bras. Med. Trop ; 54: e02122021, 2021. tab, graf
Article in English | LILACS | ID: biblio-1288097

ABSTRACT

Abstract INTRODUCTION: This study evaluated the epidemiology of American cutaneous leishmaniasis in the immediate region of Ji-Paraná, Rondônia State. METHODS: Samples and epidemiological data were collected from 105 patients. RESULTS: Leishmania infection was observed in 58 (55.2%) patients, and Leishmania braziliensis was present in 82.9% of the 41 sequenced samples. Infected patients were predominantly male (93.1%). Leishmania infection was twice as prevalent among rural inhabitants versus urban inhabitants. Lesions were more frequent in the upper limbs (arms/hands, 41.82%). CONCLUSIONS: The present data corroborate the zoonotic profile of cutaneous leishmaniasis; this information could help to improve surveillance and control strategies.


Subject(s)
Humans , Male , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/epidemiology , Rural Population , Brazil/epidemiology , Base Sequence
3.
Mem. Inst. Oswaldo Cruz ; 115: e200113, 2020. tab, graf
Article in English | LILACS, SES-SP | ID: biblio-1135235

ABSTRACT

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.


Subject(s)
Animals , Psychodidae/parasitology , Leishmania braziliensis/genetics , Proteomics , Leishmania/genetics , Cell Line , Transcriptome
4.
Rev. Soc. Bras. Med. Trop ; 53: e20200255, 2020. tab, graf
Article in English | LILACS, ColecionaSUS, SES-SP | ID: biblio-1143862

ABSTRACT

Abstract INTRODUCTION: In the Belém Metropolitan Region (BMR), Pará State, Brazil, American cutaneous leishmaniasis (ACL) is endemic; however, very little is known regarding its causative agents. Therefore, we used our standard diagnostic approach combined with an RNA polymerase II largest subunit (RNAPOIILS)-polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphism (PCR-RFLP) to identify Leishmania spp. ACL agents in this region. METHODS: Thirty-two Leishmania spp. isolates from patients with ACL in the BMR during 1995-2018 were analyzed. Leishmania spp. DNA samples were amplified using the primers RPOR2/RPOF2, and the 615-bp PCR products were subjected to enzymatic digestion using TspRI and HgaI endonucleases. RESULTS: ACL etiological agents in the BMR comprised Leishmania (Viannia) lindenbergi (43.7%) followed by Leishmania (Viannia) lainsoni (34.4%), Leishmania (Leishmania) amazonensis (12.5%), and Leishmania (Viannia) braziliensis (9.4%). CONCLUSIONS: To our knowledge, the results of the study revealed for the first time that L. (V.) lindenbergi and L. (V.) lainsoni are the main ACL agents in BMR.


Subject(s)
Humans , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/epidemiology , Leishmania/genetics , United States , Brazil/epidemiology , Leishmaniasis, Cutaneous
5.
Rev. bras. parasitol. vet ; 29(2): e003520, 2020. tab, graf
Article in English | LILACS | ID: biblio-1138090

ABSTRACT

Abstract Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.


Resumo Amostras de sangue e swabs da conjuntiva ocular e oral foram obtidas de 64 gatos. Das 64 amostras de soro, 19 foram positivas para anticorpos contra Leishmania por ELISA (29,80%). Oito gatos foram positivos por PCR (12,5%) em amostras de swab da boca e / ou mucosa ocular. Demonstrou-se baixa concordância kappa entre os resultados sorológicos e moleculares (k = 0,16). Das cinco amostras positivas para PCR, uma era L. braziliensis e quatro eram L infantum. A análise filogenética realizada com os cinco isolados de Leishmania, mostrou que amostras de L. infantum, isoladas dos gatos, eram filogeneticamente próximas às isoladas de cães domésticos do Brasil enquanto L. braziliensis era muito semelhante ao descrito em humanos na Venezuela. O estudo demonstrou que, apesar da alta soropositividade para Leishmania, em gatos que vivem na região do estudo, pouca concordância entre os resultados sorológicos e moleculares indica que a sorologia positiva não é indicativa de infecção por Leishmania em gatos. O DNA do parasita pode ser detectado na conjuntiva ocular e nas zaragatoas orais de gatos, indicando que essas amostras podem ser usadas para o diagnóstico. . Resultados de análises filogenéticas mostram que L. infantum, circulando no Brasil, é capaz de infectar diferentes hospedeiros, demonstrando a capacidade do parasita de superar a barreira interespécies.


Subject(s)
Animals , Cats , Leishmania braziliensis/isolation & purification , Cat Diseases/parasitology , Leishmaniasis/parasitology , Leishmania infantum/isolation & purification , Phylogeny , Leishmania braziliensis/genetics , Leishmania braziliensis/immunology , Enzyme-Linked Immunosorbent Assay/veterinary , Antibodies, Protozoan/blood , Cat Diseases/diagnosis , Leishmaniasis/diagnosis , Polymerase Chain Reaction/veterinary , DNA, Protozoan/analysis , Leishmania infantum/genetics , Leishmania infantum/immunology
6.
Mem. Inst. Oswaldo Cruz ; 115: e190413, 2020. tab, graf
Article in English | LILACS | ID: biblio-1101274

ABSTRACT

BACKGROUND The leishmaniases are complex neglected diseases caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis in the New World. In recent studies, genomic changes such as chromosome and gene copy number variations (CNVs), as well as transcriptomic changes have been highlighted as mechanisms used by Leishmania species to adapt to stress situations. OBJECTIVES The aim of this study was to determine the effect of short-term minor temperature shifts in the genomic and transcriptomic responses of L. braziliensis promastigotes in vitro. METHODS Growth curves, genome and transcriptome sequencing of L. braziliensis promastigotes were conducted from cultures exposed to three different temperatures (24ºC, 28ºC and 30ºC) compared with the control temperature (26ºC). FINDINGS Our results showed a decrease in L. braziliensis proliferation at 30ºC, with around 3% of the genes showing CNVs at each temperature, and transcriptomic changes in genes encoding amastin surface-like proteins, heat shock proteins and transport proteins, which may indicate a direct response to temperature stress. MAIN CONCLUSIONS This study provides evidence that L. braziliensis promastigotes exhibit a decrease in cell density, and noticeable changes in the transcriptomic profiles. However, there were not perceptible changes at chromosome CNVs and only ~3% of the genes changed their copies in each treatment.


Subject(s)
Animals , Temperature , Leishmania braziliensis/genetics , Adaptation, Physiological/genetics , DNA Copy Number Variations/genetics , Transcriptome/genetics , Adaptation, Physiological/physiology , Gene Expression Profiling , Genetic Profile
7.
Mem. Inst. Oswaldo Cruz ; 114: e190111, 2019. tab, graf
Article in English | LILACS | ID: biblio-1020081

ABSTRACT

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.


Subject(s)
Humans , Leishmania braziliensis/drug effects , Leishmania braziliensis/genetics , Drug Resistance/drug effects , Leishmaniasis, Cutaneous/parasitology , Aquaglyceroporins/metabolism , Antimony/pharmacology , Drug Resistance/genetics , Real-Time Polymerase Chain Reaction
8.
Mem. Inst. Oswaldo Cruz ; 114: e180438, 2019. tab, graf
Article in English | LILACS | ID: biblio-1040619

ABSTRACT

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.


Subject(s)
Leishmania braziliensis/genetics , DNA, Protozoan/genetics , Sequence Analysis, DNA
9.
Mem. Inst. Oswaldo Cruz ; 114: e190147, 2019. tab, graf
Article in English | LILACS | ID: biblio-1040618

ABSTRACT

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.


Subject(s)
Animals , Mice , Leishmania braziliensis/chemistry , Calpain/genetics , Macrophages, Peritoneal/metabolism , Genome, Protozoan/genetics , Leishmania braziliensis/genetics , Leishmania braziliensis/metabolism , Leishmania braziliensis/ultrastructure , Immunohistochemistry , Calpain/drug effects , Calpain/metabolism , Calpain/ultrastructure , Cysteine Proteinase Inhibitors/pharmacology , Gene Expression Regulation , Blotting, Western , Reverse Transcriptase Polymerase Chain Reaction , Virulence Factors , Microscopy, Electron, Transmission , Dipeptides/pharmacology , Flow Cytometry , Mice, Inbred BALB C
10.
Rev. Soc. Bras. Med. Trop ; 51(3): 376-381, Apr.-June 2018. tab, graf
Article in English | LILACS | ID: biblio-1041460

ABSTRACT

Abstract INTRODUCTION This study proposes to identify the Leishmania species found in the skin lesions of cutaneous leishmaniasis (CL) patients from Brasiléia municipality (Acre). METHODS Skin biopsy imprints or biopsy fragments were assayed via kDNA-PCR/RFLP and FRET-real-time PCR. RESULTS Of individuals with suspected CL, 18 were positive for Leishmania kDNA. Leishmania (Viannia) braziliensis (61.1%) and Leishmania (Viannia) guyanensis (5.5%) were identified in the positive samples. CONCLUSIONS These results are congruent with the previous reports in Acre and Bolivia, revealing L. braziliensis as the most prevalent species. L. guyanensis identification also corroborates with the epidemiology of the disease in the Amazon Basin.


Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Child, Preschool , Child , Adolescent , Adult , Young Adult , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/diagnosis , Leishmania guyanensis/genetics , Biopsy , Polymorphism, Restriction Fragment Length , Brazil/epidemiology , DNA, Protozoan/genetics , Leishmaniasis, Cutaneous/epidemiology , DNA, Kinetoplast/genetics , Endemic Diseases , Real-Time Polymerase Chain Reaction
11.
Rev. Soc. Bras. Med. Trop ; 51(1): 108-110, Jan.-Feb. 2018. graf
Article in English | LILACS | ID: biblio-897042

ABSTRACT

Abstract Cutaneous leishmaniasis is usually transmitted by infected phlebotomine sand fly bites that initiate local cutaneous lesions. Few reports in the literature describe other modes of transmission. We report a case of a previously healthy 59-year-old woman who underwent electrocoagulation to remove seborrheic keratosis confirmed by dermatoscopy. Three months later, a skin fragment tested positive for Leishmania culture; the parasite was identified as L. (V.) braziliensis. Trauma may generate inflammatory cascades that favor Leishmania growth and lesion formation in previously infected patients. American cutaneous leishmaniasis is a dynamic disease with unclear pathophysiology because of continually changing environments, demographics, and human behaviors.


Subject(s)
Humans , Female , Leishmania braziliensis/isolation & purification , Leishmaniasis, Cutaneous/etiology , Electrocoagulation/adverse effects , Leishmania braziliensis/genetics , Polymerase Chain Reaction , Leishmaniasis, Cutaneous/diagnosis , Middle Aged
12.
Mem. Inst. Oswaldo Cruz ; 112(9): 617-625, Sept. 2017. tab, graf
Article in English | LILACS | ID: biblio-894879

ABSTRACT

BACKGROUND Leishmanolysins have been described as important parasite virulence factors because of their roles in the infection of promastigotes and resistance to host's defenses. Leishmania (Viannia) braziliensis contains several leishmanolysin genes in its genome, especially in chromosome 10. However, the functional impact of such diversity is not understood, but may be attributed partially to the lack of structural data for proteins from this parasite. OBJECTIVES This works aims to compare leishmanolysin sequences from L. (V.) braziliensis and to understand how the diversity impacts in their structural and dynamic features. METHODS Leishmanolysin sequences were retrieved from GeneDB. Subsequently, 3D models were built using comparative modeling methods and their dynamical behavior was studied using molecular dynamic simulations. FINDINGS We identified three subgroups of leishmanolysins according to sequence variations. These differences directly affect the electrostatic properties of leishmanolysins and the geometry of their active sites. We identified two levels of structural heterogeneity that might be related to the ability of promastigotes to interact with a broad range of substrates. MAIN CONCLUSION Altogether, the structural plasticity of leishmanolysins may constitute an important evolutionary adaptation rarely explored when considering the virulence of L. (V.) braziliensis parasites.


Subject(s)
Humans , Leishmania braziliensis/genetics , Metalloendopeptidases/genetics , Protein Conformation , Genetic Variation , Models, Molecular
13.
Article in English | LILACS | ID: biblio-842775

ABSTRACT

ABSTRACT Traditional diagnostic methods used to detect American Tegumentary Leishmaniasis, such as histopathology using biopsy samples, culture techniques, and direct search for parasites, have low sensitivity and require invasive collection procedures. This study evaluates the efficiency of noninvasive sampling methods (swab) along with Polymerase Chain Reaction (PCR) for diagnosing American Tegumentary Leishmaniasis using skin and mucous samples from 25 patients who had tested positive for leishmaniasis. The outcome of the tests performance on swab samples was compatible with PCR results on biopsy samples. The findings have also shown that PCR-kDNA test is more efficient than PCR-HSP70 and qPCR tests (sensitivity of 92.3%, 40.7%, and 41%, respectively). Given the high sensitivity of the tests and the fact that the sampling method using swabs affords greater patient comfort and safety, it could be said that this method is a promising alternative to conventional biopsy-based methods for the molecular diagnosis of leishmaniasis.


Subject(s)
Humans , DNA, Kinetoplast/genetics , DNA, Protozoan/genetics , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/diagnosis , Biopsy , Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity , Skin Tests/methods , Specimen Handling
14.
Mem. Inst. Oswaldo Cruz ; 111(5): 349-354, May 2016. graf
Article in English | LILACS | ID: lil-782047

ABSTRACT

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.


Subject(s)
DNA Damage/genetics , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , Escherichia coli/genetics , Leishmania braziliensis/genetics , Mutation/genetics , Amino Acid Sequence , Escherichia coli Proteins/genetics , Escherichia coli/enzymology , Molecular Sequence Data
15.
Rev. Soc. Bras. Med. Trop ; 48(5): 555-559, Sept.-Oct. 2015. graf
Article in English | LILACS | ID: lil-763336

ABSTRACT

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.


Subject(s)
Female , Humans , Male , Leishmania braziliensis/genetics , Leishmania guyanensis/genetics , Leishmaniasis, Mucocutaneous/parasitology , Mucous Membrane/parasitology , DNA, Protozoan/analysis , Leishmania braziliensis/isolation & purification , Leishmania guyanensis/isolation & purification , Paraffin , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Protozoan Proteins/genetics
16.
Rev. Inst. Med. Trop. Säo Paulo ; 57(3): 257-262, May-Jun/2015. tab, graf
Article in English | LILACS | ID: lil-752598

ABSTRACT

Introduction: American tegumentary leishmaniasis (ATL) can be caused by Leishmania (Viannia) braziliensis complex. The evolution of ATL initially results in lesions and can develop into disseminated or diffuse forms. The genetic diversity of L. (V.) braziliensis in some endemic areas of Brazil has been poorly studied, such as in the state of São Paulo. This study analyzed the genetic diversity of L. (V.) braziliensis isolates collected from patients and dogs with LTA from the state of São Paulo. Methods: Leishmaniasis diagnosis was determined by PCR. The 132 biopsies were collected in different regions of Sao Paulo State, Brazil (36 municipalities). The genetic characterization of L. (V.) braziliensis isolates was tested by RFLP-PCR using DNA extracted from biopsies. The primer set amplified a specific region of Leishmania internal transcribed spacers of the ribosomal DNA locus. Results: Of the 132 samples, 52 (40%) were completely genotyped by RFLP-PCR (44 from human patients and eight from dogs). The results showed nine distinct patterns. The majority of the genotyped samples were from Sorocaba (30), and the others were distributed among 14 other municipalities. The first pattern was more frequent (29 samples), followed by pattern 2 (nine samples) and pattern 3 (three samples). Patterns 4, 6, 7, 8 and 9 were composed of two samples each and pattern 5 of one sample. Conclusion: These results suggest that polymorphic strains of L. (V.) braziliensis circulate in the state of São Paulo. These data agree with studies from other regions of Brazil, showing great variability among the natural populations of endemic foci. .


Introdução: A leishmaniose tegumentar americana (LTA) é causada pelo sub-gênero Leishmania (Viannia) braziliensis. A evolução da LTA resulta com a evolução das lesões iniciais. A diversidade genética de L. (V.) braziliensis em algumas áreas endêmicas brasileiras, como no estado de São Paulo, é pouco conhecida. Assim, este estudo teve como objetivo analisar a variabilidade genética de isolados de L. (V.) braziliensis coletados de biopsias de pacientes e cães com LTA no estado de São Paulo. Métodos: O diagnóstico da leishmaniose foi realizado por PCR. As 132 biópsias analisadas foram coletadas em diferentes regiões do Estado de São Paulo, Brasil (36 municípios). A caracterização genética de L. (V.) braziliensis foi realizada por RFLP-PCR utilizando DNA extraído das biopsias. O conjunto de iniciadores utilizado amplificou a região ITS de Leishmania. Resultados: Das 132 amostras analisadas, 52 (40%) foram completamente genotipadas por RFLP-PCR (44 de pacientes e oito de cães). Os resultados mostraram nove padrões distintos. A maioria das amostras genotipadas foi de Sorocaba (30), e as demais foram distribuídas entre 14 outros municípios. O primeiro padrão foi mais frequente (29 amostras), seguido pelo padrão 2 (nove amostras), padrão 3 (três amostras). Padrões 4, 6, 7, 8 e 9 foram compostos de duas amostras de cada um e o padrão 5, com uma amostra. Conclusão: Estes resultados sugerem que cepas polimórficas de L. (V.) braziliensis circulam no estado de São Paulo. Estes dados são concordantes com estudos em outras regiões do Brasil, mostrando grande variabilidade destas populações naturais de focos endêmicos. .


Subject(s)
Animals , Dogs , Humans , DNA, Protozoan/genetics , Genetic Variation , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/parasitology , Biopsy , Brazil , Genotype , Leishmania braziliensis/isolation & purification , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
17.
Rev. Soc. Bras. Med. Trop ; 48(2): 224-227, mar-apr/2015. tab
Article in English | LILACS | ID: lil-746225

ABSTRACT

Toxoplasmosis and leishmaniasis are two worldwide zoonoses caused by the protozoan parasites Toxoplasma gondii and Leishmania spp., respectively. This report describes the clinical and laboratorial findings of a co-infection with both parasites in a 4-year-old female dog suspected of ehrlichiosis that presented anemia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, tachyzoite-like structures to the lung imprints, and polymerase chain reaction (PCR) results positive for T. gondii (kidney, lung, and liver) and Leishmania spp. Co-infection with Toxoplasma gondii and Leishmania braziliensis was confirmed by sequencing; restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) confirmed an atypical T. gondii genotype circulating in dogs that has been reported to cause human congenital toxoplasmosis.


Subject(s)
Animals , Dogs , Female , Dog Diseases/parasitology , Leishmania braziliensis/genetics , Leishmaniasis/veterinary , Toxoplasma/genetics , Toxoplasmosis, Animal/parasitology , Coinfection/parasitology , Coinfection/veterinary , DNA, Protozoan , Dog Diseases/diagnosis , Genotype , Leishmaniasis/diagnosis , Leishmaniasis/parasitology , Polymorphism, Restriction Fragment Length , Polymerase Chain Reaction/veterinary , Toxoplasmosis, Animal/diagnosis
18.
Belo Horizonte; s.n; 2015. 48 p.
Thesis in Portuguese | LILACS, ColecionaSUS | ID: biblio-942652

ABSTRACT

Leishmania RNA vírus (LRVs) são comumente encontrados infectando espécies de Leishmania, especialmente as do subgênero Viannia. O LRV1 é um vírus RNA de cadeia dupla (Totiviridae) descrita pela primeira vez em cepas de Leishmania guyanensis e Leishmania braziliensis da região amazônica. Dados anteriores demonstraram que cepas infectados com LRV1 provoca um perfil próinflamatório mais Exacerbado parcialmente causada pela activação de receptoresToll like 3 (TLR3) através do cDNA viral. A presença de cepas infectadas pelo LRV1 já foi detectado em biópsia de pacientes com leishmaniose cutânea (LC) da cidade de Caratinga, Minas Gerais. No entanto, não há informações da frequência de LRV1 para outras regiões endêmicas do Estado. O principal objetivo deste estudo é a prospecção da presença de LRV1 em cepas de Leishmania braziliensis isoladas de regiões no estado de Minas Gerais incluindo São João das Missões, onde são relatados muitos casos de CL na reserva indígena de Xacriabá e também formas atípicas de leishmaniose cutânea causa das por L. braziliensis que nunca anteshaviam sido prospectadas para o LRV1. As reacções de PCR utilizaram iniciadores para a cápsideo viral e o cDNA de L. guyanensis (MHOM/BR /75/M4147) foi utilizado como controlo positivo. Foram prospectadas 41 cepas de L. braziliensis de várias regiões do Estado de Minas Gerais e alguns outros de outras regiões. A presença deLVR1 em parasitos isolados a partir de pacientes não foi observada. Esses resultados sugerem que a frequência de LRV1 em cepas de L. braziliensis parecem ser muito baixa na região Sudeste do Brasil.


Subject(s)
Male , Female , Humans , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/genetics , Molecular Biology/methods
19.
Belo Horizonte; s.n; 2015. 107 p.
Thesis in Portuguese | LILACS, ColecionaSUS | ID: biblio-941938

ABSTRACT

No Brasil, de todas as espécies do gênero Leishmania, a mais frequentemente encontrada parasitando o homem é a Leishmania (Viannia) braziliensis. Esta espécie pode causar um amplo espectro de manifestações, desde lesões únicas ao envolvimento mucoso, sendo esta última a complicação mais séria. Análises que visam acessar a variabilidade genética de Leishmania (Viannia) braziliensis são essenciais para o estudo de possíveis correlações entre manifestações clínicas de leishmaniose tegumentar americana (LTA) com parasitos geneticamente variantes e sua origem geográfica. O objetivo do estudo é analisar a variabilidade genética de isolados de L. braziliensis provenientes de diversas regiões de Minas Gerais. Foi realizado o diagnóstico clínico e molecular de indivíduos portadores de manifestações típicas e atípicas de várias regiões endêmicas do estado e os isolados separados em dois grupos amostrais: o grupo 1 contendo amostras de variadas macrorregiões do estado; e grupo 2 composto por amostras isoladas na terra indígena Xakriabá, em São João das Missões. A identificação específica de todas as amostras como L. braziliensis foi confirmada utilizando a técnica de PCR-g6pd. A análise da variabilidade genética foi realizada para os marcadores genéticos hsp70, Cpb, ITS1, g6pd e 6pgd. Na PCR-RFLP do hsp70 foram observados dois perfis de restrição: todas as amostras do grupo 1 e as cepas MG15 e MG16 do grupo 2 tiveram perfil de restrição indistinguível ao da cepa L. braziliensis referência, enquanto a maioria as amostras do grupo 2 exibiram perfil de restrição variante.


As cepas MG19 e MG27 (grupo 2) exibiram perfis de restrição diferentes das cepas referência utilizadas. O sequenciamento do fragmento da PCR-6pgd exibiu polimorfismos que diferenciam entre as espécies L. braziliensis e L. guyanensis nas amostras estudadas. Os perfis de restrição e as sequências foram utilizadas em análises estatísticas de classificação por similaridade por partição e hierárquico. A análise de partição corroborou a divisão das amostras em dois grupos, sugerindo uma maior variabilidade genética entre as amostras do grupo 2. As análises aglomerativas suportaram a de partição onde foi observada associação do grupo 2 com a origem geográfica e presença de manifestações atípicas de LTA não sendo observada associação com número de lesões. As sequências foram utilizadas em análises filogenéticas onde foi observado que tanto utilizando somente L. guyanensis quanto outras espécies filogeneticamente mais distantes de L. braziliensis como outgroup, a divisão em grupos proposta foi suportada. A partir do painel de amostras de L. braziliensis estudado conclui-se que em Minas Gerais observamos a presença de um grupo de amostras geneticamente variantes, associadas ao perfil atípico de lesões e provenientes da região norte do estado.


O fragmento obtido pela PCR do hsp70 foi sequenciado e foram observados polimorfismos inclusive no sítio de restrição da enzima HaeIII. Na PCR-RFLP do Cpb, as amostras do grupo 1 e as cepas MG15 e MG16 do grupo 2 tiveram perfil de restrição indistinguível ao da cepa referência, enquanto a maioria das amostras do grupo 2 apresentaram perfil de restrição correspondente à demais espécies do subgênero Viannia. O sequenciamento do fragmento revelou polimorfismos inclusive no sítio de restrição da enzima TaqI. Na PCR-RFLP do ITS1 foi observado que as amostras do grupo 1 e as cepas MG15 e MG16 exibiram perfil de restrição semelhante a L. guyanensis, enquanto as amostras do grupo 2 perfil de L. braziliensis.


Subject(s)
Male , Female , Humans , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/genetics , Polymerase Chain Reaction
20.
Belo Horizonte; s.n; 2015. 107 p.
Thesis in Portuguese | LILACS | ID: lil-773229

ABSTRACT

No Brasil, de todas as espécies do gênero Leishmania, a mais frequentemente encontrada parasitando o homem é a Leishmania (Viannia) braziliensis. Esta espécie pode causar um amplo espectro de manifestações, desde lesões únicas ao envolvimento mucoso, sendo esta última a complicação mais séria. Análises que visam acessar a variabilidade genética de Leishmania (Viannia) braziliensis são essenciais para o estudo de possíveis correlações entre manifestações clínicas de leishmaniose tegumentar americana (LTA) com parasitos geneticamente variantes e sua origem geográfica. O objetivo do estudo é analisar a variabilidade genética de isolados de L. braziliensis provenientes de diversas regiões de Minas Gerais. Foi realizado o diagnóstico clínico e molecular de indivíduos portadores de manifestações típicas e atípicas de várias regiões endêmicas do estado e os isolados separados em dois grupos amostrais: o grupo 1 contendo amostras de variadas macrorregiões do estado; e grupo 2 composto por amostras isoladas na terra indígena Xakriabá, em São João das Missões. A identificação específica de todas as amostras como L. braziliensis foi confirmada utilizando a técnica de PCR-g6pd. A análise da variabilidade genética foi realizada para os marcadores genéticos hsp70, Cpb, ITS1, g6pd e 6pgd. Na PCR-RFLP do hsp70 foram observados dois perfis de restrição: todas as amostras do grupo 1 e as cepas MG15 e MG16 do grupo 2 tiveram perfil de restrição indistinguível ao da cepa L. braziliensis referência, enquanto a maioria as amostras do grupo 2 exibiram perfil de restrição variante...


Subject(s)
Humans , Male , Female , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/genetics , Polymerase Chain Reaction
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