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1.
Rev Bras Parasitol Vet ; 29(2): e003520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520088

RESUMO

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.


Assuntos
Doenças do Gato/parasitologia , Leishmania braziliensis/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose/parasitologia , Animais , Anticorpos Antiprotozoários/sangue , Doenças do Gato/diagnóstico , Gatos , DNA de Protozoário/análise , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Filogenia , Reação em Cadeia da Polimerase/veterinária
2.
Mem Inst Oswaldo Cruz ; 115: e190413, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348407

RESUMO

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.


Assuntos
Adaptação Fisiológica/genética , Variações do Número de Cópias de DNA/genética , Leishmania braziliensis/genética , Temperatura , Transcriptoma/genética , Adaptação Fisiológica/fisiologia , Animais , Perfilação da Expressão Gênica , Perfil Genético
3.
Rev Soc Bras Med Trop ; 53: e20190433, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348430

RESUMO

INTRODUCTION: As highly specific molecular biology-based techniques may not be sensitive enough for the diagnosis of American tegumentary leishmaniasis (ATL), clinicians frequently rely on immunological tests before treatment initiation. Hence, the correct combination of diagnostic tests is imperative for ATL diagnosis. We aimed to evaluate the accuracy of the Montenegro (Leishmanin) skin test (MST) in polymerase chain reaction (PCR)-negative patients to accurately detect ATL. METHODS: Patients with a clinical picture compatible with ATL were divided into ATL (confirmed by lesion smear, culture indirect immunofluorescence, and/or histopathology) and no-ATL (diseases that can mimic leishmaniasis) groups. Conventional PCR for the minicircle kDNA of Leishmania was performed, and the MST was carried out for PCR-negative patients. RESULTS: Ninety-nine patients were included in this study, including 79 diagnosed with ATL (6 with mucocutaneous leishmaniasis) and 20 without ATL (no-ATL group). The MST showed a high sensitivity of 90.0% (95% confidence interval [CI] = 69.90-97.21) in PCR-negative patients that was 10% higher than the sensitivity reported in PCR-positive population (79.66%; 95% CI = 67.73-87.96). CONCLUSIONS: One of the most important reasons for PCR negativity among patients with active ATL is the presence of a strong cellular immunological response, especially in chronic and mucocutaneous leishmaniasis. This reinforces the considerable utility of the tests that detect cellular responses against Leishmania antigens such as the MST in PCR-negative patients when the performance in screening situations is questionable.


Assuntos
Testes Intradérmicos/métodos , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/diagnóstico , Adulto , Idoso , Doença Crônica , Estudos Transversais , DNA de Protozoário/genética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania braziliensis/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sensibilidade e Especificidade
4.
Parasit Vectors ; 13(1): 9, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31915065

RESUMO

BACKGROUND: Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice. METHODS: LbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. RESULTS: Leishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. CONCLUSIONS: LbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.


Assuntos
Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Interações Hospedeiro-Parasita , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania braziliensis/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandinas/biossíntese , Proteínas de Protozoários/genética
5.
Mem Inst Oswaldo Cruz ; 114: e190147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31553371

RESUMO

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.


Assuntos
Calpaína/genética , Genoma de Protozoário/genética , Leishmania braziliensis/química , Macrófagos Peritoneais/metabolismo , Animais , Western Blotting , Calpaína/efeitos dos fármacos , Calpaína/metabolismo , Calpaína/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Imuno-Histoquímica , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmania braziliensis/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Virulência
6.
Biomedica ; 39(Supl. 2): 58-65, 2019 08 01.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-31529834

RESUMO

INTRODUCTION: Mucosal leishmaniasis has a progressive course and can cause deformity and even mutilation in the affected areas. It is endemic in the American continent and it is mainly caused by Leishmania (Viannia) braziliensis. OBJECTIVE: To describe a series of mucosal leishmaniasis cases and the infectious Leishmania species. MATERIALS AND METHODS: We included 50 patients with a clinical diagnosis of mucosal leishmaniasis and parasitological confirmation, and we described their clinical and laboratory results. We performed species typing by PCR-RFLP using the miniexon sequence and hsp70 genes; confirmation was done by sequencing. RESULTS: The median time of disease evolution was 2.9 years (range: 1 month to 16 years). The relevant clinical findings included mucosal infiltration (94%), cutaneous leishmaniasis scar (74%), total loss of the nasal septum (24%), nasal deformity (22%), and mucosal ulceration (38%). The symptoms reported included nasal obstruction (90%), epistaxis (72%), rhinorrhea (72%), dysphonia (28%), dysphagia (18%), and nasal pruritus (34%). The histopathological study revealed a pattern compatible with leishmaniasis in 86% of the biopsies, and amastigotes were identified in 14% of them. The Montenegro skin test was positive in 86% of patients, immunofluorescence in 84%, and culture in 8%. Leishmania (V.) braziliensis was identified in 88% of the samples, L. (V) panamensis in 8%, and L. (V.) guyanensis and L. (L.) amazonensis in 2% respectively. CONCLUSION: In this study, we found a severe nasal disease with destruction and deformity of the nasal septum in 25% of the cases, probably associated with late diagnosis. Leishmania (V.) braziliensis was the predominant species. We described a case of mucosal leishmaniasis in Colombia caused by L. (L.) amazonensis for the first time.


Assuntos
Leishmania braziliensis/isolamento & purificação , Leishmania guyanensis/isolamento & purificação , Leishmaniose Mucocutânea/parasitologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Colômbia/epidemiologia , DNA de Protozoário/genética , Feminino , Genes de Protozoários , Proteínas de Choque Térmico HSP70/genética , Humanos , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania guyanensis/classificação , Leishmania guyanensis/genética , Leishmaniose Mucocutânea/complicações , Leishmaniose Mucocutânea/epidemiologia , Leishmaniose Mucocutânea/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Pele/parasitologia , Especificidade da Espécie , Adulto Jovem
7.
Mem Inst Oswaldo Cruz ; 114: e190111, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31433006

RESUMO

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.


Assuntos
Antimônio/farmacologia , Resistência a Medicamentos/genética , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/genética , Tripanossomicidas/farmacologia , Aquagliceroporinas/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Humanos , Leishmania braziliensis/genética , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
8.
BMC Infect Dis ; 19(1): 747, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455227

RESUMO

BACKGROUND: Leishmaniasis caused by different species of Leishmania affect 98 countries worldwide. Visceral Leishmaniasis (VL) is the mortal clinical presentation of the disease that causes the dead to more than 90% of the patients who suffer it. The diagnosis of VL is made by the direct observation of the parasite in bone marrow, spleen and/or liver aspirates that requires complex proceedings. Therefore, serum samples are submitted to Indirect Immunofluorescence to identify the presence of anti-Leishmania antibodies. Despite the variability in the diagnostic performance of the Immunochromatographic Tests (ICTs), there are many evidences that suggest that ICTs can be used for epidemiological screening. However, in Colombia there are not any evidence about the performance of the ICTs for VL diagnosis, both for human and canine serum samples. Therefore, this study evaluated the diagnostic performance of 4 ICTs for VL (2 ICTs in human sera and 2 ICTs in canine sera) in samples from endemic areas of Colombia. METHODS: We selected a total of 156 human serum samples (82 positive and 74 negative for VL) and 126 canine serum samples (71 positive and 54 negative) diagnosed by in house Indirect Immunofluorescence (IIF). The samples were submitted to the ICTs following the manufacturers' instructions. Statistical analysis was performed to evaluate the diagnostic performance of each ICT in comparison with the IIF. PCR for HSP70 gene and sanger sequencing was performed in samples with negative results for both ICTs. RESULTS: The sensitivity (S) of both ICTs for human samples (Ad-bio Leishmania IgG/IgM Combo Rapid Test and Kalazar Detect™) was 91.5% and specificity (E) were 93.2 and 89.2% respectively, while for the ICTs tested on canine samples (Kalazar Detect™ Rapid Test, Canine and DPP® CVL rapid test) we found S values between 82.9 and 85.7% and E values between 79.6 and 92.6%. We found L. infantum by PCR and sequencing in 2 human samples, and L. braziliensis and L. amazonensis in canine serum samples that were negative by both ICTs. CONCLUSIONS: We conclude that both tests evaluated on human samples have a similar diagnostic performance, while the Kalazar Detect™ Rapid Test, Canine showed a better diagnostic performance than the DPP® CVL rapid test evaluated on canine samples. Also, we suggest that it is necessary to design tests with antigens of the circulating strains to increase its diagnostic utility.


Assuntos
Doenças do Cão/diagnóstico , Imunoensaio/métodos , Leishmaniose Visceral/diagnóstico , Animais , Colômbia , Testes Diagnósticos de Rotina , Doenças do Cão/parasitologia , Cães , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
9.
Am J Trop Med Hyg ; 101(4): 780-788, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31407656

RESUMO

American tegumentary leishmaniasis is an endemic anthropozoonosis undergoing expansion on the American continent. The disease is caused by several Leishmania species and it is manifested as cutaneous and mucocutaneous leishmaniasis. In this study, we evaluate the viability of high-resolution melt polymerase chain reaction (HRM-PCR) analysis to differentiate four closely related Leishmania species as a routine tool for the diagnosis of leishmaniasis. For this purpose, biopsy specimens from cutaneous and mucocutaneous lesions were taken from 132 individuals from endemic and non-endemic areas for leishmaniasis. Each sample was processed for parasitological, histopathological, and molecular analysis. Positive biopsy samples were analyzed by HRM-PCR of a 144-bp heat-shock protein (hsp70) gene fragment, and new cases were confirmed by sequencing. Of the 132 samples analyzed, 36 (27%) were positive for Leishmania spp., of which 86% were from cutaneous lesions and 14% from mucocutaneous lesions. We identified Leishmania (Viannia) braziliensis (84%), Leishmania (Leishmania) infantum (13%), and Leishmania (Leishmania) amazonensis (3%) in cutaneous lesions, and L. (V.) braziliensis (40%), L. (L.) infantum (20%), L. (L.) amazonensis (20%), and Leishmania (Viannia) guyanensis (20%) in mucocutaneous lesions. The main purpose of this research was to report for the first time in Paraguay the presence of L. (L.) amazonensis and L. (V.) guyanensis in patients with cutaneous and mucocutaneous lesions, using the HRM-PCR technique. In addition, we report the presence of additional new cases of L. (L.) infantum in cutaneous lesions.


Assuntos
Leishmania braziliensis/isolamento & purificação , Leishmania guyanensis/isolamento & purificação , Leishmania infantum/isolamento & purificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Mucocutânea/epidemiologia , Adulto , Idoso , Feminino , Geografia , Humanos , Leishmania braziliensis/genética , Leishmania guyanensis/genética , Leishmania infantum/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/patologia , Masculino , Pessoa de Meia-Idade , Paraguai/epidemiologia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Pele/parasitologia , Pele/patologia
10.
Artigo em Inglês | MEDLINE | ID: mdl-31355149

RESUMO

Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1ß, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.


Assuntos
Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/imunologia , Ativação de Macrófagos , Receptor 4 Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Técnicas de Inativação de Genes , Glicoesfingolipídeos/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico , Psychodidae/parasitologia , Receptor 4 Toll-Like/genética , Fatores de Virulência
11.
PLoS Negl Trop Dis ; 13(7): e0007532, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31310601

RESUMO

BACKGROUND: Cutaneous leishmaniasis (CL), caused by Leishmania braziliensis, is the most important presentation of tegumentary leishmaniasis (TL) in Latin American. While the role of dogs as reservoirs of Leishmania infantum, and the clinic features of canine visceral leishmanisis are well described, little is known about the importance of dogs in the transmission of L. braziliensis to humans. In the present study, we determine the frequency of L. braziliensis infection in dogs with cutaneous and mucosal ulcers in an endemic area of CL. We also describe the clinical manifestations and histopathologic features, and determine if the parasites isolated from dogs are genetically similar to those found in humans. METHODOLOGY: This is a cross sectional study in which 61 dogs living in an endemic area of CL and presenting ulcerated lesions were evaluated. Detection of L. braziliensis DNA by polymerase chain reaction (PCR) in skin biopsies, serology and leishmania skin test (LST) with soluble L. braziliensis antigen were performed. The clinical and histopathologic features were described, and we compared the genotypic characteristics of isolates obtained from dogs and humans. PRINCIPAL FINDINGS: The sensitivity of the three tests together to detect exposure was 89% and the concordance between the tests was high. The skin lesions were most frequent in the ears, followed by scrotal sac. The PCR was positive in 41 (67%) of animals, and the lesions in the snout, followed by the scrotal sac and ears were the sites where parasite DNA was most detected. There were genotype similarities between L.braziliensis isolates from dogs and humans. CONCLUSIONS: The high frequency of L. braziliensis infection in dogs with ulcers and the similarities between the isolates of L. braziliensis and cutaneous leishmaniasis in dogs and humans in an endemic area of TL, raise the possibility of an important role of dogs in the transmission chain of L. braziliensis.


Assuntos
Reservatórios de Doenças/parasitologia , Doenças do Cão/parasitologia , Leishmania braziliensis/genética , Leishmaniose Cutânea/veterinária , Pele/patologia , Animais , Brasil , Estudos Transversais , DNA de Protozoário/genética , Cães , Doenças Endêmicas , Feminino , Leishmaniose Cutânea/patologia , Masculino , Técnicas de Diagnóstico Molecular , Sensibilidade e Especificidade , Testes Sorológicos , Pele/parasitologia
12.
PLoS Negl Trop Dis ; 13(6): e0007382, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170148

RESUMO

Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis (CL) in Latin America, is characterized by major differences in basic biology in comparison with better-known Leishmania species. It is also associated with a high phenotypic and possibly genetic diversity that need to be more adequately defined. Here we used whole genome sequences to evaluate the genetic diversity of ten L. braziliensis isolates from a CL endemic area from Northeastern Brazil, previously classified by Multi Locus Enzyme Electrophoresis (MLEE) into ten distinct zymodemes. These sequences were first mapped using the L. braziliensis M2904 reference genome followed by identification of Single Nucleotide Polymorphisms (SNPs). A substantial level of diversity was observed when compared with the reference genome, with SNP counts ranging from ~95,000 to ~131,000 for the different isolates. When the genome data was used to infer relationship between isolates, those belonging to zymodemes Z72/Z75, recovered from forested environments, were found to cluster separately from the others, generally associated with more urban environments. Among the remaining isolates, those from zymodemes Z74/Z106 were also found to form a separate group. Phylogenetic analyses were also performed using Multi-Locus Sequence Analysis from genes coding for four metabolic enzymes used for MLEE as well as the gene sequence coding for the Hsp70 heat shock protein. All 10 isolates were firmly identified as L. braziliensis, including the zymodeme Z26 isolate previously classified as Leishmania shawi, with the clustering into three groups confirmed. Aneuploidy was also investigated but found in general restricted to chromosome 31, with a single isolate, from zymodeme Z27, characterized by extra copies for other chromosomes. Noteworthy, both Z72 and Z75 isolates are characterized by a much reduced heterozygosity. Our data is consistent with the existence of distinct evolutionary groups in the restricted area sampled and a substantial genetic diversity within L. braziliensis.


Assuntos
Ecótipo , Variação Genética , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Brasil , Humanos , Leishmania braziliensis/isolamento & purificação , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
14.
Int J Mol Sci ; 20(6)2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30875904

RESUMO

Serine proteinases in Leishmania (Viannia) braziliensis promastigotes were assessed in this work. This study included the investigation of the enzymatic activity of subcellular fractions obtained from benzamidine affinity chromatography, reverse transcription polymerase chain reactions, and in silico assays of subcellular localization of subtilisin. Promastigote serine proteinases showed gelatinolytic activity with molecular masses of 43 kDa to 170 kDa in the cytosolic fraction and 67 kDa to 170 kDa in the membranous fraction. Serine proteinase activities were detected using N-benzyloxycarbonyl-l-phenylalanyl-l-arginine 7-amino-4-methylcoumarin (Z-FR-AMC) and N-succinyl-l-alanine-l-phenylalanine-l-lysine 7-amino-4-methylcoumarin (Suc-AFK-AMC) as substrates in the cytosolic fraction (Z-FR-AMC = 392 ± 30 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 252 ± 20 µmol.min-1 mg of protein-1) and in the membranous fraction (Z-FR-AMC = 53 ± 5 µmol.min-1 mg of protein-1 and Suc-AFK-AMC = 63.6 ± 6.5 µmol.min-1 mg of protein-1). Enzyme specificity was shown by inhibition with aprotinin (19% to 80% inhibition) and phenylmethanesulfonyl fluoride (3% to 69%), depending on the subcellular fraction and substrate. The expression of subtilisin (LbrM.13.0860 and LbrM.28.2570) and tryparedoxin peroxidase (LbrM.15.1080) genes was observed by the detection of RNA transcripts 200 bp, 162 bp, and 166 bp long, respectively. Subsequent in silico assays showed LbrM.13.0860 can be located in the cytosol and LbrM.28.2570 in the membrane of the parasite. Data obtained here show the subcellular distribution and expression of serine proteinases, including the subtilisin-like serine proteinases in L. (V.) braziliensis promastigotes.


Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Leishmania braziliensis/enzimologia , Serina Proteases/genética , Serina Proteases/metabolismo , Cromatografia de Afinidade , Simulação por Computador , Regulação da Expressão Gênica , Leishmania braziliensis/genética , Peso Molecular , Peroxidases/genética , Peroxidases/metabolismo , Transporte Proteico , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sensibilidade e Especificidade , Subtilisina/genética , Subtilisina/metabolismo
15.
Int J Parasitol Drugs Drug Resist ; 11: 139-147, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30850347

RESUMO

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites.


Assuntos
Resistência a Medicamentos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose Cutânea/parasitologia , Fosforilcolina/análogos & derivados , Transporte Biológico , Perfilação da Expressão Gênica , Humanos , Leishmania braziliensis/genética , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologia , Proteínas de Protozoários/genética , Análise de Sequência de RNA
16.
Acta Trop ; 193: 12-17, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30772331

RESUMO

In Brazil, the visceral leishmaniasis (VL) is caused by Leishmania infantum, while the tegumentary leishmaniasis (TL) etiological agents are mainly Leishmania braziliensis and Leishmania amazonensis. The canine visceral leishmaniasis (CVL) diagnosis is an important step of the VL control program in Brazil, which involves the elimination of infected dogs, the main urban VL reservoirs. The current serology-based diagnostic tests have shown cross-reactivity between these three species, whereas molecular diagnosis allows high sensitivity and specie identification. In the present study, 349 dogs of the metropolitan region of Belo Horizonte (Minas Gerais state) were screened by conjunctival swab and the samples analyzed by ITS-1 nested PCR. Thirty dogs (8.5%) tested positive. The RFLP of amplicons using HaeIII demonstrated that 17/30 samples presented a banding pattern compatible with L. infantum, 4/30 matched with L. amazonenis, 1/30 with L. braziliensis and 8/30 showed a mixed infection pattern. The samples that were distinct of L. infantum or presented a mixed pattern were submitted to RFPL with HaeIII and RsaI enzymes that confirmed the mixed pattern. Such patterns were also confirmed by Sanger Sequencing. The results pointed eight dogs with mixed infections and the establishment of TL causing species in the Belo Horizonte dog population. These findings highlight the need for more comprehensive epidemiological studies, since the TL transmission profile might be changing. This study also shows the potential of the ITS1-nPCR associated with RFLP for the proper Leishmania diagnosis and typing in the dog population.


Assuntos
Coinfecção/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Leishmaniose Cutânea/veterinária , Leishmaniose Visceral/veterinária , Leishmaniose/veterinária , Animais , Brasil , Coinfecção/diagnóstico , Coinfecção/parasitologia , Cães , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmania infantum/genética , Leishmania infantum/imunologia , Leishmaniose/diagnóstico , Leishmaniose/parasitologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Testes Sorológicos
17.
BMC Genomics ; 20(1): 118, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30732584

RESUMO

BACKGROUND: The leishmaniasis are parasitic diseases caused by protozoans of the genus Leishmania, highly divergent eukaryotes, characterized by unique biological features. To survive in both the mammalian hosts and insect vectors, these pathogens make use of a number of mechanisms, many of which are associated with parasite specific proteases. The metalloprotease GP63, the major Leishmania surface antigen, has been found to have multiple functions required for the parasite's survival. GP63 is encoded by multiple genes and their copy numbers vary considerably between different species and are increased in those from the subgenus Viannia, including L. braziliensis. RESULTS: By comparing multiple sequences from Leishmania and related organisms this study sought to characterize paralogs in silico, evaluating their differences and similarities and the implications for the GP63 function. The Leishmania GP63 genes are encoded on chromosomes 10, 28 and 31, with the genes from the latter two chromosomes more related to genes found in insect or plant parasites. Those from chromosome 10 have experienced independent expansions in numbers in Leishmania, especially in L. braziliensis. These could be clustered in three groups associated with different mRNA 3' untranslated regions as well as distinct C-terminal ends for the encoded proteins, with presumably distinct expression patterns and subcellular localizations. Sequence variations between the chromosome 10 genes were linked to intragenic recombination events, mapped to the external surface of the proteins and predicted to be immunogenic, implying a role against the host immune response. CONCLUSIONS: Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes, possibly related to the pathogenesis of L. braziliensis and closely related species within the mammalian host. They also indicate different functions associated to genes mapped to different chromosomes. For the chromosome 10 genes, variable subcellular localizations were found to be most likely associated with multiple functions and target substrates for this versatile protease.


Assuntos
Simulação por Computador , Variação Genética , Evasão da Resposta Imune/genética , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Metaloendopeptidases/genética , Sequência de Aminoácidos , Cromossomos/genética , Epitopos de Linfócito B/imunologia , Evolução Molecular , Leishmania braziliensis/patogenicidade , Metaloendopeptidases/química , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Virulência/genética
18.
Parasit Vectors ; 12(1): 60, 2019 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-30683152

RESUMO

BACKGROUND: Glycosylphosphatidylinositol is a surface molecule important for host-parasite interactions. Mannosyltransferase (GPI-14) is an essential enzyme for adding mannose on the glycosylphosphatidyl group. This study attempted to overexpress the GPI-14 gene in Leishmania braziliensis to investigate its role in the antimony-resistance phenotype of this parasite. RESULTS: GPI-14 mRNA levels determined by quantitative real-time PCR (qRT-PCR) showed an increased expression in clones transfected with GPI-14 compared to its respective wild-type line. In order to investigate the expression profile of the surface carbohydrates of these clones, the intensity of the fluorescence emitted by the parasites after concanavalin-A (a lectin that binds to the terminal regions of α-D-mannosyl and α-D-glucosyl residues) treatment was analyzed. The results showed that the clones transfected with GPI-14 express 2.8-fold more mannose and glucose residues than those of the wild-type parental line, indicating effective GPI-14 overexpression. Antimony susceptibility tests using promastigotes showed that clones overexpressing the GPI-14 enzyme are 2.4- and 10.5-fold more resistant to potassium antimonyl tartrate (SbIII) than the parental non-transfected line. Infection analysis using THP-1 macrophages showed that amastigotes from both GPI-14 overexpressing clones were 3-fold more resistant to SbIII than the wild-type line. CONCLUSIONS: Our results suggest the involvement of the GPI-14 enzyme in the SbIII-resistance phenotype of L. braziliensis.


Assuntos
Antimônio/farmacologia , Antiprotozoários/farmacologia , Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Manosiltransferases/metabolismo , Resistência a Medicamentos , Glicosilfosfatidilinositóis/metabolismo , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/genética , Manosiltransferases/genética , Fenótipo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
19.
Clin Microbiol Infect ; 25(4): 515.e5-515.e7, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30616010

RESUMO

OBJECTIVES: We aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis. METHODS: A cross-sectional study was performed in all individuals with CL without nasal lesions (n = 153) attended within 2 years in an endemic area of L. (Viannia) braziliensis in Bahia (Brazil). An otorhinolaryngologist assessed the clinical status of the nasal mucosa by anterior rhinoscopy and endoscopic examinations. Swab samples were collected for parasite DNA detection by PCR from all individuals before standard treatment for leishmaniasis. A second evaluation 3 months after treatment was performed to assess clinical outcomes. RESULTS: Parasite DNA was detected in 7.8% (12/153) of clinically healthy nasal mucosa of individuals with CL. Interestingly, DNA was more frequently identified in individuals with more skin lesions (median 1.5, interquartile range (IQR) 1-3.5 versus 1.0, IQR 1-1.5; p 0.044), or larger injuries (median 2.7, IQR 2-3.8 versus 1.6, IQR 1-2.5; p 0.013). Additionally, the disease of those individuals with positive PCR evolved more frequently to unusual forms of leishmaniasis (recidiva cutis and disseminated) (45.5% (5/11) versus 11.5% (14/122); p 0.009), and required more cycles of treatment to reach clinical cure (median 2, IQR 1-4 versus 1, IQR 1-2; p 0.05). CONCLUSION: These findings suggest an early parasite tropism to nasal mucosa in L. (Viannia) braziliensis infection and a clinical phenotype of CL cases associated with parasite DNA in nasal mucosa. Future studies should evaluate whether PCR of nasal swab samples could serve as a prognostic tool for individuals at risk of mucocutaneous leishmaniasis.


Assuntos
DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Mucosa Nasal/química , Adulto , Brasil , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tropismo/fisiologia , Adulto Jovem
20.
RNA Biol ; 16(5): 639-660, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30689499

RESUMO

Leishmaniasis is a worldwide public health problem caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the most important species responsible for tegumentary leishmaniases in Brazil. An understanding of the molecular mechanisms underlying the success of this parasite is urgently needed. An in-depth study on the modulation of gene expression across the life cycle stages of L. braziliensis covering coding and noncoding RNAs (ncRNAs) was missing and is presented herein. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). Gene ontology (GO) analysis indicated stage-specific enriched biological processes. A computational pipeline and 5 ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions and locations; many of them had a distinct expression profile compared to that of their protein-coding neighbors. Thirty-five putative ncRNAs were submitted to northern blotting analysis, and one or more hybridization-positive signals were observed in 22 of these ncRNAs. This work presents an overview of the L. braziliensis transcriptome and its adjustments throughout development. In addition to determining the general features of the transcriptome at each life stage and the profile of protein-coding transcripts, we identified and characterized a variety of noncoding transcripts. The novel putative ncRNAs uncovered in L. braziliensis might be regulatory elements to be further investigated.


Assuntos
Perfilação da Expressão Gênica/métodos , Leishmania braziliensis/crescimento & desenvolvimento , RNA de Protozoário/genética , Análise de Sequência de RNA/métodos , Animais , Brasil , Biologia Computacional/métodos , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Humanos , Insetos/parasitologia , Leishmania braziliensis/genética , Mamíferos/parasitologia , RNA não Traduzido/genética
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